Oligomerization and Endocytosis of the α-Factor Receptor: A Dissertation

α-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. The ability of α-factor receptors to form oligomeric complexes with each other and with other proteins was investigated. Both in vivo and in vitroevidence was obtained that suggests homo-oligomerization of receptors in the plasma membrane. When the membranes from cells coexpressing two differentially-tagged receptors were solubilized with detergent and subjected to immunoprecipitation, the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Treatment of cultures with α-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind α-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. Therefore, α-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. A crosslinking and immunoprecipitation strategy was used to capture and characterize the transient complexes that contain the α-factor receptor Ste2. Tagged receptors were crosslinked to form at least three high molecular weight complexes and the complexes were immunoprecipitated with antibodies against the tag. Western blotting analysis of the precipitated material revealed the presence of β and γ subunits of the heterotrimeric G protein, Ste4 and Stel8. Similar results were obtained when the cultures had been treated with α-factor prior to analysis. A truncated receptor missing most of the cytoplasmic C-terminal tail was also active in binding Ste4. Overall, these results constitute the first biochemical evidence for a physical association between the α-factor receptor and its cognate G-protein. Endocytic signals in the C-terminal tail (residues 297-431) of the α-factor receptor were analyzed. One signaling element, SINNDAKSS, (residues 331-339) is known to be sufficient (but not necessary) for endocytosis. Internal deletions of the STE2 gene were constructed that remove sequences encoding SINNDAKSS and selected regions of the C-terminal tail. Strains containing these alelles were then assayed for endocytosis in the presence and absence of α-factor. Residues from 360 to 431 were sufficient to mediate both constitutive and ligand-mediated endocytosis of the receptor even though 63 residues including the SINNDAKSS motif had been removed. Structural features of this region that were investigated further were the highly-ubiquitinated Lys374, the neighboring Lys387, and the GPFAD motif (residues 392-396). Lys374 and Lys387 were unnecessary for the element to promote exit from the plasma membrane; however, Lys374 may play some role in intracellular trafficking. The GPFAD motif was not sufficient to promote endocytosis, since the residues 360-399 provided no detectable endocytic activity. Overall, these results suggest that a new region in the C-terminal of the α-factor receptor, redundant with the SINNDAKSS motif, is sufficient to mediate the constitutive endocytosis as well as the ligand-mediated endocytosis of the receptor

Excess cholesterol induces mouse egg activation and may cause female infertility

The HDL receptor scavenger receptor, class B type I (SR-BI) controls the structure and fate of plasma HDL. Female SR-BI KO mice are infertile, apparently because of their abnormal cholesterol-enriched HDL particles. We examined the growth and meiotic progression of SR-BI KO oocytes and found that they underwent normal germinal vesicle breakdown; however, SR-BI KO eggs, which had accumulated excess cholesterol in vivo, spontaneously activated, and they escaped metaphase II (MII) arrest and progressed to pronuclear, MIII, and anaphase/telophase III stages. Eggs from fertile WT mice were activated when loaded in vitro with excess cholesterol by a cholesterol/methyl-β-cyclodextrin complex, phenocopying SR-BI KO oocytes. In vitro cholesterol loading of eggs induced reduction in maturation promoting factor and MAPK activities, elevation of intracellular calcium, extrusion of a second polar body, and progression to meiotic stages beyond MII. These results suggest that the infertility of SR-BI KO females is caused, at least in part, by excess cholesterol in eggs inducing premature activation and that cholesterol can activate WT mouse eggs to escape from MII arrest. Analysis of SR-BI KO female infertility raises the possibility that abnormalities in cholesterol metabolism might underlie some cases of human female infertility of unknown etiology.National Institutes of Health (U.S.)National Institutes of Health (U.S.) (Pre-doctoral Training Grant T32GM007287)Massachusetts Institute of Technology (International Science and Technology Initiatives Chile Cooperative Grant

Follicular fluid high density lipoprotein-associated micronutrient levels are associated with embryo fragmentation during IVF

