Podcasts about gtpases

BUFFALO, NY- November 27, 2023 – A recent #review paper was #published in Oncotarget's Volume 14 on July 1, 2023, entitled, “Targeting Ras with protein engineering.” Ras proteins are small GTPases that regulate cell growth and division. Mutations in Ras genes are associated with many types of cancer, making them attractive targets for cancer therapy. Despite extensive efforts, targeting Ras proteins with small molecules has been extremely challenging due to Ras's mostly flat surface and lack of small molecule-binding cavities. These challenges were recently overcome by the development of the first covalent small-molecule anti-Ras drug, sotorasib, highlighting the efficacy of Ras inhibition as a therapeutic strategy. However, this drug exclusively inhibits the Ras G12C mutant, which is not a prevalent mutation in most cancer types. Unlike the G12C variant, other Ras oncogenic mutants lack reactive cysteines, rendering them unsuitable for targeting via the same strategy. In this review, researchers Atilio Tomazini and Julia M. Shifman from The Hebrew University of Jerusalem discuss protein engineering as a promising emergent method to target Ras, since engineered proteins have the ability to recognize various surfaces with high affinity and specificity. “While the development of small-molecule Ras inhibitors has been reviewed elsewhere [40], we focus our review on protein-based Ras inhibitors, describing the methods for their engineering, various scaffolds used for inhibitor design, and prospects for delivery of the designed Ras inhibitors into the cellular cytoplasm, where Ras is located.” Over the past few years, scientists have engineered antibodies, natural Ras effectors, and novel binding domains to bind to Ras and counteract its carcinogenic activities via a variety of strategies. These include inhibiting Ras-effector interactions, disrupting Ras dimerization, interrupting Ras nucleotide exchange, stimulating Ras interaction with tumor suppressor genes, and promoting Ras degradation. In parallel, significant advancements have been made in intracellular protein delivery, enabling the delivery of the engineered anti-Ras agents into the cellular cytoplasm. “These advances offer a promising path for targeting Ras proteins and other challenging drug targets, opening up new opportunities for drug discovery and development.” DOI - https://doi.org/10.18632/oncotarget.28469 Correspondence to - Julia M. Shifman - jshifman@mail.huji.ac.il Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28469 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - Ras oncogene, anti-Ras therapeutics, Ras targeting, protein engineering, protein design About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

