Scap structures highlight key role for rotation of intertwined luminal loops in cholesterol sensing.

The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.

Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes.

Sphingomyelin and cholesterol are essential lipids that are enriched in plasma membranes of animal cells, where they interact to regulate membrane properties and many intracellular signaling processes. Despite intense study, the interaction between these lipids in membranes is not well understood. Here, structural and biochemical analyses of ostreolysin A (OlyA), a protein that binds to membranes only when they contain both sphingomyelin and cholesterol, reveal that sphingomyelin adopts two distinct conformations in membranes when cholesterol is present. One conformation, bound by OlyA, is induced by stoichiometric, exothermic interactions with cholesterol, properties that are consistent with sphingomyelin/cholesterol complexes. In its second conformation, sphingomyelin is free from cholesterol and does not bind OlyA. A point mutation abolishes OlyA's ability to discriminate between these two conformations. In cells, levels of sphingomyelin/cholesterol complexes are held constant over a wide range of plasma membrane cholesterol concentrations, enabling precise regulation of the chemical activity of cholesterol.

Retrospective on Cholesterol Homeostasis: The Central Role of Scap.

Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.

A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells.

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

Epigenetic perspectives associated with COVID-19 infection and related cytokine storm: an updated review.

PURPOSE: The COVID-19 pandemic caused by the novel Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has put the world in a medical crisis for the past three years; nearly 6.3 million lives have been diminished due to the virus outbreak. This review aims to update the recent findings on COVID-19 infections from an epigenetic scenario and develop future perspectives of epi-drugs to treat the disease. METHODS: Original research articles and review studies related to COVID-19 were searched and analyzed from the Google Scholar/PubMed/Medline databases mainly between 2019 and 2022 to brief the recent work. RESULTS: Numerous in-depth studies of the mechanisms used by SARS-CoV-2 have been going on to minimize the consequences of the viral outburst. Angiotensin-Converting Enzyme 2 receptors and Transmembrane serine protease 2 facilitate viral entry to the host cells. Upon internalization, it uses the host machinery to replicate viral copies and alter the downstream regulation of the normal cells, causing infection-related morbidities and mortalities. In addition, several epigenetic regulations such as DNA methylation, acetylation, histone modifications, microRNA, and other factors (age, sex, etc.) are responsible for the regulations of viral entry, its immune evasion, and cytokine responses also play a major modulatory role in COVID-19 severity, which has been discussed in detail in this review. CONCLUSION: Findings of epigenetic regulation of viral pathogenicity open a new window for epi-drugs as a possible therapeutical approach against COVID-19.

Identification of a degradation signal at the carboxy terminus of SREBP2: A new role for this domain in cholesterol homeostasis.

Lipid homeostasis in animal cells is maintained by sterol regulatory element-binding proteins (SREBPs), membrane-bound transcription factors whose proteolytic activation requires the cholesterol-sensing membrane protein Scap. In endoplasmic reticulum (ER) membranes, the carboxyl-terminal domain (CTD) of SREBPs binds to the CTD of Scap. When cholesterol levels are low, Scap escorts SREBPs from the ER to the Golgi, where the actions of two proteases release the amino-terminal domains of SREBPs that travel to the nucleus to up-regulate expression of lipogenic genes. The CTD of SREBP remains bound to Scap but must be eliminated so that Scap can be recycled to bind and transport additional SREBPs. Here, we provide insights into how this occurs by performing a detailed molecular dissection of the CTD of SREBP2, one of three SREBP isoforms expressed in mammals. We identify a degradation signal comprised of seven noncontiguous amino acids encoded in exon 19 that mediates SREBP2's proteasomal degradation in the absence of Scap. When bound to the CTD of Scap, this signal is masked and SREBP2 is stabilized. Binding to Scap requires an arginine residue in exon 18 of SREBP2. After SREBP2 is cleaved in Golgi, its CTD remains bound to Scap and returns to the ER with Scap where it is eliminated by proteasomal degradation. The Scap-binding motif, but not the degradation signal, is conserved in SREBP1. SREBP1's stability is determined by a degradation signal in a different region of its CTD. These findings highlight a previously unknown role for the CTD of SREBPs in regulating SREBP activity.

