Analysis of the Transcriptional Regulation and Expression of TRPML2

描述(申请人提供)：IV型粘脂沉积症(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、眼科和胃部异常。ML-IV是由粘蛋白-1(TRPML1)蛋白功能异常引起的。TRPML1属于瞬时受体电位(Trp)离子通道超家族中的粘蛋白(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转运、内体-溶酶体生物发生和自噬中发挥作用。已经报道了三个TRPML蛋白亚基之间的异构体相互作用和功能冗余。事实上，我们最近已经表明，TRPML1和TRPML2具有非常相似的电生理特征，而其他研究表明，TRPML2或TRPML3的缺失会产生在ML-IV细胞中常见的表型。我们还发现，TRPML1的缺失有助于TRPML2转录本的组织特异性下调表达。有趣的是，佛波酯(PMA；一种有效的蛋白激酶C[PKC]激活剂)和离子霉素(细胞内钙激动剂)显著上调TRPML2的mRNA表达。这个建议的第一个目的是利用双荧光素酶基因报告和凝胶迁移率改变分析来确定和定位参与TRPML2转录激活的核心启动子区域。其次，我们将确定Micro-RNAs在TRPML2表达的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分析和质谱仪来确定参与调节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印迹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于蛋白质水平。这项建议将为我们提供许多研究途径，以促进我们对TRPML2离子通道转录激活和调控的了解，同时，它将为未被充分代表的本科生创造研究机会。从本质上说，从拟议的研究中获得的知识将是我们实现长期目标的第一步，即利用功能冗余的概念验证方法，并用TRPML2或TRPML3蛋白取代ML-IV患者TRPML1功能的丧失。 公共卫生相关性：第四型粘脂沉积症(ML-IV)是一种人类遗传性疾病，由粘蛋白-1(TRPML1)蛋白功能障碍引起。ML-IV在儿童发育早期表现出来，会产生衰弱的症状，包括但不限于精神运动里程碑异常、消化问题、白内障形成和失明。该提案将研究一个密切相关的成员TRPML2的激活过程。从这项研究中获得的知识可能使我们有可能取代TRPML1蛋白的丢失，作为ML-IV的治疗方法。

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.