References Mol Biol Cell. 2001 Sep; 12(9): 2711–2720. Small GTPases. 2014; 5: e28579. Cells. 2021 Jul 20;10(7):1831. J Investig Med. 2023 Feb;71(2):113-123 --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536397v1?rss=1 Authors: Huang, Y., Dong, X., Sun, S. Y., Lim, T.-K., Lin, Q., He, C. Y. Abstract: Arl13b and Arl3 are ciliary GTPases implicated in human Joubert Syndrome, affecting ciliary membrane and axoneme organization. Although the mechanism of Arl13b as a guanine nucleotide exchange factor (GEF) of Arl3 and the function of Arl13b and Arl3 in ciliary membrane protein transport are well established, their role in axoneme biogenesis is unclear. In Trypanosoma brucei, TbArl13 acts as a GEF for two distinct TbArl3 proteins, TbArl3A and TbArl3C. Here, we identified the T. brucei homolog of ODA16, a cargo adapter facilitating intraflagellar transport (IFT) of motile ciliary components, as an effector of both TbArl3A and TbArl3C. Depletion of TbArl3 GTPases stabilized TbODA16 interaction with IFT, while active TbArl3 variants displaced TbODA16 from IFT, demonstrating a mechanism of TbArl3 in motile ciliary cargo transport. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535147v1?rss=1 Authors: Hladyshau, S., Stoop, J. P., Kamada, K., Nie, S., Tsygankov, D. V. Abstract: Rho-GTPases are central regulators within a complex signaling network that controls the cytoskeletal organization and cell movement. This network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, and their numerous effectors that provide mutual regulation and feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling using a simulation model which couples GTPase signaling with cell morphodynamics to capture the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of the time-lapsed recordings of cell dynamics and GTPase activity. This approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.28.534499v1?rss=1 Authors: Rehl, K. M., Selvakumar, J., Hoang, D., Arumugam, K., Gorfe, A., Cho, K.-J. Abstract: Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new editorial paper was published in Oncotarget's Volume 14 on February 25, 2023, entitled, “Mitochondria engage the integrated stress response to promote tumor growth.” In this new editorial, researchers Dillon P. Boulton and M. Cecilia Caino from the University of Colorado School of Medicine discussed prostate cancer (PCa)—the most diagnosed and second deadliest cancer among men in the United States, with an estimated 268,490 new cases and 34,500 deaths in 2022 (ACS Cancer Facts and Figures 2022). “While the prognosis for men with early-stage disease remains extremely favorable (>99% 5-year overall survival, OS), men diagnosed with metastatic PCa have a 30% 5-year OS, clearly demonstrating a need for therapeutic options for these patients (ACS Cancer Facts and Figures 2022).” Due to a strong reliance on androgens to drive PCa, first and second courses of therapy involve androgen deprivation therapy or targeting the androgen receptor directly in combination with several other cytotoxic agents [1–3]. Unfortunately, some tumors develop resistance to these androgen axis therapies and progress to castrate resistant and metastatic PCa, which drives the majority of PCa deaths. This underscores a strong need to identify and characterize actionable targets within these tumors. Of interest, mitochondria are emerging as critical organelles that promote tumorigenesis and metastasis. “​​Along this line, we have recently described a novel signaling pathway where mitochondria promote castrate resistant metastatic PCa growth by acting as a signaling platform to facilitate efficient stress signaling [11]. This pathway is centered around mitochondrial Rho GTPase 2 (MIRO2), an outer-mitochondrial membrane protein in the Ras superfamily of GTPases [12, 13].” Full editorial: DOI: https://doi.org/10.18632/oncotarget.28372 Correspondence to: M. Cecilia Caino - Cecilia.caino@cuanschutz.edu Keywords: mitochondria, prostate cancer, integrated stress response About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.15.528749v1?rss=1 Authors: Maxson, M. E., Huynh, K., Grinstein, S. Abstract: While it has been known for decades that luminal acidification is required for normal traffic along the endocytic pathway, the precise underlying mechanism(s) remain unknown. We found that dissipation of the endomembrane pH gradient resulted in acute formation of large Rab5- or Rab7-positive vacuoles. Vacuole formation was associated with and required hyperactivation of the Rabs, which was attributable to impaired GTPase activity, despite normal recruitment of cognate GAPs. Surprisingly, LRRK2 -a kinase linked to Parkinsons disease-was recruited to endomembranes and markedly activated upon dissipation of luminal acidification. LRRK2 phosphorylated Rab GTPases, rendering them insensitive to deactivation. Importantly, genetic deletion of LRRK2 prevented the {Delta}pH-induced vacuolation, implying that the kinase is required to modulate vesicular traffic. We propose that by dictating the state of activation of LRRK2 and in turn that of Rab GTPases, the development of a progressive luminal acidification serves as a timing device to control endocytic maturation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.28.526044v1?rss=1 Authors: Cason, S. E., Holzbaur, E. L. F. Abstract: Neuronal autophagosomes, "self-eating" degradative organelles, form at presynaptic sites in the distal axon and are transported to the soma to recycle their cargo. During transit, autophagic vacuoles (AVs) mature through fusion with lysosomes to acquire the enzymes necessary to breakdown their cargo. AV transport is driven primarily by the microtubule motor cytoplasmic dynein in concert with dynactin and a series of activating adaptors that change depending on organelle maturation state. The transport of mature AVs is regulated by the scaffolding proteins JIP3 and JIP4, both of which activate dynein motility in vitro. AV transport is also regulated by ARF6 in a GTP-dependent fashion. While GTP-bound ARF6 promotes the formation of the JIP3/4-dynein-dynactin complex, RAB10 competes with the activity of this complex by increasing kinesin recruitment to axonal AVs and lysosomes. These interactions highlight the complex coordination of motors regulating organelle transport in neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Battling cancer takes place in many parts of the world and our next guest has led initiatives to do just that. In Part One of this Oncology, Etc. Podcast episode, Dr. Richard Sullivan, Professor of Cancer and Global Health at King's College London, shares with us his intriguing life trajectory, encompassing a childhood in various parts of the world, aspirations for a veterinary career that turned to basic science, medicine, health policy (4:27), and even a long-term stint with the British Army Intelligence (12:22). Dr. Sullivan, who served as Director of Cancer Research UK for nearly a decade also discusses traits he looks for in a cancer investigator (19:21), and how to be happy (21:16)! Guest Disclosures Dr. Richard Sullivan: Honoraria – Pfizer; Consulting or Advisory Role – Pfizer Dr. David Johnson: Consulting or Advisory Role – Merck, Pfizer, Aileron Therapeutics, Boston University Dr. Patrick Loehrer: Research Funding – Novartis, Lilly Foundation, Taiho Pharmaceutical If you liked this episode, please follow. To explore other episodes, as well as courses visit https://education.asco.org. Contact us at education@asco.org. TRANSCRIPT  Pat Loehrer: Hi, I'm Pat Loehrer. I'm director of the Center of Global Oncology and Health Equity at Indiana University Cancer Center.   Dave Johnson: And I'm Dave Johnson at UT Southwestern in Dallas, Texas.   Pat Loehrer: And this is Oncology, Etc. Dave, what book have you read this last month?   Dave Johnson: I have one I wanted to recommend to you. It's very interesting. It's by Steven Johnson, not of the syndrome fame. It's entitled Extra Life: A Short History of Living Longer. You may have heard of this because PBS made a special documentary about this particular book. But in it, Johnson talks about the remarkable increase in human lifespan, especially over the 20th century, and the various factors that contributed to increased years of life from on average in the United States of about 48-49 in 1900 to just about 80 in the year 2000. So that beats anything in the history of mankind before.   And he has a chapter about each of the factors that contribute to this, and some of which I think we all recognize. Things like antibiotics playing a role, but some of the things that I hadn't thought about were improved drug regulation and the development of randomized controlled trials, which all of us have participated in. How important that is.   He also talked about, at least in the United States, the importance of automotive safety. And I'm sure some of us on this podcast are old enough to remember cars that did not have safety belts and certainly not other safety maneuvers that have really improved lifespan in that regard. So I found it a fascinating book. I think our listeners who are interested in medical history would also enjoy this text.   Pat Loehrer: Did he mention this podcast?   Dave Johnson: No, actually it wasn't mentioned, and I thought that was a tremendous oversight. So, I've sent him a letter and recommended that he add it.   Pat Loehrer: We may not live longer, but it just seems like we're living longer. When you listen to this podcast, time stands still.   Pat Loehrer: Well, it's my real great pleasure to introduce our interviewee today, Richard Sullivan. I met Richard several years ago through the late Professor Peter Boyle in Leon, and it's one of the greatest highlights of my life to be able to know Richard.   Professor Richard Sullivan's Research Group studies health systems and particularly chronic disease policy and the impact of conflict on health. He's a professor of cancer and Global Health at King's College in London and director of the Institute of Cancer Policy and Co-director of Conflict and Health Research Group. As well as holding a number of visiting chairs, Richard is an NCD advisor to the WHO, a civil military advisor to the Save the Children Foundation, and a member of the National Cancer Grid of India. His research focuses on global cancer policy and planning and health system strengthening, particularly in conflict ecosystems. He's principal investigative research programs ranging from automated radiotherapy planning for low resource settings to the use of augmented or virtual reality for cancer surgery through the political economy to build affordable equitable cancer control plans around the world.   Richard has led more Lancet Oncology commissions than anyone else. In fact, Lancet is talking about calling it the Sullivan Commissions. He's led five Lancet Oncology commissions and worked on four others. He's currently co-leading the Lancet Oncology Commission on the Future of Cancer Research in Europe and Cancer Care and Conflict in the conflict systems. His research teams have had major programs in capacity building in conflict regions across the Middle East and North Africa. He's done studies on the basic packages of health services in Afghanistan and worked in Pakistan, Syria, and the Democratic Republic of Congo. He's been a member of the British Army, intelligence and security, and in that capacity he's worked many years in biosecurity and counterterrorism issues. I think in some ways, this is the most interesting man in the world, and it's our pleasure today to have Richard join us. Richard, thank you for coming.   Richard Sullivan: Pat, Dave, you're really too kind. Marvelous to be with you. Thank you for the invitation.   Pat Loehrer: Can you tell us a little about your upbringing and early life before you became Dr. James Bond?   Richard Sullivan: I'm not sure that's anywhere close to the truth, sadly. But, yeah, I have had a very interesting, eclectic life. I was born in Aden just on the cusp of where the British Aden Protectorate met a country which actually no longer exists, the People's Democratic Republic of Yemen. Because after the British left Aden, essentially the East Germans, and what was then the Soviet Union took over southern Yemen. So I was born in a very unusual part of the world, which sadly, since then has just deteriorated. I spent many years of my life with my parents, who were in the diplomatic service and doing other things, wandering around the globe, mainly in the Middle East and East Africa. We spent quite a lot of time, strangely enough, we washed up on the shores in the USA once as well. Dayton, Ohio, and eventually-   Pat Loehrer:  Not to interrupt you, Richard, there are no shores in Dayton, Ohio. So just correct you there.   Richard Sullivan: That is so true. My memory - cornfields everywhere. I had a wonderful dog then, that's how I remember it so well. And I didn't really come back to the UK until, oh, gosh, I was nearly 10-11 years old. So, coming back to the UK was actually a bit of a culture shock for me. And then relatively classical in terms of the UK, sort of minor public school and then into medical school. In the old days when it was in the 80's. I had a fabulous childhood, going all over the place, seeing lots of things, being exposed to lots of different cultures. I think it remained with me all my life. I never really feel a foreigner in a foreign land. That's nice. That's really unique and it's been marvelous being able to tie in the passion for global health with my upbringing as well. So, yeah, I had a wonderful childhood.   Dave Johnson: Would you mind expanding on your medical training, Richard? Tell us a little bit about that.   Richard Sullivan: Yeah, so when I, when I went to medical school in the UK, we were still running the old system. And by the old system, I mean, you know, these small medical schools with entries of, you know, 70, 80 individuals, particularly in London, you had that St. Mary's Hospital Medical School, which is where I went, Charing Cross, Guy's, St. Thomas', and they were all individual medical schools. Now, most of these now have merged together into these super medical schools. But certainly when I went to medical school, I'll be absolutely honest with you, I wanted to be a vet to begin with, but actually discovered I wasn't bright enough to be a vet. It was harder to become a vet than it was to become a doctor. In my day going into medicine, and people listening to this, or some people who understand the A level system in the UK will recognize if you're offered a BCD, that's quite low grades to get into medical school. So I went to Mary's, to be absolutely honest with you, because I heard that they took people that played rugby, and I came from a rugby-playing school. And sure enough, 90% of the interview was based on my rugby prowess, and that was St. Mary's Hospital Medical School. So it was wonderful.   And we'd already had people going there who were big rugby players. And again, it was, I remember thinking to myself, am I making the right decision here? But it was interesting, as soon as I went into medical school, I realized that was the life for me. I had done myself a favor by not going into veterinary science, which I would have been awful at. We had six years of very, very intensive pre-medicine, the classical medical rotations, and then that movement into the old schools of pre registration house officers, registrar jobs. We were quite an early stage. I kind of slightly went off-piste and started doing more academic work. Interestingly, most of my academic early days academic work was not in health policy and research. It was actually in very hard core cell signaling. So my doctorate was in biochemistry, and we worked on small GTPases, calcium-sensing proteins.   There were some really extraordinary heady days, and I'm talking here about the early nineties and the mid-nineties of tremendous discovery, real innovation. I was at UCL at the time, but mixing and matching that up with a sort of surgical training, and again, surgical training in those days was pretty classical. You went into your general surgery, then sort of specialized. It was really, really interesting but it was full on. I mean, you spent your entire life working. Morning to night so these were the days of 100 hours week rotations. You were doing one in twos, one in threes. That's every other night and every other weekend on call. It was incredibly intense, but there was a lot more diversity and plasticity in those days. You could dip in and out of medicine because of the way you were chosen and how you were recruited. So it suited my personality because I liked moving around and doing different things and that sort of took me through, really until the late 1990s.   Pat Loehrer: You became a urologist, right?   Richard Sullivan: That's right. Exactly. So I trained up until the late 1990s, it was all pretty standard, I would say. And then I decided I was bored and moved into the pharmaceutical industry and I went to work in for Merck Damstadt at the time, which was relatively small. I was going to say family owned, but it was quite family-owned pharmaceutical company that was just moving into oncology. And because I'd done the background in cell signaling and cell signaling was really the backbone of the new era of targeted therapies, this seemed like a great move. To be absolutely blunt with you, I didn't last very long, less than a couple of years, I think, mainly because I just found the whole environment way too constraining. But what it did provide me with was a springboard to meet the wonderful late Gordon McVie, who I met at a conference. And he said to me, ‘You're absolutely wasting your time and life by staying in the pharmaceutical industry. Why don't you come out, get an academic job at University College London and become my head of clinical programs?” - for what was then the Cancer Research Campaign. This Cancer Research Campaign and the Imperial Cancer Research Fund were the forerunners of Cancer Research UK. So, you know, this was an offer that was too good to be true.   So I jumped ship immediately, went back into academic life and joined CRC. And really the next ten years was this extraordinary blossoming of the merger of CRC with the Imperial College Research Fund, the creation of Cancer Research UK, and that was Paul Nurse, and obviously Gordon and me, bringing that all together. And it was the heady days of that resurgence of cancer, the importance of cancer care and research in the UK. And coupled with that, of course, it was the blossoming of my interest, really then into the global health aspects of cancer, which really, Gordon, people like you mentioned already, the late, wonderful Peter Boyle, all those individuals were already engaged in and they were the ones that really kind of catapulted me into a more international scene.   Dave Johnson: Did you know Dr. McVie before you met him at this conference, or was it just a chance encounter?   Richard Sullivan: No, he actually met me via John Mendelson, because John had picked up a paper I'd been writing on basically the very early versions of Rituximab that we were working on and we were looking for pharmacodynamic endpoints. And of course, one of the things I noticed with the patients is they were getting all these skin rashes on their faces, and I thought, that's terrific. Just seemed to be the skin rashes seemed to be together with those individuals that had better responses. And I remember writing this paper for Signal, which was a kind of relatively minor journal, and I think it was John Mendelson who picked it up and must have mentioned something to Gordon. Gordon hunted me out down at a particular conference, said, "How on earth do you know about this, that you're not anything more than a surgeon?" He was absolutely right about, goodness sake, what do you know about pharmacodynamic endpoints, and I kind of had to sort of confess that I've gone kind of slightly off-piste by doing biochemistry and cells signaling and working with these extraordinary people. And that's how I essentially met Gordon. He was very good for spotting slightly unusual, eclectic human beings.   Pat Loehrer: I'm very curious about the intersection of your work and how you got into the British Army and Intelligence with medicine and how that even may continue even today. So explain that story, that part of your life a little bit to us.   Richard Sullivan: Yeah, it was very early on, as I went into medical school, one of the key concerns was making money. I looked around for ways of doing something interesting to make money, and most of the jobs on offer were bar jobs, et cetera. Then I thought, what about the Territorial Army, which, in the early days of the 1980s, was, and still is, a very large component of the UK Armed Forces. So I actually joined the Royal Army Medical Corps, as you would expect for someone going into medicine. I thought, okay, I'll join the Royal Army Medical Corps, and I was a combat Medical Training Technician, et cetera. So I went along, signed up, and I think I was about three months into training when I was at a place called Kew Barracks and some chap came up to me and handed me a little bit of paper. It said "Intelligence Security Group" and gave a phone number. He said, "This is more your line of work. Why don't you give them a ring?"   It was interesting because, in those early days, they were looking for analysts who could work on lots of different areas. In those days, most of the work was domestic.. Of course, there was counterterrorism with Northern Ireland, but there was also the Soviet Union, and the fallout from the Warsaw Pact, so they were still actively recruiting into that area. There are lots of details I can't talk about, but it was relatively, to begin with, quite hard work and low level. It was a lot of learning foreign equipment recognition. It was what we consider to be standard combat intelligence. But the more time you spend in it, the more interesting it gets.   One of the areas they were looking to recruit into, which I didn't realize at the time but only later, was bioweapons and biosecurity. They needed people who understood biotechnology and the language of science, and who could be taught the language of infectious disease on top of that. That is quite a difficult combination to find. It's very easy to teach people trade craft and intelligence, it's very hard to teach them subject matter expertise. And they were really missing people who specialized in that area.   