OSBPL9

Scientists have found how human cells distribute and maintain their cholesterol levels, aiding in research in neurodegenerative diseases such as Alzheimer's disease, as well as cardiovascular diseases. Scientists at Nanyang Technological University, Singapore (NTU Singapore) have found how human cells distribute and maintain their cholesterol levels, aiding in research in neurodegenerative diseases such as Alzheimer's disease, as well as cardiovascular diseases. All the cells in the human body contain cholesterol, a waxy, fat-like substance and use it for vital body functions, such as building new cells, producing hormones, and even producing substances that help fight against pathogens. Maintaining normal cholesterol levels within the central nervous system is essential for various processes, including the brain's development and daily functions. The cells in our body either produce cholesterol or acquire it through our diet and maintaining the appropriate levels and distribution of cholesterol within our cells is crucial, as failure to do so can lead to various diseases, including heart attacks and dementia. Using a highly sensitive cholesterol probe in tests involving human cells, the team identified the key proteins involved in regulating and transporting cholesterol within cells, called OSBP1, ORP92, and gram domain-containing proteins 1 (GRAMD1s). The study provides new insights into the precise mechanisms for maintaining cholesterol distribution within cells. Lead author Associate Professor Yasunori Saheki, from NTU Singapore's Lee Kong Chian School of Medicine (LKCMedicine), who led the study, said: "Our findings shed light on the critical mechanisms underlying cellular cholesterol distribution and their potential implications for various health conditions. This offers critical insights into the mechanisms underlying the maintenance of cellular cholesterol distribution. The study is of particular significance as disruptions in this process have been strongly associated with a wide range of neurodegenerative disorders, including Alzheimer's disease." Assoc Prof Saheki is also a cell biologist and a medical doctor. The study's first author Dr Tomoki Naito, Research Fellow from NTU LKCMedicine, said: "While cholesterol is essential for our cells, the mechanisms governing its distribution have long been a mystery. We found critical regulators of cholesterol distribution within our cells. Our findings mark a major milestone in fundamental cell biology." The study, which represents an advance in understanding the underlying biology of how the human body functions and becomes susceptible to diseases, reflects NTU's commitment to responding to the needs and challenges of healthy living and ageing, which is one of four humanity's grand challenges that the University seeks to address through its NTU 2025 strategic plan. The findings were published in the scientific journal Nature Communications in September. Everything in moderation, including moderation Through using a cholesterol biosensor to measure levels of cholesterol, the researchers found that the proteins called ORP9 and OSBP regulate the distribution and abundance of cholesterol within the cell. They found that when ORP9 is deactivated, OSBP becomes hyperactive, resulting in an excess of cholesterol transported to other parts of the cell. The team also found that the GRAMD1 protein was responsible for regulating the distribution of cholesterol in the cell, as they observed that cells without this specific protein had an abnormal distribution of cholesterol in crucial parts of the cell. Assoc Prof Saheki explained: "A build-up of cholesterol inside cells might harm them by disrupting their normal functions. Too much cholesterol inside cells can also lead to them being oxidised into oxysterols, which can be toxic to cells. Not just that -- regulating the amount of cholesterol is important as it affects the flexibility and fluidity of cell membranes. A healthy and low amount of cholesterol keeps them flexible and fluid, while a higher cholesterol level makes them stiff. When dysregulation of cholesterol takes place in brain cells, it has been linked to diseases such as Alzheimer's disease and dementia. In the cardiovascular system, it could lead to heart attacks." Dr Naito added: "We are working hard to determine how the cholesterol regulators we identified in this study affect human health. I hope our future research findings will contribute to developing new therapeutic approaches to tackle diseases that arise from cholesterol dysregulation." The NTU team will be conducting further research to explore how to adjust the activities of these proteins within damaged cells to help them better regulate cholesterol, possibly leading to therapies that could one day help with dementia and heart attacks.

