Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, Cardiac and Cardiovascular Surgical Procedures, FDA Regulatory Affairs, Frontiers in Cardiology and Cardiovascular Disorders, Pharmacotherapy of Cardiovascular Disease on January 4, 2016| Leave a Comment »
Authors: Stuart L. Cantor, PhD, and Kadriye Ciftci, PhD
Stuart L. Cantor, PhD, and Kadriye Ciftci, PhD, discuss the road to development and regulatory approval associated with two of the leading therapeutic focus markets: cardiology and oncology.
SOURCE
Posted in Diabetes Mellitus on January 4, 2016| Leave a Comment »
Reporter: Stuart Cantor, PhD
Leptin:
Leptin is termed the “satiety hormone” and decreases appetite. It is made by adipose cells and other cells and regulates energy balance. It works opposite to the hormone ghrelin, known as the “hunger hormone.” Obese patients have elevated blood levels of leptin and have reduced leptin sensitivity. Thus, obese patients can be unable to feel satiety despite having high energy stores. Leptin levels are decreased by exercise and increased by insulin. Fasting or a very low calorie diet will also decrease levels of leptin.
A 2010 study by Wang M-Y et al. showed that in non-obese uncontrolled diabetic mice with Type 1 diabetes, recombinant leptin therapy alone or combined with low dose insulin reversed the catabolic state by suppressing elevated glucagon levels in the blood (and without increasing body fat). Leptin can normalize hemoglobin A1c with far less glucose variability. Results showed that leptin may have multiple short- and long-term advantages over insulin monotherapy for Type 1 diabetes. However – they also stated that well-controlled diabetic patients with normal or increased levels of adipocytes MAY be LESS responsive to leptin therapy.
In 2014, FDA approved Myalept (metreleptin for injection) to treat leptin deficiency (affects ~ 200 patients) & lipodystrophy (can be caused by repeated insulin injections in the same place on the body). The drug is marketed now by Astra Zeneca. FDA is requiring 7 post-marketing studies, including the assessment for immunogenicity (antibody formation), which is a potential serious risk. In clinical trials (48 patients), a common side effect observed was hypoglycemia.
Betatrophin:
Wang L, et al. Circulating Levels of Betatrophin and Irisin Are Not Associated with Pancreatic β-Cell Function in Previously Diagnosed Type 2 Diabetes Mellitus Patients. J Diabetes Res. 2016;2016:2616539. doi: 10.1155/2016/2616539. Epub 2015 Nov 16.
Wang et al. concludes that betatrophin and irisin were not associated with β-cell function in previously diagnosed T2DM patients.
Betatrophin is also called Angiopoietin-like protein 8 (ANGPTL8). Harvard stem cell researcher Doug Melton published a paper on the supposed ability of betatrophin to increase the production of beta cells. His work has been cited 59 times, according to Thomson Scientific’s Web of Knowledge, however, the results have been called into question by research from an independent group, as well as follow-up work from the original team.
Gusarova et al paper says that no, ANGPTL8 does not have an effect on beta-cell replication and Melton agrees with them. Melton and co-authors say “the conclusion from Yi et al. must be corrected and modified with respect to the magnitude of the effect [..] some mice respond strongly to ANGPTL8/betatrophin expression but many do not. When all mice are taken into account the results show a modest average increase in beta cell replication.
PPAR-gamma:
PPARG regulates fatty acid storage and glucose metabolism. This article mentions the use of pomegranate flower having dual alpha/gamma PPAR activating properties.
Medagama AB. . The glycaemic outcomes of Cinnamon, a review of the experimental evidence and clinical trials. Nutr J. 2015 Oct 16;14(1):108. doi: 10.1186/s12937-015-0098-9.
This work was done in Sri Lanka. There already is a marketed water-soluble cinnamon extract product developed in 2006 sold under the name Cinnulin PF (IN ingredients).
Cinnamon is currently marketed as a remedy for obesity, glucose intolerance, diabetes mellitus and dyslipidaemia. Integrative medicine is a new concept that combines conventional treatment with evidence-based complementary therapies.
The aim of this review is to critically evaluate the experimental evidence available for cinnamon in improving glycaemic targets in animal models and humans.
Insulin receptor auto-phosphorlylation and de-phosphorylation, glucose transporter 4 (GLUT-4 ) receptor synthesis and translocation, modulation of hepatic glucose metabolism through changes in Pyruvate kinase (PK) and Phosphenol Pyruvate Carboxikinase (PEPCK), altering the expression of PPAR (γ) and inhibition of intestinal glucosidases are some of the mechanisms responsible for improving glycaemic control with cinnamon therapy. We reviewed 8 clinical trials that used Cinnamomum cassia in aqueous or powder form in doses ranging from 500 mg to 6 g per day for a duration lasting from 40 days to 4 months as well as 2 clinical trials that used cinnamon on treatment naïve patients with pre-diabetes. An improvement in glycaemic control was seen in patients who received Cinnamon as the sole therapy for diabetes, those with pre-diabetes (IFG or IGT) and in those with high pre-treatment HbA1c. In animal models, cinnamon reduced fasting and postprandial plasma glucose and HbA1c.
Cinnamon has the potential to be a useful add-on therapy in the discipline of integrative medicine in managing type 2 diabetes. At present the evidence is inconclusive and long-term trials aiming to establish the efficacy and safety of cinnamon is needed. However, high coumarin content of Cinnamomum cassia is a concern, but Cinnamomum zeylanicum with its low coumarin content would be a safer alternate.
Han, JM. Effects of Lonicera japonica Thunb. on Type 2 Diabetes via PPAR-γ Activation in Rats. Phytother Res. 2015 Oct;29(10):1616-21. doi: 10.1002/ptr.5413. Epub 2015 Jul 14.
Lonicera japonica Thunb. (Caprifoliaceae) is a traditional herbal medicine and has been used to treat diabetic symptoms. Notwithstanding its use, the scientific basis on anti-diabetic properties of L. japonica is not yet established. This study is designed to investigate anti-diabetic effects of L. japonica in type 2 diabetic rats. L. japonica was orally administered at the dose of 100 mg/kg in high-fat diet-fed and low-dose streptozotocin-induced rats. After the treatment of 4 weeks, L. japonica reduced high blood glucose level and homeostatic model assessment of insulin resistance in diabetic rats. In addition, body weight
and food intake were restored by the L. japonica treatment. In the histopathologic examination, the amelioration of damaged β-islet in pancreas was observed in L. japonica-treated diabetic rats. The administration of L. japonica elevated peroxisome proliferator-activated receptor gamma and insulin receptor subunit-1 protein expressions. The results demonstrated that L. japonica had anti-diabetic effects in type 2 diabetic rats via the peroxisome proliferator-activated receptor gamma regulatory action of L. japonica as a potential mechanism.
Gu C, et al. Astragalus polysaccharides affect insulin resistance by regulating the hepatic SIRT1-PGC-1α/PPARα-FGF21 signaling pathway in male Sprague Dawley rats undergoing catch-up growth. Mol Med Rep. 2015 Nov;12(5):6451-60. doi: 10.3892/mmr.2015.4245. Epub 2015 Aug 25.
The present study investigated the effects of Astragalus polysaccharides (APS) on insulin resistance by modulation of hepatic sirtuin 1 (SIRT1)‑peroxisome proliferator‑activated receptor (PPAR)‑γ coactivator (PGC)‑1α/PPARα‑fibroblast growth factor (FGF)21, and glucose and lipid metabolism. Thirty male Sprague Dawley rats were divided into three groups: A normal control group, a catch‑up growth group and an APS‑treated (APS-G) group. The latter two groups underwent food restriction for 4 weeks, prior to being provided with a high fat diet, which was available ad libitum. The APS‑G group was orally treated with APS for 8 weeks, whereas the other groups were administered saline. Body weight was measured and an oral glucose tolerance test (OGTT) was conducted after 8 weeks. The plasma glucose and insulin levels obtained from the OGTT were assayed, and hepatic morphology was observed by light and transmission electron microscopy. In addition, the mRNA expression levels of PGC‑1α/PPARα, and the protein expression levels of SIRT1, FGF21 and nuclear factor‑κB were quantified in the liver and serum. APS treatment suppressed abnormal glycolipid metabolism and insulin resistance following 8 weeks of catch‑up growth by improving hepatic SIRT1‑PPARα‑FGF21 intracellular signaling and reducing chronic inflammation, and by partially attenuating hepatic steatosis. The suppressive effects of APS on liver acetylation and glycolipid metabolism‑associated molecules contributed to the observed suppression of insulin resistance. However, the mechanism underlying the effects of APS on insulin resistance requires further research in order to be elucidated. Rapid and long‑term treatment with APS may provide a novel, safe and effective therapeutic strategy for type 2 diabetes.
Posted in Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, Pharmaceutical R&D Informatics, Pharmacogenomics on January 4, 2016| Leave a Comment »
Reporter: Aviva Lev-Ari, PhD, RN
Press release
04 January 2016
Genomics and Vertex Collaborate to Identify Target Therapeutic Pathways
Genomics’ Proprietary Statistical Analysis Tools and Integrated Multi-Phenotype Database to be used to Support Research and Development at Vertex Pharmaceuticals
Oxford, UK, 04 January 2016: Genomics plc (“Genomics”), a leading analysis company developing algorithms, data resources, and software solutions to uncover the relationships between genetic variation and human disease, today announced that Vertex Pharmaceuticals will use Genomics’ integrated database and state-of-the-art analysis tools to inform its drug research and development. These tools aim to provide confidence in the rationale for targeting Vertex’s pathways of interest for the treatment of certain diseases and to identify potential safety concerns and repositioning opportunities.
Genomics has developed a unique analytical platform for genome analysis and interpretation. The platform combines proprietary algorithms and software with the Company’s integrated genome-phenome database and analytical expertise to learn about human biology. Genomics has several existing partnerships with large pharmaceutical companies, and in clinical genomics is a Platform Partner for Genomics England, the company undertaking the 100,000 Genomes Project in the UK.