Cholesterol access in cellular membranes controls Hedgehog signaling.

The Hedgehog (Hh) signaling pathway coordinates cell-cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function.

Ostreolysin A and anthrolysin O use different mechanisms to control movement of cholesterol from the plasma membrane to the endoplasmic reticulum.

Recent studies using two cholesterol-binding bacterial toxin proteins, perfringolysin O (PFO) and domain 4 of anthrolysin O (ALOD4), have shown that cholesterol in the plasma membranes (PMs) of animal cells resides in three distinct pools. The first pool comprises mobile cholesterol, accessible to both PFO and ALOD4, that is rapidly transported to the endoplasmic reticulum (ER) to signal cholesterol excess and maintain cholesterol homeostasis. The second is a sphingomyelin (SM)-sequestered pool inaccessible to PFO and ALOD4 but that becomes accessible by treatment with SM-degrading sphingomyelinase (SMase). The third is an essential pool also inaccessible to PFO and ALOD4 that cannot be liberated by SMase treatment. The accessible cholesterol pool can be trapped on PMs of live cells by nonlytic ALOD4, blocking its transport to the ER. However, studies of the two other pools have been hampered by a lack of available tools. Here, we used ostreolysin A (OlyA), which specifically binds SM/cholesterol complexes in membranes, to study the SM-sequestered cholesterol pool. Binding of nonlytic OlyA to SM/cholesterol complexes in PMs of live cells depleted the accessible PM cholesterol pool detectable by ALOD4. Consequently, transport of accessible cholesterol from PM to ER ceased, thereby activating SREBP transcription factors and increasing cholesterol synthesis. Thus, OlyA and ALOD4 both control movement of PM cholesterol, but through different lipid-binding mechanisms. We also found that PM-bound OlyA was rapidly internalized into cells, whereas PM-bound ALOD4 remained on the cell surface. Our findings establish OlyA and ALOD4 as complementary tools to investigate cellular cholesterol transport.

Pharmacogenomic phase transition from personalized medicine to patient-centric customized delivery.

Personalized medicine has been a booming area in clinical research for the past decade, in which the detailed information about the patient genotype and clinical conditions were collected and considered to optimize the therapy to prevent adverse reactions. However, the utility of commercially available personalized medicine has not yet been maximized due to the lack of a structured protocol for implementation. In this narrative review, we explain the role of pharmacogenetics in personalized medicine, next-generation personalized medicine, i.e., patient-centric personalized medicine, in which the patient's comfort is considered along with pharmacogenomics to be a primary factor. We extensively discuss the classifications, strategies, tools, and drug delivery systems that can support the implementation of patient-centric personalized medicine from an industrial perspective.

How Collaborative Mentoring Networks Are Building Capacity in Primary Care.

The need for increased capacity in primary care to treat the growing numbers of patients with complex chronic health conditions is well established (Roberts et al. 2015). Meeting that need requires not only more family physicians but also more support and resources to handle challenging cases. The Collaborative Mentoring Networks (CMNs), created in 2001 by the Ontario College of Family Physicians and funded by the Ontario government, have provided that support and proven particularly successful in improving physicians' competence and confidence in caring for patients struggling with mental health, addictions and chronic pain. The networks give family physicians timely, ongoing access to mentors with greater clinical expertise. In 2017, the networks expanded from two to seven, spreading support to palliative and end-of-life care and medical assistance in dying and focusing on leadership in primary care, early years in practice and rural medicine. CMNs' early impact involved increased primary care capacity in family practice, better-supported family physicians treating more patients with complex conditions, fewer specialist referrals, less isolation and greater retention.

Cholesterol-induced conformational changes in the sterol-sensing domain of the Scap protein suggest feedback mechanism to control cholesterol synthesis.

