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Identify proinsulin regulators using CRISPR screen and mouse genetics [RNA-Seq]

ABSTRACT: Proinsulin is the precursor of insulin in pancreatic beta cells. Altered proinsulin and proinsulin to insulin ratio mark beta cell dysfunction, predictuing disease progression into type 1 and type 2 diabetes. Of essential role for beta cell function, knowledge about proinsulin production and its role in disease are currently very limited. Using genome wide CRISPR screen, we identified 84 proinsulin regulators including classical protein convertases Pcsk1 and Cpe, and novel factors like Pdia6. Among the list 29 proinsulin regulators were trajectory genes involved in disease progression in obesity and type 2 diabetes in humans. In vivo mouse genetics study revealed unique genetic architecture and quantitative trait loci (QTLs) modulating plasma proinsulin levels. Integrative analyzing and mapping of the QTL signals directly pinpointed to proinsulin regulators identified from the CRISPR screen, which in return greatly improved resolution of the mouse genetic study. 4 out of 5 overlapped genes can be individually validated. Knocking down the leading hits Pdia6 leads to decreased proinsulin content and remarkable loss of proinsulin granules in beta cells. Consequently, proinsulin secretion was greatly decreased. Mechanistically, protein translation rate was greatly impaired after knocking down Pdia6. Our study demonstrated the power of combining in vitro functional genomics with in vivo mouse genetics study to identify proinsulin regulatory network in pancreatic beta cells.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE245844 | GEO | 2023/11/15

REPOSITORIES: GEO

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Identify proinsulin regulators using CRISPR screen and mouse genetics [CRISPR screen]

Project description:Proinsulin is the precursor of insulin in pancreatic beta cells. Altered proinsulin and proinsulin to insulin ratio mark beta cell dysfunction, predictuing disease progression into type 1 and type 2 diabetes. Of essential role for beta cell function, knowledge about proinsulin production and its role in disease are currently very limited. Using genome wide CRISPR screen, we identified 84 proinsulin regulators including classical protein convertases Pcsk1 and Cpe, and novel factors like Pdia6. Among the list 29 proinsulin regulators were trajectory genes involved in disease progression in obesity and type 2 diabetes in humans. In vivo mouse genetics study revealed unique genetic architecture and quantitative trait loci (QTLs) modulating plasma proinsulin levels. Integrative analyzing and mapping of the QTL signals directly pinpointed to proinsulin regulators identified from the CRISPR screen, which in return greatly improved resolution of the mouse genetic study. 4 out of 5 overlapped genes can be individually validated. Knocking down the leading hits Pdia6 leads to decreased proinsulin content and remarkable loss of proinsulin granules in beta cells. Consequently, proinsulin secretion was greatly decreased. Mechanistically, protein translation rate was greatly impaired after knocking down Pdia6. Our study demonstrated the power of combining in vitro functional genomics with in vivo mouse genetics study to identify proinsulin regulatory network in pancreatic beta cells.

2023-11-15 | GSE245845 | GEO

Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2

Project description:Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8¬¬+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation. These results indicate that insulin-derived peptides, presented by HLA-class I molecules at the cell surface, originate from ER-resident proinsulin that has been dislocated to the cytosol for subsequent degradation. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future therapies that may cure or even prevent T1D.

2024-03-20 | PXD049996 | Pride

Pancreatic β cell SERCA2 deficiency leads to impaired proinsulin processing and reduced ER to Golgi protein trafficking

Project description:Impaired proinsulin processing is observed in both type 1 and type 2 diabetes. We have previously shown that reductions in endoplasmic reticulum (ER) calcium (Ca2+) in the pancreatic β cell arising from impaired activity of the Sarcoendoplasmic Reticulum Ca2+ ATPase (SERCA) pump are associated with increased proinsulin secretion. However, the mechanisms responsible for reduced proinsulin processing in the context of SERCA2 deficiency remain incompletely understood. To test this, we developed mice with β cell specific SERCA2 deletion (βS2KO mice) and S2KO INS1 cells. βS2KO mice exhibited age-dependent glucose intolerance and reduced glucose-stimulated insulin secretion without evidence of impaired insulin sensitivity. ER Ca2+ levels in islets from βS2KO mice were significantly reduced, while serum proinsulin/insulin (PI/I) ratios and whole pancreas PI/I content were elevated. Immunoblot analysis of βS2KO islets and S2KO INS-1 cells revealed reduced active forms of the proinsulin processing enzymes, PC1/3, PC2 and CPE. Restoration of SERCA2b via adenoviral transduction in S2KO INS1 cells was sufficient to restore PC1/3 and PC2 maturation and enzyme activity. Brefeldin A treatment in INS1 cells recapitulated the impairments in PC1/3 and PC2 maturation observed in S2KO cells, suggesting a disturbance in protein trafficking between the ER and Golgi. Consistent with this, trafficking assays were performed using a vesicular stomatitis virus G (VSVG) protein construct and revealed a significantly slower rate of VSVG movement from the ER to the Golgi in S2KO INS1 cells. Moreover, pancreas sections from βS2KO mice showed increased co-localization of proinsulin and ProPC2 in the early compartments of the secretory pathway. Taken together, these data suggest that loss of SERCA2 activity and ER Ca2+ loss in the pancreatic β cell leads to impaired proinsulin processing via reduced maturation and trafficking of proinsulin processing enzymes.

