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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.06.26.546598v1?rss=1 Authors: Shangguan, J., Rock, R. S. Abstract: Myosin 10 (Myo10) is a vertebrate-specific motor protein well known for its role in filopodia formation. Although Myo10-driven filopodial dynamics have been characterized, there is no information about the numbers of Myo10 in filopodia. To better understand molecular stoichiometries and packing restraints in filopodia, we measured Myo10 abundance in these structures. Here we combined SDS-PAGE analysis with epifluorescence microscopy to quantitate HaloTag-labeled Myo10 in U2OS cells. About 6% of total intracellular Myo10 localizes to filopodia, where it tends to be enriched at opposite ends of the cell. Hundreds of Myo10 are found in a typical filopodium, and their distribution across filopodia is log-normal. Some filopodial tips even contain more Myo10 than accessible binding sites on the actin filament bundle. Our estimates of Myo10 molecules in filopodia provide insight into the physics of packing Myo10, its cargo, and other filopodia-associated proteins in narrow membrane deformations in addition to the numbers of Myo10 required for filopodia initiation. Our protocol provides a framework for future work analyzing Myo10 abundance and distribution upon perturbation. 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/2022.12.04.519021v1?rss=1 Authors: Gil, B., Rose, J., Demurtas, D., Mancini, G.-F., Sordet-Dessimoz, J., Sorrentino, V., Rudinskiy, N., Frosh, M. P., Hyman, B. T., Moniatte, M., Spires-Jones, T. L., Herron, C. E., Schmid, A. W. Abstract: In Alzheimer's disease (AD), Amyloid-beta (A{beta}) oligomers are considered an appealing therapeutic- and diagnostic target. However, to date, the molecular mechanisms associated with the pathological accumulation or structure of A{beta} oligomers remains an enigma to the scientific community. Here we demonstrate the strong seeding properties of unique A{beta} fragment signatures and show that the truncated A{beta}peptides of residues A{beta}1-23, A{beta}1-24 and A{beta}1-25, rapidly seed to form small, SDS-PAGE stable assemblies of ~5kDa to ~14kDa molecular mass range. Mass spectrometry analysis of SDS-PAGE fractionated and gel extracted oligomers revealed that the truncated A{beta} isoforms of residues 1-23 to 1-25 form stable entities with low molecular weight (LMW) oligomers, which strongly resemble the regularly reported A{beta} entities of putative dimeric or trimeric assemblies found in human post-mortem AD and Tg mouse brain extracts. Furthermore, electrophysiological recordings in the mouse hippocampus indicate that LMW A{beta} assemblies formed by fragments A{beta}1-23 to A{beta}1-25 significantly impair long-term-potentiation (LTP) in the absence of full-length A{beta}1-42. Extensive antibody screening highlights the important observation, that the LMW A{beta} assemblies formed by these truncated A{beta} peptides escape immuno-detection using conventional, conformation specific antibodies but, more importantly, the clinical antibody aducanumab. Our novel findings suggest that there are new A{beta} target loopholes which can be exploited for the development of therapeutic antibodies with binding properties against stable target hotspots present in A{beta} oligomers. We provide here a first example of a new class of monoclonal antibody with unique binding properties against LMW A{beta} oligomers, in the absence of binding to large fibrillar A{beta} assemblies, or dense amyloid plaques. Our research supports a novel, unparalleled approach for targeting early, pathological A{beta} species during the insidious phase of AD and prior to the appearance of large oligomeric or protofibrilar assemblies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

#28 — SDS-PAGE gels can be run at constant current, constant voltage, or constant power. But which is best? Listen to this episode of Mentors at Your Benchside to discover the differences between current, voltage, and power, and how they affect how your gels run. Visit the original article for a summary table of running your SDS-PAGE gels at a constant voltage, current, or power. [1] We've also got tips and tricks for casting the perfect SDS-PAGE gel, [2] or read a refresher on how SDS-PAGE works. [3] Resources: 1. Constant Current or Voltage in SDS-PAGE: The Great Debate. Available at: https://bitesizebio.com/51744/constant-current-or-voltage-in-sds-page/. 2. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/. 3. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/.

#20 — Have you ever wondered how Laemmli buffer actually works? In this episode of Mentors At Your Benchside, we talk through the different components of Laemmli buffer, what they do and why they are essential for your SDS-PAGE experiments. Read the full article to learn more about this buffer and get a handy Laemmli buffer recipe. [1] Looking for more information on SDS-PAGE? Discover the theory behind SDS-PAGE and get advice on how you can cast the perfect SDS-PAGE gel. [2,3] You can also download our useful SDS-PAGE protocol PDF that contains simple buffer recipes, gel recipes, and a neat casting protocol. [4] Resources: 1. Laemmli Buffer: What Is It for Anyway? Available at: https://bitesizebio.com/44540/laemmli-buffer-what-is-it-for-anyway/ 2. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 3. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/ 4. Bitesize Bio's SDS-PAGE Protocol PDF Cheat Sheet. Available at: https://bitesizebio.com/sds-page-protocol-pdf/

#18 — Why are SDS-PAGE gels run vertically? What would happen if we ran them horizontally? Has anyone ever tried? Discover the answers to these questions and more in this episode of Mentors At Your Benchside. To see what others are saying and join in the conversation, head over to the comments section in the original article on why SDS-PAGE is run vertically. [1] If this episode has ignited your desire for answers, Check out some of our related articles to learn the how SDS-PAGE works and discover whether running your SDS-PAGE gels with constant current or constant voltage is best. [2,3] Resources: 1. Why Is SDS-PAGE Run Vertically? Here are 3 Great Answers. Available at: https://bitesizebio.com/10699/why-is-sds-page-run-vertically/ 2. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 3. Constant Current or Voltage in SDS-PAGE: The Great Debate. Available at: https://bitesizebio.com/51744/constant-current-or-voltage-in-sds-page/

#8 — You probably have or will use SDS-PAGE at some point to separate proteins, but do you really understand how this technique works? Knowing how SDS-PAGE works means you can tweak and troubleshoot your technique as well as impress your supervisor and lab mates. In this episode, we take you through how SDS-PAGE works, including what SDS does, why you need to use a reducing agent like DTT or beta-mercaptoethanol, and the critical importance of the stacking gel. Read the full How SDS-PAGE Works article to see helpful visuals for how this technique works and access the table showing the protein sizes that different acrylamide percentages can separate. [1] Expand your knowledge by buffing up on Laemmli buffer and get our Guide to Gradient Gels. [2,3] If you pour your own SDS-PAGE gels, take a deep dive into the chemistry of how gels work and learn how to pour perfect gels every time with our Simple SDS-PAGE Gel Recipe with 10-Step Casting Protocol. [4] Resources: 1. How SDS-PAGE Works. Available at: https://bitesizebio.com/580/how-sds-page-works/ 2. Laemmli Buffer: What Is It for Anyway? Available at: https://bitesizebio.com/44540/laemmli-buffer-what-is-it-for-anyway/ 3. A Guide to Gradient Gels: The Why's and How's. Available at: https://bitesizebio.com/47184/gradient-gels/ 4. A Simple SDS-PAGE Gel Recipe and 10-Step Casting Protocol for Perfect Gels. Available at: https://bitesizebio.com/59429/sds-page-gel-recipe/

Aydogdu talks us through how the accurate and quantitative analysis of product purity is a fundamental component of effective development of a biotherapeutic protein. But without consistent techniques and high-quality, reproducible results, it is difficult to precisely assess the product's efficacy and/or safety, potentially delaying, even jeopardising, its approval. He also reveals how Bio-Techne overcame this issue for a particular customer.