John Colenutt, CEO, Genomics plc, said: “Pharmaceutical and biotech companies are increasingly using human genetic data in research to increase the chance of success in drug development. We are excited that Vertex has chosen to use Genomics’ proprietary technology, integrated database and tools to support them in this aim.”
Paul de Bakker, Ph.D., Head of Computational Genomics for Vertex said: “Vertex is focused on advancing research programs where disease mechanisms are validated by human biology. Our collaboration with Genomics is aimed at obtaining insights into the genetic underpinnings of specific targets and diseases to help predict which potential medicines may have success moving from discovery research toward patients.”
ENDS
Photo: John Colenutt, CEO, Genomics plc. For a high resolution image please contact lorna.cuddon@zymecommunications.com
For further information please contact:
Zyme Communications
Lorna Cuddon
Tel: +44 (0)7811996942
Email: lorna.cuddon@zymecommunications.com
About Genomics plc http://www.genomicsplc.com/
Genomics was founded by four leading Oxford academics, including Professor Peter Donnelly, Director of The Wellcome Trust Centre for Human Genetics, and Professor Gil McVean, Director of The Big Data Institute. The Company has developed a unique platform for genomic sequence data analysis and interpretation which combines world-leading expertise in statistical analysis and data mining with a unique integrated database linking genotypes and phenotypes. Genomics England, the company running the UK project to undertake whole genome sequencing of 100,000 patients in the National Health Service, has appointed Genomics plc as a Platform Partner and has also awarded the Company three SBRI grants. Genomics plc is also working with four major pharmaceutical companies to bring the benefits of genomic analysis to their drug development processes. The Company is supported by major investors, including IP Group, Invesco Perpetual, Woodford Investment Management and Lansdowne Partners.
SOURCE
From: Lorna Cuddon <lorna.cuddon@zymecommunications.com>
Reply-To: <lorna.cuddon@zymecommunications.com>
Date: Monday, January 4, 2016 at 4:11 AM
To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>
Subject: Genomics and Vertex Collaborate to Identify Target Therapeutic Pathways
Posted in Intellectual Property, Intellectual Property, Innovations, Commercialization, Investment in technological breakthrough, Patents, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmaceutical Drug Discovery, Pharmaceutical Industry Competitive Intelligence, Pharmaceutical R&D Informatics, Pharmaceutical R&D Investment on January 4, 2016| Leave a Comment »
Reporter: Aviva Lev-Ari, PhD, RN
| NAME | COUNTRY | PREVIOUS WINNER | PREVIOUS WINNER | PREVIOUS WINNER |
| Abbott | USA |
2014 |
2013 | |
| Bayer | GERMANY |
2011 |
||
| Boehringer
Ingelheim |
GERMANY | |||
| Brinstol-Myers Squibb | USA |
2011 |
||
| J&J | USA |
2014 |
2013 |
|
| Novartis | Switzerland |
2014 |
||
| Roche | Switzerland |
2014 |
2013 |
2012, 2011 |
SOURCE
New in 2015:
Top Bay Area Innovators For the first time, Thomson Reuters analysts studied Silicon Valley, known as the technology and innovation corridor in the US, to see which companies are leading there. Following a methodology similar to that of the Top 100 Global Innovators, except for the Volume criteria, all companies headquartered or with a major subsidiary in that region were investigated. The Top Bay Area Innovators list can be found on page 19. There are 11 companies that overlap with the Top 100 Global Innovators; meaning 31 percent of the leading US innovators and 11 percent of the world’s top innovators are located in the Bay Area.
Absentees:
The United Kingdom is absent from the list yet again this year. Innovation incentives introduced in the UK, such as Patent Box legislation, do not have enough legacy yet to have had an impact. Additionally, the UK spends much less on R&D as a percentage of Gross Domestic Product (GERD) than the Top 100 Global Innovator countries do. The UK’s GERDis 1.63 percent, whereas, for example, Japan’s is 3.47 percent.5 The region’s underuse of its patent system and lack of significant commercialization keep the UK from making the list once again.
China is also absent from the 2015 list. It joined the innovation-leader ranks in 2014, for the first time, via Huawei, however wasn’t able to replicate that performance to join again in 2015. A big factor contributing to China’s shortcoming is the fact that most of its innovation is domestic and therefore is not realized outside of its borders. In fact, only about six percent of China’s innovation activity is protected, and commercialized, outside of China. In order for China to see more organizations join this prestigious group, it will need to think more internationally and look to bring its inventions to market around the world. There are 27 companies that dropped from the prior year (see Table 1 on page 12), including AT&T, IBM, Siemens and Xerox. While these companies are still innovating at noteworthy levels, their respective scores across all of the metrics did not advance them to the Top 100. It’s expected that we will see them again in the future.
Patent Reform
There’s been some influential intellectual property legislation that is shaping how companies innovate, where they seek protection and when. Some of these initiatives include the America Invents Act and the Patent Trial & Appeal Board; the European unitary patent and unified patent court; the UK’s Patent Box legislation; and impactful court rulings, such as Alice 101 in the US. The landscape is ripe with reform as patent offices and filers grapple with how best to implement these changes given their goals and needs. Despite these changes, one thing remains certain: the patent system is vital to protecting innovation and to the economic wellbeing of organizations, nations and our world. OECD statistics confirm that nations with higher GDPs have similarly high patent filing rates (aka strong patent infrastructures), whereas the converse holds equally true. One way for developing nations to propel their economies forward is to invest in innovation and building a reliable intellectual property infrastructure.
Methodology
The Thomson Reuters Top 100 Global Innovator methodology analyzes patent and citation data across four main criteria:
using Thomson Reuters solutions including Derwent World Patents Index (DWPI), Thomson Innovation and Derwent Patent Citations Index (PCI).
Volume
Volume is the first criteria. An organization must have at least 100 unique inventions protected by a granted patent over the most recent five year period to advance for further analysis. A unique invention is defined as one instance of a published application or granted patent for an idea for which protection is sought. In DWPI, these are called “basic” patents. DWPI provides access to 50 patentissuing authorities. Subsequent filings for the same invention are recorded as equivalents and collated into patent families which, for this analysis, were not included. Once an organization passes the volume stage gate, it is measured across the next three criteria: success, globalization and influence.
Success
The success metric covers the ratio of inventions described in published applications (those patents which are filed and publicly published by the patent office but not yet granted) to inventions protected with granted patents over the most recent five years. Not all patent applications pass through the examination process and are granted.
Globalization
Globalization has to do with the value an organization places on an invention by protecting it across the major world markets. The premise being that inventions protected in all four of the Thomson Reuters Quadrilateral Patent Index authorities: the Chinese Patent Office, the European Patent Office, the Japanese Patent Office and the United States Patent & Trademark Office, are deemed to be of significant value to the organization. A ratio is created of the inventions protected across the Quadrilateral Patent Index authorities versus the total volume for that period. Influence Finally,
Influence
influence is the downstream impact of an invention, measured by how often it is cited by other organizations. Via the Derwent Patent Citation Index, citations to an organization’s patents are counted over the most recent five years, excluding self citations. Scores for each of these areas are tallied and combined to produce the Top 100 Global Innovator list.