It was interesting because it was still a relatively open domain. There was still a lot of work going on in the counterterrorism front with biological weapons, and a lot around the Verification of the Biological Weapons and Toxin Convention. And it was an interesting, and I'd almost say parallel life. But your medical knowledge and the scientific knowledge I had already gained and was gaining was what was being looked for. So that was very early on and it has expanded over the years. More and more now we talk about health security and intelligence so that goes beyond what you would consider classic medical intelligence or Armed Forces - this is more about putting together the disciplines of intelligence with the securitized issues of, for example Ebola. That is a classic example. The big outbreaks in West Africa, the DRC, these are sort of the classic security intelligence issues - even COVID 19 for example - and mostly around the world, what we've seen is the intelligence apparatus taking front and center in that, whether you're looking at states like South Korea, et cetera. So I've moved more into that, and we do a lot of work and research into this as well. So we look at, particularly now, how to improve human intelligence in this area, the pros and cons of signal intelligence collection. And we go as far as to kind of ask sort of deep ethical and moral issues, for example, about how far should these sorts of apparatus of state be applied to public good issues like health. Because at the end of the day, when you're talking about the armed forces security sector, their primary job is for defense of the realm. So applying them in other areas obviously comes with a whole load of moral and ethical challenges. So, yes, it's been a fascinating journey, which, as I said, it extends all the way back to the late 1980s. It's been both complementary and different.   Dave Johnson: So, Richard, there's so many things in your resume that warrant exploration, but you served as Clinical Director of Cancer Research UK for nearly a decade. What was that experience like, and what accomplishment are you most proud of?   Richard Sullivan: It was an enormous privilege. In your life, you always look at some jobs and you think, “How lucky I was to be there at that time with those people.” I think, first of all, enormous respect for the people that ran both Cancer Research Campaign, Imperial Cancer Research Fund – I mean, Paul Nurse and Gordon McVeigh, Richard Treisman – I mean, some extraordinary people who were leading both of these charities. And so to be there at that moment when they both came together, but more importantly as well, they had this most amazing global network of literally the illuminati of cancer research, spanning from basic science all the way through to epidemiology, public health, health systems. And in those days, of course, those individuals would come on site visits to the UK to look at the different units and evaluate them. So you can imagine when you're bringing those sorts of individuals across, you get a chance to go out with them, go drinking, talk to them, learn about their research, and also learn about the extraordinary breadth of research that was there in the UK. So you're condensing almost a lifetime's worth of learning into a few years. It was an absolute privilege to have been able to serve the community like that.   What I'm most proud of? Gosh, I like to think I suspect that most proud of trying to help a lot of the fellows get through to where they were going to actually get the most out of their careers. When I look back, there are lots and lots of names of people who started at a very early stage with funding from Cancer Research Campaign or the Imperial College Research Fund, who are now very, very senior professors and global research leaders. And I like to think that we did a little bit to help them along that way and also help to support individual research programs actually reach their full potential. Because I think research management and planning is often overlooked. People think of this as very transactional – it's not transactional. It's an incredibly important, serious discipline. It requires very careful handling to get the very best out of your research ecosystem. You've really, really got to get under the skin and really have a clear view of how you're going to help people. So I think that's what I'm most proud of – is the individuals who made it all the way through and now these great leaders out there.   But it was also, let's be honest, it was halcyon days. Great innovations, great discoveries, new networks growing, incredible expansion of funding in the UK, in Europe, in the USA. They were very, very good days. And it was, as I said, it was a real privilege to be there almost at the center for nearly a decade.   Dave Johnson: Let me follow up on that, if I may, just for a moment. You have had such an incredible influence. What characteristics do you think are most desired in a cancer investigator? What sorts of things do you look for, especially when you're thinking about funding someone?   Richard Sullivan: Creativity. I think creativity is really important. We talk about the word innovation a lot, and it's an interesting engineering term, but creativity is that spark that you can see it in people, the way they talk about what they're doing. They have this really creative approach. And with that, I think you have to have the passion. Research careers are long and difficult, and I'd probably suggest there's probably more downs than there are ups, and you have to have that passion for it. And I think along with that passion is the belief in what you're doing – that first of all, you have that belief that actually drives you forward, that what you know you're doing is good work, and that you're really dedicated to it. But obviously, hand on heart, when you're looking at researchers, it's that passion and that creativity.   I think it's a brave person to judge how any person's career or program is going to go. I don't think any of us are prophets. Even in our own land. We might be able to see slightly into the future, but there are so many elements that make up  “success”. It's funny when I look back and I think those who've been successful, it's people who've also been generally happy in their lives. They've found their careers in whatever shape or form, fulfilling, and they've generally been happy human beings, and they've managed to create a life around research which has given them meaning.   Pat Loehrer: Richard, you have reinvented yourself a number of times – this transition of going from like a basic scientist, a surgeon, moving into public policy and global policy. Tell me a little bit about the journey that's been in terms of academics. How do you learn? What were the transition points in each of these things to get you now to be, as I mentioned before, kind of the key person for Lancet's commissions to somebody who was a rugby player?   Richard Sullivan: I suppose if you're being mean, you say, he clearly gets bored easily. But it's not that. Actually, I'm not very instrumental about life either. I mean, there are many people you will meet who have got their lives and strategies mapped out. They know they're going to do X next year, Y the following year. And for me, it's never been like that. For me, it's that excitement, that creativity of working on new and interesting things, but also knowing when you've run out of road in a particular area, where it no longer gets you out of bed in the morning, where you no longer feel happy, where you no longer feel you're contributing. All of us talking today have the great privilege of having choice about our lives, about what direction our lives should take. And it's not a privilege one should squander lightly because many people do not have choices about their lives. It's all about chance. And having that choice to be able to move into different areas is really important because I said you can stick in the same thing because you think you have to. And you can become an unhappy, miserable human being. And that makes you a miserable researcher to be around. It makes you a terrible doctor. Probably makes you a terrible person, actually, generally, if you're having a miserable life.   So finding new things, that really you're passionate about how you do it, there's no shortcut in this. It's hard work. Readily admit I went back to law school of economics, retaught myself lots of things. There are no shortcuts for. Deciding if you're going to a new area is learning, learning, practice, practice, practice, and just doing the hard work. I think that's an ethos that was probably drilled into us quite early anyway in medical school, because that's how you approach medicine. That's how you approach science when I was growing up. And it was that idea of humility that you can never have enough learning, you will always learn off other people. That's probably what drove me and how I've managed to change and as I say, who knows what the future is? I don't know. Maybe one day I'll think about doing a bit of poetry.   Dave Johnson: Your comments about happiness and work resonate with Pat and me. I think we both feel like humor is really important for happiness and career success. And, you know, Osler once said, “The master word of medicine is work.” You can't get around that. It is what it is. And I think you just reaffirmed that.   Well, this concludes part one of our interview with Richard Sullivan, professor of Cancer and Global Health at King's College, London and director of the King's Institute of Cancer Policy and co-director of the Conflict and Health Research Group. In the second part of this episode, Professor Sullivan will speak about the progress of global health, especially in conflict areas, and the need for young people to enter into the world of oncology and oncology research.   Thank you to all of our listeners for tuning into Oncology, Etc. This is an ASCO educational podcast where we will talk about just about anything and everything. So if you have an idea for a topic or a guest you would like us to interview, please email us at education@asco.org. Thank you again for listening.  Thank you for listening to the ASCO Education podcast. To stay up to date with the latest episodes, please click subscribe. Let us know what you think by leaving a review. For more information, visit the Comprehensive Education Center at education ASCO.org. The purpose of this podcast is to educate and to inform. This is not a substitute for professional medical care and is not intended for use in the diagnosis or treatment of individual conditions. Guests on this podcast express their own opinions, experience and conclusions. Statements on the podcast do not express the opinions of ASCO. The mention of any product, service, organization, activity or therapy should not be construed as an ASCO endorsement.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.07.515393v1?rss=1 Authors: Kounoupa, Z., Tivodar, S., Theodorakis, K., Kyriakis, D., Denaxa, M., Karagogeos, D. Abstract: Rho GTPases, among them Rac1 and Rac3, are major transducers of extracellular signals and are involved in multiple cellular processes. In cortical interneurons, the neurons that control excitation/inhibition balance of cortical circuits, Rac1 and Rac3 are essential for their development. Ablation of both, leads to a severe reduction in the numbers of mature interneurons found in the murine cortex, which is partially due to abnormal cell cycle progression of interneuron precursors and defective formation of their growth cones. Here we present new evidence that upon Rac1 and Rac3 ablation, centrosome, Golgi complex and lysosome positioning are significantly perturbed, thus affecting both interneuron migration and axon growth. Moreover, for the first time we provide evidence of altered expression and localization of the two-pore channel 2 (TPC2) voltage-gated ion channel that mediates Ca2+ release. Pharmacological inhibition of TPC2 negatively affected axonal growth and migration of interneurons. Our data taken together suggest that TPC2 contributes to the severe phenotype in axon growth initiation, extension and interneuron migration in the absence of Rac1 and Rac3. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514532v1?rss=1 Authors: Aljiboury, A. A., Ingram, E., Krishnan, N., Ononiwu, F., Pal, D., Manikas, J., Taveras, C., Hall, N. A., Da Silva, J., Freshour, J., Hehnly, H. Abstract: An essential process for cilia formation during epithelialization is the movement of the centrosome to dock with the cell's nascent apical membrane. Our study examined centrosome positioning during the development of Danio rerio's left-right organizer (Kupffer's Vesicle, KV). We found that when KV mesenchymal-like cells transition into epithelial cells that are organizing into a rosette-like structure, KV cells move their centrosomes from random intracellular positions to the forming apical membrane in a Rab11 and Rab35 dependent manner. During this process, centrosomes construct cilia intracellularly that associated with Myo-Va while the centrosomes repositioned towards the rosette center. Once the centrosomes with associated cilia reach the rosette center, the intracellular cilia recruit Arl13b until they extend into the forming lumen. This process begins when the lumen reaches an area of approximately 300 m2. Using optogenetic and depletion strategies, we identified that the small GTPases, Rab11 and Rab35, regulate not only cilia formation, but lumenogenesis, whereas Rab8 was primarily involved in regulating cilia length. These studies substantiate both conserved and unique roles for Rab11, Rab35, and Rab8 function in cilia formation during lumenogenesis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.514611v1?rss=1 Authors: Plazen, L., Al Rahbani, J., Brown, C. M., Khadra, A. Abstract: In mesenchymal cell motility, several migration patterns have been observed, including directional, exploratory and stationary. Two key members of the Rho-family of GTPases, Rac and Rho, along with an adaptor protein called paxillin, have been particularly implicated in the formation of such migration patterns and in regulating adhesion dynamics. Together, they form a key regulatory network that involves the mutual inhibition exerted by Rac and Rho on each other and the promotion of Rac activation by phosphorylated paxillin. Although this interaction is sufficient to generating wave-pinning that underscores cellular polarization comprised of cellular front (high active Rac) and back (high active Rho), it remains unclear how they interact collectively to induce other modes of migration detected in Chinese hamster Ovary (CHO-K1) cells. We previously developed a 6D reaction-diffusion model describing the interactions of these three proteins (in their active/phosphorylated and inactive/unphosphorylated forms) along with other auxiliary proteins, to decipher their role in generating wave-pinning. In this study, we explored, through computational modeling and image analysis, how differences in timescales within this molecular network can potentially produce the migration patterns in CHO-K1 cells and how switching between them could occur. To do so, the 6D model was reduced to an excitable 4D spatiotemporal model possessing three different timescales. The model produced not only wave-pinning in the presence of diffusion, but also mixed-mode oscillations (MMOs) and relaxation oscillations (ROs). Implementing the model using the Cellular Potts Model (CPM) produced outcomes in which protrusions in cell membrane changed Rac-Rho localization, resulting in membrane oscillations and fast directionality variations similar to those seen in CHO-K1 cells. The latter was assessed by comparing the migration patterns of CHO-K1 cells with CPM cells using four metrics: instantaneous cell speed, exponent of mean square-displacement (called -value), directionality ratio and protrusion rate. Variations in migration patterns induced by mutating paxillin's serine 273 residue was also captured by the model and detected by a machine classifier, revealing that this mutation alters the dynamics of the system from MMOs to ROs or nonoscillatory behaviour through variation in the concentration of an active form of an adhesion protein called p21-Activated Kinase 1 (PAK). These results thus suggest that MMOs and adhesion dynamics are the key ingredients underlying CHO-K1 cell motility. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.28.514275v1?rss=1 Authors: Plazen, L., Khadra, A. Abstract: Mesenchymal cell motility is mainly regulated by two members of the Rho-family of GTPases, called Rac and Rho. The mutual inhibition exerted by these two proteins on each other's activation and the promotion of Rac activation by an adaptor protein called paxillin have been implicated in driving cellular polarization comprised of front (high active Rac) and back (high active Rho) during cell migration. Mathematical modeling of this regulatory network has previously shown that bistability is responsible for generating a spatiotemporal pattern underscoring cellular polarity called wave-pinning when diffusion is included. We previously developed a 6D reaction-diffusion model of this network to decipher the role of Rac, Rho and paxillin (along with other auxiliary proteins) in generating wave-pinning. In this study, we simplify this model through a series of steps into an excitable 3D ODE model comprised of one fast variable (the scaled concentration of active Rac), one slow variable (the maximum paxillin phosphorylation rate - turned into a variable) and a very slow variable (a recovery rate - also turned into a variable). We then explore, through slow-fast analysis, how excitability is manifested by showing that the model can exhibit relaxation oscillations (ROs) as well as mixed-mode oscillations (MMOs) whose underlying dynamics are consistent with a delayed Hopf bifurcation. By reintroducing diffusion and the scaled concentration of inactive Rac into the model, we obtain a 4D PDE model that generates several unique spatiotemporal patterns that are relevant to cell motility. These patterns are then characterized and their impact on cell motility are explored by employing the cellular potts model (CPM). Our results reveal that wave pinning produces purely very directed motion in CPM, while MMOs allow for meandering and non-motile behaviours to occur. This highlights the role of MMOs as a potential mechanism for mesenchymal cell motility. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.19.389494v1?rss=1 Authors: Shan, Y., Zhang, X., Song, C., Liu, Y., Ke, P., Kuo, Y.-C., Wang, Y. Abstract: Plexins are semaphorin receptors that play essential roles in neuronal axon guidance and in many other important biological processes. Plexin signaling depends on a semaphorin-induced dimerization mechanism, and is modulated by small signaling GTPases of the Rho family, of which RND1 serves as a plexin activator yet its close homolog RhoD an inhibitor. Using molecular dynamics (MD) simulations we showed that RND1 reinforces plexin dimerization interface whereas RhoD destabilizes it due to their differential interaction with cell membrane. Upon binding plexin dimers at the Rho-GTPase binding (RBD) domains, RND1 and RhoD interact differently with the inner leaflet of cell membrane, and exert opposite effects on the dimerization interface via an allosteric network involving the RBD domain, RBD linkers, and a buttress segment adjacent to the dimerization interface. The differential membrane interaction is attributed to the fact that, unlike RND1, RhoD features a short C-terminal tail and a positively-charged membrane interface. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.387449v1?rss=1 Authors: Underwood, R. N., Wang, B., Pathak, A., Volpicelli-Daley, L., Yacoubian, T. A. Abstract: Parkinson's disease and Dementia with Lewy Bodies are two common neurodegenerative disorders marked by proteinaceous aggregates composed primarily of the protein -synuclein. -Synuclein is hypothesized to have prion-like properties, by which misfolded -synuclein induces the pathological aggregation of endogenous -synuclein and neuronal loss. Rab27a and Rab27b are two highly homologous Rab GTPases that regulate -synuclein secretion, clearance, and toxicity in vitro. In this study, we tested the impact of Rab27a/b on the transmission of pathogenic -synuclein. Double knockout of both Rab27 isoforms eliminated -synuclein aggregation and neuronal toxicity in primary cultured neurons exposed to fibrillary -synuclein. In vivo, Rab27 double knockout mice lacked fibril-induced -synuclein inclusions, dopaminergic neuron loss, and behavioral deficits seen in wildtype mice with fibril-induced inclusions. Studies using AlexaFluor488-labeled -synuclein fibrils revealed that Rab27a/b knockout prevented -synuclein internalization without affecting bulk endocytosis. Rab27a/b knockout also blocked the cell-to-cell spread of -synuclein pathology in multifluidic, multichambered devices. This study provides critical insight into the role of Rab GTPases in Parkinson's disease and identifies Rab27s as key players in the progression of synucleinopathies. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.17.386177v1?rss=1 Authors: Cuevas-Navarro, A., Van, R., Cheng, A., Urisman, A., Castel, P., McCormick, F. Abstract: The spindle assembly checkpoint (SAC) is an evolutionarily conserved safety mechanism that maintains genomic stability. However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of GTPases. Here we show that RIT1, a Ras-related GTPase, is essential for timely progression through mitosis and proper chromosome segregation. Furthermore, pathogenic levels of RIT1 silence the SAC, accelerate transit through mitosis, and promote chromosome segregation errors through direct association with SAC proteins MAD2 and p31comet. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.372730v1?rss=1 Authors: Novev, J. K., Heltberg, M. L., Jensen, M. H., Doostmohammadi, A. Abstract: How cells sense and respond to mechanical stimuli remains an open question. Recent advances have identified the translocation of Yes-associated protein (YAP) between nucleus and cytoplasm as a central mechanism for sensing mechanical forces and regulating mechanotransduction. We formulate a spatiotem-poral model of the mechanotransduction signalling pathway that includes coupling of YAP with the cell force-generation machinery through the Rho family of GTPases. Considering the active and inactive forms of a single Rho protein (GTP/GDP-bound) and of YAP (non-phosphorylated/phosphorylated), we study the cross-talk between cell polarization due to active Rho and YAP activation through its nuclear localization. For fixed mechanical stimuli, our model predicts stationary nuclear-to-cytoplasmic YAP ratios consistent with experimental data at varying adhesive cell area. We further predict damped and even sustained oscillations in the YAP nuclear-to-cytoplasmic ratio by accounting for recently reported positive and negative YAP-Rho feedback. Extending the framework to time-varying mechanical stimuli that simulate cyclic stretching and compression, we show that the YAP nuclear-to-cytoplasmic ratio's time dependence follows that of the cyclic mechanical stimulus. The model presents one of the first frameworks for understanding spatiotemporal YAP mechanotransduction, providing several predictions of possible YAP localization dynamics, and suggesting new directions for experimental and theoretical studies. Copy rights belong to original authors. Visit the link for more info