This clip is from the Separation and Purification Techniques podcast. Enjoy!   Questions, comments, concerns? Email me: MCATPodcast@medschoolcoach.com

This month on Episode 17 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the September 25 and October 9 issues of Circulation Research. This episode features an in-depth conversation with Drs David Dichek, Sina Gharib and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. Article highlights: Cai, et al. Single Cell RNA-Seq in Arteriosclerosis Schuhmann, et al. CD84 in Ischemic Stroke VanOudenhove, et al. Gene Regulatory Dynamics of Developing Human Heart Nie, et al. Periostin is a Target to Treat PH Dr Cindy St. Hilaire: Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh, and today I'll be highlighting four articles selected from the late September and early October issues of CircRes. I'll also be interviewing Drs David Dichek, Sina Gharib, and Tomáš Vaisar regarding their study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. The first article I want to share is titled Single Cell RNA Sequencing of Allograft Cells in Transplant Arteriosclerosis. The first author is Jingjing Cai and the corresponding author is Qingbo Xu from Zhejiang university in Hangzhou, China. Arteriosclerosis is a major contributor to organ transplant failure. The thickening and stiffening of arteries within the grafts results in diminished blood flow supplies and diminished organ function. While it is well understood that atherosclerosis is an inflammatory disease, the details of the cellular and molecular players on this transplant-specific pathology are lacking. Now, Cai and colleagues used single cell RNA sequencing to identify a consensus of cells and cytokines in sclerotic transplanted aortas in mice. Two weeks after transplant, the grafted vessels exhibited signs of arteriosclerosis and by four weeks, this remodeling had worsened significantly. Analyzing the RNA transcripts of over 12,000 individual cells isolated at both two and four weeks, the team discovered that the number of T-cells was greatly increased throughout the process. An early abundance of macrophages gave way to a later wave of B cells and there was also evidence of the development of tertiary lymphoid tissue. They further found that chemokine CCL121 was up regulated after transplant, both the mRNA in the tissues, as well as the protein levels in the animal's blood. The authors then went on to show that blocking CCL121 or its attracting partner, CXC3, significantly delayed arteriosclerosis in the grafted vessel. Hence, this work not only defines the cellular and molecular drivers of arteriosclerosis in grafted vessels, but highlights potential molecular targets for future therapeutic interventions. The second article I want to share is titled CD84 Links T-cell and Platelet Activity in Cerebral Thrombo-inflammation in Acute Stroke. The first author is Michael Schuhmann and the corresponding author is David Stegner, and the work was completed at University of Würzburg in Germany. Ischemic stroke is caused by the occlusion of cerebral blood vessels and it is a leading cause of death and disability worldwide. Despite treatments to degrade or remove clots such as mechanical thrombectomy, infarct size itself can continue to grow even when blood perfusion is re-established. Thrombo-inflammatory processes are thought to mediate this worsening injury, with both T-cells and activated platelets playing a role. Because both T-cells and activated platelets express CD84, which is a self-binding adhesion molecule involved in lymphocyte activation, this team tested the hypothesis that CD84 might mediate stroke inflammatory processes. They went on to show that mice lacking CD84 have smaller infarct sizes with reduced T-cell inflammation after stroke than wild-type animals. Furthermore, mice that specifically lack CD84 in either T-cells or platelets also experienced smaller infarcts. The team went on to show that CD84 promotes T cell migration in vitro. And then in patients with stroke, high expression of CD84 in platelets was associated with poor outcomes. Together, these results suggest that activated CD84-secreting platelets encourage inflammatory T cell migration to the infarct site. And that blocking CD84 activity could be a novel therapeutic strategy for minimizing inflammatory injury after stroke. The third article I want to share is titled Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis. The first author is Jennifer VanOudenhove and the corresponding author is Justin Cotney. And they're from the University of Connecticut. Congenital heart defects, or CHDs, are common birth abnormalities and while some genes have been linked to congenital heart defects, the majority, close to 60%, have unknown etiologies. It's thought that multiple genetic and environmental factors contribute to congenital heart defects. One of which could be variations in both cis and trans regulatory regions of the genome. To find such heart specific regulatory regions, this team examined heart tissue from human embryos obtained four to eight weeks after conception. They performed chromatin immunoprecipitation experiments to scour the heart genomes for histone modifications associated with increased or decreased gene transcription. They also performed transcriptome analysis to see whether the genomic regions identified by chip corresponded with the activity status of nearby genes. In total, the team found more than 12,000 previously unknown enhancers that were enriched for binding sites for heart specific transcription factors, some of which included GATA, MEF2 and Nkx. These binding sites tended to be close to genes activated in the heart. Many of the regions also contain sequence variations that have been associated with atrial fibrillation. These newly identified sites are potential congenital heart defect candidate loci and the authors have now made their data readily available so that other investigators may study it. The last article I want to share with you before we switch to our interview is titled Periostin: a Potential Therapeutic Target for Pulmonary Hypertension? The first author is Xiaowei Nie from the Shenzhen Third People's Hospital and the corresponding authors are Jingyu Chen and Jin-Song Bian from the Wuxi People's Hospital and the National University of Singapore, respectively. Pulmonary hypertension, or PH for short, is a life-threatening disease where an excess in the proliferation of vascular smooth muscle cells and the deposition of extracellular matrix thickens the walls of the lung vasculature, which leads to an increase in pulmonary blood pressure and ultimately contributes to right heart failure. Vasodilatory medications can be used to treat the symptoms of the disease. However, these medications do not prevent or reverse the underlying pathogenic remodeling. This study now suggests that drugs targeting the secreted extracellular matrix protein, periostin, might be a potential therapeutic strategy for the treatment of pulmonary hypertension. Periostin is an abundant protein in the lung arteries of pulmonary hypertension patients. And it is thought to be involved in cell adhesion and wound healing mechanisms, such as the proliferation and the migration of smooth muscle cells. The team confirmed increased production of periostin in patient lungs, and also found the same to be true for mice with an induced model of pulmonary