Biology subjects

Scharfmann, R.

Publications and source records attributed to Scharfmann, R..

9 recordsLinked to original sources

bioRxiv2026

Villin-SNAP25 interaction regulates insulin secretion downstream of ICA512 signaling

Pancreatic islet {beta}-cells step-up insulin production and secretion in response to the elevation of blood glucose levels. ICA512/PTPRN is a transmembrane cargo of the insulin secretory granules (SGs). Upon SG exocytosis, its Ca2+/calpain mediated cleavage at the plasma membrane generates an ICA512-cleaved cytosolic fragment (ICA512-CCF) which as a decoy phosphatase prolongs phospho-STAT5/3 activities, thereby enhancing the transcription of mRNAs for SG cargoes, including insulin and its own, to replenish SG stores. In addition, ICA512 positively regulates the expression of F-actin modifier villin. Villin, in turn, modulates the size of actin cages surrounding cortical SGs, hence regulating SG mobility and exocytosis. Here we show that villin controls the number of SG docking sites for exocytosis by directly interacting at low glucose with the t-SNARE SNAP-25, thus restricting in this condition SG access to fusion sites and insulin release. Replacement of ICA512-CCF N-terminal ubiquitin-acceptor lysine 609 (K609) with valine (V) stabilized ICA512-CCF in insulinoma cells and homozygous ICA512K609V mice and enhanced the levels of phospho-STAT5/3 and villin. In ICA512K609V female mice these molecular traits correlated with reduced body weight, improved insulin sensitivity, reduced basal insulin secretion and onset time for glucose stimulated insulin secretion. Taken together, these data demonstrates that SG exocytosis induced ICA512 retrograde signaling acts in concert with the novel SNARE complex regulator villin to plastically adapt {beta}-cell actin cytoskeleton and access to SG docking sites for optimal control of insulin secretion in response to variations in extracellular glucose levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/699220v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@37d835org.highwire.dtl.DTLVardef@5389f1org.highwire.dtl.DTLVardef@855d98org.highwire.dtl.DTLVardef@1c3c639_HPS_FORMAT_FIGEXP M_FIG Graphical abstract Model of ICA512 and STAT3/5-dependent regulation of -cell function. At basal conditions, villin limits secretory granule (SG) docking by interacting with SNAP-25, restricting insulin release (1). Increased metabolic load triggers hyperglycemia-induced ICA512 processing and growth factor-mediated STAT3/5 phosphorylation (2-4), enhancing ISG mRNAs, granule biogenesis, and villin expression (5,6). Villin modulates SNARE protein availability, controlling SG docking and secretion. Together, ICA512 retrograde signaling and villin-mediated SNARE regulation dynamically remodel the -cell actin cytoskeleton, adjusting SG docking site access to ensure appropriate insulin secretion in response to fluctuating glucose levels. C_FIG

cell biology↗

bioRxiv2025

GGA2-depletion in beta cells by Enteroviruses causes Golgi acidification, premature activation of cathepsins and alters the MHC class I immunopeptidome

Enteroviruses (EVs), such as Coxsackievirus B5 (CVB5), are linked to pancreatic beta cell autoimmunity in type 1 diabetes (T1D). CVB5 infection of beta cells blocks their cap-dependent translation but spares cap-independent translation of insulin secretory granule (SG) cargoes, including major T1D autoantigens. Despite this, insulin SG stores are depleted. We show that CVB5 protease 2A rapidly depletes Golgi-associated sorting factor GGA2 in beta cells due to its short half-life. Immunostaining of pancreas sections from recent-onset T1D donors confirmed reduced GGA2 in beta cells expressing EV capsid protein VP1. Both GGA2 depletion and CVB5 2A expression impair SG biogenesis at the TGN without affecting MHC class I trafficking. They also disrupt the sorting of vacuolar ATPase and cathepsins, leading to TGN acidification and premature lysosomal hydrolase activation, which typically generate peptides presented in HLA-II alleles. Notably, the unique immunopeptidome of EV-infected, GGA2-depleted ECN90 beta-like cells is mainly presented via HLA-B alleles with an average isoelectric point (pI) of [~]5, compared to a pI of [~]7 of the prevailing HLA-I A allele immunopeptidome of non-infected cells. We propose that delayed sorting at the acidified TGN promotes cathepsin-mediated processing of in-transit secretory proteins, including SG cargoes, thereby generating novel peptides that may replace HLA class I antigens due to the acidic environment. This novel cross-presentation mechanism could have implications for T1D pathogenesis and antigen presentation in general.

