Biology subjects

Shearn, C. T.

Publications and source records attributed to Shearn, C. T..

3 recordsLinked to original sources

bioRxiv2025

Inflammatory Oxidative Stress Compounds Inhibit Insulin Secretion through Rapid Protein Carbonylation

Pancreatic {beta}-cells in pre-type 1 diabetes (T1D) experience stress due to islet inflammation, which accompanies early defects in insulin secretion that precede autoimmune destruction. One product of inflammatory stress is protein carbonylation (PC), brought on by reactive oxygen species (ROS) combining with lipids to produce reactive aldehydes such as 4-hydroxynonenal (4-HNE) that irreversibly modify Cys, His, and Lys sidechains. In this study, we used proteomics to measure patterns of PC in pancreatic islets from 10-week-old pre-diabetic NOD mice and in cultured insulin-secreting cells treated with either 4-HNE or pro-inflammatory cytokines. All three stress conditions increased carbonylation of proteins central to {beta}-cell function including Rab GTPases and other proteins that are essential for vesicle trafficking. Gene ontology analysis indicates that the affected proteins and pathways in pre-diabetic NOD islets reflect a combination of those impacted by 4-HNE and cytokine treatment. Furthermore, both 4-HNE and cytokines significantly inhibited insulin secretion by [~]50% in cultured MIN6 and INS-1-GRINCH cells. In particular, exposure to 4-HNE for as little as 5 minutes suppressed insulin secretion and increased the carbonylation of over 1000 proteins. Overall, the observed PC pattern in pre-T1D islets is consistent with a model in which {beta}-cells experience multiple sources of oxidative stress, including ROS generation within {beta}-cells themselves and reactive compounds released by infiltrating immune cells. The latter exogenous source may represent a novel rapid mechanism for inhibiting insulin secretion.

biochemistry↗

bioRxiv2023

Endocrine pancreas-specific Gclc gene deletion causes a severe diabetes phenotype

Reduced glutathione (GSH) is an abundant antioxidant that regulates intracellular redox homeostasis by scavenging reactive oxygen species (ROS). Glutamate-cysteine ligase catalytic (GCLC) subunit is the rate-limiting step in GSH biosynthesis. Using the Pax6-Cre driver mouse line, we deleted expression of the Gclc gene in all pancreatic endocrine progenitor cells. Intriguingly, Gclc knockout (KO) mice, following weaning, exhibited an age-related, progressive diabetes phenotype, manifested as strikingly increased blood glucose and decreased plasma insulin levels. This severe diabetes trait is preceded by pathologic changes in islet of weanling mice. Gclc KO weanlings showed progressive abnormalities in pancreatic morphology including: islet-specific cellular vacuolization, decreased islet-cell mass, and alterations in islet hormone expression. Islets from newly-weaned mice displayed impaired glucose-stimulated insulin secretion, decreased insulin hormone gene expression, oxidative stress, and increased markers of cellular senescence. Our results suggest that GSH biosynthesis is essential for normal development of the mouse pancreatic islet, and that protection from oxidative stress-induced cellular senescence might prevent abnormal islet-cell damage during embryogenesis.

physiology↗

bioRxiv2020

Multivalent lipid targeting by the calcium-independent C2A domain of Slp-4/granuphilin

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS), but much weaker when either the background anionic lipids or PIP2 are removed. Through computational and experimental approaches, we show that this high affinity membrane interaction arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, there exists a larger cationic surface surrounding the cluster which contributes substantially to the affinity for physiologically relevant lipid compositions. While mutations at the PIP2-selective site decrease affinity for PIP2, multiple mutations are needed to decrease binding to physiologically relevant lipid compositions. Docking and molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant in the bacterially expressed protein, which arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and has a low membrane affinity. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

biophysics↗