hypertension. They also showed that genetic deletion of periostin attenuated pulmonary hypertension in mice, while suppression of periostin via RNA inhibition could even reverse pathological vessel thickening and the subsequent right ventricle hypertrophy. The team went on to identify factors HIF-1a and TrkB as factors that mediate periostin's effects in cultured arterial cells. And they suggest that blocking either of these factors or by blocking periostin itself could be a novel strategy for the treatment of pulmonary hypertension patients. Drs Tomáš Vaisar, Sina Gharib, and David Dichek from the University of Washington in Seattle, Washington are here with me today and we're going to discuss their recent study titled Parallel Murine and Human Plaque Proteomics Reveals Pathways of Plaque Rupture. Thank you all so much for joining me today and congratulations on this beautiful and interesting study. So this is an atherosclerotic study, but unlike many in the field, it's really looking at the end stage event called plaque rupture. So for those listeners who are unfamiliar with the term, plaque rupture is when an atherosclerotic plaque degrades and its contents are exposed to the circulation, which can then induce a clotting event and lead to all sorts of adverse pathologies, myocardial infarction, transient ischemic events, stroke. So I'm wondering if we if maybe you can give us a little bit of background about what's known and what really is unknown at least before your study regarding plaque rupture. Dr David Dichek: So the pathogenesis of acute myocardial infarction and stroke was really unknown for many years. And the idea that it was due to acute thrombosis was really confirmed by a study probably 30 years ago, that did angioscopy in the coronary arteries, proximal to a myocardial infarction, and visualized actual clot so that the clot was confirmed to be associated with the acute event. At that point, the question became why would a coronary artery form a clot? And that led to identification of, or histologic studies that identified ruptured caps of atherosclerotic plaques, exposure of the blood to the thrombogenic contents of the lesion, and a thrombus. However, it was not known what the initiating event was in rupture of the plaque cap. And there were a lot of hypotheses and a lot of nice work, but it does still remain an unknown. A significant amount of focus has been devoted to the possibility that proteolysis is the initiating event. And that was sort of the takeoff point from our study because we had developed a mouse model where proteolysis clearly was associated with rupture of plaque caps. And we decided we wanted to get more into the biochemistry of what was going on and go beyond the histology. So that was really what led up to our study. Dr Cindy St. Hilaire: It's really interesting. So, mice are really good and obviously really useful, very well-known model systems to study atherosclerosis and particularly the initial drivers and maybe the mechanisms of the disease pathogenesis, but like many models systems, they're not perfect. So I'm wondering if you could discuss the limitations of murine model systems and specifically for this study, how you were able to overcome some of those limitations. Dr David Dichek: So the limitations of mouse models of plaque rupture are that essentially none of them duplicate the histology of human plaque rupture, particularly the thrombus that occurs on top of the plaque rupture. So there are various mouse models where caps are disrupted, but there's not acute thrombosis. It has been argued in the vascular community as to whether these models are authentic models of plaque rupture, because they don't have the superimposed thrombosis. And the counter argument is well, mice aren't people, they have different hemostatic and coagulation factors that may be differentially regulated. The hemodynamics of small mouse arteries is different from human mouse arteries. And the fact that you don't get a thrombus doesn't necessarily mean that you're not modeling the process that would cause it. So we really accepted that argument as being valid and felt that the occurrence of frank plaque rupture, and that was in our Circulation paper in 2010, in these lesions in the mice, really validated it as an authentic model of cap disruption. And so I agree it's arguable, whether this is an authentic model. But we actually took that issue head on by saying, well, is the biochemistry of the ruptured plaque similar to the biochemistry of a ruptured human plaque? And that if there were similarities that we would gain more confidence in our model being an authentic model of plaque rupture and that it matched not only the histology, but also the biochemistry. Dr Cindy St. Hilaire: One of the main tools you used, you used shotgun proteomics, which I think is just a great name for it. And also a algorithmic learning tool or analysis tool called proteomaps. I was wondering if you could give us a little bit of background about the proteomic aanalysis involved, what does that entail? Especially, when you're comparing a teeny tiny mouse plaque to a larger human plaque and then how that analysis was done? Dr Tomáš Vaisar: So the shotgun proteomics term was coined by John Yates, but way back in the early 1990s. And it's essentially the way how you enumerate the proteins and in more recent forms, you even quantify the abundance of the proteins in a very complex mixture. So shotgun proteomics essentially takes a protein sample. And in this case it was an extract of the tissue and uses protease, trypsin typically, to cut the proteins down to peptides, which are relatively small and relatively well behaved compared to intact proteins. And then using tandem mass spectrometry combined with liquid chromatography separation, basically aims to sequence every single of those peptides or majority of the peptides. And then based from the identification of the sequence of individual peptides piece back together like a jigsaw puzzle, what was the protein present in the original mixture? Dr Cindy St. Hilaire: That's so interesting. I know it's been around for a while. I'm always impressed by it. Dr Tomáš Vaisar: And then the other approach we use it's called Proteomap developed by Ben Cravatt, collaborator on this paper at La Jolla. And that approach uses basically the shotgun approach but as a first step uses gel electrophoresis, SDS PAGE electrophoresis to fractionate the very complex mixture to size segments. Dr Tomáš Vaisar: So you run your complex mixture on a gel, you slice it by size and then run shotgun proteomics experiment on each of those slices after gel digestion. And then Ben developed a set of tools where you identify the proteins and their abundance in each of the bands based on the size and the way it's applied to formation or mapping of proteolytic events is based on the idea of that intact protein will show up at the molecular weight of the intact protein but if it was cleaved by a protease, it will also show up at the molecular weight, which is smaller corresponding to the fragments formed by proteolysis. Then you use set of bioinformatics tools to piece this all together and generate the Proteomaps. Dr Cindy St. Hilaire: You pick it apart, throw it in the machine and then put it back together. That's so cool. It's so amazing. So what were the main findings that you were able to pull out of your comparisons? And I think