Feeds:
Posts
Comments

Archive for the ‘Proteomics’ Category

Aging Protein Signature

Posted in Biomarkers & Medical Diagnostics, Biomedical Measurement Science, Cardiovascular Research, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Curation, Diagnostics and Lab Tests, Disease Biology, Epigenetics and Cardiovascular Risks, Innovation in Immunology Diagnostics, Proteins, Proteomics, Translational Science, tagged , on October 23, 2015| Leave a Comment »

Aging Protein Signature

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

Anti-Aging Protein GDF11: Does it Work?

October 22, 2015 by
https://beyondthedish.wordpress.com/2015/10/22/anti-aging-protein-gdf11-does-it-work/
The age old riddle remains somewhat a riddle.  Is there a fountain of youth?

The protein is called GDF11 and some scientists claim that is can rejuvenate older laboratory animals and make them healthier. Sounds like science fiction, but could it be true?

Several decades ago, in the 1950s, some creative and enterprising scientists connected the circulatory systems of two inbred mice, one of which was old and the second of which was young. The blood from the young mouse seemed to rejuvenate the older mouse. That led to a question: “If blood from younger mouse rejuvenated the older mouse, what was it in the blood that did it?” Further work has landed on GDF11 as the rejuvenating protein, but the experimental path to this protein has been fraught with false starts, bumps, and wrong turns. New work by a team of Harvard University scientists hopes to set the record straight on GDF11.

Work by Harvard stem cell biologist Amy Wagers, cardiologist Richard Lee and the members of their laboratories and their collaborators have discovered that the blood concentrations of GDF11 drop in mice as they age. Such a finding is a correlation, which might be suggestive, but it fall short of proving that GDF11 is an anti-aging protein. However, Wagers and Lee and their colleagues also showed that when older mice are injected with GDF11, the protein partially reverses the thickening of the heart that comes with age. Wagers and her team also showed in two papers that were published in the journal Science that administration of GDF11 can rejuvenate the muscles and brains of older mice.

Wagers’ findings, however, received some push-back in May, 2015. According to Jocelyn Kaiser, writing at the Science web site, David Glass, who works at the Novartis Institutes for Biomedical Research in Cambridge, Massachusetts, and his colleagues have made use of an antibody that specifically binds to GDF11 to detect the protein and measure its concentration in the blood and tissues. Experiments with the anti-GDF11 antibody revealed that blood levels of GDF11 increase as rats and people get older. Also, in the hands of Glass and his team, injected GDF11 protein inhibited muscle regeneration in young mice. Furthermore, work from Steven Houser’s group at Temple University in Philadelphia, Pennsylvania, has shown that injections of GDF11 do not decrease the age-related thickening of the hearts of older mice. Now we have a genuine scientific controversy: so who’s right?

Wagers and Lee have concluded that the specific assay Novartis used to detect GDF11 and a related protein (GDF8 or myostatin) did not work properly. In their own experiments, the combined efforts of the Wagers and Lee teams showed that the main protein detected by the antibody test designed and used by the Glass group is immunoglobulin (antibodies). The levels of antibody proteins in the blood are known to rise in the blood as people get older. As a control, when the Wagers and Lee group used the Novartis-designed test to measure the proteins levels of laboratory mice that do not possess the gene that encodes antibodies, the blood of those mice tested negative. According to Jocelyn Kaiser, these data were published in a paper that appeared in the journal Circulation Research.

Wagers summarized the results of her and Lee’s laboratories, “They actually had very consistent findings to ours with respect to the blood levels of GDF11/8 with the antibody we all used.” However, according to Wagers, “their interpretation was confused by this case of mistaken identity.” To corroborate your point, Wagers cited a recently published study by scientists from the University of California, San Francisco, who found that GDF11/8 blood levels decline as people age, and are low in heart disease patients. These results support the hypothesis that GDF11 has antiaging activity.

The Harvard team’s paper also examined the results from the Houser laboratory. According to Wagers, Houser and his colleagues utilized commercially purchased GDF11, and this source of protein can vary in activity and levels. Wagers noted that it “wasn’t something that affected us early on, but we figured out it was an issue. The variability of commercially purchased GDF11 might explain why Houser and his colleagues were unable to see any results from injected GDF11. Houser and his team were quite careful to make sure that they injected the same dose of GDF11 as the Wagers and Lee. However, Wagers pointed out that if only a fraction of the protein was as active as the protein used by Wagers and Lee, then it is likely that Houser and his group actually used a lower effective dose than the Harvard group. Lee has also noted that he and his group have data that suggests that the GDF11 dose they used was actually higher than they initially thought.

Wagers and others also showed that daily injections of GDF11 can shrink heart muscle in both old and new mice, and, incredibly, the mice also lost weight. “We don’t have much insight into that right now, but we’re looking into it,” Wagers says. Wagers suspects that GDF11 only works within a particular therapeutic concentration, outside of which is will not work and above which it might cause side effects that are harmful.

What does the competition think? Houser thinks that Wager and Lee are probably correct that at least one of the assays used by the Novartis team to measure GDF11 detected immunoglobulin. However, both Houser David Glass have pointed out that the Novartis team used a different GDF11 detection assay whose accuracy was not challenged by the work in this new paper.

Houser remains sanguine about finding molecules that can delay aging.  “I’m going to be 65 in a couple months. I’d love to have something that improves my heart, brain, and muscle function,” said Houser. “I think the field is going to figure this out and this is another piece of the puzzle.”

The jury is still out when it comes to GDF11, but Wagers and Lee have made a positive contribution to a robust and thrillingly interesting scientific discussion.

Read Full Post »

lncRNAs in Human Cancers

Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Clinical & Translational, Clinical Genomics, Curation, Disease Biology, Proteomics, RNA Biology, tagged , on October 13, 2015| Leave a Comment »

lncRNAs in Human Cancers

Curator: Larry H. Bernstein, MD, FCAP

UPDATED 9/26/2021

 

Characterizing lncRNAs in Human Cancers

Changes in long non-coding RNA expression levels are highly indicative of specific cancer types, perhaps even more so than protein-coding genes, according to a new study published today in Cancer Cell.

https://www.genomeweb.com/cancer/penn-scientists-lead-study-characterizing-lncrnas-human-cancers?

Scientists led by first authors Xiaohui Yan, Zhongyi Hu, and Yi Feng and senior authors Chi Dan and Lin Zhang, all from the University of Pennsylvania’s Perelman School of Medicine, characterized alterations in long non-coding RNAs (lncRNAs) in 5,037 tumor specimens comprising 13 cancer types from The Cancer Genome Atlas. They looked at differential changes to lncRNAs at the transcriptional, genomic, and epigenomic level to identify cancer-driving lncRNAs and predict their function.

The study found 2,316 lncRNAs altered in all 13 cancer types. As part of a more detailed analysis, the study looked at disregulation of lncRNA expression in seven cancers. Expression could be both up-regulated and down-regulated compared to normal cells, with, on average, 15 percent being up-regulated and 11 percent being down-regulated in the cancers.

UPDATED 9/26/2021

Here is some recent literature on various lncRNAs that are linked to colorectal cancer growth.

The long noncoding RNA LUCAT1 promotes colorectal cancer cell proliferation by antagonizing Nucleolin to regulate MYC expression.

Wu, R., Li, L., Bai, Y. et al. The long noncoding RNA LUCAT1 promotes colorectal cancer cell proliferation by antagonizing Nucleolin to regulate MYC expression. Cell Death Dis 11, 908 (2020). https://doi.org/10.1038/s41419-020-03095-4

Abstract

The long noncoding RNA (lncRNA) LUCAT1 was recently reported to be upregulated and to play an essential role in multiple cancer types, especially colorectal cancer (CRC), but the molecular mechanisms of LUCAT1 in CRC are mostly unreported. Here, a systematic analysis of LUACT1 expression is performed with data from TCGA database and clinic CRC samples. LUCAT1 is identified as a putative oncogene, which is significantly upregulated in CRC and is associated with poor prognosis. Loss of LUCAT1 restricts CRC proliferative capacities in vitro and in vivo. Mechanically, NCL is identified as the protein binding partner of LUCAT1 by using chromatin isolation by RNA purification coupled with mass spectrometry (ChIRP-MS) and RNA immunoprecipitation assays. We also show that NCL directly binds to LUCAT1 via its putative G-quadruplex-forming regions from nucleotides 717 to 746. The interaction between LUCAT1 and NCL interferes NCL-mediated inhibition of MYC and promote the expression of MYC. Cells lacking LUCAT1 show a decreased MYC expression, and NCL knockdown rescue LUCAT1 depletion-induced inhibition of CRC cell proliferation and MYC expression. Our results suggest that LUCAT1 plays a critical role in CRC cell proliferation by inhibiting the function of NCL via its G-quadruplex structure and may serve as a new prognostic biomarker and effective therapeutic target for CRC.

Long noncoding RNA CMPK2 promotes colorectal cancer progression by activating the FUBP3–c-Myc axis

Gao, Q., Zhou, R., Meng, Y. et al. Long noncoding RNA CMPK2 promotes colorectal cancer progression by activating the FUBP3–c-Myc axis. Oncogene 39, 3926–3938 (2020). https://doi.org/10.1038/s41388-020-1266-8

Abstract

Long noncoding RNAs (lncRNAs) have been shown to play crucial roles in cancer long noncoding RNAs (lncRNAs) have been known to play crucial roles in cancer development and progression by regulating chromatin dynamics and gene expression. However, only a few lncRNAs with annotated functions in the progression of colorectal cancer (CRC) have been identified to date. In the present study, the expression of lncCMPK2 was upregulated in CRC tissues and positively correlated with clinical stages and lymphatic metastasis. The overexpression of lncCMPK2 promoted the proliferation and cell cycle transition of CRC cells. Conversely, the silencing of lncCMPK2 restricted cell proliferation both in vitro and in vivo. lncCMPK2 was localized to the nucleus of CRC cells, bound to far upstream element binding protein 3 (FUBP3), and guided FUBP3 to the far upstream element (FUSE) of the c-Myc gene to activate transcription. lncCMPK2 also stabilized FUBP3. These results provide novel insights into the functional mechanism of lncCMPK2 in CRC progression and highlight its potential as a biomarker of advanced CRC and therapeutic target.

Long Noncoding RNA MIR17HG Promotes Colorectal Cancer Progression via miR-17-5p

Jie Xu, Qingtao Meng, Xiaobo Li, Hongbao Yang, Jin Xu, Na Gao, Hao Sun, Shenshen Wu, Giuseppe 

Familiari, Michela Relucenti, Haitao Zhu, Jiong Wu and Rui ChenDOI: 10.1158/0008-5472.CAN-18-3880 Published October 2019

Abstract

Immune dysregulation plays a vital role in colorectal cancer initiation and progression. Long noncoding RNAs (lncRNA) exhibit multiple functions including regulation of gene expression. Here, we identified an immune-related lncRNA, MIR17HG, whose expression was gradually upregulated in adjacent, adenoma, and colorectal cancer tissue. MIR17HG promoted tumorigenesis and metastasis in colorectal cancer cells both in vitro and in vivo. Mechanistically, MIR17HG increased the expression of NF-κB/RELA by competitively sponging the microRNA miR-375. In addition, RELA transcriptionally activated MIR17HG in a positive feedback loop by directly binding to its promoter region. Moreover, miR-17-5p, one of the transcribed miRNAs from MIR17HG, reduced the expression of the tumor suppressor B-cell linker (BLNK), resulting in increased migration and invasion of colorectal cancer cells. MIR17HG also upregulated PD-L1, indicating its potential role in immunotherapy. Overall, these findings demonstrate that MIR17HG plays an oncogenic role in colorectal cancer and may serve as a promising therapeutic target.

Significance: These findings provide mechanistic insight into the role of the lncRNA MIR17HG and its miRNA members in regulating colorectal cancer carcinogenesis and progression.

Read Full Post »

Protein folding

Posted in Amino acids, Cell Biology, Proteins, Proteomics, tagged , , on September 9, 2015| Leave a Comment »

Protein folding

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Innovation

Series E. 2; 4.8

Protein folding is the process by which a protein structure assumes its functional shape or conformation. It is the physical process by which a polypeptide folds into its characteristic and functional three-dimensional structure from random coil.

The importance of protein folding

Joachim Pietzsch

Proteins are the biological workhorses that carry out vital functions in every cell. To carry out their task, proteins must fold into a complex three-dimensional structure, but what tells a protein which shape it should be and how does it achieve this?

Of all the molecules found in living organisms, proteins are the most important. They are used to support the skeleton, control senses, move muscles, digest food, defend against infections and process emotions. Proteins come in all shapes and sizes (Fig. 1)

  • they can be round (like haemoglobin), long (like collagen), strong (like spectrin c which protects erythrocytes (the cells that carry oxygen from the lungs to our tissues) from the powerful shearing forces they’re exposed to), or elastic (like titin, which controls muscle stretching and contraction). The name protein is derived from the Greek word prôtos, meaning – primary, or first rank of importance, and with good reason.

These are the most abundant component within a cell, more than half the dry weight of a cell is made up of proteins, and they have a range of indispensable roles; for example, enzymes, the biocatalysts that carry out crucial biochemical reactions in every cell that would otherwise be too slow to sustain life.

What is remarkable is that the more than 100,000 proteins in our bodies are produced from a set of only 20 building blocks, known as amino acids. All amino acids have the same basic structure, an amino group, a carboxyl group and a hydrogen atom, but differ due to the presence of a side-chain (known as R; (Fig. 2)). This side-chain varies dramatically between amino acids, from a simple hydrogen atom in the amino acid glycine to a complex structure found in tryptophan. Depending on the nature of the side-chain, an amino acid can be hydrophilic (water-attracting) or hydrophobic (water-repelling), acidic or basic; and it is this diversity in side-chain properties that gives each protein its specific character.

Creating a functional protein

The sequence of amino acids in a protein defines its primary structure. The blueprint for each amino acid is laid down by sets of three letters known as base triplets that are found in the coding regions of genes. These base triplets are recognized by ribosomes, the protein building sites of the cell, which create and successively join the amino acids together. This is a remarkably quick process: a protein of 300 amino acids will be made in little more than a minute.

The result is a linear chain of amino acids, but this only becomes a functional protein when it folds into its three-dimensional (tertiary structure) form. This occurs through an intermediate form, known as secondary structure, the most common of which are the rod-like a-helix and the plate-like b-pleated sheet (Fig. 3). These secondary structures are formed by a small number of amino acids that are close together, which then, in turn, interact, fold and coil to produce the tertiary structure that contains its functional regions (called domains).

Although it is possible to deduce the primary structure of a protein from a gene’s sequence, its tertiary structure cannot be determined (although it should become possible to make predictions when more tertiary sequences are submitted to databases). It can only be determined by complex experimental analyses and, at present, this information is only known for about 10% of proteins. It is therefore not yet known how an amino-acid chain folds into its tertiary structure in the short time scale (fractions of a second) that occurs in the cell. So, there is a huge gap in our knowledge of how we move from protein sequence to function in living organisms: the line of sight from the genetic blueprint for a protein to its biological function is blocked by the impenetrable jungle of protein folding, and some researchers believe that clearing this jungle is the most important task in biochemistry at present.

The quest to understand protein folding

One of the most important results in understanding the process of protein folding was a thought-provoking experiment that was carried out by Christian Anfinsen and colleagues in the early 1960s. They investigated a protein called ribonuclease, which they isolated from the pancreatic tissue of cattle. This enzyme, made up of 124 amino acids, cleaves any ribonucleic acid (RNA) that could be harmful to the cell, such as truncated RNA that would not make a fully operational protein. To do this, although this was not known in Anfinsen’s time, it briefly binds RNA in a binding site and requires several sulphur-containing amino-acid cysteine residues in the protein, which form bonds with each other (called disulphide bridges) and hold the protein structure together.

Ribonuclease can be denatured by adding certain chemicals or by heat. The disulphide bridges break and other forces of attraction between amino acids disappear, which makes the enzyme collapse into a tangled, useless ball. In various studies, Anfinsen showed that this denaturation process could be completely reversed by removing these denaturing chemicals or by lowering the temperature. The ribonuclease then folds back to its natural functional state on its own. So, Anfinsen concluded that the amino-acid sequence determines the shape of a protein, a finding for which Anfinsen received the Nobel Prize in Chemistry in 1972.

But, if this is true, how do proteins find the right conformation out of the simply endless number of potential three-dimensional forms that it could randomly fold into? After all, the folding of a protein is not a chemical reaction, with a bond breaking here and a new one forming there. It is more like the weaving of an intertwined molecular pattern, the stability of which is defined by innumerable forces between atoms. Indeed, Cyrus Levinthal calculated in 1969 that finding the strongest attraction by simple trial and error would be impossible. He said that even if a protein only consisted of 100 amino acids and each of these flexible residues could only take on two different spatial orientations, the protein could theoretically adopt as many as 1030 possible conformations. Assuming a protein could try out 100 billion different conformations per second, it would still take 100 billion years to try all possibilities. So, Levinthal suggested that nature must have devised more effective methods to achieve this and postulated the existence of defined sets of folding pathways by which protein folding can take place rapidly.

However, we now know that such fixed protein folding pathways do not seem to exist. Various protein folding pathways that have been investigated experimentally and theoretically in recent years have thrown up interesting hypotheses, but have remained hard to prove in working models. In the case of proteins of less than 100 amino acids, only two levels of folding can be observed, the unfolded protein (which occurs in numerous forms) and the finished, folded, functional protein. For larger proteins, three steps can be observed. The intermediate is either a so-called molten globule, which is formed by a process called hydrophobic collapse (in which all hydrophobic side-chains suddenly slide inside the protein or clump together) or a structure in which the secondary structures of the protein are already fully formed. However, there is disagreement about whether these intermediates are formed en route to the correct folding pattern, or whether they represent structural cul-de-sacs.

Finding the energy to fold

As with all processes in nature, protein folding also needs energy, the process has to obey the laws of thermodynamics. A protein always folds so that it achieves the lowest possible energy, just as we always try to adopt the most comfortable position, in which we need to move about least, when going to sleep. It is thought that this is achieved by using an energy gradient or funnel along the path from the random tangle to the folded protein. Alan Fersht of Cambridge University used the following analogy to illustrate this model: if you blindfold a golfer and let him hit the ball in any direction he likes, the probability that he will hole the ball is almost infinitesimal. The same is true of a protein finding the right form by chance. However, if all parts of the golf course slope toward the hole, which is at the lowest point in the area, even a blindfolded golfer has a good chance of finding the hole. So, fixed reaction pathways are not necessary, as each protein seeks out its natural shape through a funnel of declining energy; it can take many folding routes and still reach its target of the completed tertiary structure.

A helping hand

This understanding of protein folding was obtained from computer models (in silico) or from experiments in the laboratory (in vitro) in which an individual protein was denatured to observe it folding back into its original form. But, the situation is considerably more complex in the living cell (in vivo). Although the fundamental energy rules also apply here, folding at least of large proteins rarely takes place spontaneously, as the ribosomes do not synthesize only one protein at a time. Instead, cells contain a vast number of proteins and other biomolecules at the extraordinarily high concentration of 340 grams per litre. Ordered protein folding in this cramped chaos is only possible under the supervision of specialized molecules, called chaperones, which accompany proteins and make sure that those that are being formed at the ribosomes do not clump together prematurely (Fig. 4). Chaperones do not merely oversee the folding of the protein, they also protect its tertiary structure in situations in which the cell is under stress; for example, elevated body temperature, so these chaperones have also been classified as heat-shock proteins (HSPs).

The HSP70s, so called because they have a molecular weight of 70 kilodaltons, are the most important class of chaperones. They bind to the developing protein chain and protect those parts of the newly formed protein that are particularly sensitive to premature reaction with the environment and therefore to malformation. When they let go of the new protein chain it is ready to fold. It is now taken over by a chaperonin, a molecule shaped like a double ring, which fits round the protein chain like a cylinder so that it can fold undisturbed inside (Fig. 4). Although the cylindrical folding cage opens every ~10 seconds, the protein only leaves the chaperonin when it has achieved its required form.

We now understand better than ever how protein folding, both in vitro and in vivo, takes place. And this, in turn, has given us a better understanding of the origin and course of diseases that are associated with defective protein folding. But why the normal folding of every protein always runs towards a predetermined goal and what this goal looks like, that is, what the instructions in the primary structure are that determine the correct tertiary structure is still a great mystery, even though the number of proposed models has dramatically increased. Computer simulations cannot yet solve the folding code that is hidden in the primary structure by simply calculating the molecular dynamics atom by atom, as to work through just 50 milliseconds of folding would take even the fastest computer around 30,000 years. Any realistic hope of cracking the folding code, such as to produce special designer proteins that evolution had not planned, is probably a very long way off. However, our improved understanding of the route that a protein must take from its synthesis to the correct folded form already enables us to contemplate better treatments or even cures for diseases in which proteins have departed from the correct folding route (see Treating protein folding diseases).

