Biology subjects

Ushakumary, M. G.

Publications and source records attributed to Ushakumary, M. G..

5 recordsLinked to original sources

bioRxiv2025

Phosphoproteomics unveils the signaling dynamics in neuronal cells stimulated with insulin and insulin-like growth factors

BackgroundGiven the role of metabolism in brain health and disease, investigating the role of insulin (INS) and insulin-like growth factors (IGFs) as potential therapeutic strategies for neurodegenerative diseases is currently underway. Yet, the signaling pathways associated with INS and IGFs in the brain remain elusive, particularly for the human brain. Unraveling these pathways is critical for harnessing their therapeutic potential in metabolism-associated brain disorders. MethodsThis study employed phosphoproteomics using human neuroblastoma cell line, SH-SY5Y, to unravel the signaling network of INS, IGF-1, and IGF-2. Briefly, cells were stimulated at 10 and 60-minutes with the ligands, followed by protein extraction, trypsin digestion, tandem mass tag (TMT)-labelling and phosphopeptides enrichment using an immobilized metal affinity chromatography (IMAC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Data was processed using R statistical software. Protein annotations were obtained from the UniprotKB database, and pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). ResultsPhosphoproteomics performed at 10 and 60 minutes identified 34358 phosphosites of which 3284 were significant at 10 min and 2374 at 60 min (p.adj <0.05) across all three ligands. Ligand stimulation induced modulation in phosphorylation at both the receptor level and downstream signaling targets at serine (S), threonine (T) and tyrosine (Y) residues. Phosphorylation of LIMA1-Y229, a regulator of actin-cytoskeletal function, was the most prominent Y phosphosite across all ligands. IPA identified Rho GTPase, the molecular switches that regulate actin cytoskeletal dynamics, as the most significantly enriched pathway, with IGF-1 predominantly driving phosphorylation of Rho GTPase effectors such as Rho Guanine nucleotide exchange factors (ARHGEFs), Rho GTPase activating proteins (ARHGAPs) and CDC42. Myocardin related transcription factor A (MRTFA), a transcriptional target of Rho GTPase, was increased in ligand-stimulated cells at 10 min, and inhibition of Rho/SRF pathway by CCG1423 prevents nuclear localization of IGF-1-induced MRTFA. ConclusionsThis study demonstrates that INS, IGF-1 and IGF-2 regulate Rho GTPase and MRTFA activation, thereby contributing to the control of actin cytoskeletal dynamics in neuronal cells. Given the role of INS and IGFs in neuronal survival and neurodegenerative conditions, elucidating the mechanisms is of critical importance, as it offers insights into disease pathogenesis and potential therapeutic targets.

biochemistry↗

bioRxiv2025

Sexually distinct multi omic responses to progressive endurance exercise training in the rat lung. Findings from MoTrPAC

Despite the lungs being essential for ventilation and aerobic exercise capacity, conventionally the lungs are not thought to adapt to exercise training. Endurance exercise is key to pulmonary rehabilitation programs, which also displays sex-specific differences in therapeutic efficacy. Given the molecular underpinnings of sex-specific lung adaptations to endurance exercise are uncharacterized, we used a multi-omics approach to study sex differences in the lungs of 6-month-old Fischer 344 rats in response to an 8 week progressive endurance treadmill training protocol. This was accomplished by reannotating publicly accessible data from the Molecular Transducers of Physical Activity Consortium (MoTrPAC) and integrating newly-analyzed acetylome data to assess multi-omic sex differences in sedentary and progressively trained states. Female rats displayed enrichment in immune-related features and pathways at the transcriptome and proteome level that were maintained with training. Conversely, in the male rat lung there was an overall decrease in immune pathways following 8 weeks of training. Sexually conserved responses to training included increased enrichment in transcriptomic pathways related to type I alveoli and proteomic pathways related to cilia, and decreased mitochondrial protein acetylation. In both sexes, features known to be enriched in lung diseases were attenuated with training. Together our findings provide novel insight into sex specific responses to endurance exercise training in the rat lung and may offer translational insight into sex-specific differences in lung disease pathogenesis and treatment.