you had three main groups, if I understood it right? There's the transgenic mouse that has the plaques that don't rupture, and then there's the atherosclerotic mouse that had the transgenic bone marrow, and then you had the human. Can you tell us a little bit about the different groups you compared and then what ultimately you found? Dr David Dichek: Sure. Yes. You are absolutely right. We had what we refer to as the straight transgenic mice that are either transgenic for macrophage overexpressed uPA or not. We also had older mice who had advanced atherosclerosis and receive bone marrow transplants from mice that either had uPA overexpression in bone marrow or not and then we had the human plaque. So those were the three groups. So what we found was that looking at the proteome of those three groups, we were able to find some common biological processes, and this was really Sina's work, but taking the proteomics data and analyzing it with sophisticated bioinformatics tools. We looked not only at the overlap in specific proteins among the models, but the overlap in biological processes, because it may be in different species that there are different proteins, different actors carrying out the same roles. And that's been described in other systems as well. So we were able to identify not only common biological processes, but surprisingly, we were able to identify decreases in specific category of proteins, basement membrane proteins that were common to two of the models, the straight transgenic and the human model and loss of these proteins certainly has a plausible role in precipitating plaque rupture. So I think one aspect of the analysis that's worthy of note is that we initially thought we would observe more profound changes in the bone marrow transplant mice because they had more advanced atherosclerosis. And in fact, we found fewer changes than in the straight transgenic mice, but thinking about it after letting the data talk to us, rather than trying to impose our own on the data- Dr Cindy St. Hilaire: Always a good idea. Dr David Dichek: ... was that the straight transgenic mice were telling us we've been overexpressing urokinase for 20 weeks since we were conceived, and the bone marrow transplant mice had it for only eight weeks. And indeed they had far less loss of basement membrane proteins and far fewer changes in their plaques than the mice that had expressed it for a longer time. And so when one placed the three groups in a chronology of exposure to protease activity with the bone marrow transplant mice, being the shortest exposure than the straight transgenic mice, and then the humans who've had decades of exposure, it really tells you a nice chronological story about the biological processes leading to plaque rupture. And I think that's a generally applicable lesson and can be applied to other problems in cardiovascular biology. And that is when you have a biological process for which you can't get human tissue until after it's occurred because you can't go in and biopsy. Dr Cindy St. Hilaire: I have that problem with valve calcifications that you can't take your valve out early. Dr David Dichek: If you can get a mouse model that duplicates the pathology, then you have access to the steps leading up to the event. And that's what we tried to construct in this study. And really it was really Sina’s analyses that allowed us to make those connections. Dr Sina Gharib: Yeah. Of course, David was kind of the mastermind behind the design of the experiments on the developing of genetic models and Tomáš is a renowned expert in proteomics analysis. And I kind of joined more on the bioinformatics component of this study, tried to put some of the large data that was being generated together. And as David and Tomáš mentioned, of course atherosclerosis is a very complex disease with many, many components. And then of course the mouse model doesn't quite capture all the different pathophysiological events that happen. So one of the aims of this study was to try to integrate and merge the findings from these model without coming a priori with a bias or a pathway or a candidate gene, we decided to do a relatively unbiased shotgun proteomics approach, we actually do for everything. So the challenge then was how to put it all together. And as Tomáš mentioned, there are statistical tools to try to identify a relative abundance of proteins. But, a few things that pure biologists often don't have to encounter is, you're not one or two different proteins, you're looking at thousands of proteins. So there are issues, statistical issues, such as multiple comparisons. If you looked for changes, you're going to find changes just by random chance. So a lot of statistical adjustments had to be made to ensure that those were adjusted for. This are also pathways and processes that were coming out of these results. There's many different pathways that were interrogated. And again, statistically, you want to adjust for the fact that many of those could have been there by random chance. So there's a fair amount of statistical methods that need to be applied for this data. We also did somewhat more sophisticated pathway analysis where we develop networks based on the differential expressed proteins between the ruptured and unruptured plaques to identify connection among these proteins and identify hubs which are highly connected nodes that could potentially drive the biology of a network. So other types of kind of deeper statistical analysis was done, which are maybe more hypothesis-generating because we actually did not follow up on some of these candidates, but I think they really do provide a map or framework to then pursue more mechanistic experiments to see what happens if we knocked out this highly connected node at the plaque rupture site to see if we can either stabilize or manipulate the biology as plaque rupture. Dr Cindy St. Hilaire: Yeah. I mean, that's really the strength of these unbiased approaches is you can come up with so many more novel targets and pathways that might be contributing. So they're just really great. So one thing I found really interesting, you mentioned that you saw a clear distinction in the proteome and I think it was specifically talking about the human samples because they were large enough to see ruptured area versus non ruptured area, but you really saw a distinct difference in the proteomes of the ruptured area of the plaque versus the non ruptured area of the plaque. And obviously the models you were using are overexpressing a protease. So of course there's a role for proteolysis in this process, which you've now firmly established, but I'm wondering if there are other processes that might also erode the basement membrane. And did you pull up anything that might suggest of other things that are happening or even are there other hypothesis out there that we could test with an approach like yours? Dr David Dichek: I think the pathways that came up have probably all been implicated previously. We have processes like inflammation and complement activation, immune response, thrombosis. That's a post-hoc event. I think what was most unexpected was the decrease in the abundance to basement membrane proteins rather than collagens. So collagen has become the sort of signature protein of stable and unstable plaques and used as a surrogate, people do Picrosirius red staining. It's easy to detect with a histochemical stain, you