The Anfinsen Experiment in Protein Folding

http://sandwalk.blogspot.com/2007/02/anfinsen-experiment-in-protein-folding.html

Chaperones, which help proteins find their native, active conformation

Kazutoshi Mori and Peter Walter

PNAS 2014; 111(50): 17696–17697 http://dx.doi.org:/10.1073/pnas.1419343111

Kazutoshi Mori and Peter Walter

Albert Lasker
Basic Medical Research Award

For discoveries concerning the unfolded protein response — an intracellular quality control system that detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.

http://www.laskerfoundation.org/awards/2014_b_interview_mori.htm

Proteins that are secreted from the cell or inserted into the plasma membrane, transit through the endoplasmic reticulum where they are properly folded and assembled and may undergo post-translational modification. Walter tells the story of the exciting discovery made in his lab of the “unfolded protein response”, a feedback pathway that ensures that the cell makes enough ER to properly modify all the secreted proteins in the cell.

https://youtu.be/cvE6Kia0T9E

Kazutoshi Mori and Peter Walter
For discoveries concerning the unfolded protein response — an intracellular quality control system that detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.

The 2014 Albert Lasker Basic Medical Research Award honors two scientists for their discoveries concerning the unfolded protein response, an intracellular quality-control system that detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures. Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) identified core components of this process and unveiled unexpected aspects of its mechanism.

Approximately one third of cellular proteins pass through the endoplasmic reticulum (ER), a netlike labyrinth of membrane-bound tubes and flattened sacs inside the cell. Work in the 1960s revealed that the ER sorts and transports proteins that are destined for export or the cell’s surface, and we now know that the ER allows cargo to pass only after applying stringent standards. In particular, proteins must assume correct three-dimensional shapes to perform their jobs, and the ER fosters this outcome. Furthermore, when unfolded proteins accumulate in this compartment, the cell bolsters the ER’s folding capacity. This phenomenon forms the linchpin of the unfolded protein response (UPR).

The first clues about this system’s existence emerged in the late 1970s, when researchers discovered that glucose starvation drives cells to boost production of particular proteins. Amy Lee (University of Southern California) reported in 1983 that the rise stems from an increase in the quantity of messenger RNA (mRNA) templates for these glucose-regulated proteins, or GRPs.

Three years later, Hugh Pelham (Medical Research Council, Cambridge) established that one of the GRPs, GRP78, resides in the ER and resembles a protein that prevents heat-damaged proteins from clumping. When glucose supplies drop, sugars that normally decorate some proteins are no longer available. Pelham proposed that the resulting sugar-deficient proteins stick together, perhaps because they misfold, and that GRP78, like its molecular relative, thwarts protein aggregation. Pelham also found that GRP78 was identical to another protein, BiP, that associates with partially assembled antibody molecules in the ER. In parallel, Mary-Jane Gething and Joseph Sambrook (University of Texas Southwestern Medical Center) showed that BiP attaches to misfolded forms of a different protein in the ER.

These findings hinted that BiP helps proteins fold; if true, manufacture of BiP in response to unfolded proteins would serve a clear benefit. The connection between glucose starvation and folding remained murky, however, and the model relied on that link. In 1988, Gething and Sambrook established that misfolded protein rather than sugar-adornment defects sends the alert to ramp up BiP output.

In 1989, yeast BiP surfaced. Its quantities also climb in response to unfolded ER proteins. Mori joined Gething and Sambrook’s lab as a postdoctoral fellow, and the group identified a short series of DNA letters that abuts the BiP gene. This sequence spurs molecular machinery to copy, or transcribe, the BiP DNA into mRNA when unfolded proteins accumulate in the ER; the sequence, when placed next to a different gene, similarly turns on its transcription.

Together, these observations suggested that cells must somehow monitor the abundance of unfolded proteins in the ER and transmit that information to the nucleus, which houses the genes. These events spark production of BiP and other proteins that promote folding, which reverse the problem. But no one knew how the nuclear equipment senses the ER environment.

The complexity of Ire1
Independently, Mori, in Texas, and Walter, in San Francisco, placed the DNA stretch that Mori had uncovered next to a gene whose product makes a blue substance. When unfolded proteins accumulate in the ER and the engineered yeast cells send the usual signal to the nucleus, it stimulates not only typical UPR targets, but also the gene that turns the yeast blue. Yeast with defects in the UPR system would not change color, the researchers reasoned.

In 1993, the investigators used this scheme to isolate white yeast strains and thus tracked down the faulty genes whose normal versions presumably contribute to the UPR. One encodes a protein called Ire1.

Sequence analysis of Ire1 suggested that it is a kinase—an enzyme that adds phosphates to itself and/or other proteins. Additional work by Walter and Mori confirmed and extended this prediction. They found that Ire1 lies in the ER membrane with its kinase portion in the cytoplasm. In this orientation, the ER region could detect an unfolded protein signal and the other end could convey the message to cytoplasmic partners.

Mammalian kinases were well known to monitor environmental cues and, by adding phosphates to themselves or other molecules, trigger adaptive physiological changes. Perhaps, Mori and Walter reasoned, Ire1 would behave similarly.

To figure out how Ire1 delivers the unfolded-protein message, Walter and Mori (by then an independent investigator in Japan) set out to identify the presumptive courier that picks up the signal and carries it to the nucleus. They sought a protein that binds to the DNA sequences adjacent to UPR target genes and provokes transcription. The investigators captured the component they sought, a protein that previously had been named Hac1.

Their results, reported in 1996, contradicted expectation. In the simplest scenario, the theoretical protein to which Ire1 affixes a phosphate would be ready for action upon stimulation. Hac1, however, is not ready for anything; rather, it is manufactured only after the UPR alarm rings.

A crucial clue to explain this result came from the observation that the Hac1-encoding mRNA shrinks when unfolded proteins accumulate. Instead of adding a phosphate to another protein, Ire1 prompts removal of a chunk of Hac1’s mRNA. Additional work by Walter, which was confirmed and extended by Mori, established thatHAC1 precursor mRNA contains an internal stretch of 252 genetic letters that is eliminated to supply the blueprint for active Hac1.

A canonical molecular machine splices sequences from precursor mRNAs and operates in the nucleus. The plot thickened when Walter showed that this apparatus does not act on HAC1 mRNA. Instead, he found, the severed HAC1 mRNA is stitched together by a cytoplasmic enzyme—tRNA ligase—that normally joins the two components of a different type of RNA, transfer RNA.

The search was now on for an enzyme that excises the middle piece of the HAC1 precursor mRNA. Inspired by a related protein’s behavior, Walter showed that the cytoplasmic segment of Ire1, which contains the kinase and an additional stretch of protein, could cut HAC1 precursor mRNA at the expected sites. Then he demonstrated that the splicing reaction could occur in the test tube with only two enzymes: Ire1 cleaves the HAC1 precursor mRNA at both splice junctions, and the transfer RNA ligase sews them together.

Mammalian systems unfold
As these details of the yeast UPR were materializing, researchers were struggling to gain traction in the mammalian system. In 1998, Mori unearthed a sequence that was common only to genes that fire up in response to unfolded ER proteins. This element rouses several UPR target genes, he found. Furthermore, a human protein called ATF6 binds to this DNA motif and activates adjacent genes.

Mori noticed that an overabundance of unfolded proteins incites conversion of full-length ATF6 to a smaller version; the large form dwells in the ER, whereas the trimmed one resides in the nucleus. This and other work suggested that excess unfolded proteins trigger release of a portion of ER membrane-bound ATF6. The liberated fragment travels to the nucleus and activates transcription of UPR target genes.

While Mori was discovering and elucidating ATF6’s role in the UPR, David Ron (New York University School of Medicine) and Randal Kaufman (University of Michigan Medical Center) found mammalian versions of Ire1, which share fundamental functional features with their yeast cousin. Three years later, Mori and Ron identified the human and worm versions of yeast Hac1, a protein known as XBP1.

In the meantime, near the beginning of 1999, David Ron and Ron Wek (Indiana University School of Medicine) had independently uncovered a third arm of the UPR, which depends on a protein called PERK. Like Ire1 and ATF6, PERK also lies across the ER membrane. Furthermore, its ER domain resembles that of Ire1. On the cytoplasmic side, a protein kinase segment of PERK adds phosphates to a particular protein, which then impedes translation of mRNAs. As a result, fewer proteins enter the ER, thus lightening the folding load.

Strength in numbers
In the last ten years, Walter, with UCSF colleague Robert Stroud, has peered more closely at Ire1 activation with X-ray crystallography. Previous work by Mori, Walter, and others had suggested that UPR induction causes Ire1 molecules to snuggle up in the membrane. By studying yeast Ire1, Walter and Stroud provided an atomic-level rationale for those results and illuminated details of the reaction.

In addition to providing assistance during protein folding, BiP attaches to Ire1 on the side that lies within the ER; when BiP falls off, naked Ire1 molecules pair up and create grooves that bind the unfolded proteins, Walter and Stroud suggest. Multiple Ire1 duos then congregate to form higher order structures; such association rearranges their cytoplasmic segments, positioning them so they can grab and then snip the HAC1/XBP1 mRNA, according to the model.

Researchers are still uncovering layers in the UPR. For example, Ire1 governs ER membrane synthesis and a system that shuttles recalcitrant unfolded proteins from the ER to a cellular incinerator. Even with these additional components, the unfolded protein burden sometimes surpasses the cell’s management capacity. That situation can trigger cell suicide, which obliterates unhealthy cells that might otherwise wreak havoc. Investigators are deciphering how the Ire1, ATF6, and PERK branches of the pathway help cells make life-and-death decisions.

Many scientists are now pursuing ways to harness the UPR for medical advantage. Certain forms of some inherited diseases that cause elevated cholesterol levels, cystic fibrosis, and retinitis pigmentosa produce abnormal proteins that do not fold properly and overwhelm the UPR.

Walter and Mori have unraveled a process with numerous unusual features. Their work has unlocked a multi-layered, highly choreographed system that lies at the heart of normal cellular function.

by Evelyn Strauss

http://www.laskerfoundation.org/awards/2014_b_description.htm

http://www.jci.org/articles/view/78419

Peter Walter: Winner of the 2015 Vilcek Prize in Biomedical Science

https://youtu.be/3lSsNdP_Mmc

Kazutoshi Mori of Kyoto University in Japan and Peter Walter of the University of California, San Francisco, have won the 2014 Lasker Award for basic medical research. Mori and Walter are being honored by the Albert and Mary Lasker Foundation for their work related to the unfolded protein response—a cellular stress response that has been implicated in several protein-folding diseases.

In its announcement, the foundation said that “Mori and Walter’s work has led to a better understanding of inherited diseases such as cystic fibrosis, retinitis pigmentosa, and certain elevated cholesterol conditions in which unfolded proteins overwhelm the unfolded protein response.”

Three years ago, the Lasker Foundation honored Franz-Ulrich Hartl and Arthur Horwich for their protein-folding work with its 2011 basic research award.

Meanwhile, Alim Louis Benabid of Joseph Fourier University in Grenoble, France, and Mahlon DeLong of the Emory University School of Medicine in Atlanta, Georgia, have won the this year’s Lasker-DeBakey Clinical Medical Research Award for their deep-brain stimulation work that has been used to help restore and motor function in patients with advanced Parkinson’s disease.

And the University of Washington’s Mary-Claire King has won the 2014 Lasker-Koshland Special Achievement Award in Medical Science for “bold, imaginative, and diverse contributions to medical science and human rights” related to her work to reunite missing persons or their remains with their families, as well as her discovery of the cancer-related BRCA1 gene locus. In a commentary published in JAMA today (September 8), King and her colleagues advocated for population-based screening for cancer-related genetic variants. “Population-wide screening will require significant efforts to educate the public and to develop new counseling strategies, but this investment will both save women’s lives and provide a model for other public health programs in genomic medicine,” they wrote.

This year’s recipients will receive a $250,000 honorarium per category. The awards will be presented on Friday, September 19, in New York City.

http://www.the-scientist.com/?articles.view/articleNo/40946/title/Lasker-Winners-Announced/

2014Life Science and MedicineProfessor Kazutoshi Mori and Professor Peter Walter

http://www.shawprize.org/en/shaw.php?tmp=4&twoid=97&threeid=238&fourid=428

https://youtu.be/NgW6WvMgj2s

World’s Largest Protein Interaction Map Created

GEN News  Sep 8, 2015
http://www.genengnews.com/gen-news-highlights/world-s-largest-protein-interaction-map-created/81251700/

A veritable tree-of-life investigation shows how proteins fit together and interact. [iStock/xrender]

http://www.genengnews.com/Media/images/GENHighlight/thumb_Sep8_2015_iStock_26507385_protein5412011712.jpg

An international research team reports that it has studied cells from numerous organisms, from amebae to worms to mice to humans, to show how proteins fit together to build different tissues and bodies. They say they uncovered tens of thousands of new protein interactions, accounting for about a quarter of all estimated protein contacts in a cell.

When even a single one of these interactions is lost it can lead to disease, and the map is already helping scientists spot individual proteins that could be at the root of complex human disorders, the team points out in an article (“Panorama of ancient metazoan macromolecular complexes”) in Nature. They add that their data will be available through open access databases.

Proteins work in teams by sticking to each other to carry out their jobs. Many proteins come together to form so called molecular machines that play key roles, such a building new proteins or recycling those no longer needed by literally grinding them into reusable parts. But for the vast majority of proteins, and there are tens of thousands of them in human cells, we still don’t know what they do.

This is where the map created by researchers led by Andrew Emili, Ph.D., from the University of Toronto’s Donnelly Centre and Edward Marcotte, Ph.D., from the University of Texas at Austin map comes in. Using a technique developed by the groups, the scientists were able to fish thousands of protein machineries out of cells and count individual proteins they are made of. They then built a network that, similar to social networks, offers clues into protein function based on which other proteins they hang out with. For example, a new and unstudied protein, whose role we don’t yet know, is likely to be involved in fixing damage in a cell if it sticks to cell’s known “handymen” proteins.

The study gathered information on protein machineries from nine species that represent the tree of life: baker’s yeast, amebae, sea anemones, flies, worms, sea urchins, frogs, mice, and humans. The new map expands the number of known protein associations over 10-fold, and gives insights into how they evolved over time.

“For me the highlight of the study is its sheer scale. We have tripled the number of protein interactions for every species. So across all the animals, we can now predict, with high confidence, more than 1 million protein interactions [which is] a fundamentally ‘big step’ moving the goal posts forward in terms of protein interactions networks,” says Dr. Emili, who is also Ontario Research Chair in Biomarkers in Disease Management and a professor in the department of molecular genetics.

The researchers discovered that tens of thousands of protein associations remained unchanged since the first ancestral cell appeared, one billion years ago, preceding all of animal life on Earth.

“Protein assemblies in humans were often identical to those in other species. This not only reinforces what we already know about our common evolutionary ancestry, it also has practical implications, providing the ability to study the genetic basis for a wide variety of diseases and how they present in different species,” says Dr. Marcotte, who notes that the map is already proving useful in pinpointing possible causes of human disease.

One example is a newly discovered molecular machine, dubbed Commander, which consists of about a dozen individual proteins. Genes that encode some of Commander’s components had previously been found to be mutated in people with intellectual disabilities but it was not clear how these proteins worked.

Because Commander is present in all animal cells, the scientists went on to disrupt its components in tadpoles, revealing abnormalities in the way brain cells are positioned during embryo development and providing a possible origin for a complex human condition.

“With tens of thousands of other new protein interactions, our map promises to open many more lines of research into links between proteins and disease, which we are keen to explore in depth over the coming years,” says Dr. Emili.

Hormone Injections May Have Spread ‘Seeds’ of Alzheimer’s

by Seth Augenstein

http://www.biosciencetechnology.com/news/2015/09/hormone-injections-may-have-spread-seeds-alzheimers-study-says?et_cid=4808735&et_rid=535648082&type=cta

The “seeds” of Alzheimer’s and related brain diseases may be transmitted by direct tissue transplantation, according to a new study.

The brains of eight people showed development of Creutzfeldt-Jakob disease, caused by treatments with contaminated growth hormones from human cadavers decades before, said the scientists, who published their findings in this week’sNature. Six of the eight also had the amyloid buildup that is a tell-tale sign of Alzheimer’s.

“This is the first evidence of real-world transmission of amyloid pathology,” John Hardy, a University College London molecular neuroscientist, reportedly told the journal.

The hormone treatments were administered to people who were short in height. The samples were taken from cadavers’ pituitary glands that contaminated with prions. The treatments started in 1958, and were halted in 1985 due to reports of Creutzfeldt-Jakob. By the year 2000, 38 of the patients had developed the disease. By 2012, the number had grown to 450 cases among the recipients of the cadaver-derived HGH and some other medical procedures, like transplants and brain surgery.

The study last week showed that the newly-understood Multiple System Atrophy could be spread by direct contact with re-used surgical instruments.

“You can’t kill a protein, and it can stick tightly to stainless steel, even when the surgical instrument is cleaned,” said Kurt Giles, a UCSF researcher and one of the authors of that MSA study.

Brain-eating cannibals from Papua New Guinea were the subject of a June study analyzing their genetic adaptations allowing them to survive a prion disease called kuru. The authors of the newest paper on the hormone spread of Creutzfeldt-Jakob were also cautious in pointing out particular dangers. 

Researchers Uncover More About The Elusive Pericyte-Tumour Interaction.

Sept 11, 2015     by Healthinnovations       Reported by Aviva Lev-Avi, PhD, RN

Tumours are known to evade the immune system by a variety of mechanisms, one of which is the recruitment of ‘myeloid-derived suppressor cells’ (MDSC). MDSCs suppress the ability of the immune system’s killer T-cells to destroy cancer cells. It is known that the more MDSCs present, the worse the prognosis or therapy response of the patient. Tumours secrete signal molecules such as interleukin-6 (IL-6) that help in recruiting MDSCs, however; the mechanisms behind IL-6 tumour secretion are quite unknown.

Now, a study from researchers at Karolinska Institutet is the first to suggest that cells in the tumour blood vessels contribute to a local environment that protects the cancer cells from these tumour-killing immune cells. The team state that their study can contribute to the development of better immune-based cancer therapies.  The study is published in the Journal of the National Cancer Institute.

Chemists solve major piece of cellular mystery

08/28/2015 Kimm Fesenmaier, California Institute of Technology

http://www.rdmag.com/videos/2015/08/chemists-solve-major-piece-cellular-mystery?et_cid=4775862&et_rid=535648082&location=top

Not just anything is allowed to enter the nucleus, the heart of eukaryotic cells where, among other things, genetic information is stored. A double membrane, called the nuclear envelope, serves as a wall, protecting the contents of the nucleus.

The NPC is targeted by a number of diseases, including some aggressive forms of leukemia and nervous system disorders such as a hereditary form of Lou Gehrig’s disease. Now a team led by André Hoelz, assistant professor of biochemistry at Caltech, has solved a crucial piece of the puzzle.

Hoelz and his colleagues published a paper describing the atomic structure of the NPC’s coat nucleoporin complex, a subcomplex that forms what they now call the outer rings. The team has now solved the architecture of the pore’s inner ring, a subcomplex that is central to the NPC’s ability to serve as a barrier and transport facilitator. They built up the complex and then systematically dissected it. Then they validated how it works in vivo, in a species of fungus.

“Using an interdisciplinary approach, we solved the architecture of this subcomplex and showed that it cannot change shape significantly,” says Hoelz. “It is a relatively rigid scaffold that is incorporated into the pore and basically just sits as a decoration, like pom-poms on a bicycle. It cannot dilate.”

 

Folding Funnels Reveal Protein’s Inner Life

Tue, 03/22/2016 – 12:17pm
Rick Kubetz, University of Illinois at Urbana-Champaign
http://www.rdmag.com/news/2016/03/folding-funnels-reveal-proteins-inner-life
The molecular folding funnel contains all of the stable molecular states and folding pathways between them, providing important information about structure and mechanisms that can reveal how a polymer or protein folds, or aid in the design of drug molecule or ligands with a particular shape.
The molecular folding funnel contains all of the stable molecular states and folding pathways between them, providing important information about structure and mechanisms that can reveal how a polymer or protein folds, or aid in the design of drug molecule or ligands with a particular shape.

Proteins are the molecules of life. They are chemically programmed by their amino acid sequence to fold into highly organized conformations that underpin all of biological structure (e.g., hair, scales) and function (e.g., enzymes, antibodies). Understanding the sequence-structure-function relationship—the “protein folding problem”—is one of the great, unsolved problems in physical chemistry, and is of inestimable scientific value in exposing the inner workings of life and the rational design of molecular machines.

“This work lays the foundations to recover the protein folding landscapes directly from experimental data, providing a route to new understanding and rational design of proteins,” explained Andrew Ferguson, an assistant professor of materials science and engineering at the University of Illinois at Urbana-Champaign. “While we remain far from this goal, our understanding of protein folding was revolutionized by the ‘new view’ that envisages molecular folding as a conformational search over a funneled free energy surface.”

According to Ferguson, the single-molecule free energy surface encodes all of the thermodynamics and pathways of folding, dictating protein structure and dynamics. Each point on the landscape corresponds to an ensemble of similar protein conformations, and the height of the landscape prescribes their stability. It is a key goal of physical chemistry to determine molecular folding landscapes.

