Mereena George Ushakumary

In type 1 diabetes (T1D), insulin-producing β cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. β-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in β-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFNγ + IL-1β + TNFα identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced β-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for β-cell metabolism and insulin production in the context of islet inflammation.

The study is the first to perform proteomics on sorted pulmonary epithelial and endothelial populations from bronchopulmonary dysplasia (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. Finally, our data suggest the modulation of cell proliferation and redox homeostasis in BPD providing potential mechanisms for the reduced vascular growth associated with BPD.

Background Given 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. Methods This 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). Results Phosphoproteomics performed at 10 and 60 minutes identified 34358 phosphosites of which 3284 were significant at 10 min and 2374 at 60 min (p.adj Conclusions This 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.

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 & Noteworthy: The 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.

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-β1/ 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.

Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha–positive (PDGFRA ⁺ ) fibroblasts are critical for alveolarization and that PDGFRA ⁺ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population–targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O ₂ postnatal days 0–7, PN0–PN7) and performed studies on sorted PDGFRA ⁺ cells during injury and room air recovery. After hyperoxia injury, PDGFRA ⁺ matrix and myofibroblasts decreased and PDGFRA ⁺ lipofibroblasts increased by transcriptional signature and population size. PDGFRA ⁺ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA ⁺ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA ⁺ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA ⁺ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-β1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-β1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-β1.

Developing, regenerating, and repairing a lung all require interstitial resident fibroblasts (iReFs) to direct the behavior of the epithelial stem cell niche. During lung development, distal lung fibroblasts, in the form of matrix-, myo-, and lipofibroblasts, form the extra cellular matrix (ECM), create tensile strength, and support distal epithelial differentiation, respectively. During de novo septation in a murine pneumonectomy lung regeneration model, developmental processes are reactivated within the iReFs, indicating progenitor function well into adulthood. In contrast to the regenerative activation of fibroblasts upon acute injury, chronic injury results in fibrotic activation. In murine lung fibrosis models, fibroblasts can pathologically differentiate into lineages beyond their normal commitment during homeostasis. In lung injury, recently defined alveolar niche cells support the expansion of alveolar epithelial progenitors to regenerate the epithelium. In human fibrotic lung diseases like bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis (IPF), and chronic obstructive pulmonary disease (COPD), dynamic changes in matrix-, myo-, lipofibroblasts, and alveolar niche cells suggest differential requirements for injury pathogenesis and repair. In this review, we summarize the role of alveolar fibroblasts and their activation stage in alveolar septation and regeneration and incorporate them into the context of human lung disease, discussing fibroblast activation stages and how they contribute to BPD, IPF, and COPD.