don't even need an antibody. And surprisingly, we found very few differences in collagens and actually no differences in the type 1 or type 3 collagen, which are thought to be the primary stabilizers of the plaque cap. They weren't significantly different in between ruptured and stable areas of the same plaque. So that was certainly a big surprise. Dr Cindy St. Hilaire: Yeah. Because that would indicate that it's not necessary... We always say thinning of the cap, which obviously we know that there's remodeling, it can get thinner. But you kind of found that the contents were the same, but it's the basement really that was eroded. Dr David Dichek: Yeah. The basement membrane proteins were lost. It used to be said in physiology that if you discovered something new, you should just go to the German literature and go back 30 years and it had already been described. And so, looking back in the literature, there are actually the work of Jean-Baptiste Michelle in France and a scientist in Finland, Petri Kovanen, have actually focused on the potential role of basement membrane in unstable atherosclerosis many years ago, but it was kind of buried in the collagen hypothesis. And I think it needs resurrection. Dr Cindy St. Hilaire: Well, I think this paper has done that so well done there. That's great. Dr Tomáš Vaisar: Would be worthwhile to know that of course, the way you prepare the samples may affect what exactly you're seeing. But we've done very careful characterization of the sample preparation of the extraction procedure to focus, to enrich the exosomal matrix proteins because of this collagen hypothesis. And even with that, we basically saw no difference. Dr David Dichek: Yeah. I think that's an excellent point. If we hadn't found collagen in our extracts, we would not be able to conclude a lot about it. And how you do the extraction, how you process the samples here can really influence what you find. We call it unbiased, but there are technical biases that enter, especially in sample preparation, but our extraction process really was able to extract collagens as well as elastin, which is really infamous for being a- Dr Cindy St. Hilaire: Difficult. Dr David Dichek: ... I really think we were getting a good sampling of the matrix here. Dr Cindy St. Hilaire: I don’t know iif there is an answer for this question, but it's something I'm always thinking about. We always talk about athero being so prevalent because there's no kind of evolutionarily the way to tamp it down, it happens later in life. But can you think of any advantage that the vasculature would have in eroding the basement membrane or altering proteases in a response? I was just trying to think, is this just harnessing a wound healing process that's gone awry or could this ever be protective at all in any way? Dr David Dichek: Well, I think you hit the nail on the head, at least according to my bias. It's a healing response gone awry and that you can really draw out the pathways, basement membrane digestion release of chemotactic peptides as part of the inflammatory response, attraction of more inflammatory cells and then a potential healing response that unfortunately results in digestion of the matrix, which has a morbid or fatal consequence rather than physiologic remodeling. And you're right, that's not selected against. It's selected for, in settings in earlier life infections, for example, perhaps neoplasia, but it's not selected against in late life because people are post reproductive. Dr Cindy St. Hilaire: So what's next for these studies? What questions are you going to attack next with either these models or with some of your proteomic findings? Dr David Dichek: Well, we were just talking about that recently, Tomáš and I, and I think we'd like to look at... For one study, we're interested in doing, plaques that are high risk based on MRI imaging, which is really very well developed here at the University of Washington. And many of those patients have endarterectomies and they don't have ruptured plaques. So they are in a high risk group. So they undergo a endarterectomy for that. Not because they've had a plaque rupture and those plaques might be particularly instructive because they're pre event and won't have the healing response to thrombotic response. And it would be really interesting to see if our studies were confirmed. So that's one direction we're going in. Dr Cindy St. Hilaire: That would be amazing. Luckily, you have access to a whole bunch of human tissue for those kinds of really high impact studies. Dr Sina Gharib: I just wanted to point out that one of the advantages of doing proteomics and being part of the scientific community is that we made all this data available in the manuscript for other researchers to access and confirm. So, really probably the best way to procced with this is to have other investigators replicate our findings and expand on it. So I just want to bring that up because all of that data that was generated has been included within the supplements of this manuscripts and it's accessible to any scientist who wants to pursue further. Dr David Dichek: Yeah, I would add one other direction we'd like to go is we still like to know what the substrates are. We think their disappearance based on their abundance is due to proteolysis. But boy, would it be exciting if we could detect fragments. We were unable to do that in the study, probably because they were lost either in vivo or in processing. Technical advancements in that area, and maybe Tomáš can speak to that, might enable us to actually find more direct evidence of proteolysis. Dr Tomáš Vaisar: Yeah, I mean, to start with, it's really hard to determine physiological substrates of proteases. There's a huge amount of literature identifying proteolytic substrates in vitro, but the physiological substrates are really extremely hard to determine, and especially physiologic in vivo confirming that because in vitro, in a tube, you can mix whatever you want and you modify the ratio of proteins to protease substrate, and you can cleave almost everything with anything. It's a little exaggeration, but it's close. While the physiology substrates in the really complex milia of tissues is extremely hard. And so there has been several approaches developed and one of them is the Proteomaps. The other one is an approach called TAILS developed by Chris Overall at UBC that uses the idea of formation of the neo termini and then tagging the neo termini. So that in the actual sample, you can specifically detect these neo termini formed. But even with that approach, it's really hard to determine what are actual physiological substrates. And on top of that, what are the cleavage sites of the proteases? Dr Cindy St. Hilaire: And I guess the third being, if those substrates are cleaved, are they circulating and can we detect them in a blood sample? That would be, I guess, the gold standard. Well, thank you all so much for joining me today. Congratulations on this really very cool study. Being into human and translational work, I always love mouse studies that bring in lots of human samples. So congratulations on that. And I look forward to your future publications on this. Dr Tomáš Vaisar: Thanks a lot. Dr David Dichek: Thanks. Dr Cindy St. Hilaire: That's it for the highlights from the late September and early October issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs David Dichek, Sina