Scap is a polytopic protein of endoplasmic reticulum (ER) membranes that transports sterol regulatory element-binding proteins to the Golgi complex for proteolytic activation. Cholesterol accumulation in ER membranes prevents Scap transport and decreases cholesterol synthesis. Previously, we provided evidence that cholesterol inhibition is initiated when cholesterol binds to loop 1 of Scap, which projects into the ER lumen. Within cells, this binding causes loop 1 to dissociate from loop 7, another luminal Scap loop. However, we have been unable to demonstrate this dissociation when we added cholesterol to isolated complexes of loops 1 and 7. We therefore speculated that the dissociation requires a conformational change in the intervening polytopic sequence separating loops 1 and 7. Here we demonstrate such a change using a protease protection assay in sealed membrane vesicles. In the absence of cholesterol, trypsin or proteinase K cleaved cytosolic loop 4, generating a protected fragment that we visualized with a monoclonal antibody against loop 1. When cholesterol was added to these membranes, cleavage in loop 4 was abolished. Because loop 4 is part of the so-called sterol-sensing domain separating loops 1 and 7, these results support the hypothesis that cholesterol binding to loop 1 alters the conformation of the sterol-sensing domain. They also suggest that this conformational change helps transmit the cholesterol signal from loop 1 to loop 7, thereby allowing separation of the loops and facilitating the feedback inhibition of cholesterol synthesis. These insights suggest a new structural model for cholesterol-mediated regulation of Scap activity.

Augmented anticancer activity of naringenin-loaded TPGS polymeric nanosuspension for drug resistive MCF-7 human breast cancer cells.

Naringenin (NAR) is a naturally occurring plant flavonoid, found predominantly in citrus fruits, possesses a wide range of pharmacological properties. However, despite the therapeutic potential of NAR, its clinical development has been hindered due to low aqueous solubility and inefficient transport across biological membranes resulting in low bioavailability at tumor sites. In our previous studies, nanosuspension of naringenin (NARNS) was prepared using high pressure homogenization method using different polymers. D-α-Tocopheryl polyethylene glycol succinate 1000 (TPGS) was added as a co-stabilizer. All formulation characterization studies were performed. As a continuation of our previous research, current study has further evaluated the ability of the TPGS-coated NARNS, to reverse drug-resistance of P-gp-over expressing MCF-7 human breast adenocarcinoma cell line and animal model. MTT-based colorimetric assay revealed higher cytotoxic efficacy of NARNS than free NAR in MCF-7 cells. NARNS treatment significantly increased intracellular ROS level, mitochondrial membrane potential, caspase-3 activity, lipid peroxidation status (TBARS) and decreased GSH levels when compared to free NAR treatment in MCF-7 cells. It has been also noticed that the presence of apoptotic indices (membrane blebbing, nuclear fragmentation) in NARNS treated cancer cells. Further, NARNS exhibited dose-dependent in vitro antitumor activity with DLA cells. A significant increase in the life span and a decrease in the cancer cell number and tumor weight were noted in the tumor-induced mice after treatment with NARNS.

Direct Demonstration That Loop1 of Scap Binds to Loop7: A CRUCIAL EVENT IN CHOLESTEROL HOMEOSTASIS.

Cholesterol homeostasis is mediated by Scap, a polytopic endoplasmic reticulum (ER) protein that transports sterol regulatory element-binding proteins from the ER to Golgi, where they are processed to forms that activate cholesterol synthesis. Scap has eight transmembrane helices and two large luminal loops, designated Loop1 and Loop7. We earlier provided indirect evidence that Loop1 binds to Loop7, allowing Scap to bind COPII proteins for transport in coated vesicles. When ER cholesterol rises, it binds to Loop1. We hypothesized that this causes dissociation from Loop7, abrogating COPII binding. Here we demonstrate direct binding of the two loops when expressed as isolated fragments or as a fusion protein. Expressed alone, Loop1 remained intracellular and membrane-bound. When Loop7 was co-expressed, it bound to Loop1, and the soluble complex was secreted. A Loop1-Loop7 fusion protein was also secreted, and the two loops remained bound when the linker between them was cleaved by a protease. Point mutations that disrupt the Loop1-Loop7 interaction prevented secretion of the Loop1-Loop7 fusion protein. These data provide direct documentation of intramolecular Loop1-Loop7 binding, a central event in cholesterol homeostasis.

