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Identification of HIPs in the islets of mice and non-diabetic human donors

ABSTRACT: Hybrid insulin peptides (HIPs) result from the linkage of an insulin C-peptide fragment and another peptide via a traditional peptide bond to generate a sequence that is not encoded in the genome. Here, we sought to identify HIPs naturally present in the pancreatic islets of non-obese diabetic (NOD) mice, BALB/c mice, and non-diabetic human donors by mass spectrometry.

INSTRUMENT(S): Agilent instrument model

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Pancreatic Islet, Islet Of Langerhans

DISEASE(S): Disease Free

SUBMITTER: Timothy Wiles

LAB HEAD: Thomas Delong

PROVIDER: PXD011606 | Pride | 2019-01-07

REPOSITORIES: Pride

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			Action	DRS
	BALB1_E3.d.zip	Other
	BALB1_E3.pep.xml	Pepxml
	BALB1_E3_exclusion.d.zip	Other
	BALB1_E3_exclusion.pep.xml	Pepxml
	BALB2_E3.d.zip	Other

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Publications

Identification of Hybrid Insulin Peptides (HIPs) in Mouse and Human Islets by Mass Spectrometry.

Wiles T. Aaron TA Powell Roger R Michel Cole R Beard K. Scott KS Hohenstein Anita A Bradley Brenda B Reisdorph Nichole N Haskins Kathryn K Delong Thomas T

Journal of proteome research 20190103 3

We recently discovered hybrid insulin peptides (HIPs) as a novel class of post-translationally modified peptides in murine-derived beta cell tumors, and we demonstrated that these molecules are autoantigens in type 1 diabetes (T1D). A HIP consists of an insulin fragment linked to another secretory granule peptide via a peptide bond. We verified that autoreactive CD4 T cells in both mouse and human autoimmune diabetes recognize these modified peptides. Here, we use mass spectrometric analyses to ...[more]

PMID: 30585061

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NOD Mouse Islet Peptide Content Identification

Project description:Beta-cells produce hybrid insulin peptides (HIPs) by linking insulin fragments to other peptides through peptide bonds. HIPs have unique amino acid sequences and are targeted by autoreactive T cells in type 1 diabetes (T1D). Individuals with recent-onset T1D have significantly higher levels of HIP-reactive T cells in their blood compared to non-diabetic control subjects. HIP-reactive T cells have also been found in the residual pancreatic islets of deceased T1D organ donors. In non-obese diabetic (NOD) mice, a major T1D animal model, several CD4 T cell clones that trigger diabetes have been shown to target HIPs. Through mass spectrometry, a subgroup of HIPs containing N-terminal amine groups of various peptides linked to aspartic acid residues of insulin C-peptide has been detected in NOD islets. Our research reveals that these HIPs form spontaneously in beta-cells via an aspartic anhydride intermediate mechanism. This process leads to the creation of a regular HIP with a standard peptide bond and a HIP-isomer (isoHIP) with an isopeptide bond linked to the carboxylic acid side-chain of the aspartic acid residue. Our mass spectrometric analyses confirmed the presence of both HIP isomers in murine islets, thereby validating the occurrence of this new reaction mechanism in beta-cells. The spontaneous formation of neoepitopes through the development of new peptide bonds within cells may contribute to the pathogenesis of T1D and other autoimmune diseases.

2023-11-16 | PXD041616 | Pride

Gene expression in the islets of 12 wk old Non-Obese Diabetic (NOD) mice and healthy control NOD.B10 mice

Project description:Gene expression in the islets of NOD and NOD.B10 mice at 12 weeks of age, prior to the onset of destructive insulitis. The pancreata of NOD and NOD.B10 mice (n=6) were frozen and islets were isolated by laser capture microdissection. Cryosections (8 M-NM-<m) were cut and stained using the Arcturus HistoGene Frozen Section Staining kit (Applied Biosystems) and laser dissection was performed using the Leica AS LMD and the Leica IM 1000 Image Manager Basic software. Sections from at least 40 individual islets were collected and RNA was extracted using the RNeasy micro kit (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Pico Reagent Kit (Agilent). Samples were preamplified using the TrueLabeling-PicoAMP kit (SA Biosciences), post-labeled with Cy5 and run against a Cy3-labeled mouse Universal RNA control (SA Biosciences). Microarrays were performed using the Whole Mouse Genome Microarray Kit, 4M-CM-^W44K 2-color arrays (Agilent Technologies).

2013-04-10 | E-GEOD-45897 | biostudies-arrayexpress

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Project description:Hybrid insulin peptides (HIPs) form in pancreatic beta-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs, which have been confidently identified in pancreatic islets by mass spectrometry, are targeted by CD4 T cells in subjects with Type 1 Diabetes (T1D), as well as by disease-triggering CD4 T cell clones in non-obese diabetic (NOD) mice. The mechanism(s) of HIP formation are currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs that are targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient of cathepsin L (CatL), another protease that was implied in the formation of disease-relevant HIPs, contain elevated levels of HIPs, signifying a primary role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow down the autoimmune destruction of beta-cells mediated by HIP-reactive CD4 T cells in T1D.

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Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants (RNA-seq)

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A discovery-based proteomics approach identifies protein disulfide isomerase (PDIA1) as a biomarker of β cell stress in type 1 diabetes

Project description:Background: Activation of stress pathways intrinsic to the β cell are thought to both accelerate β cell death and increase β cell immunogenicity in type 1 diabetes (T1D). However, information on the timing and scope of these responses is lacking. Methods: To identify temporal and disease-related changes in islet β cell protein expression, SWATH-MS/MS proteomics analysis was performed on islets collected longitudinally from NOD mice and NOD-SCID mice rendered diabetic through T cell adoptive transfer. Findings: In islets collected from female NOD mice at 10, 12, and 14 weeks of age, we found a time-restricted upregulation of proteins involved in the maintenance of β cell function and stress mitigation, followed by loss of expression of protective proteins that heralded diabetes onset. Pathway analysis identified EIF2 signaling and the unfolded protein response, mTOR signaling, mitochondrial function, and oxidative phosphorylation as commonly modulated pathways in both diabetic NOD mice and NOD-SCID mice rendered acutely diabetic by adoptive transfer, highlighting this core set of pathways in T1D pathogenesis. In immunofluorescence validation studies, β cell expression of protein disulfide isomerase A1 (PDIA1) and 14-3-3b were found to be increased during disease progression in NOD islets, while PDIA1 plasma levels were increased in pre-diabetic NOD mice and in the serum of children with recent-onset T1D compared to age and sex-matched non-diabetic controls. Interpretation: We identified a common and core set of modulated pathways across distinct mouse models of T1D and identified PDIA1 as a potential human biomarker of β cell stress in T1D.

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2013-11-08 | E-GEOD-51311 | biostudies-arrayexpress