Gharib, and Tomáš Vaisar. This podcast is produced by Rebecca McTavish and Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.29.318196v1?rss=1 Authors: Bierig, T., Collu, G., Blanc, A., Poghosyan, E., Benoit, R. M. Abstract: 2019-nCoV is the causative agent of the serious, still ongoing, worldwide COVID-19 pandemic. High quality recombinant virus proteins are required for research related to the development of vaccines and improved assays, and to the general understanding of virus action. The receptor-binding domain (RBD) of the 2019-nCoV spike (S) protein contains disulfide bonds and N-linked glycosylations, therefore, it is typically produced by secretion. Here, we describe a construct and protocol for the expression and purification of yellow fluorescent protein (YFP) labeled 2019-nCoV spike RBD. The fusion protein, in the vector pcDNA 4/TO, comprises an N-terminal interferon alpha 2 (IFN2) signal peptide, an eYFP, a FLAG-tag, a human rhinovirus 3C protease cleavage site, the RBD of the 2019-nCoV spike protein and a C-terminal 8x His-tag. We stably transfected HEK 293 cells. Following expansion of the cells, the fusion protein was secreted from adherent cells into serum-free medium. Ni-NTA IMAC purification resulted in very high protein purity, based on analysis by SDS-PAGE. The fusion protein was soluble and monodisperse, as confirmed by size-exclusion chromatography (SEC) and negative staining electron microscopy. Deglycosylation experiments confirmed the presence of N-linked glycosylations in the secreted protein. Complex formation with the peptidase domain of human angiotensin-converting enzyme 2 (ACE2), the receptor for the 2019-nCoV spike RBD, was confirmed by SEC, both for the YFP-fused spike RBD and for spike RBD alone, after removal of YFP by proteolytic cleavage. Possible applications for the fusion protein include binding studies on cells or in vitro, fluorescent labeling of potential virus-binding sites on cells, the use as an antigen for immunization studies or as a tool for the development of novel virus- or antibody-detection assays. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.296756v1?rss=1 Authors: Budiardjo, S. J., Ikujuni, A. P., Firlar, E., Cordova, A., Kaelber, J., Slusky, J. S. G. Abstract: Overexpression of tripartite efflux pump systems in gram-negative bacteria are a principal component of antibiotic resistance. High-yield purification of the outer membrane component of these systems will enable biochemical and structural interrogation of their mechanisms of action and allow testing of compounds that target them. However, preparation of these proteins is typically hampered by low yields requiring laborious large-scale efforts. If refolding conditions can be found, refolding these proteins from inclusion bodies can lead to increased yields as compared to membrane isolations. Here, we develop a concentration-dependent folding protocol for refolding TolC, the outer membrane component of the antibiotic efflux pump from Escherichia coli. We show that by our method of re-folding, homotrimeric TolC remains folded in SDS-PAGE, retains binding to an endogenous ligand, and recapitulates the known crystal structure by single particle cryoEM analysis. We find that a key factor in successful re-folding is a concentration dependence of TolC oligomerization. We extended the scheme to CmeC, a homologous protein from Campylobacter jejuni, and find that concentration-dependent oligomerization is a general feature of these systems. Because outer-membrane efflux pump components are ubiquitous across gram-negative species, we anticipate that incorporating a concentration step in re-folding protocols will promote correct refolding allowing for reliable, high-yield preparation of this family of proteins. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.24.219790v1?rss=1 Authors: Sahadevan, P., Nawaito, S. A., Trepanier, J., Benamar, S., Sahmi, F., Theberge-Julien, G., Villeneuve, L. R., Gaestel, M., Tardif, J.-C., Allen, B. G. Abstract: MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGF{beta}, or H2O2 and this was prevented by inhibition of p38alpha/beta. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38alpha/beta inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38alpha and/or p38beta are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts. Copy rights belong to original authors. Visit the link for more info

Die vorliegende Arbeit befasst sich mit der Entwicklung und Charakterisierung monoklonaler Antikörper (mAk) gegen die C-Komponente des Non-haemolytic Enterotoxin (Nhe)-Komplexes von Bacillus cereus sowie deren Einsatz zur Analyse der Rolle des NheC im Hinblick auf die Wirkungsweise von Nhe. NheC wurde in Escherichia coli rekombinant exprimiert und mittels Immunaffinitätschromatographie aufgereinigt. Nach Überprüfung durch SDS-PAGE und Quantifizierung mittels SYPRO® Ruby Protein Gel Färbung wurde das gereinigte Toxin als Immunogen eingesetzt. Nachfolgend konnten drei mAk vom IgG-Subtyp (mAk 2G8, 1E12, 2F10) und ein mAk vom IgM-Subtyp (mAk 3D6) gegen NheC hergestellt werden. Die Charakterisierung der mAk erfolgte mittels Enzymimmuntest (EIA), Western Immunblot und Immunfluoreszenzmikroskopie. Bei Verwendung eines indirekten EIAs erlaubten die mAk den sensitiven Nachweis von rNheC im unteren Nanogrammbereich (Nachweisgrenze 10 – 15 ng/ml). Untersuchungen zur Spezifität der mAk innerhalb des Nhe-Komplexes mittels indirekten EIAs und Western Immunblots zeigten, dass die mAk 2G8 und 1E12 substantielle Kreuzreaktionen mit der strukturverwandten NheB-Komponente aufweisen. Dies konnte durch Epitopanalysen und kompetitive Bindungstests noch bestätigt werden. Während die mAk 2G8, 1E12 und 3D6 mit allen überprüften B. cereus-Stämmen reagierten, weist das von dem mAk 2F10 erkannte Epitop eine stammspezifische Variabilität auf. In Zellkulturtests war keiner der vier mAk in der Lage die zytotoxische Aktivität des Nhe-Komplexes zu neutralisieren. Demgegenüber gelang es mit allen mAk mittels Immunfluoreszenz erstmals die direkte Bindung von NheC an die Zielzelle darzustellen. Unter Verwendung eines polyklonalen Kaninchen-Antiserums gegen NheC als Fangantikörper und der vier mAk gegen NheC als Nachweisantikörper wurden hochempfindliche Sandwich-EIAs für den Nachweis von gereinigtem NheC entwickelt. In natürlichen B. cereus-Überständen konnten jedoch nur geringe Mengen (< 10 %) des theroretisch vorhandenen NheC nachgewiesen werden. Die auf diesen Ergebnissen basierende Hypothese, dass NheC in Lösung an NheB gebunden vorliegt, konnte zunächst in artifiziellen Systemen bestätigt werden. Mangels SDS-Stabilität der NheB-NheC-Komplexe, erfolgte der Nachweis mittels intermolekularem Cross-Linking und Dot Blot-Analysen. Durch Kombination des NheC-spezifischen mAk 3D6 als Fangantikörper und des HRP-gekoppelten mAk 1E11 gegen NheB als Nachweisantikörper konnte ein spezifischer NheC/B-Sandwich-EIA zum Nachweis von NheB-NheC-Komplexen in B. cereus-Kulturüberständen etabliert werden. Zur Analyse der Funktion der NheB-NheC-Komplexe wurde zum einen die Komplexbildung mit der Zytotoxizität verglichen, und zum anderen per Durchflusszytometrie deren Zellbindung mittels NheB-Quantifizierung (durch mAk 1E11) bestimmt. Fazit ist, dass scheinbar sowohl eine definierte Menge an NheB-NheC-Komplexen, als auch ausreichend freies NheB vorhanden sein müssen, damit effiziente Zellbindung und Zytotoxizität von Nhe gewährleistet sind.

