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Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

基本信息

批准号：
7734931
负责人：
rosa puertollano
金额：
$ 134.27万
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/7734931
关键词：
Accounting Achlorhydria Address Amino Acids Apoptotic Ashkenazim Autophagocytosis Autophagosome Biogenesis C-terminal Cations Cell Death Cell Line Cell Maturation Cell division Cell membrane Cells Cellular Stress Characteristics Clathrin Adaptors Confocal Microscopy Cornea Cysteine Cytoplasm Cytosol Data Defect Detergents Disease Electron Microscopy Ensure Epithelial Cells Family Fibroblasts Functional disorder Ganglioside Sialidase Deficiency Disease Gangliosides Genetic Carriers Glycosaminoglycans Hair Cells Human Pathology Hydrolysis Immunofluorescence Immunologic Leucine Light Lipids Localized Lysosomes Mammals Mediating Melanosomes Mitochondria Modeling Monovalent Cations Mus Muscle Tonus Muscle hypotonia Mutation Nerve Degeneration Neurodegenerative Disorders Neurons Organelles Pathway interactions Patients Phenotype Phospholipids Phosphorylation Pigmentation physiologic function Play Population Post-Translational Protein Processing Predisposition Process Production Protein Overexpression Proteins Psyche structure Rate Reactive Oxygen Species Regulation Relative (related person)Reporting Resistance Retinal Retinal Degeneration Role Screening procedure Sphingolipids Stimulus Strabismus Structure Suggestion Tail Transcription Factor AP-1 Transcription Factor AP-2 Alpha Visual Yeasts base bis(monoacylglyceryl)phosphate founder mutation hearing impairment hepatocyte growth factor-regulated tyrosine kinase substrate human disease in vivo interest late endosome member molecular mass palmitoylation prevent protein aggregate receptor research study trafficking yeast two hybrid system