This episode builds on content covered in episode 3, so be sure to check it out if you haven't listened to it already. This week, we explore an example of the GTPase content we discussed last time. Specifically, we look at the diploid mating of baker's yeast in stressful environments. Source for this episode: 1) Alberts, Johnson, Lewis Raff, Roberts and Walter (2008), Molecular Biology of the Cell, Fifth Edition. Abingdon: Garland Science, Taylor and Francis Group LLC.

On the podcast today, we scratch the surface of cell polarity and how enzymes known as GTPases are linked to this process. Sources for this episode: 1) Alberts, Johnson, Lewis Raff, Roberts and Walter (2008), Molecular Biology of the Cell, Fifth Edition, Abingdon: Garland Science, Taylor and Francis Group LLC. 2) Thain, M. And Hickman, M. (2014), The Penguin Dictionary of Biology, 11th edition. London: Penguin Publishing Group. 3) Some of the discussion is also based on my studies.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.20.344556v1?rss=1 Authors: Osmak, G., Kiselev, I., Baulina, N., Favorova, O. Abstract: MicroRNAs (miRNAs) are short single-stranded non-coding RNA molecules, which are involved in regulation of main biological processes, such as apoptosis, cell proliferation and differentiation, through sequence-specific interaction with target mRNAs. In this study we propose a workflow for predicting miRNAs function by analyzing the structure of the network of their target genes. This workflow was applied to study the functional role of miR-375 in the heart muscle (myocardium), since this miRNA was previously shown to be associated with heart diseases and data on its function in myocardium are mostly unclear. We identified PIK3CA, RHOA, MAPK3, PAFAH1B1, CTNNB1, MYC, PRKCA, ERBB2, and CDC42 as key genes in the miR-375 regulated network and predicted the possible function of miR-375 in the heart muscle, consisting mainly in the regulation of the Rho-GTPases-dependent signalling pathways. We implemented our algorithm for miRNA function prediction into Python module, which is available at GitHub (https://github.com/GJOsmak/miRNET). Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.11.293589v1?rss=1 Authors: Emond, M. R., Biswas, S., Morrow, M. L., Jontes, J. Abstract: Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead to PCDH19 Female-limited epilepsy (PCDH19 FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19 in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.03.281675v1?rss=1 Authors: Terrey, M., Adamson, S. I., Gibson, A. L., Deng, T., Ishimura, R., Chuang, J. H., Ackerman, S. L. Abstract: Ribosome-associated quality control pathways respond to defects in translational elongation to recycle arrested ribosomes and degrade aberrant polypeptides and mRNAs. Loss of an individual tRNA gene leads to ribosomal pausing that is resolved by the translational GTPase GTPBP2, and in its absence causes neuron death. Here we show that loss of the homologous protein GTPBP1 during tRNA deficiency in the mouse brain also leads to codon-specific ribosome pausing and neurodegeneration, suggesting that these non-redundant translational GTPases function in the same pathway to mitigate ribosome pausing. Ribosome stalling in the mutant brain led to activation of the integrated stress response (ISR) mediated by GCN2 and decreased mTORC1 signaling. However, in contrast to the ISR, which enhanced neuron survival, reduced mTORC1 signaling increased neuronal death. Our data demonstrate that GTPBP1 functions as an important quality control mechanism during translation elongation and suggest that translational signaling pathways intricately interact to regulate neuronal homeostasis during defective translation elongation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.16.253419v1?rss=1 Authors: Batra, S., Kumar, A., Prakash, B. Abstract: GTP hydrolysis is the underlying basis for functioning of 'biological switches' or GTPases. Extensively studied GTPases, Ras and EF-Tu, use a conserved Gln/His that facilitates the activation of attacking water for nucleophilic attack. However, this is insufficient to explain catalysis in Hydrophobic Amino acid Substituted (HAS)-GTPases that naturally possess a hydrophobic residue in lieu of Gln/His. We had previously reported a bridging water-chain mediated catalytic mechanism for HAS-GTPase FeoB; which utilizes two distantly-located but conserved glutamates. Curiously, mutating these does not abolish GTP hydrolysis. Similarly, in this study we report our observations on another HAS-GTPase Era, wherein the mutants of catalytically important residues continue to hydrolyze GTP. We attempt to rationalize these inquisitive observations on GTP hydrolysis by FeoB and Era mutants. We propose a general theory that appears common to at least three classes of GTPases, where 'alternative mechanisms' emerge when the primary mechanism is disrupted. Based on the analysis of crystal structures of FeoB and Era mutants, bound to the transition state analogue GDP.AlFx, this work suggests that in the absence of catalytically important residues, the active site waters in both FeoB and Era undergo re-arrangements, which in turn helps in sustaining GTP hydrolysis. Similar employment of alternative mechanisms was also suggested for the catalytic mutants of hGBP1. Importantly, such alternatives underscore the robustness of GTP hydrolysis mechanisms in these systems, and raise important questions regarding the need for persistent GTP hydrolysis and the physiological relevance of structural plasticity seen here. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.15.252247v1?rss=1 Authors: Motiwala, Z., Sandholu, A. S., Sengupta, D., Kulkarni, K. Abstract: Ras superfamily GTPases are molecular switches which regulate critical cellular processes. Extensive structural and computational studies on this G protein family have tried to establish a general framework for their switching mechanism. The current understanding of the mechanism is that two loops, Switch I and Switch II, undergo conformational changes upon GTP binding and hydrolysis, which results in alteration of their functional state. However, because of variation in the extent of conformational changes seen across the members of the Ras superfamily, there is no generic modus operandi defining their switching mechanism, in terms of loop conformation. Here, we have developed a novel method employing wavelet transformation to dissect the structures of these molecular switches to explore indices that defines the unified principle of working. Our analysis shows that the structural coupling between the Switch I and Switch II regions is manifested in terms of wavelet coherence phases. The resultant phase pertaining to these regions serve as a functional identity of the GTPases. The coupling defined in terms of wavelet coherence phases is conserved across the Ras superfamily. In oncogenic mutants of the GTPases the phase coupling gets disentangled, this perhaps provides an alternative explanation for their aberrant function. Although similar observations were made using MD simulations, there was no structural parameter to define the coupling, as delineated here. Furthermore, the technique reported here is computationally inexpensive and can provide significant functional insights on the GTPases by analyzing as few as two structures. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.28.225524v1?rss=1 Authors: Fromm, S., Lawrence, R. E., Hurley, J. H. Abstract: The mechanistic target of rapamycin complex 1 (mTORC1) couples cell growth to nutrient, energy and growth factor availability1-3. mTORC1 is activated at the lysosomal membrane when amino acids are replete via the Rag guanosine triphosphatases (GTPases)4-6. Rags exist in two stable states, an inactive (RagA/BGDP:RagC/DGTP) and active (RagA/BGTP:RagC/DGDP) state, during low and high cellular amino acid levels4,5. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator7,8, and the FLCN:FNIP (FLCN-interacting protein) GTPase activating protein (GAP) complex9, and prevents activation of the Rag dimer during amino acid starvation10,11. How the LFC is released upon amino acid refeeding is a major outstanding question in amino-acid dependent Rag activation. Here we show that the cytoplasmic tail of the lysosomal solute carrier family 38 member 9 (SLC38A9), a known Rag activator12-14, destabilizes the LFC. By breaking up the LFC, SLC38A9 triggers the GAP activity of FLCN:FNIP toward RagC. We present the cryo electron microscopy (cryo-EM) structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre and post GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.12.147975v1?rss=1 Authors: Kavalali, E. T., Afuwape, O., Chanaday, N., Kasap, M., Monteggia, L. M. Abstract: Dynamins are GTPases required for pinching vesicles off the plasma membrane once a critical curvature is reached during endocytosis. Here, we probed dynamin function in central synapses by depleting all three dynamin isoforms in postnatal hippocampal neurons. We found a decrease in the propensity of evoked neurotransmission as well as a reduction in synaptic vesicle numbers. Using the fluorescent reporter vGluT1-pHluorin, we observed that compensatory endocytosis after 20 Hz stimulation was arrested in ~40% of presynaptic boutons, while remaining synapses showed only a modest effect suggesting the existence of a dynamin-independent endocytic pathway in central synapses. Surprisingly, we found that the retrieval of single synaptic vesicles, after either evoked or spontaneous fusion, was largely impervious to disruption of dynamins. Overall, our results suggest that classical dynamindependent endocytosis is not essential for retrieval of synaptic vesicle proteins after quantal single synaptic vesicle fusion. Copy rights belong to original authors. Visit the link for more info

