Biology subjects

Setty, S. R. G.

Publications and source records attributed to Setty, S. R. G..

4 recordsLinked to original sources

bioRxiv2025

Interaction and co-phase separation of SARS-CoV-2 nucleocapsid protein and human hnRNPA1 and its implications for viral life cycle

The nucleocapsid (N) protein of SARS-CoV-2 is central to viral assembly and replication. It binds viral RNA to form a helical nucleocapsid and enabling genome packaging and release into host cells. Human hnRNPA1, one of the most abundant RNA-binding proteins in eukaryotes, regulates key aspects of RNA metabolism, including splicing, transcription, localisation, and transport. Here, we report a direct physical interaction between SARS-CoV-2 N protein and hnRNPA1 with moderate affinity (Kd [~] 0.18 M), primarily mediated through their intrinsically disordered regions. Furthermore, we found that these proteins co-phase separate in vitro and co-localise within stress granules in cells. In vivo studies reveal that hnRNPA1 suppresses viral replication, suggesting that the N protein - hnRNPA1 interaction plays an important role in modulating the viral life cycle.

biochemistry↗

bioRxiv2025

STX1A localizes to the lysosome and controls its exocytosis

Lysosome exocytosis is one of the critical functions of lysosomes in maintaining cellular homeostasis and plasma membrane repair. The SNAREs regulating the lysosome fusion with the cell surface at the basal level have been poorly defined. Here, we identified STX1A, a Qa- SNARE localized majorly to lysosomes and a cohort to the plasma membrane in HeLa cells. Overexpression of GFP-STX1A in HeLa cells causes decreased lysosome number and their peripheral dispersion. In line, STX1A knockdown resulted in the accumulation of lysosomes beneath the cell surface and showed reduced lysosome exocytosis in HeLa cells. TIRF imaging microscopy demonstrated an enhanced enrichment of LAMP1-positive vesicles at the cell surface in STX1A depleted compared to control cells. Moreover, STX1A depletion reduces the proteolytic activity without affecting the lysosome content or acidity and increases peripheral lysosome dispersion compared to control cells. Consistently, these cells display an enhanced number of autophagosomes and accumulate autolysosomes. Functionally, GFP-STX1A also localizes to LLOMe-induced GAL3-positive damaged lysosomes and reduces their number by enhancing exocytosis. Biochemically, STX1A forms a SNARE complex with SNAP23 or SNAP25 (Qbc) and VAMP2 (R), and their knockdown in HeLa cells mimics the STX1A- depletion phenotypes. Overall, these studies demonstrate a unique function of STX1A in regulating lysosomal exocytosis by localizing to these degradative organelles. Key pointsO_LISTX1A localizes to lysosomes and plasma membrane in HeLa cells C_LIO_LISTX1A overexpression decreases the lysosome number and their dispersion and facilitates the exocytosis of LLOMe-damaged lysosomes C_LIO_LISTX1A knockdown enhances the lysosome dispersion, accumulates beneath the cell surface in HeLa cells, and displays decreased lysosome exocytosis C_LIO_LISTX1A biochemically interacts with SNAP23/SNAP25, VAMP2 and their individual knockdown phenocopy their phenotypes each other C_LI

cell biology↗

bioRxiv2022

Golgi localized Arl15 regulates cargo transport, cell adhesion and motility

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Recent studies indicate a possible role for Arl15 in multiple physiological processes, including magnesium homeostasis. However, the molecular function of Arl15 is poorly defined. We evaluated the role of Arl15 in vesicular transport using techniques to quantify cargo trafficking, to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization to the Golgi and cell surface, including filopodia, and a cohort to recycling endosomes. The dissociation of Golgi, using small molecular inhibitors or the expression of Arf1 dominant-negative mutant, completely mislocalized Arl15 to the cytosol. Interestingly, site-directed mutagenesis analysis identified a novel V80A mutation in the GTP-binding domain that turns Arl15 into a dominant-negative form with reduced number of filopodia. Depletion of Arl15 in HeLa cells caused mislocalization of cargo, such as caveolin-2 and STX6, from the Golgi. Arl15 knockdown cells displayed reduced filopodial number, altered focal adhesion kinase organization, and enhanced soluble and receptor-mediated cargo uptake without affecting the TfR recycling. Arl15 knockdown decreased cell migration and enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion as compared to control cells. Collectively, these studies demonstrated a functional role for Arl15 in the Golgi, which includes regulating cargo transport to organize membrane domains at the cell surface. Key pointsO_LIArl15 primarily localizes to Golgi and plasma membrane, including filopodia C_LIO_LIMembrane localization of Arl15 is dependent on Golgi integrity or Arf1 activation C_LIO_LIArl15 knockdown mislocalizes STX6-dependent Golgi localized cargo required for cell surface organization and reduces the filopodial number C_LIO_LIArl15 is involved in cell spreading, adhesion, and migration C_LI

cell biology↗

bioRxiv2022

Restoration of beta-GC trafficking improves the lysosome function in Gaucher disease

Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic route. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gauchers disease (GD), the lysosomes accumulate glucosylceramide due to a lack of {beta}-glucocerebrosidase ({beta}-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant {beta}-GC to lysosomes may delay the progression of GD. RNAi screen using high throughput based lysosomal enzyme activity assay followed by reporter trafficking assay utilizing {beta}-GC-mCherry lead to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the {beta}-GC activity by increasing the folding and trafficking of Gauchers mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their function upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity possibly delays the GD and forms an alternative therapeutic strategy for this genetic disease. Key pointsO_LIPhosphatome RNAi screen identified both activators and inhibitors of cellular glucocerebrosidase activity C_LIO_LIDepletion of selective phosphatases in HeLa cells improved the folding and trafficking of mutant {beta}-glucocerebrosidase to lysosomes C_LIO_LIKnockdown of selective phosphatases restored the low basal {beta}-glucocerebrosidase activity to that of wild-type in primary cells derived from Gauchers disease patients C_LIO_LIDepletion of selective phosphatases displayed variable {beta}-GC activity in neuropathic and non-neuropathic Gauchers disease patient cells C_LI

cell biology↗