Molecular and Functional Characterization of Mucolipin-1

描述(由申请人提供)：这个有指导的职业发展奖的主要目标是为申请人在神经退行性疾病和钙(2+)信号研究方面的职业生涯做好准备。申请人表现出对生物医学研究事业的坚定承诺，并在细胞和分子生物学、生物化学和溶酶体储存疾病的基因靶向方面获得了广泛的先前培训和专业知识，但她相信，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独立科学家的竞争力。申请者的环境非常适合支持她的职业发展，拥有最先进的核心设施和现场科学专家。她的导师穆阿利姆博士是钙(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。候选人的近期目标是阐明Trp-ML1(ML1)的细胞功能，并确定这一溶酶体膜离子通道的缺陷是如何导致IV型粘脂病(MLIV)的，这是一种脂质储存神经退行性疾病。MLIV在细胞水平上的定义是脂类的溶酶体堆积，以及晚期内体/混合细胞器形成成熟溶酶体的障碍。ML1的确切功能尚不清楚。在初步研究中，我们已经证明ML1是一种H+选择性阳离子通道，它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，可能起到调节脂肪酶活性的作用。我们现在假设ML1是一个双功能蛋白，通过调节溶酶体pH的变化来调节溶酶体的脂代谢。在缺乏功能性ML1的情况下，脂质代谢异常。以下特定目标将检验以下假设：1)表征溶酶体靶向和蛋白水解性切割的分子决定因素，2)鉴定哪些脂肪酶被ML1激活，3)表征ML1与MLS以及与可能在复合体中发挥作用的其他蛋白质的相互作用。这些研究的结果将为这种疾病的细胞和治疗基础提供依据。

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.