2023-10-11 | GSE207498 | GEO

Eye imaginal disc expressing transgenic human proinsulin in Drosophila eye under regulation of GMR-GAL4

2012-12-23 | GSE43128 | GEO

A reference map of the human proinsulin biosynthetic interaction network

Project description:A monoclonal antibody specific for human proinsulin (20G11) was generated and utilized for affinity purification mass spectrometry (AP-MS) for unbiased profiling of proinsulin protein:protein interactions in human islets. Half of each islet prep was treated with the cytokines IL1-beta and IFN-gamma. Immunoprecipitated proinsulin or control IgG immunoprecipitates from human islets were then subjected to state of the art mass spectrometry revealing a rich proinsulin biosynthetic network that is remarkably conserved across a diverse group of donors.

2019-07-03 | MSV000084057 | MassIVE

Eye imaginal disc expressing transgenic human proinsulin in Drosophila eye under regulation of GMR-GAL4

Project description:We used microarrays to detail the global program of gene expression in response to expression of either mutant (C96Y) or wild-type human proinsulin and identified distinct classes of up-regulated genes. Results provides insight into the molecular mechanisms underlying a form of neonatal diabetes. Drosophila eye imaginal discs were selected at the wandering stage of 3rd instar larval development for RNA extraction and hybridization on Affymetrix microarrays.

2012-12-23 | E-GEOD-43128 | biostudies-arrayexpress

Proinsulin dependent interaction between ENPL-1/GRP94 and ASNA-1 in neurons is required to maintain insulin secretion in C. elegans.

Project description:Maturation of insulin is crucial for insulin secretion and function. ENPL[1]1/GRP94/HSP90B1 plays an important role in this process. ASNA-1/TRC40/GET3 and ENPL-1/GRP94 are conserved insulin secretion regulators in Caenorhabditis elegans and mammals and mouse mutants display type 2 diabetes. ENPL-1 and GRP94 bind proinsulin and regulate proinsulin levels in C. elegans and cultured cells. Here we found that ASNA-1 and ENPL-1 co-operated to regulate insulin secretion in worms via a physical interaction that required pro-DAF-28/insulin but occurred independently of the insulin binding site of ENPL-1. ASNA-1 acted in neurons to promote DAF-28/insulin secretion. The interaction occurred in insulin expressing neurons and was sensitive to changes in pro-DAF-28 levels. The chaperone form of ASNA-1 is likely bound to ENPL-1. Loss of asna-1 disrupted Golgi trafficking pathways. ASNA-1 localization was affected in enpl-1 mutants and ENPL-1 overexpression partially bypassed ASNA[1]1 requirement. Taken together, we find a functional interaction between ENPL-1 and ASNA-1 which is necessary to maintain proper insulin secretion in C. elegans and provides insights about how their loss might produce diabetes in mammals.

2023-03-10 | PXD038504 | Pride

Gene expression changes in response to doxycyline-induced expression of proinsulin (C96Y)-GFP in an INS-1 insulinoma cell line

Project description:A doxycyline-inducible INS-1 insulinoma cell line expressing proinsulin (C96Y)-GFP was engineered. Addition of doxycyline causes the production of the proinsulin (C96Y)-GFP, which is retained in the endoplasmic reticulum. This study analyzes the gene expression changes that occur after doxycyline-induced expression of proinsulin (C96Y)-GFP for 24h, 48h and 5 days. Expression changes were compared between control un-induced cells and cells treated with doxycyline. Three replicates (experiments) were performed for each time point.

2010-06-24 | GSE22537 | GEO

Single-cell RNA sequencing enables transcriptomic analysis of iPSC-derived beta-cells in a model of neonatal diabetes caused by insulin mutations.

Project description:Insulin gene mutations are a leading cause of neonatal diabetes. They can lead to proinsulin misfolding and its retention in endoplasmic reticulum (ER). This results in increased ER-stress suggested to trigger beta-cell apoptosis. In humans, the mechanisms underlying beta-cell failure remain unclear. Here we show that misfolded proinsulin impairs developing beta-cell proliferation without increasing apoptosis. We generated iPSCs from diabetics carrying insulin mutations, engineered isogenic CRISPR-Cas9 mutation-corrected lines and differentiated them to beta-like cells using a 3D-suspension differentiation protocol. Single-cell RNA-sequencing analysis showed increased ER-stress and reduced proliferation in INS-mutant beta-like cells compared with corrected controls. Upon transplantation to mice, INS-mutant grafts presented reduced insulin secretion and aggravated ER-stress. Cell size, mTORC1 signaling, and respiratory chain subunit expression were all reduced in INS-mutant beta-like cells, yet apoptosis was not increased at any stage. Our results demonstrate that neonatal diabetes-associated INS-mutations lead to defective beta-cell mass expansion, contributing to diabetes development.

2018-12-01 | GSE115257 | GEO

Proteomics analysis of rough endoplasmic reticulum in pancreatic beta cells

Project description:Pancreatic beta cells have well-developed endoplasmic reticulum (ER) to accommodate for the massive production and secretion of insulin. ER homeostasis is vital for normal beta cell function. Perturbation of ER homeostasis contributes to beta cell dysfunction in both type 1 and type 2 diabetes. To systematically identify the molecular machinery responsible for proinsulin biogenesis and maintenance of beta cell ER homeostasis, a widely used mouse pancreatic beta cell line, MIN6 cell was used to purify rough ER. Two different purification schemes were utilized. In each experiment, the ER pellets were solubilized and analyzed by one dimensional SDS-PAGE coupled with HPLC-MS/MS. A total of 1467 proteins were identified in three experiments with ≥95% confidence, among which 1117 proteins were found in at least two separate experiments. Gene ontology analysis revealed a comprehensive profile of known and novel players responsible for proinsulin biogenesis and ER homeostasis. This dataset defines a molecular environment in the ER for proinsulin synthesis, folding and export and laid a solid foundation for further characterizations of altered ER homeostasis under diabetes-causing conditions.

2015-04-15 | PXD001081 | Pride

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