Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 8149460
• 负责人: rosa puertollano
• 金额: $ 43.14万
• 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/8149460
• 关键词: human disease; trafficking

Mucolipins constitute a family of 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). MLIV is an autosomal recessive disease characterized by mental and psychomotor retardation, diminished muscle tone or hypotonia, achlorhydria, and visual problems including corneal clouding, retinal degeneration, sensitivity to light, and strabismus. Analysis of fibroblasts from MLIV patients by electron microscopy revealed the presence of enlarged vacuolar structures that accumulate mucopolysaccharides and lipids forming characteristic multiconcentric lamellae. These enlarged vacuoles are present not only in fibroblasts but in every tissue and organ of MLIV patients, suggesting a general impairment of the lysosomal function. Interestingly, and in contrast with other lysosomal storage diseases, lysosomal hydrolases are functional and correctly transported to lysosomes in MLIV, indicating that MCOLN1 might be necessary for the correct trafficking of protein and lipids along the late endosomal/lysosomal pathway. This idea is supported by experiments in Caenorhabditis elegans showing that knockout of cup-5, the orthologue of MCOLN1, results in formation of enlarged hybrid organelles that contain both late-endosomal and lysosomal markers. Additional roles for MCOLN1 in lysosomal acidification, lysosomal secretion, and lysosomal iron release have been proposed. Electrophysiological studies indicate that MCOLN1 is an inwardly (from lumen to cytoplasm) rectifying channel permeable to Ca2+, Na+, K+ and Fe2+/ Mn2+, whose activity is modulated by pH and Ca2+ Our previous studies focused in the characterization of the regulation and cellular distribution of MCOLN1. We found that, consistent with the proposed role of this protein in the late endocytic pathway, MCOLN1 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. In addition, the C-terminal tail of MCOLN1 undergoes post-translational modifications that regulate its activity and trafficking. 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. The mechanisms that regulate activation of MCOLN1 under physiological conditions remain unknown. For this reason, we 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. We further show that MCOLN1 and ALG-2 co-localize to enlarged endosomes induced by over-expression of an ATPase-defective dominant negative form of Vps4B (Vps4BE235Q). 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. Overall, our 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. To better understand the cellular function of MCOLN1, a split-ubiquitin yeast two-hybrid screen was performed with the purpose of revealing new MCOLN1 interactors. We have identified several promising candidates implicated in neuronal development, intracellular trafficking, and apoptosis. We will use a combination of confocal microscopy, cellular biology, and biochemistry to characterize the relevance of these interactions and their implication in human disease.

黏脂蛋白是一个阳离子通道家族，与瞬时受体电位超家族具有同源性。在哺乳动物中，粘蛋白家族包括三个成员，即粘蛋白-1、-2和-3(MCOLN1-3)。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是一种常染色体隐性遗传病，其特征是智力和精神运动发育迟缓、肌肉张力降低或低眼压、失弛缓症以及包括角膜混浊、视网膜变性、对光敏感和斜视在内的视觉问题。对MLIV患者成纤维细胞的电子显微镜分析显示，存在大量的空泡结构，这些结构积聚了粘多糖和脂类，形成了具有特征的多同心片层。这些扩大的空泡不仅存在于成纤维细胞中，而且存在于MLIV患者的每个组织和器官中，表明溶酶体功能普遍受损。有趣的是，与其他溶酶体储存疾病相比，溶酶体水解酶具有功能并正确地运输到MLIV中的溶酶体，表明MCOLN1可能是沿着晚期内酶体/溶酶体途径正确运输蛋白质和脂类所必需的。这一观点得到了秀丽隐杆线虫实验的支持，该实验表明，敲除MCOLN1的同源基因CUP-5会导致形成同时包含晚期内体和溶酶体标记的扩大的杂交细胞器。MCOLN1在溶酶体酸化、溶酶体分泌和溶酶体铁释放中的其他作用已被提出。电生理学研究表明，MCOLN1是一种内向(从腔到细胞质)对钙、钠、钾和Fe2+/Mn2+具有通透性的整流通道，其活性受pH和Ca2+的调节 我们以前的研究主要集中在MCOLN1的调控和细胞分布的表征上。我们发现，与该蛋白在晚期内吞途径中的作用一致，MCOLN1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。此外，MCOLN1的C-末端尾部经历了翻译后的修饰，以调节其活动和贩运。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。 在生理条件下调节MCOLN1激活的机制尚不清楚。为此，我们寻找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。我们进一步证明了MCOLN1和ALG-2共同定位于由ATPase缺陷的显性阴性形式Vps4B(Vps4BE235Q)的过度表达诱导的扩大的内体。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。总体而言，我们的数据为调节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。 为了更好地了解MCOLN1的细胞功能，进行了裂解泛素酵母双杂交筛选，目的是揭示新的MCOLN1相互作用因子。我们已经确定了几个与神经元发育、细胞内转运和细胞凋亡有关的有希望的候选基因。我们将使用共聚焦显微镜、细胞生物学和生物化学的组合来描述这些相互作用的相关性及其在人类疾病中的意义。