3M Company USA Chemical 2011, 2012, 2013, 2014
Abbott Laboratories USA Pharmaceutical 2013, 2014
Advanced Micro Devices USA Semiconductor & Electronic Components 2011, 2012, 2013, 2014
Air Products USA Chemical 2013
Aisin Seiki Japan Automotive 2014
Alcatel-Lucent France Telecommunication & Equipment 2011, 2012, 2013, 2014
Alstom France Electrical Power
Amazon USA Media Internet Search & Navigation Systems
Analog Devices USA Semiconductor & Electronic Components 2011, 2012, 2013
Apple USA Telecommunication & Equipment 2011, 2012, 2013, 2014
Arkema France Chemical 2011, 2012, 2013, 2014
Avago Technologies (previously LSI) USA Semiconductor & Electronic Components 2011,2012, 2013, 2014
BASF Germany Chemical 2011, 2014
Bayer Germany Pharmaceutical 2011
Becton Dickinson USA Medical Devices
Blackberry Canada Telecommunication & Equipment 2013, 2014
Boehringer Ingelheim Germany Pharmaceutical
Boeing USA Aerospace 2011, 2012, 2013, 2014
Bridgestone Japan Automotive
Bristol-Myers Squibb USA Pharmaceutical 2011
Canon Japan Imaging 2011, 2012, 2013, 2014
Casio Computer Japan Computer Hardware 2014
Chevron USA Oil & Gas 2011, 2012, 2013
CNRS, The French National Center for Scientific Research France Scientific Research 2011, 2012, 2013, 2014
CEA–The French Alternative Energies and Atomic Energy Commission France Scientific Research 2011, 2012, 2013, 2014
Daikin Industries Japan Industrial 2011, 2014
Dow Chemical Company USA Chemical 2011, 2012, 2013, 2014
DuPont USA Chemical 2011, 2012, 2013, 2014
Emerson Electric USA Electrical Products 2012, 2013, 2014
Ericsson Sweden Telecommunication & Equipment 2011, 2012, 2013, 2014
Exxon Mobil USA Oil & Gas 2011, 2012, 2013
Fraunhofer Germany Scientific Research 2013, 2014
Freescale Semiconductor USA Semiconductor & Electronic Components 2013, 2014
Fujifilm Japan Imaging 2012, 2013, 2014
Fujitsu Japan Computer Hardware 2011, 2012, 2013, 2014
Furukawa Electric Japan Electrical Products 2014
General Electric USA Consumer Products 2011, 2012, 2013, 2014
Google (now Alphabet Inc.) USA Media Internet Search & Navigation Systems 2012, 2013, 2014
Hitachi Japan Computer Hardware 2011, 2012, 2013, 2014
Honda Motor Japan Automotive 2011, 2012, 2013, 2014
Honeywell International USA Electrical Products 2011, 2012, 2013, 2014
Idemitsu Kosan Japan Oil & Gas
IFP Energies Nouvelles France Scientific Research 2011, 2012, 2013, 2014
Intel USA Semiconductor & Electronic Components 2011, 2012, 2013, 2014
InterDigital USA Telecommunication & Equipment
Japan Science and Technology Agency (JST) Japan Scientific Research
Johnson & Johnson USA Pharmaceutical 2013, 2014
Johnson Controls USA Automotive
JTEKT Japan Automotive Kawasaki Heavy Industries Japan Industrial
Kobe Steel Japan Primary Metals 2014
Komatsu Japan Industrial 2014
Kyocera Japan Electrical Products 2014
LG Electronics S Korea Consumer Products 2011, 2012, 2013, 2014
Lockheed Martin USA Transportation Equipment 2012, 2013, 2014
LSIS S Korea Electrical Power 2011, 2012, 2013, 2014
Makita Corporation Japan Machinery
Marvell USA Semiconductor & Electronic Components 2012, 2013, 2014
MediaTek Taiwan Semiconductor & Electronic Components 2014
Medtronic USA Medical Devices 2014
Micron USA Semiconductor & Electronic Components 2012, 2013, 2014
Microsoft USA Computer Software 2011, 2012, 2013, 2014
Mitsubishi Electric Japan Electrical Products 2011, 2012, 2013, 2014
Mitsubishi Heavy Industries Japan Machinery 2012, 2013, 2014
Mitsui Chemicals Japan Chemical NEC Japan Computer Hardware 2011, 2012, 2013, 2014
Nike USA Consumer Products 2012, 2013, 2014
Nippon Steel & Sumitomo Metal Japan Primary Metals 2012, 2013, 2014
Nissan Motor Japan Automotive 2013, 2014
Nitto Denko Japan Chemical 2011, 2012, 2013, 2014
Novartis Switzerland Pharmaceutical 2014 2015
NTT Japan Telecommunication & Equipment 2011, 2012, 2013, 2014
Olympus Japan Healthcare Products 2011, 2012, 2013, 2014
Oracle USA Computer Software 2013, 2014
Panasonic Japan Consumer Products 2011, 2012, 2013, 2014
Philips Netherlands Electrical Products 2011, 2013, 2014
Qualcomm USA Semiconductor & Electronic Components 2011, 2012, 2013, 2014
Roche Switzerland Pharmaceutical 2011,2012,2013, 2014
Safran France Transportation Equipment 2013, 2014
Saint-Gobain France Industrial 2011, 2012, 2013, 2014
Samsung Electronics S Korea Semiconductor & Electronic Components 2011, 2012, 2013, 2014
Seagate USA Computer Hardware 2012, 2013, 2014
Seiko Epson Japan Imaging 2011, 2012, 2013, 2014
Shin-Etsu Chemical Japan Chemical 2011, 2012, 2013, 2014
Showa Denko Japan Chemical
Solvay Belgium Chemical 2012
Sony Japan Consumer Products 2011, 2012, 2013, 2014
Sumitomo Electric Japan Industrial 2011, 2013, 2014
Symantec USA Computer Software 2011, 2012, 2013, 2014
TE Connectivity Switzerland Semiconductor & Electronic Components 2011, 2012, 2013, 2014
Thales France Transportation Equipment 2012, 2013
Toray Japan Chemical
Toshiba Japan Computer Hardware 2011, 2012, 2013, 2014
Toyota Motor Japan Automotive 2011, 2012, 2013, 2014
Valeo France Automotive 2012, 2013
Xilinx USA Semiconductor & Electronic Components 2012, 2013, 2014
Yamaha Japan Consumer Products 2011, 2014
Yamaha Motor Japan Automotive
Yaskawa Electric Japan Industrial
Yazaki Japan Automotive
SOURCE
Posted in CANCER BIOLOGY & Innovations in Cancer Therapy, tagged cancer gene, cancer genes, gene expression, gene mutations on January 4, 2016| Leave a Comment »
Reporter: Evelina Cohn, PhD
Personalized genomics is the next research in cancer. However not all the mutations found in this disease are targeted equally by the researchers.
“Genetics is changing oncology for the good,” says Benjamin Kipp, an expert in clinical genetics at the Mayo Clinic in Rochester, Minn. “But overinterpretation can harm the patient.”
Genetic profile of tumors offers opportunities for both cancer diagnostics and treatment. For example, bowel cancer tumors with mutations in the KRAS gene respond poorly to the drug Cetuximab, while the drug Vemurafenib works only in melanomas that have a particular mutation in BRAF gene. But such genetic testing can be misleading if it isn’t conducted alongside tests of healthy cells from the same person, says oncologist Victor Velculescu of the Johns Hopkins University School of Medicine. He led a vast analysis comparing the genetic profiles of tumors and normal tissue of more than 800 cancer patients and found that nearly two-thirds of mutations in the studied tumors — many of which might be used to guide treatment — also showed up in patients’ healthy tissues . Thus, there are many “false positive” mutations that appear to contribute to cancer but in reality they are showing up elsewhere in an individual’s health tissue. Sampling both tumor and healthy tissues might provide a way to sort out truly cancerous mutations, the scientists report.
A team of researchers in Baltimore tested tumor tissue and healthy tissue from 815 patients who had various cancers. Using only the tumor analysis, the tests spotted an average of 382 mutations per case that appeared associated with cancer. But nearly two-thirds of these variations, on average, also showed up in healthy tissues, suggesting that they weren’t driving the cancer, the authors report in the April 15 Science Translational Medicine.
For those patients, the mutations were probably just benign variants unrelated to the cancer. Analyzing healthy tissue can also reveal whether mutations found in tumors are heritable or not, Velculescu says, which is important for deciding whether a cancer patient’s family should receive genetic counseling.
Even mutation that have been linked to the cancer not always manifest as cancer making this interpretation even worse. A study published in May examining eyelid skin discovered numerous cancer-associated mutations in normal, healthy patches of the skin. Researchers had previously thought that the types of mutations that fuel tumor growth were rare and happened just before a cell becomes cancerous. But a study of the eyelids of four people who don’t have cancer reveals that such mutations “are staggeringly common in normal skin,” says Philip Jones, a clinical scientist at the University of Cambridge. Thus, Jones and his colleagues collected 234 skin samples from four people ages 55 to 73 who had plastic surgery to correct droopy eyelids. DNA sequencing showed that about 20 percent of the skin cells had mutations in the NOTCH1 gene, the team reports in the May 22 Science. When mutated, that gene is a driving force in some cancers, including skin cancers called squamous cell carcinomas.
As genetic testing of tumors becomes more widespread, best practices will emerge, as will a better understanding of the disease. “We are trying to change the way we look at cancer,” says Sameek Roychowdhury, a medical oncologist at the Ohio State University Comprehensive Cancer Center in Columbus. “But we are just seeing the tip of the iceberg.”
Conclusion: We have to be really careful when are making interpretation of mutated genes that may cause cancer and identify those mutations in both healthy and cancer tissues as well as find the expression of those genes that may lead to cancer, being said that only cancer mutations that are expressed may have an importance in cancer appearance.
Posted in Uncategorized on January 3, 2016| Leave a Comment »
Two-part journey brings a package of medicines to an Appalachian free clinic
Sourced through Scoop.it from: pharmaceuticalcommerce.com
Posted in Anticancer Resistance, Biological Networks, Curation, Proteomics, tagged Protein folding, transformer proteins on January 3, 2016| Leave a Comment »
Transformer Proteins against Cancer
Larry H. Bernstein, MD, FCAP, Curator
LPBI
Revision 1/13/20165
GEN News Highlights: “Transformer” Proteins with Role in Cancer
http://www.genengnews.com/gen-news-highlights/transformer-proteins-with-role-in-cancer/81252133/
Investigators at the Scripps Research Institute (TSRI) and St. Jude Children’s Research Hospital say they have found how a protein involved in cancer twists and morphs into different structures.
“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said Ashok Deniz, Ph.D., associate professor at TSRI.
The study (“Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding”), published in Angewandte Chemie, focuses on the nucleophosmin (NPM1) protein, which has many functions and, when mutated, has been shown to interfere with cells’ normal tumor suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.
Previous research led by study collaborators Richard Kriwacki, Ph.D., and Diana Mitrea, Ph.D., at St. Jude had demonstrated that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.
Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell. To shed light on how this transformation occurred, Dr. Deniz and his colleagues used a combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET), and circular dichroism, which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.
The researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. Such phosphorylation prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.
When NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.
Priya Banerjee, Ph.D., an American Heart Association-supported postdoctoral research associate at TSRI and the first author of the study, compared these complicated transitions to the morphing of a “Transformers” toy, where a robot can become a car and then a jet. “Phosphorylation and partner-binding are like different cellular switches driving these changes,” said Dr. Banerjee.
According to Dr. Banerjee, the new study also reveals many intermediate states between monomer and pentamer structures and that these states can be manipulated or “tuned” by changing conditions such as salt levels, phosphorylation, and partner binding, which may explain how cells regulate the protein’s multiple functions. The researchers said future studies could shed more light on the biological functions of these different structures and how they might be used in future cancer therapies.
The team added that combining the three techniques used in this study, plus a novel protein-labeling technique for single-molecule fluorescence, could be a useful strategy for studying other unstructured, “intrinsically disordered proteins” (IDPs), which are involved in a host of cellular functions, as well as neurodegenerative disease, heart disease, infectious disease, type 2 diabetes and other conditions.
