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The activity-dependent bulk endocytosis proteome reveals a key presynaptic role for Rab11

ABSTRACT: Activity-dependent bulk endocytosis (ADBE) is the dominant mode of synaptic vesicle (SV) endocytosis during high frequency stimulation, suggesting it should play key roles in neurotransmission during periods of intense neuronal activity. However efforts in elucidating the physiological role of ADBE have been hampered by the lack of identified molecules which are unique to this endocytosis mode. To address this, we performed proteomic analysis on purified bulk endosomes, which are a key intermediate in ADBE. Bulk endosomes were enriched via two independent approaches, a classical subcellular fractionation method and isolation via magnetic nanoparticles. There was a 79 % overlap in proteins identified via the two protocols and these molecules formed the ADBE proteome. Bioinformatic analysis revealed a strong enrichment in cell adhesion, cytoskeletal and signalling molecules, in addition to expected SV and trafficking proteins. Network analysis identified rab GTPases as a central hub within the ADBE proteome. Subsequent investigation of a subset of these rabs revealed that constitutively active rab11 both facilitated ADBE and accelerated clathrin-mediated endocytosis. This first result suggests that the ADBE proteome will provide a rich resource for the future study of presynaptic function.

INSTRUMENT(S): Orbitrap Fusion ETD

ORGANISM(S): Rattus Norvegicus (rat)

SUBMITTER: Matthias Trost

LAB HEAD: Matthias Trost

PROVIDER: PXD009745 | Pride | 2018-10-15

REPOSITORIES: Pride

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			Action	DRS
	20160211_MC_Dim_MG_R1.raw	Raw
	20160211_MC_Dim_MG_R2.raw	Raw
	20160211_MC_Dim_MG_R3.raw	Raw
	20160211_MC_Dim_Nyco_R2.raw	Raw
	20160211_MC_Dim_Nyco_R3.raw	Raw

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Project description:The pathogenesis of rheumatoid arthritis (RA) is complicated and involves both innate and adaptive immunity [5]. The innate immunity such as macrophages or fibroblast-like synoviocytes (FLS) in the synovium can generate inflammatory mediators, including TNF-α, IL-1, IL-6, IL-17, IFN-γ, and chemo kines that lead to synovial inflammation, bone erosion, and cartilage damage [6-8]. The IL-17 signaling mediates the autoantibody production in collagen-induced arthritis (CIA) model [9]. However, the treatment response with an anti-IL17 monoclonal antibody in RA patients shows a high degree of heterogeneity [10]. The inhibitors of the Janus kinase (JAK) pathway are approved for RA patients [11]. These data suggest that the targeted multiple cytokines through the JAK pathway are useful for RA treatment. Disturbance of type I IFNs (IFNs-I) signaling and production drive autoimmune development [12]. The presymptomatic RA patients display an increase of IFNs-I before the onset of symptoms [13]. The RA patients also show the elevation of IFN- α in the synovial fluid and high expression of IFNs-I regulated gene in peripheral blood mononuclear cells (PBMC) [14]. However, the role of IFNs-I in arthritis and bone homeostasis has suggested the accelerating effect of arthritis and bone damage. The interferon-alpha receptor knockout mice develop arthritis severity higher than wild-type mice in the model of antigen-induced arthritis [15]. IFNs-I also affects the bone homeostasis by inhibiting osteoclastogenesis via receptor activator of nuclear factor-kappa B (RANK) pathway, and reduction of c-FOS expression [16-18]. Therefore, the goal of RA treatment with antagonizing the IFNs-I pathway has to be optimized between efficacy and potentially adverse effect. STING is a cytosolic DNA sensor that initiates the production of IFNs-I. STING functions have been reported as both pro-inflammatory signaling and negative regulator against inflammation [19-21]. The mutation in exon 5 of the STING gene results in gain function leading to initiate inflammation and cause the Sting associated vasculopathy with onset in infancy (SAVI) [22]. Loss of STING function rescues DNaseII-deficient mice from lethality and polyarthritis [23]. However, Sting-deficient lupus mice (MRL/Lpr mice) show higher and earlier mortality than Sting-sufficient MRL/Lpr mice [24]. The pathology also shows lymphoid hypertrophy with a higher amount of macrophages and granulocytes infiltration, autoantibodies, and cytokine [24]. The objective of this study was to identify the role of STING in the pathogenesis of rheumatoid arthritis using collagen-induced arthritis (CIA) model as a representative model of the human RA.

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The activity-dependent bulk endocytosis proteome reveals a key presynaptic role for Rab11

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