Biology subjects

Shetty, M.

Publications and source records attributed to Shetty, M..

11 recordsLinked to original sources

bioRxiv2026

Bioenergetic and Proteomic Profiling of ARID1A-Deficient Ovarian Clear Cell Carcinoma under Metabolic Challenge

ARID1A loss is associated with increased mitochondrial respiration in ovarian clear cell carcinoma (OCCC), but whether this phenotype is maintained under metabolic challenge remains unclear. Here, we examined mitochondrial function and electron transport chain (ETC) protein abundance in isogenic ARID1A wild-type (WT) and ARID1A-knockout (KO) OCCC cells cultured under physiological glucose, galactose, or the Complex I inhibitor IACS-010759. Across all conditions, KO cells maintained higher basal, ATP-linked, and maximal respiration, greater spare respiratory capacity, and higher coupling efficiency than WT cells, despite lower steady-state ATP levels. In human OCCC tumors, ARID1A-negative cases showed a higher bioenergetic cellular (BEC) index than ARID1A-positive cases, and high BEC was associated with poorer cancer-specific survival. Quantitative proteomics revealed a trend toward higher ETC complex abundance in KO cells, although whole-complex differences did not reach statistical significance. ARID1A loss was associated with selective increases in Complex III and V proteins, whereas metabolic challenge was associated with increases in Complex I and IV proteins. Together, these findings show that the ARID1A-associated mitochondrial phenotype persists across distinct metabolic challenges and is not explained simply by coordinated increases in ETC protein abundance. The association of mitochondrial bioenergetics with clinical outcomes further supports its relevance in human OCCC.

cancer biology↗

bioRxiv2025

UNC-45A Drives ATP-Independent Microtubule Destabilization via Defect Recognition and Repair Inhibition Contributing to Neurite Dystrophy

UNC-45A is the only known ATP-independent microtubule severing protein. Using in vitro reconstitution and TIRF microscopy, we show that, unlike canonical severing enzymes such as katanin, spastin, and fidgetin, which hydrolyze ATP to remove tubulin dimers and promote lattice repair, UNC-45A selectively binds to pre-existing microtubule defects and inhibits GTP-tubulin incorporation. This mechanism prevents the formation of stabilized hot spots that typically protect microtubules from disassembly, resulting in persistent lattice damage and net microtubule loss, even in the presence of physiological levels of free GTP-tubulin. We further demonstrate that UNC-45A localizes near amyloid deposits in both mouse models and human cases of Alzheimers disease (AD). In cultured neurons, UNC-45A accumulates in axonal swellings--regions of pronounced microtubule disruption and experimental surrogates for dystrophic neurites in AD--and exacerbates their size and number, particularly under conditions mimicking microtubule damage. Notably, this is the first report of a microtubule severing protein that both localizes near amyloid plaques in tissue and accumulates in neurite swellings in cultured neurons, where it modulates their pathology. Together, our findings establish the mechanism of ATP-independent, damage-responsive severing pathway that couple defects recognition to repair inhibition, defining a new paradigm in microtubule quality control with broad implications for cytoskeletal integrity and remodeling in health and disease.

cell biology↗

bioRxiv2025

Tumor Cells Enriched for Interferon and Inflammatory Programs Pre-Exist in High Grade Serous Ovarian Cancer and are Proportionately Significantly Increased Post Chemotherapy

Drug-tolerant, high-grade serous ovarian cancer (HGSOC) cells that persist after first-line chemotherapy and subsequently relapse often retain sensitivity to secondary treatment, suggesting a therapeutic window before stable chemoresistance emerges. We performed single-cell RNA sequencing (scRNA-seq) on seven matched pairs of tumors, collected pre- and post-chemotherapy, to define vulnerabilities in these reversibly-resistant cells. Treatment induced a marked enrichment of tumor and stromal cell populations expressing correlated interferon (IFN) and inflammatory (IFM) gene signatures, with a concurrent depletion of proliferation-related and MYC-associated states in the tumor cells. Cross-cohort single cell sequencing analysis of >130 treatment-naive tumors revealed heterogeneity in the abundance of IFN/IFM-expressing cells. Multiplex immunofluorescence imaging of IFN-stimulated gene (ISG) products confirmed the presence of spatially clustered ISG-positive tumor cells in all cases, as well as in serous tubal intraepithelial carcinomas (STIC lesions), the presumptive HGSOC precursors. ISG expression correlated strongly with ORF1P, a protein encoded by the endogenous retrotransposon LINE1. These data suggest that early oncogenic events drive LINE1 derepression and innate immune activation, establishing an IFN-rich transcriptional state that persists in tumor subpopulations and is strongly enhanced by chemotherapy.