This month on Episode 5 of the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from the September 27 and October 11, 2019 issues of Circulation Research and talks with Sarvesh Chelvanambi and Matthias Clauss  about their article HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients.   Article highlights:   Stamatelopoulos, et al. Reactive Vasodilation in AL Amyloidosis   Cao, et al. Miro2-Mediated Cardiac Mitochondrial Communication   Georgakis, et al. Circulating MCP-1 Levels and Incident Stroke   Sun, et al. Body Mass Index and DNA Methylation   Tan, et al. Yy1 Suppresses DCM Through Bmp7 and Ctgf Transcript Cindy St. H:                       Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the September 27th and October 11th issues of Circulation Research.                                            We'll also have an in-depth conversation with Drs Matthias Clauss and Sarvesh Chelvanambi, who are the lead authors in one of the exciting discoveries from our October 11th issue.                                            The first article I want to share with you is titled, Reactive Vasodilation Predicts Mortality in Primary Systemic Light Chain Amyloidosis. The first authors are Drs Kimon Stamatelopoulos, Georgios Georgiopoulos, and the corresponding author is Dr Efstathios Kastritis. And the studies were conducted at the National Kapodistrian University of Athens School of Medicine in Athens, Greece.                                            So we hear about amyloids a lot in things like Alzheimer's, but amyloids are really just aggregates of protein that fold into shapes. And the nature of these shapes allows these individual protein molecules to bind and form many copies that form these fibers that are rather sticky. And the fibers then aggregate into larger and larger globules. And light chain amyloidosis is the most common type of amyloidosis. It's a rare but deadly disease, and it's caused by antibody-producing cells that are aberrantly churning out parts of antibodies called light chains. And it's these light chains that will aggregate and form sticky fibers.                                            So these fibers aggregate and form amyloid deposits, and these deposits build up and damage the organs and the tissue in which they're accumulating. And because it's dependent on where the aggregates are accumulating, AL amyloidosis can present with a wide variety of symptoms. However, symptoms of heart dysfunction and low blood pressure correlate with poor prognosis.                                            And because vascular dysfunction can contribute to hypotension or low blood pressure, this group decided to examine the vascular health of patients by conducting a measurement called flow-mediated vasodilation. And so this is a measurement where the diameter of the brachial artery, which is located in your arm, is measured before and then after a brief period of lower arm ischemia. And they formed a cohort of 115 newly diagnosed AL patients and another cohort of 115 matched controls. This study found that in AL patients, flow-mediated vasodilation was higher than in aged, sex, and cardiovascular risk factor-matched controls. The mean follow-up time for this study was 54 months, and in that time, the authors went on to find that high values of FMD in the amyloidosis patients was strongly predictive of mortality. In fact, high FMD values were more predictive of death than some measures of cardiovascular health. These results suggest that flow-mediated vasodilation may be a superior means of identifying AL patients most at risk and for assessing potential benefits of therapeutic interventions.                                            The next article I'd like to highlight is titled, Miro2 Regulates Inter-Mitochondrial Communication in the Heart and Protects Against TAC-Induced Cardiac Dysfunction. The first author is Yangpo Cao, and the corresponding author is Ming Zheng. And the work was conducted at Peking University, Beijing, China, Key Laboratory of Molecular Cardiovascular Science at the Ministry of Education, also in Beijing, China.                                            Beating heart cells have very high energy requirements, and thus they need lots of fully functioning mitochondria. And as we all know from our good old high school biology days, mitochondria are the powerhouse of the cell. Mitochondrial health and performance is directly dependent on the ability of individual mitochondria to be able to communicate with each other. In many cells, this mitochondrial communication occurs via the fusion of mitochondria into a giant network. However, in cardiomyocytes, the mitochondrial movement is much more constrained. In cardiomyocytes, mitochondria communicate by briefly connecting with neighboring mitochondria, which is often called kissing, mitochondrial kissing, or by nanotunneling, which is when the mitochondria create a sustained connection by means of long nanometer-sized tubular protrusions called nanotubes. And it's thought that the proper health of the cell is dependent on proper mitochondrial communication.                                            Miro2 is a Rho GTPase on the outer mitochondrial membrane and it harbors a calcium sensing domain. Miro2 can interact with transport proteins to promote mitochondrial transport along microtubules in a calcium-dependent manner. This group wanted to investigate whether Miro2 regulates cardiac inter- mitochondrial communication. To do this, they used transverse aortic constriction or TAC or they used an Ang II infusion model to induce hypertrophy in murine hearts. Using these two models, they found Miro2 expression was decreased via Parkin-mediated ubiquitination, and they also found that inter-mitochondrial communication was disrupted.                                            By contrast, transgenic mice over-expressing Miro2 were more resistant to hypertrophy, and they were able to do this by maintaining proper cardiac function than their wild type counterparts. Together these results reveal a novel role for Miro2 in mitochondrial communication and show that maintaining such communication may mitigate effects of hypertrophy.                                            The next paper I want to highlight is titled, Circulating Monocyte Chemoattractant Protein-1 or MCP-1 and the Risk of Stroke: A Meta-Analysis of Population-Based Studies Involving 17,180 individuals. That is a huge study. The first author is Marios Georgakis, and the corresponding author is Martin Dichgans. And they are from the University of Munich in Munich, Germany.                                            A major component of atherosclerosis is chronic inflammation and inhibiting the activity of proinflammatory cytokines has been identified as a potential therapeutic strategy to help slow the disease progression. One such cytokine under study is monocyte chemoattractant protein-1 or MCP-1, and animal studies have shown that blocking MCP-1 limits, or boosting MCP-1, accelerates atherosclerosis. However, large scale observational studies of MCP-1 in humans are lacking. To address this gap in knowledge, this group performed a meta-analysis of previously unpublished data from six population cohorts, which totaled over 17,000 individuals.                                            These individuals were followed for an average of 16 years, which when you think about it, this is an absolutely huge study. So in looking at this cohort of patients, the team identified a significant association between high baseline MCP-1 levels and the likelihood of suffering a future ischemic stroke. Interestingly, this effect was not seen with hemorrhagic stroke, which is typically not associated with atherosclerosis. These findings not only support the previous animal studies, but also support a recent study in humans in which a genetic predisposition for high levels of MCP-1 was associated with an increased risk of coronary artery disease and stroke. This study also suggests that future studies should explore the potential of lowering MCP-1 levels as a possible prevention strategy. Perhaps there could be another CANTOS-like trial where we use something to block MCP-1 signaling. Maybe that would have much broader effects. I guess we'll have to wait and see what the data says.                                            The next paper I want to highlight is titled, Body Mass Index Drives Changes in DNA Methylation, a Longitudinal Study. The first authors are Dianjianyi Sun, Tao Zhong and Shaoyong Su, and the corresponding authors are Shengxu Li and Wei Chen. And they're from the Children's Minnesota Research Institute, Children's Hospitals and Clinics of Minnesota in Minneapolis, Minnesota and The Peking University Health Science Center in Beijing, China, respectively.                                            So it's well appreciated that obesity is increasing worldwide. And obesity contributes to a whole host of cardiovascular morbidity, and ultimately contributes to mortality. It's also well known that environmental factors such as the food we eat and the air we breathe, as well as genetic factors, can influence a person's risk of obesity. And recently there have been studies that suggest that perhaps epigenetic factors also contribute to obesity. And just to remind you what epigenetics is, DNA is the genetic code, and mutations can happen on DNA that can alter either gene expression or maybe protein folding or whether a protein is made at all. But epigenetic factors are not as permanent as DNA mutations.                                            Epigenetic factors are alterable modifications that can happen to DNA itself or that can happen to the proteins on which the DNA is wrapped around. And epigenome-wide association studies have shown that DNA methylation at certain loci is linked to an increase in body mass index, or BMI. However, it's unknown whether these methylations are a cause or consequence of obesity.                                            So to get to the bottom of this, this group performed a large-scale longitudinal study. They examined thousands of DNA methylation sites in 995 white individuals and 490 black individuals. And they also determined the subjects' BMIs. They did this at a baseline measurement and then approximately six years later, they collected the same data in the same patient cohort. What they found was that only a handful of methylation sites were shared between the two ethnicities. And in both groups, however, there was a similar unidirectional link between BMI and methylation. Very interestingly, baseline BMI could predict methylation at a number of genetic loci. However, the team found that none of those baseline methylation sites could predict future BMI. From this data, the authors are able to conclude that it's obesity driving the methylation at certain genetic loci as opposed to certain genetic loci driving obesity, which I think is just extremely interesting. Really nice study.                                            The last article I want to highlight for you is a paper titled, Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf. The first author is Chia Yee Tan, and the corresponding author is Jianming Jiang, and they're from the National University of Singapore.                                            Dilated cardiomyopathy or DCM is characterized by left ventricle enlargement and associated contractile dysfunction and fibrosis. Patients with DCM are at risk of arrhythmia and also of sudden death. And there's actually a huge number of genetic variants that have been linked to DCM, but the most common one or the most well-studied are mutations that affect the nuclear lamin gene or LMNA. So LMNA knockout mice are used to study the role of this gene in DCM, and these animals exhibit not only cardiac defects but also systemic defects. And those systemic defects include things like shorter lifespan, growth retardation, muscular dystrophy, neuropathy, and lipodystrophy.                                            Recently, LMNA-related dilated cardiomyopathy was linked to the deregulation of cardiac cell cycle. Meaning there was issues in how these cardiac cells are proliferating. So in this study, Tan and colleagues showed that boosting expression of a protein involved in cell cycle regulation, this protein is called Yin Yang 1, so boosting this gene's expression actually reversed the dilated cardiomyopathy symptoms in mice with heart-specific LMNA deficiency. Compared with untreated mice, mice receiving Yy1 via an adenoviral vector exhibited improved cardiac function and also reduced fibrosis after four weeks. The team then went on to show that Yy1 treatment prompted suppression of the extracellular matrix factor, Ctgf, and the upregulation of the growth factor, Bmp7.                                            Now, neither of these factors alone could rescue the symptoms of LMNA lacking mice. However, when both of these factors were manipulated together, they mimicked Yy1 treatment. These results highlight that Yin Yang 1 and its downstream targets Bmp7 and Ctgf are key players and potential therapeutic targets that can be harnessed for tackling LMNA-driven dilated cardiomyopathy.                                            Okay, so now we're going to have our interview with Drs Matthias Clauss and Sarvesh Chelvanambi. And they are from Indiana University School of Medicine in Indianapolis, Indiana. And their title of their paper is, HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients. So thank you both very much for joining me. Sarvesh C:                         Thank you so much, Cindy. Matthias C:                       Thanks for having us here. Cindy St. H:                       Could you both introduce yourselves and tell us a little bit about your background? Sarvesh C:                         My name is Sarvesh Chelvanambi. I grew up in Chennai, India. I did my undergraduate degree at Miami University in Oxford, Ohio. I got a Bachelor of Arts in Zoology with a minor in Finance. I then went to the Pennsylvania State University where I got my Masters in Biotechnology before coming over to Indiana University in 2014 to do my PhD work. And then I joined the lab of Dr Matthias Clauss, and in 2016, I got an American Heart Association predoctoral fellowship to study this project specifically. Cindy St. H:                       Wow! Congratulations. That's wonderful. Sarvesh C:                         Thank you so much. Cindy St. H:                       And now you completed the circle by publishing your AHA grant in Circulation Research. Sarvesh C:                         Exactly. Cindy St. H:                       And Matthias, how about you? Matthias C:                       I'm a Research Professor at IU School of Medicine, and my research interests focus in understanding how stressors connected with endothelium in this way contribute to vascular disease. These stressors include cigarette smoke and viral infections. Regarding viral agents, we are studying both acute infections and chronic infections and that is HIV. This HIV interest started actually 12 years ago in collaboration with Dr Samir Gupta who is also of course on this paper. We started off with a simple question, why are there so many cardiovascular events in patients, in HIV patients, with interrupted antiretroviral therapy? Cindy St. H:                       So it's not just the fact that they're HIV positive, it's that they were on therapy and then went off it? Matthias C:                       Yes. And this was part of this SMART study and this study was then actually halted because of the safety issues. Cindy St. H:                       So you're starting with the idea that patients with HIV who go off this antiviral therapy are more prone or get more cardiovascular events. So what did you start with, with this particular study? Matthias C:                       Well, our overarching idea was that the HIV virus could also do damage in the era of the combined antiretroviral therapy. And we started up with two questions, one was, is there an HIV protein which is persistent? And the other question, how is this HIV protein, if there's any one which is persistent, performing this? And this may be then leading over to your specific way to address these questions. Sarvesh C:                         That's kind of where we are starting with this project. Because back in 2016, the START trial came out saying, "We need to change the way we treat HIV patients," because initially the previous regimen of our drugs had a lot of metabolic side effects, but the current regimen of integrase inhibitors is actually really good and has very low metabolic effects. So there was a New England Journal Of Medicine paper that said, "Well, if a patient walks into the clinic, they're diagnosed as being HIV positive, put them on antiretroviral therapy right away."                                            But even in this era when everybody is on ART and there's almost no viral replication, you still see the persistence of a lot of comorbidities. And especially those associated with vascular events, whether it's peripheral arterial disease, coronary arterial disease, and a lot of other vascular diseases in the lung, or the kidney or the brain. So that kind of is what set us up, is there an element in the blood of these patients that is contributing towards vascular dysfunction? Cindy St. H:                       And so the protein that you are talking about in this paper is a protein called Nef, and is that where you come in,  Sarvash? Sarvesh C:                         Yes, because the project before I joined the lab, that's kind of where it led off, saying that Nef can get to the endothelium and it's very good at killing endothelial cells, but the mechanism through which it transfers into endothelial cells and the signaling pathways that Nef hijacks to induce this apoptosis was not clearly elucidated. A lot of work is done in Nef in monocytes and macrophages because as an HIV protein, it was studied in CD40 cells and the whole immune system as a whole, but we were the first to leverage all of those findings within an endothelial context and answer the questions, so what does Nef do and how does it get there? Cindy St. H:                       All right, so tell us a little bit what does it do and how does it get there? Sarvesh C:                         So we started doing some experiments with starting with HIV patient blood. So we took two fragments, we took the PBMC fraction, that Dr Clauss was talking about, which we knew had Nef within many of those cells. We also took the extracellular vesicle fraction, and we chose to look at this because there's a lot of literature out there saying that this fraction could not only disseminate particles throughout the body but also help signal through that. So in both of these fractions we added to the endothelial cells, we found increased apoptosis in HIV patients when compared to HIV negative patients.                                            And we were excited, but then we went and asked which of these patients had HIV Nef positivity in their blood, and then using that information when we stratified our apoptosis results, we made the surprising observation that the HIV positive, Nef positive patients were more prone to endothelial cell apoptosis. And this sparked a lot of conversation, so how do we target this and what is the signaling pathway it gets into? And that is kind of what led to most of the work in this paper, where you're showing that the transfer is mediated by extracellular, because this is such a nice tool, for HIV I guess, to spread itself into literally every cell type. Because while the HIV virus can only infect very few cell types, extracellular vesicles can be taken up by anything.                                            And the second observation we made was within endothelial cells, we found the signaling pathways that Nef was able to hijack to induce cell death. And that became the focus of this paper. Cindy St. H:                       That was one thing I wanted you to clarify, because I think what a really interesting aspect of this study is that it's the immune cells that are infected. The endothelial cells themselves are healthy and really they're getting this damage from the vesicles. That is,…wow! I don't know. It's just a really, really neat study. So can you tell us a little bit about the techniques you used in this paper? Sarvesh C:                         Yes, so we did a lot of assays to evaluate endothelial cell stress. So we started by looking at apoptosis, and a lot of those studies were done by looking at caspase-3 activity, which is a classic marker for cell death. We also did a lot of microscopy work where we took out extracellular vesicles out of those vesicles on the endothelial cells to show the uptake of Nef protein and thereby hammer that extracellular vesicles are indeed a mechanism of transfer for this protein in particular.                                            Now, one of the interesting experiments that we actually ended up doing, which was not a part of this paper really, was we wanted to see if chemotaxis was being affected by this. So we took an endothelial monolayer and separated T-cells that are expressing Nef using a Transwell membrane. And I had this huge problem where I couldn't read for a week because instead of using the 4-micron filters that allow T-cells to transfer, I was using 0.4 micron filters that T-cells cannot transfer through. But I still went about it and did my whole experiment because I didn't make that realization until a week later, because when I looked at the bottom of these chambers, there were no T-cells at all. But when I looked at the endothelial cells, I observed cytoplasmic transfer and Nef transfer, and we had a couple of conversations going, why is this happening? Did the T-cells all die or did they disappear?                                            And that's when we went back and looked in literature and found that Nef is very good at making virion particles. And those are the similar pathways that extracellular vesicle trafficking comes from. And so that was a huge shift in the way this project was designed and where we then started looking into the modes of transfer, the protein and the subsequent apoptosis that that transfer can cause. Cindy St. H:                       I love this story. So essentially your mistaken filter created this paper and this finding of the vesicles affecting the endothelial cells. Matthias C:                       Yeah, that's a typical finding for practitioner Chelvanambi, because he has this gift to turn negative things into positive things. So we have a lot of fun, and this mistake was really the beginning of a great study. Cindy St. H:                       That's wonderful. Really beautiful images, as well. So a little bit digging into, I guess, the next step. So first off, how were the endothelial cells getting damaged? They're getting damaged from these extracellular vesicles, but then what's Nef doing in the endothelial cell? What's happening there? Sarvesh C:                         So that was a very big question because if you look at it, Nef is a very small protein with almost no known enzymatic function. And yet it is able to interact with a lot of host proteins, which I guess makes it a very good viral protein. So when I went back and looked at literature, there were a host of studies in the 90s to show that Nef interacts with this kinase and that small GTPases, and there was a giant list for us to go after. And we were kind of left a bit fuddled, because we were like, which signaling pathway do we start with? Cindy St. H:                       Right. It's almost like there's too many. Sarvesh C:                         Exactly. And so what we ended up doing was we started looking into one of the various mutants of Nef that we had access to. And one of these mutants was a mutant that was incapable of PAK2 activation, and we showed that that doesn't have a lot of these stress damages. So we asked, "What is directly upstream of PAK2?" And that is where Rac1 came into the picture. And the small GTPase Rac1 is a nice candidate because it is also a master of many, many trades. Cindy St. H:                       I love this because it's such an interesting multidisciplinary approach to addressing the question, why are patients with HIV getting more cardiovascular events? What do you think evolutionarily is going on? Why would this be beneficial? Why would damaging the endothelium be beneficial? What are your thoughts on that? Sarvesh C:                         Personally, I think this is a side effect because HIV is never meant to exist in the era of ART. One of the analogies I always like to use is from Harry Potter, where HIV is Voldemort, which is the big bad villain. And what we have done is a really good job of banishing Voldemort. But what we have failed to do as a field is target its Death Eater, Nef. And I think with what we are suggesting, this paper with additional statins and other strategies that focus this, we can get to that point where we not only block HIV expansion but also the expansion of its minions, Nef. Cindy St. H:                       I love this analogy. I think you should redo your graphical abstract in a Harry Potter theme. Matthias C:                       Yeah, but I like your question. But also in this regard, I think it may be an example of a novel mechanism, how viral infections work in a different way than just infecting cells. And there's evidence from lots of viruses that they make toxic proteins, and why they are doing this, we don't know. But we noticed that the systemic effect of Nef may have some advantage for the infectious agent, because it makes T-cells more sticky, it makes them stick and transmigrate through the endothelium, and that is also shown in our paper. Cindy St. H:                       You have evidence that perhaps statins would be beneficial to give to these HIV patients on ART therapy. Can you tell us a little bit about that and how that would work? Sarvesh C:                         So based on what we did on our mouse studies that was a part of this paper, even after there is endothelial dysfunction, treatment with statins was able to restore endothelial function. Currently, there is a study going on called The Reprieve Trial where they're giving a statin called pitavastatin to HIV patients. The interesting part here is that these are HIV patients who don't have dyslipidemia. And the long-term goal is that statin treatment can help prevent the development of cardiovascular events. We're eagerly awaiting the results of this trial. Cindy St. H:                       Well done. Well thank you so much for speaking with me today. It was a pleasure to speak with you, Dr Chelvanambi and Dr Clauss. And congratulations again on this beautiful project, this beautiful story. And really, the implications for helping patients with HIV is really profound. HIV used to always be in the news and now that we have the ART therapy it's not talked about as much, but these patients are still in danger and I think your study is really doing a lot to highlight that and maybe even help them. So thank you very much and congratulations. Matthias C:                       Thank you. Sarvesh C:                         Thank you so much for the opportunity. Cindy St. H:                       So that's it for highlights from the September 27th and October 11th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams.                                            I'm your host, Dr Sidney St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.  

