Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 7968954
• 负责人: rosa puertollano
• 金额: $ 98.84万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/7968954
• 关键词: Achlorhydria; Acute; Address; Agreement; Amino Acids; Autophagocytosis; Autophagosome; Behavior; Binding; Biogenesis; C-terminal; CCL4 gene; Cell membrane; Cells; Charge; Clathrin Adaptors; Color; Cornea; Cysteine; Cytoplasm; Data; Defect; Disease; EF Hand Motifs; EGF gene; Endosomes; Epidermal Growth Factor Receptor; Epithelial Cells; Event; Exhibits; Family; Family member; Functional disorder; Ganglioside Sialidase Deficiency Disease; Goals; Head; Human; Ion Channel; Lead; Leucine; Life; Light; Lipids; Location; Lysosomes; Mammals; Mediating; Membrane; Membrane Fusion; Membrane Protein Traffic; Mental Retardation; Modeling; Molecular; Monovalent Cations; Motor; Mus; Mutate; Mutation; Nerve Degeneration; Neurologic; Organelles; Pathway interactions; Patients; Pentas; Phenotype; Phosphorylation; Play; Post-Translational Protein Processing; Process; Protein Family; Proteins; Regulation; Reporting; Research; Retinal Degeneration; Role; Sorting - Cell Movement; Strabismus; Suggestion; Symptoms; Tail; Transcription Factor AP-1; Transcription Factor AP-2 Alpha; Vacuole; Visual; hearing impairment; human disease; in vivo; insight; late endosome; lipid transport; member; novel; palmitoylation; patch clamp; protein expression; receptor; research study; sensor; trafficking

Mucolipins are a family of ion channels that belongs to the superfamily of transient receptor potential (TRP) channels. In mammals, the mucolipin family includes three members, mucolipin-1, -2, and -3 (MCOLN1-3), that exhibit a common six-membrane-spanning topology. 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). MLIV is a lysosomal storage disorder characterized by acute neurological and ophthalmologic defects. Symptoms appear during the first year of life and include mental retardation, delayed motor milestones, achlorhydria, and visual problems such us corneal clouding, retinal degeneration, sensitivity to light, and strabismus. The accumulation of enlarged vacuoles that contain different types of undigested lipids is a hallmark of MLIV cells and has lead to the suggestion that MCOLN1 might regulate membrane trafficking at the late endocytic pathway. More specifically, MCOLN1 has been suggested to participates in several Ca2+-dependent processes including fusion of lysosomes with the plasma membrane, fusion of late endosomes and autophagosomes with lysosomes, and lysosomal biogenesis. Consistent with the proposed role of MCOLN1 in lysosomal function, we have previously described that the protein localizes to late endosomes/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. Thus, for example, palmitoylation of three cysteine residues (Cys565, Cys566, and Cys567) increases the rate of MCOLN1 internalization from the plasma membrane, while PKA-mediated phosphorylation of Ser557 and Ser559 negatively regulates MCOLN1 channel activity in vivo. We have also found that the alterations of the late endosomal/lysosomal pathway observed in MCOLN1 deficient cells cause defective autophagy and lead to the accumulation of protein inclusions and ubiquitinated aggregates that might contribute to the neurodegeneration observed in MLIV patients. To better understand the mechanisms that regulate MCOLN1 function we have searched for proteins that interact with MCOLN1 in a Ca2+-dependent manner. We found that the penta-EF-hand protein ALG-2 binds to the NH-terminal cytosolic tail of MCOLN1. The interaction is direct, strictly dependent on Ca2+, and mediated by a patch of charged and hydrophobic residues located between MCOLN1 residues 37-49. In agreement with the proposed role of MCOLN1 in the regulation of fusion/fission events, we found that over-expression of MCOLN1 caused accumulation of enlarged, aberrant endosomes that contain both early and late endosomes markers. Interestingly, aggregation of abnormal endosomes was greatly reduced when the ALG-2-binding domain in MCOLN1 was mutated, suggesting that ALG-2 regulates MCOLN1 function. These data provide new insight into the molecular mechanisms that regulate MCOLN1 activity. We propose that ALG-2 acts as a Ca2+ sensor that modulates the function of MCOLN1 along the late endosomal-lysosomal pathway. MCOLN3 shows about 75% amino acid similarity with MCOLN1. Mutations in MCOLN3 are the cause of the varitint-waddler phenotype in mice, characterized by hearing loss, vestibular dysfunction (circling behavior, head-bobbing, waddling), and coat color dilution. Whole cell patch clamp experiments revealed that TRPML3 is an inwardly (from lumen into cytoplasm) rectifying monovalent cation channel that is permeable to Ca2+ and suppressed by low pH. We have addressed the function of MCOLN3 in epithelial cells. We found that MCOLN3 primarily localizes to early and late endosomes in ARPE-19 cells. This distribution at the less acidic portions of the endocytic pathway is consistent with the reported inactivation of the channel by low pH. Furthermore, we found that over-expression of MCOLN3 caused dramatic alterations in the endosomal pathway, including enlargement of Hrs-positive endosomes, delayed degradation of EGF and EGFR, and defective autophagosome maturation. Interestingly, similar defects have been described upon over-expression of proteins implicated in endosome biogenesis, such as ESCRT and Hrs. We have also found that endosomal pH is higher in cells over-expressing MCOLN3 and propose a model in which Ca2+ release from endosomes mediated by MCOLN3 might be important for efficient endosomal acidification. Therefore, MCOLN3 is a novel Ca2+ channel that plays a crucial role in the regulation of cargo trafficking along the endosomal pathway. Overall, our data reveal new evidence indicating that mucolipins distribute to specific locations along the endosomal pathway and play an important regulatory role in the sorting of lipids and proteins.

