Biology subjects

Thakkar, N. P.

Publications and source records attributed to Thakkar, N. P..

3 recordsLinked to original sources

bioRxiv2025

Splicing factor proline- and glutamine-rich (SFPQ) protein causes transcriptional repression of SNAIL to counteract TGF-β signaling

TGF-{beta} is known to regulate several embryonic and adult signaling pathways. Moreover, this signaling pathway regulates several cellular functions including differentiation, cell division, angiogenesis, hematopoiesis, and cell migration. However, studies suggest that an uncontrolled activation of TGF-{beta} signaling may contribute to many human diseases. Therefore, counter-regulatory mechanism(s) to restrain abrupt TGF-{beta} activation during cellular homeostasis is necessary to maintain an adequate balance of TGF-{beta} downstream signaling. TGF-{beta} through Smad complex activation causes transcriptional regulation of many transcription factors including Snail which act as an immediate-early response gene in TGF-{beta} signaling. Herein, for the first time, we report that Splicing factor proline- and glutamine-rich (SFPQ), an RNA binding paraspeckles-associated protein works as a transcriptional repressor of Snail. We first confirmed a significant reduction in the expression level of SFPQ in the kidney glomeruli of rats that underwent subtotal nephrectomy. Endothelial cells (EC) treated with TGF-{beta} exhibited loss of SFPQ protein level without altering its transcript level. Inhibition of proteasomal or autophagosome-lysosome pathway revealed ubiquitination-dependent proteasomal degradation of SFPQ upon TGF-{beta} challenge. Prior to degradation, TGF-{beta} treatment resulted in the cytosolic export of SFPQ thereby diminishing nuclear SFPQ level. Knockdown of SFPQ augmented TGF-{beta}-dependent increase in Snail level while overexpression of SFPQ reversed TGF-{beta} induced Snail expression. Although SFPQ exhibited association with many transcription factors including Smad2/3, Smad4, and N1-ICD which regulate Snail gene expression, TGF-{beta} failed to alter the association of SFPQ with these transcription factors. Instead, through ChIP-qPCR analysis, we confirmed the enrichment of SFPQ in E-box promoter region and coding region proximal to TSS of the Snail gene. This study is the first to report SFPQ as a transcriptional repressor of Snail thereby regulating TGF-{beta} signaling during cellular homeostasis.

biochemistry↗

bioRxiv2024

S-nitrosylation of EZH2 at C329 and C700 interplay with PRC2 complex assembly, methyltransferase activity, and EZH2 stability to regulate endothelial functions

Nitric oxide (NO), a versatile bio-active molecule modulates cellular function through diverse mechanisms including S-nitrosylation of proteins. However, the role of this post-translational modification in regulating epigenetic pathways was very limitedly explored. Herein, we report that NO causes S-nitrosylation of selected cysteine residues of EZH2 in endothelial cells (EC) resulting in SUZ12 dissociation from EZH2 bound PRC2 complex, reduced methyltransferase activity, and diminished nuclear localization eventually hampering its stability. We detected a significant reduction in H3K27me3 upon exposure to NO as contributed by the early dissociation of SUZ12 from the PRC2 complex. Longer exposure to NO donors caused EZH2 cytosolic translocation, its ubiquitination, and further degradation primarily through the autophagosome-lysosome pathway. Through in silico S-nitrosylation prediction analysis and site-directed mutagenesis assay, we identified three cysteine residues namely at locations 260, 329, and 700 in EZH2 and further determined that S-nitrosylation of cysteine 329 induced EZH2 instability while S-nitrosylation of cysteine 700 abrogated EZH2s catalytic activity. A double mutant of EZH2 containing mutations at Cysteine 329 and 700 remained undeterred to NO exposure. Furthermore, reinforcing H3K27me3 in NO exposed EC through the use of an inhibitor of H3K27me3 demethylase, we confirmed a significant contribution of the EZH2-H3K27me3 axis in defining NO-mediated regulation of endothelial gene expression and migration. Molecular dynamics simulation study revealed SUZ12s inability in efficiently binding to the SAL domain of EZH2 upon S-nitrosylation of C329 and C700. Taken together, our study for the first-time reports that S-nitrosylation dependent regulation of EZH2 and its associated PRC2 complex influences endothelial homeostasis.

cell biology↗

bioRxiv2023

Regulation of shelterin proteins TERF2IP and TRF2 by H3K4me3-p65 axis drives hyperglycemia dependent endothelial senescence

BackgroundEndothelial senescence has been linked to several cardiovascular diseases. Dysregulation of proteins of the shelterin complex including TRF2 and TERF2IP causes senescence as it hampers DNA repair and cell proliferation. However, whether exposure to hyperglycemia interplays with proteins of the shelterin complex thus further dictates the senescent phenotype of endothelial cells (EC) remain to be explored. Approach and ResultsIn this study, we observed elevated levels of p21 and p53 in endothelial cells upon exposure to intermittent hyperglycemia. We also noted hyperglycemia exposure increased the levels of TERF2IP and TRF2, part of the shelterin complex. No change in the level of TRF1 and TPP1 were detected. Furthermore, a robust induction was detected in p65 level upon intermittent hyperglycemia challenge. ChIP-qPCR analysis revealed enhanced H3K4me3 enrichment in the promoter regions of p65, TERF2IP and TRF2. Inhibition of catalysis of H3K4me3 either by pharmacological inhibitor or siRNA-mediated knockdown of MLL2 attenuated increase in p65, TERF2IP and TRF2 levels including reversal of senescence markers p53 and p21. Interestingly, pharmacological inhibition of NF-{kappa}B signaling also diminished abrupt increase in TERF2IP and TRF2 levels thus further reversed intermittent hyperglycemia-induced p53 and p21 levels. More importantly, co-immunoprecipitation and co-localization analysis revealed an interaction between nuclear p65 and MLL2 in EC stimulated with hyperglycemia. Further knockdown of either TERF2IP or TRF2 impaired intermittent hyperglycemia-induced p53 and p21 expression and associated endothelial senescence. ConclusionOverall, the present study describes an interplay of epigenetics in defining NF-{kappa}B signaling and shelterin proteins expression which further govern the biochemical and functional state of endothelial senescence in hyperglycemic milieu.

biochemistry↗