Biology subjects

Arvan, P.

Publications and source records attributed to Arvan, P..

At least 19 recordsLinked to original sources

bioRxiv2025

SEL1L-HRD1 ER-Associated Degradation Facilitates Prohormone Convertase 2 Maturation and Glucagon Production in Islet α Cells

Proteolytic cleavage of proglucagon by prohormone convertase 2 (PC2) is required for islet cells to generate glucagon. However, the regulatory mechanisms underlying this process remain largely unclear. Here, we report that SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD), a highly conserved protein quality control system responsible for clearing misfolded proteins from the ER, plays a key role in glucagon production by regulating turnover of the nascent proform of the PC2 enzyme (proPC2). Using a mouse model with SEL1L deletion in proglucagon-expressing cells, we observed a progressive decline in stimulated glucagon secretion and a reduction in pancreatic glucagon content. Mechanistically, we found that endogenous proPC2 is a substrate of SEL1L-HRD1 ERAD, and that degradation of misfolded proPC2 ensures the maturation of activation-competent proPC2 protein. These findings identify ERAD as a novel regulator of PC2 biology and an essential mechanism for maintaining cell function.

cell biology↗

bioRxiv2024

Small molecule FICD inhibitors suppress endogenous and pathologic FICD-mediated protein AMPylation

The AMP transferase, FICD, is an emerging drug target finetuning stress signaling in the endoplasmic reticulum (ER). FICD is a bi-functional enzyme, catalyzing both AMP addition (AMPylation) and removal (deAMPylation) from the ER resident chaperone BiP/GRP78. Despite increasing evidence linking excessive BiP/GRP78 AMPylation to human diseases, small molecules to inhibit pathogenic FICD variants are lacking. Using an in-vitro high-throughput screen, we identify two small-molecule FICD inhibitors, C22 and C73. Both molecules significantly inhibit FICD-mediated BiP/GRP78 AMPylation in intact cells while only weakly inhibiting BiP/GRP78 deAMPylation. C22 and C73 also efficiently inhibit pathogenic FICD variants and improve proinsulin processing in {beta} cells. Our study identifies and validates FICD inhibitors, highlighting a novel therapeutic avenue against pathologic protein AMPylation.

cell biology↗

bioRxiv2023

Permanent Neonatal Diabetes-causing Insulin Mutations have Dominant Effects on Proinsulin Processing and Beta Cell Identity

Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM) that results from beta cell failure. We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations. Using b and mutated hESCs, we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on the in vivo performance of patient-derived SC-beta cells after transplantation into NSG mice. These insulin mutations derange endoplasmic reticulum (ER) homeostasis, and result in the loss of beta-cell mass and function. In addition to anticipated apoptosis, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. These results highlight both known and novel mechanisms contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

cell biology↗

bioRxiv2022

Differential roles of beta-cell IP3R and RyR ER Ca2+ channels in ER stress-induced alterations of beta-cell Ca2+ homeostasis

Pancreatic beta cells maintain glucose homeostasis by secreting pulses of insulin in response to a rise in glucose. Pulsatile secretion occurs due to glucose-induced oscillations in beta-cell cytosolic Ca2+. The endoplasmic reticulum (ER) helps regulate beta-cell cytosolic Ca2+, and ER stress can lead to ER Ca2+ depletion, beta-cell dysfunction and an increased risk of type 2 diabetes. To determine the effects of tunicamycin-induced ER stress on ER inositol 1,4,5-triphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) and their involvement in subsequent Ca2+ dysregulation, INS-1 832/13 cells and primary mouse islets were treated with tunicamycin. This increased RyR1 mRNA and potentiated RyR-mediated Ca2+ signaling without affecting RyR2 mRNA. TM treatment also enhanced IP3R function, while it decreased IP3R1 and IP3R3 mRNA. Stress reduced ER Ca2+, triggered oscillations in cytosolic Ca2+ under subthreshold glucose conditions, and increased apoptosis; these changes were prevented by cotreatment with the RyR1 inhibitor dantrolene. In contrast, inhibiting IP3Rs with xestospongin-C failed to suppress the cytosolic Ca2+ oscillations due to tunicamycin treatment and did not protect beta cells from tunicamycin-induced apoptosis, although xestospongin-C inclusion prevented ER Ca2+ depletion. Taken together, changes in RyR1 function were shown to play a critical role in ER stress induced Ca2+ dysfunction and beta-cell apoptosis.