To investigate whether follicular fluid lipid-soluble micronutrients are associated with embryo morphology parameters during IVF. Follicle fluid and oocytes were obtained prospectively from 81 women. Embryo morphology parameters were used as surrogate markers of oocyte health. HDL lipids and lipid-soluble micronutrients were analyzed by high-pressure liquid chromatography. Non-parametric bi-variate analysis and multivariable ordinal logistic regression models were employed to examine associations between biochemical and embryo morphology parameters. Follicular fluid HDL cholesterol (r = −0.47, p < 0.01), α-tocopherol (r = −0.41, p < 0.01), δ-tocopherol (r = −0.38, p < 0.05) and β-cryptoxanthine (r = −0.42, p < 0.01) are negatively correlated with embryo fragmentation. Ordinal logistic regression models indicate that a 0.1 μmol/L increase in β-cryptoxanthine, adjusted for γ-tocopherol, is associated with a 75% decrease in the cumulative odds of higher embryo fragmentation (p = 0.010). Follicular fluid HDL micronutrients may play an important role in the development of the human oocyte as evident by embryo fragmentation during IVF

Challenges in Using Cultured Primary Rodent Hepatocytes or Cell Lines to Study Hepatic HDL Receptor SR-BI Regulation by Its Cytoplasmic Adaptor PDZK1

Background: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Methodology/Principal Findings: Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI’s C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Conclusions/Significance: Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.National Institutes of Health (U.S.) (Grant HL052212)National Institutes of Health (U.S.) (Grant HL066105)National Institutes of Health (U.S.) (Grant ES015241)National Institutes of Health (U.S.) (Grant GM068762

Internalization Dissociates β2-Adrenergic Receptors

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that β2-adrenergic receptors (β2ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of β2ARs between subcellular compartments. BRET between β2ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between β2ARs and endosome markers increases. Energy transfer between β2ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled β2ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that β2ARs associate transiently with each other in the plasma membrane, or that β2AR dimers or oligomers are actively disrupted during internalization

Dietary Manipulation and Social Isolation Alter Disease Progression in a Murine Model of Coronary Heart Disease

Background: Mice with a deficiency in the HDL receptor SR-BI and low expression of a modified apolipoprotein E gene (SR-BI KO/ApoeR61h/h) called ‘HypoE’ when fed an atherogenic, ‘Paigen’ diet develop occlusive, atherosclerotic coronary arterial disease (CHD), myocardial infarctions (MI), and heart dysfunction and die prematurely (50% mortality ~40 days after initiation of this diet). Because few murine models share with HypoE mice these cardinal, human-like, features of CHD, HypoE mice represent a novel, small animal, diet-inducible and genetically tractable model for CHD. To better describe the properties of this model, we have explored the effects of varying the composition and timing of administration of atherogenic diets, as well as social isolation vs. group housing, on these animals. Methodology/Principal Findings: HypoE mice were maintained on a standard lab chow diet (control) until two months of age. Subsequently they received one of three atherogenic diets (Paigen, Paigen without cholate, Western) or control diet for varying times and were housed in groups or singly, and we determined the plasma cholesterol levels, extent of cardiomegaly and/or survival. The rate of disease progression could be reduced by lowering the severity of the atherogenic diet and accelerated by social isolation. Disease could be induced by Paigen diets either containing or free of cholate. We also established conditions under which CHD could be initiated by an atherogenic diet and then subsequently, by replacing this diet with standard lab chow, hypercholesterolemia could be reduced and progression to early death prevented. Conclusions/Significance: HypoE mice provide a powerful, surgery-free, diet-‘titratable’ small animal model that can be used to study the onset of recovery from occlusive, atherosclerotic CHD and heart failure due to MI. HypoE mice can be used for the analysis of the effects of environment (diet, social isolation) on a variety of features of cardiovascular disease.National Institutes of Health (U.S.)National Heart, Lung, and Blood Institut

