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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（由申请方提供）：IV型粘脂沉积症（ML-IV）是一种影响关键发育里程碑的人溶酶体贮积症。ML-IV的临床表现包括严重的神经运动、眼和胃异常。ML-IV是由粘脂蛋白-1（TRPML1）蛋白的功能异常引起的。TRPML1属于离子通道的瞬时受体电位（TRP）超家族的粘脂（TRPML）亚家族。TRPML亚家族由TRPML 1、TRPML-2和TRPML-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起作用。已经报道了三个TRPML蛋白亚基之间的异聚亚基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，而其他人已经表明TRPML2或TRPML3的缺失产生通常在ML-IV细胞中看到的表型。我们还发现，TRPML1的损失有助于TRPML2转录本的组织特异性下调表达。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的表达。本建议的第一个目的是确定和映射的核心启动子区域参与TRPML2的转录激活使用双荧光素酶基因报告和凝胶迁移率变动分析。其次，我们将确定微小RNA在TRPML2表达的转录后调节中的潜在作用。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离子通道的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，即采用概念验证方法来利用功能冗余并替代ML-IV患者中TRPML1功能丧失的TRPML2或TRPML3蛋白。公共卫生相关性：粘脂沉积症IV（ML-IV）是一种由粘脂-1（TRPML1）蛋白功能障碍引起的人类遗传性疾病。ML-IV在儿童早期发育过程中表现出来，并产生使人衰弱的症状，包括但不限于异常的心理里程碑、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。从研究中获得的知识可以使我们有可能取代TRPML1蛋白的丢失作为ML-IV的治疗方法。