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Posts Tagged ‘intracellular and extracellular transport’


A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

 

There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.

 

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The transport of molecules across membranes

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Cellular Transport and the Nobel Prize for Medicine

Extracted  from October 8, 2013 | by

The 2013 Nobel Prize in Physiology or Medicine was  awarded to  to Randy W. Schekman, at the University of California at Berkeley; James E. Rothman,  at Yale University in New Haven, Connecticut; and Thomas C. Südhof,  at Stanford University, for their discoveries of machinery regulating vesicle traffic, a major transport system in cells.three U.S. scientists for their work on how the cell coordinates its transport system to shuttle proteins and other molecules from one location to another.

The organization and transport of molecules across cellular mmembranes is accomplished via vesicles that shuttle cargo between organelles or fuse to other structures to release their cargo outside the cell. The vesicle transport system is critical for a variety of physiological processes, ranging from signaling in the brainto release of hormones and immune cytokines.

Schekman identified three classes of genes that control different facets of the cell’s transport system.

Vesicle fusion

http://www.highlighthealth.com/wp-content/uploads/2013/10/vesicle-fusion.jpg

This was followed by James Rothman’s discovery that a protein complex enables vesicles to fuse with their target membranes (pictured in orange above). This lock and key mechanism ensures that the vesicle fuses at the right location and that cargo molecules are delivered to the correct destination.

Also in the 1990s, Thomas Südhof was studying how nerve cells communicate in the brain. Calcium ions were known to be involved in vesicle cargo release, and Südhof searched for calcium sensitive proteins in nerve cells. He identified the molecular machinery (pictured in purple above) that responds to an influx of calcium ions (Ca2+) and triggers vesicle fusion.

Extracellular vesicles are  participate in the pathogenesis of various diseases, most notably neurodegenerative disorders, and extracellular vesicles are likely to have therapeutic applications in large-molecule drug delivery.

References

  1. The Nobel Prize in Physiology or Medicine 2013 – Press Release. Nobelprize.org. 7 Oct 2013.
  2. Andaloussi et al. Extracellular vesicles: biology and emerging therapeutic opportunities. Nature Reviews Drug Discovery 2013 Vol: 12(5):347-357. DOI: 10.1038/nrd3978
    View abstract
  3. Anderson et al. Role of extracellular membrane vesicles in the pathogenesis of various diseases, including cancer, renal diseases, atherosclerosis, and arthritis. Lab Invest. 2010 Nov;90(11):1549-57. DOI: 10.1038/labinvest.2010.152. Epub 2010 Aug 30.
    View abstract

 

Machinery Regulating Vesicle Traffic, A Major Transport System in our Cells

http://www.nobelprizemedicine.org/wp-content/uploads/2013/10/Scientific-Background-Zierath-and-Lindahl.pdf

Together, Rothman, Schekman and Südhof have transformed the way we view transport of molecular cargo to specific destinations inside and outside the cell. Their discoveries explain a long-standing enigma in cell biology and also shed new light on how disturbances in this machinery can have deleterious effects and contribute to conditions such as neurological diseases, diabetes, and immunological disorders.

Eukaryotic cells differ from prokaryotic cells by their more complex intracellular organization. In eukaryotes, specific cellular functions are compartmentalized into the cell nucleus and organelles surrounded by intracellular membranes. This compartmentalization vastly improves the efficiency of many cellular functions and prevents potentially dangerous molecules from roaming freely within the cell. But when distinct cellular processes are compartmentalized, a problem emerges. Different compartments need to exchange specific molecules (Figure 1). Furthermore, certain molecules need to be exported to the cell exterior. Most molecules are too large to directly pass through membranes, thus a mechanism that ensures specific delivery of this molecular cargo is required.

Figure 1: Each cell in the body has a complex organization where specific cellular functions are separated into different compartments called organelles. Molecules produced in the cell are packaged in vesicles and transported with special and temporal precision to the correct locations within and outside the cell.

Mysteries of cellular compartmentalization have long intrigued scientists. Improved light microscopy techniques aided in the understanding of intracellular organization in eukaryotic cells, but the advent of electron microscopy and new staining techniques, combined with subcellular fractionation assays using differential ultracentrifugation procedures, led to a deeper understanding of the cell’s inner life. Albert Claude, George Palade and Christian de Duve, who received the Nobel Prize in Physiology or Medicine 1974*, were pioneers in this area and have shed light on how the cell is organized and compartmentalized. Secretory proteins were shown to be produced on ribosomes in the endoplasmic reticulum (ER) and trafficked to the Golgi complex (named after the 1906 Nobel Laureate Camillo Golgi) (Figure 1). Progress was also made in deciphering how proteins find their appropriate destination. Günter Blobel was awarded the 1999 Nobel Prize in Physiology or Medicine* for his discoveries that proteins have intrinsic signals that govern their transport and localization in the cell. Yet, a lingering question remained. How are molecules, including hormones, transport proteins, and neurotransmitters, correctly routed to their appropriate destination? From the work of Palade, the traffic of secretory proteins from the ER was understood to be carried out using small membrane-surrounded vesicles that bud from one membrane and fuse with another, but how precision could be acquired in this process remained enigmatic.