The Gram-negative bacterium Legionella pneumophila naturally parasitises environmental amoebae, but is also able to infect human alveolar macrophages in a mechanistically similar manner. This can result in the mild "Pontiac fever", a flu-like illness, or a potentially lethal pneumonia termed Legionnaires' disease". Crucial for establishing an intracellular replication niche is the Icm/Dot type IV secretion system (T4SS), which translocates approximately 300 different "effector" proteins into the host cell. These substrates enhance uptake efficiency into phagocytes and direct formation of a replication-permissive compartment, called the Legionella-containing vacuole (LCV), and ultimately the egress of the bacteria. Some of the effectors interfere with small GTPases, phosphoinositide metabolism or the ubiquitination machinery, and modulate host cell signalling and vesicle trafficking. We developed a method to isolate intact LCVs by using immuno-magnetic separation with an LCV-specific antibody followed by density gradient centrifugation. Proteomic analysis of the purified phagosomes together with findings of previous studies showed, that the vacuoles harbour markers of the endosomal network, associate with mitochondria, early secretory vesicles and the endoplasmic reticulum, but avoid fusion with lysosomes. Our investigations of the novel L. pneumophila effector RidL revealed that the LCV also communicates with the retrograde vesicle trafficking pathway of infected cells. This pathway recycles amongst others acid-hydrolase receptors, such as the cation-independent mannose 6-phosphate receptor (CIMPR), from the tubular endosomal network back to the trans-Golgi. This transport requires the multiprotein "retromer" complex, which consists of two major subunits: the heterotrimeric cargo-selective subcomplex comprising the proteins Vps26, Vps29 and Vps35 and the membrane-deforming heterodimeric subcomplex composed of any combination of the phosphoinositide (PI)-binding sorting nexins SNX1 or SNX2 plus SNX5 or SNX6. Pull-down experiments with lysates of RAW 264.7 macrophages or D. discoideum amoebae revealed Vps26, Vps29 and Vps35 to be retained by the then uncharacterised protein RidL, which represented an intriguing, novel effector interaction. Like most T4SS substrate mutants, L. pneumophila lacking ridL showed no phenotype for growth in liquid AYE medium and uptake into phagocytes compared to wild-type bacteria. However, intracellular replication was strongly impaired for the mutant strain in several host cell lines. RidL is preferentially expressed in the late post-exponential growth phase and translocated in an T4SS-dependent manner at early time-points of the infection, suggesting a role shortly after the uptake of the bacteria. The effector exhibited a bipolar localisation on the LCV membrane, but upon overexpression the protein covered the entire vacuole. Interestingly, RidL bound the lipid phosphatidylinositol 3-phosphate (PtdIns(3)P), a known eukaryotic endosomal membrane anchor, and also specifically bound to the retromer subunit Vps29. Although the protein had no effect on the acquisition of Vps26, Vps29 and Vps35, the percentage of LCVs positive for the retrograde cargo receptors CIMPR or sortilin was reduced in presence of RidL, suggesting interference with the retrograde transport pathway. Furthermore, significantly less SNX1- and SNX2-positive LCVs were detected in cells infected with wild-type L. pneumophila compared to the ridL mutant strain. Moreover, RidL competed with SNX1 for binding at PtdIns(3)P-positive membranes. To directly examine the influence of RidL on retrograde trafficking, the retromer-dependent transport of cholera and Shiga toxin inside cells was analysed in macrophages infected with wild-type or ridL L. pneumophila, and in HeLa cells ectopically producing RidL, respectively. In both cases, the trafficking was inhibited by RidL, and for cholera toxin the transport was arrested at the endosomal stage. In line with these findings, siRNA knockdown experiments revealed that a functional retrograde pathway restricted intracellular growth of L. pneumophila. Taken together, we postulate that RidL (Retromer interactor decorating LCVs) inhibits retrograde trafficking at endosomes by binding to the retromer subunit Vps26 and/or by competition with sorting nexins, thus promoting intracellular replication of L. pneumophila. Collectively, the results obtained in this thesis shed light on the host factor composition of LCVs and provide mechanistic insights into a novel L. pneumophila effector protein.