“Molecular folding landscapes can be inferred from long computer simulations in which the positions of all atoms in the molecule are known,” said Jiang Wang, a graduate research assistant and first author of the paper, “Nonlinear reconstruction of single-molecule free energy surfaces from univariate time series,” published in the March 21, 2016 issue of Physical Review E.

“Experimental techniques such as single molecule Förster resonance energy transfer (FRET) can measure distances between covalently-grafted fluorescent dye molecules to track the size of the molecule as a function of time, but it has so far not been possible to reconstruct folding funnels from experimental measurements of single coarse-grained observables,” Ferguson explained. “In this work, we have integrated nonlinear machine learning and statistical thermodynamics with Takens’ Theorem from dynamical systems theory to demonstrate in computer simulations of a hydrophobic polymer chain that it is possible to determine molecular folding landscapes from time series of a single experimentally-accessible observable.”

“The information loss associated with its reconstruction from a single observable means that the topography of the reconstructed funnel may be perturbed—the heights and depths of the free energy peaks and valleys may be altered—but it faithfully preserves the topology of the true funnel—the locality, continuity, and connectivity of molecular configurations,” Wang noted. “This means that the folding funnel determined from measurements of, in this case, the head-to-tail distance of the chain is geometrically and topologically identical and contains precisely the same molecular states and transition pathways as that computed from knowledge of all the atomic positions,” Ferguson added.

“We are very excited by this idealized proof of principle for computer simulations of a polymer chain, and are currently working to extend our analyses to simulations of biologically realistic peptides and proteins, and partner with single molecule biophysicists to apply our technique to experimental measurements of real proteins,” Ferguson said.

 

Nonlinear reconstruction of single-molecule free-energy surfaces from univariate time series

Jiang Wang and Andrew L. Ferguson
Phys. Rev. E 93, 032412 – Published 21 March 2016     http://dx.doi.org/10.1103/PhysRevE.93.032412
The stable conformations and dynamical fluctuations of polymers and macromolecules are governed by the underlying single-molecule free energy surface. By integrating ideas from dynamical systems theory with nonlinear manifold learning, we have recovered single-molecule free energy surfaces from univariate time series in a single coarse-grained system observable. Using Takens’ Delay Embedding Theorem, we expand the univariate time series into a high dimensional space in which the dynamics are equivalent to those of the molecular motions in real space. We then apply the diffusion map nonlinear manifold learning algorithm to extract a low-dimensional representation of the free energy surface that is diffeomorphic to that computed from a complete knowledge of all system degrees of freedom. We validate our approach in molecular dynamics simulations of a C24H50 n-alkane chain to demonstrate that the two-dimensional free energy surface extracted from the atomistic simulation trajectory is – subject to spatial and temporal symmetries – geometrically and topologically equivalent to that recovered from a knowledge of only the head-to-tail distance of the chain. Our approach lays the foundations to extract empirical single-molecule free energy surfaces directly from experimental measurements.

Read Full Post »

Roeder – the coactivator OCA-B, the first cell-specific coactivator, discovered by Roeder in 1992, is unique to immune system B cells

Posted in Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Curation, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Gene Regulation, Genetics & Pharmaceutical, Human Immune System in Health and in Disease, Immuno-Oncology & Genomics, Infectious Disease Immunodiagnostics, Innovation in Immunology Diagnostics, Metabolism, Molecular Genetics & Pharmaceutical, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Proteins, Proteomics, Signaling & Cell Circuits, Transcriptomics, Translational Science, tagged , , , , on September 7, 2015| Leave a Comment »

Roeder – the coactivator OCA-B, the first cell-specific coactivator, discovered by Roeder in 1992, is unique to immune system B cells

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence
The B-cell-specific transcription coactivator OCA-B/OBF-1/Bob-1 is essential for normal production of immunoglobulin isotypes

Unkyu Kim*, Xiao-Feng Qin†, Shiaoching Gong†, Sean Stevens*, Yan Luo*, Michel Nussenzweig† & Robert G. Roeder*
Nature 383, 542 – 547 (10 October 1996);  http://dx.doi.org:/10.1038/383542a0
* Laboratory of Biochemistry and Molecular Biology, and † Laboratory of Molecular Immunology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021, USA

OCA-B was initially identified as a B-cell-restricted coactivator that functions with octamer binding transcription factors (Oct-1 and Oct-2) to mediate efficient cell type-specific transcription of immunoglobulin promoters in vitro 1–3. Subsequent cloning studies led to identification of the coactivator as a single poly-peptide, designated either as OCA-B (ref. 3), OBF-1 (ref. 4) or Bob-1 (ref. 5). OCA-B itself does not bind to DNA directly, but interacts with either Oct-1 or Oct-2 to potentiate transcriptional activation1–5. To determine the biological role of OCA-B, we generated OCA-B-deficient mice by gene targeting. Mice lacking OCA-B undergo normal antigen-independent, B-cell differentiation, including appropriate expression of both immunoglobulin genes and other early B-cell-restricted genes. However, antigen-dependent maturation of B cells is greatly affected. The pro- liferative response to surface IgM crosslinking is impaired, and there is a severe deficiency in the production of secondary immunoglobulin isotypes including IgGl, IgG2a, IgG2b, IgG3, IgA and IgE in OCA-B-deficient B cells. This defect is not due to a failure of the isotype switching process, but rather to reduced levels of transcription from normally switched immunoglobulin heavy-chain loci. In accord with the defective isotype production, germinal centre formation is absent in these mutant mice.

References

1. Pierani, A., Heguy, A., Fujii, H. & Roeder, R. G. Mol. Cell. Biol. 10, 6204−6215 (1990). | PubMed | ChemPort |
2. Luo, Y., Fujii, H., Gerster, T. & Roeder, R. G. Cell 71, 231−241 (1992). | Article | PubMed | ISI | ChemPort |
3. Luo, Y. & Roeder, R. G. Mol. Cell. Biol. 15, 4115−4124 (1995). | PubMed | ISI | ChemPort |
4. Strubin, M., Newell, J. W. & Matthias, P. Cell 80, 497−506 (1995). | Article | PubMed | ISI | ChemPort |
5. Gstaiger, M., Knoepfel, L., Georgiev, O., Schaffner, W. & Hovens, C. M. Nature 373, 360−362 (1995). | Article | PubMed | ISI | ChemPort |
6. Yancopoulos, G. D. & Alt, F. W. Cell 40, 271−281 (1985). | Article | PubMed | ISI | ChemPort |
7. Yancopoulos, G. D. & Alt, F. W. Annu. Rev. Immunol. 4, 339−368 (1986). | Article | PubMed | ISI | ChemPort |
8. Rajewsky, K. Curr. Opin. Immunol. 4, 171−176 (1992). | Article | PubMed | ChemPort |
9. Rolink, A. & Melchers, F. Adv. Immunol. 53, 123−156 (1993). | PubMed | ISI | ChemPort |
10. Gold, M. R. & DeFranco, A. L. Adv. Immunol. 55, 221−295 (1994). | PubMed | ISI | ChemPort |
11. Coffman, R. L., Lebman, D. A. & Rothman, P. Adv. Immunol. 54, 229−270 (1993). | PubMed | ISI | ChemPort |
12. Parker, D. C. Annu. Rev. Immunol. 11, 331−360 (1993). | Article | PubMed | ISI | ChemPort |
13. Wang, H. Y., Paul, W. E. & Keegan, A. Immunity 4, 113−122 (1996). | Article | PubMed | ISI | ChemPort |
14. McLennan, I. C. Annu. Rev. Immunol. 12, 117−139 (1994). | Article | PubMed | ISI | ChemPort |
15. Lutzker, S. & Alt, F. W. Mol. Cell. Biol. 8, 1849−1852 (1988). | PubMed | ISI | ChemPort |
16. Stavnezer, J. et al. Proc. Natl Acad. Sci. USA 85, 7704−7708 (1988). | PubMed | ChemPort |
17. Staudt, L. M. & Lenardo, M. J. Annu. Rev. Immunol. 9, 373−398 (1991). | Article | PubMed | ISI | ChemPort |
18. Dariavach, P., Williams, G. T., Campbell, K., Pettersson, S. & Neuberger, M. S. Eur. J. Immunol. 21, 1499−1504 (1991). | PubMed | ISI | ChemPort |
19. Grant, P. A., Thompson, C. B. & Pettersson, S. EMBO J. 14, 4501−4513 (1995). | PubMed | ISI | ChemPort |
20. Bain, G., Gruenwald, S. & Murre, C. Mol. Cell. Biol. 13, 3522−3529 (1993). | PubMed | ISI | ChemPort |
21. Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. & Mulligan, R. C. Cell 65, 1153−1163 (1991). | Article | PubMed | ISI | ChemPort |
22. DeFranco, A. L. J. Exp. Med. 155, 1523−1536 (1982). | Article | PubMed | ISI | ChemPort |
23. Bottaro, A. et al. EMBO J. 13, 665−674 (1994). | PubMed | ISI | ChemPort |
24. Cogne, M. et al. Cell 77, 737−747 (1994). | Article | PubMed | ISI |
25. Gong, S. & Nussenzweig, M. C. Science 272, 411−414 (1996). | PubMed | ISI | ChemPort |
26. Qin, X. F. et al. EMBO J. 13, 5967−5976 (1994). | PubMed | ISI | ChemPort |
27. Li, S. C. et al. Int. Immunol. 6, 491−497 (1994). | PubMed | ChemPort |

 

Cloning, Functional Characterization, and Mechanism of Action of the B-Cell-Specific Transcriptional Coactivator

Oca-B Yan Luo & Robert G. Roeder*
Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021
Molecular And Cellular Biology, Aug. 1995;  15(8):4115–4124 0270-7306/95/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC230650/pdf/154115.pdf

Biochemical purification and cognate cDNA cloning studies have revealed that the previously described transcriptional coactivator OCA-B consists of a 34- or 35-kDa polypeptide with sequence relationships to known coactivators that function by protein-protein interactions. Studies with a recombinant protein have proved that a single OCA-B polypeptide is the main determinant for B-cell-specific activation of immunoglobulin (Ig) promoters and provided additional insights into its mechanism of action. Recombinant OCA-B can function equally well with Oct-1 or Oct-2 on an Ig promoter, but while corresponding POU domains are sufficient for OCA-B interaction, and for octamer-mediated transcription of a histone H2B promoter, an additional Oct-1 or Oct-2 activation domain(s) is necessary for functional synergy with OCA-B. Further studies show that Ig promoter activation by Oct-1 and OCA-B requires still other general (USA-derived) cofactors and also provide indirect evidence that distinct Oct-interacting cofactors regulate H2B transcription.

A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors

Yan Luo, Hiroshi Fujii, Thomas Gerster, Robert G. Roeder

Laboratory of Biochemistry and Molecular Biology The Rockefeller University New York, New York 10021 USA
★Present address: Department of Biochemistry, Niigata University School of Medicine, Niigata 951, Japan.
†Present address: Biozentrum der Universität Basel, Abteilung Zellbiologie, 4056 Basel, Switzerland.
Cell Oct 1992 71(2):231–241    http://dx.doi.org/10.1016/0092-8674(92)90352-D
A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters.

Identification of transcription coactivator OCA-B dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses

Unkyu Kim*, Rachael Siegel*, Xiaodi Ren*, Cary S. Gunther*, Terry Gaasterland†, and Robert G. Roeder*‡
*Laboratory of Biochemistry and Molecular Biology and †Laboratory of Computational Genomics, The Rockefeller University, 1230 York Avenue, New York, NY 10021

PNAS  http://www.pnas.org/content/100/15/8868.full.pdf

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

 

Identification and Characterization of a Novel OCA-B Isoform: Implications for a Role in B Cell Signaling Pathways

Xin Yu, Lu Wang†, Yan Luo, Robert G. Roeder
Immunity Feb 2001; 14(2): 157–167   http://dx.doi.org:/10.1016/S1074-7613(01)00099-1

OCA-B is a B lymphocyte–specific transcription coactivator that mediates tissue- and stage-restricted transcription of immunoglobulin genes. Earlier genetic studies revealed that OCA-B is essential for germinal center formation and production of secondary immunoglobulin isotypes. Biochemically purified OCA-B contains p35 and p34 isoforms, and a further analysis has now revealed that p35 is derived from a newly found isoform, p40. More importantly, it has been found that p35 is myristoylated in vivo and that this leads to dramatic changes (including localization to membrane compartments) in its properties. These results suggest that the p35 isoform of OCA-B has functions distinct from those of the nuclear p34 and that it might be a component of a signaling pathway that is required for late-stage B cell development.

 

The B cell–restricted function of immunoglobulin (Ig) promoters is mediated mainly by an octamer element (5′-ATGCAAAT-3′) that is conserved in virtually all Ig heavy (H) and light (L) chain gene promoters, as well as in some Ig enhancers (reviewed by Staudt and Lenardo, 1990). However, this same element is also a key central element for transcription of differentially regulated genes that include ubiquitously expressed small nuclear RNA genes (snRNA) and cell cycle-regulated histone H2B genes (reviewed inLuo et al., 1992). The regulatory functions of octamer elements, therefore, are likely dependent on transcription factors that bind this DNA sequence. The well-characterized octamer binding transcription factors include the ubiquitous Oct-1 and the B cell–enriched Oct-2, both of which belong to the POU family and share a conserved DNA binding structure called the POU domain (reviewed by Herr et al. 1988 and Wegner et al. 1993). It was originally thought that Oct-2 would account for the tissue-specific activity of Ig promoters, whereas Oct-1 would facilitate transcription of the ubiquitously expressed genes regulated through octamer elements (e.g., snRNA and histone H2B genes) Staudt et al. 1986, Cockerill and Klinken 1990 and Murphy et al. 1992. However, subsequent biochemical Pierani et al. 1990 and Luo et al. 1992 and genetic (Corcoran et al., 1993)analyses clearly demonstrated that this was not the case. Instead, the promoter specificity was shown to be due to an Oct-1 interacting factor called OCA-B (Luo et al., 1992), and the purification of related p35 and p34 isoforms with apparently equivalent activity in vitro (Luo and Roeder, 1995) set the stage for further studies of the structure and function of OCA-B.

Subsequent to the biochemical identification of OCA-B and its mechanism of action, cognate cDNAs were cloned using both biochemical (Luo and Roeder, 1995) and genetic screening Gstaiger et al. 1995 and Strubin et al. 1995 methods. Analyses of recombinant OCA-B (p34) function in cell-free systems and in transfection assays confirmed both physical and functional interactions with Oct-1 and Oct-2 (via their POU domains) on Ig promoters Gstaiger et al. 1995, Luo and Roeder 1995 and Strubin et al. 1995 and led to the definition of an N-terminal OCA-B domain that interacts with the Oct POU domainCepek et al. 1996, Gstaiger et al. 1996, Babb et al. 1997 and Chaseman et al. 1999 and a C-terminal activation domain that acts synergistically with Oct activation domains to recruit additional coactivators (Luo et al., 1998).

The physiological roles of OCA-B were further investigated by genetic disruption of OCA-B expression in mice Kim et al. 1996, Nielson et al. 1996 and Schubart et al. 1996. These studies showed that, although not required for early B cell development, OCA-B functions are essential both for germinal center formation and for efficient secondary Ig isotype production (including IgGs, IgA, and IgE). In accordance with the biochemical function of OCA-B in activating Ig promoter transcription, it has been found that the decrease of secondary antibody production in OCA-B-deficient mice is largely due to reduced levels of transcription from normally switched IgH chain loci, rather than a reduced capacity for class switching events per se Kim et al. 1996 and Schubart et al. 1996. Recent results further demonstrated that OCA-B plays an essential role in efficient transcription from switched IgH loci by directly regulating 3′ IgH enhancer function in conjunction with Oct-1 or Oct-2 Tang and Sharp 1999 and Stevens et al. 2000b. On the other hand, the lack of germinal center formation in OCA-B-deficient mice cannot be explained by reduced Ig isotype production, since these are two independent events in B cell development (Vajdy et al., 1995). Therefore, OCA-B may regulate germinal center formation by activating the expression of other target genes or by mediating signal pathways that in turn trigger a specific genetic program. At least two lines of evidence support this idea: (1) B cells lacking OCA-B are defective in the proliferative response to surface IgM cross-linking (Kim et al., 1996); (2) OCA-B expression, which is very low in early B cells but high in activated B cells in vivo, can be dramatically and synergistically induced in naive B cells by B cell stimuli (CD40L, Ig cross-linking, and IL4) that are required for germinal center formation (Qin et al., 1998).

Our findings in this report raise the possibility that OCA-B may be directly involved in B cell signaling pathways through novel mechanisms. We report the presence of a novel isoform of OCA-B (p40) that results from utilization of an upstream alternative translation initiation codon and that serves as a precursor to the p35 isoform of OCA-B. Relative to the conventional p34 OCA-B isoform, p35 shows distinct protein modification, subcellular localization, and transcriptional coactivator properties. The unique features of p35 suggest a novel function for this molecule in signal transduction.

 

Synergism with the Coactivator OBF-1 (OCA-B, BOB-1) Is Mediated by a Specific POU Dimer Configuration

Alexey Tomilin1, 2, #, Attila Reményi2, 4, #, Katharina Lins1, Hanne Bak2, Sebastian Leidel1, Gerrit Vriend3, Matthias Wilmanns4, Hans R Schöler1, 2
Cell   Dec 2000; 103(6):853–864  doi:10.1016/S0092-8674(00)00189-6

POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the PORE (ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact. An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Our findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts.

Development of multicellular organisms is characterized by an intricate series of genetic and epigenetic events that generate the complex adult body from the unicellular zygote. A refined and sophisticated regulatory network that is established during embryogenesis reflects the complexity of organisms. Although embryonic development is a multistep process characterized by the sequential activation and repression of many genes, only a relatively small number of transcription factors are responsible for regulating the expression of developmental genes. This diversity in transcriptional control by a limited array of transcription factors is achieved through a complex network of interactions between these proteins and specific DNA sequences found in promoters and enhancers of developmental genes. The primary structure of these DNA elements defines the composition and architecture of the transcriptional activation complexes that ultimately control gene expression in the appropriate temporo-spatial context of the developing organism. For example, nonsteroid members of the nuclear receptor superfamily that possess a zinc-finger DNA binding domain operate by binding to the hormone response elements (HREs). HREs consist of two minimal core hexad sequences, AGGTCA, which can be configured into various functional motifs. The orientation and spacing between these two hexamers as well as subtle differences in their sequence dictate the identity and the mode (monomer, hetero-, or homodimer) of nuclear receptor binding that results in diverse effects on transcription (Mangelsdorf and Evans 1995).

The operation of members of the POU domain family of transcription factors is also highly dependent on the nature of cognate DNA elements. The 160 amino-acid-long DNA binding domain of these proteins is composed of two structurally independent subdomains: the POU-type homeodomain (POU-homeo or POUH), and the POU-specific domain (POUS) that are connected by a flexible linker region (27 and 36). POU domain proteins demonstrate impressive versatility in how they regulate transcription. This is due to several, often interdependent, factors: (1) flexible amino acid–base interaction, (2) variable orientation, spacing, and positioning of DNA-tethered POU subdomains relative to each other, (3) posttranslational modification, and (4) interaction with heterologous proteins (Herr and Cleary 1995).

POU domain proteins are able to bind to DNA cooperatively, thus conferring additional functional variability. The homo- and heterodimerization of Oct-1 and Oct-2 on immunoglobulin (Ig) heavy chain promoters (VH) provided evidence of cooperativity, with a yet unknown dimer arrangement (13, 16 and 23). The cis-elements are considered to consist of low-affinity heptamer and high-affinity octamer sites separated by two nucleotides (Full-size image (<1 K)ATFull-size image (<1 K)).

The pituitary-specific POU domain protein Pit-1 binds to DNA either as a homodimer or as a heterodimer with Oct-1 (Voss et al. 1991). Crystallographic studies determined the structure of a Pit-1 homodimer assembled on the synthetic motif ATGTATATACAT (referred to here as PitD) that had been derived from the natural Pit-1 cognate element within the prolactin gene promoter (ATATATATTCAT) (Jacobson et al. 1997). The structure of the Pit-1 POUS and POUH domains, and their docking onto DNA, are very similar to that observed in the cocrystal of the Oct-1 POU domain monomer with the octamer site (ATGCAAAT, Klemm et al. 1994). The Oct-1 POUS domain recognizes the ATGC subsite whereas the Pit-1 POUS domain binds to the sequence ATAC. However, the latter subsite lies on the opposite strand and, as a consequence, the orientation of POUS relative to the POUH domain is inverted (Jacobson et al. 1997).

Another mechanism outlining cooperative DNA binding by POU proteins was recently determined during the course of an Oct-4 target gene characterization (Botquin et al. 1998). The P alindromic O ct factor R ecognition E lement (PORE), ATTTGAAATGCAAAT (15 bp), of the Osteopontin (OPN) enhancer interacts with an Oct-4 dimer, thereby mediating strong transcriptional activation in preimplantation mouse embryos. Homo- and heterodimerization of other Oct factors like Oct-1 and Oct-6 on the PORE has also been demonstrated.