cell biology↗

bioRxiv2023

Interferon-α promotes neo-antigen formation andpreferential HLA-B-restricted antigen presentation in pancreatic β-cells

Interferon (IFN)- is the earliest cytokine signature observed in individuals at risk for type 1 diabetes (T1D), but its effect on the repertoire of HLA Class I (HLA-I)-bound peptides presented by pancreatic {beta}-cells is unknown. Using immunopeptidomics, we characterized the peptide/HLA-I presentation in in-vitro resting and IFN--exposed {beta}-cells. IFN- increased HLA-I expression and peptide presentation, including neo-sequences derived from alternative mRNA splicing, post-translational modifications - notably glutathionylation - and protein cis-splicing. This antigenic landscape relied on processing by both the constitutive and immune proteasome. The resting {beta}-cell immunopeptidome was dominated by HLA-A-restricted ligands. However, IFN- only marginally upregulated HLA-A and largely favored HLA-B, translating into a major increase in HLA-B-restricted peptides and into an increased activation of HLA-B-restricted vs. HLA-A-restricted CD8+ T-cells. A preferential HLA-B hyper-expression was also observed in the islets of T1D vs. non-diabetic donors, and we identified islet-infiltrating CD8+ T-cells from T1D donors reactive to HLA-B-restricted granule peptides. Thus, the inflammatory milieu of insulitis may skew the autoimmune response toward epitopes presented by HLA-B, hence recruiting a distinct T-cell repertoire that may be relevant to T1D pathogenesis.

immunology↗

bioRxiv2023

Coxsackievirus infection induces direct pancreatic β-cell killing but poor anti-viral CD8+ T-cell responses

Coxsackievirus B (CVB) infection of pancreatic {beta} cells is associated with {beta}-cell autoimmunity. We investigated how CVB impacts human {beta} cells and anti-CVB T-cell responses. {beta} cells were efficiently infected by CVB in vitro, downregulated HLA Class I and presented few, selected HLA-bound viral peptides. Circulating CD8+ T cells from CVB-seropositive individuals recognized only a fraction of these peptides, and only another sub-fraction was targeted by effector/memory T cells that expressed the exhaustion marker PD-1. T cells recognizing a CVB epitope cross-reacted with the {beta}-cell antigen GAD. Infected {beta} cells, which formed filopodia to propagate infection, were more efficiently killed by CVB than by CVB-reactive T cells. Thus, our in-vitro and ex-vivo data highlight limited T-cell responses to CVB, supporting the rationale for CVB vaccination trials for type 1 diabetes prevention. CD8+ T cells recognizing structural and non-structural CVB epitopes provide biomarkers to differentially follow response to infection and vaccination.

immunology↗

bioRxiv2021

PCSK9 affects expression of key surface proteins in human pancreatic beta cells through intra- and extracellular regulatory circuits