项目摘要

Mucolipins constitute a family of cation channels with homology to the transient receptor potential superfamily. In mammals, the mucolipin family includes three members (MCOLN1-3). MCOLN1 is the best-characterized member of the mucolipin family as mutations in this protein have been associated with mucolipidosis type IV (MLIV). MLIV is an autosomal recessive disease characterized by severe mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. MLIV is relatively frequent among Ashkenazi Jews, with 1/100 of the population estimated to be a genetic carrier. Two founder mutations that result in the absence of MCOLN1 account for 95% of the cases in this group. MCOLN1 is a 580-amino acid protein and has a molecular mass of 65 kDa. The predicted topology of the protein consists of six transmembrane-spanning domains with the N- and C- terminal tails oriented within the cytosol. To gain information on the function of MCOLN1, we addressed its intracellular distributions and trafficking. We have previously described that MCOLN1 localized to late endosomes and lysosomes. Two di-leucine motifs cooperate to regulate delivery of MCOLN1 to lysosomes through interactions with the clathrin adaptors AP1, AP2, and AP3. The C-terminal tail of MCOLN1 undergoes post-translational modifications that regulate its activity and trafficking. For example, palmitoylation of three cysteine residues (Cys565, Cys566, and Cys567) increases the rate of MCOLN1 internalization from the plasma membrane. In addition, PKA-mediated phosphorylation of Ser557 and Ser559 negatively regulates MCOLN1 channel activity in vivo. To a cellular level, MLIV is characterized by accumulation of enlarged vacuolar structures containing phospholipids, sphingolipids, mucopolysaccharides, and gangliosides. This accumulation of undigested material in lysosomes led to the suggestion that MCOLN1 plays a role in organelle biogenesis or regulation of lysosomal hydrolysis. However, the mechanism by which defects in MCOLN1 function result in mental and psychomotor retardation remains largely unknown. Recent studies reveal an interesting connection between lysosomal function, autophagy, and neurodegeneration. Autophagy is a crucial clearance mechanism that protects against the accumulation of toxic protein aggregates and damaged organelles. The last step of autophagy requires fusion of autophagosomes with late endosomes/lysosomes to ensure degradation of the autophagosomes content. We have examined the autophagic process in fibroblasts from MLIV patients. We reasoned that the defects on the late endosomal/lysosomal pathway observed in MLIV might have a profound impact on the formation and/or degradation of autophagosomes. Using multiple approaches, including LC3 localization, LC3-II/LC3-I westernblot, electron microscopy, and LC3/CD63 co-localization analysis, we find increased basal autophagy in MLIV fibroblasts. Autophagosome accumulation in MCOLN1-deficient cells is due to decreased autophagosome degradation and increased autophagosome formation. We also find increased levels of p62, a protein commonly found in protein inclusions associated with neurodegenerative disorders, as well as accumulation of ubiquitinated aggregates. Importantly, the majority of p62 accumulated in MLIV cells is resistant to extraction with detergents, an indication that p62 is part of aggregates or protein inclusions. Based on these data, we propose a model in which trafficking defects along the endosomal/lysosomal pathway due to absence of MCOLN1 impairs lysosomal function and results in inefficient autophagosome degradation. This in turn, causes accumulation of ubiquitinated aggregates that generate cellular stress and further induces autophagosome formation and accumulation. Defects in autophagy may not be especially detrimental in fibroblasts, where the rapid division of the cells helps to prevent the accumulation of misfolded or aggregated proteins. However, the accumulation of these products in neurons may result in cell death and, as the neurons cannot be replaced, neurodegeneration occurs. In addition, autophagic dysfunction also causes accumulation of damaged mitochondria. These abnormal mitochondria are deficient in ATP production, produce increased amounts of reactive oxygen species, and increase susceptibility of cells to pro-apoptotic stimuli. In summary, our data demonstrates the impact of lysosomal function on autophagic activity and suggest that, analogous to other lysosomal storage disorders, accumulation of ubiquitinated protein inclusions due to defective autophagy may contribute to the neurodegeneration observed in MLIV patients. MCOLN3 is another member of the mucolipin family that might play a role in different human pathologies. Mutations in MCOLN3 are responsible for the varitint-waddler mouse phenotype characterized by defects in pigmentation and hearing loss. MCOLN3 is located in hair cells, and it has been suggested that it may be implicated in hair cell maturation and melanosome trafficking. Several recent reports showed that TRPML3 is an inwardly (from lumen into cytoplasm) rectifying monovalent cation channel that is permeable to Ca2+ and suppressed by low pH. To better understand the function of MCOLN3 we decided to study its subcellular localization in the retinal epithelial cell line ARPE19. Using immunofluorescence and confocal microscopy we found that heterologously expressed GFP-tagged-MCOLN3 (GFP-MCOLN3) localized to the plasma membrane and to the endosomal pathway in ARPE19 cells. Overexpressed GFP-MCOLN3 showed extensive co-localization with the early endosomal markers Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), while it co-localized to less extent with late endosomal markers such as LBPA (lysobisphosphatidic acid). These results show a clear difference in the subcellular distribution between MCOLN3 and its relative MCOLN1, which is mostly lysosomal. The localization of MCOLN3 within the upper, less acidic, portions of the endocytic pathway is consistent with the reported inhibition of the channel at low pH characteristic of lysosomes. Ongoing experiments will help us to determine the function of MCOLN3 within the endocytic pathway.

粘磷脂构成了一个与瞬时受体电位超家族同源的阳离子通道家族。在哺乳动物中，粘脂蛋白家族包括三个成员（MCOLN1-3）。MCOLN1是粘脂蛋白家族中最具特征的成员，因为该蛋白的突变与IV型粘脂病（MLIV）有关。MLIV是一种常染色体隐性遗传病，其特征是严重的精神和精神运动迟缓，肌肉张力降低或张力低下，胃酸过少，以及包括角膜混浊，视网膜变性，对光敏感和斜视在内的视力问题。MLIV在德系犹太人中比较常见，估计有1/100的人口是遗传携带者。导致MCOLN1缺失的两种始祖突变占该组病例的95%。MCOLN1是一种含有580个氨基酸的蛋白质，分子量为65 kDa。该蛋白的预测拓扑结构由6个跨膜结构域组成，其N-和C-末端在细胞质内定向。为了获得有关MCOLN1功能的信息，我们研究了它在细胞内的分布和转运。我们之前已经描述过MCOLN1定位于晚期核内体和溶酶体。两个二亮氨酸基序通过与网格蛋白接头AP1、AP2和AP3相互作用，共同调节mcln1向溶酶体的传递。MCOLN1的c端尾部经过翻译后修饰，调节其活性和转运。例如，三个半胱氨酸残基（Cys565、Cys566和Cys567）的棕榈酰化增加了MCOLN1从质膜内化的速率。此外，pka介导的Ser557和Ser559的磷酸化在体内负调控MCOLN1通道活性。