The aforementioned examples provide evidence of the various ways in which POU domain proteins are able to cooperatively bind to substrate DNA. The particular mode of binding employed is primarily defined by the DNA sequence. To address the question of whether diversity in cooperative binding is reflected in transcriptional regulation, we have assessed and compared the ability of two different types of POU dimers to interact with the coactivator OBF-1 (OCA-B, Bob-1). This coactivator synergistically interacts with Oct-1 and Oct-2 monomers bound to the octamer motif (18, 9, 17 and 33). We have investigated one type of POU dimer that is formed on the PORE and another that is formed on another palindromic DNA motif called MORE (M ore P ORE), ATGCATATGCAT. The data presented in this study provide an example of how POU domain molecules that bind to DNA in the same stoichiometry but in different configurations can differentially recruit a transcriptional coactivator to the promoter resulting in differential transcriptional activation.

B-cell-specific Coactivator OCA-B: Biochemical Aspects, Role in B-Cell Development and Beyond

Cold Spring Harb Symp Quant Biol 1999 64: 119-132;
http://dx.doi.org:/10.1101/sqb.1999.64.119

Read Full Post »

A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

Posted in Cell Biology, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Myocardial adenine nucleotide metabolism, Myocardial Metabolism, Na-K-ATPase, Neurological Diseases, Proteins, Proteomics, tagged , , , on September 6, 2015| Leave a Comment »

A new way of moving – Michael Sheetz, James Spudich, Ronald Vale

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.5

 

J Clin Invest. 2012 Oct 1; 122(10): 3374–3377.

http://dx.doi.org:/10.1172/JCI66361

The MBI community congratulates Michael Sheetz upon winning the prestigious Albert Lasker Basic Medical Research Award. Michael Sheetz, Director of the Mechanobiology Institute, Singapore, Distinguished Professor of the Department of Biological Sciences, NUS and William R Kenan, Junior Professor at Columbia University, shares this award with two of his collaborators, James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco). The Award was presented at a ceremony on Friday, September 21, 2012, in New York City.

 

The Albert Lasker Basic Medical Research Award was given to Prof Sheetz, Prof Spudich and Prof Vale in honor of seminal contributions made in establishing methods to study cytoskeletal motor proteins. These developments paved the way to study molecular motors and enabled the discovery of the motor protein, kinesin. The landmark achievements in deciphering new components of cellular motors, which helped explain how these motors worked, were pivotal in understanding the basic fundamental process of energy conversion within the cell. These have led to explorations of these physiologically relevant molecules, as potential drug targets in a variety of disease conditions.

 

Many basic cellular functions depend on the directed movement of cytoplasmic organelles, macromolecules, membranes or chromosomes from one place to another within the cell. The transport of this intracellular cargo is achieved by molecular motor proteins, such as myosin and kinesin, which provide force and movement through the conversion of chemical energy (ATP) into mechanical energy. Molecular motor proteins move along scaffolds made of specific protein polymers, with kinesins moving along microtubules and myosins along actin filaments, in order to carry their cargo to the appropriate destination within the cell.
Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out. Michael Sheetz and colleagues, Ronald Vale and Thomas Reese, carried out pivotal experiments that ultimately led to the discovery of kinesins, a novel and hitherto unknown family of motor proteins. These experiments involved the development of an in vitromotility assay, whereby proteins from the cytoplasm of neuronal cells were shown to power the movement of microtubules across the surface of glass coverslips. This technique was found to be a sensitive and rapid assay for testing the activity of kinesin and was adopted by numerous labs following these crucial initial experiments.

For more details of the award winning contribution towards understanding the basics of cellular machinery, please go to http://www.laskerfoundation.org/media/index.htm and also http://www.laskerfoundation.org/media/pdf/2012citation_basic.pdf

 

Michael Sheetz, along with James Spudich and Ronald Vale strongly believe in an open culture of scientific exchange. ‘The most interesting scientific insights result from collaborative, interdisciplinary adventures’, has been the one common theme of Michael Sheetz career. A firm believer of an open laboratory concept where students from different labs and backgrounds, share bench space and often ideas, he emulated the Open Lab model (learn more about MBI’s open labs) at the Mechanobiology Institute, Singapore. This new model of open laboratory environment in interdisciplinary institutes provides an excellent way to encourage fast paced discovery process.

“My greatest excitement comes from considering the puzzle provided by an unexpected result when new technology is applied to an old problem, says Professor Sheetz.„

In his acceptance essay, which can be read here (PDF), Michael Sheetz refers to the importance of collaborations, where the parties are learning from each other and also ‘encourages young scientists to perform speculative experiments whenever they have such an idea, even if most of them fail; since an experiment, even a flawed on, can reveal the solution to an important problem’.

 

The Mechanobiology Institute is delighted to announce that Michael Sheetz has been selected as a Massry Prize Laureate for 2013.

michaelSheetz_WB_9079Shared with James Spudich (Stanford University) and Ronald Vale (University of California, San Francisco), the award to the trio is a recognition of their work defining molecular mechanisms of ‘intracellular motility.’

This process involves the deployment of molecular machines to move cargo on molecular tracks which are a part of the cellular skeleton.

The discovery of a novel family of motor proteins, the kinesins, by Michael Sheetz, Ronald Vale and Thomas Reese and the methodology developed for the same, proved to be pivotal and the technique developed led to many further discoveries by different laboratories.

Subsequently, Sheetz and Spudich worked out an in-vitro method for visualizing actin filaments creeping along myosin coated surfaces, and this system still remains the gold-standard assay for studying myosin movement. With a read-out in hand, many details of the mechanism of action of the motor molecules within the cell were worked out.

 

 

 

 

 

 

The Lasker Awards: motors take centre stage

Nature Cell Biology | Editorial
Nature Cell Biology 14,1113(2012)  http://dx.doi.org:/10.1038/ncb2618

Michael Sheetz, James Spudich and Ronald Vale have now joined the list of Lasker laureates, having jointly received the 2012 Albert Lasker Basic Medical Research Award for their “discoveries concerning cytoskeletal motor proteins, machines that move cargoes within cells, contract muscles, and enable cell movements”1.

Although the mechanism of action and the cellular functions performed by force-generating cytoskeletal motors, including their roles in intracellular trafficking, cell migration, cell division and muscle contraction, are now a fundamental part of cell biology, in the 1970s and 1980s they were still mostly a mystery. Following a postdoctoral fellowship under the guidance of Hugh Huxley, a pioneer of muscle contraction studies, James Spudich established his independent work at the University of California San Francisco (UCSF) and later at Stanford University on what was, at the time, the largely unchartered territory of myosin activity and function. A fortuitous crossing of paths occurred in 1982, when Michael Sheetz joined the Spudich laboratory on sabbatical from his own independent research at the University of Connecticut Health Center. Working together, Spudich and Sheetz demonstrated myosin movement on actin filaments using the Nitella axillaris alga as a model, and later established an in vitro reconstitution system that demonstrated the ability of purified myosin to move on purified actin filaments in the presence of adenosine triphosphate at rates consistent with those of muscle contraction. This seminal work was published in Nature in 19832 and 19853.

Spurred by the exciting work on myosin-based movement, Ronald Vale, then a graduate student at Stanford University, decided with Michael Sheetz to define the particle movement observed in squid axons. Their experiments at the Marine Biology Laboratory in Woods Hole led to a series of groundbreaking Cell publications in 19854, 5, 6, 7, 8, which determined that axonal movement was not driven by myosin on actin filaments as they had anticipated, but was instead occurring on microtubules and was powered by a then-uncharacterized factor that they purified and named kinesin.

These initial efforts investigating myosin- and kinesin-powered motility, and the in vitro assays developed to characterize cytoskeletal motor activities, opened up new and fascinating avenues of research and have become a corner-stone of cell biology today. Following these key discoveries, Spudich went on to define many other aspects of myosin activity and function. Vale continued his work on molecular motors and their cellular roles in his independent research at UCSF, and Sheetz followed a varied research career ranging from motility studies to work on cell adhesion and mechanosensing at Columbia University and the Mechanobiology Institute in Singapore.

In honouring the early work of Sheetz, Spudich and Vale, the Lasker Foundation recognizes the significance of the cytoskeletal motor field in biology, and also the importance of understanding the principles underlying cellular motor function in human diseases in which such activities are deregulated. Indeed, the characterization of normal myosin and kinesin activity and function has served as the spring-board for studies on their impaired or aberrant action in disease, with the goal of developing therapies for heart conditions in the case of myosins, and neurological disorders and malignancy in the case of kinesins.

It should also be noted that the discoveries acknowledged by the Lasker Award and the subsequent scientific careers of the three awardees were the outcome of an inspired mix of cell and molecular biology, biochemistry and physics, among other disciplines, and are thus a testament to the importance of fostering multidisciplinary science. Moreover, as the three award recipients eloquently noted in their Lasker Award acceptance remarks, the motivating force during the exciting times of their initial research on motors was not only a thirst for discovery and a passion for science, but also a strong collaborative spirit. As a fundamentally creative and adventurous endeavour, science is often seen by outsiders as a solitary pursuit of inquiry and testing one’s own ideas. However, the reality of a bustling laboratory reveals that teamwork, discussion and brainstorming, and a successful combination of different personalities, are just as important as individual intellect and drive. In that respect, the dedication, creativity and collaborative efforts of Sheetz, Spudich and Vale should be an inspiration to scientists everywhere.

References

  1. www.laskerfoundation.org/awards/2012_b_description.htm
  2. Sheetz, M. P. & Spudich, J. A. Nature 303, 31–35 (1983).
  3. Spudish, J. A., Kron, S. J. & Sheetz, M. P. Nature 315, 584–586 (1985).
  4. Vale, R. D., Schnapp, B. J., Reese, T. S. & Sheetz, M. P. Cell 40, 449–454 (1985).

One path to understanding energy transduction in biological systems

James A Spudich

http://www.laskerfoundation.org/awards/pdf/2012_b_spudich.pdf

Who is not fascinated by the myriad biological movements that define life? From cell migration, cell division and a network of translocation activities within cells to highly specialized muscle contraction, molecular motors operate by burning ATP as fuel. Three types of molecular motors—myosin, kinesin and dynein— and nearly 100 different subtypes transduce that chemical energy into mechanical movements to carry out a wide variety of cellular tasks. Understanding the molecular basis of energy transduction by these motors has taken decades. Our understanding of molecular motors could be viewed as beginning with the two 1954 papers in Nature by Hugh Huxley and Jean Hanson and Andrew Huxley and Rolf Niedergerke, respectively, where the authors proposed the sliding-filament theory of muscle contraction. But a good place to start my story is 1969, when Hugh Huxley, on the basis of his remarkable X-ray diffraction experiments on live muscle coupled with electron microscopy, postulated the swinging crossbridge hypothesis of muscle contraction1. Thus, more than 40 years ago, he proposed the basic concepts of how the myosin molecule produces the sliding of actin filaments to produce contraction. Hugh Huxley laid the foundation for the molecular motor field, and we are all indebted to him. My beginnings in myosin research began as a postdoctoral fellow in Hugh’s laboratory at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England, coincidentally in 1969. But my fascination with science began much earlier.

Neither of my parents was college educated, but they both had keen intellects, positive and enthusiastic outlooks and profound work ethics. My father was intrigued by how things work and shared that interest with my brother John and me. After the coal mines closed, my father taught himself electrical engineering, founded the Spudich Electric Company and patented one of his inventions. He often told John and me, “do whatever excites you, but do it well and be respectful of people you interact with.”

I was captivated with chemistry from a young age. Beginning at the age of six, I mastered every chemistry set I could get. The myriad chemical reactions that could be created using everyday materials, sometimes with marvelously explosive results, fed my excitement for chemistry. It was a world unfamiliar to my parents, but they respected my preoccupations and cleared the pantry of our modest home for me to set up a lab with discarded equipment given to me by my high school chemistry teacher Robert Brandsmark. My brother John has also followed the allure of science into an exciting and distinguished career in basic research. His work has established the molecular basis of signaling in an important class of rhodopsins that he discovered in 1982 (ref. 2). John was my first collaborator.

A chance encounter with Woody Hastings at the University of Illinois launched my experimental-science career. Throughout my undergraduate years, I worked with Woody on bioluminescence in Vibrio fischeri3. I was inspired by his high-spirited fascination with biology and was fortunate to be invited to help him teach in the physiology course at the Marine Biological Laboratory (MBL) in Woods Hole (Fig. 1). At the MBL, I was introduced to the breadth and potential of many biological systems, including muscle contraction.

In 1963 I joined the PhD program in the new Department of Biochemistry at Stanford University, founded by Arthur Kornberg. One of the many remarkable aspects of the biochemistry department was that, although Arthur was my thesis advisor, all the faculty members were my mentors. This unique environment shaped the way I do research and taught me how to be a responsible colleague and a mentor to others (Fig. 2). I learned how important it is to reduce complex biological systems to their essential components and create quantitative in vitro assays for the function of interest. Those years also made it clear to me that interdisciplinary approaches would be key to understanding complex biological processes. So I decided to do postdoctoral work in both genetics and structural biology. I spent one year at Stanford with another influential role model, Charley Yanofsky, working on the genetics of the Escherichia coli tryptophan operon. I then joined Hugh Huxley’s laboratory in Cambridge.

I chose to study the  unanswered questions in cell biology at the time when I established my own laboratory – how the chemical energy of ATP hydrolysis brings about mechanical movement and what roles a myosin-like motor might have in nonmuscle cells.

The essential first steps were to develop a quantitative in vitro motility assay for myosin movement on actin, which is crucial for understanding the molecular mechanism of energy transduction by this system, and to develop a model organism to unravel the molecular basis of the myriad nonmuscle-cell movements that are apparent by light microscopy. We explored Neurospora crassa, Saccharomyces cerevisiae, Physarum polycephalum, Dictyostelium discoideum, Nitella axillaris and other organisms, all unfamiliar to me at the time. The giant cells of the alga Nitella were particularly intriguing because of their striking intracellular cytoplasmic streaming that was visible under a simple light microscope. Although not suitable for biochemistry or genetics, Nitella would assume an important role in my lab a decade later, after Yolande Kersey in Norm Wessells’s laboratory in the Department of Biological Sciences at Stanford showed oriented actin cables lying along chloroplast rows in these cells 5. The slime mold Dictyostelium proved best for our initial biochemical approach 6. Margaret Clarke, my postdoctoral fellow, identified a myosin in Dictyostelium. We also showed that actin is associated with the cell membrane in this organism, and we isolated membranecoated polystyrene beads with actin filaments emanating from them. We were tremendously excited about the possibilities these results presented as a small step along the way to an in vitro motility assay where these actin-coated particles could move along a myosin-coated surface (Fig. 3).

Figure 3 Dictyostelium has a muscle-like myosin and membrane-associated actin. (a) A possible scheme for pulling two membranes together (redrawn from ref. 6). (b) Margaret Clarke discovered myosin II in Dictyostelium and showed that it forms bipolar thick filaments, similar to muscle myosin. (c) Phagocytized polystyrene beads offered an opportunity to explore one version of an in vitro motility assay where the beads may be pulled along by myosin. Taken from my laboratory notebook, 21 January 1973.

Figure 4 One approach to an in vitro motility assay from a totally defined system. (a) The concept was to observe myosin-coated beads moving along fixed actin filaments oriented by buffer flow. The actin filaments had biotinylated severin bound to their barbed ends; the barbed ends were attached to an avidin-coated surface by way of the tight avidin-biotin link. The filaments were oriented by buffer flow. B, biotin; S, severin. (b) Myosin-coated beads were observed by light microscopy to move upstream toward the barbed end of the surface-attached actin filaments. The position of each of the three bead aggregates is shown as a function of time. This was the first demonstration of quantitative, directed movement of myosin along actin with a totally defined system (taken from ref. 11). ATP binding releases the myosin Myosin binds to actin ATP ADP Pi ADP .

In 1977 I joined the Department of Structural Biology at Stanford. In the next years we extensively characterized the actin-myosin system in Dictyostelium. My student Arturo De Lozanne made the chance discovery that genes in Dictyostelium can be knocked out by homologous recombination and provided the first genetic proof that myosin II is essential for time that myosin II drove the forward movement of cells. Dietmar Manstein, Meg Titus and Arturo then extended these experiments to create a myosin-null cell8, which was crucial to our later work using mutational analysis to define the structure-function relationships of the myosin molecule and for important experiments in support of the swinging cross-bridge hypothesis9. Interestingly, reports from a number of laboratories between 1969 and 1980 did not support the swinging cross-bridge model, and it was more imperative than ever to develop a quantitative in vitro motility system to test the various models under consideration. In 1981 we identified and purified Dictyostelium severin, a protein that tightly binds the ‘barbed ends’ of actin filaments. This provided an opportunity to try another version of an in vitro motility assay. Using biotinylated severin, we attached the actin filament barbed ends to an avidin-coated slide and flowed aqueous solution over them. Long filaments attached to the surface at one end would be expected to orient in the direction of the flowing solution (Fig. 4a). We placed myosin-coated beads on these actin-coated slides and added ATP but saw only sporadic movements. In retrospect, we probably did not have sufficient alignment of filaments; we were not monitoring filament alignment at that time by electron microscopy, as we did later.

A key breakthrough occurred in 1982 when Mike Sheetz came to my laboratory on sabbatical. Not certain what component of our system might be limiting our approach, we took advantage of the known orientation of actin filaments in Nitella5 to overcome the actin filament alignment problem. Peter Sargent, a neurobiologist in the Structural Biology Department at that time, helped us cut open a Nitella cell, and we attached it to a surface to expose the actin fibers. We added myosin-coated beads and eureka! We saw robust ATP-dependent unidirectional movement along chloroplast rows, which mark the actin fibers 10.

Armed with the Nitella results, Mike left my lab and went to the MBL to explore whether myosin-coated vesicles may account for the particle movements observed in squid axons. Ron Vale, then a graduate student at Stanford with Eric Shooter, was fascinated by the movement of organelles in nerve axons and joined Mike at the MBL. To their great surprise, they found that movement in axons is not myosin driven. Instead, they discovered the new molecular motor kinesin, a discovery that completely energized the field and opened up years of exciting work from their laboratories and many others. ..

 

The combination of the in vitro motility assay and the Dictyostelium myosin-null cell provided powerful tools for Kathy Ruppel, Taro Uyeda, Dietmar Manstein, William Shih, Coleen Murphy, Meg Titus, Tom Egelhoff and others in my lab to use mutations along myosin to define the biochemical, biophysical and assembly properties of the molecule. Our results were consistent with the proposed actin-activated myosin chemomechanical cycle derived largely from the elegant biochemical kinetic studies from Edward Taylor’s laboratory in the early 1970’s (ref. 15) (Fig. 5). Then, in 1993, Ivan Rayment and his colleagues16 obtained a high-resolution crystal structure of myosin S1. Ivan’s pivotal work allowed us to place our mutational analyses in a myosin structure-function context.

Figure 5 The actin-activated myosin chemomechanical cycle. This cycle, extensively studied by many researchers over several decades, was derived from kinetic studies of Lymn and Taylor 15. A mechanical stroke only occurs when the myosin is strongly bound to actin. Our mutational analyses of Dictyostelium myosin II probed each of the steps shown and provided structure-function analyses that helped define how the myosin motor works. ADP-Pi , ADP and inorganic phosphate, the products of ATP hydrolysis, remain bound to the active site until actin binds to the myosin.

Figure 6 In vitro motility taken to the single-molecule level using the physics of laser trapping. (a) The Kron in vitro motility assay observing fluorescent actin filaments (yellow) moving on a myosin-coated (red) surface. (b) Two polystyrene beads attached to the ends of a single actin filament are trapped in space by laser beams. The filament is lowered onto a single myosin molecule on a bump on the surface (gray sphere). (c) Jeff Finer building the dual-beam laser trap in around 1990.

Fundamental issues still remained— primarily to establish the step size that the myosin takes for each ATP hydrolysis, which was under considerable debate.

more…

One of my great satisfactions is that the more detailed understanding of energy transduction by myosin has led to potential clinical therapies. A small molecule that binds and activates b-cardiac myosin is now in clinical trials for the treatment of heart failure, and another small molecule currently in clinical trials activates skeletal muscle contraction and may aid patients with amyotropic lateral sclerosis and other diseases.

