Biology subjects

Sedlyarov, V.

Publications and source records attributed to Sedlyarov, V..

7 recordsLinked to original sources

bioRxiv2025

Genetic identification of the RAS proteostatic machinery and its failure to regulate oncogenic variants

The regulation of cellular homeostasis, differentiation, and proliferation is safeguarded by proteostatic mechanisms, which are crucial for maintaining cellular function. We used endogenously affinity-tagged KRAS cells in a fluorescence-activated cell sorting (FACS)-based CRISPR knockout screen to map genome-wide genetic requirements for proteostatic KRAS regulation. Three regulatory modules emerged, specifically cullin E3 ligase activity (CUL3, NAE1, UBE2M, CAND1, and UBE2L3), LZTR1 protein function (LZTR1, NUDCD3, and ZRSR2), and RAS GTPase modulation and processing (NRAS, HRAS, FNTB, RCE1, ICMT, and GOLGA7), all critical for regulating KRAS abundance. This expands our knowledge on the machinery controlling ubiquitin-meditated RAS family member regulation. Combining endogenous affinity tagging with genetic variant introduction, we found that the oncogenic KRAS G12D mutant exhibits reduced regulation by CRL3LZTR1, potentially contributing to oncogenic transformation and proliferation. In summary, our study genetically defines the machinery regulating RAS GTPase protein abundance, providing a foundation for a deeper molecular understanding and potential therapeutic exploitation of CRL3LZTR1-RAS GTPase regulation in human disease.

cell biology↗

bioRxiv2024

Data- and knowledge-derived functional landscape of human solute carriers

Research on the understudied solute carrier (SLC) superfamily of membrane transporters would greatly profit from a comprehensive knowledgebase, synthesizing data and knowledge on different aspects of SLC function. We consolidated multi-omics data sets with selected curated information from the public domain, such as structure prediction, substrate annotation, disease association and subcellular localization. This SLC-centric knowledge is made accessible to the scientific community via a web portal, featuring interactive dashboards and a tool for family-wide, tree-based visualization of SLC properties. Making use of the systematically collected and curated data sets, we selected eight feature-dimensions to compute an integrated functional landscape of human SLCs. This landscape represents various functional aspects, harmonizing local and global features of the underlying data sets, as demonstrated by inspecting structural folds and subcellular locations of selected transporters. Based on all available data sets and their integration, we assigned a biochemical/biological function to each SLC, making it one of the largest systematic annotations of human gene function and likely acting as a blueprint for future endeavours.

systems biology↗

bioRxiv2024

Metabolic mapping of the human solute carrier superfamily

Solute carrier (SLC) transporters govern most of the chemical exchange across cellular membranes and are integral to metabolic regulation, which in turn is linked to cellular function and identity. Despite their key role, individual functions of the members of the SLC superfamily were not evaluated systematically. We determined the metabolic and transcriptional profiles upon SLC overexpression in knock-out or wild-type isogenic cell backgrounds. Targeted metabolomics provided a fingerprint of 189 intracellular metabolites, while transcriptomics offered insights into cellular programs modulated by SLC expression. Beyond the metabolic profiles of 102 SLCs directly related to their known substrates, we also identified putative substrates or metabolic pathway connections for 71 SLCs without previously annotated bona fide substrates, including SLC45A4 as a new polyamine transporter. By comparing the molecular profiles, we identified functionally related SLC groups, including some with distinct impacts on osmolyte balancing and glycosylation. The assessment of functionally related human genes presented here may serve as a blueprint for other systematic studies of human gene function and supports future investigations into the functional roles of SLCs.

systems biology↗

bioRxiv2022

A genome-wide CRISPR functional survey of the human phagocytosis molecular machinery

Phagocytosis, the process of engulfing large particles by cells, is a multilayered biological activity driving tissue clearance and host defense. Dysregulation of phagocytosis is connected to autoimmunity, accumulation of toxic disease proteins, and increased risks for infections. Despite its importance and multiple roles, we lack a full understanding of the cellular machinery involved in executing and regulating the process, including the coordination with other cellular events. To create a functional map in human cells, we performed a reporter- and FACS-based genome-wide CRISPR/Cas9 knock-out screen that identified 716 genes. Mapping the gene hits to a comprehensive protein-protein interaction network annotated for functional cellular processes, allowed to highlight those protein complexes identified multiple times, to identify missing components of the cellular phagocytosis network, and to suggest functional partition among complexes. We validate complexes known to be involved, such as the Arp2/3 complex, the vacuolar-ATPase-Rag machinery, and the Wave-2 complex, as well as processes previously not or only poorly associated with phagocytosis. Among the novel, phagocytosis-relevant cellular functions validated are the oligosaccharyltransferase complex (MAGT1/SLC58A1, DDOST, STT3B, and RPN2) as well as the hypusine pathway (eIF5A, DHPS, and DOHH). Overall, our network of phagocytosis regulators and effectors maps elements of cargo uptake, cargo shuffling and cargo biotransformation through the cell, providing a valuable resource for the identification of potential novel drivers for diseases of the endo-lysosomal system. We further propose that our approach of mining and integrating publicly available protein-protein interaction data with datasets derived from reporter-based genome-wide screens offers a broadly applicable way to functionally map biological processes onto the molecular machinery of the cell. Summary blurbThe validation and interpretation of a FACS reporter-based genome-wide CRISPR/Cas9 knock-out screen through protein-protein interaction data yields a comprehensive view of the molecular network regulating and executing phagocytosis in human cells.