The work of  Rothman, Schekman and Südhof represents a paradigm shift in our understanding of how the eukaryotic cell, with its complex internal compartmentalization, organizes the routing of molecules packaged in vesicles to various intracellular destinations, as well as to the outside of the cell. Specificity in the delivery of molecular cargo is essential for cell function and survival. This specificity is required for the release of neurotransmitters into the presynaptic region of a nerve cell to transmit a signal to a neighboring nerve cell. Likewise, specificity is required for the export of hormones such as insulin to the cell surface. While vesicles within the cell were long known to be critical components of this transportation scheme, the precise mechanism by which these vesicles found their correct destination and how they fused with organelles or the plasma membrane to deliver the cargo remained mysterious. The work of the three 2013 Laureates radically altered our understanding of this aspect of cell physiology. Randy W. Schekman used yeast genetics to identify a set of genes critical for vesicular trafficking. He showed that these genes were essential for life and could be classified into three categories regulating different aspects of vesicle transport. James E. Rothman embarked on a biochemical approach and identified proteins that form a functional complex controlling cell fusion. Proteins on the vesicle and target membrane sides bind in specific combinations, ensuring precise delivery of molecular cargo to the right destination. Thomas C. Südhof became interested in how vesicle fusion machinery was controlled. He unraveled the mechanism by which calcium ions trigger release of neurotransmitters, and identified key regulatory components in the vesicle fusion machinery.

Schekman discovered genes encoding proteins that are key regulators of vesicle traffic. Comparing normal with genetically mutated yeast cells in which vesicle traffic was disturbed, he identified genes that control transport to different compartments and to the cell surface

Rothman published a series of papers where he reconstituted the intracellular transport of the VSV-G protein within the Golgi complex. He then used the assay to study both vesicle budding and fusion, and purified proteins from the cytoplasm that were required for transport. The first protein to be purified was the Nethylmaleimide-sensitive factor (NSF). Rothman’s discovery of NSF paved the way for the subsequent identification of other proteins important for the control of vesicle fusion, and the next one in line was SNAP (soluble NSFattachment protein). SNAPs bind to membranes and assist in the recruitment of NSF.

One of the yeast mutants, sec18, corresponded to NSF, which also revealed that the vesicle fusion machinery was evolutionarily ancient. Furthermore, Rothman and Schekman collaboratively cloned sec17 and provided evidence of its functional equivalence to SNAP. Other sec genes were shown to correspond to genes encoding fusion proteins were identified by other methods.

Using the NSF and SNAP proteins as bait, Rothman next turned to brain tissue, from which he purified proteins that he later named SNAREs (soluble NSF-attachment protein receptors). Intriguingly, three SNARE proteins, VAMP/Synaptobrevin, SNAP-25 and syntaxin, were found in stoichiometric amounts, which suggested to Rothman that they functioned together in the vesicle and target membranes. The three proteins had previously been identified by several scientists, including Richard Scheller, Kimio Akagawa, Reinhard Jahn and Pietro de Camilli, and localized to the presynaptic region, but their function was largely unknown. VAMP/Synaptobrevin resided on the vesicle, whereas SNAP-25 and syntaxin were found at the plasma membrane. This prompted Rothman to propose a hypothesis – the SNARE hypothesis – which stipulated that target and vesicle SNAREs (t-SNAREs and v-SNAREs) were critical for vesicle fusion through a set of sequential steps of synaptic docking, activation and fusion.

Thomas C. Südhof originally trained at the Georg-August-Universität and the Max-Planck Institute for Biophysical Sciences in Göttingen, Germany, and was a postdoctoral fellow with Michael Brown and Joseph Goldstein (Nobel Prize 1985) at University of Texas Southwestern Medical School in Dallas. As a junior group leader, he set out to study how synaptic vesicle fusion was controlled. Rothman and Schekman had provided fundamental machinery for vesicle fusion, but how vesicle fusion was temporally controlled remained enigmatic. Vesicular fusions in the body need to be kept carefully in check, and in some cases vesicle fusion has to be executed with high precision in response to specific stimuli. This is the case for example for neurotransmitter release in the brain and for insulin secretion from the endocrine pancreas.

The neurophysiology field was electrified by the discoveries of Bernard Katz, Ulf von Euler and Julius Axelrod who received the Nobel Prize in Physiology or Medicine 1970* for their discoveries concerning the humoral transmittors in the nerve terminals and the mechanism for their storage, release and inactivation. Südhof was intrigued by the rapid exocytosis of synaptic vesicles, which is under tight temporal control and regulated by the changes in the cytoplasmic free calcium concentration. Südhof elucidated how calcium regulates neurotransmitter release in neurons and discovered that complexin and synaptotagmin are two critical proteins in calcium-mediated vesicle fusion.

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