…

 

Read Full Post »

Proteins that control neurotransmitter release

Posted in Academic Publishing, Biological Networks, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Clinical & Translational, Clinical Genomics, Disease Biology, Gene Regulation, Genetics & Pharmaceutical, Genome Biology, Innovations in Neurophysiology & Neuropsychology, Intellectual Property, International Global Work in Pharmaceutical, Investment in Technological Breakthrough, Molecular Genetics & Pharmaceutical, Neurological Diseases, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Analytics, Pharmaceutical Drug Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmacogenomics, Population Health Management, Proteins, Proteomics, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Schizophrenia, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged , , , , on September 6, 2015| Leave a Comment »

Proteins that control neurotransmitter release

Author & Curator: Larry H. Bernstein, MD, FCAP

Richard H. Scheller, PhD

The sec6/8 Complex Is Located at Neurite Outgrowth and Axonal Synapse-Assembly Domains

Christopher D. Hazuka, Davide L. Foletti, Shu-Chan Hsu, Yun Kee, F. Woodward Hopf, and Richard H. Scheller
Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305-5428

The Journal of Neuroscience, February 15, 1999, 19(4):1324–1334   http://www.jneurosci.org/content/19/4/1324.full.pdf

The molecules that specify domains on the neuronal plasma membrane for the delivery and accumulation of vesicles during neurite outgrowth and synapse formation are unknown. We investigated the role of the sec6/8 complex, a set of proteins that specifies vesicle targeting sites in yeast and epithelial cells, in neuronal membrane trafficking. This complex was found in layers of developing rat brain undergoing synaptogenesis. In cultured hippocampal neurons, the sec6/8 complex was present in regions of ongoing membrane addition: the tips of growing neurites, filopodia, and growth cones. In young axons, the sec6/8 complex was also confined to periodic domains of the plasma membrane. The distribution of synaptotagmin, synapsin1, sec6, and FM1–43 labeling in cultured neurons suggested that the plasma membrane localization of the sec6/8 complex preceded the arrival of synaptic markers and was downregulated in mature synapses. We propose that the sec6/8 complex specifies sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis. Key words: synaptogenesis; neurotransmission; secretion; exocytosis; synaptic vesicle; vesicle targeting

Targeting of vesicles to synaptic sites during development may use similar mechanisms as those involved in vesicle fusion underlying membrane outgrowth. Before contact with a postsynaptic target, axons possess mobile vesicle clusters bearing synaptotagmin, which fuse with the plasma membrane after stimulation (Matteoli et al., 1992; Kraszewski et al., 1995; Dai and Peng, 1996). Thus, growing axons must contain the molecular machinery required for constitutive exocytosis, endocytosis, and activitydependent vesicle release. However, it is unclear how vesicles become clustered at synapses. Although vesicle fusion in axons might occur anywhere along the plasma membrane, there must be membrane targets that signal the clustering of vesicles for synapse formation. Furthermore, it is unclear how sites of vesicle exocytosis are modified as the neuron forms stable contacts with postsynaptic partners.

Identification of a Novel Rab11/25 Binding Domain Present in Eferin and Rip Proteins

Rytis Prekeris*, Jason M. Davies*, and Richard H. Scheller#
JBC Papers in Press. Published on July 31, 2001 as Manuscript M106133200
http://www.jbc.org/content/early/2001/07/31/jbc.M106133200.full.pdf

Rab11, a low molecular weight GTP binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the TGN. In this study we describe a novel Rab11 effector, EF hands containing Rab11 interacting protein (eferin). In addition, we identify a 20 amino acid domain that is present at the C-terminus of eferin and other Rab11/25 interacting proteins, such as Rip11 and nRip11. Using biochemical techniques we demonstrate that this domain is necessary and sufficient for Rab11 binding in vitro and that it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for the binding to Rab11/25 possibly accounting for the diversity of Rab11 functions.

Members of the Rab/Ypt GTPase family have emerged as important regulators of vesicular trafficking (1). Rab proteins have been proposed to mediate a variety of functions, including vesicle translocation and docking at a specific fusion sites. Like all small GTPases, Rabs cycle between active (GTP bound) and inactive (GDP bound) conformations (2). In the GTP bound state, Rab proteins can bind a variety of downstream effector proteins, while GTP hydrolysis leads to a conformational change in the “switch” region that renders the Rab GTPase unrecognizable to its effector proteins (3,4). A key question in understanding the interactions between Rabs and their effectors concerns the mechanisms by which Rab GTPases specifically bind a diverse spectrum of effectors and how this is regulated by the common structural motif used as a GTP switch. Biochemical and genetic studies have identified several hypervariable regions that might be involved in determining Rab specificity, including N- and C-termini, as well as α3/β5 by guest on September 6, 2015 http://www.jbc.org/ Downloaded from loop (5,6). Indeed, the recently reported structure of Rab3a bound to a putative effector, rabphillin-3a, revealed that Rab3a/rabphillin-3a complex interacts through two main regions (7). The first consists of conformationally sensitive “switch” regions of Rab3a bound to the a1 helix and the C-terminal part of rabphillin-3a. The second involves the SGAWFF domain of rabphillin-3a which fits into a pocket formed by the three hypervariable complementary determining regions (CDRs) of Rab3a, corresponding to the N- and C-termini and α3/β5 loop. Thus, it appears that the hypervariable RabCDR are involved in determining the specificity of effector binding, while the conserved “switch” regions impart GTP dependency and binding. It remains to be determined, however, whether this paradigm also applies to other Rab/effector complexes. Rab11a, -11b, and -25 are closely related members of Rab GTPase family that have been implicated in regulating a variety of different post-Golgi trafficking pathways, such as protein recycling (8), phagocytosis (9), insulin-stimulated Glut4 insertion in the plasma membrane (10), and membrane trafficking from early endosomes to the transGolgi network (11). During the last few years several Rab11/25 interacting proteins have been identified, including Rab11BP/Rabphilin-11, Rip11, nRip11, and myosin Vb (12- 15). However, the mechanisms of their function, as well as molecular aspects of their interactions with Rab11, remain to be fully understood. In the present study, we report the identification of EF-hands containing Rab11/25 interacting protein (eferin). Furthermore, we characterized a Rab binding domain (RBD11) which is present at the Cterminus of eferin as well as other Rab11/25 binding proteins, such as Rip11 and nRip11. Using biochemical techniques we demonstrated that RBD11 is the region which encodes the specificity for Rab11/25, but is distinct from the region interacting with Rab “switch” domain, since its interactions with the Rab11/25 are not GTP-dependent.

The functional significance of the differences in Rip and eferin interactions with Rab11/25 remains to be determined. One possibility is that additional cellular factors can regulate the affinity of Rab11/25 binding to its effectors. Indeed, the recombinant full length Rip11 binds poorly to Rab11a in pull down and yeast-two hybrid assays as compared to full length endogenous Rip11 from cellular TX-100 extracts (data not shown). Furthermore, it has been previously shown that Rip11 can also interact with γSNAP and cytoskeleton (13,24). Thus, the interactions of Rips and eferin with different factors could be used as a means of differentially regulating Rab11/25 binding. Alternatively, the Rab11/25 binding motif in eferin and Rip11 might be conformationlly hidden and require activation before binding to Rab11/25. We have previously demonstrated that phosphorylation of Rip11 plays an important role in its trafficking (13). Thus, differential phosphorylation on Rab11/25 binding motifs could also play a role in regulating the binding of Rip11 and eferin to Rab GTPases. Despite to recent progress in understanding the roles of Rabs and their effectors in regulating membrane trafficking, we are only beginning to unravel the structural determinants of their function. Identification and characterization of the Rab11/25 binding regions in Rip and Eferin proteins will be of a crucial importance in understanding the molecular mechanisms involved in differential regulation of the variety of Rab11-dependent trafficking pathways.

Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs.
J. Immunol. Methods
J Immunol Methods 2008 Mar 14;332(1-2):41-52. Epub 2008 Jan 14.
Jagath R Junutula, Sunil Bhakta, Helga Raab, Karen E Ervin, Charles Eigenbrot, Richard Vandlen, Richard H Scheller,Henry B Lowman
Genentech Inc., 1DNA Way, South San Francisco, California, 94080, United States. jagath@gene.com
Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system
Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index.
Nat. Biotechnol.
Nat Biotechnol 2008 Aug 20;26(8):925-32. Epub 2008 Jul 20.
Jagath R Junutula, Helga Raab, Suzanna Clark, Sunil Bhakta, Douglas D Leipold, Sylvia Weir, Yvonne Chen, Michelle Simpson, Siao Ping Tsai, Mark S Dennis, Yanmei Lu, Y Gloria Meng, Carl Ng, Jihong Yang, Chien C Lee, Eileen Duenas,Jeffrey Gorrell, Viswanatham Katta, Amy Kim, Kevin McDorman, Kelly Flagella, Rayna Venook, Sarajane Ross, Susan D Spencer, Wai Lee Wong, Henry B Lowman, Richard Vandlen, Mark X Sliwkowski, Richard H Scheller, Paul Polakis, William Mallet
Genentech Inc, South San Francisco, California 94080, USA.

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding.

Isolation and characterization of a Drosophila neuropeptide gene.
Neuron
Neuron 2008 Nov;60(3):400-1
Richard H Scheller, John R Nambu
Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer.
Clin. Cancer Res.
Clin Cancer Res 2010 Oct 30;16(19):4769-78. Epub 2010 Aug 30.
Jagath R Junutula, Kelly M Flagella, Richard A Graham, Kathryn L Parsons, Edward Ha, Helga Raab, Sunil Bhakta, Trung Nguyen, Debra L Dugger, Guangmin Li, Elaine Mai, Gail D Lewis Phillips, Hajime Hiraragi, Reina N Fuji, Jay Tibbitts,Richard Vandlen, Susan D Spencer, Richard H Scheller, Paul Polakis, Mark X Sliwkowski
Genentech, Inc., South San Francisco, California 94080, USA. jagath@gene.com

Antibody drug conjugates (ADCs) combine the ideal properties of both antibodies and cytotoxic drugs by targeting potent drugs to the antigen-expressing tumor cells, thereby enhancing their antitumor activity. Successful ADC development for a given target antigen depends on optimization of antibody selection, linker stability, cytotoxic drug potency, and mode of linker-drug conjugation to the antibody. Here, we systematically examined the in vitro potency as well as in vivo preclinical efficacy and safety profiles of a heterogeneous preparation of conventional trastuzumab-mcc-DM1 (TMAb-mcc-DM1) ADC with that of a homogeneous engineered thio-trastuzumab-mpeo-DM1 (thioTMAb-mpeo-DM1) conjugate.

Primary cilia membrane assembly is initiated by Rab11 and transport protein particle II (TRAPPII) complex-dependent trafficking of Rabin8 to the centrosome.
Proc. Natl. Acad. Sci. U.S.A.
Proc Natl Acad Sci U S A 2011 Feb 27;108(7):2759-64. Epub 2011 Jan 27.
Christopher J Westlake, Lisa M Baye, Maxence V Nachury, Kevin J Wright, Karen E Ervin, Lilian Phu, Cecile Chalouni,John S Beck, Donald S Kirkpatrick, Diane C Slusarski, Val C Sheffield, Richard H Scheller, Peter K Jackson
Genentech, South San Francisco, CA 94080, USA.

Sensory and signaling pathways are exquisitely organized in primary cilia. Bardet-Biedl syndrome (BBS) patients have compromised cilia and signaling. BBS proteins form the BBSome, which binds Rabin8, a guanine nucleotide exchange factor (GEF) activating the Rab8 GTPase, required for ciliary assembly.

Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.
Nat. Biotechnol.
Nat Biotechnol 2012 Feb 22;30(2):184-9. Epub 2012 Jan 22.
Ben-Quan Shen, Keyang Xu, Luna Liu, Helga Raab, Sunil Bhakta, Margaret Kenrick, Kathryn L Parsons-Reponte, Janet Tien, Shang-Fan Yu, Elaine Mai, Dongwei Li, Jay Tibbitts, Jakub Baudys, Ola M Saad, Suzie J Scales, Paul J McDonald,Philip E Hass, Charles Eigenbrot, Trung Nguyen, Willy A Solis, Reina N Fuji, Kelly M Flagella, Darshana Patel, Susan D Spencer, Leslie A Khawli, Allen Ebens, Wai Lee Wong, Richard Vandlen, Surinder Kaur, Mark X Sliwkowski, Richard H Scheller, Paul Polakis, Jagath R Junutula
Genentech Inc., 1 DNA Way, S. San Francisco, California, USA.

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione.

Rac and Rab GTPases dual effector Nischarin regulates vesicle maturation to facilitate survival of intracellular bacteria.
EMBO J.
EMBO J 2013 Mar 5;32(5):713-27. Epub 2013 Feb 5.
Coenraad Kuijl, Manohar Pilli, Suresh K Alahari, Hans Janssen, Poh-Sim Khoo, Karen E Ervin, Monica Calero,Sobhanaditya Jonnalagadda, Richard H Scheller, Jacques Neefjes, Jagath R Junutula
Genentech Inc., South San Francisco, CA 94080, USA. kuijlc@gene.com

The intracellular pathogenic bacterium Salmonella enterica serovar typhimurium (Salmonella) relies on acidification of the Salmonella-containing vacuole (SCV) for survival inside host cells. The transport and fusion of membrane-bound compartments in a cell is regulated by small GTPases, including Rac and members of the Rab GTPase family, and their effector proteins. However, the role of these components in survival of intracellular pathogens is not completely understood.

In search of the molecular mechanism of intracellular membrane fusion and neurotransmitter release.
Nat. Med.
Nat Med 2013 Oct;19(10):1232-5
Richard H Scheller
Genentech Research and Early Development, 1 DNA Way, San Francisco, California, USA.
An improved and robust DNA immunization method to develop antibodies against extracellular loops of multi-transmembrane proteins.
MAbs
MAbs 2014 Jan-Feb;6(1):95-107
Meredith Hazen, Sunil Bhakta, Rajesh Vij, Steven Randle, Dara Kallop, Vicki Chiang, Isidro Hötzel, Bijay S Jaiswal, Karen E Ervin, Bing Li, Robby M Weimer, Paul Polakis, Richard H Scheller, Jagath R Junutula, Jo-Anne S Hongo
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry.
Site-specific trastuzumab maytansinoid antibody-drug conjugates with improved therapeutic activity through linker and antibody engineering.
J. Med. Chem.
J Med Chem 2014 Oct 18;57(19):7890-9. Epub 2014 Sep 18.
Thomas H Pillow, Janet Tien, Kathryn L Parsons-Reponte, Sunil Bhakta, Hao Li, Leanna R Staben, Guangmin Li, Josefa Chuh, Aimee Fourie-O’Donohue, Martine Darwish, Victor Yip, Luna Liu, Douglas D Leipold, Dian Su, Elmer Wu, Susan D Spencer, Ben-Quan Shen, Keyang Xu, Katherine R Kozak, Helga Raab, Richard Vandlen, Gail D Lewis Phillips, Richard H Scheller, Paul Polakis, Mark X Sliwkowski, John A Flygare, Jagath R Junutula
Genentech, Inc. , 1 DNA Way, South San Francisco, California 94080, United States.

Antibody-drug conjugates (ADCs) have a significant impact toward the treatment of cancer, as evidenced by the clinical activity of the recently approved ADCs, brentuximab vedotin for Hodgkin lymphoma and ado-trastuzumab emtansine (trastuzumab-MCC-DM1) for metastatic HER2+ breast cancer. DM1 is an analog of the natural product maytansine, a microtubule inhibitor that by itself has limited clinical activity and high systemic toxicity. However, by conjugation of DM1 to trastuzumab, the safety was improved and clinical activity was demonstrated.

Richard H Scheller, PhD

Published on 16 Sep 2014

The Keck School of Medicine of USC is the first medical school in the nation to host the Lasker Lectures, featuring recipients of the prestigious 2013 Albert Lasker Basic Medical Research Award. In this installment, Richard H. Scheller, PhD, executive vice president of Genentech research and early development, discusses breakthoughs in drug development that are turning the tide in the war against cancer.

https://www.youtube.com/watch?v=Fx54EVJMcxM

Kavli Prize 2015

Xenon Pharmaceuticals Appoints Dr. Richard H. Scheller to Its Board of Directors

Biopharmaceutical company Xenon Pharmaceuticals (NasdaqGM:XENE) reported on Monday the addition of Richard H. Scheller, PhD to its board of directors.

Most recently, Dr Scheller has served as chief science officer and head of Therapeutics at 23andMe.

Previously Dr Scheller was the executive vice president at Genentech Research and Early Development & a member of the Roche Corporate Executive Committee; chief scientific officer, executive vice president of Research and senior vice president of Research at Genentech; as well as a professor of Molecular and Cellular Physiology and of Biological Sciences at Stanford University Medical Center and an investigator of the Howard Hughes Medical Institute.

Dr Scheller is currently an adjunct professor in the Department of Biochemistry and Biophysics, School of Medicine at the University of California, San Francisco.

He has been a Director at Xenon Pharmaceuticals Inc. since March 16, 2015 and Medrio, Inc. since November 2012. He serves as a Member of the Medical and Scientific Review Board of Evotec (US), Inc. (Renovis Inc.). In 2014, he was named a trustee of Caltech. He served as a Member of Scientific Advisory Board of Intra-Cellular Therapies, Inc. and Rinat Neuroscience Corporation.

He served on numerous advisory boards including the National Advisory Mental Health Council of the National Institutes of Health. Dr. Scheller served as chairman of the Genentech Foundation’s board of directors. He is a globally recognized leader in biomedical research.

He has published over 200 papers in scientific journals, and worked in cell biology. He has received several additional awards for his work elucidating the molecular mechanisms governing neurotransmitter release, including the 2013 Albert Lasker Basic Medical Research Award, the 2014 California Institute of Technology’s Caltech Distinguished Alumni Award, the 2010 Kavli Prize in Neuroscience, and the 1997 U.S. National Academy of Sciences Award in Molecular Biology. He is a Fellow of the American Academy of Arts and Sciences. Dr. Scheller holds a Doctorate in Chemistry from the California Institute of Technology in 1980, where he was also a Postdoctoral Fellow, Division of Biology. He was also a Postdoctoral Fellow at Columbia University, College of Physicians & Surgeons. He has Bachelor’s Degree in Biochemistry in 1975 at the University of Wisconsin, Madison.

Education: 1971-1975 University of Wisconsin-Madison B.S. – Biochemistry with Honors 1975-1980 California Institute of Technology Ph.D. – Chemistry – Advisor: Eric H. Davidson 1980-1981 California Institute of Technology Postdoctoral Fellow-Division of Biology Advisor: Eric H. Davidson 1981-1982 Columbia University-College of Physicians & Surgeons Postdoctoral Fellow-Molecular Neurobiology Advisors: Richard Axel and Eric R. Kandel Industry Positions: 2001-2003 Senior Vice President – Research Genentech, Inc. 2003-2009 Executive Vice President – Research Genentech, Inc. 2008-2009 Chief Scientific Officer and Executive Vice President – Research Genentech, Inc. 2009- Executive Vice President – Genentech Research and Early Development (gRED) and Member of the Enlarged Roche Corporate Executive Committee Academic Appointments: 1982-1987 Assistant Professor, Department of Biological Sciences, Stanford University 1987-1990 Associate Professor, Department of Biological Sciences, Stanford University 1990-1993 Associate Professor, Department of Molecular and Cellular Physiology, Stanford University Associate Professor (by courtesy), Department of Biological Sciences, Stanford University

Read Full Post »

Ubiquitin researchers win Nobel

Posted in Proteins, Proteomics, Signaling, Signaling & Cell Circuits, Technology Transfer: Biotech and Pharmaceutical, Ubiquitinylation, tagged , , on September 5, 2015| Leave a Comment »

Ubiquitin researchers win Nobel

Larry H. Bernstein, MD, FCAP, Curator

 

Ciechanover, Hershko, and Rose awarded for discovery of ubiquitin-mediated proteolysis

http://www.the-scientist.com/?articles.view/articleNo/23111/title/Ubiquitin-researchers-win-Nobel/

Nature Cell Biology 2, E171 (2000) http://dx.doi.org:/10.1038/35036412

The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2004 “for the discovery of ubiquitin-mediated protein degradation” jointly to

Aaron Ciechanover
Technion – Israel Institute of Technology, Haifa, Israel,

Avram Hershko
Technion – Israel Institute of Technology, Haifa, Israel and

Irwin Rose
University of California, Irvine, USA

 

Proteins labelled for destruction

Proteins build up all living things: plants, animals and therefore us humans. In the past few decades biochemistry has come a long way towards explaining how the cell produces all its various proteins. But as to thebreaking down of proteins, not so many researchers were interested. Aaron Ciechanover, Avram Hershko and Irwin Rose went against the stream and at the beginning of the 1980s discovered one of the cell’s most important cyclical processes, regulated protein degradation. For this, they are being rewarded with this year’s Nobel Prize in Chemistry.

Aaron Ciechanover, Avram Hershko and Irwin Rose have brought us to realise that the cell functions as a highly-efficient checking station where proteins are built up and broken down at a furious rate. The degradation is not indiscriminate but takes place through a process that is controlled in detail so that the proteins to be broken down at any given moment are given a molecular label, a ‘kiss of death’, to be dramatic. The labelled proteins are then fed into the cells’ “waste disposers”, the so called proteasomes, where they are chopped into small pieces and destroyed.

 

Avram Hershko is an Israeli biochemist and winner of the 2004 Nobel Prize for Chemistry.

