Molecular and Functional Characterization of Mucolipin-1

描述（由申请人提供）：本指导职业发展奖的主要目标是为申请人作为神经退行性和Ca（2+）信号研究的独立研究者做好准备。申请人已经坚定地致力于生物医学研究事业，并在细胞和分子生物学、生物化学和溶酶体贮积病的基因靶向方面获得了广泛的培训和专业知识，然而，她认为在这项拟议研究的背景下，在概述的成像和电生理技术方面进行一段时间的额外培训将极大地提高她作为一名独立科学家的竞争力。申请人的环境非常适合支持她的持续职业发展，可以使用最先进的核心设施和现场科学专家。她的导师Muallem博士是Ca（2+）信号和离子通道功能调控方面的专家，在指导年轻研究人员方面拥有丰富的经验。候选人的直接目标是阐明TRP-ML1 （ML1）的细胞功能，并确定这种溶酶体膜离子通道的缺陷如何导致IV型粘脂质沉积症（MLIV），一种脂质储存性神经退行性疾病。MLIV在细胞水平上由溶酶体的脂质积累和晚期内体/杂交细胞器形成成熟溶酶体的阻滞来定义。ML1的确切功能尚不清楚。在初步研究中，我们已经证明ML1是一个H+选择性阳离子通道，在溶酶体中被特异性切割。裂解形式主要见于天然细胞和组织中，可能具有调节脂肪酶活性的功能。我们现在假设ML1是一种双重功能蛋白，通过介导溶酶体ph的改变来调节溶酶体脂质代谢。在缺乏功能性ML1的情况下，脂质代谢是异常的。以下具体目标将测试以下假设：1)表征溶酶体靶向和蛋白水解裂解的分子决定因素，2)确定哪些脂肪酶被ML1激活，以及3)表征ML1与MLS和其他可能在复合体中起作用的蛋白质的相互作用。这些研究结果将为该疾病的细胞和治疗提供依据。

DESCRIPTION (provided by applicant): The primary goal of this Mentored Career Development Award is to prepare the applicant for a career as an independent investigator in neurodegenerative and Ca(2+) signaling research. The applicant has shown a firm commitment to a biomedical research career and has acquired extensive prior training and expertise in cell and molecular biology, biochemistry, and gene targeting of lysosomal storage diseases, however, she believes that a period of additional training in the outlined imaging and electrophysiological techniques in the context of this proposed study will dramatically improve her competitiveness as an independent scientist. The applicant's environment is ideally suited to support her continued career development, with access to state-of-the-art core facilities and on-site scientific experts. Her mentor, Dr. Muallem, is an expert in Ca(2+) signaling and in the regulation of ion channel functions and has had extensive experience in mentoring young investigators. The candidate's immediate goal is to elucidate the cellular functions of TRP-ML1 (ML1), and to determine how defects in this lysosomal membrane ion channel lead to mucolipidosis type IV (MLIV), a lipid storage neurodegenerative disorder. MLIV is defined at the cellular level by a lysosomal accumulation of lipids and by a block in the formation of mature lysosomes from late endosomes/hybrid organelles. The exact function of ML1 is unknown. In preliminary studies, we have shown that ML1 is an H+ selective cation channel that is specifically cleaved in the lysosomes. The cleaved form is predominantly seen in native cells and tissues and may function to regulate lipase activity. We now hypothesize that ML1 is a dual function protein that regulates lysosomal lipid metabolism by mediating alterations in lysosomal pH. In the absence of a functional ML1, lipid metabolism is aberrant. The following Specific Aims will test the following hypothesis: 1) Characterize the molecular determinants of lysosomal targeting and proteolytic cleavage, 2) identify which lipases are activated by ML1, and 3) characterize the interactions of ML1 with MLS and with other proteins that may function in a complex. Results from these studies will provide the cellular and therapeutic basis of this disease.