Single ‘Transformer’ Proteins
http://www.technologynetworks.com/Proteomics/news.aspx?ID=186996
“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said TSRI Associate Professor Ashok Deniz, senior author of the new study along with Richard Kriwacki, faculty member at St. Jude.
The study focuses on a protein called nucleophosmin (NPM1). This protein has many functions and, when mutated, has been shown to interfere with cells’ normal tumor-suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.
Previous research led by study collaborators Kriwacki and Diana Mitrea at St. Jude had shown that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.
Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell.
To shed light on how this transformation occurred, Deniz and his colleagues used an innovative combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET) and circular dichroism—which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.
Remarkably, the researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. This modification, called phosphorylation, prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.
Interestingly, when NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.
Priya Banerjee, an American Heart Association-supported postdoctoral research associate at TSRI and the first author of the study, compared these complicated transitions to the morphing of a “Transformers” toy, where a robot can become a car and then a jet. “Phosphorylation and partner-binding are like different cellular switches driving these changes,” said Banerjee.
Banerjee said the new study also reveals many intermediate states between monomer and pentamer structures—and that these states can be manipulated or “tuned” by changing conditions such as salt levels, phosphorylation and partner binding, which may explain how cells regulate the protein’s multiple functions. The researchers said future studies could shed more light on the biological functions of these different structures and how they might be used in future cancer therapies.
The researchers added that combining the three techniques used in this study, plus a novel protein-labeling technique for single-molecule fluorescence, could be a useful strategy for studying other unstructured, “intrinsically disordered proteins” (IDPS). IDPS are involved in a host of cellular functions, as well as neurodegenerative disease, heart disease, infectious disease, type 2 diabetes and other conditions.
TSRI and St. Jude Scientists Study Physics of Single ‘Transformer’ Proteins with Role in Cancer
http://www.scripps.edu/news/press/2015/20151221deniz.html
December 21, 2015 – A new study led by scientists at The Scripps Research Institute (TSRI) and St. Jude Children’s Research Hospital shows how a protein involved in cancer twists and morphs into different structures.
“We’re studying basic biophysics, but we believe the complexity and rules we uncover for the physics of protein disorder and folding could one day also be used for better designs of therapeutics,” said TSRI Associate Professor Ashok Deniz, senior author of the new study along with Richard Kriwacki, faculty member at St. Jude.
The study, published recently in the journal Angewandte Chemie, focuses on a protein called nucleophosmin (NPM1). This protein has many functions and, when mutated, has been shown to interfere with cells’ normal tumor suppressing ability. NPM1 has been implicated in cancers such as non-Hodgkin lymphoma and acute myelogenous leukemia.
Previous research led by study collaborators Kriwacki and Diana Mitrea at St. Jude had shown that a section of NPM1, called the N-terminal domain (Npm-N), doesn’t have a defined, folded structure. Instead, the protein morphs between two forms: a one-subunit disordered monomer and a five-subunit folded pentamer.
Until now, the mechanism behind this transformation was unknown, but scientists believed this monomer-pentamer equilibrium could be important for the protein’s location and functioning in the cell.
To shed light on how this transformation occurred, Deniz and his colleagues used an innovative combination of three techniques—single-molecule biophysics, fluorescence resonance energy transfer (FRET) and circular dichroism—which enabled them to study individual molecules and collections of molecules. Single-molecule methods are especially useful for such studies because they can uncover important information that remains hidden in conventional studies.
Remarkably, the researchers found that the transformation can proceed through more than one pathway. In one pathway, the transformation begins when the cell sends signals to attach phosphoryl groups to NPM1. This modification, called phosphorylation, prompts the ordered pentamer to become disordered and likely causes NPM1 to shuttle outside the cell’s nucleus. A meeting with a binding partner can mediate the reverse transformation to a pentamer.
Interestingly, when NPM1 does become a pentamer again under these conditions, which likely causes it to move back to the nucleolus, it takes a different path instead of just retracing its earlier steps.
Asymmetric Modulation of Protein Order-Disorder Transitions by Phosphorylation and Partner Binding
Priya R. Banerjee, Diana M. Mitrea, Richard W. Kriwacki, Ashok, A. Deniz
http://doi.org/f3kbj7 http://dx.doi.org:/10.1002/anie.201507728
As for many intrinsically disordered proteins, order–disorder transitions in the N-terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm-N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single-molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm-N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway-specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm-N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm-N order–disorder transition enabled a complex interplay of phosphorylation and partner-binding steps to modulate its folding landscape.
Conditionally and Transiently Disordered Proteins: Awakening Cryptic Disorder To Regulate Protein Function
Related articles:
Discovery of Small Molecules that Inhibit the Disordered Protein, p27(Kip1).
Iconaru LI, Ban D, Bharatham K, Ramanathan A, Zhang W, Shelat AA, Zuo J, Kriwacki RW.
Sci Rep. 2015 Oct 28;5:15686. doi: 10.1038/srep15686.
Design, Synthesis and Evaluation of 2,5-Diketopiperazines as Inhibitors of the MDM2-p53 Interaction.
Pettersson M, Quant M, Min J, Iconaru L, Kriwacki RW, Waddell MB, Guy RK, Luthman K, Grøtli M.
PLoS One. 2015 Oct 1;10(10):e0137867. doi: 10.1371/journal.pone.0137867. eCollection 2015.
Discoveries and controversies in BCL-2 protein-mediated apoptosis.
Zheng JH, Viacava Follis A, Kriwacki RW, Moldoveanu T.
FEBS J. 2015 Sep 28. doi: 10.1111/febs.13527. [Epub ahead of print] Review.
Pin1-Induced Proline Isomerization in Cytosolic p53 Mediates BAX Activation and Apoptosis.
Follis AV, Llambi F, Merritt P, Chipuk JE, Green DR, Kriwacki RW.
Mol Cell. 2015 Aug 20;59(4):677-84. doi: 10.1016/j.molcel.2015.06.029. Epub 2015 Jul 30.
A phosphorylation switch controls the spatiotemporal activation of Rho GTPases in directional cell migration
Xuan Cao, Tomonori Kaneko, Jenny S. Li, An-Dong Liu, et al.
Nature Communications 2015; 6(7721) http://dx.doi.org:/10.1038/ncomms8721
Although cell migration plays a central role in development and disease, the underlying molecular mechanism is not fully understood. Here we report that a phosphorylation-mediated molecular switch comprising deleted in liver cancer 1 (DLC1), tensin-3 (TNS3), phosphatase and tensin homologue (PTEN) and phosphoinositide-3-kinase (PI3K) controls the spatiotemporal activation of the small GTPases, Rac1 and RhoA, thereby initiating directional cell migration induced by growth factors. On epidermal growth factor (EGF) or platelet-derived growth factor (PDGF) stimulation, TNS3 and PTEN are phosphorylated at specific Thr residues, which trigger the rearrangement of the TNS3–DLC1 and PTEN–PI3K complexes into the TNS3–PI3K and PTEN–DLC1 complexes. Subsequently, the TNS3–PI3K complex translocates to the leading edge of a migrating cell to promote Rac1 activation, whereas PTEN–DLC1 translocates to the posterior for localized RhoA activation. Our work identifies a core signalling mechanism by which an external motility stimulus is coupled to the spatiotemporal activation of Rac1 and RhoA to drive directional cell migration.
Protein Phosphorylation is an Important Tool… Chapter 13. in Protein Phosphorylation in Human Health
Roberta Fraschini, Erica Raspelli and Corinne Cassani
http://cdn.intechopen.com/pdfs-wm/38809.pdf
Protein phosphorylation is a reversible posttranslational modification that can modulate protein role in several physiological processes in almost every possible way. These include modification of its intrinsic biological activity, subcellular location, half-life and binding with other proteins. Protein phosphorylation is particularly important for the regulation of key proteins involved in the control of cell cycle progression.
Protein phosphorylation is the covalent binding of a phosphate group to some critical residues of the polypeptide. The phosphorylation state of a protein is given by a balance between the activity of protein kinases and protein phosphatases. Eukaryotic protein kinases transfer phosphate groups (PO43-) from ATP to an hydroxyl group of the lateral chain of specific serine, threonine or tyrosine residues on peptide substrates. In simple eukaryotic cells, like yeasts, Ser/Thr kinases are more common, while more complex eukaryotic cells, like human cells, have many Tyr kinases. Protein kinases recognize their substrates specifically and their active site consists of an activation loop and a catalytic loop between which substrates bind. Protein kinases differ from each other in the structure of their catalitic domain. A second class of enzymes, protein phosphatases, is responsible for the reverse reaction in which phosphate groups are removed from a protein. Phosphatases gain specificity by binding protein cofactors which facilitate binding to specific phosphoproteins. The active phosphatase often consists of a complex of the phosphatase catalytic subunit and a regulatory subunit.
The phosphorylation of specific residues induces structural changes that regulate protein functions by modulating protein folding, substrate affinity, stability and activity. For example, phosphorylation can cause switch-like changes in protein function, which can also lead to major modifications in the catalytic function of enzymes, including kinases and phosphatases. In addition, protein phosphorylation often leads to rearrangement in the structure of the protein that can induce changes in interacting partners or subcellular localization.
Phosphorylation acts as a molecular switch for many regulatory events in signalling pathways that drive cell division, proliferation, differentiation and apoptosis. In order to ensure an appropriate balance of protein phosphorylation, the cell can compartmentalize both protein kinases and phosphatases. Another kind of regulation can be achieved by the spatial distribution of kinases and phosphatases, that creates a gradient of phosphorylated substrates across different subcellular compartments. This spatial separation can also control the activity of other proteins or enzymes and the occurrence of other posttranslational modifications.
Molecular Biology of the Cell. 4th edition. Protein Function. http://www.ncbi.nlm.nih.gov/books/NBK26911/
We have seen that each type of protein consists of a precise sequence of amino acids that allows it to fold up into a particular three-dimensional shape, or conformation. But proteins are not rigid lumps of material. They can have precisely engineered moving parts whose mechanical actions are coupled to chemical events. It is this coupling of chemistry and movement that gives proteins the extraordinary capabilities that underlie the dynamic processes in living cells.
