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Analysis of the Transcriptional Regulation and Expression of TRPML2

TRPML2转录调控及表达分析

基本信息

批准号：
8101779
负责人：
MATH P CUAJUNGCO
金额：
$ 33.89万
依托单位：
CALIFORNIA STATE UNIVERSITY FULLERTON
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-02-01 至 2015-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8101779
关键词：
3&apos Untranslated Regions Acetates Affect Antibodies Autophagocytosis Base Pairing Biogenesis Bioinformatics Biological Assay Blindness Calcium Cataract Cations Cell Line Cells Child Development Clinical Cloning Cytolysis DNA Sequence Data Development Disease Embryo Exhibits Firefly Luciferases Fluorescence Functional disorder Ganglioside Sialidase Deficiency Disease Gene Expression General Transcription Factors Genes Genetic Genomics Goals Hereditary Disease Human Human Cell Line Human Genetics Image Imaging Techniques Initiator Codon Ion Channel Ionomycin Kidney Knowledge Luc Gene Luciferases Lymphoid Cell Maps Mass Spectrum Analysis Mediating Membrane Protein Traffic Messenger RNA MicroRNAs Molecular Motor Mus Patients Pattern Phenotype Phorbol Esters Physiological Play Point Mutation Post-Transcriptional Regulation Process Promoter Regions Protein Kinase C Protein Subunits Proteins RNA Interference Regulation Reporter Reporting Research Role Sampling Splenocyte Stomach Students Symptoms Therapeutic Time Tissues Transcript Transcription Initiation Site Transcriptional Activation Transcriptional Regulation Translating Western Blotting activating transcription factor base chromatin immunoprecipitation fluorescence imaging gel mobility shift assay kidney cell mRNA Expression member mutant phorbol-12-myristate promoter protein expression receptor trafficking transcription factor vector

项目摘要

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.

描述（由申请人提供）：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贮积症。 ML-IV 的临床表现包括严重的神经运动、眼部和胃部异常。 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运输、内体-溶酶体生物发生和自噬中发挥作用。三个 TRPML 蛋白亚基之间的异聚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 ML-IV 细胞中常见的表型。我们还发现 TRPML1 的缺失会导致 TRPML2 转录本的组织特异性表达下调。有趣的是，佛波醇 12-肉豆蔻酸酯 13-乙酸酯（PMA；一种有效的蛋白激酶 C [PKC] 激活剂）和离子霉素（细胞内钙动员剂）显着上调 TRPML2 mRNA 表达。该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率变化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 micro-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测定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，并使用蛋白质印迹和荧光成像技术将其与我们的基因报告数据关联起来，因为 mRNA 转录物水平的变化不一定直接对应于蛋白水平。该提案将为研究开辟许多途径，以增进我们对 TRPML2 离子通道转录激活和调节的了解，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，从拟议研究中获得的知识将是实现我们长期目标的第一步，即利用功能冗余并用 TRPML2 或 TRPML3 蛋白替代 ML-IV 患者中 TRPML1 功能的丧失的概念验证方法。公共健康相关性：粘脂沉积症 IV (ML-IV) 是一种由粘脂蛋白-1 (TRPML1) 蛋白功能障碍引起的人类遗传性疾病。 ML-IV 在儿童早期发育期间表现出来，并产生衰弱症状，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能够替代 TRPML1 蛋白的丢失作为 ML-IV 的治疗方法。