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Regulation of the Endo/Lysosomal pathway

内切/溶酶体途径的调节

基本信息

批准号：
10699712
负责人：
rosa puertollano
金额：
$ 70万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10699712
关键词：
Address Agonist Antigen Presentation Apoptosis Autophagosome Bacteria CCL2 gene Calcium Cations Cell Death Cell physiology Cells Cholesterol Cholesterol Homeostasis Collaborations Color Data Defect Dominant-Negative Mutation Dynein ATPase Endosomes Epidermal Growth Factor Receptor Event Family Family member Functional disorder Ganglioside Sialidase Deficiency Disease Genetic Transcription Germany Homeostasis Human Pathology Immune response Immunofluorescence Immunologic Impairment In Vitro Injections Innate Immune Response Integral Membrane Protein Ions Kidney Knockout Mice Laboratories Ligands Link Lysosomal Storage Diseases Lysosomes Malignant Neoplasms Mammals Measures Mediating Membrane Messenger RNA Minus End of the Microtubule Molecular Conformation Movement Mus Mutation Nerve Degeneration Organ Organelles Pathway interactions Peripheral Peritoneal Phenotype Physiological Physiological Processes Play Positioning Attribute Production Proteins Recycling Regulation Rest Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Starvation Stress Surface System TFE3 gene TRP channel Tissues cell type chemokine gain of function mutation hearing impairment human disease in vivo interest knock-down late endosome lymphoid organ macrophage member migration novel overexpression prevent receptor recruit response trafficking

项目摘要

Mucolipins (or TRPMLs) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3). MCOLN1 is the best-characterized member of the family due to the fact that mutations in this protein are associated with a human disease known as mucolipidosis type IV (MLIV). We and others have shown that the primary role of MCOLN1 in cells is to mediate calcium efflux from late endosomes and lysosomes, thus promoting organelle fusion and regulating endosomal trafficking. Gain-of-function mutation in MCOLN3 causes the varitint-waddler (Va) phenotype in mice, which is characterized by hearing loss, vestibular dysfunction, and coat color dilution. The Va phenotype results from a punctual mutation (A419P) in the pore region of MCOLN3 that locks the channel in an open conformation causing massive entry of calcium inside cells and inducing cell death by apoptosis. Overexpression of wild-type MCOLN3 produces severe alterations of the endosomal pathway, including enlargement and clustering of endosomes, delayed EGF receptor degradation, and impaired autophagosome maturation, thus suggesting that MCOLN3 plays an important role in the regulation of endosomal function. To understand better the physiological role of MCOLN3, we inhibited MCOLN3 function by expression of a channel-dead dominant negative mutant (458DD/KK) or by knockdown of endogenous MCOLN3 and measure several endosomal parameters including luminal calcium, pH, and endosomal fusion. We found impairment of MCOLN3 activity caused a significant accumulation of luminal calcium at endosomes. This accumulation led to severe defects in endosomal acidification as well as to increased endosomal fusion. Our findings reveal a prominent role for MCOLN3 in regulating calcium homeostasis at the endosomal pathway and confirm the importance of luminal calcium for proper acidification and membrane trafficking. The cellular function of MCOLN2 is far less characterized. To address MCOLN2 function in a physiologically relevant cell type, we first analyzed MCOLN2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of MCOLN2 at the transcriptional level. While MCOLN2 expression was negligible in resting macrophages, its mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, MCOLN1 and MCOLN3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous MCOLN2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with MCOLN1 and MCOLN3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of MCOLN2, we generated a MCOLN2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in MCOLN2-knockout mice. Furthermore, MCOLN2-knockout mice displayed impaired recruitment of peripheral macrophages in response to intra peritoneal (IP) injections of LPS and live bacteria, suggesting a potential defect in the immune response. These observations were further expanded in a recent collaboration with the laboratory of Dr. Christian Grimm from the Ludwig-Maximilians-Universitt in Germany, confirming treatment with ML2-SA1, a novel MCOLN2-specific agonist, increased CCL2 secretion in LPS-stimulated macrophages and promoted migration. Furthermore, MCOLN2 functions as an hypotonicity/mechanosensitive endolysosomal cation channel that enhances fast recycling and secretion pathways in activated macrophages. Overall, our study reveals interesting differences in the regulation and distribution of the members of the MCOLN family and identifies a novel role for MCOLN2 in the innate immune response. Recent evidence suggests that lysosomal distribution is linked to the role of lysosomes in many cellular functions, including autophagosome degradation, cholesterol homeostasis, antigen presentation, and cell invasion. Moreover, alterations in lysosomal positioning contribute to different human pathologies, such as cancer, neurodegeneration, and lysosomal storage diseases. We have identified a novel mechanism of lysosomal trafficking regulation. We found that the lysosomal transmembrane protein TMEM55B recruits JIP4 to the lysosomal surface, inducing dynein-dependent transport of lysosomes toward the microtubules minus-end. TMEM55B overexpression causes lysosomes to collapse into the cell center, whereas depletion of either TMEM55B or JIP4 results in dispersion toward the cell periphery. TMEM55B levels are transcriptionally upregulated following TFEB and TFE3 activation by starvation or cholesterol-induced lysosomal stress. TMEM55B or JIP4 depletion abolishes starvation-induced retrograde lysosomal transport and prevents autophagosome-lysosome fusion. These data reveal that the TFEB/TMEM55B/JIP4 axis coordinates lysosome movement in response to a variety of stress conditions.

Mucolipins（或trpml）是一个内体阳离子通道家族，与瞬时受体电位超家族同源。在哺乳动物中，粘脂蛋白家族包括三个成员，粘脂蛋白-1、-2和-3 （MCOLN1-3）。MCOLN1是该家族中最具特征的成员，因为该蛋白的突变与一种称为黏液脂质病IV型（MLIV）的人类疾病有关。我们和其他人已经表明，MCOLN1在细胞中的主要作用是介导钙从晚期核内体和溶酶体流出，从而促进细胞器融合和调节核内体运输。