In this section, we explain how proteins bind to other selected molecules and how their activity depends on such binding. We show that the ability to bind to other molecules enables proteins to act as catalysts, signal receptors, switches, motors, or tiny pumps. The examples we discuss in this chapter by no means exhaust the vast functional repertoire of proteins. However, the specialized functions of many of the proteins you will encounter elsewhere in this book are based on similar principles.
All Proteins Bind to Other Molecules
The biological properties of a protein molecule depend on its physical interaction with other molecules. Thus, antibodies attach to viruses or bacteria to mark them for destruction, the enzyme hexokinase binds glucose and ATP so as to catalyze a reaction between them, actin molecules bind to each other to assemble into actin filaments, and so on. Indeed, all proteins stick, or bind, to other molecules. In some cases, this binding is very tight; in others, it is weak and short-lived. But the binding always shows great specificity, in the sense that each protein molecule can usually bind just one or a few molecules out of the many thousands of different types it encounters. The substance that is bound by the protein—no matter whether it is an ion, a small molecule, or a macromolecule— is referred to as a ligand for that protein (from the Latin word ligare, meaning “to bind”).
The ability of a protein to bind selectively and with high affinity to a ligand depends on the formation of a set of weak, noncovalent bonds—hydrogen bonds, ionic bonds, and van der Waals attractions—plus favorable hydrophobic interactions (see Panel 2-3, pp. 114–115). Because each individual bond is weak, an effective binding interaction requires that many weak bonds be formed simultaneously. This is possible only if the surface contours of the ligandmolecule fit very closely to the protein, matching it like a hand in a glove (Figure 3-37).
The selective binding of a protein to another molecule. Many weak bonds are needed to enable a protein to bind tightly to a second molecule, which is called aligand for the protein. A ligand must therefore fit precisely into a protein’s binding (more…)
The region of a protein that associates with a ligand, known as the ligand’s binding site, usually consists of a cavity in the protein surface formed by a particular arrangement of amino acids. These amino acids can belong to different portions of the polypeptide chain that are brought together when the protein folds (Figure 3-38). Separate regions of the protein surface generally provide binding sites for different ligands, allowing the protein’s activity to be regulated, as we shall see later. And other parts of the protein can serve as a handle to place the protein in a particular location in the cell—an example is the SH2 domain discussed previously, which is often used to move a protein containing it to sites in theplasma membrane in response to particular signals.
The binding site of a protein. (A) The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. This crevice contains a set of amino acid side chains disposed in such a way that they can make noncovalent bonds only (more…)
Although the atoms buried in the interior of the protein have no direct contact with the ligand, they provide an essential scaffold that gives the surface its contours and chemical properties. Even small changes to the amino acids in the interior of a protein molecule can change its three-dimensional shape enough to destroy a binding site on the surface.
Proteins have impressive chemical capabilities because the neighboring chemical groups on their surface often interact in ways that enhance the chemical reactivity of amino acid side chains. These interactions fall into two main categories.
First, neighboring parts of the polypeptide chain may interact in a way that restricts the access of water molecules to aligand binding site. Because water molecules tend to form hydrogen bonds, they can compete with ligands for sites on the protein surface. The tightness of hydrogen bonds (and ionic interactions) between proteins and their ligands is therefore greatly increased if water molecules are excluded. Initially, it is hard to imagine a mechanism that would exclude a molecule as small as water from a protein surface without affecting the access of the ligand itself. Because of the strong tendency of water molecules to form water–water hydrogen bonds, however, water molecules exist in a large hydrogen-bonded network (see Panel 2-2, pp. 112–113). In effect, a ligand binding site can be kept dry because it is energetically unfavorable for individual water molecules to break away from this network, as they must do to reach into a crevice on a protein’s surface.
Second, the clustering of neighboring polar amino acid side chains can alter their reactivity. If a number of negatively charged side chains are forced together against their mutual repulsion by the way the protein folds, for example, the affinity of the site for a positively charged ion is greatly increased. In addition, when amino acid side chains interact with one another through hydrogen bonds, normally unreactive side groups (such as the –CH2OH on the serine shown inFigure 3-39) can become reactive, enabling them to enter into reactions that make or break selected covalent bonds.
An unusually reactive amino acid at the active site of an enzyme. This example is the “catalytic triad” found in chymotrypsin, elastase, and other serine proteases (see Figure 3-14). The aspartic acid side chain (Asp 102) induces the histidine (more…)
The surface of each protein molecule therefore has a unique chemical reactivity that depends not only on which amino acid side chains are exposed, but also on their exact orientation relative to one another. For this reason, even two slightly different conformations of the same protein molecule may differ greatly in their chemistry.
As we have described previously, many of the domains in proteins can be grouped into families that show clear evidence of their evolution from a common ancestor, and genome sequences reveal large numbers of proteins that contain one or more common domains. The three-dimensional structures of the members of the same domain family are remarkably similar. For example, even when the amino acid sequence identity falls to 25%, the backbone atoms in a domain have been found to follow a common protein fold within 0.2 nanometers (2 Å).
These facts allow a method called “evolutionary tracing” to be used to identify those sites in a protein domain that are the most crucial to the domain’s function. For this purpose, those amino acids that are unchanged, or nearly unchanged, in all of the known protein family members are mapped onto a structural model of the three-dimensional structure of one family member. When this is done, the most invariant positions often form one or more clusters on the protein surface, as illustrated in Figure 3-40A for the SH2 domain described previously (see Panel 3-2, pp. 138–139). These clusters generally correspond to ligand binding sites.
The evolutionary trace method applied to the SH2 domain. (A) Front and back views of a space-filling model of the SH2 domain, with evolutionarily conserved amino acids on the protein surface colored yellow, and those more toward the protein interior colored (more…)
The SH2 domain is a module that functions in protein–protein interactions. It binds the protein containing it to a second protein that contains a phosphorylated tyrosine side chain in a specific amino acid sequence context, as shown in Figure 3-40B. The amino acids located at the binding site for the phosphorylated polypeptide have been the slowest to change during the long evolutionary process that produced the large SH2 family of peptide recognition domains. Becausemutation is a random process, this result is attributed to the preferential elimination during evolution of all organisms whose SH2 domains became altered in a way that inactivated the SH2-binding site, thereby destroying the function of the SH2 domain.
In this era of extensive genome sequencing, many new protein families have been discovered whose functions are unknown. By identifying the critical binding sites on a three-dimensional structure determined for one family member, the above method of evolutionary tracing is being used to help determine the functions of such proteins.
Proteins can bind to other proteins in at least three ways. In many cases, a portion of the surface of one protein contacts an extended loop of polypeptide chain (a “string”) on a second protein (Figure 3-41A). Such a surface–string interaction, for example, allows the SH2 domain to recognize a phosphorylated polypeptide as a loop on a second protein, as just described, and it also enables a protein kinase to recognize the proteins that it will phosphorylate (see below).
Three ways in which two proteins can bind to each other. Only the interacting parts of the two proteins are shown. (A) A rigid surface on one protein can bind to an extended loop of polypeptide chain (a “string”) on a second protein. (B)(more…)
A second type of protein–protein interface is formed when two α helices, one from each protein, pair together to form acoiled-coil (Figure 3-41B). This type of protein interface is found in several families of gene regulatory proteins, as discussed in Chapter 7.
The most common way for proteins to interact, however, is by the precise matching of one rigid surface with that of another (Figure 3-41C). Such interactions can be very tight, since a large number of weak bonds can form between two surfaces that match well. For the same reason, such surface–surface interactions can be extremely specific, enabling aprotein to select just one partner from the many thousands of different proteins found in a cell.
Molecular Motors
Perhaps the most fascinating proteins that associate with the cytoskeleton are the molecular motors called motor proteins. These remarkable proteins bind to a polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move steadily along it. Dozens of different motor proteins coexist in every eucaryotic cell. They differ in the type of filament they bind to (either actin or microtubules), the direction in which they move along the filament, and the “cargo” they carry. Many motor proteins carry membrane-enclosed organelles—such as mitochondria, Golgi stacks, or secretory vesicles—to their appropriate locations in the cell. Other motor proteins cause cytoskeletal filaments to slide against each other, generating the force that drives such phenomena as muscle contraction, ciliary beating, and cell division.
Cytoskeletal motor proteins that move unidirectionally along an oriented polymer track are reminiscent of some other proteins and protein complexes discussed elsewhere in this book, such as DNA and RNA polymerases, helicases, and ribosomes. All of these have the ability to use chemical energy to propel themselves along a linear track, with the direction of sliding dependent on the structural polarity of the track. All of them generate motion by coupling nucleosidetriphosphate hydrolysis to a large-scale conformational change in a protein, as explained in Chapter 3.
The cytoskeletal motor proteins associate with their filament tracks through a “head” region, or motor domain, that binds and hydrolyzes ATP. Coordinated with their cycle of nucleotide hydrolysis and conformational change, the proteins cycle between states in which they are bound strongly to their filament tracks and states in which they are unbound. Through a mechanochemical cycle of filament binding, conformational change, filament release, conformational relaxation, and filament rebinding, the motor protein and its associated cargo move one step at a time along the filament (typically a distance of a few nanometers). The identity of the track and the direction of movement along it are determined by the motor domain (head), while the identity of the cargo (and therefore the biological function of the individual motor protein) is determined by the tail of the motor protein.
In this section, we begin by describing the three groups of cytoskeletal motor proteins. We then describe how they work to transport membrane-enclosed organelles or to change the shape of structures built from cytoskeletal filaments. We end by describing their action in muscle contraction and in powering the whiplike motion of structures formed from microtubules.
