Analysis of the Transcriptional Regulation and Expression of TRPML2

描述（由申请人提供）：IV型粘脂病（ML-IV）是一种影响关键发育里程碑的人类溶酶体储存障碍。ML-IV的临床表现包括严重的神经运动、眼和胃异常。ML-IV是由mucolipin-1 （TRPML1）蛋白功能异常引起的。TRPML1属于离子通道瞬时受体电位（TRP）超家族的粘脂（TRPML）亚家族。TRPML亚家族由TRPML1、-2和-3蛋白组成。TRPML蛋白被认为在细胞内运输、内核溶酶体生物发生和自噬中发挥作用。三种TRPML蛋白亚基之间的异质亚基相互作用和功能冗余已被报道。事实上，我们最近表明TRPML1和TRPML2具有非常相似的电生理特征，而其他人则表明TRPML2或TRPML3的缺失会产生一种典型的ML-IV细胞表型。我们还发现TRPML1的缺失导致了TRPML2转录物的组织特异性下调表达。有趣的是，phorbol 12-肉豆蔻酸13-乙酸酯（PMA，一种有效的蛋白激酶C [PKC]激活剂）和Ionomycin（细胞内钙动员剂）显著上调TRPML2 mRNA的表达。本提案的第一个目的是使用双荧光素酶基因报告基因和凝胶迁移转移测定来确定和绘制参与TRPML2转录激活的核心启动子区域。其次，我们将确定微rna在TRPML2表达转录后调控中的潜在作用。最后，我们将使用染色质免疫沉淀法和质谱法确定参与调节TRPML2转录水平的转录因子蛋白。我们还将评估TRPML2蛋白水平，并使用Western blot和荧光成像技术将其与我们的遗传报告数据相关联，因为mRNA转录水平的变化不一定直接对应蛋白水平。该提案将为研究开辟许多途径，以提高我们对TRPML2离子通道转录激活和调控的认识，同时，它将为代表性不足的本科生创造研究机会。从本质上讲，从拟议的研究中获得的知识将是我们长期目标的第一步，即利用功能冗余和替代TRPML2或TRPML3蛋白来弥补ML-IV患者TRPML1功能缺失的概念验证方法。

DESCRIPTION (provided by applicant): Mucolipidosis type IV (ML-IV) is a human lysosomal storage disorder affecting critical developmental milestones. The clinical manifestation of ML-IV includes severe neuro-motor, opthalmic and gastric abnormalities. ML-IV is caused by abnormal function of the mucolipin-1 (TRPML1) protein. TRPML1 belongs to the mucolipin (TRPML) subfamily of the transient receptor potential (TRP) superfamily of ion channels. The TRPML subfamily consists of TRPML1, -2 and -3 proteins. The TRPML proteins are believed to play a role in intracellular trafficking, endosomal-lysosomal biogenesis, and in autophagy. Heteromeric subunit interactions and functional redundancy between the three TRPML protein subunits have been reported. Indeed, we have recently shown that TRPML1 and TRPML2 share a very similar electro-physiological profile, while others have shown that loss of TRPML2 or TRPML3 produces a phenotype typically seen in ML-IV cells. We also found that loss of TRPML1 contributes to the tissue-specific down-regulated expression of TRPML2 transcripts. Interestingly, phorbol 12- myristate 13-acetate (PMA; a potent protein kinase C [PKC] activator) and Ionomycin (intracellular calcium mobilizer) significantly up-regulate TRPML2 mRNA expression. The first aim of this proposal is to determine and map the core promoter region involved in the transcriptional activation of TRPML2 using a dual-luciferase gene reporter and gel mobility shift assays. Secondly, we will determine the potential role of micro-RNAs in its potential role in the post-transcriptional regulation of TRPML2 expression. Finally, we will determine the transcription factor protein involved in regulating TRPML2 transcript levels using chromatin immunoprecipitation assays and mass spectrometry. We will also assess TRPML2 protein levels and correlate it with our genetic reporter data using Western blot and fluorescence imaging techniques, since changes in mRNA transcript levels do not necessarily correspond directly to protein levels. This proposal will open many avenues for research to advance our knowledge on the transcriptional activation and regulation of the TRPML2 ion channel, and at the same time, it will create research opportunities for under-represented undergraduate students. In essence, the knowledge gained from the proposed studies will be the first step to our long-term goal of a proof-of-concept approach to exploit functional redundancy and substitute TRPML2 or TRPML3 protein for the loss of TRPML1 function in ML-IV patients. PUBLIC HEALTH RELEVANCE: Mucolipidosis IV (ML-IV) is a human genetic disorder caused by the dysfunction of the mucolipin-1 (TRPML1) protein. ML-IV is manifested during early child development, and produces debilitating symptoms that include, but are not limited to, abnormal psychomotor milestones, digestive problems, cataract formation, and blindness. This proposal will study the activation process of a closely related member, TRPML2. Knowledge gained from the study could allow us to potentially replace the loss of TRPML1 protein as therapeutic approach for ML-IV.