Proteomics Analysis of Insulin Secretory Granules

Brunner, Yannick; Couté, Yohann; Iezzi, Mariella; Foti, Michelangelo; Fukuda, Mitsunori; Hochstrasser, Denis F.; Wollheim, Claes B.; Sanchez, Jean‐Charles

doi:10.1074/mcp.m600443-mcp200

Cited by 138 publications

(189 citation statements)

References 71 publications

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“…5A and B). Consistent with the above results, recent studies have shown that Rab37 regulates the release of secretory granules from mast cells in vitro [27], and in a rat insulin-secreting cell line [28]. Therefore, these data indicated that Rab37 regulates trafficking and TNF-a-containing secretory vesicles in macrophages.…”

Section: Inflammationsupporting

confidence: 86%

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

et al. 2011

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Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-a secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/ linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-a-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-a secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-a secretion from activated macrophages.Key words: Macrophage . Munc13-1 . Rab37 . Skin wound healing . TNF-a Supporting Information available online IntroductionIn response to skin tissue damage, inflammatory cells rapidly infiltrate the wound site to protect against infection. In the acute phase of inflammation, neutrophils, the first responder inflammatory cells, migrate toward wound sites to internalize and eliminate pathogens. Subsequently, macrophages preferentially infiltrate to wound sites after neutrophil recruitment, where they release cytokines, chemokines, and growth factors to control skin wound healing by stimulating the activation and proliferation of fibroblasts and keratinocytes, and by promoting wound contraction and angiogenesis [1]. Although this inflammatory response 3230is part of the wound-healing process, wound models in several organisms have shown that the inflammatory response can cause fibrotic diseases (i.e. scarring) in the skin and other organs [2]. Recently, we have shown that osteopontin is secreted by activated fibroblasts in response to platelet-derived growth factor secreted from macrophages, and that silencing of osteopontin at skin wound sites reduces scarring and improves the quality of healing in vivo [3]. Therefore, the pathways controlling inflammatory mediator secretion could provide novel molecular therapeutic targets [4].One such inflammatory mediator, TNF-a, is a pleiotropic proinflammatory cytokine expressed by macrophages and neutrophils at skin wound sites [5,6]. It has been reported that signaling via TNF-a negatively regulates skin wound healing, because TNF-a receptor p55 KO mice showed rapid skin wound healing coincident with reduced inflammatory respons...

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Section: Inflammationsupporting

confidence: 86%

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

et al. 2011

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“…This is of importance, as first-phase GSIS is much reduced in patients with type 2 diabetes, and this has been attributed in part to greatly reduced islet SYN-1A levels [23], along with reduction of cognate SNAREs (SNAP25 and VAMP2) and the Sec1/Munc18-like protein (SM) Munc18a. SYN-3 was shown in pancreatic acinar cells to preferentially form complexes with VAMP8 [20] and Munc18b [38,39], which are also present in beta cells [40][41][42]. We recently reported, employing the Vamp8-knockout mice [43], that VAMP8 also mediated the recruitment and fusion of newcomer SGs, and was the putative cognate v-SNARE that binds SYN-3.…”

Section: Discussionmentioning

confidence: 99%

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

et al. 2012

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Aims/hypothesis The molecular basis of the exocytosis of secretory insulin-containing granules (SGs) during biphasic glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells remains unclear. Syntaxin (SYN)-1A and SYN-4 have been shown to mediate insulin exocytosis. The insulinsecretory function of SYN-3, which is particularly abundant in SGs, is unclear. Methods Mouse pancreatic islets and INS-1 cells were treated with adenovirus carrying Syn-3 (also known as Stx3) or small interfering RNA targeting Syn-3 in order to examine insulin secretion by radioimmunoassay. The localisation and distribution of insulin granules were examined by confocal and electron microscopy. Dynamic single-granule fusion events were assessed using total internal reflection fluorescence microscopy (TIRFM). Results Depletion of endogenous SYN-3 inhibited insulin release. TIRFM showed no change in the number or fusion competence of previously docked SGs but, instead, a marked reduction in the recruitment of newcomer SGs and their subsequent exocytotic fusion during biphasic GSIS. Conversely, overexpression of Syn-3 enhanced both phases of GSIS, owing to the increase in newcomer SGs and, remarkably, to increased SG-SG fusion, which was confirmed by electron microscopy. Conclusions/interpretation In insulin secretion, SYN-3 plays a role in the mediation of newcomer SG exocytosis and SG-SG fusion that contributes to biphasic GSIS.

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“…Notably, mRab37 co-localizes with TNF-a-containing vesicles in activated macrophages 16 . In addition, mRab37 is involved in the control of insulin exocytosis in murine pancreatic b-cells 17,18 . However, more investigation is required to identify the cargos of hRAB37 and to dissect the defect mechanism of hRAB37 in human disease such as cancer.…”

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confidence: 99%

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis

et al. 2014

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Rab small GTPases are master regulators of membrane trafficking and guide vesicle targeting. Recent publications show that Rab-controlled trafficking pathways are altered during tumorigenesis. However, whether any of the Rabs plays a metastasis suppressor role is least explored. Here we address the metastasis suppressive function of human Rab37 (hRAB37) using secretomics, cell, animal and clinical analyses. We show that tissue inhibitor of metalloproteinase 1 (TIMP1), a secreted glycoprotein that inhibits extracellular matrix turnover, is a novel cargo of hRAB37. hRAB37 regulates the exocytosis of TIMP1 in a nucleotide-dependent manner to inactivate matrix metalloproteinase 9 (MMP9) migration axis in vitro and in vivo. Dysfunction of hRAB37 or TIMP1 abrogates metastasis suppression. Lung cancer patients with metastasis and poor survival show low hRAB37 protein expression coinciding with low TIMP1 in tumours. Our findings identify hRAB37 as a novel metastasis suppressor Rab that functions through the TIMP1-MMP9 pathway and has significant prognostic power.

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Proteomics Analysis of Insulin Secretory Granules

Cited by 138 publications

References 71 publications

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

Small GTPase Rab37 targets tissue inhibitor of metalloproteinase 1 for exocytosis and thus suppresses tumour metastasis

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