Hershko (born December 31, 1937) was born as Hersko Ferenc in Karcag, Hungary. In 1950, Hershko and his family emigrated from Hungary to Israel, where he adopted the name Avram. Hershko received his M.D. and Ph.D. from the Hadassah Medical School of the Hebrew University. In 1965-67, Hershko worked as a physician in the Israel Defense Forces.

In 1969-72, Hershko was a postdoctoral fellow with the late Dr. Gordon Tomkins at the University of California, San Francisco.

In 1987, Hershko was awarded the Weizmann Prize for Sciences, an honor given to top Israeli scientists. In 1994, he won the Israeli Prize for his contributions to Israeli society through biochemistry and medicine.

In 2004, Hershko was awarded the Nobel Prize in Chemistry “for the discovery of ubiquitin-mediated protein degradation.”

Ciechanover was born in Haifa, a year before the establishment of Israel. He is the son of Bluma (Lubashevsky), a teacher of English, and Yitzhak Ciechanover, an office worker.[1] His family were Jewish immigrants from Poland before World War II.

He earned a master’s degree in science in 1971 and graduated from Hadassah Medical School in Jerusalem in 1974. He received his doctorate in biochemistry in 1981 from the Technion – Israel Institute of Technology in Haifa before conducting postdoctoral research in the laboratory of Harvey Lodish at the Whitehead Instituteat MIT from 1981-1984. He is currently a Technion Distinguished Research Professor in the Ruth and Bruce Rappaport Faculty of Medicine and Research Institute at the Technion.

Ciechanover is a member of the Israel Academy of Sciences and Humanities, the Pontifical Academy of Sciences, and is a foreign associate of the United States National Academy of Sciences.

As one of Israel’s first Nobel Laureates in Science, he is honored in playing a central role in the history of Israel and in the history of the Technion – Israel Institute of Technology

 

  • Ciechanover, A., Hod, Y. and Hershko, A. (1978). A Heat-stable Polypeptide Component of an ATP-dependent Proteolytic System from Reticulocytes. Biochem. Biophys. Res. Commun. 81, 1100–1105.
  • Ciechanover, A., Heller, H., Elias, S., Haas, A.L. and Hershko, A. (1980). ATP-dependent Conjugation of Reticulocyte Proteins with the Polypeptide Required for Protein Degradation. Proc. Natl. Acad. Sci. USA 77, 1365–1368.
  • Hershko, A. and Ciechanover, A. (1982). Mechanisms of intracellular protein breakdown. Annu. Rev. Biochem. 51, 335–364.

Interview Transcript

Transcript from an interview with the 2004 Nobel Laureates in Chemistry Aaron Ciechanover, Avram Hershko and Irwin Rose, on 9 December 2004. Interviewer is Joanna Rose, science writer.

Aaron Ciechanover, Avram Hershko and Irwin Rose during the interview

Aaron Ciechanover, Avram Hershko and Irwin Rose during the interview.

Dr Ciechanover, Dr Hershko and Dr Rose, my congratulations to the Nobel Prize and welcome to this interview. I know that you two started as medical doctors but you are in science now, and you get the prize for scientific research. How come you left medicine?

Avram Hershko: Well, I started out as a medical student, I wanted to be a doctor. And during my medical studies I studied biochemistry. That was one of the subjects that every medical student studies, so I liked it very much. I liked, you know, the whole concept of biochemistry, of looking for chemical processes in cells, so we had, we could take off one year from the studies to spend in research in the lab. I also found a very good teacher, Jacob Mager, and I wanted to spend it with him, so I did. That’s how I got involved in biochemistry. Afterwards, I finished my medical studies but already, I, after that one year, I knew that I will go to biochemistry and not to practical medicine. That’s how I started. So, it’s, it’s, like all things in life, it starts by some kind of accident or so, that was the accident, I met a subject during my studies that I liked.

And a good teacher.

Avram Hershko: And a very good teacher.

Was it also a topic, an issue that you were interested in?

Avram Hershko: No, no, not yet, not yet. Mager was interested in many subjects so that was … Actually, I continued with him after my army service as a doctor, and during the course of a couple of years I evolved in four completely different subjects, protein, synthesis, purine metabolism, and a certain disease called glucose-6-phosphate dehydrogenase deficiency, because he was interested in many things, so that gave me a very good background, a very, very, you know, very good basic background.

What about you, Dr Ciechanover?

I fell in love with biochemistry …

Aaron Ciechanover: Surely you can repeat the story verbatim. The same very story, I started in the same medical school, and after four years I decided to try and taste, I fell in love with biochemistry, too.

Like ten years later.

Aaron Ciechanover: Exactly ten years later, and I also decided to taste it, and at that time at medical school they let students take one year off for medical studies, try some research, so I went into biochemistry, same very story, different mentor. And, a wonderful mentor, and I studied lipids.

Also.

Aaron Ciechanover: Not proteins at all, and then exactly, made a decision, that that’s it. But I had, because of obligations to serve in Israel in the military as a physician. I completed my medical studies, went to serve in the army, but meanwhile, in between, I was looking already for a future mentor, in biochemistry, and Avram was at the time abroad, in the University of California in San Francisco, and I got rave recommendation, that he is a great teacher and a great biochemist, and I wrote him, and he was ready to accept me, and there started this story. More or less.

So did you go to the States?

Aaron Ciechanover: No, no, he came here. He returned to /- – -/ fellow, he started a new department in Haifa, which was a new medical school, I joined him, not initially on this project, on a different one because I still had to serve in the army. It’s a little bit complicated date-wise, but basically it’s the same very story, mentorship, the same footstep, without knowing where I am going.

You will never know.

Aaron Ciechanover: I never know, but it’s basically, ten years later the same very footsteps.

Oh, that’s funny. What about you, Dr Rose? How did you get …

Irwin Rose: I have an anomalist’s story. It doesn’t, there is no precedent for this. We moved from the east coast to the town of Spokane, Washington, when I was about 13 years old, and I did not adapt very well to the, to the style of the place, and I spent most of my time in the public library. And I enjoyed the company of the Journal of Biological Chemistry, because it was the book shaped thing, in those days, you know, it was the small journal …

Avram Hershko: At the age of 13?

Irwin Rose: No, you know, like a couple of years, you know, I was very unpopular with the other students, and so I read the Journal of Biological … the small, the small Journal of Biological Chemistry, and I found an article I thought I understood. And I read it and I thought I understood it to the point where I could make some suggestions as to how it would be, the experiment might work, and then I was very satisfied with that, and then I … I didn’t spend much time in science at that point. Went into the navy, got out of the navy, tried to go to the University of California at Berkeley, but due to the failure to find the bulletin board announcing the laboratory time of organic chemistry, I couldn’t do my organic chemistry there.

So I said OK, I’ll be a biochemist …

So I went back to the State College of Washington and there I was influenced, I would say, by the embryology teacher, who was a very strong personality in terms of academic research, he tried to encourage his students. Then I went to the University of Chicago and there was a big shock to learn all the new kinds of things that they were teaching there, in organic chemistry and that sort of stuff, and very attractive concepts, and things began to come together in my mind as to how chemistry worked and how I might be able to exploit some of the early kinds of techniques that were being used in organic chemistry into biochemistry, which was something I was attracted to, due to my reading of the Journal of Biological Chemistry. So at that point I signed up, there was a big gymnasium, and people were signing people up for which major you were going to go into. So I said OK, I’ll be a biochemist.

So I entered into the department of biochemistry, never saw the chairman of biochemistry because he was the appointed ambassador to Britain for the United States. So I floated around in the department of biochemistry and learned some interesting things, and then I began to … I never wanted to work with a mentor, because I always wanted to have my own reputation and be free to do what I wanted to do. So I worked with the weakest people in the department. Don’t make that public. No, I don’t mention the names, but … so I did that sort of thing and that way I came to learn some more independence, and once in a while I did a good experiment, and so I had more confidence that I could do research, and so that’s how it got started.

Avram Hershko: Can I mention the story that you did your PhD or eight counts per minute or …

Irwin Rose: Oh yes, well, in those days people weren’t counting, people counted on planchettes. And you …

Avram Hershko: Puckered.

Irwin Rose: Well, it could be, depends on they were flat.

Avram Hershko: You dried them, didn’t you?

Irwin Rose: Yes, you dried them out, depending … yes, that’s right. You had to dry them out, it depends on what the compound was, but if it was trillium you had to get an infinitely thin layer so that you wouldn’t get self-absorption.

Avram Hershko: It’s common, self-absorption on a planchette.

Irwin Rose: Did you guys do that, too?

Aaron Ciechanover: Yeah, yeah, yeah.

Avram Hershko: We had a counter with only three /- – -/ so we moved it like that …

Irwin Rose: Oh yeah, yeah.

Avram Hershko: … it was a big excitement.

Irwin Rose: So I wasn’t that primitive. You were doing these things in Israel, an advanced state.

Aaron Ciechanover: You came to our country.

Irwin Rose: I did. I came to Israel. But anyway, yes, so we did those things. And even if you had eight counts above background, if there were eight, there were eight. That’s right. So you could do some experiments. That’s how it worked out.

So how did you meet together?

Avram Hershko: Well, that’s another story. I got interested in protein degradation during my post-doc fellowship in San Francisco, and when I came back to Israel I continued with that, and at that time it was a very obscure field, you know. People, there were all kinds of, not too many people were interested in it. Those that were interested were not very good. So I looked for somebody, and so my first time I think I came up and I looked for somebody to spend a sabbatical with. I couldn’t find anybody that attracted me. So then I met Ernie at a meeting in 1976, one year before, before my sabbatical was due. And do you remember, we met in the breakfast, so I said can I, just began to talk …

Irwin Rose: It’s alright, I forgot.

… it turned out that he was interested in protein degradation. And that was a secret …

Avram Hershko: … breakfast table, so I knew who he was, he was very well known for his work on enzyme mechanism. That I knew, but then I asked him what are you interested in, in other things? So it turned out that he was interested in protein degradation. And that was a secret, it was a secret because he never published anything on it, and I asked him how come you never published anything, and so he said there is nothing worth publishing on protein degradation. So that’s what he said.

Irwin Rose: Yeah, that was my opinion. Well, because I hadn’t done anything, you don’t say it right.

Avram Hershko: OK. Well, that’s how I remember it. And anyhow, I liked that attitude very much, and asked, I asked him can I spend my sabbatical with you? And he said yes, so that’s how it started, and then Aaron, the same year he started his PhD with me, and after my sabbatical the following, the summer after my sabbatical, Aaron joined us, and then he joined us for a couple of summers afterwards, so that’s how, that’s how the whole connection started.

But how come you pick up an obscure field in science, to work on?

Irwin Rose: Well, I’ll tell you, because when I first worked at Yale, the guy who had a lab next to me had made the original observation that there was a protein, there was an energy dependent on protein breakdown. Now, nobody believed him, but he had made some pretty strong observations that if you …

Avram Hershko: Here, we could mention names.

Irwin Rose: Yes, Melvin Simpson. He made these important observations.

Aaron Ciechanover: He hardly believed himself, because when you go into discussion on the paper, you kind of come to a convoluted argument whether it’s a direct requirement or indirect. We can do the conclusion that it’s indirect.

When was it?

Avram Hershko: 1953, so …

Irwin Rose: So I didn’t read the paper, but I had this man in the laboratory next to me and he said, he made this observation and I got very interested in it. And worked on it for, on sabbatical, and when I went to England and when I went to Israel I got mice from Mager, it turned out the same guy, but he wasn’t there at the time, and … but I never found an energy dependence on the protein breakdown. And it turns out later on that a fellow named Art Haas who had been a post doc with me, made the observation that if you’re not careful when you break cells, there’s a lysosomal enzyme that degrades the ubiquitin. So I never would have found it, you know. Somebody else had to make the observation that you could make a self-resistent that … that would show an ATP dependence on protein breakdown. It was not for me, but I did work on it earlier, and that’s the, that’s why I told you that I’d never made any important observations.

But you three work together. How does it work, to do things together?

Irwin Rose: I don’t do anything.

You do nothing? Who is the worker?

Avram Hershko: Well, that’s, first of all, that’s not true. I remember that you made some ubiquitin preparation …

Irwin Rose: I did.

Avram Hershko: Yes, and it fell on the floor, and then you collected it up from the floor … yeah, yeah. That first step is to boil the extra, because ubiquitin is heat stable, so you boiled it but then it fell on the floor, but you picked it up and it was good, yeah.

Irwin Rose: It was good, nothing could destroy it.

Irwin Rose: It was a licence only enzyme.

Aaron Ciechanover: The /- – -/ can take it, but not the floor.

Avram Hershko: But, yeah, but when I came to his lab we already had his first step, which was the fractionation, well, the reticulocyte cell-free system system was actually established in the laboratory of somebody else, Alfred Goldberg in Harvard, but they didn’t …

Aaron Ciechanover: /Inaudible./

Avram Hershko: No, no, but, yeah, but he made it first, he made it first.

Aaron Ciechanover: The first publication was from Harvard, no doubt.

Avram Hershko: But then he didn’t progress, but then he didn’t do what he should have done, which is fractionation. It’s hard to purify right away, but ATP dependent enzyme, he never found it. And what we did was fractionation and constitution, so we already had this first step of separating it into two, two fractions, fraction one and fraction two.

… we didn’t really understand that it’s binding …

So during these two years between the beginning of ’77 when I write to your lab and December of ’79, when we made the breakthrough in your lab, we purified the component from fraction one, we found it a heat stable protein, and then you had a part in that, you also boiled ubiquitin, and then in Haifa we found that it gets … when we labelled it with iodine and we found it gets bound to proteins and ATP dependent reaction, but we didn’t really understand that it’s binding, its co-herent binding the substate until that summer in 1971 in the laboratory of Rose where you invited me, together with Aaron who was then my graduate student in /- – -/ who was there. 1979, 1979. So that is when, when the discovery that ubiquitin …

Irwin Rose: Shall I tell the story about the ubiquitin?

Avram Hershko: Yes. I think I have finished. So then, that’s how I remember it, and how …

Irwin Rose: OK, well, here they had a heat stable factor that was required, and they made the observation that the ubiquitin went on to proteins. And so one of my post docs went to a post doc of another student, of another faculty member at the Fox Chase Cancer Centre, and said, there was a conversation, and do you know of any examples of a protein covalently linked to a protein? And this post doctoral fellow said yes, there is in the nucleus, a protein called ubiquitin that’s covalently linked to histone. And so they rushed to look at the amino acid composition of that so-called ubiquitin, and they compared it to the amino acid composition which you had published, I guess …

Aaron Ciechanover: No, not yet.

Irwin Rose: Not yet published.

Aaron Ciechanover: But in the end it was published back to back with JBC.

Irwin Rose: No, no, no. But how did they know the conversation …

Aaron Ciechanover: No, because they knew, the end story is that the Wilkinson paper came back to back with ours on the /- – -/.

Avram Hershko: OK. Let’s not go into the detail.

Irwin Rose: Well, for some reason or other, they found confidence…

Avram Hershko: They knew that I published that.

Irwin Rose: Really, and I was not a leak.

Avram Hershko: No, no, you were not.

Aaron Ciechanover: No, he was in the lab, he was free and did this. We didn’t hide anything.

Irwin Rose: OK, you’re getting the inside story here. Now, wait a second.

I have a statement from your colleague. “At first nobody cared about your work, and those that knew something about it, they didn’t believe it.” Was it so …?

Irwin Rose: Who said that?

Avram Hershko: That was, that was Fred Goldberg, yeah.

Aaron Ciechanover: Let’s not mention names.

Avram Hershko: Oh! No, we didn’t mention names.

That citation is right.

Aaron Ciechanover: I’ll tell you, I’ll tell you a funny story. I left the lab in ’81, basically after my PhD was completed I submitted it and I went to Harvard, I went to MIT to do a post doc fellow, and Harvard carried out weekly seminars. And in this weekly seminar, one of the founders in the field of proteolysis, one of the originally, not the founder, but it doesn’t matter. A famous scientist in the field presented the weekly seminar at Harvard. I knew of him because he was our competitor for many years, and I went to hear the seminar, so I crossed the river by the bus, I took the shuttle bus that goes /- – -/ and I was sitting in the very back bench. And this was probably about two weeks before you came to visit, it was the very beginning of my, do you remember when I met you, I came to the airport to pick you up.

Avram Hershko: Yeah, yeah.

Aaron Ciechanover: And then, near me, was sitting a very famous scientist that I only later realised that his name is Arthur Dee, a very famous scientist, and after this presentation of the professor, this was only ’81 when we had like eight or nine papers already in the literature with a huge amount of information there. And he was a protein researcher and he raised his hand, I remember very well, and the other guy, when we were both  /- – -/ he said, you know, I have a question to ask you. There is a fellow in Haifa by the name of Hershko, and another one with a very complicated Polish name that I cannot even pronounce, that published a series of papers on a small protein that is attached to other proteins and marks them for degradation, can you comment on it? And he basically dismissed it as an artefact.

… it adds to our benefit, because they left us alone for seven successive years …

And I don’t, I don’t criticise him, all I’m telling you it was symbolic for me enough for after eight papers in the literature, this was the spirit in the field from people who worked in the field, and there were very few. As a matter of fact, it adds to our benefit, because they left us alone for seven successive years, even after I left the lab to work out basically the entire system. The next scientist to join the field was a scientist at MIT, Alex Varshavsky, who joined in ’84, ’83, but published in ’84, and given I was there and collaborated, so for seven successive years they let us lay the entire stone down in the literature so I don’t criticise him, actually I appreciate him tremendously for letting us do it. You know, in retrospect.

But I wonder, how do you survive as a scientist when nobody believes you somehow? Nobody’s interested. You become kind of non-visible.

Irwin Rose: You’re making observations, and the observations get published, so the observations are true. Whether anybody will say that belongs to a big story like it turns out to be is not predictable, but so you don’t make claims like that. You say that this is very interesting and so on and so on and so on, and you keep following it up, and it doesn’t necessarily become the centre of attention yet, until you build a big enough story. I think that’s the way it works.

We all survive because funding for research was generous in those days, you know. It’s been less generous now, and we have a peer review system which is more critical and so I think you have to, you have to add successively to the picture you’re trying to portray. It’s not sufficient to just provide data. So I think that’s part of it. But I agree that it’s important to be left alone for a sufficient amount of time in order to be able to do it, and not feel that you’re in the middle of a big activity already, so you know, you need to do that sort of thing.

So do you think you would get support today for such work, which was kind of apart?

Avram Hershko: Well, I hope the fund /- – -/ look up your website and will hear these things. Because it’s … yeah, Joe Goldstein, you know, a Nobel Laureate and a good one, wrote a nice article about this year’s Lasker Award, in which he compared science to a sculpture by this British sculptor who had his stone, it was a huge stone of two and a half ton, on which another stone, and another stone, and another stone, and at the end is a little stone, so he said that in science there are big stones and small stones. The important science is the opposite. When you have a little stone, and on top of it you put a bigger stone and then a bigger stone. If you throw out a big stone at the beginning so there’s a lot of publicity sometimes nothing comes out of it, and the scientist, to find his little stone, on which the other stones can be built. So I recommend to read his article.

Now you find the small stones, Dr Rose, in your kitchen, as I understand it. You have a small laboratory there?

Irwin Rose: You want to talk about my kitchen?

Yeah. Your laboratory, I would say.

Irwin Rose: Well, when I retired from Fox Chase I took my spectrophotometer and a lot of my chemicals, based on a sort of suggestion of Dr … his recommendation. So I took all my chemicals and my spectrophotometer and my constant temperature bath and so forth with me to Irvine, and when the person whose laboratory I was sitting decided to retire, I had to do something with the spectrophotometer and so I found a place in my kitchen for it. And this was very convenient because it saved me a lot of time. I didn’t have to go to work every day and if I had a little experiment to do I could do it in my kitchen. So that was very good, although I’ve got a lot of chemicals that I have no use for and I’d like to take them back.

Aaron Ciechanover: Send them over, send them over.

Irwin Rose: I’ll send them over. I’ll get a box.

Avram Hershko: But I worry that you don’t have an ice machine. You need an ice machine.

Irwin Rose: No, I don’t have an ice machine. But I have a freezer and I can make ice cubes and I can break them up.

It’s kind of worrying, in science. So you can work when everything’s /- – -/ ?

Irwin Rose: Yeah, that’s right, exactly.

So are you the kind of scientists that work all day and all night long, kind of nerd scientists?

So that’s my recommendation, do not retire. Do not retire fellas. …

Irwin Rose: I think we all work all day and all night long. I do. I don’t have any hobbies, you know, I’m very embarrassed when people ask me what are my hobbies, I don’t have any hobbies. I mean, it’s just enough to keep up with the things I’m trying to solve. You know, I used to work on little puzzles and so on and so forth. Each puzzle requires attention and, so you get an idea. You get your ideas at different times. Sometimes your wife makes a statement and you say: aha, maybe you’re right. And so you go off to your kitchen, and do a little experiment, so you try to, that’s the way you make progress, if you continue these things. So that’s my recommendation, do not retire. Do not retire fellas.

Avram Hershko: I won’t.

Aaron Ciechanover: I’m never going to.