Actin-based Motor Proteins Are Members of the Myosin Superfamily
The first motor protein identified was skeletal muscle myosin, which is responsible for generating the force for muscle contraction. This myosin, called myosin II (see below) is an elongated protein that is formed from two heavy chains and two copies of each of two light chains. Each of the heavy chains has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long amino acid sequence that forms an extended coiled-coil that mediates heavy chain dimerization (Figure 16-51). The two light chains bind close to the N-terminal head domain, while the long coiled-coil tail bundles itself with the tails of other myosin molecules. These tail-tail interactions result in the formation of large bipolar “thick filaments” that have several hundred myosin heads, oriented in opposite directions at the two ends of the thick filament (Figure 16-52).
Myosin II. (A) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long (green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization (more…)
The myosin II bipolar thick filament. (A) Electron micrograph of a myosin II thick filament isolated from frog muscle. Note the central bare zone, which is free of head domains. (B) Schematic diagram, not drawn to scale. The myosin II molecules aggregate (more…)
Each myosin head binds and hydrolyses ATP, using the energy of ATP hydrolysis to walk toward the plus end of anactin filament. The opposing orientation of the heads in the thick filament makes the filament efficient at sliding pairs of oppositely oriented actin filaments past each other. In skeletal muscle, in which carefully arranged actin filaments are aligned in “thin filament” arrays surrounding the myosin thick filaments, the ATP-driven sliding of actin filaments results in muscle contraction (discussed later). Cardiac and smooth muscle contain myosins that are similarly arranged, although they are encoded by different genes.
When a muscle myosin is digested by chymotrypsin and papain, the head domain is released as an intact fragment (called S1). The S1 fragment alone can generate filament sliding in vitro, proving that the motor activity is contained completely within the head (Figure 16-53).
Direct evidence for the motor activity of the myosin head. In this experiment, purified S1 myosin heads were attached to a glass slide, and then actin filaments labeled with fluorescent phalloidin were added and allowed to bind to the myosin heads. (A) (more…)
It was initially thought that myosin was present only in muscle, but in the 1970’s, researchers found that a similar two-headed myosin protein was also present in nonmuscle cells, including protozoan cells. At about the same time, other researchers found a myosin in the freshwater amoeba Acanthamoeba castellanii that was unconventional in having a motor domain similar to the head of muscle myosin but a completely different tail. This molecule seemed to function as a monomer and was named myosin I (for one-headed); the conventional myosin was renamed myosin II (for two-headed).
Subsequently, many other myosin types were discovered. The heavy chains generally start with a recognizable myosin motor domain at the N-terminus, and then diverge widely with a variety of C-terminal tail domains (Figure 16-54). The new types of myosins include a number of one-headed and two-headed varieties that are approximately equally related to myosin I and myosin II, and the nomenclature now reflects their approximate order of discovery (myosin III through at least myosin XVIII). The myosin tails (and the tails of motor proteins generally) have apparently diversified during evolution to permit the proteins to dimerize with other subunits and to interact with different cargoes.
Myosin superfamily tree. (A) A family tree for a few of the many known members of the myosin superfamily. The length of the lines separating individual family members indicates the amount of difference in the amino acid sequence of the motor domain. Groups (more…)
Some myosins (such as VIII and XI) have been found only in plants, and some have been found only in vertebrates (IX). Most, however, are found in all eucaryotes, suggesting that myosins arose early in eucaryotic evolution. The yeastSaccharomyces cerevisiae contains five myosins: two myosin Is, one myosin II, and two myosin Vs. One can speculate that these three types of myosins are necessary for a eucaryotic cell to survive and that other myosins perform more specialized functions in multicellular organisms. The nematode C. elegans, for example, has at least 15 myosin genes, representing at least seven structural classes; the human genome includes about 40 myosin genes.
All of the myosins except one move toward the plus end of an actin filament, although they do so at different speeds. The exception is myosin VI, which moves toward the minus end.
The exact functions for most of the myosins remain to be determined. Myosin II is always associated with contractile activity in muscle and nonmuscle cells. It is also generally required for cytokinesis, the pinching apart of a dividing cell into two daughters (discussed in Chapter 18), as well as for the forward translocation of the body of a cell during cell migration. The myosin I proteins contain a second actin–binding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization and the protrusion of actin-rich structures at the cell surface. Myosin V is involved in vesicle and organelle transport. Myosin VII is found in the inner ear in vertebrates, and certain mutations in the gene coding for myosin VII cause deafness in mice and humans.
There Are Two Types of Microtubule Motor Proteins: Kinesins and Dyneins
Kinesin is a motor protein that moves along microtubules. It was first identified in the giant axon of the squid, where it carries membrane-enclosed organelles away from the neuronal cell body toward the axon terminal by walking toward the plus end of microtubules. Kinesin is similar structurally to myosin II in having two heavy chains and two light chains per active motor, two globular head motor domains, and an elongated coiled-coil responsible for heavy chain dimerization. Like myosin, kinesin is a member of a large protein superfamily, for which the motor domain is the only common element (Figure 16-55). The yeastSaccharomyces cerevisiae has six distinct kinesins. The nematode C. elegans has 16 kinesins, and humans have about 40.
Kinesin and kinesin-related proteins. (A) Structures of four kinesin superfamily members. As in the myosin superfamily, only the motor domains are conserved. Conventional kinesin has the motor domain at the N-terminus of the heavy chain. The middle domain (more…)
There are at least ten families of kinesin-related proteins, or KRPs, in the kinesin superfamily. Most of them have the motor domain at the N-terminus of the heavy chain and walk toward the plus end of the microtubule. A particularly interesting family has the motor domain at the C-terminus and walks in the opposite direction, toward the minus end of the microtubule. Some KRP heavy chains lack a coiled-coil sequence and seem to function as monomers, analogous to myosin I. Some others are homodimers, and yet others are heterodimers. At least one KRP (BimC) can self-associate through the tail domain, forming a bipolar motor that slides oppositely oriented microtubules past one another, much as a myosin II thick filament does for actin filaments. Most kinesins carry a binding site in the tail for either a membrane-enclosed organelle or another microtubule. Many of the kinesin superfamily members have specific roles in mitotic and meiotic spindle formation and chromosome separation during cell division.
The dyneins are a family of minus-end-directed microtubule motors, but they are unrelated to the kinesin superfamily. They are composed of two or three heavy chains (that include the motor domain) and a large and variable number of associated light chains. The dynein family has two major branches (Figure 16-56). The most ancient branch contains thecytoplasmic dyneins, which are typically heavy-chain homodimers, with two large motor domains as heads. Cytoplasmic dyneins are probably found in all eucaryotic cells, and they are important for vesicle trafficking, as well as for localization of the Golgi apparatus near the center of the cell. Axonemal dyneins, the other large branch, include heterodimers and heterotrimers, with two or three motor-domain heads, respectively. They are highly specialized for the rapid and efficient sliding movements of microtubules that drive the beating of cilia and flagella (discussed later). A third, minor, branch shares greater sequence similarity with cytoplasmic than with axonemal dyneins but seems to be involved in the beating of cilia.
Dyneins. Freeze-etch electron micrographs of a molecule of cytoplasmic dynein and a molecule of ciliary (axonemal) dynein. Like myosin II and kinesin, cytoplasmic dynein is a two-headed molecule. The ciliary dynein shown has three heads. Note that the (more…)
Dyneins are the largest of the known molecular motors, and they are also among the fastest: axonemal dyneins can move microtubules in a test tube at the remarkable rate of 14 μm/sec. In comparison, the fastest kinesins can move their microtubules at about 2–3 μm/sec.
The motor domain of myosins is substantially larger than that of kinesins, about 850 amino acids compared with about 350. The two classes of motor proteins track along different filaments and have different kinetic properties, and they have no identifiable amino acid sequence similarities. However, determination of the three-dimensional structure of the motor domains of myosin and kinesin has revealed that these two motor domains are built around nearly identical cores (Figure 16-57). The central force-generating element that the two types of motor proteins have in common includes the site of ATP binding and the machinery necessary to translate ATP hydrolysis into an allosteric conformational change. The differences in domain size and in the choice of track can be attributed to large loops extending outward from this central core. These loops include the actin-binding and microtubule-binding sites, respectively.
X-ray crystal structures of myosin and kinesin heads. The central nucleotide-binding domains of myosin and kinesin (shaded in yellow) are structurally very similar. The very different sizes and functions of the two motors are due to major differences (more…)
An important clue to how the central core is involved in force generation has come from the observation that the motor core also bears some structural resemblance to the nucleotidebinding site of the small GTPases of the Ras superfamily. As discussed in Chapter 3 (see Figure 3-74), these proteins exhibit distinct conformations in their GTP-bound (active) and GDP-bound (inactive) forms: mobile “switch” loops in the nucleotide-binding site are in close contact with the γ-phosphate in the GTP-bound state, but these loops swing out when the hydrolyzed γ-phosphate is released. Although the details of the movement are different for the two motor proteins, and ATP rather than GTP is hydrolyzed, the relatively small structural change in the active site—the presence or absence of a terminal phosphate—is similarly amplified to cause a rotation of a different part of the protein. In kinesin and myosin, a switch loop interacts extensively with those regions of the protein involved in microtubule and actin binding, respectively, allowing the structural transitions caused by the ATP hydrolysis cycle to be relayed to the polymer-binding interface. The relay of structural changes between the polymer-binding site and the nucleotide hydrolysis site seems to work in both directions, since theATPase activity of motor proteins is strongly activated by binding to their filament tracks.