physiology↗

bioRxiv2022

Retrograde mitochondrial signaling governs the identity and maturity of metabolic tissues

Mitochondrial damage is a hallmark of metabolic diseases, including diabetes and metabolic dysfunction-associated steatotic liver disease, yet the consequences of impaired mitochondria in metabolic tissues are often unclear. Here, we report that dysfunctional mitochondrial quality control engages a retrograde (mitonuclear) signaling program that impairs cellular identity and maturity across multiple metabolic tissues. Surprisingly, we demonstrate that defects in the mitochondrial quality control machinery, which we observe in pancreatic {beta} cells of humans with type 2 diabetes, cause reductions of {beta} cell mass due to dedifferentiation, rather than apoptosis. Utilizing transcriptomic profiling, lineage tracing, and assessments of chromatin accessibility, we find that targeted deficiency anywhere in the mitochondrial quality control pathway (e.g., genome integrity, dynamics, or turnover) activate the mitochondrial integrated stress response and promote cellular immaturity in {beta} cells, hepatocytes, and brown adipocytes. Intriguingly, pharmacologic blockade of mitochondrial retrograde signaling in vivo restores {beta} cell mass and identity to ameliorate hyperglycemia following mitochondrial damage. Thus, we observe that a shared mitochondrial retrograde response controls cellular identity across metabolic tissues and may be a promising target to treat or prevent metabolic disorders.

cell biology↗

bioRxiv2022

Restoration of PITPNA in Type 2 diabetic human islets reverses pancreatic beta-cell dysfunction

Defects in insulin processing and granule maturation are linked to pancreatic beta-cell failure during type 2 diabetes (T2D). Phosphatidylinositol transfer protein alpha (PITPNA) stimulates activity of phosphatidylinositol (PtdIns) 4-OH kinase to produce sufficient PtdIns-4-phosphate (PtdIns-4-P) in the trans-Golgi network to promote insulin granule maturation. PITPNA in beta-cells of T2D human subjects is markedly reduced suggesting its depletion accompanies beta-cell dysfunction. Conditional deletion of Pitpna in the beta-cells of Ins-Cre;Pitpnaflox/flox mice leads to hyperglycemia resulting from decreased glucose-stimulated insulin secretion (GSIS) and reduced pancreatic beta-cell mass. Furthermore, PITPNA silencing in human islets confirmed its role in PtdIns-4-P synthesis and led to impaired insulin granule maturation and docking, GSIS, and proinsulin processing with evidence of ER stress. Restoration of PITPNA in islets of T2D human subjects reversed these beta-cell defects and identify PITPNA as a critical target linked to beta-cell failure in T2D.

cell biology↗

bioRxiv2022

SERCA2 regulates proinsulin processing and processing enzyme maturation in the pancreatic β cell

Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in both type 1 and type 2 diabetes; however, the mechanisms underlying impaired proinsulin processing remain incompletely understood. Here, we identify the sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) pump and {beta} cell ER Ca2+ as key regulators of systemic glucose tolerance and proinsulin processing. We generated mice with a {beta} cell-specific SERCA2 deletion ({beta}S2KO) and SERCA2 deficient INS-1 cells to show that SERCA2 loss increases systemic and pancreatic levels of proinsulin protein and leads to aberrant localization of proinsulin within the proximal {beta} cell secretory pathway. These defects in proinsulin processing were linked to reduced maturation of the proinsulin processing enzymes PC1/3 and PC2, suggesting a model whereby chronic ER Ca2+ depletion in the {beta} cell, which is observed in many pathological conditions, impairs the spatial regulation of prohormone trafficking, processing, and maturation within the {beta} cell secretory pathway.

