Endocytic Trafficking and Human Diseases

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，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。MLIV是一种常染色体隐性遗传病，其特征是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。通过电镜分析MLIV患者的成纤维细胞，发现存在增大的液泡结构，积聚粘多糖和脂质，形成特征性的多同心片。这些增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，与其他溶酶体贮积性疾病相比，在MLIV中，溶酶体水解酶是功能性的，并能正确地转运到溶酶体，这表明MCOLN1可能是沿内体/溶酶体晚期途径正确转运蛋白质和脂质的必要条件。这一观点得到了线虫实验的支持，实验表明，敲除MCOLN1的同源基因cup-5，会导致杂交细胞器的形成，这些细胞器既包含内体晚期标志物，也包含溶酶体标志物。已提出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。电生理研究表明，MCOLN1是一个向内（从管腔到细胞质）渗透Ca2+、Na+、K+和Fe2+/ Mn2+的整流通道，其活性受pH和Ca2+的调节

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.