Endocytic Trafficking and Human Diseases

粘脂构成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，粘脂家族包括三个成员，粘脂-1、粘脂-2和粘脂-3（MCOLN 1 -3）。MCOLN 1是该家族中最具特征的成员，因为这种蛋白质的突变与称为IV型粘脂沉积症（MLIV）的人类疾病相关。MLIV是一种常染色体隐性遗传疾病，其特征在于智力和精神发育迟缓、肌张力降低或张力减退、无氯血症和视觉问题，包括角膜混浊、视网膜变性、对光敏感和斜视。通过电子显微镜分析MLIV患者的成纤维细胞，发现存在扩大的空泡结构，积累粘多糖和脂质，形成特征性的多中心层。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，表明溶酶体功能的普遍损害。有趣的是，与其他溶酶体储积疾病相反，溶酶体水解酶具有功能并正确转运至MLIV中的溶酶体，这表明MCOLN 1可能是蛋白质和脂质沿着晚期内体/溶酶体途径正确转运所必需的。这一想法得到了秀丽隐杆线虫实验的支持，该实验表明，敲除Cup-5（MCOLN 1的直向同源物）导致形成含有晚期内体和溶酶体标记物的扩大的杂交细胞器。已经提出了MCOLN 1在溶酶体酸化、溶酶体分泌和溶酶体铁释放中的其他作用。电生理研究表明，MCOLN 1是一个内向整流通道，可通过Ca ~（2+）、Na ~+、K ~+和Fe ~（2+）/Mn ~（2+），其活性受pH和Ca ~（2+）的调节 我们以前的研究集中在MCOLN 1的调控和细胞分布的表征。我们发现，与该蛋白在晚期内吞途径中的作用一致，MCOLN 1定位于晚期内体/溶酶体。两个双亮氨酸基序通过与网格蛋白衔接子AP 1、AP 2和AP 3的相互作用协同调节MCOLN 1向溶酶体的递送。此外，MCOLN 1的C-末端尾经历了调节其活性和运输的翻译后修饰。棕榈酰化的三个半胱氨酸残基（Cys 565，Cys 566，和Cys 567）增加MCOLN 1从质膜内化的速率，而PKA介导的Ser 557和Ser 559的磷酸化负调节体内MCOLN 1通道活性。我们还发现，在MCOLN 1缺陷细胞中观察到的晚期内体/溶酶体途径的改变导致有缺陷的自噬，并导致蛋白质内含物和泛素化聚集体的积累，这可能有助于在MLIV患者中观察到的神经变性。 在生理条件下调节MCOLN 1激活的机制仍然未知。因此，我们寻找以Ca 2+依赖性方式与MCOLN 1相互作用的蛋白质。我们发现五EF手蛋白ALG-2结合到MCOLN 1的NH-末端胞质尾部。这种相互作用是直接的，严格依赖于Ca 2+，并通过位于MCOLN 1残基37-49之间的一片带电和疏水残基介导。我们进一步表明，MCOLN 1和ALG-2共定位于由ATP酶缺陷型显性负性Vps 4 B（Vps 4 BE 235 Q）过度表达诱导的扩大的内体。与MCOLN 1在融合/分裂事件调节中的拟议作用一致，我们发现MCOLN 1的过度表达引起了包含早期和晚期内体标记物的扩大的异常内体的积累。有趣的是，当MCOLN 1中的ALG-2结合结构域突变时，异常内体的聚集大大减少，表明ALG-2调节MCOLN 1功能。总的来说，我们的数据为调节MCOLN 1活性的分子机制提供了新的见解。我们认为ALG-2作为一个钙离子传感器，调节MCOLN 1的功能，沿着晚期内体-溶酶体途径。 为了更好地了解MCOLN 1的细胞功能，进行了分裂泛素酵母双杂交筛选，目的是揭示新的MCOLN 1相互作用子。我们已经确定了几个有前途的候选人参与神经元发育，细胞内运输，和凋亡。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表征这些相互作用的相关性及其在人类疾病中的意义。

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.