physiology↗

bioRxiv2022

Inflammatory cytokines rewire the proinsulin interaction network in human islets

Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both Type I and Type II diabetes (T1D, T2D). Inflammatory cytokines are implicated in pancreatic islet stress in both forms of diabetes but the mechanisms remain unclear. Here we examined how the diabetes associated cytokines interleukin-1{beta} and interferon-{gamma} alter proinsulin interactions with proteins that regulate its folding, trafficking, and secretion. Human islets treated with cytokines exhibited secretion of proinsulin, IL6 and nitrite, as well as evidence of endoplasmic reticulum (ER) stress. Unbiased proinsulin Affinity Purification-Mass Spectrometry revealed a proinsulin interactome reshaped by cytokines relative to controls. Cytokine treatment increased proinsulin binding to multiple ER chaperones and oxidoreductases, including the major ER chaperone BiP. Moreover, increased BiP binding was an adaptive response required to maintain proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and T1D and T2D GWAS candidate proteins not previously known to interact with proinsulin (e.g., Ataxin-2) and these GWAS proteins formed a tight network with each other. Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins. Consistent with a role for these proteins in proinsulin trafficking and release, chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data quantitatively map the proinsulin interactome rewired by cytokines, shedding new light on how human proinsulin biosynthesis is dysregulated by an inflammatory environment.

cell biology↗

bioRxiv2021

Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain -helix: one neonatal in DM onset (ValB18[->]Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated -helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding ({Delta}{Delta}Gu 1.2({+/-}0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

biochemistry↗

bioRxiv2021

Predisposition to Proinsulin Misfolding as a Genetic Risk to Diet-Induced Diabetes

Throughout evolution, proinsulin has exhibited significant sequence variation in both C-peptide and insulin moieties. As the proinsulin coding sequence evolves, the gene product continues to be under selection pressure both for ultimate insulin bioactivity and for the ability of proinsulin to be folded for export through the secretory pathway of pancreatic {beta}-cells. The substitution proinsulin-R(B22)E is known to yield a bioactive insulin, although R(B22)Q has been reported as a mutation that falls within the spectrum of Mutant INS-gene induced Diabetes of Youth (MIDY). Here we have studied mice expressing heterozygous (or homozygous) proinsulin-R(B22)E knocked into the Ins2 locus. Neither females nor males bearing the heterozygous mutation develop diabetes at any age examined, but subtle evidence of increased proinsulin misfolding in the endoplasmic reticulum is demonstrable in isolated islets from the heterozygotes. Moreover, males have indications of glucose intolerance and within a few week exposure to a high-fat diet, they develop frank diabetes. Diabetes is more severe in homozygotes, and the development of disease parallels a progressive heterogeneity of {beta}-cells with increasing fractions of proinsulin-rich/insulin-poor cells, as well as glucagon-positive cells. Evidently, sub-threshold predisposition to proinsulin misfolding can go undetected, but provides genetic susceptibility to diet-induced {beta}-cell failure.

cell biology↗

bioRxiv2021

Adaptation to chronic ER stress enforces pancreatic β-cell plasticity

Pancreatic {beta}-cells undergo high levels of endoplasmic reticulum (ER) stress due to their role in insulin secretion. Hence, they require sustainable and efficient adaptive stress responses to cope with the stress. Whether duration and episodes of chronic ER stress directly compromises {beta}-cell identity is largely unknown. We show that under reversible, chronic ER stress, {beta}-cells undergo a distinct transcriptional and translational reprogramming. During reprogramming, expression of master regulators of {beta}-cell function and identity and proinsulin processing is impaired. Upon recovery from stress, {beta}-cells regain their identity, highlighting a high-degree of adaptive {beta}-cell plasticity. Remarkably, when stress episodes exceed a certain threshold, {beta}-cell identity is gradually lost. Single cell RNA-seq analysis of islets from type 1 diabetes (T1D) patients, identifies the severe deregulation of the chronic stress-adaptation program, and reveals novel biomarkers for progression of T1D. Our results suggest {beta}-cell adaptive exhaustion ({beta}EAR) is a significant component of the pathogenesis of T1D.