Tue, 15 Oct 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17526/ https://edoc.ub.uni-muenchen.de/17526/1/Leroy_Celine.pdf Leroy, Céline ddc:610, ddc:600, Medizinische Fakultät

Insights into the developmental processes during which the brain forms from the neuroepithelium may provide a deeper understanding how the brain works. The Rho family of small GTPases is known for its many cell biological functions such as regulation of the cytoskeleton, gene expression, cell migration, adhesion, cell polarity and the cell cycle. All of these functions are of importance during the formation of the cerebral neocortex, which consists of the generation of its different cell types, their migration to their destination and their maturation to a functional network. These roles have been mostly established in vitro using dominant negative or constitutively active constructs. Since these approaches are often not entirely specific for single pathways, this work used the Cre/loxP system to genetically delete an individual member of the Rho family, RhoA, to examine its role following a loss-of-function approach. Specifically, we examined a mouse line where part of the RhoA gene has been deleted by means of the Emx1::Cre mouse line. This idea is based on previous experiences with the deletion of Cdc42 in the developing neocortex, which leads to a loss of apical progenitors. RhoA often works as a functional antagonist to Cdc42. Using immunofluorescence, we could detect a loss of RhoA at embryonic day 12 (E12) in Emx1::Cre-positive offspring carrying the floxed RhoA-construct in both alleles (cKO). At E14, we detected an increase in mitotic cells to 160% (±25%, p

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

Vesicle traffic in eukaryotic cells is a tightly organized process involving a multitude of regulatory proteins. Key regulators of this traffic are small GTPases called Rabs. With about 60 members in the human genome, they constitute the largest subgroup in the superfamily of Ras like monomeric GTPases. They recruit effector proteins to specific membranes and thus define the identity of organelles. Rabs switch between an active, GTP bound state and an inactive GDP bound state. Key regulators of this conversion are RabGAPs, which accelerate the hydrolysis of bound GTP. All RabGAPs are characterized by the presence of a TBC domain. In the human genome 40 RabGAPs were identified, most of which had not been studied so far. To assign them to their specific Rab proteins, a novel reverse yeast two-hybrid screening method was developed. This identified a GAP for Rab5 termed RabGAP-5. RabGAP-5 stimulated the GTPase activity of Rab5. Its expression inactivated Rab5 and redistributed the Rab5 effector EEA1 from early endosomes to the cytoplasm. RabGAP-5 also blocked the Rab5 dependent uptake of EGF and transferrin from the plasma membrane. When RabGAP-5 was depleted, the size of endosomes was increased, indicating elevated Rab5-GTP levels. Endocytosed EGF was unable to exit the endosome, indicating that trafficking through endosomes was also blocked. To identify GAPs and Rabs implicated in the regulation of early secretory events simultaneously, a second novel screening method was established. It involved the analysis of phenotypes caused by the inactivation of endogenous target Rabs via the overexpression of RabGAPs. Changes in Golgi morphology, ERGIC organisation and the proceeding of secretion were only observed with one candidate RabGAP, the highly conserved protein TBC1D20. TBC1D20 showed activity towards Rab1 and Rab2 in vitro, and acted primarily on Rab1 in vivo. In contrast to all other RabGAPs it has a transmembrane domain, which localises it to the ER. TBC1D20 interacts with RTN-1 on ER membranes. This interaction modulates the activity of TBC1D20. These data indicate a novel function for Rab1 in regulating ER exit, and thus extend the classical view of RabGAPs as regulators of active Rab lifetime.

Fri, 22 Feb 2008 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/8283/ https://edoc.ub.uni-muenchen.de/8283/1/Evelyn_Fuchs.pdf Fuchs, Evelyn ddc:500, ddc:570, Fakultät f

Rac1 is a ubiquitously expressed member of the Rho family of small GTPases, which acts as a molecular switch by shuttling in a highly regulated manner between an active (GTP-bound) and an inactive (GDP-bound) state. Different signalling pathways, which involve integrins, growth factor receptors, cadherins as well as other Rho GTPases, can induce Rac1 activation. Only in the GTP bound form, Rac1 can associate with different effector molecules to initiate cellular responses. Initially described as an important regulator of the actin cytoskeleton, Rac1 was later found to be also involved in the modulation of other processes such as cell adhesion, proliferation, survival, differentiation and migration. In epithelial cells, Rac1 was shown to regulate the formation and maintenance of cadherin dependent cell cell contacts, which are essential for the establishment of the polarized cell morphology. Before this project was initiated, almost all knowledge about the function of Rac1 was based on in vitro studies. As constitutive deletion of the murine rac1 gene leads to early embryonic lethality, mice allowing for a conditional inactivation of the rac1 gene were generated in this study to enable the analysis of the function of Rac1 in selected tissues. To investigate the role of Rac1 in the epidermis and hair follicles and to determine its function in the establishment and maintenance of cell cell contacts between epithelial cells in vivo, mice with a keratinocyte-restricted ablation of the rac1 gene were generated and analyzed. The results obtained in this study showed that the absence of Rac1 in the murine epidermis leads to a progressive hair loss but surprisingly has no effect on the maintenance of the epidermis. The hair loss is caused by the inability of hair follicle keratinocytes to maintain their differentiation state, which leads to the phagocytic removal of the non permanent parts of the hair follicles by infiltrating macrophages. In contrast, differentiation and proliferation of epidermal keratinocytes as well as the formation and maintenance of cell-cell and cell-matrix contacts, and the deposition of the basement membrane in the epidermis are not affected by the loss of Rac1. Biochemical analysis of epidermal lysates demonstrated that the absence of epidermal defects in vivo is not a result of compensatory upregulation of closely related members of the Rho family of GTPases, further indicating that the function of Rac1 in epithelial cells in vivo is limited. Also, the analysis of the formation of the embryoid bodies from Rac1-deficient embryonic stem cells showed that the presence of Rac1 is not required for the establishment of cell cell contacts during differentiation of the polarized primitive ectoderm and for the formation of epithelial sheets, supporting the conclusion that the function of Rac1 in the regulation of cell cell adhesion between epithelial cells is dispensable. However, the re epithelialization after wounding was impaired in the mutant epidermis, demonstrating that Rac1 plays an important role in pathological conditions. The delayed wound closure in the absence of Rac1 is caused by impaired cell migration and proliferation of neo epidermal keratinocytes. Another interesting finding of this study was the observation that, in contrast to the steady state in vivo situation, isolated Rac1 deficent primary keratinocytes display severe defects in cell culture, which lead to their detachment from the matrix. While the initial adhesion is only mildly affected by the lack of Rac1, mutant keratinocytes are unable to spread, show an impaired organization of the actin cytoskeleton and fail to form mature focal adhesions. The differences between in vivo and in vitro effects resulting from the inactivation of the rac1 gene indicate that the function of Rac1 in epithelial cells depends on the complexity of the cellular system and emphasize the importance of performing in vivo studies to fully understand its role. Taken together, the data presented in this study show that Rac1 plays an important role in the maintenance of hair follicles and during epidermal wound healing, but that it is not essential for the homeostasis of the epidermis in physiological conditions and for the formation and maintenance of cell cell contacts between epithelial cells in vivo.