genomics↗

bioRxiv2022

Gain-of-function genetic screens in human cells identify SLC transporters overcoming environmental nutrient restrictions

Solute carrier (SLC) transporters control fluxes of nutrients and metabolites across membranes and thereby represent a critical interface between the microenvironment and cellular and subcellular metabolism. Because of substantial functional overlap, the interplay and relative contributions of members of this family in response to environmental stresses remain poorly elucidated. In order to infer functional relationships between SLCs and metabolites, we developed a strategy to identify human SLCs able to sustain cell viability and proliferation under growth-limiting concentrations of essential nutrients. One-by-one depletion of 13 amino acids required for cell proliferation enabled gain-of-function genetic screens using a SLC-focused CRISPR/Cas9-based transcriptional activation approach to uncover transporters relieving cells from the growth-limiting metabolic bottleneck. We identified the cationic amino acid transporter SLC7A3 as a gene that, when upregulated, overcame low availability of arginine and lysine by increasing their uptake. SLC7A5 (LAT1), on the other hand, was able to sustain cellular fitness upon deprivation of several neutral amino acids. A genome-wide screen identified SLC7A3 as the single main gene product able to rescue cell survival in the limiting arginine conditions tested, demonstrating the potentially decisive role of transporters in overcoming nutrient limitations. Moreover, we identified metabolic compensation mediated by the glutamate/aspartate transporters SLC1A2 and SLC1A3 under glutamine-limiting conditions. Overall, this gain-of-function approach using human cells led to the definition of functional transporter-nutrient relationships and revealed that upregulation of transport activity may be sufficient to overcome environmental metabolic restrictions.

cell biology↗

bioRxiv2022

PCYT2 controls muscle health and muscle aging

Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing, affecting hundreds of millions of people. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases represents an important goal in improving human health. Here, we show that phosphatidylethanolamine cytidyltransferase (PCYT2/ECT), the critical enzyme of the Kennedy branch of phosphatidylethanolamine (PE) synthesis pathway, has an essential role in muscle health. Human genetic deficiency in PCYT2 causes a severe disease with failure to thrive and progressive muscle weakness. Pcyt2 mutant zebrafish recapitulate the patient phenotypes, indicating that the role of PCYT2/PE in muscle is evolutionary conserved. Muscle specific Pcyt2 knockout mice exhibited failure to thrive, impaired muscle development, progressive muscle weakness, muscle loss, accelerated ageing, and reduced lifespan. Mechanistically, Pcyt2 deficiency affects mitochondrial bioenergetics and physicochemical properties of the myofiber membrane lipid bilayer, in particular under exercise strain. We also show that PCYT2 activity declines in the aging muscles of humans and mice. AAV-based delivery of PCYT2 rescued muscle weakness in Pcyt2 knock-out mice and, importantly, improved muscle strength in old mice, offering a novel therapeutic avenue for rare disease patients and muscle aging. Thus, PCYT2 plays a fundamental, specific, and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2 synthesized PE lipids to severe muscle dystrophy, exercise intolerance and aging.

genetics↗

bioRxiv2020

A systematic genetic interaction map of human solute carriers assigns a role to SLC25A51/MCART1 in mitochondrial NAD uptake

Solute Carriers (SLCs) represent the largest family of human transporter proteins, consisting of more than 400 members1,2. Despite the importance of these proteins in determining metabolic states and adaptation to environmental changes, a large proportion of them is still orphan and lacks associated substrates1,3,4. Here we describe a systematic mapping of genetic interactions among SLCs in human cells. Network-based identification of correlated genetic interaction profile neighborhoods resulted in initial functional assignments to dozens of previously uncharacterized SLCs. Focused validation identified SLC25A51/MCART1 as the SLC enabling mitochondrial import of NAD(H). This functional interaction map of the human transportome offers a route for systematic integration of transporter function with metabolism and provides a blueprint for elucidation of the dark genome by biochemical and functional categories.

cell biology↗