黏脂蛋白是一个离子通道家族，属于瞬时受体电位(Trp)通道超家族。在哺乳动物中，黏脂蛋白家族包括三个成员，即黏脂蛋白-1、-2和-3(MCOLN1-3)，它们表现出共同的六膜跨膜拓扑结构。 MCOLN1是该家族中特征最好的成员，因为这种蛋白的突变与一种称为IV型粘脂沉积症(MLIV)的人类疾病有关。MLIV是一种溶酶体储存障碍，以急性神经和眼科缺陷为特征。症状出现在生命的第一年，包括智力低下、运动里程碑延迟、酸中毒和视觉问题，如角膜混浊、视网膜变性、对光敏感和斜视。MLIV细胞的一个特征是积累含有不同类型未消化脂质的增大的空泡，这导致了MCOLN1可能调节晚期内吞途径的膜运输。更具体地说，MCOLN1被认为参与了几个钙依赖的过程，包括溶酶体与质膜的融合，晚期内吞体和自噬小体与溶酶体的融合，以及溶酶体的生物发生。与MCOLN1在溶酶体功能中的作用一致，我们先前已经描述了该蛋白定位于晚期内小体/溶酶体。两个二亮氨酸基序通过与网状蛋白适配器AP1、AP2和AP3的相互作用来调节MCOLN1向溶酶体的传递。MCOLN1的C末端尾巴经过翻译后修饰，调节其活动和贩运。因此，例如，三个半胱氨酸残基(Cys565、Cys566和Cys567)的棕榈酰化增加了MCOLN1从质膜内化的速度，而PKA介导的Ser557和Ser559的磷酸化负调控体内MCOLN1通道的活性。我们还发现，在MCOLN1缺陷细胞中观察到的晚期内体/溶酶体途径的改变导致自噬缺陷，并导致蛋白质包涵体和泛素化聚集体的积累，这可能导致MLIV患者观察到的神经变性。 为了更好地了解调控MCOLN1功能的机制，我们寻找了以钙离子依赖的方式与MCOLN1相互作用的蛋白质。我们发现5-EF-Hand蛋白ALG-2与MCOLN1的NH末端胞液尾巴结合。这种相互作用是直接的，严格依赖于钙离子，并由位于MCOLN1残基37-49之间的一块带电和疏水残基介导。与MCOLN1在融合/裂变事件调控中的作用一致，我们发现MCOLN1的过度表达导致了包含早期和晚期内吞体标记物的增大的、异常的内体的积累。有趣的是，当MCOLN1的ALG-2结合域发生突变时，异常内体的聚集大大减少，这表明ALG-2调节MCOLN1的功能。这些数据为调节MCOLN1活性的分子机制提供了新的见解。我们认为ALG-2作为一种钙感受器，沿着晚期内体-溶酶体途径调节MCOLN1的功能。 MCOLN3与MCOLN1的氨基酸同源性约为75%。MCOLN3基因突变是导致小鼠Variint-Waddler表型的原因，其特征是听力损失、前庭功能障碍(旋转行为、摇头、摇摆)和毛色变淡。全细胞膜片钳实验表明，TRPML3是一种内向整流型单价阳离子通道，对钙离子具有通透性，受低pH值抑制。我们已经研究了MCOLN3在上皮细胞中的功能。我们发现MCOLN3主要定位于ARPE-19细胞的早期和晚期内吞体内。这种分布在内吞途径的较弱酸性部分与报道的低pH使通道失活是一致的。此外，我们还发现MCOLN3的过表达导致了内体途径的显著变化，包括HRs阳性的内体增大，EGF和EGFR的延迟降解，以及自噬小体成熟缺陷。有趣的是，类似的缺陷已经被描述为与内体生物发生有关的蛋白质的过度表达，如ESCRT和HRS。我们还发现在过度表达MCOLN3的细胞中，内体的pH更高，并提出了一个模型，在该模型中，MCOLN3介导的内体释放钙可能是有效的内体酸化的重要因素。因此，MCOLN3是一种新的钙通道，在调节内体途径的货物运输中起着至关重要的作用。 总体而言，我们的数据揭示了新的证据表明，粘脂分布在内体途径的特定位置，并在脂类和蛋白质的分类中发挥重要的调节作用。