Article

Improved characterization of the insulin secretory granule proteomes.

Biomedical Proteomics Research Group, Department of Human Protein Sciences, University Medical Center, Geneva, Switzerland.
Journal of proteomics (Impact Factor: 5.07). 04/2012; 75(15):4620-31. DOI: 10.1016/j.jprot.2012.04.023
Source: PubMed

ABSTRACT Insulin secretory granules (ISGs) are pivotal organelles of pancreatic ß-cells and represent a key participant to glucose homeostasis. Indeed, insulin is packed and processed within these vesicles before its release by exocytosis. It is therefore crucial to acquire qualitative and quantitative data on the ISG proteome, in order to increase our knowledge on ISG biogenesis, maturation and exocytosis. Despites efforts made in the past years, the coverage of the ISG proteome is still incomplete and comprises many potential protein contaminants most likely coming from suboptimal sample preparations. We developed here a 3-step gradient purification procedure combined to Stable Isotope Labeling with Amino acids in Cell culture (SILAC) to further characterize the ISG protein content. Our results allowed to build three complementary proteomes containing 1/ proteins which are enriched in mature ISGs, 2/ proteins sharing multiple localizations including ISGs, and finally 3/ proteins sorted out from immature ISGs and/or co-purifying contaminants. As a proof of concept, the ProSAAS, a neuronal protein found in ISGs was further characterized and its granular localization proved. ProSAAS might represent a novel potential target allowing to better understand the defaults in insulin processing and secretion observed during type 2 diabetes progression. This article is part of a special issue entitled: Translational Proteomics.

0 Bookmarks
 · 
123 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: In the present study, we performed an extensive qualitative characterization of the platelet granule proteome using subcellular fractionation followed by mass spectrometry analysis and functional annotation. Eight-hundred-and-twenty-seven proteins were identified, most of them being associated to granules and to the granule's secretory machinery. Functional pathway analysis revealed 30 pathways, including the major histocompatibility complex class 1 (MHC I) presenting antigen pathway. This pathway was of particular interest for its potential interrelation between platelets and the immune system. Key proteins belonging to this metabolic route such as β-2-microglobulin, 26S protease regulatory subunit 10B from the proteasome and proteins 1 and 2 of the transporter associated with antigen processing were shown to co-localize with von Willebrand factor in resting platelets and to be located on the plasma membrane when platelets were activated. Key proteins of the MHC1 antigen-presenting pathway are located in platelet secretory granules. These results suggest a possible functional role of platelet granules in platelet-related immune modulation. In this study, we described the largest dataset related to platelet granule proteins. We performed a functional pathway analysis that evidenced several expected granule-related pathways. We also highlighted the "Antigen processing and presentation" pathway that drawn our attention. Using immunofluorescence technique, we confirmed the presence of several key proteins for antigen presentation in platelet granules. This study suggests a putative functional role of MHC1 and platelet granules in the immune modulation.
    Journal of proteomics 02/2014; · 5.07 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The deposition of fibrillated human islet β-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure-function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.
    FEBS letters 09/2013; · 3.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: β Cells represent one of many cell types in heterogeneous pancreatic islets and play the central role in maintaining glucose homeostasis, such that disrupting β-cell function leads to diabetes. This review summarizes the methods for isolating and characterizing β cells, and describes integrated 'omics' approaches used to define the β cell by its transcriptome and proteome. RNA sequencing and mass spectrometry-based protein identification have now identified RNA and protein profiles for mouse and human pancreatic islets and β cells, and for β-cell lines. Recent publications have outlined these profiles and, more importantly, have begun to assign the presence or absence of specific genes and regulatory molecules to β-cell function and dysfunction. Overall, researchers have focused on understanding the pathophysiology of diabetes by connecting genome, transcriptome, proteome, and regulatory RNA profiles with findings from genome-wide association studies. Studies employing these relatively new techniques promise to identify specific genes or regulatory RNAs with altered expression as β-cell function begins to deteriorate in the spiral toward the development of diabetes. The ultimate goal is to identify the potential therapeutic targets to prevent β-cell dysfunction and thereby better treat the individual with diabetes. http://links.lww.com/COE/A5.
    Current opinion in endocrinology, diabetes, and obesity 02/2014; · 3.77 Impact Factor

Full-text

Download
49 Downloads
Available from
May 26, 2014