systems biology↗

bioRxiv2021

Distinct states of proinsulin misfolding in MIDY

A precondition for efficient proinsulin export from the endoplasmic reticulum (ER) is that proinsulin meets ER quality control folding requirements, including formation of the Cys(B19)-Cys(A20) "interchain" disulfide bond, facilitating formation of the Cys(B7)-Cys(A7) bridge. The third proinsulin disulfide, Cys(A6)-Cys(A11), is not required for anterograde trafficking, i.e., a "lose-A6/A11" mutant [Cys(A6), Cys(A11) both converted to Ser] is well secreted. Nevertheless, an unpaired Cys(A11) can participate in disulfide mispairings, causing ER retention of proinsulin. Among the many missense mutations causing the syndrome of Mutant INS gene-induced Diabetes of Youth (MIDY), all seem to exhibit perturbed proinsulin disulfide bond formation. Here we have examined a series of seven MIDY mutants [including G(B8)V, Y(B26)C, L(A16)P, H(B5)D, V(B18)A, R(Cpep+2)C, E(A4)K], six of which are essentially completely blocked in export from the ER in pancreatic {beta}-cells. Three of these mutants, however, must disrupt the Cys(A6)-Cys(A11) pairing to expose a critical unpaired cysteine thiol perturbation of proinsulin folding and ER export, because when introduced into the proinsulin lose-A6/A11 background, these mutants exhibit native-like disulfide bonding and improved trafficking. This maneuver also ameliorates dominant-negative blockade of export of co-expressed wild-type proinsulin. A growing molecular understanding of proinsulin misfolding may permit allele-specific pharmacological targeting for some MIDY mutants.

biochemistry↗

bioRxiv2020

In vivo imaging of individual islets across the mouse pancreas reveals a heterogeneous insulin secretion response to glucose

While numerous techniques can be used to measure and analyze insulin secretion in isolated islets in culture, assessments of insulin secretion in vivo are typically indirect and only semiquantitative. The CpepSfGFP reporter mouse line allows the in vivo imaging of insulin secretion from individual islets after a glucose challenge, in live, anesthetized mice, addressing secretion from the pancreas as a whole. Imaging the whole pancreas at high resolution in live mice includes numerous technical challenges. Using rapid-fire imaging in high dynamic range and tiling combined with computer-assisted masked morphometry, we have developed a method to overcome motion blur, and the effects of anesthesia, to be able to monitor and quantify insulin (CpepSfGFP) content simultaneously and longitudinally for the first time in hundreds of individual islets, throughout a glucose challenge. Through this approach we demonstrate that while isolated islets respond homogeneously to glucose in culture, their response profile differs significantly in vivo. Independent of size or location, some islets respond sharply to a glucose stimulation while others barely secrete at all. This platform therefore provides a powerful approach to study the impact of disease, diet, surgery or pharmacological treatments on insulin secretion in the intact pancreas in vivo.

physiology↗

bioRxiv2020

The type 2 diabetes gene product STARD10 is a phosphoinositide binding protein that controls insulin secretory granule biogenesis

ObjectiveRisk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the {beta}-cell, impaired glucose-induced insulin secretion and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids, and thus the pathways through which STARD10 regulates {beta}-cell function, are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the {beta}-cell and its effect on proinsulin processing and insulin granule biogenesis and maturation. MethodsWe used isolated islets from mice deleted selectively in the {beta}-cell for Stard10 ({beta}StarD10KO) and performed electron microscopy, pulse-chase, RNA sequencing and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay were performed on purified STARD10 protein. Results{beta}StarD10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased. Amongst the differentially expressed genes in {beta}StarD10KO islets, expression of the phosphoinositide binding proteins Pirt and Synaptotagmin 1 were decreased while lipidomic analysis demonstrated changes in phosphatidyl inositol levels. The inositol lipid kinase PIP4K2C was also identified as a STARD10 binding partner. STARD10 bound to inositides phosphorylated at the 3 position and solution of the crystal structure of STARD10 to 2.3 [A] resolution revealed a binding pocket capable of accommodating polyphosphoinositides. ConclusionOur data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis and insulin processing.

cell biology↗

bioRxiv2020

Establishing cell-intrinsic limitations in cell cycle progression controls graft growth and promotes differentiation of pancreatic endocrine cells