With seven known human coronaviruses and many others still resident in animal populations, the risk of interspecies transmission and the emergence of new strains affecting humans is an ever-present danger. Yet, more than one year into the global COVID-19 pandemic, prevention and treatment options are limited. Broad-spectrum anti-coronavirus could address the great medical needs and help contain future outbreaks resulting from interspecies transmission. Identifying the common host factors that are essential for coronavirus replication, therefore, is an important element in developing broad-spectrum, antiviral therapeutics. A genome-wide CRISPR study, reported in Nature Genetics and conducted by a research group from the Rega Institute and the Vlaams Instituut voor Biotechnologie, both in Belgium, identified both virus-specific and shared host factors that may be exploited for drug development against SARS-CoV-2 and the human coronavirus 229E (one of the viruses responsible for the common cold). “We discovered that SARS-CoV-2 requires the lysosomal protein TMEM106B to infect human cell lines and primary lung cells,” the researchers wrote. “TMEM106B overexpression enhanced SARS-CoV-2 infection as well as pseudovirus infection, suggesting a role in viral entry.” The expression of that protein correlates with COVID-19 infection, the researchers concluded after examining the results of single-cell RNA sequencing from the airway cells of COVID-19 patients. “TMEM106B is a 274-amino acid transmembrane protein that resides in endosomes and lysosomes, controlling lysosome size, number, mobility, and trafficking,” the paper explained. “TMEM106B is poorly characterized and only recently received attention because of its role in frontotemporal dementia, the second leading cause of pre-senile neurodegeneration.” Previous research characterized the entry step of coronaviruses, but later steps in the viral life cycle, including receptor binding, endocytosis, fusion, viral protein translation, genome replication, virion assembly, and release, are less well-understood. Those functions each may offer opportune targets for drug development. Using CRISPR-based genetic screens, the researchers, including corresponding authors Jim Baggen and Dirk Daelemans, also “identified phosphoinositide 3-kinase (PI3K) type 3 as a common host factor for SARS-CoV-2, HCoV-229E, and HCoV-OC43.” The latter is involved in the common cold as well as encephalitis. Targeting PI3K, therefore, could potentially inhibit infections by those coronaviruses. The researcher team “performed a CRISPR-based genome-wide knockout screen in the human Huh7 cell line, without introducing an exogenous receptor, because our SARS-CoV-2 strain induced a clear cytopathic effect in these cells,” the paper explained. In addition to finding the “significantly enriched” TMEM106B gene encoding the “poorly characterized” TMEM106B protein and the PI3K host factor, a low-stringency screen identified a large number of other enriched genes. Although many of those genes were discounted, a handful stood out. They included the PIK3C3 gene, which is involved in endocytic trafficking and autophagy; TMEM41B, involved in autophagy; EXT1 and ITGB6, each of which is required for cell surface receptor expression; OSBPL9, PTDSS1, and TMEM30A, which are involved in lipid homeostasis; and CCZ1B, PTPN23, and RSG1, which are involved in membrane and/or vesicle trafficking. Validation showed the knockout of TMEM106B caused the strongest resistance to SARS-CoV. sgRNAs targeting PIK3C3, TMEM41B, EXT1, OSBPL9, and TMEM30A, also exhibited significant resistance, the researchers noted. Notably, some genes were resistant to the viruses only when using a pool of sgRNAs. Knocking out EXT1 – but not ITGB6 – also was protective, the paper noted. Additionally, it pointed out that EXT1 encodes exostosis-1, which is involved in heparan sulfate synthesis. Therefore, the researchers suggested, as others also have noted, that heparan sulfate may help control COVID-19. No one gene holds the key to preventing infection by SARS-CoV-2, of course. For a therapy to be most effective, therefore, it should involve TMEM106B and also “the genes implicated in heparan sulfate synthesis (EXT1), autophagy (PIK3C3 and TMEM41B), and lipid transport (OSBPL9 and TMEM30A),” the team noted. Additional experiments determined that TMEM106B is “a bona fide proviral host factor required for productive SARS-CoV2 infection of human cells.” It is clearly implicated in spike-mediated entry into human cells. While this research was underway, six other groups performed similar genome-wide CRISPR screens that identify host factors for SARS-CoV-2 replication. Those studies involved multiple different host cells. Not surprisingly, screens involving the same cell types have the greatest similarly of results. Of the 50 genes most often identified in the studies, 33 were identified in two or more screens, and eight of those were identified in the Belgian study. Perhaps more pertinently, four of the screens identified ACE2. Three identified CTSL, in addition to the EXT1, ARPC4, and TMEM106B genes identified by the Belgian group, thus independently confirming those results. Two other studies examined coronavirus replication. They also identified the ANPEP, PIK3C3, and TMEM41B genes as being pivotal to replication. The authors suggest that TMEM106B was not found in the A549 screens because it, perhaps, “might not be required when cells are saturated with high ACE2 levels,” adding that ACE2 levels “may have been lower in our cells” than in the A549 cells used in those screens. Likewise, TMEM106B was not identified in VeroE6 cells, suggesting they may be specific to human cells and not, therefore, present in the African green monkeys from which the Vero E6 cells were extracted. The importance of TMEM106B expression in the infection pathway for SARS-CoV-2 could allow it to serve as a prognostic biomarker for COVID-19 severity, the researchers speculated, based on the possibility that overexpression enabled the virus to enter normal cells. Alternatively, the higher expression levels of TMEM106B may be caused by the SARS-CoV-2 infection itself, they wrote. A third option is that TMEM106B may promote endosomal acidification, easing the entry of the SARS-CoV-2 genome into a cell’s cytoplasm. Clearly, much more remains to be learned. These nine studies, taken together, suggest that SARS-CoV-2 may involve more cellular receptors than initially believed. Therefore, multiple screens and combined data will be vital to building a comprehensive understanding of the host factors and their dependencies in the context of SARS-CoV-2 and other coronavirus infections.