cancer biology↗

bioRxiv2025

Transient Suppression of Dopamine Transporter Palmitoylation by Methamphetamine: Implications for Transport Regulation

The dopamine transporter (DAT) exerts temporal and spatial control over dopaminergic neurotransmission through reuptake of extracellular dopamine (DA). The functional capacity of DAT is under the control of signaling inputs and post-translational modifications that confer acute presynaptic regulation of reuptake in response to physiological needs, and dysregulation of these processes may contribute to DA imbalances in mood disorders and drug addiction. A key modification of DAT is palmitoylation, a lipid adduction that enhances transport velocity, is suppressed by protein kinase C, and opposes protein kinase C-mediated down-regulation. Here we now show in rat striatum and heterologous cells that transporter palmitoylation is also linked to methamphetamine (METH), undergoing rapid and transient reductions in response to the drug. The time course and other characteristics of palmitoylation reduction parallel those of METH-induced transport down-regulation, and a palmitoylation-deficient DAT mutant shows enhanced down-regulation to METH, supporting a mechanistic link between reduction of the modification and reduced reuptake activity. Recovery rates differed, however, with palmitoylation returning to starting levels more rapidly than reuptake, indicating that down-regulation mechanisms remain engaged with transporters that have undergone repalmitoylation. These results support palmitoylation as a rapid response mechanism that modulates DAT entry into METH-induced down-regulation states and suggest a broader role for the modification in control of reuptake in additional physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2023

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the Integrated Stress Response

Dephosphorylation of pSer51 of the subunit of translation initiation factor 2 (eIF2P) terminates signalling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a PP1 catalytic subunit, the conserved C-terminally located [~]70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential co-factor) efficiently dephosphorylate eIF2P in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-Ray crystallography and biochemistry to uncover binding of the {gamma} subunit of eIF2 to a short helical peptide repeated four times in the functionally-important N-terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an -helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2{gamma} in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2P by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

biochemistry↗

bioRxiv2023

The Microtubule Severing Protein UNC-45A Counteracts the Microtubule Straightening Effects of Taxol

UNC-45A is the only known ATP-independent microtubule (MT) severing protein. Thus, it severs MTs via a novel mechanism. In vitro and in cells UNC-45A-mediated MT severing is preceded by the appearance of MT bends. While MTs are stiff biological polymers, in cells, they often curve, and the result of this curving can be breaking off. The contribution of MT severing proteins on MT lattice curvature is largely undefined. Here we show that UNC-45A curves MTs. Using in vitro biophysical reconstitution and TIRF microscopy analysis, we show that UNC-45A is enriched in the areas where MTs are curved versus the areas where MTs are straight. In cells, we show that UNC-45A overexpression increases MT curvature and its depletion has the opposite effect. We also show that this effect occurs is independent of actomyosin contractility. Lastly, we show for the first time that in cells, Paclitaxel straightens MTs, and that UNC-45A can counteracts the MT straightening effects of the drug. Significance: Our findings reveal for the first time that UNC-45A increases MT curvature. This hints that UNC-45A-mediated MT severing could be due to the worsening of MT curvature and provide a mechanistic understanding of how this MT-severing protein may act. UNC-45A is the only MT severing protein expressed in human cancers, including paclitaxel-resistant ovarian cancer. Our finding that UNC-45A counteracts the paclitaxel-straightening effects of MTs in cells suggests an additional mechanism through which cancer cells escape drug treatment.

cell biology↗

bioRxiv2022

Fecal Microbiota Restoration Modulates the Microbiome in Inflammation-Driven Colorectal Cancer

Chronic inflammation of the colon (colitis) is a known risk factor for inflammatory-driven colorectal cancers (idCRC), and intestinal microbiota has been implicated in the etiology of id-CRC. Manipulation of the microbiome is a clinically viable therapeutic approach to limiting id-CRC. To understand the microbiome changes that occur over time in id-CRC, we used a mouse model of idCRC with the treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS) and measured the microbiome over time using 16S rRNA amplicon sequencing. We included cohorts where the microbiome was restored using cage bedding swapping and where the microbiome was depleted using antibiotics to compare to untreated animals. We identified consistent increases in Akkermansia in mice receiving horizontal microbiome transfer (HMT), while the control cohort had consistent longitudinal increases in Anaeroplasma and Alistipes. Additionally, fecal lipocalin-2 (Lcn-2) concentrations were elevated in unrestored animals compared to restored and antibiotic-treated counterparts following HMT. These observations suggest a potential role for Akkermansia, Anaeroplasma, and Alistipes in regulating colonic inflammation in id-CRC.