Limitations in cell proliferation are a key barrier to reprogramming differentiated cells to pluripotent stem cells, and conversely, acquiring these limitations may be important to establish the differentiated state. The pancreas, and beta cells in particular have a low proliferative potential, which limits regeneration, but how these limitations are established is largely unknown. Understanding proliferation potential is important for the safty of cell replacement therapy with cell products made from pluripotent stem cell which have unlimited proliferative potential. Here we test a novel hypothesis, that these limitations are established through limitations in S-phase progression. We used a stem cell-based system to expose differentiating stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest, impairing S-phase entry, or S-phase completion. Upon release from these molecules, we determined growth potential, differentiation and function of insulin-producing endocrine cells both in vitro and after grafting in vivo. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells towards insulin-producing cells, improved the stability of the differentiated state, and protected mice from diabetes without the formation of cystic growths. Therefore, a compromised ability to enter S-phase and replicate the genome is a functionally important property of pancreatic endocrine differentiation, and can be exploited to generate insulin-producing organoids with predictable growth potential after transplantation.

cell biology↗

bioRxiv2019

Role of Proinsulin Self-Association in Mutant INS gene-induced Diabetes of Youth

Abnormal interactions between misfolded mutant and wild-type (WT) proinsulin in the endoplasmic reticulum (ER) drive the molecular pathogenesis of Mutant-INS-gene induced Diabetes of Youth (MIDY). How these abnormal interactions are initiated remains unknown. Normally, proinsulin-WT dimerizes in the ER. Here, we suggest that the normal proinsulin-proinsulin contact surface, involving the B-chain, contributes to dominant-negative effects of misfolded MIDY mutants. Specifically, we find that proinsulin Tyr-B16, which is a key residue in normal proinsulin dimerization, helps confer dominant-negative behavior of MIDY mutant proinsulin-C(A7)Y. Substitutions of Tyr-B16 with ether Ala, Asp, or Pro in proinsulin-C(A7)Y each decrease the abnormal interactions between the MIDY mutant and proinsulin-WT, rescuing proinsulin-WT export, limiting ER stress, and increasing insulin production in {beta}-cells and human islets. This study reveals the first evidence indicating that noncovalent proinsulin-proinsulin contact initiates dominant-negative behavior of misfolded proinsulin, pointing to a novel therapeutic target to enhance bystander proinsulin export for increased insulin production.

cell biology↗

bioRxiv2019

A reference map of the human proinsulin biosynthetic interaction network

The beta-cell protein synthetic machinery is dedicated to the production of insulin, which plays a critical role in organismal homeostasis. Insulin synthesis requires the proper folding and trafficking of its precursor, proinsulin, yet the precise network of proinsulin protein interactions in the secretory pathway remains poorly defined. In the present study we conducted unbiased profiling of the proinsulin interactome in human islets, utilizing a human proinsulin-specific monoclonal antibody for affinity purification and mass spectrometry. Stringent analysis identified a central node of interactions between human proinsulin and sequential secretory pathway proteins that is remarkably conserved across 3 ethnicities and both genders. Among the most prominent proinsulin interactions was with ER-localized peroxiredoxin-4 (PRDX4). A functional role for PRDX4 in beta-cells was demonstrated by gene silencing that rendered proinsulin susceptible to misfolding, particularly in response to oxidative stress. Conversely, exogenous PRDX4 improved proinsulin folding. Notably, oxidative stress and even high glucose treatment alone induced proinsulin misfolding in human islets and MIN6 cells, and this was accompanied by sulfonylation of PRDX4, a modification known to inactivate peroxiredoxins. This finding prompted PRDX4 analysis in a panel of human islet samples that revealed significantly higher levels of sulfonylated (inactive) PRDX4 in islets from patients with T2D compared to that of healthy individuals. Taken together, these data highlight the importance of elucidating the complete proinsulin interactome in human islets in order to understand critical steps controlling insulin biosynthesis, beta cell function, and T2D.

biochemistry↗

bioRxiv2019

Abnormalities in Proinsulin Processing in Islets from Individuals with Longstanding T1D

Work by our group and others has suggested that elevations in circulating proinsulin relative to C-peptide is associated with development of Type 1 diabetes (T1D). We recently described the persistence of detectable serum proinsulin in a large majority (95.9%) of individuals with longstanding T1D, including individuals with undetectable serum C-peptide. Here we describe analyses performed on human pancreatic sections from the nPOD collection (n=30) and isolated human islets (n=10) to further explore mechanistic etiologies of persistent proinsulin secretion in T1D. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with an inflammatory cytokine cocktail reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced {beta} cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.

pathology↗