Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 7321762
• 负责人: rosa puertollano
• 金额: --
• 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/7321762
• 关键词: Endocytic; Trafficking; Human; Diseases

Mucolipins constitute a family of cation channels with homology to the transient receptor potential (TRP) superfamily. In mammals, mucolipin family includes three members named mucolipin-1 (MCOLN1), mucolipin-2 (MCOLN2), and mucolipin-3 (MCOLN3) that exhibit a common six-membrane-spanning molecular organization. Homologous of mammalian mucolipins have also been described in Drosophila and C. elegans. MCOLN-1 is the best-characterized member of the mucolipin family as mutations in this protein have been linked to mucolipidosis type IV (MLIV), a recessive lysosomal storage disease characterized by severe neurological and ophthalmologic abnormalities. It has been suggested that mutations in MCOLN1 channel causes defects on late-endosomal/lysosomal trafficking resulting in the accumulation of enlarged vacuolar structures that contain phospholipids, mucopolysacharides, and gangliosides. In agreement with this idea, loss-of-function mutations in cup-5, the C. elegans orthologue of mucolipin-1, caused formation of large endosome?lysosome hybrid organelles that contain both late-endosomal and lysosomal markers. MCOLN-3 might also play a role in different human pathologies, as mutations in this gene are responsible for the varitint-waddler mousse phenotype that is characterized by defects on pigmentation and hearing loss. MCOLN3 is located in hair cells and it could be implicated in hair cell maturation and melanosomes trafficking. In contrast, the function and location of MCOLN2 remains to be characterized. One approach to gain information on the function of a protein is to study its trafficking and cellular distribution. For example, we have previously described that, consistent with its role in lysosomal biogenesis, MCOLN1 has specific sorting motifs that mediate interaction with clathrin adaptors and the subsequent delivery of MCOLN1 to lysosomes. Posttranslational modifications may also play an important role in the regulation of protein function. We have established that the C-terminal tail of MCOLN1 is palmitoylated and that this palmitoylation promotes efficient internalization of MCOLN1 from the plasma membrane. We have also found that MCOLN1 is phosphorylated both in vitro and in vivo. We have been able to identify the residues and the kinase responsible for this phosphorylation and are now analyzing the effect of this modification on the activity of MCOLN1 channels in vivo. We have also address the function and cellular distribution of MCOLN2 in HeLa cells. We found that MCOLN2 traffics via the Arf6-dependent pathway and co-localizes with MHCI in both vesicles and long tubular structures. Expression of Arf6Q67L or activation of endogenous Arf6 by transfection with EFA6 or treatment with AlF caused accumulation of MCOLN2 in enlarged vacuoles that also contain MHCI and CD59. Moreover, over-expression of MCOLN2 promoted efficient activation of Arf6. Finally, depletion of endogenous MCOLN2 by expression of a specific shRNA caused missorting of cargo proteins that travel through the Arf6-regulated pathway. Finally, we are analyzing the presence of specific sorting motifs in MCOLN3 and its contribution to the cellular distribution of this protein. We are also addressing the participation of MCOLN3 in the trafficking of melanosomes. Therefore our data reveal that all three members of the mucolipin family may be important regulators of specific intracellular trafficking events.

粘脂构成与瞬时受体电位（TRP）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，即粘脂蛋白-1（MCOLN 1）、粘脂蛋白-2（MCOLN 2）和粘脂蛋白-3（MCOLN 3），它们具有共同的六跨膜分子结构。哺乳动物粘脂的同源性也在果蝇和C.优美的MCOLN-1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型粘脂沉积症（MLIV）相关，MLIV是一种以严重神经和眼科异常为特征的隐性溶酶体贮积病。已经表明MCOLN 1通道的突变导致晚期内体/溶酶体运输的缺陷，导致含有磷脂、粘多糖和神经节苷脂的扩大的空泡结构的积累。与这一观点一致，cup-5中的功能丧失突变，C。elegans的mucolipin-1的直系同源物，引起大的内体的形成？溶酶体杂合细胞器，包含晚期内体和溶酶体标记物。MCOLN-3也可能在不同的人类病理学中发挥作用，因为该基因的突变导致varitint-waddler慕斯表型，其特征在于色素沉着和听力损失的缺陷。MCOLN 3位于毛细胞中，并且其可能涉及毛细胞成熟和黑素体运输。与此相反，MCOLN 2的功能和位置仍有待确定。 获得蛋白质功能信息的一种方法是研究其运输和细胞分布。例如，我们之前已经描述了，与其在溶酶体生物发生中的作用一致，MCOLN 1具有特异性分选基序，其介导与网格蛋白衔接子的相互作用以及随后将MCOLN 1递送至溶酶体。翻译后修饰也可能在蛋白质功能的调节中发挥重要作用。我们已经确定，MCOLN 1的C-末端尾部是棕榈酰化的，并且这种棕榈酰化促进了MCOLN 1从质膜的有效内化。我们还发现MCOLN 1在体外和体内都被磷酸化。我们已经能够识别负责这种磷酸化的残基和激酶，现在正在分析这种修饰对体内MCOLN 1通道活性的影响。 我们还讨论了MCOLN 2在HeLa细胞中的功能和细胞分布。我们发现，MCOLN 2交通通过Arf 6依赖的途径和共定位与MHC I在囊泡和长管状结构。Arf 6 Q67 L的表达或通过用EFA 6转染或用AlF处理的内源性Arf 6的活化引起MCOLN 2在也含有MHCI和CD 59的扩大的空泡中的积累。此外，MCOLN 2的过表达促进Arf 6的有效激活。最后，通过表达特定的shRNA耗尽内源性MCOLN 2导致通过Arf 6调节途径的货物蛋白的错误分选。 最后，我们正在分析MCOLN 3中特定分选基序的存在及其对该蛋白质细胞分布的贡献。我们还在研究MCOLN 3参与黑素体贩运的问题。 因此，我们的数据显示，所有三个成员的粘脂家族可能是重要的调节器的特定的细胞内运输事件。