Aims/hypothesisProprotein convertase subtilisin/kexin 9 (PCSK9) is involved in the degradation of LDLR. However, PCSK9 can target other proteins in a cell-type specific manner. While PCSK9 has been detected in pancreatic islets, its expression in insulin-producing pancreatic beta cells is debated. Herein, we studied PCSK9 expression, regulation and function in the human pancreatic beta cell line EndoC-{beta}H1. MethodsWe assessed PCSK9 expression in mouse and human pancreatic islets, and in the pancreatic beta cell line EndoC-{beta}H1. We also studied PCSK9 regulation by cholesterol, lipoproteins, Mevastatin, and by SREBPs transcription factors. To evaluate PCSK9 function in pancreatic beta cells, we performed PCSK9 gain-and loss-of-function experiments in EndoC-{beta}H1 using siPCSK9 or recombinant PCSK9 treatments, respectively. ResultsWe demonstrate that PCSK9 is expressed and secreted by pancreatic beta cells. In EndoC-{beta}H1 cells, PCSK9 expression is regulated by cholesterol and by SREBPs transcription factors. Importantly, PCSK9 knockdown results in multiple transcriptome, proteome and secretome deregulations and impaired insulin secretion. By gain- and loss-of-function experiments, we observed that PCSK9 regulates the expression levels of LDLR and VLDLR through an extracellular mechanism while CD36, PD-L1 and HLA-ABC are regulated through an intracellular mechanism. Conclusions/interpretationCollectively, these results highlight PCSK9 as an important regulator of CD36, PD-L1 and HLA-ABC cell surface expression in pancreatic beta cells. Data availabilityRNA-seq data have been deposited to GEO database with accession number GSE182016. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the following identifiers: PXD027921, PXD027911 and PXD027913.

cell biology↗

bioRxiv2021

Glucose controls co-translation of structurally related mRNAs via the mTOR and eIF2 pathways in human pancreatic beta cells

Pancreatic beta cell response to glucose is critical for the maintenance of normoglycemia. A strong transcriptional response was classically described in rodent models but, interestingly, not in human cells. In this study, we exposed human pancreatic beta cells to an increased concentration of glucose and analysed at a global level the mRNAs steady state levels and their translationalability. Polysome profiling analysis showed an early acute increase in protein synthesis and a specific translation regulation of more than 400 mRNAs, independently of their transcriptional regulation. We clustered the co-regulated mRNAs according to their behaviour in translation in response to glucose and discovered common structural and sequence mRNA features. Among them mTOR- and eIF2-sensitive elements have a predominant role to increase mostly the translation of mRNAs encoding for proteins of the translational machinery. Furthermore, we show that mTOR and eIF2 pathways are independently regulated in response to glucose, participating to a translational reshaping to adapt beta cell metabolism. The early acute increase in the translation machinery components prepare the beta cell for further protein demand due to glucose-mediated metabolism changes. AUTHOR SUMMARYAdaptation and response to glucose of pancreatic beta cells is critical for the maintenance of normoglycemia. Its deregulation is associated to Diabetic Mellitus (DM), a significant public health concern worldwide with an increased incidence of morbidity and mortality. Despite extensive research in rodent models, gene expression regulation in response to glucose remains largely unexplored in human cells. In our work, we have tackled this question by exposing human EndoC-BH1 cells to high glucose concentration. Using polysome profiling, the gold standard technique to analyse cellular translation activity, we observed a global protein synthesis increase, independent from transcription activity. Among the specific differentially translated mRNAs, we found transcripts coding for ribosomal proteins, allowing the cell machinery to be engaged in a metabolic response to glucose. Therefore, the regulation in response to glucose occurs mainly at the translational level in human cells, and not at the transcriptional level as described in the classically used rodent models. Furthermore, by comparing the features of the differentially translated mRNAs, and classifying them according to their translational response, we show that the early response to glucose occurs through the coupling of mRNA structure and sequence features impacting translation and regulation of specific signalling pathways. Collectively, our results support a new paradigm of gene expression regulation on the translation level in human beta cells.

genomics↗

bioRxiv2021

Characterization of the secretome, transcriptome and proteome of human β cell line EndoC-βH1