The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor

Sensing of Dietary Lipids by Enterocytes: A New Role for SR-BI/CLA-1

BACKGROUND: The intestine is responsible for absorbing dietary lipids and delivering them to the organism as triglyceride-rich lipoproteins (TRL). It is important to determine how this process is regulated in enterocytes, the absorptive cells of the intestine, as prolonged postprandial hypertriglyceridemia is a known risk factor for atherosclerosis. During the postprandial period, dietary lipids, mostly triglycerides (TG) hydrolyzed by pancreatic enzymes, are combined with bile products and reach the apical membrane of enterocytes as postprandial micelles (PPM). Our aim was to determine whether these micelles induce, in enterocytes, specific early cell signaling events that could control the processes leading to TRL secretion. METHODOLOGY/PRINCIPAL FINDINGS: The effects of supplying PPM to the apex of Caco-2/TC7 enterocytes were analyzed. Micelles devoid of TG hydrolysis products, like those present in the intestinal lumen in the interprandial period, were used as controls. The apical delivery of PPM specifically induced a number of cellular events that are not induced by interprandial micelles. These early events included the trafficking of apolipoprotein B, a structural component of TRL, from apical towards secretory domains, and the rapid, dose-dependent activation of ERK and p38MAPK. PPM supply induced the scavenger receptor SR-BI/CLA-1 to cluster at the apical brush border membrane and to move from non-raft to raft domains. Competition, inhibition or knockdown of SR-BI/CLA-1 impaired the PPM-dependent apoB trafficking and ERK activation. CONCLUSIONS/SIGNIFICANCE: These results are the first evidence that enterocytes specifically sense postprandial dietary lipid-containing micelles. SR-BI/CLA-1 is involved in this process and could be a target for further study with a view to modifying intestinal TRL secretion early in the control pathway