You worked together in the beginning, you were the graduate student of Dr Hershko, how was it to separate from each other?

Aaron Ciechanover: Well, it’s the nature of science, I think, because you know, you graduate, you go your post doctorate fellowship, and Avram was gracious enough to bring me back, but now is independent and that’s the entire idea, if you bring a young scientist back, you give him a bench, start up funds, and then you tell him now in five years, come back in five years, and show the committees that you worked for something. So actually, you know, it would be unnatural if we would have continued to work together. So, each of us is independent. Now we’re in the same institute and that’s the whole idea of children that grow up, students that become their own, scientists on their own, I think that’s the way.

Do you compete with each other?

Avram Hershko: No, there is enough to do in the ubiquitin field, we don’t feel that we had to compete. There are different aspects of the ubiquitin field. I am working on cell cycle and he works on …

Aaron Ciechanover: /- – -/. Completely different.

How is it to live in a small country with big problems and to get funds for science?

Avram Hershko: It is not easy, it is not easy. You have to know the daily tension which is of course distractive. The funds are small, some funds for science are small. Graduate students have to go to serve in the army and things like that, so it’s more difficult than elsewhere, but it’s possible, it’s possible.

And now everybody’s happy. About the Nobel Prize. So thank you very much for sharing your thoughts with us, and being with us.

 

See a Video of the Interview
26 min.

 

Share this:

To cite this page
MLA style: “Transcript from an interview with the 2004 Nobel Laureates in Chemistry Aaron Ciechanover, Avram Hershko and Irwin Rose, on 9 December 2004”. Nobelprize.org. Nobel Media AB 2014. Web. 5 Sep 2015. <http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2004/ciechanover-hershko-rose-interview-transcript.html>

http://www.youtube.com/watch%3Fv%3DB1et3Lt_ikc

 

Other related articles published in this Open Access Online Scientific Journal include the following:

 

Innovations in Israel – Nobel Prize in Chemistry 2004, 2011

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/08/18/innovations-in-israel-nobel-prize-in-chemistry-2004-2011/

 

Ubiquitin Pathway Involved in Neurodegenerative Diseases

Larry H Bernstein, MD,  FCAP

https://pharmaceuticalintelligence.com/2013/02/15/ubiquitin-pathway-involved-in-neurodegenerative-diseases/

 

Ubiquitin-Proteosome pathway, Autophagy, the Mitochondrion, Proteolysis and Cell Apoptosis: Part III

Curator: Larry H Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2013/02/14/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis-reconsidered/

 

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes-in-serous-endometrial-tumors/

 

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-in-transcription-ubiquitination-and-dna-repair/

 

Exome sequencing of serous endometrial tumors shows recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes

Reporter and Curator: Dr. Sudipta Saha, Ph.D

https://pharmaceuticalintelligence.com/2012/12/18/exome-sequencing-of-serous-endometrial-tumors-shows-recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes/

 

Read Full Post »

Small but mighty RNAs

Posted in Innovations, Proteomics, RNA Biology, tagged , on September 5, 2015| Leave a Comment »

Small but mighty RNAs

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 3.5

Revised 9/30/2015

Albert Lasker
Basic Medical Research Award

Victor Ambros, David Baulcombe, and Gary Ruvkun

For discoveries that revealed an unanticipated world of tiny RNAs that regulate gene function in plants and animals

The 2008 Albert Lasker Award for Basic Medical Research honors three scientists who discovered an unanticipated world of tiny RNAs that regulate gene activity in plants and animals. Victor R. Ambros (University of Massachusetts Medical School, Worcester) and Gary B. Ruvkun (Massachusetts General Hospital, Boston, Harvard Medical School) unearthed the first example of this type of molecule in animals and demonstrated how the RNAs turn off genes whose activities are crucial for development. David C. Baulcombe (University of Cambridge) established that small RNAs silence genes in plants as well, thus catalyzing discoveries of many such RNAs in a wide range of living things. His findings led to the identification of the biochemical machinery that unifies numerous processes by which small RNAs govern gene activity.

Ambros, Baulcombe, and Ruvkun did not set out to unveil small regulatory RNAs. Ambros and Ruvkun were studying how the worm Caenorhabditis elegans develops from a newly hatched larva into an adult. Baulcombe, in a seemingly unrelated line of inquiry, was probing how plants defend themselves against viruses. All three investigators possessed the open mindedness, wisdom, and experimental finesse to entertain the possibility—and then verify—that tiny RNAs could perform momentous feats. Their work has led to the realization that these molecules are pivotal regulators of normal physiology as well as disease.

RNA—the little molecule that could
In the early 1980s, Ambros joined the laboratory of Robert Horvitz at the Massachusetts Institute of Technology as a postdoctoral fellow. He wanted to outline the means by which genes choreograph the construction of fully formed adults from single cells. Analyses of flies had revealed that certain genes instruct embryos where to place body parts—for example, wings belong on each side and legs belong on the bottom. But Ambros was intrigued by the notion that other genes might specify the timing—rather than the location—of developmental events; alterations in such genes might cause cells and tissues to adopt fates that are normally associated with earlier or later stages of development.

He directed his attention toward one of the first-known genes of this type, called lin-4, which had been identified earlier in the laboratory of Sydney Brenner (Lasker Special Achievement Award, 2000) and subsequently characterized by Horvitz, Martin Chalfie, and John Sulston. Ambros recognized that, during worms’ trek toward adulthood, those with inactive lin-4 get stuck repeating early larval stages. Consequently, they lack cell types and structures typical of fully formed animals and instead contain extra copies of cells ordinarily produced only at early stages. These observations suggested that normal lin-4 allows immature worms to advance past a particular developmental stage; animals with the defective version cannot overcome that hurdle. Ambros discovered that worms lacking a different gene—lin-14—were the antithesis of those with inactive lin-4. The animals skip early steps in development and prematurely acquire characteristics that normally appear later. These and other results suggested that lin-4 and lin-14 exert opposite effects in worm cells.

To dig further into lin-14‘s function and its possible relationship with lin-4, Ruvkun, who by this time (1982) had joined Horvitz’s laboratory as a postdoctoral fellow, collaborated with Ambros to isolate the lin-14 gene. After the investigators set up independent laboratories in the mid 1980s, Ruvkun, at Massachusetts General Hospital in Boston, established that the protein product of lin-14 is abundant during early larval stages and then its quantities plummet. Under conditions in which it unnaturally remains plentiful, early steps repeat, suggesting that the normal drop in the lin-14 protein allows worms to proceed to later stages. Ambros, at Harvard University, found that lin-4 dampens lin-14 activity and thus a picture emerged about how the genes collaborate. At the appropriate time, lin-4 blocks lin-14 and thus allows worms to continue their developmental trajectory.

Ruvkun sought to identify the portion(s) of lin-14 that lin-4 targets, so he tracked down certain genetic anomalies in lin-14‘s sequence that underlie excess production of the lin-14 protein. He found that these alterations reside in the area of the gene that follows the protein blueprint, a span called the 3′ untranslated region (3′ UTR). The perturbations do not influence amounts of the protein’s messenger RNA (mRNA), the molecule that carries genetic information from DNA to the cell’s protein-making factory, Ruvkun showed. Rather, they alter protein quantities. Therefore, molecules that turn off lin-14 after early stages of development presumably exert their effects through the 3′ UTR region of the lin-14 mRNA and prevent the cell from translating its code into protein.

In the meantime, Ambros’s laboratory was isolating the lin-4 gene, which they assumed encoded a protein; although a few RNAs were known to control gene activity in bacteria, conventional wisdom held that, in animal cells, proteins alone enjoy such powers. The team homed in on smaller and smaller pieces of DNA from normal animals that restore typical developmental behavior to a worm that lacks lin-4. Stretches of DNA that were far shorter than standard genes worked. Eventually, the researchers began considering the possibility that its product was an RNA, but they still assumed that the regulatory molecule they sought would be a respectable size. The smallest RNAs known to do anything important in cells contained about 75 nucleotide (nt) building blocks. Eventually, however, their experiments led them to a tiny RNA, composed of about 22 nucleotides. A larger—61 nt—molecule that contained the smaller RNA appeared as well and Ambros noticed that it could fold into a double-stranded “hairpin”—a structure whose significance would become clear years later.

In an exciting exchange of data, Ambros and Ruvkun realized that the 22-nt lin-4 RNA matched sections within the 3′ UTR of the lin-14 mRNA: These sequences could bind one another by the same base-pairing rules that hold together the Watson and Crick DNA strands. In this view, the tiny lin-4 RNA settles on the target lin-14mRNA—in its 3′ UTR—and the resulting double-stranded structure somehow interferes with translation of the lin-14 mRNA’s genetic information into protein (see illustration).

Image of microRNA
Small but mighty.
This scheme shows how one type of tiny RNA, a microRNA (miRNA), silences genes. It is cut out of a precursor hairpin-shaped pre-miRNA to form a mature miRNA, which binds to the 3′ untranslated region (3′ UTR) of a target gene’s messenger RNA and turns off its activity. [Credit: Carin Cain. Based on an illustration from Victor Ambros]

Despite verification that lin-4 was a tiny RNA with huge regulatory powers, these 1993 findings constituted a mere blip on most biologists’ radar screens: lin-4 resided only in worms, so the phenomenon seemed like an oddity that most organisms did not exploit. Worms were exotic in many ways, experts reasoned, and the observation only fueled that attitude.

Branching out to plants and beyond
Across the Atlantic, David Baulcombe, then of the Sainsbury Laboratory in Norwich, UK, was studying how plants resist viruses. When he and others added to viral-infected plants unusual versions of viral genes, the mRNA copies of the normal genes as well as the newly introduced ones disappeared. Similarly, experimentally added non-viral genes suppressed activity of plant genes that contained similar sequences. Baulcombe proposed that such gene silencing occurs when RNAs embrace target mRNA—through typical Watson-Crick base-pairing—and promote destruction of the mRNA or interfere with its translation into protein. However, no one could find such RNAs.

Baulcombe reasoned that the predicted RNAs might have eluded researchers because the molecules were shorter than anyone imagined and thus, experiments had not been designed to detect them. In 1999, he and a postdoctoral fellow in his laboratory, Andrew Hamilton, devised a hunt specifically for small RNAs. They added test genes to plants and found 25-nt long RNAs that matched; furthermore, these small RNAs appeared only under conditions in which target mRNA activity was shut off. The stunning similarity in size between the plant and worm RNAs suggested that small regulatory RNAs exist in many organisms. Furthermore, it hinted at the presence of cellular machinery that dedicates itself to creating these precisely sized molecules and then uses them to quash gene activity.

In 2000, Ruvkun’s laboratory discovered a second tiny regulatory RNA in worms of exactly the same size as thelin-4 RNA and in the same genetic pathway. Similar to the lin-4 RNA, this let-7 RNA dampens activity of its target gene through its 3′ UTR. Furthermore, its sequence too resides within a larger molecule that folds up on itself to form a double-stranded hairpin structure. Later that year, Ruvkun found that many other creatures, including humans, fruit flies, chickens, frogs, zebrafish, mollusks and sea urchins, carry their own versions of let-7, which could also fold into hairpins. The apparent binding site for let-7 RNA in its target was conserved in some of these organisms as well. Moreover, let-7 RNA appeared and disappeared at similar points during development in many of the animals.

The small RNAs, now called microRNAs (miRNAs), had broken through their designation as “worm curiosities.” Researchers realized that the miRNAs likely execute vital functions during growth and development of other creatures as well. Multiple teams raced to expose regulatory RNAs of approximately 22 nucleotides in length. In 2001, Ambros’s group, now at Dartmouth Medical School, in Hanover, as well as those of David Bartel (Massachusetts Institute of Technology) and Thomas Tuschl (Max Planck Institute for Biophysical Chemistry, G�ttingen) discovered almost 100 of these small regulatory RNAs in flies, humans, and worms.

In addition to revealing that small regulatory RNAs dwell in organisms other than worms, Baulcombe’s finding caught many researchers’ attention because it seemed related to a process called RNA interference (RNAi), which had recently exploded onto the biological scene. In RNAi, long RNAs injected into cells hamper gene activity from similar sequences. No one knew why organisms possessed this ability, but presumably it played some role in normal physiology. In 1998, Andrew Fire (Carnegie Institution of Washington, Baltimore) and Craig Mello (University of Massachusetts Medical School, Worcester), published a watershed paper that defined the fundamental features of RNAi (which garnered them the Nobel Prize in 2006). That work yielded the surprising insight that the process depends on double-stranded RNA. However, the means by which double-stranded RNA triggered silencing remained mysterious.

Experiments from Baulcombe’s laboratory provided the crucial clues. Production of the silencing RNA strand depended on the presence of the other strand, he had noticed. This observation suggested that, at some point during manufacture of the small regulatory RNA, it exists as part of a double-stranded molecule. Suddenly it seemed possible that Baulcombe’s tiny RNAs arose by trimming longer molecules of the type that Fire and Mello had discovered. Furthermore, this notion suggested that the hairpin-like lin-4 and let-7 RNAs similarly gave rise to the mature, 22-nt entities.

Scientists wondered whether the cell deployed the same biochemical machinery to create and use RNA molecules that subdued gene activity in all of these gene-silencing systems. However, the mechanisms of the worm miRNAs seemed to differ from those of the plant molecules as well as RNAi. Unlike the system that Ambros and Ruvkun had been untangling, which allowed mRNA to accumulate but thwarted cells’ abilities to translate the information it contained into protein, the plant system and RNAi destroyed mRNA. For that reason and others, many people doubted that the processes were connected. Still the possibility that they shared a common mechanism and machinery tantalized researchers.

In 2001, the Mello, Ruvkun, and Fire groups collaborated to show that efficient liberation of the lin-4 and let-7RNAs from the hairpin molecules relies on the C. elegans version of Dicer, an enzyme that Gregory Hannon (Cold Spring Harbor Laboratory) discovered and named for its ability to chop dsRNA into uniformly sized, small RNAs that direct mRNA destruction during RNAi. These results and others, including similar ones generated by Philip Zamore (University of Massachusetts Medical School, Worcester), cemented the connection between miRNAs and RNAi, thus providing one biological “reason” for the RNAi machinery. Moreover, they identified the apparatus by which cells generate miRNAs and harness them for key pursuits.

Studies in the past several years have indicated that the human genome contains more than 500 and perhaps as many as 1000 miRNAs that could collectively control a third of all of our protein-producing genes. These regulatory molecules have been implicated in a wide range of normal and pathological activities. They play roles not only in embryonic development, but in blood-cell specialization, cancer, muscle function, heart disease, viral infections, and possibly neurological signaling and stem-cell behavior. Researchers are exploring the possibility of using miRNAs “signatures” for diagnosis and prognosis and are considering manipulating their quantities for therapeutic purposes.

Looking where no one had looked before, Ambros, Baulcombe, and Ruvkun spied an unforeseen universe of potent molecules. Their work has elevated these hitherto unrecognized agents into the spotlight of biology and medicine.

by Evelyn Strauss, Ph.D.

2014 Gruber Genetics Prize
Trio honored for pioneering discoveries of microRNAs
By Jim Fessenden
UMass Medical School Communications

Victor R. Ambros, PhD, professor of molecular medicine, has been awarded the 2014 Gruber Genetics Prize, along with longtime collaborator Gary Ruvkun, PhD, professor of genetics at Massachusetts General Hospital and Harvard Medical School, and David Baulcombe, PhD, professor of botany at the University of Cambridge. They received the prize for their pioneering discoveries of the existence and function of microRNAs and small interfering RNAs, molecules that are now known to play a critical role in gene expression. Dr. Ambros is the Silverman Chair in Natural Sciences and co-director of the RNAi Therapeutics Institute.

Gary Ruvkun, PhD, was awarded the Breakthrough Prize in Life Sciences on November 9, along with Victor Ambros for their work on the discovery of microRNAs and their broad use in biology.

The Breakthrough Prize Foundation announced the recipients of the 2015 Breakthrough Prizes in Fundamental Physics and Life Sciences. These distinguished winners, along with previously announced recipients in the Mathematics category, each receive a $3 million prize.

https://breakthroughprize.org/?controller=Page&action=news&news_id=21

 

Gary Ruvkun, PhD, of the Center for Computational and Integrative Biology and the Department of Molecular Biology, has been awarded the 2014 Gruber Genetics Prize from the Gruber Foundation through Yale University for his work with Victor Ambros, PhD, University of Massachusetts, identifying the existence of microRNAs in animals that control the activity of other genes.

http://gruber.yale.edu/genetics/2014/gary-ruvkun

 

Phillip A. Sharp, PhD
Koch Institute Professor of Integrative Cancer Research

The Sharp Lab focuses on the biology and technology of small RNAs and other types of non-coding RNAs.  RNA interference (RNAi) has dramatically expanded the possibilities for genotype/phenotype analysis in cell biology and for therapeutic intervention.  MicroRNAs (miRNAs) are encoded by endogenous genes and regulate primarily at the stage of translation over half of all genes in mammalian cells.  The Sharp laboratory is working to identify physically the target mRNAs for particular miRNAs.  His laboratory has recently discovered a new class of microRNAs that are produced from sequences adjacent to transcription start sites (TSS-miRNAs).  The functions of the small RNAs are a subject of investigation.  His laboratory is also investigating the relationship between gene regulation by miRNAs and angiogenesis and cellular stress.  Most promoters and enhancers in mammalian cells are transcribed divergently with RNA polymerases initiating in both directions.  Divergent transcription generates thousands of long non-coding RNAs.  The extent of elongation by polymerase in either the sense direction or the antisense direction is controlled by recognition of the nascent RNA by U1 snRNP, a spliceosome component.  The function of the divergent non-coding transcripts is being investigated as well as the relationship of RNA splicing, chromatin modifications and transcription.

 

Noncoding RNAs: A Cache of Cancer Clues?

Rummaging through the Noncoding RNA Attic Has Brought to Light Interesting Baubles—miRNAs and lncRNAs

Kathy Liszewski    GEN Sep  1, 2015 (Vol. 35, No. 15)
http://www.genengnews.com/gen-articles/noncoding-rnas-a-cache-of-cancer-clues/5561/

 

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

http://www.genengnews.com/Media/images/Article/thumb_Cornell_graphics1_Neat1Signature1436235247.jpg

 

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

In the postgenomic era, the numerous and diverse noncoding RNA species once dismissed as “junk RNA” are increasingly seen as treasure. Noncoding RNAs, we now know, have diverse functions in health and disease.

Some in the field believe that we have only started to appreciate the riches of noncoding RNA. The ultimate jewels? They may prove to be previously hidden connections with cancer.

Almost as numerous as newly discovered RNA baubles are the newly organized RNA conferences. One such event, Molecular and Cellular Biology: MicroRNAs and Noncoding RNAs in Cancer, was held June 7–12 in Keystone, CO. This event, a Keystone Symposia conference, focused on the complex universe of RNA biology that is disturbed in cancer.

Providing a perspective on the field was John L. Rinn, Ph.D., an associate professor of stem cell and regenerative biology, Harvard Medical School. He said that if you are not reading a new textbook, your ideas about RNA may be wrong.

“This is a dynamic and fast-moving field,” he insisted. “Recent advances in RNA sequencing technologies have disclosed the existence of thousands of previously unknown noncoding transcripts, including many long noncoding RNAs (lncRNAs) whose functions remain mostly undetermined. However, there are an increasing number of examples that show they are not only key regulators of gene expression, but also direct targets of cancer pathways.”

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

http://www.genengnews.com/Media/images/Article/thumb_Harvard_oncolncRNA1512422915.jpg

 

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

Noncoding RNAs include the well-known microRNAs (miRNAs) and the lesser-known lncRNAs. Usually defined on the basis of their size, the single-stranded short miRNAs consist of about 22 nucleotides. They regulate gene expression via translation inhibition or degradation of their mRNA targets. Long ncRNAs refer to transcripts that consist of more than 200 nucleotides and lack extended open reading frames. This arbitrary cutoff excludes most known, yet still poorly understood, classes of small RNAs, such as tRNAs and short interfering RNAs.

Recent studies have provided an intriguing hypothesis: Long ncRNAs may be the missing links in cancer. According to Dr. Rinn, “We now know that lncRNAs constitute an important layer of genome regulation over a diverse array of biological processes and diseases, such as cancer.”

Since the ultimate cause of cancer is altered homeostasis of cellular networks and gene expression programs, even the slightest perturbation of these pathways can result in malignant cellular transformation. “These cell circuits are fine-tuned and largely maintained by the coordinated functioning of proteins as well as ncRNAs,” explained Dr. Rinn. “But, beyond the layer of the well-known protein-coding RNAs and miRNAs, lies the realm of lncRNAs that are fast emerging as critical components and regulators of tumor-suppressor and oncogenic pathways.”

Regulator of Metastasis

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

http://www.genengnews.com/Media/images/Article/thumb_UnivMN_Precancerous1072313061.jpg

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

The major specific hallmarks of cancer include malignant cell migration, invasion, and metastasis. The latter is the primary cause of cancer recurrence and subsequent death.