physiology↗

bioRxiv2025

Sorted-Cell Proteomics Reveals an AT1-Associated Epithelial Cornification Phenotype and Suggests Endothelial Redox Imbalance in Human Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease characterized by inflammation and scarring leading to long-term tissue damage. Previous whole tissue proteomics identified BPD-specific proteome changes and cell type shifts. Little is known about the proteome-level changes within specific cell populations in disease. Here, we sorted epithelial (EPI) and endothelial (ENDO) cell populations based on their differential surface markers from normal and BPD human lungs. Using a low-input compatible sample preparation method (MicroPOT), proteins were extracted and digested into peptides and subjected to Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) proteome analysis. Of the 4,970 proteins detected, 293 were modulated in abundance or detection in the EPI population and 422 were modulated in ENDO cells. Modulation of proteins associated with actin-cytoskeletal function such as SCEL, LMO7, and TBA1B were observed in the BPD EPIs. Using confocal imaging and analysis, we validated the presence of aberrant multilayer-like structures comprising SCEL and LMO7, known to be associated with epidermal cornification, in the human BPD lung. This is the first report of accumulation of cornification-associated proteins in BPD. Their localization in the alveolar parenchyma, primarily associated with alveolar type 1 (AT1) cells, suggests a role in the BPD post-injury response. In the ENDOs, redox balance and mitochondrial function pathways were modulated. Alternative mRNA splicing and cell proliferative functions were elevated in both populations suggesting potential dysregulation of cell progenitor fate. This study characterized the proteome of epithelial and endothelial cells from the BPD lung for the first time, identifying population-specific changes in BPD pathogenesis. New & NoteworthyThe study is the first to perform proteomics on sorted pulmonary epithelial and endothelial populations from BPD and age-matched control human donors. We identified an increase in cornification-associated proteins in BPD (e.g., SCEL and LMO7), and evidenced the presence of multilayered structures unique to BPD alveolar regions, associated with alveolar type 1 (AT1) cells. By changing the nature and/or biomechanical properties of the epithelium, these structures may alter the behavior of other alveolar cell types potentially contributing to the arrested alveolarization observed in BPD. Lastly, our data suggest the modulation of cell proliferation and redox homeostasis in BPD providing potential mechanisms for the reduced vascular growth associated with BPD.

biochemistry↗

bioRxiv2022

Metformin attenuates hyperglycaemia-stimulated pro-fibrotic gene expression in vascular adventitial fibroblasts via inhibition of Discoidin Domain Receptor 2

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a collagen receptor tyrosine kinase, has an obligate regulatory role in collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and over-expression approaches, the present study examined whether DDR2 is a target of metformin, and whether, by targeting DDR2, it inhibits fibronectin and collagen expression in vascular adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-{beta}1/ Smad2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of fibronectin and collagen, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a major mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis, and possibly, cardiac fibrosis associated with diabetic cardiomyopathy.

molecular biology↗

bioRxiv2022

Matrix fibroblast function during alveolarization is dependent on GATA6

Alveolarization is dependent on myo-, matrix- and lipo- fibroblast functions by interstitial PDGFRa+ fibroblasts. While these fibroblasts are derived from GLI and PDGFRa expressing fibroblasts, the transcriptional control of their functional specification remains unknown. Perinatally, the transcription factor GATA6 is upregulated in PDGFRa+ fibroblasts. To study the role of GATA6 during fibroblast differentiation, we generated PDGFRaCreER/GATA6flx/flx mice and deleted GATA6 in the perinatal period and in adult mice prior to left lobe pneumonectomy. Loss of GATA6 in the PDGFRa+-fibroblasts impaired alveolarization, and extracellular matrix deposition, in association with increased TCF21 expression and lipofibroblast differentiation. Loss of GATA6 in PDGFRa+ fibroblasts resulted in loss of alveolar type 1 (AT1) cells and gain of transitional alveolar type 2 (AT2) cells. Loss of GATA6 was associated with reduced WNT signaling. Restoration of WNT signaling in GATA6 deficient alveolar lung organoids restored AT2 and AT1 cell differentiation. GATA6 induces matrix fibroblast functions and represses lipofibroblast functions, serving as key regulator of fibroblast differentiation during alveolarization and regeneration. Present findings link matrix fibroblast functions with the ability of transitional AT2 cells to differentiate into AT1 cells. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/494950v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@47375forg.highwire.dtl.DTLVardef@184630borg.highwire.dtl.DTLVardef@8285b3org.highwire.dtl.DTLVardef@117e47a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO C_FIG

developmental biology↗