Comprendre la désinformation et promouvoir l'information fiable

Americans are more worried about misinformation than about sexism, racism, terrorism, and climate change. Fears over misinformation on social media are overblown. Misinformation represents a minute proportion of the news that people consume online (~ 1%), and a small minority of people account for most of the misinformation consumed and shared online. People, on average, are good at detecting fake news and identifying reliable sources of information. People do not believe everything they see and read on the internet. Instead, they are active consumers of information who domesticate technologies in unexcepted ways. It’s very unlikely that social media exacerbates the misinformation problem, that fake news contributes to important political events or that falsehoods spread faster than the truth. Yet, some fake news stories do go viral, and understanding why, despite their inaccuracy, they go viral is important. In a series of experiments, we identified a factor that, alongside accuracy, drives the sharing of true and fake news: the ‘interestingness-if-true’ of a piece of news, e.g. if alcohol was a cure against COVID-19, the pandemic would end in an unprecedented international booze-up. In three experiments (N = 904), participants were more willing to share news they found more interesting-if-true, as well as news they deemed more accurate. They rated fake news less accurate but more interesting-if-true than true news. People may not share news of questionable accuracy by mistake, but instead because the news has qualities that compensate for its potential inaccuracy, such as being interesting-if-true. Despite these qualities, why are most people are reluctant to share fake news? To benefit from communication, receivers should trust less people sharing fake news. And the costs of sharing fake news should be higher than the reputational benefits of sharing true news. Otherwise we would end up trusting people misleading us half of the time. Four experiments (N = 3,656) support this hypothesis: sharing fake news hurts one’s reputation in a way that is difficult to fix, even for politically congruent fake news. Most participants asked to be paid to share fake news (even when politically congruent), and asked for more when their reputation was at stake. During the second part of my PhD, I tested solutions to inform people efficiently. I found that discussing in small groups the scientific evidence on Genetically Modified (GM) food safety and the usefulness of vaccines changed people’s minds in the direction of the scientific consensus. To scale up the power of discussion, we created a chatbot that emulated the most important traits of discussion. We found that rebutting the most common counterarguments against GMOs with a chatbot led to more positive attitudes towards GMOs than a non-persuasive control text and a paragraph highlighting the scientific consensus. However, the dialogical structure of the chatbot seemed to have mattered more than its interactivity. During the pandemic, we deployed a chatbot to inform the French population about COVID- 19 vaccines. Interacting a few minutes with this chatbot, which answered the most common questions about COVID-19 vaccines, increased people’s intention to get vaccinated and had a positive impact on their attitudes towards the vaccines. In the end, people are not stupid. When provided with good arguments, they change their mind in the direction of good arguments. Most people avoid sharing misinformation because they care about their reputation. We do not live in a post-truth society in which people disregard the truth. Overall, we should probably be more concerned about the large portion of people who do not trust reliable sources and are uninformed because they do not follow the news, rather than the minority of people who trust unreliable sources and are misinformed.Les fausses nouvelles affolent. Les Américains sont plus préoccupés par la désinformation que par le sexisme, le racisme, et le changement climatique. Ces craintes sont très largement exagérées. La désinformation ne représente qu'une infime portion des nouvelles consommées en ligne (~ 1 %) et une petite minorité de gens est à l'origine de la majorité des fausses informations consommées et partagées en ligne. En moyenne, les gens sont capables de reconnaître les fausses nouvelles et d'identifier les sources d'information fiables. Les gens ne croient pas tout ce qu'ils voient et lisent sur l'internet. Il est peu probable que les réseaux sociaux exacerbent le problème de la désinformation, que les fausses nouvelles aient contribué à des événements politiques importants ou que les fausses nouvelles se répandent plus vite que la vérité. Cependant, certaines fausses nouvelles sont virales, et il est intéressant de comprendre pourquoi, malgré leur manque de fiabilité, ces fausses nouvelles deviennent virales. Au cours d'une série d'expériences, nous avons identifié un facteur qui motive le partage des vraies et des fausses nouvelles : "l'intérêt-si-vrai" d'une nouvelle, e.g. si l’alcool était un remède contre la COVID-19 il suffirait de faire la fête pour se protéger du virus. Au cours de trois expériences en ligne (N = 904), les participants étaient plus disposés à partager des nouvelles qu'ils trouvaient plus intéressantes-si-vraies, ainsi que des nouvelles qu'ils jugeaient plus fiables. Ils considéraient les fausses nouvelles comme moins fiables mais plus intéressantes-si-vraies que les vraies nouvelles. Les gens pourraient partager des fausses nouvelles non pas par erreur, mais plutôt parce que ces nouvelles possèdent des qualités qui compensent pour leur manque de fiabilité, comme le fait d'être intéressantessi- vraies. Malgré ces qualités, pourquoi la plupart des gens sont-ils réticents à partager des fausses nouvelles ? Quatre expériences (N = 3 656) montrent que le partage de fausse nouvelle nuit à la réputation de son transmetteur d'une manière difficile à compenser par le partage de vraies nouvelles. La plupart des participants demandèrent à être payés pour partager des fausses nouvelles, et ce montant était d’autant plus important que leur réputation était en jeu. Durant le deuxième parti de mon doctorat j’ai mesuré l’efficacité d’interventions pour informer efficacement les gens. J’ai montré que discuter en petits groupes des preuves scientifiques portant sur la sûreté des organismes génétiquement modifiés (OGM) et de l'utilité des vaccins, influençait l’opinion des gens en direction du consensus scientifique. Pour étendre le pouvoir persuasif de la discussion, nous avons développé un chatbot simulant les caractéristiques les plus importantes d’une discussion. Interagir avec ce chatbot réfutant les contre-arguments les plus courants contre les OGMs entraîna des attitudes plus positives à l'égard des OGMs que plusieurs conditions de contrôle (N = 1306). Pendant la pandémie, nous avons déployé un chatbot répondant aux questions les plus courantes sur les vaccins COVID-19. Interagir quelques minutes avec ce chatbot augmenta l'intention des gens de se faire vacciner et eu un impact positif sur leurs attitudes envers les vaccins. Au final, les gens ne sont pas stupides. Lorsqu'on leur présente de bons arguments, ils changent d'avis en direction de ces bons arguments. La plupart des gens évitent de partager des fausses nouvelles par souci pour leur réputation. L’ère de la « post-vérité » n’existe pas, la fiabilité de l’information est aussi importante aujourd’hui que par le passé. Dans l'ensemble, il est probablement plus important de se préoccuper du grand nombre de gens qui ne font pas confiance aux sources fiables et ne sont pas informés parce qu'ils ne suivent pas l'actualité, plutôt que de la minorité de gens qui font trop confiance aux sources douteuses et sont mal informées

A Love-Hate Relationship: Cholesterol in Health and Disease

In this class, we will examine cholesterol's role in the cell and in the body as a whole, from its function as a structural component of the membrane to its function in signaling. We will discuss mechanisms of cholesterol sensing, mechanisms of feedback regulation in cells, cholesterol in the brain, cholesterol in the circulation, 'good cholesterol' and 'bad cholesterol,' cholesterol-related human disorders, and the drugs that deal with some of these disorders. This course is one of many&nbsp;Advanced Undergraduate Seminars&nbsp;offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the&nbsp;Advanced Undergraduate Seminars&nbsp;are postdoctoral scientists with a strong interest in teaching