Although the cytoskeletal motor proteins and GTP-binding proteins both use structural changes in their nucleoside-triphosphate-binding sites to produce cyclic interactions with a partner protein, the motor proteins have a further requirement: each cycle of binding and release must propel them forward in a single direction along a filament to a newbinding site on the filament. For such unidirectional motion, a motor protein must use the energy derived from ATP binding and hydrolysis to force a large movement in part of the protein molecule. For myosin, each step of the movement along actin is generated by the swinging of an 8.5-nm-long α helix, or lever arm (see Figure 16-57), which is structurally stabilized by the binding of light chains. At the base of this lever arm next to the head, there is a piston-like helix that connects movements at the ATP-binding cleft in the head to small rotations of the so-called converter domain. A small change at this point can swing the helix like a long lever, causing the far end of the helix to move by about 5.0 nm. These changes in the conformation of the myosin are coupled to changes in its binding affinity for actin, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another. The full mechanochemical cycle of nucleotide binding, nucleotide hydrolysis, and phosphate release (which causes the “power stroke”) produces a single step of movement (Figure 16-58). In the myosin VI subfamily of myosins, which move backward (toward the minus end of the actin filament), the converter domain probably lies in a different orientation, so that the same piston-like movement of the small helix causes the lever arm to rotate in the opposite direction.
The cycle of structural changes used by myosin to walk along an actin filament. (Based on I. Rayment et al., Science 261:50–58, 1993. © AAAS.)
In kinesin, instead of the rocking of a lever arm, the small movements of switch loops at the nucleotide–binding siteregulate the docking and undocking of the motor head domain to a long linker region that connects this motor head at one end to the coiled-coil dimerization domain at the other end. When the front (leading) kinesin head is bound to amicrotubule before the power stroke, its linker region is relatively unstructured. On the binding of ATP to this bound head, its linker region docks along the side of the head, which throws the second head forward to a position where it will be able to bind a new attachment site on the protofilament, 8 nm closer to the microtubule plus end than the binding site for the first head. The nucleotide hydrolysis cycles in the two heads are closely coordinated, so that this cycle of linker docking and undocking can allow the two-headed motor to move in a hand-over-hand (or head-over-head) stepwise manner (Figure 16-59A).
Comparison of the mechanochemical cycles of kinesin and myosin II. The shading in the two circles representing the hydrolysis cycle indicates the proportion of the cycle spent in attached and detached states for each motor protein. (A) Summary of the (more…)
The coiled-coildomain seems both to coordinate the mechanochemical cycles of the two heads (motor domains) of thekinesin dimer and to determine its directionality of movement. Recall that whereas most members of the kinesin superfamily, with their motor domains at the N-terminus, move toward the plus end of the microtubule, a few superfamily members have their motor domains at the C-terminus and move toward the minus end. Since the motor domains of these two types of kinesins are essentially identical, how can they move in opposite directions? The answer seems to lie in the way in which the heads are connected. In high-resolution images of forward-walking and backward-walking members of the kinesin superfamily bound to microtubules, the heads that are attached to the microtubule are essentially indistinguishable, but the second, unattached heads are oriented very differently. This difference in tilt apparently biases the next binding site for the second head, and thereby determines the directionality of motor movement (Figure 16-60).
Orientation of forward- and backward-walking kinesin superfamily proteins bound to microtubules. These images were generated by fitting the structures of the free motor-protein dimers (determined by x-ray crystallography) onto a lower resolution image (more…)
Although both myosin and kinesin undergo analogous mechanochemical cycles, the exact nature of the coupling between the mechanical and chemical cycles is different in the two cases (see Figure 16-60). For example, myosin without any nucleotide is tightly bound to its actin track, in a so-called “rigor” state, and it is released from this track by the association of ATP. In contrast, kinesin forms a rigor-like tight association with a microtubule when ATP is bound to the kinesin, and it is hydrolysis of ATP that promotes release of the motor from its track.
Thus, cytoskeletal motor proteins work in a manner highly analogous to GTP-binding proteins, except that in motor proteins the small protein conformational changes (a few tenths of a nanometer) associated with nucleotide hydrolysis are amplified by special protein domains—the lever arm in the case of myosin and the linker in the case of kinesin—to generate large-scale (several nanometers) conformational changes that move the motor proteins stepwise along their filament tracks. The analogy between the GTPases and the cytoskeletal motor proteins has recently been extended by the observation that one of the GTP-binding proteins—the bacterial elongation factorG—translates the chemical energy of GTP hydrolysis into directional movement of the mRNAmolecule on the ribosome.
The motor proteins in the myosin and kinesin superfamilies exhibit a remarkable diversity of motile properties, well beyond their choice of different polymer tracks. Most strikingly, a single dimer of conventional kinesin moves in a highly processive fashion, traveling for hundreds of ATPase cycles along a microtubule without dissociating. Skeletal muscle myosin II, in contrast, cannot move processively and makes just one or a few steps along an actin filament before letting go. These differences are critical for the motors’ various biological roles. A small number of kinesin molecules must be able to transport a mitochondrion all the way down a nerve cellaxon, and therefore require a high level of processivity. Skeletal muscle myosin, in contrast, never operates as a single molecule but rather as part of a huge array of myosin II molecules. Here processivity would actually inhibit biological function, since efficient muscle contraction requires that each myosin head perform its power stroke and then quickly get out of the way, to avoid interfering with the actions of the other heads attached to the same actin filament.
There are two reasons for the high degree of processivity of kinesin movement. The first is that the mechanochemical cycles of the two motor heads in a kinesin dimer are coordinated with each other, so that one kinesin head does not let go until the other is poised to bind. This coordination allows the motor protein to operate in a hand-over-hand fashion, never allowing the organelle cargo to diffuse away from the microtubule track. There is no apparent coordination between the myosin heads in a myosin II dimer. The second reason for the high processivity of kinesin movement is that kinesin spends a relatively large fraction of its ATPase cycle tightly bound to the microtubule. For both kinesin and myosin, the conformational change that produces the force-generating working stroke must occur while the motor protein is tightly bound to its polymer, and the recovery stroke in preparation for the next step must occur while the motor is unbound. But as we have seen in Figure 16-59, myosin spends only about 5% of its ATPase cycle in the tightly bound state and is unbound the rest of the time.
What myosin loses in processivity it gains in speed; in an array in which many motor heads are interacting with the sameactin filament, a set of linked myosins can move their filament a total distance equivalent to 20 steps during a single cycle time, while kinesins can move only two. Thus, myosins can typically drive filament sliding much more rapidly than kinesins, even though they hydrolyze ATP at comparable rates and take molecular steps of comparable length.
Within each motor protein class, movement speeds vary widely, from about 0.2 to 60 μm/sec for myosins, and from about 0.02 to 2 μm/sec for kinesins. These differences arise from a fine-tuning of the mechanochemical cycle. The number of steps that an individual motor molecule can take in a given time, and thereby the velocity, can be increased by either increasing the motor protein’s intrinsic ATPase rate or decreasing the proportion of cycle time spent bound to the filament track. Moreover, the size of each step can be changed by either changing the length of the lever arm (for example, the lever arm of myosin V is about three times longer than the lever arm of myosin II) or the angle through which the helix swings (Figure 16-61). Each of these parameters varies slightly among different members of the myosin and kinesin families, corresponding to slightly different protein sequences and structures. It is assumed that the behavior of each motor protein, whose function is determined by the identity of the cargo attached through its tail-domain, has been fine-tuned during evolution for speed and processivity according to the specific needs of the cell.
The effect of lever arm length on the step size for a motor protein. The lever arm of myosin II is much shorter than the lever arm of myosin V. The power stroke in the head swings their lever arms through the same angle, so myosin V is able to take a (more…)
A major function of cytoskeletal motors in interphase cells is the transport and positioning of membrane-enclosed organelles. Kinesin was originally identified as the protein responsible for fast axonal transport, the rapid movement of mitochondria, secretory vesicle precursors, and various synapse components down the microtubule highways of theaxon to the distant nerve terminals. Although organelles in most cells need not cover such long distances, their polarized transport is equally necessary. A typical microtubule array in an interphase cell is oriented with the minus ends near the center of the cell at the centrosome, and the plus ends extending to the cell periphery. Thus, centripetal movements of organelles toward the cell center require the action of minus-end-directed motor proteins such as cytoplasmic dynein, whereas centrifugal movements toward the periphery require plus-end-directed motors such as kinesins.
The role of microtubules and microtubule motors in the behavior of intracellular membranes is best exemplified by the part they play in organizing the endoplasmic reticulum (ER) and the Golgi apparatus. The network of ER membranetubules aligns with microtubules and extends almost to the edge of the cell, whereas the Golgi apparatus is located near the centrosome. When cells are treated with a drug that depolymerizes microtubules, such as colchicine or nocodazole, the ER collapses to the center of the cell, while the Golgi apparatus fragments and disperses throughout the cytoplasm(Figure 16-62). In vitro, kinesins can tether ER-derived membranes to preformed microtubule tracks, and walk toward the microtubule plus ends, dragging the ER membranes out into tubular protrusions and forming a membranous web very much like the ER in cells. Likewise, the outward movement of ER tubules toward the cell periphery is associated with microtubule growth in living cells. Conversely, dyneins are required for positioning the Golgi apparatus near the cell center, moving Golgi vesicles along microtubule tracks toward minus ends at the centrosome.
Effect of depolymerizing microtubules on the Golgi apparatus. (A) In this endothelial cell, the microtubules are labeled in red, and the Golgi apparatus is labeled in green (using an antibody against a Golgi protein). As long as the system of microtubules (more…)
The different tails and their associated light chains on specific motor proteins allow the motors to attach to their appropriate organelle cargo. For example, there is evidence for membrane-associated motor receptors, sorted to specific membrane-enclosed compartments, that interact directly or indirectly with the tails of the appropriate kinesin family members. One of these receptors seems to be the amyloid precursor protein, APP, which binds directly to a light chainon the tail of kinesin-I and is proposed to be a transmembrane motor proteinreceptormolecule in nerve-cell axons. It is the abnormal processing of this protein that gives rise to Alzheimer’s disease, as discussed in Chapter 15.