Etioplasten sind hochspezialisierte pflanzliche Organelle der Plastidenfamilie, die während der Skotomorphogenese von Pflanzen gebildet werden. Die Morphologie der Etioplasten unterscheidet sich grundlegend von Chloroplasten, die während der Photomorphogenese gebildet werden. Durch Belichtung von Pflanzen, die im Dunkeln angezogen worden sind, kommt es zur Induktion der Transformation von Etioplasten zu Chloroplasten. Die unmittelbar vor Induktion des biologischen Systems bestehende Zusammensetzung der Proteine und Proteinkomplexe des Etioplasten ist allerdings bislang kaum untersucht worden. Im Rahmen dieser Arbeit erfolgten mehrere spezifische Analysen von plastidären Subproteomen. Ausgewählte Subproteome der inneren Membranen von Etioplasten der Gerste wurden im Vergleich zum Proteom der Thylakoidmembran von Chloroplasten analysiert. Durch die Kombination verschiedener gelelektrophoretischer Trennmethoden für Einzelproteine und Proteinkomplexe mit massenspektrometrischen Analysemethoden gelangen sensitivste Nachweise niedrig konzentrierter Untereinheiten von Membranproteinkomplexen. Darüber hinaus gelangen der Nachweis niedermolekularer membranintegraler Proteine und die spezifische Charakterisierung von Einzelproteinen. Im ersten Teil der Arbeit wurden die N-Termini von NADPH:Protochlorophyllid-Oxidoreduktase (POR) A und B durch ein LC-MS basiertes Verfahren bestimmt. Es erfolgte die Entwicklung einer Methode zur selektiven Isolation N-terminaler Peptide mittels Höchstdruckflüssigkeitschromatographie (UPLC). Dazu wurden zwei chemische Reaktionsschritte auf Protein- und Peptidebene durchgeführt, wodurch das N-terminale Peptid nach einem tryptischen Verdau ausschließlich acetyliert vorlag und interne Peptide durch eine weitere Modifikation mit 2,4,6-Trinitrobenzolsulfonsäure abgetrennt wurden. Dadurch konnte gezeigt werden, dass die N-Termini von PORA und PORB homolog zueinander sind und eine vergleichbare Erkennungssequenz für die prozessierende(n) Protease(n) vorliegt. Das Transitpeptid von PORA ist somit deutlich kürzer, als bislang vermutet, wodurch neue Rückschlüsse bezüglich einer möglichen Bindestelle von Protochlorophyllid gezogen werden konnten, da eine von Reinbothe et al. 2008 beschriebene Bindestelle nicht im Bereich des Transitpeptids, sondern in Bereich der maturen PORA liegt. Bei PORB konnten neben einem dominierenden N-terminalen Peptid zwei weitere um jeweils ein Alanin verkürzte N-terminale Peptide mit geringerer Signalintensität nachgewiesen werden. Dies deutet auf eine unpräzise N-terminale Prozessierung hin. Im zweiten Teil der Arbeit gelang die bislang umfassendste massenspektrometrische Charakterisierung des NAD(P)H-Dehydrogenase-Komplexes aus einer C3-Pflanze. In Etioplasten konnten sechs plastidär kodierte und mindestens fünf kernkodierte Untereinheiten des NDH-Komplexes identifiziert werden. Dies gelang durch die Isolation des Komplexes mittels nativer PAGE als 1. Dimension und die anschließende Aufkonzentrierung der Untereinheiten in einer SDS-PAGE als konzentrierende 2. Dimension. Dadurch konnte gezeigt werden, dass der NDH-Komplex bereits in Etioplasten neben dem membranintegralen Subkomplex aus mindestens zwei löslichen Subkomplexen aufgebaut ist. Aufgrund dieser umfangreichen Assemblierung ist eine physiologische Funktion wahrscheinlich und erste Versuche zur NAD(P)H-Dehydrogenase Aktivität lieferten Hinweise auf eine mögliche enzymatische Aktivität. Im dritten Teil der Arbeit gelang in Etioplasten erstmals der Nachweis aller bekannten membranintegralen, niedermolekularen Untereinheiten von Photosystem II, nicht aber von Photosystem I. Die Untereinheiten von PSI konnten ausschließlich in Chloroplasten nachgewiesen werden. Von PSII konnten 13 niedermolekulare Untereinheiten mit jeweils einer Transmembrandomäne nachgewiesen werden. Diese Untereinheiten konnten im Gegensatz zu Chloroplasten nicht in höhermolekularen Komplexen, sondern ausschließlich nahe der Lauffront einer BN-PAGE im Bereich der freien Proteine nachgewiesen werden. Der Nachweis von PsbN war ausschließlich in Etioplasten möglich. Aus diesen Ergebnissen wurde geschlossen, dass ausschließlich nicht-chlorophyllbindende Untereinheiten von PSII in Etioplasten akkumuliert werden und die Anreicherung von chlorophyllbindenden Untereinheiten von PSI und PSII von der Anwesenheit von Chlorophyll abhängt. Darüber hinaus konnten die vier niedermolekularen, membranintegralen Untereinheiten des Cytochrom b6f-Komplexes in Etioplasten und in Chloroplasten sowohl in der monomeren, als auch dimeren Assemblierungsstufe nachgewiesen werden. Ermöglicht wurden diese Nachweise durch eine neu entwickelte Methode zur Extraktion von Proteinen aus einem Polyacrylamid-Gel mit organischen Lösungsmitteln und der anschließenden massenspektrometrischen Charakterisierung mittels offline ESI-MS.

Protein expression profiling of human glatiramer acetate (GA) and myelin basic protein (MBP) specific T cell lines from a multiple sclerosis (MS) patient and healthy donors Investigations in MS and the animal model experimental autoimmune encephalomyelitis (EAE) indicate that autoreactive T cells play a pivotal role in the pathogenesis. Putative auto antigens include e.g. MBP. One approved therapy of patients with relapsing-remitting MS is GA (Copaxone®), which induces a shift in the phenotype of CD4+ T cells from TH1- to TH2-type. As the molecular mechanisms involved are largely unknown, we were interested in the regulated proteome of GA- and MBP-specific human T cell lines. We generated GA- and MBP-specific T cell lines from three healthy donors and GA- specific T cell lines from a MS patient before and after 6 months of GA therapy. Proteome analysis of activated and resting GA- and MBP-specific, CD3+ CD4+ CD8- T cells was done by SDS-PAGE and MALDI-TOF mass spectrometry. A total of 120 regulated protein spots were detected on the gels. 69 were identified as different proteins by mass spectrometry. A single protein pathway analysis of these proteins led us to five proteins. One of them is known to be a negative regulator of growth factors. Three proteins known for their anti-inflammatory properties were upregulated in GA-specific T cells, one protein with pro-inflammatory characteristics was found upregulated in encephalitogenic MBP-specific T cells. Based on their expression profile and their characteristics these proteins may be involved in immunological mechanisms relevant in MS and/or possibly during GA treatment.