microbiology↗

bioRxiv2021

GLS1 is a Protective Factor Rather than a Molecular Target in ARID1A-Mutated Ovarian Clear Cell Carcinoma

Targeting glutamine metabolism has emerged as a novel therapeutic strategy for several human cancers, including ovarian cancer. The primary target of this approach is the kidney isoform of glutaminase, glutaminase 1 (GLS1), a key enzyme in glutamine metabolism that is overexpressed in several human cancers. A first-in-class inhibitor of GLS1, called CB839 (Telaglenastat), has been investigated in several clinical trials, with promising results. The first clinical trial of CB839 in platinum-resistant ovarian cancer patients is forthcoming. ARID1A-mutated ovarian clear cell carcinoma (OCCC) is a relatively indolent and chemoresistant ovarian cancer histotype. In OCCC-derived cells in vitro and mouse models, loss of ARID1A leads to upregulation of GLS1. Thus, targeting of GLS1 with CB839 has been suggested as a targeted approach for OCCC patients with tumors harboring ARID1A-mutations. Here, we investigated whether GLS1 is differentially expressed between OCCC patients whose tumors are ARID1A positive and patients whose tumors are ARID1A negative. In clinical specimens of OCCC, we found that GLS1 overexpression was not correlated with ARID1A loss. In addition, GLS1 overexpression was associated with better clinical outcomes. Our findings suggest that GLS1 expression in OCCC may be a protective factor and that caution should be taken when considering the use of CB839 to treat OCCC patients.

cancer biology↗

bioRxiv2021

Targeting Mitochondrial Metabolism in Clear Cell Carcinoma of the Ovaries

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A a member of the SWI/SNF chromatin remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells, however the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here we show that ARID1A-loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated to increase of C-myc and to increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide the for targeting mitochondrial activity in ARID1A-mutanted OCCCs.

cancer biology↗

bioRxiv2020

UNC-45A Destabilizes MTs Independent of its Effect on Non-Muscle Myosin II

In invertebrates, UNC-45 regulates myosin stability and functions. Vertebrates have two distinct isoforms of the protein: UNC-45B, expressed in muscle cells only and UNC-45A, expressed in all cells and implicated in regulating both Non-Muscle Myosin II (NMII)- and microtubule (MT)-associated functions. Here we show for the first time that: a) in vitro UNC-45A binds to the MT lattice and weakens its integrity leading to MT bending, breakage and depolymerization, b) in cells, UNC-45A overexpression causes loss of MT mass and increase in MT breakages, c) both in vitro and in cells, UNC-45A destabilizes MTs independent of its NMII C-terminal binding domain and destabilization occurs even in presence of the NMII inhibitor blebbistatin. These findings are consistent with a not mutually exclusive but rather dual role of UNC-45A in regulating NMII activity and MT stability. Because many human diseases, from cancer to neurodegenerative diseases, are caused by or associated with deregulation of MT stability our findings have profound implications in both, the biology of MTs as well as the biology of human diseases and possible therapeutic implications for their treatment.

cell biology↗

bioRxiv2019

UNC-45A is preferentially expressed in epithelial cells and binds to and co-localizes with interphase MTs

UNC-45A is a ubiquitously expressed protein highly conserved throughout evolution. Most of what we currently know about UNC-45A pertains to its role as a regulator of the actomyosin system. However, emerging studies from both our and other laboratories support a role of UNC-45A outside of actomyosin regulation. This includes studies showing that UNC-45A: regulates gene transcription, co-localizes and biochemically co-fractionates with gamma tubulin and regulates centrosomal positioning, is found in the same subcellular fractions where MT-associated proteins are, and is a mitotic spindle-associated protein with MT destabilizing activity in absence of the actomyosin system.\n\nHere, we extended our previous findings and show that UNC45A is variably expressed across a spectrum of cell lines with the highest level being found in HeLa cells and in ovarian cancer cells inherently paclitaxel-resistant. Furthermore, we show that UNC-45A is preferentially expressed in epithelial cells, localizes to mitotic spindles in clinical tumor specimens of cancer and co-localizes and co-fractionates with MTs in interphase cells independent of actin or myosin.\n\nIn sum, we report alteration of UNC45A localization in the setting of chemotherapeutic treatment of cells with paclitaxel, and localization of UNC45A to MTs both in vitro and in vivo. These findings will be important to ongoing and future studies in the field that further identify the important role of UNC45A in cancer and other cellular processes.

cell biology↗