For dynein, attachment to membranes is known to be mediated by a large macromolecular assembly. Cytoplasmic dynein is itself a huge proteincomplex, and it requires association with a second large protein complex called dynactinto translocate organelles effectively. The dynactin complex includes a short actinlike filament that is made of the actin-related protein Arp1 (distinct from Arp2 and Arp3, the components of the ARP complex involved in the nucleation of conventional actin filaments). Membranes of the Golgi apparatus are coated with the proteins ankyrin and spectrin, which have been proposed to associate with the Arp1 filament in the dynactin complex to form a planar cytoskeletal array reminiscent of the erythrocyte membranecytoskeleton (see Figure 10-31). The spectrin array probably gives structural stability to the Golgi membrane, and—via the Arp1 filament—it may mediate the regulatable attachment of dynein to the organelle (Figure 16-63).
A model for the attachment of dynein to a membrane-enclosed organelle. Dynein requires the presence of a large number of accessory proteins to associate with membrane-enclosed organelles. Dynactin is a large complex (red)that includes components that (more…)
Motor proteins also have a significant role in organelle transport along actin filaments. The first myosin shown to mediate organelle motility was myosin V, a two-headed myosin with a large step size (see Figure 16-61). In mice, mutations in the myosin V gene result in a “dilute” phenotype, in which fur color looks faded. In mice (and humans),membrane-enclosed pigment granules, called melanosomes, are synthesized in cells called melanocytes beneath the skin surface. These melanosomes move out to the ends of dendritic processes in the melanocytes, from where they are delivered to the overlying keratinocytes that form the skin and fur. Myosin V is associated with the surface of melanosomes, and it is able to mediate their actin-based movement in a test tube (Figure 16-64). In dilute mutant mice, the melanosomes are not delivered to the keratinocytes efficiently, and pigmentation is defective. Other myosins, including myosin I, are associated with endosomes and a variety of other organelles.
Myosin V on melanosomes. (A) Phase-contrast image of a portion of a melanocyte isolated from a mouse. The black spots are melanosomes, which are membrane-enclosed organelles filled with the skin pigment melanin. (B) The same cell labeled with a fluorescent (more…)
The cell can regulate the activity of motor proteins, allowing it to change either the positioning of its membrane-enclosed organelles or its whole-cell movements. One of the most dramatic examples is provided by fish melanocytes. These giant cells, which are responsible for rapid changes in skin coloration in several species of fish, contain large pigment granules that can alter their location in response to neuronal or hormonal stimulation (Figure 16-65). These pigment granules aggregate or disperse by moving along an extensive network of microtubules. The minus ends of these microtubules are nucleated by the centrosome and are located in the center of the cell, while the plus ends are distributed around the cell periphery. The tracking of individual pigment granules reveals that the inward movement is rapid and smooth, while the outward movement is jerky, with frequent backward steps (Figure 16-66). Both dynein and kinesinare associated with the pigment granules. The jerky outward movements apparently result from a tug-of-war between the two motor proteins, with the stronger kinesin winning out overall. When the kinesin light chains become phosphorylated after a hormonal stimulation that signals skin color change, kinesin is inactivated, leaving dynein free to drag the pigment granules rapidly toward the cell center, changing the fish’s color. In a similar way, the movement of other membrane organelles coated with particular motor proteins is controlled by a complex balance of competing signals that regulate both motor protein attachment and activity.
Regulated melanosome movements in fish pigment cells. These giant cells, which are responsible for changes in skin coloration in several species of fish, contain large pigment granules, or melanosomes (brown). The melanosomes can change their location (more…)
Bidirectional movement of a melanosome on a microtubule. An isolated melanosome (yellow) moves along a microtubule on a glass slide, from the plus end toward the minus end. Halfway through the video sequence, it abruptly switches direction and moves from (more…)
Myosin activity can also be regulated by phosphorylation. In nonmuscle cells, myosin II can be phosphorylated on a variety of sites on both heavy and light chains, affecting both motor activity and thick filament assembly. The myosin II can exist in two different conformational states in such cells, an extended state that is capable of forming bipolar filaments, and a bent state in which the tail domain apparently interacts with the motor head. Phosphorylation of the regulatory light chain by the calcium-dependent myosin light-chain kinase (MLCK) causes the myosin II to preferentially assume the extended state, which promotes its assembly into a bipolar filament and leads to cell contraction (Figure 16-67). Myosin light-chain phosphorylation is an indirect target of activated Rho, the small GTPasediscussed previously whose activation causes a reorganization of the actincytoskeleton into contractile stress fibers. The MLCK is also activated during mitosis, causing myosin II to assemble into the contractile ring that is responsible for dividing the mitotic cell into two. Regulation of other members of the myosin superfamily is not as well understood, but control of these myosins is also likely to involve site-specific phosphorylations.
Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments. (A) The controlled phosphorylation by the enzyme myosin light-chain kinase (MLCK) of one of the two light chains (the so-called regulatory light chain, shown (more…)
Motor proteins use the energy of ATP hydrolysis to move along microtubules or actin filaments. They mediate the sliding of filaments relative to one another and the transport of membrane-enclosed organelles along filament tracks. All known motor proteins that move on actin filaments are members of the myosin superfamily. The motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family. The myosin and kinesin superfamilies are diverse, with about 40 genes encoding each type of protein in humans. The only structural element shared among all members of each superfamily is the motor “head” domain. These heads can be attached to a wide variety of “tails,” which attach to different types of cargo and enable the various family members to perform different functions in the cell. Although myosin and kinesin walk along different tracks and use different mechanisms to produce force and movement by ATP hydrolysis, they share a common structural core, suggesting that they are derived from a common ancestor.
Two types of specialized motility structures in eucaryotic cells consist of highly ordered arrays of motor proteins that move on stabilized filament tracks. The myosin-actin system of the sarcomere powers the contraction of various types of muscle, including skeletal, smooth, and cardiac muscle. The dynein–microtubule system of the axoneme powers the beating of cilia and the undulations of flagella.
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Posted in LPBI Group, e-Scientific Media, DFP, R&D-M3DP, R&D-Drug Discovery, US Patents: SOPs and Team Management on January 3, 2016| Leave a Comment »
Curator: Aviva Lev-Ari, PhD, RN
| Evaluation Criterion | 2014 | 2015 | All Times Total on 1/3/2016 |
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230,000 |
300,000 |
841,700
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12-10-14 2,133 |
1/7/15 1,994 |
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| MOST popular article | The History of Hematology –
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New Encapsulation Agents for Delivery of Nitric Oxide – Dr. Aviva Lev-Ari |
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New Articles
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The Louvre Museum has 8.5 million visitors per year. This blog was viewed about 300,000 times in 2015. If it were an exhibit at the Louvre Museum, it would take about 13 days for that many people to see it.
Posted in 3D Plotting Scaffolds, 3D Printing for Medical Application, Biological Engineering, Biopolymer Blend Open Porous, Nanotechnology for Drug Delivery, tagged biodegradable porous scaffold, Cell growth, cell-cell interactions, magnetic field, magnetic nanoparticles, n3D Biosciences, nanoparticles, spheroids on January 3, 2016| Leave a Comment »
Reporter: Irina Robu
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In magnetic levitation, cells are magnetized with NanoShuttle-PL (which consists of gold, iron oxide, and poly-L-lysine and magnetizes cells by electrostatically attaching to cell membranes) through overnight incubation and dispensed into a cell-repellent, multiwell plate, where they are levitated off the bottom of the dish by a magnet above the plate. In levitating cells off the bottom of a multiwell plate, the magnetic forces work as an invisible scaffold that rapidly aggregates cells, and induces cell-cell interactions and ECM synthesis. The 3D culture is formed without any artificial substrate or specialized media or equipment and can be cultured long-term. Additionally, adding and removing solutions is made easy by the use of magnets to hold down 3D cultures when removing solutions, limiting culture loss. 3D cultures can also be picked up and transferred between vessels using magnetic tools such as the MagPen.
SOURCE
Cell culture is essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces a two dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo. In some cases, cells may or may not adhere to the tissue culture dishes.
Technology developed by n3D Biosciences brings cells together in a dish using magnetic nanoparticles to levitate them in a magnetic field. This procedure allows cells to develop into spheroids, but if they touch the bottom of the dish, they spread out in a single layer.
The technology developed by n3D Biosciences provide an alternative to biodegradable porous scaffold and protein matrices. Conventionally, biodegradable scaffold may suffer from slow or delayed propagation of cells and establishment of cell-cell interactions. The technology allows adaptable magnetic-based cell levitation and can provide an improved three-dimensional cell growth condition in certain settings.
To achieve this, the first step is to attach magnetic nanoparticles to the cell’s surface which is done by crosslinking the gold and iron oxide nanoparticles with polylysine. In the second step, the cells are brought together while levitating them off the dish. In this condition, the cells can develop in surroundings mirroring growth inside a body where the cells are in contact with each other. The media exchange is also relatively simple because a magnet holds the tissue in place.
Souza, the founder of n3D Biosciences states that microtissues grown with cell levitation have morphology closer to in vivo tissues than conventional cell culture. According to Souza “One huge hurdle is the standardization of the handling of tissue culture cells. Ideally, new tissues for a patient wouldbe grown from their own cells, avoiding the issues of “rejection” of tissue as not the body’s own. One problem is that cells are very sensitive to changes in temperature during the freezing, thawing, and recovery process. ”
Source
http://www.biocision.com/blog/7459/levitating-cells-nanoparticles
Posted in Uncategorized on January 2, 2016| Leave a Comment »
By Diana Negrín da Silva(*)
Dedicated to the interconnected paths of Juan Negrín, Sasha Shulgin and Silviano Camberos.
The texture, color and flower of the peyote remain a vivid childhood memory.
I grew up in a unique…
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