“Deregulated lncRNAs may impact a diverse array of human cancers, especially their progression,” said David L. Spector, Ph.D., a professor at the Cold Spring Harbor Laboratory. “One of these lncRNAs is the cancer-associated MALAT1 [metastasis-associated lung adenocarcinoma transcript 1]. It’s not only very abundant in many types of human cells; it is also highly conserved across many mammalian species.”

Dr. Spector’s laboratory identified a novel mechanism for 3′-end processing of this nucleus-restricted lncRNA and is dissecting its mechanism of action: “Since MALAT1 is upregulated in several human cancers, it may play an important role during tumor progression. Because its physiological function at the tissue and organismal levels was unknown, we developed a Malat1 loss-of-function genetic mouse model. Since our in vivo studies demonstrated that Malat1 isn’t essential for mouse development and does not affect global gene expression, we are currently pursuing whether this is due to redundancy or context dependency.”

The team of Sven Diederichs at the German Cancer Research Center DKFZ, in collaboration with the Spector lab, examined the role of MALAT1 by knocking it out in human lung tumor cells. They incorporated an RNA-destabilizing element using zinc finger nucleases. This resulted in a unique loss-of-function model with more than a 1,000-fold silencing. When these cells were utilized in a xenograft mouse model, they found that MALAT1-deficient cells had impaired migration and homing to the lungs. This study supports a role of MALAT1 as a regulator of cell migration that is important in gene expression governing the metastasis of lung cancer cells.

These findings have therapeutic implications, according to Dr. Spector. “MALAT1 could represent a predictive marker of disease and use of antisense oligonucleotides could provide a potential therapeutic strategy,” he concluded.

To extend these studies, Dr. Spector’s group is now examining how altered levels of MALAT1 might impact breast cancer initiation and progression.

Noncoding RNAs: A Cache of Cancer Clues?

Rummaging through the Noncoding RNA Attic Has Brought to Light Interesting Baubles—miRNAs and lncRNAs

http://www.genengnews.com/gen-articles/noncoding-rnas-a-cache-of-cancer-clues/5561/

Cornell_graphics1_Neat1Signature1436235247

http://www.genengnews.com/Media/images/Article/Cornell_graphics1_Neat1Signature1436235247.jpg

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

  • In the postgenomic era, the numerous and diverse noncoding RNA species once dismissed as “junk RNA” are increasingly seen as treasure. Noncoding RNAs, we now know, have diverse functions in health and disease.
  • Some in the field believe that we have only started to appreciate the riches of noncoding RNA. The ultimate jewels? They may prove to be previously hidden connections with cancer.
  • Almost as numerous as newly discovered RNA baubles are the newly organized RNA conferences. One such event, Molecular and Cellular Biology: MicroRNAs and Noncoding RNAs in Cancer, was held June 7–12 in Keystone, CO. This event, a Keystone Symposia conference, focused on the complex universe of RNA biology that is disturbed in cancer.
  • Providing a perspective on the field was John L. Rinn, Ph.D., an associate professor of stem cell and regenerative biology, Harvard Medical School. He said that if you are not reading a new textbook, your ideas about RNA may be wrong.
  • “This is a dynamic and fast-moving field,” he insisted. “Recent advances in RNA sequencing technologies have disclosed the existence of thousands of previously unknown noncoding transcripts, including many long noncoding RNAs (lncRNAs) whose functions remain mostly undetermined. However, there are an increasing number of examples that show they are not only key regulators of gene expression, but also direct targets of cancer pathways.”

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

  • Noncoding RNAs include the well-known microRNAs (miRNAs) and the lesser-known lncRNAs. Usually defined on the basis of their size, the single-stranded short miRNAs consist of about 22 nucleotides. They regulate gene expression via translation inhibition or degradation of their mRNA targets. Long ncRNAs refer to transcripts that consist of more than 200 nucleotides and lack extended open reading frames. This arbitrary cutoff excludes most known, yet still poorly understood, classes of small RNAs, such as tRNAs and short interfering RNAs.
  • Recent studies have provided an intriguing hypothesis: Long ncRNAs may be the missing links in cancer. According to Dr. Rinn, “We now know that lncRNAs constitute an important layer of genome regulation over a diverse array of biological processes and diseases, such as cancer.”
  • Since the ultimate cause of cancer is altered homeostasis of cellular networks and gene expression programs, even the slightest perturbation of these pathways can result in malignant cellular transformation. “These cell circuits are fine-tuned and largely maintained by the coordinated functioning of proteins as well as ncRNAs,” explained Dr. Rinn. “But, beyond the layer of the well-known protein-coding RNAs and miRNAs, lies the realm of lncRNAs that are fast emerging as critical components and regulators of tumor-suppressor and oncogenic pathways.”
  • Regulator of Metastasis

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

A Dangerous PartnershipThe major specific hallmarks of cancer include malignant cell migration, invasion, and metastasis. The latter is the primary cause of cancer recurrence and subsequent death.

  • “Deregulated lncRNAs may impact a diverse array of human cancers, especially their progression,” said David L. Spector, Ph.D., a professor at the Cold Spring Harbor Laboratory. “One of these lncRNAs is the cancer-associated MALAT1 [metastasis-associated lung adenocarcinoma transcript 1]. It’s not only very abundant in many types of human cells; it is also highly conserved across many mammalian species.”
  • Dr. Spector’s laboratory identified a novel mechanism for 3′-end processing of this nucleus-restricted lncRNA and is dissecting its mechanism of action: “Since MALAT1 is upregulated in several human cancers, it may play an important role during tumor progression. Because its physiological function at the tissue and organismal levels was unknown, we developed a Malat1 loss-of-function genetic mouse model. Since our in vivo studies demonstrated that Malat1 isn’t essential for mouse development and does not affect global gene expression, we are currently pursuing whether this is due to redundancy or context dependency.”
  • The team of Sven Diederichs at the German Cancer Research Center DKFZ, in collaboration with the Spector lab, examined the role of MALAT1 by knocking it out in human lung tumor cells. They incorporated an RNA-destabilizing element using zinc finger nucleases. This resulted in a unique loss-of-function model with more than a 1,000-fold silencing. When these cells were utilized in a xenograft mouse model, they found that MALAT1-deficient cells had impaired migration and homing to the lungs. This study supports a role of MALAT1 as a regulator of cell migration that is important in gene expression governing the metastasis of lung cancer cells.
  • These findings have therapeutic implications, according to Dr. Spector. “MALAT1 could represent a predictive marker of disease and use of antisense oligonucleotides could provide a potential therapeutic strategy,” he concluded.
  • To extend these studies, Dr. Spector’s group is now examining how altered levels of MALAT1 might impact breast cancer initiation and progression.

One lncRNA, PVT1, is keeping bad company, at least according to new studies linking it to the key cancer-causing oncogene, MYC. This unexpected partnership has stirred up much interest in the scientific community, especially since MYC is linked to a majority of human cancers.

Anindya Bagchi, Ph.D., an assistant professor of genetics, cell biology and development, University of Minnesota, reported that her group began by looking at structural alterations in cancer genome. “[Of particular interest is the loss or gain of particular segments of the genome that occurs recurrently in cancer,” he notes. “One such region that is of immense interest to us is 8q24, a genomic region often found to be gained in a number of cancers.

“The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region. We found that in cancer, MYC is consistently co-gained with an adjacent ‘gene desert’ of about 2 megabases that includes the lncRNA gene PVT1.”

Dr. Bagchi and colleagues utilized chromosomal engineering in mice to construct three iterations to model: MYC only, MYC plus this surrounding area, and the surrounding region alone. “Surprisingly, we found that MYC enhanced tumor growth only when the surrounding region was included,” Dr. Bagchi pointed out. “This verified that MYC is not acting alone.

“We next utilized primary human cancer cell lines and found that PVT1 RNA and MYC protein expression were correlated. Further, we determined that copy number of PVT1 was increased in more than 98% of cancers with MYC gain.”

Finally, Dr. Bagchi’s group definitively fingered PVT1 as the co-conspirator with MYC. The investigators knocked it out of MYC-driven colon cancer cells and found the tumors virtually disappeared. According to Dr. Bagchi, this study complements previous studies and establishes an important finding: Long ncRNA PVT1 interacts with MYC in the nucleus and protects the MYC protein from degradation, probably by reducing phosphorylation of its threonine 58 residue.

“What makes this finding so exciting is that we now may have a much needed tool to target the notoriously elusive MYC protein that has been refractory to small-molecule inhibition,” asserted Dr. Bagchi. “Perhaps by uncoupling this dangerous partnership and targeting PVT1, we could remove the driver that amplifies a major cancer gene.”

  • Prostate Cancer and Noncoding RNA

Given the roles played by ncRNAs in a host of biological processes, it is no surprise that these species also impact prostate cancer progression and therapy resistance. Nonetheless, details of the relationship between ncRNAs and prostate cancer remain to be elucidated, said Dimple Chakravarty, Ph.D., an assistant professor of pathology and laboratory medicine at Weill Cornell Medical College.

“Deregulated or aberrant expression of steroid nuclear receptors are linked with cancer progression and thus are also major targets for therapeutic intervention,” observed Dr. Chakravarty. “But specific therapies are often inadequate.

“For example, the androgen receptor [AR] plays a central role in this malignant progression. Despite the initial effectiveness of therapeutic androgen ablation, resistance inevitably develops to both first generation anti-androgen therapies and to second-generation AR-targeted therapies. The reasons for this are unclear.”

Dr. Chakravarty and colleagues wanted to better understand the role of the estrogen receptor alpha (ERα) that is expressed in prostate cancers. “Our studies identified an ERα-specific noncoding transcriptome signature. This lured us into the noncoding world,” she disclosed.

Dr. Chakravarty and her collaborators, including Mark A Rubin, M.D., a professor of pathology and laboratory medicine at Weill Cornell, scrutinized a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data. The investigators found that the most significantly overexpressed and ERα-regulated lncRNA in prostate cancer samples was a transcript called NEAT1, the nuclear enriched abundant transcript 1.

“Our studies utilized a battery of approaches,” detailed Dr. Chakravarty. “We used qRT-PCR and RNA-ISH to examine NEAT1 mRNA levels in prostate cancer tissue and in cell lines, and we analyzed public datasets of normal versus prostate cancer with advanced disease. Epigenetic studies demonstrated that NEAT1 is recruited to the chromatin of prostate cancer genes and contributes to an epigenetic ‘on’ state.”

 

Dr. Chakravarty expressed excitement over these findings: “This study is the first of its kind to demonstrate transcriptional regulation of lncRNAs by an alternative steroid receptor in prostate cancer. We believe NEAT1 could serve as both a prognostic marker for aggressive prostate cancer and also a potential therapeutic target.

 

“Completed and ongoing studies suggest NEAT1 is a good marker for patient risk stratification and a predictor of therapy resistance. We are now exploring the possibility of knocking it out in vivo to see if there is a therapeutic benefit. It could be that targeting NEAT1 and the androgen receptor in combination may provide a unique treatment strategy for a subset of patients who have advanced prostate cancer.”

  • Mouse Models for Noncoding RNA

Genetically engineered mouse models of human cancer have been indispensable in dissecting the molecular mechanisms involved in tumorigenesis. They also provide powerful platforms for preclinically studying drug sensitivity and resistance, said Andrea Ventura, M.D., Ph.D., a cancer biologist at the Memorial Sloan Kettering Cancer Center.

“Mouse models can explore the physiological function of microRNAs such as determining how they affect development and their response to tumor treatments. It is almost impossible to do these studies otherwise,” explained Dr. Ventura. “Another way mouse models are important is for modeling noncoding RNA.”

 

Tools for Studying and Using Small RNAs: From Pathways to Functions to Therapies
This poster provides an overview of the tools that have been developed to understand the functions of small RNAs and, conversely, the use of small RNAs as tools. Tools that are based on small RNAs have been exploited to investigate gene function in cultured cells and in living animals. Small RNA biogenesis, discovery and functional roles are explored in detail.

Read Full Post »

Turning genetic information into working proteins

Posted in Amino acids, Biochemical pathways, Gene Regulation, Genome Biology, Innovations, Phosphorylase, Phosphorylation, Proteomics, RNA Biology, RNA Biology, Cancer and Therapeutics, Signaling & Cell Circuits, tagged , , , , , on September 5, 2015| Leave a Comment »

Turning genetic information into working proteins

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series 2; 3.3

James E. Darnell Jr. (1930— )
Vincent Astor Professor Emeritus
2002 Albert Lasker Award for Special Achievement in Medical Science

Responsible for the various tasks required in turning genetic information into working proteins, ribonucleic acids are one of the most essential players in the life of a cell. First discovered in 1868, RNA today remains the subject of intense scientific scrutiny. Over the course of a career dedicated to understanding the intricate workings of gene transcription, Rockefeller University scientist James E. Darnell Jr. has revealed some of RNA’s most secretive and surprising mechanisms. For his half-century of illuminating research, Dr. Darnell received the 2002 Albert Lasker Award for Special Achievement in Medical Science.

In 1963, Dr. Darnell described a phenomenon he termed “RNA processing,” a step in the process of gene transcription, which had only recently been elucidated in bacterial systems. Working with mammalian cells — which differ from bacterial cells in that they contain a nucleus, where RNA is created — Dr. Darnell observed that very long strings of RNA disappear from the cell nucleus and that subsequently, shorter RNAs resembling the absent longer ones appear in the cytoplasm. Mammalian cells, he concluded, must distill their massive, immature nuclear RNA into shorter, mature forms that are individually coded for specific purposes by specific segments of the genome.

Dr. Darnell carried the principles of his finding — which he made in ribosomal RNA, part of the construction crew that builds cellular proteins — to other long nuclear RNA, including the longest one, which he named heterogeneous nuclear RNA (hnRNA). His hypothesis, that hnRNA is the precursor of the better known messenger RNA — which carries the genetic blueprint for protein building — soon bore fruit when he found a structural correlation between the two. Certain hnRNAs and nearly all messenger RNAs have a “tail” of adenine nucleotides at one end. Dr. Darnell followed this discovery with the observation that when an hnRNA string with an adenine tail disappears from the nucleus, a messenger RNA with the same tail then appears in the cytoplasm, suggesting a causal link between the two. When he found a second similarity — a cap at the end of the string opposite the adenine tail — he faced a conundrum. Scientific dogma had it that the order of nucleotides in any RNA mirrors that of DNA, whether the RNA is modeled from somewhere in the middle of the DNA or from one of the ends. The matching of a nuclear RNA to its cytoplasmic product by two end pieces glued together was surprising, but the concept was soon proven by colleagues at other institutions and called RNA splicing.

After a brief sojourn in Paris to work in François Jacob’s lab, Darnell worked at MIT, the Albert Einstein College of Medicine, and Rockefeller University on the relationship between mRNA and hnRNA. hnRNA was believed to be the precursor to mRNA, and despite making some key discoveries, Darnell admits that he could not free his imagination from the idea of colinearity and envision an hnRNA spliced to produce a smaller mRNA.

At this time, Darnell turned his attention to the question he had pondered since Paris: how were genes regulated in animal cells? This led to the discovery of the STAT and the Jak-STAT pathway of transcription control.

With the knowledge of RNA processing and splicing, Dr. Darnell next examined how cells begin the process of transcription and how they activate particular segments of DNA. Having moved to Rockefeller University in 1974, he found in the early 1980s that cells retain their specificity only in the context of their natural environment. Away from other liver cells, for example, a single liver cell stops producing liver-specific RNA, though it continues to make RNA for more generic cellular tasks. To pinpoint the signals responsible, which he believed must be coming from outside the cell, Dr. Darnell took a closer look at interferons (IFN), proteins that warn a cell when it’s time to raise its genetic defenses against harmful microbes.

Dr. Darnell’s laboratory studies how signals from the cell surface affect transcription of genes in the nucleus. Originally using interferon as a model cytokine, the Darnell group discovered that cell transcription was quickly changed by binding of cytokines to the cell surface. Introducing IFNβ into cell cultures, he watched as a particular type of mRNA accumulated in the cytoplasm, unaccompanied by any new protein synthesis. Analyzing the mRNA led him to the segment of DNA that had been activated, and the lack of new proteins told him that the cell contained its own, usually dormant, IFN-responsive transcription factor. By isolating a particular stretch of DNA from IFN-treated cells, he was able to call out of hiding the proteins that make up that factor, which, partly because they respond to signals very quickly, he called “STATs.” Dr. Darnell then traced the chemical relay that activates the STATs after IFN contact, called the Jak-Stat pathway.

The bound interferon led to the tyrosine phosphorylation of latent cytoplasmic proteins now called STATs (signal transducers and activators of transcription) that dimerize by reciprocal phosphotyrosine-SH2 interchange. They accumulate in the nucleus, bind DNA and drive transcription. This pathway has proved to be of wide importance, with seven STATs now known in mammals that take part in a wide variety of developmental and homeostatic events in all multicellular animals. Crystallographic analysis defined functional domains in the STATs, and current attention is focused on two areas: how the STATs complete their cycle of activation and inactivation, which requires regulated tyrosine dephosphorylation; and how persistent activation of STAT3 that occurs in a high proportion of many human cancers contributes to blocking apoptosis in cancer cells. Current efforts are devoted to inhibiting STAT3 with modified peptides that can enter cells.

 

Dr. Darnell received his M.D. in 1955 from the Washington University School of Medicine. His career has included poliovirus research with Harry Eagle at the National Institute of Allergy and Infectious Diseases, research with François Jacob at the Pasteur Institute in Paris and academic appointments at the Massachusetts Institute of Technology, the Albert Einstein College of Medicine and Columbia University. In 1974 Dr. Darnell joined Rockefeller as Vincent Astor Professor, and from 1990 to 1991 he was vice president for academic affairs.

A member of the National Academy of Sciences since 1973, he has received numerous awards, including the 2012 Albany Medical Center Prize in Medicine and Biomedical Research, the 2003 National Medal of Science, the 2002 Albert Lasker Award for Special Achievement in Medical Science, the 1997 Passano Award, the 1994 Paul Janssen Prize in Advanced Biotechnology and Medicine and the 1986 Gairdner Foundation International Award.

He is the coauthor with S.E. Luria of General Virology and the founding author with Harvey Lodish and David Baltimore of Molecular Cell Biology, now in its seventh edition. His book RNA, Life’s Indispensable Molecule was published in July 2011 by Cold Spring Harbor Laboratory Press. He is a member of the American Academy of Arts and Sciences and a foreign member of The Royal Society and The Royal Swedish Academy of Sciences.

 

Read Full Post »

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Posted in Academic Publishing, Amino acids, Anaerobic Glycolysis, Biochemical pathways, Biological Networks, Biomarkers & Medical Diagnostics, Bone Disease and Musculoskeletal Disease, Ca2+ triggered activation, Cachexia, Calcium, Calcium Signaling, Calmodulin, Calmodulin Kinase and Contraction, Cancer and Current Therapeutics, CANCER BIOLOGY & Innovations in Cancer Therapy, Cancer Informatics, Cardiac & Vascular Repair Tools Subsegment, Cardiac and Cardiovascular Surgical Procedures, Cardiac muscle regeneration, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Clinical & Translational, Clinical Diagnostics, Clinical Genomics, Competition in regenerative stem cell science, Curation, Curation methodology, Cytoskeleton, Dehydrogenase, Diabetes Mellitus, Discovery process, Disease Biology, Endocrine Diseases, Enzyme Induction, Enzymes and isoenzymes, Explanatory, Fatty acids, Folate and B12, Frontiers in Cardiology and Cardiovascular Disorders, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Genomic Endocrinology, Genomic Expression, Glycobiology: Biopharmaceutical Production, Health Economics and Outcomes Research, Hexokinase, Historical relevance, Inosine nucleotides, Interviews with Scientific Leaders, Iron deficiency, Kinase, Lipid metabolism, Lipids, Liver & Digestive Diseases Research, Loss of function gene, Metabolism, Metabolomics, Methylation, Mg++, Molecular Genetics & Pharmaceutical, mtDNA, Mutant Gene Expression, Myelodysplasia, Myelofibrosis, Myocardial Ischemia, Myocardial Metabolism, Na-K-ATPase, Neurodegenerative Diseases, Neurohumoral Transmission, Nitric Oxide in Health and Disease, Nutrigenomics, Nutrition, Nutrition and Phytochemistry, Nutrition Disorders, Nutritional Supplements: Atherogenesis, Open Access Journals, Oxidative phosphorylation, Pentose monophosphate shunt, Phosphatase, Phosphorylase, Phosphorylation, PKC, Plant extract, Proteins, Proteomics, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, Pyruvate Kinase, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, RNA Biology, S-nitrosylation, Signaling, Signaling & Cell Circuits, Skeletal muscle regeneration, Small Molecules in Development of Therapeutic Drugs, Theoretic convergence, Transcriptomics, Translational Science, Ubiquitinylation, Warburg effect, tagged , , , , , , , , , , , , , , , , , on August 15, 2015| Leave a Comment »

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

Article ID #180: Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle. Published on 8/15/2015

WordCloud Image Produced by Adam Tubman

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

http://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

Read Full Post »

« Newer Posts - Older Posts »