Project Abstracts - CBDS

Rohit Kulkarni, MD, PhD, Investigator, Joslin Diabetes Center
Clayton Mathews, PhD, Investigator, University of Florida
Jason McDermott, PhD, co-Investigator, Pacific Northwest National Laboratory
Vladislav Petyuk, PhD, co-Investigator, Pacific Northwest National Laboratory
Tujin Shi, PhD, co-Investigator, Pacific Northwest National Laboratory

Start Date: September 20, 2014

End Date:  August 31, 2018

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Abstract

In Type 1 Diabetes (T1D) research there remains a critical need for pursuing a systems level understanding of early stage autoimmune-mediated molecular mechanisms which trigger the specific destruction of pancreatic β cells. Thus, the overall objectives of this application are to discover novel in vivo signaling pathways and regulatory networks that contribute to early stage β cell stress and death, and to identify potential therapeutic targets for intervention and early diagnostic biomarkers. These objectives will be pursued by applying enabling proteomics technologies focusing on posttranslational protein modifications (PTMs) to unique sets of human islet samples. Specifically, we hypothesize that posttranslational regulation, involving phosphorylation, S- nitrosylation, and S-glutathionylation, represents a fundamental triggering mechanism of β cell dysfunction preceding overt T1D. To address the limitations associated with clinical samples for studying dynamic signaling networks, our plan utilizes three complementary model systems: a) human islets isolated by laser-capture microdissection from clinical human pancreatic tissues, b) human islets treated with cytokines in vitro, and c) human islets transplanted in ‘humanized’ mice for recapitulating in vivo islet dysfunction. Studies in Aim 1 will identify signaling pathway and networks involved in early stage β cell dysfunction and apoptosis using global PTM focused proteomic technologies. In Aim 2, we will apply a targeted quantification approach to verify specific regulatory networks and PTMs of interest using samples from individual patients as well as time- course islet samples from ‘humanized’ mice. In Aim 3, we will evaluate protein targets discovered in the first two aims, determining their potential as β cell specific markers for early T1D diagnosis in serum, and their potential functional roles in β cell apoptosis through additional cultured islet studies. Together, we anticipate that this project will demonstrate a new paradigm of systems level study of posttranslational regulation of β cell dysfunction and apoptosis, and will provide a novel integrative view of the early stage regulatory networks that potentially trigger β cell apoptosis and T1D. This work will also provide a rich molecular data resource for the consortium on beta-cell death and survival (CBDS), the human islet research network (HIRN), and the diabetes community in generating new hypotheses for functional studies.

Publications

• Increased Inflammation as well as Decreased Endoplasmic Reticulum Stress and Translation Differentiate Pancreatic Islets of Pre-symptomatic Stage 1 Type 1 Diabetes and Non-diabetic Cases
• Essential roles of insulin and IGF-1 receptors during embryonic lineage development
• NKG2D Signaling Within the Pancreatic Islets Reduces NOD Diabetes and Increases Protective Central Memory CD8(+) T Cell Numbers
• Comprehensive Proteomics Analysis of Stressed Human Islets Identifies GDF15 as a Target for Type 1 Diabetes Intervention
• Increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes
• Boosting to Amplify Signal with Isobaric Labeling (BASIL) Strategy for Comprehensive Quantitative Phosphoproteomic Characterization of Small Populations of Cells
• Nanoproteomics comes of age
• Insulin receptor-mediated signaling regulates pluripotency markers and lineage differentiation
• Nanowell-mediated two-dimensional liquid chromatography enables deep proteome profiling of <1000 mammalian cells
• Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells
• Serum biomarkers for diagnosis and prediction of type 1 diabetes
• Spatially resolved proteome mapping of laser capture microdissected tissue with automated sample transfer to nanodroplets
• Fatiguing contractions increase protein S-glutathionylation occupancy in mouse skeletal muscle
• Targeted Quantification of Phosphorylation Dynamics in the Context of EGFR-MAPK Pathway
• Nanodroplet processing platform for deep and quantitative proteome profiling of 10-100 mammalian cells
• Advances in Microscale Separations Towards Nanoproteomics Applications
• GLP-1 signalling compensates for impaired insulin signalling in regulating beta cell proliferation in βIRKO mice
• Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines
• Insulin Signaling Regulates the FoxM1/PLK1/CENP-A Pathway to Promote Adaptive Pancreatic β Cell Proliferation
• Novel factors modulating human β-cell proliferation
• Advances in targeted proteomics and applications to biomedical research
• Pancreas-enriched miRNAs are altered in the circulation of subjects with diabetes: a pilot cross-sectional study
• Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway
• The current state of the art of quantitative phosphoproteomics and its applications to diabetes research
• Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function during Nanoparticle-Induced Oxidative Stress
• SerpinB1 Promotes Pancreatic β Cell Proliferation
• The Clinical Impact of Recent Advances in LC-MS for Cancer Biomarker Discovery and Verification
• Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines
• Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
• Liquid Chromatography-Mass Spectrometry Metabolic and Lipidomic Sample Preparation Workflow for Suspension-Cultured Mammalian Cells using Jurkat T lymphocyte Cells