Early diabetes research is hampered by limited availability, variable quality and instability of human pancreatic islets in culture. Little is known about the human {beta} cell secretome, and recent studies question translatability of rodent {beta} cell secretory profiles. Here, we verify representativeness of EndoC-{beta}H1, one of the most widely used human {beta} cell lines, as a translational human {beta} cell model based on omics and characterize the EndoC-{beta}H1 secretome. We profiled EndoC-{beta}H1 cells using RNA-seq, Data Independent Acquisition (DIA) and Tandem Mass Tag proteomics of cell lysate. Omics profiles of EndoC-{beta}H1 cells were compared to human {beta} cells and insulinomas. Secretome composition was assessed by DIA proteomics. Agreement between EndoC-{beta}H1 cells and primary adult human {beta} cells was ~90% for global omics profiles as well as for {beta} cell markers, transcription factors and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-{beta}H1 cells compared to adult {beta} cells. Consistently, similarity was slightly higher with benign non-metastatic insulinomas. EndoC-{beta}H1 secreted 671 proteins in untreated baseline state and 3,278 proteins when stressed with non-targeting control siRNA, including known {beta} cell hormones INS, IAPP, and IGF2. Further, EndoC-{beta}H1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. Unexpectedly, exosomes appeared to be a major mode of secretion in EndoC-{beta}H1 cells. We believe that secretion of exosomes and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/459582v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1976bc0org.highwire.dtl.DTLVardef@233c4eorg.highwire.dtl.DTLVardef@14c3350org.highwire.dtl.DTLVardef@1bcf0d5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe validate EndoC-{beta}H1 as a translational human {beta} cell model using omics. C_LIO_LIWe present the first unbiased proteomics composition of human {beta} cell line secretome. C_LIO_LIThe secretome of human {beta} cells is more extensive than previously thought. C_LIO_LIUntreated cells secreted 671 proteins and stressed cells secreted 3,278 proteins. C_LIO_LISecretion of exosomes and bioactive peptides constitute directions of future research. C_LI

bioinformatics↗

bioRxiv2018

Comparison of human and murine enteroendocrine cells by transcriptomic and peptidomic profiling

Enteroendocrine cells (EECs) produce hormones that regulate food absorption, insulin secretion and appetite. Both EECs and their peptide products are foci of drug discovery programmes for diabetes and obesity. We compared the human and mouse EEC transcriptome and peptidome to validate mouse as a model of the human enteroendocrine axis. We present the first RNA sequencing analysis of human EECs, and demonstrate strong correlation with mouse, although with outliers including some low abundance G-protein coupled receptors. Liquid chromatography mass spectrometry (LC-MS) identified peptide hormone gradients along the human and mouse gut that should enhance progress in gut physiology and therapeutics.

cell biology↗

bioRxiv2017

Electrophysiological properties of human β-cell lines EndoC-βH1 and -βH2 conform with human β-cells

The electrophysiological and secretory properties of the human {beta}-cell lines EndoC-{beta}H1 and EndoC-{beta}H2 were investigated. Both cell lines respond to glucose (6-20mM) with 2-to 3-fold stimulation of insulin secretion, an effect that was mimicked by tolbutamide (0.2mM) and reversed by diazoxide (0.5mM). Glucose-induced insulin release correlated with an elevation of [Ca2+]i, membrane depolarization and increased action potential firing. KATP channel activity at 1mM glucose is low and increasing glucose to 6 or 20mM reduced KATP channel activity to the same extent as application of the KATP channel blocker tolbutamide (0.2mM). The upstroke of the action potentials in EndoC-{beta}H1 and -{beta}H2 cells observed at high glucose principally reflects activation of L- and P/Q-type Ca2+ channels with some small contribution of TTX-sensitive Na+ channels. Action potential repolarization involves activation of voltage-gated Kv2.2 channels and large-conductance Ca2+-activated K+ channels. Exocytosis (measured by measurements of membrane capacitance) was triggered by membrane depolarizations >10ms to membrane potentials above -30mV. Both cell lines were well-granulated (6,000-15,000 granules/cell) and granules consisted of a central insulin core surrounded by a clear halo. We conclude that the EndoC-{beta}H1 and -{beta}H2 cells share many features of primary human {beta}-cells and that they represent a useful experimental model.

cell biology↗