Use of mutant 125I-perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells.

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study, we generate an (125)I-labeled mutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to (125)I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The (125)I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines, we show that this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Switch-like responses of two cholesterol sensors do not require protein oligomerization in membranes.

Many cellular processes are sensitive to levels of cholesterol in specific membranes and show a strongly sigmoidal dependence on membrane composition. The sigmoidal responses of the cholesterol sensors involved in these processes could arise from several mechanisms, including positive cooperativity (protein effects) and limited cholesterol accessibility (membrane effects). Here, we describe a sigmoidal response that arises primarily from membrane effects due to sharp changes in the chemical activity of cholesterol. Our models for eukaryotic membrane-bound cholesterol sensors are soluble bacterial toxins that show an identical switch-like specificity for endoplasmic reticulum membrane cholesterol. We show that truncated versions of these toxins fail to form oligomers but still show sigmoidal binding to cholesterol-containing membranes. The nonlinear response emerges because interactions between bilayer lipids control cholesterol accessibility to toxins in a threshold-like fashion. Around these thresholds, the affinity of toxins for membrane cholesterol varies by >100-fold, generating highly cooperative lipid-dependent responses independently of protein-protein interactions. Such lipid-driven cooperativity may control the sensitivity of many cholesterol-dependent processes.

Adaptive Mentoring Networks and Compassionate Care: A Qualitative Exploration of Mentorship for Chronic Pain, Substance Use Disorders and Mental Health.

This study undertook an exploration of how Adaptive Mentoring Networks focusing on chronic pain, substance use disorders and mental health were supporting primary care providers to engage in compassionate care. The study utilised the Cole-King & Gilbert Compassionate Care Framework to guide qualitative semi-structured interviews of participants in two Adaptive Mentoring Networks in Ontario, Canada. Fourteen physician participants were interviewed including five mentors (psychiatrists) and nine mentees (family physicians) in the Networks. The Cole-King & Gilbert Framework helped provide specific insights on how these mentoring networks were affecting the attributes of compassion such as motivation, distress-tolerance, non-judgement, empathy, sympathy, and sensitivity. The findings of this study focused on the role of compassionate provider communities and the development of skills and attitudes related to compassion that were both being supported in these networks. Adaptive Mentoring Networks can support primary care providers to offer compassionate care to patients with chronic pain, substance use disorders, and mental health challenges. This study also highlights how these networks had an impact on provider resiliency, and compassion fatigue. There is promising evidence these networks can support the "quadruple aim" for healthcare systems (improve patient and provider experience, health of populations and value for money) and play a role in addressing the healthcare provider burnout and associated health workforce crisis.

DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Implementation of explainable artificial intelligence in commercial communication systems using micro systems.

The developments in the field of artificial intelligence (AI) and decision making systems are identified as virtuous models for banking and finance sector (BFS) applications. Even though AI provides great advantage in application changes it is essential to remodel the system using explainable artificial intelligence (XAI) design system. Also the standard sensing models provides appropriate monitoring values but huge size of sensors is considered as a major drawback. Thus two diverse problems are addressed in this research work where XAI has been integrated with micro electro-mechanical systems (MEMS) for solving the problems related to BFS applications. Further the data security has been enhanced as XAI is implemented with conviction managements and real time experiments are carried out by developing a unique application by integrating new mathematical designs. To validate the effectiveness of BFS application the developed model is tested with five scenarios which includes multiple parametric arrangements with interpretability process. The tested and compared outcomes with existing models indicates that XAI and MEMS provides inordinate improvements in terms of data impairments thus increasing the transparency of the projected system to an average 96%.