Kim-1 (kidney injury molecule-1), a member of the TIM (T-cell immunglobulin mucin) family is a glykosylated type 1 transmembrane protein, which is markley upregulated during the repair of renal epithel cells after ischemia and in malignant and cystic kidney disease, while expressed at low levels in normal kidneys. In previous experiments Kim-1 has been shown to localize in primary cilia of cultured renal epithelial cells in-vitro. Primary cilia have been shown to play a role in development and recently cilial dysfunction has been shown to be the unifying defect causing polycystic kidney disease. Kim-1 interacts with Inversin, a ciliary protein, which is mutated in Type 2 nephronophthisis, and which has been shown to modulate the Wnt pathway by targeting Dishevelled (Dvl) for proteasomal degradation. The expression of Wnt proteins is necessary for many fundamental processes in differentiation, proliferation, polarity and adhesion. Wnts play an important role in the early embryonic development. There is only limited knowledge on the physiological role of Kim-1 during differentiation, proliferation and survival of tubular epithel cells after renal injury. The aim of this study was to characterize the role of Kim-1 in epithelial polarization and ciliogenesis of tubular epithel cells outside of pathological injury models. Additionally the role of Kim-1 in the Wnt signaling pathway was examined. Renal tubular cells were analyzed phenotypically with immunfluorescence staining. Protein expression levels were detected by SDS PAGE and Western blot. Signaltransduction activity was measured in luciferase reporter assays. Protein overexpression was achieved with transient transfection of plasmid DNA in cultured cells after calcium chloride precipitation. Gene reduction was achieved through lentivirus-mediated transduction of short interfering RNAs. Examination of immunfluorescence stained cryosections showed, that Kim-1 is localized in-vivo in primary cilia of tubular epithelial cells in mouse kidneys. Kim-1 expression levels correlate with the polarization of tubular epithel cells in-vitro. Reporter assays using TOP FLASH luciferase constructs revealed that Kim-1 reduced the Dvl-induced TOP FLASH activation and decreased steady-state levels of ß-Catenin in HEK 293t cells, suggesting that Kim-1 antagonizes the canonical Wnt pathway. Conversely Kim-1 expression increased AP-1 activity, which has been linked to the non-canonical Wnt pathway. Phenotypical characterization of two different immortalized renal epithelial cell lines after Kim-1 knock-down showed no changes in epithelial polarization, or ciliogenesis. These results suggest that Kim-1 is a ciliary protein that probably plays no active role in cell polarization or ciliogenesis, but may participate in the Wnt signaling pathway. Kim-1 may act as a molecular switch between the canonical and non-canonical Wnt pathway and thus may play a role in epithelial differentiation.

Cultured cardiac myocytes from adult Sprague-Dawley rats express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose 6-phosphate (IGF-II/Man6P) receptors and respond to IGF-I with a dose-dependent accumulation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,4-bisphosphate [Ins(1,4)P2]. Specific binding of [125I]IGF-I to isolated membranes from cultured cardiac myocytes amounted to 1-1.2%. Binding of [125I]IGF-I was inhibited by unlabeled IGF-I at nanomolar concentrations and insulin at much higher concentrations. These data suggest that IGF-I binds to its own receptor on rat cardiac myocytes. Competitive binding studies using isolated membranes from cardiac myocytes and [125I]IGF-II showed 2-4% specific binding. Binding of [125I]IGF-II was inhibited by IGF-II and much less potently by IGF-I and insulin. Immunoglobulin G (IgG) 3637 (an IgG directed against the IGF-II/Man6P receptor) partially inhibited binding of [125I]IGF-II whereas nonimmune IgG did not. Affinity cross-linking studies with [125I]IGF-II and cardiac myocyte membranes and subsequent analysis of the ligand-receptor complex using SDS-PAGE and autoradiography showed a radiolabeled band of approximately 250 kilodalton (kDa). The formation of the [125I]IGF-II-receptor complex was inhibited by incubation with IGF-II and IgG 3637 but not by insulin or nonimmune IgG. Western blotting of protein extracts from cultured cardiac myocytes was performed using IgG 3637 and an immunoperoxidase technique for the visualization of the IGF-II/Man6P receptor protein. A specific band at 220 kDa under nonreducing conditions was detected on the blots, providing further evidence for the expression of the IGF-II/Man6P receptor by cardiac myocytes. The effect of IGFs on the accumulation of inositol phosphates was measured by HPLC analysis of perchloric acid extracts from myo-[3H]inositol-labeled cultured cardiac myocytes. IGF-I (50 ng/ml) stimulated the accumulation both of Ins(1,4,5)P3 and Ins(1,4)P2 after 30 sec by 43% and 63%. IGF-II (up to 500 ng/ml) had no significant effect on inositol phosphate accumulation under the same conditions. However, in the presence of millimolar concentrations of Man6P, IGF-II (500 ng/ml) also increased Ins(1,4,5)P3 accumulation by 59%. We conclude that cardiac myocytes from adult rats express IGF receptors and respond to IGFs with the accumulation of Ins(1,4,5)P3 and Ins(1,4)P2. This effect seems to be mediated by an IGF-I receptor-specific pathway.

The product of the ie 1 gene, the regulatory immediate early protein pp89 of murine cytomegalovirus (MCMV), interacts with core histones, which can mediate the association of pp89 with DNA. We report the capacity of pp89 to interact directly with DNA in the absence of cellular proteins. After separation of proteins by SDS–PAGe, pp89 bound ds- and ssDNA, with a preference for ssDNA. Binding to specific DNA sequences in the MCMV genome was not detected. The DNA-binding region of pp89 was located to amino acids 438 to 534 by analysis of deletion mutants expressed as -galactosidase or TrpE fusion proteins. This region is identical to the highly acidic C-terminal region spanning amino acids 424 to 532. The human cytomegalovirus IE1 protein, which contains a similar extended C-terminal acidic region, does not react with DNA under the same experimental conditions.