Hepatocyte Growth Factor (HGF) is a pleiotropic factor acting on cells expressing the Met receptor tyrosine kinase. HGF/Met signaling has been described in detail and it is known to control cell migration, growth and differentiation in several embryonic organs and to be implicated in human cancer. Conversely, little is known about the transcriptional targets that lead to Met-mediated biological functions. Also, little is known about the physiological mechanisms that attenuate Met signaling. This work provides the results of a screen for genes transcriptionally regulated by Met in several cell lines and addresses the functions of the highly inducible gene Mig6 (Mitogen-inducible-gene6, also called Gene 33 and RALT). By the use of Met loss of function mutant mice Met is shown to be the major inducer of mig6 in hepatocytes and lungs of E13.5 embryos. Mig6 is shown in turn to negatively regulate HGF/Met-induced cell migration. The effect is observed by Mig6 overexpression and reversed by Mig6 siRNA knock down experiments indicating that endogenous Mig6 is part of a mechanism that inhibits Met signaling. Mig6 functions in cells of hepatic origin and in neurons suggesting a role for Mig6 in different cell lineages. Mechanistically, Mig6 requires an intact Cdc42/Rho interactive binding (CRIB) domain to exert its inhibitory action suggesting that Mig6 acts at least in part distally from Met possibly by sequestering Rho-like GTPases. Because Mig6 is also induced by HGF stimulation, this work provides evidence that Mig6 is part of a negative feedback loop that attenuates Met functions in different contexts and cell types.

The reaction product of phospholipase D (PLD), phosphatidic acid (PA), was found to stimulate phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity in vitro. In the present study, we have examined wether PLD affects the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led to an increase in PIP-5-kinase activity and to elevated PIP2 levels in intact cells. As both PLD and PIP-5-kinase are stimulated by the GTPases Arf1 and RhoA, we investigated in the following, if PLD is involved in the regulation of PIP2 synthesis by these GTPases. Both PLD1- and PLD2-induced PIP2 synthesis was completely blocked by coexpression of catalytically inactive Arf1 T31N. Reversely, the effect of constitutive active Arf1 Q71L was fully inhibited by catalytically inactive PLD constructs. Whereas the effects of Arf1 Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L with wild-type PLD1 led to a synergistic increase in PIP-5-kinase activity. Previously, we have shown that RhoA regulates the activity of PLD and PIP-5-kinase by its downstream effector Rho-kinase. Expression of small amounts of inactive PLD1, but not of PLD2, nearly completely abolished Rho-kinase-stimulated PIP-5-kinase activity. Also expression of a non-phosphorylatable mutant of cofilin, which participates in the signalling cascade from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the stimulating effect of Rho-kinase on PIP2 synthesis. These findings suggest that PLD1 is involved in the stimulation of PIP-5-kinase by Arf1 as well as by RhoA and Rho-kinase. After sucrose density gradient centrifugation of HEK-293 cell lysates, we isolated two separate PIP2 pools. PLD1 and Arf1 selectively control the non-caveolar PIP2 pool in the high density fraction, whereas PLD2 affected PIP2 in both pools. In summary, these data suggest that particularly PLD1, apparently by the production of PA, functions as a physiological regulator of PIP-5-kinase that controls the synthesis of cellular PIP2 downstream to Arf1 and RhoA.

The endothelium is among the largest organs in the body. Stimuli originating from the blood or from neighbouring cells, like inflammatory cytokines (IC), lead to structural and functional alterations of vascular endothelial cells (EC). These alterations are often referred to as “EC activation”. Activated EC play a key role in different physiological processes like during immune response, in menstruation and in pathological processes like inflammation, allergy, viral infections, atherosclerosis and tumour angiogenesis. The human guanylate binding protein-1 (GBP-1) is a protein of the family of large GTPases. GBP-1 is characterized by a high turnover GTPase activity. Previous work showed that GBP-1 mRNA expression is induced by IC in EC and that GBP-1 is the specific mediator of the anti-proliferative effect of IC on EC in vitro. The main goals of this work were first, to investigate whether GBP-1 may be a molecular marker of IC-activated EC at the protein level in vitro. Second, to investigate GBP-1 expression in human healthy and/or disease tissues and to determine whether GBP-1 may be a molecular marker of IC-activated EC in vivo. To this goal mono- and poly-clonal antibodies against GBP-1 were generated. In vitro studies showed that GBP-1 expression in EC is induced by IFN-, IFN-, IL-1, IL-1 or TNF- but not by other cytokines, chemokines or growth factors. Moreover, simultaneous addition of bFGF and VEGF and IC reduced the IC-induced GBP-1 expression. This indicated that GBP-1 characterizes cells that are preferentially exposed to IC. In vivo studies using immunohistochemistry and immunofluorescence showed that GBP-1 expression is highly associated with vascular EC in a broad range of human tissues. This was confirmed by the simultaneous detection of GBP-1 and the EC-associated marker CD31. Notably, GBP-1 expression was undetectable in healthy skin. In contrast, GBP-1 was highly expressed in vessels of skin diseases with a high inflammatory component including psoriasis, adverse drug reactions and Kaposi’s sarcoma. This indicated that GBP-1 characterizes IC-activated EC in vivo. Further immunohistochemical studies on Kaposi’s sarcoma demonstrated that GBP-1 expression and EC cell proliferation are inversely related. This indicated that GBP-1 may also mediate the anti-proliferative effect of IC on EC in vivo. Finally, GBP-1 was found to be secreted by EC stimulated with IFN- and IFN- in vitro. This finding was confirmed by immunoprecipitation of GBP-1 from cell culture supernatants and by a novel ELISA developed for the detection of GBP-1 in solution. Further characterization of the mechanism of secretion demonstrated that GBP-1 release is due to an 3 Summary energy-dependent mechanism and is not due to cell death. Most importantly, circulating GBP-1 could be detected in increased concentrations in the blood of patients that were subjected to IFN–-therapy or in patients with inflammatory diseases. These findings indicated that GBP-1 is a novel marker of inflammatory vessel activation. Specifically, the serological detection of GBP-1 may open new perspectives for the early detection of inflammatory activation of EC in patients with inflammatory diseases.

Profilin is an ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cellular physiology. Site-directed mutagenesis of profilin II from Dictyostelium discoideum by PCR resulted in the point mutations W3N and K114E, whereby the W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin-binding, whereas the K114E profilin shows profound decrease in its ability to interact with actin but its affinity for poly-(L)-proline remained unaltered. The in vivo properties of the point-mutated profilins were studied by expressing either W3N or K114E in the profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., 1994). Expression resulted in normal cell physiology, a reduction in the F-actin content, and a complete development. Interestingly, only cells which overexpressed W3N could restore the aberrant phenotype, while the K114E profilin with its fully functional poly-(L)-proline binding and its strongly reduced actinbinding activities rescued the phenotype at low concentrations. Both the wild-type and pointmutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities. To have a better understanding of the in vivo role of profilin, D. discoideum profilin II has been tagged at its C-terminus with the green fluorescent protein (GFP) with a 100-aa linker separating profilin and GFP. This fusion construct was introduced in D. discoideum profilinminus cells and expression of the fusion protein could restore the aberrant phenotype partially. The partial rescue might be due to the uneven expression of the fusion protein leading to mixed populations even after repeated recloning. The profilin-GFP transformants showed normal cell morphology, could be cultivated in shaking suspensions, and could develop fruiting bodies which closely resembled those of the wild-type. In vivo studies revealed the distribution of the fusion protein in highly active regions of the cells such as phagocytic cups, macropinocytotic crowns, cell cortex and at the leading edges of locomoting cells. Thus profilin appears to play a significant role in the regulation of the dynamic actinbased cellular processes. A second actin-regulatory protein from D. discoideum namely, severin, a Ca2+-dependent Factin fragmenting and capping protein, was also investigated via fusion to GFP at its C-terminus. Although severin is a very active F-actin fragmenting protein in in vitro assays, the severin null D. discoideum mutant exhibits normal growth, cell motility, chemotaxis and development. Examination of the live dynamics of severin-GFP should clarify the in vivo role of severin and other functionally redundant cytoskeletal proteins. The 70 kDa severin-GFP fusion protein has been sufficiently expressed and partially purified from the severin null cells whereby in vitro assays confirmed the ability of this fusion protein to sever F-actin only in the presence of Ca2+. Data from confocal microscopy showed that the fusion protein was transiently detected in macropinocytotic crowns, phagocytic cups, membrane ruffles, at the leading edges of motile cells and cell-cell contacts of aggregating cells in directed motion. These data suggest an in vivo role for severin in the remodulation of existing F-actin structures as supported by the in vitro data. The highly dynamic cytoskeleton also plays a significant part in the defence of the cells against pathogens. The behaviour of the actin cytoskeleton of cultured mammalian cells in response to Yersinia enterocolitica infection was examined by confocal microscopy with the aid of GFP-tagged actin, cofilin and profilin II. The translocated Yersinia outer proteins (Yops) encoded by a virulence plasmid in the wild-type bacteria have been observed to disrupt the actin microfilaments, resulting in diffuse actin staining which subsequently disappeared completely upon prolonged bacterial infection. In addition, F-actin structures resembling phagocytic cups were found at the sites of bacterial adherence, suggesting the likelihood of the involvement of the Rho family of small GTPases in the regulation of the actin cytoskeleton. The secreted Yops appeared to have no major effect on the distribution of GFP-profilin whereas the staining pattern of GFP-cofilin seemed to be modified by the Yops, resulting in a decrease in length of the actin-cofilin rods and a diffuse localization of cofilin. The exact mechanisms of interaction between the Yops and their host targets remain to be determined. However, a clearer insight into the interaction between pathogens and the host cytoskeleton will certainly aid in the cellular defence and the prevention of pathogenesis.

Fri, 1 Jan 1993 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3661/ http://epub.ub.uni-muenchen.de/3661/1/3661.pdf Hofmann, Franz; Biel, Martin; Bosse, Eva; Hullin, Roger; Ruth, Peter; Welling, A.; Flockerzi, Veit Hofmann, Franz; Biel, Martin; Bosse, Eva; Hullin, Roger; Ruth, Peter; Welling, A. und Flockerzi, Veit (1993): High voltage activated Ca2+ channel. In: Dickey, Burton F. und Birnbaumer, Lutz (Hrsg.), GTPases in Biology II. Bd. 108, Handbook of Experimental Pharmacology. Springer: Berlin u.a., pp. 225-238. Chemie und Pharmazie

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.04.21.048405v1?rss=1 Authors: Bubier, J. A., Philip, V. M., Dickson, P. E., Mittleman, G., Chesler, E. J. Abstract: Substance use disorders are prevalent and present a tremendous societal cost but the mechanisms underlying addiction behavior are poorly understood and few biological treatments exist. One strategy to identify novel molecular mechanisms of addiction is through functional genomic experimentation. However, results from individual experiments are often noisy. To address this problem, the convergent analysis of multiple genomic experiments can prioritize signal from these studies. In the present study, we examine genetic loci identified in the recombinant inbred (BXD RI) genetic reference population that modulate the locomotor response to cocaine. We then applied the GeneWeaver software system for heterogeneous functional genomic analysis to integrate and aggregate multiple studies of addiction genomics, resulting in the identification of Rab3b, as a functional correlate of the locomotor response to cocaine in rodents. This gene encodes a member of the RAB family of Ras-like GTPases known to be involved in trafficking of secretory and endocytic vesicles in eukaryotic cells. The convergent evidence for a role of Rab3b was included co-occurrence in previously published genetic mapping studies of cocaine related behaviors; methamphetamine response and Cartpt (Cocaine- and amphetamine-regulated transcript prepropeptide) abundance; evidence related to other addictive substances; density of polymorphisms; and its expression pattern in reward pathways. To evaluate this finding, we examined the effect of RAB3 complex perturbation in cocaine response. B6;129-Rab3btm1Sud Rab3ctm1sud Rab3dtm1sud triple null mice (Rab3bcd-/-) exhibited significant deficits in habituation, and increased acute and repeated cocaine responses. This previously unidentified mechanism of the behavioral predisposition and response to cocaine is an example of many that can be identified and validated using aggregate genomic studies. Copy rights belong to original authors. Visit the link for more info