Mechanism and Function of TMEM14 proteins in vertebrate heme synthesis

TMEM14蛋白在脊椎动物血红素合成中的机制和功能

基本信息

批准号：
9751281
负责人：
Yvette Y Yien
金额：
$ 15.82万
依托单位：
UNIVERSITY OF DELAWARE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9751281
关键词：
Address Adult Affinity Anemia Animals Binding Biochemical Biochemistry Biological Assay Cell Culture Techniques Cell Line Cell Respiration Cell physiology Cells Chemicals Child Complement Data Defect Development Development Plans Dietary intake Disease Drug Metabolic Detoxication Enzymes Erythrocytes Erythroid Erythroid Cells Erythropoiesis Etiology Family member Fetal Liver Fetus Genes Genetic Genetic Models Goals Health Hematopoietic Heme Hemeproteins Hemoglobin Hepatic Hepatic Tissue Hepatocyte Housekeeping Human In Vitro Individual Iron Lead Life Light Liver Measures Mitochondria Mitochondrial Matrix Mitochondrial Proteins Molecular Mus Mutation Orthologous Gene Oxidation-Reduction Oxygen PPIX Pathologic Pathology Pathway interactions Patients Penetrance Physiological Physiological Processes Physiology Play Porphyrias Porphyrins Process Production Prosthesis Protein Family Proteins Reaction Respiration Respiratory Failure Role Severities Structure Substrate Specificity Survival Rate Testing Tetrapyrroles Tissues Training Vertebrates Woman Zebrafish analog base career development cell type cofactor gain of function mutation hematopoietic tissue in utero in vivo in vivo evaluation knock-down member progenitor protoporphyrin IX public health relevance success synthetic enzyme trafficking

项目摘要

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify mitochondrial proteins that facilitate the transport of heme intermediates into and within the mitochondria and to outline the physiological processes that require the function of specific transporters of heme synthesis intermediates. I have previously shown that Tmem14c is required for terminal erythropoiesis and import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis. Tmem14c deficiency causes anemia and porphyrin accumulation in our genetic models. However, several aspects of TMEM14C function and protoporphyrinogen IX transport are still unclear and lead to two main hypotheses that are addressed in my Specific Aims. Firstly, the mechanism by which TMEM14C facilitates the transport of protoporphyrinogen IX is still not understood. As the tight structure of TMEM14C suggests that it functions as a transmembrane channel, I hypothesize that TMEM14C directly transports protoporphyrinogen IX into the mitochondrial matrix. I will test this in Specific Aim 1 by quantifying the relative affinities of TMEM14C to heme and tetrapyrrolic heme intermediates. I will also measure the rates of in vitro heme synthesis in wild-type and Tmem14c deficient mitochondria in the presence of exogenous heme intermediates. Secondly, erythroid cells lacking TMEM14C have survival rates, mitochondrial potentials and mitochondrial masses similar to wild-type cells. As mitochondrial and cellular respiration, which are essential life-sustaining processes require hemoproteins (proteins with heme-cofactors), it is probable that cells possess other protoporphyrinogen IX transporters that maintain housekeeping heme synthesis. As the structures of TMEM14 proteins are very similar, I hypothesize that other members of the TMEM14 superfamily function as protoporphyrinogen IX transporters to maintain housekeeping heme synthesis and cellular physiology. In Specific Aim 2, I will test this hypothesis by knocking down TMEM14 genes in vertebrate cell lines and quantifying the effects of the knockdown on heme synthesis. Candidate TMEM14 genes involved in heme synthesis will be knocked down in primary hepatocytes and primary hematopoietic cells to examine their effect on mitochondrial physiology and hemoglobinization. I will test the in vivo requirement for tmem14 genes in erythroid and hepatic development by knockdown studies in the zebrafish. The completion of my project will shed light on the genetics and biochemistry of the heme synthesis pathway and will contribute to our fundamental understanding of the pathological consequences that occur when the pathway is perturbed by disruptions to the transport of heme intermediates. The specific aims and career development plan described in this proposal are a logical continuation of my prior training but will provide a framework by which I will scientifically and differentiate myself from my advisors, ultimately paving the way for a successful transition to independence.

描述（由适用提供）：该提案的长期目标是确定促进血红素中间体进入线粒体内外的线粒体蛋白，并概述需要血红素合成中间体特定转运蛋白功能的物理过程。我先前已经表明，TMEM14C是末端红细胞生成所必需的，并将原磷酸蛋白IX进口到线粒体基质中以进行血红素合成所必需。 TMEM14C缺乏会在我们的遗传模型中引起贫血和卟啉积累。然而，TMEM14C功能和原晶酸IX运输的几个方面尚不清楚，并导致了我的特定目标中提到的两个主要假设。首先，尚不清楚TMEM14C促进原晶IX运输的机制。由于TMEM14C的紧密结构表明它是跨膜通道的作用，因此我假设TMEM14C直接将原甲吡啶原IX转运到线粒体基质中。我将通过量化TMEM14C与血红素和四吡咯血红素中间体的相对亲和力来在特定目标1中进行测试。我还将在存在外源性血红素中间体的情况下测量野生型和TMEM14C缺乏线粒体的体外血红素合成速率。其次，缺乏TMEM14C的红细胞细胞具有与野生型细胞相似的生存率，线粒体电位和线粒体肿块。作为必不可少的生命生命过程的线粒体和细胞呼吸，需要血蛋白（带血红素 - 表现因子的蛋白质），因此细胞具有其他蛋白质卟啉原IX转运蛋白，可维持家政管合成。由于TMEM14蛋白的结构非常相似，因此我假设TMEM14超家族的其他成员充当原磷酸蛋白原IX转运蛋白，以维持管家血红素的合成和细胞生理学。在特定的目标2中，我将通过击倒脊椎动物细胞系中的TMEM14基因并量化敲低对血红素合成的影响来检验这一假设。参与血红素合成的候选TMEM14基因将在原发性肝细胞和原代造血细胞中被击倒，以检查其对线粒体生理和血红蛋白的影响。我将通过斑马鱼中的敲低研究来测试红细胞和肝发育中TMEM14基因的体内需求。我的项目的完成将阐明血红素综合途径的遗传学和生物化学，并将有助于我们对当途径被干扰对血红素中间体的运输扰动时发生的病理后果的基本理解。本提案中描述的具体目的和职业发展计划是我先前培训的逻辑延续，但将提供一个框架，我将科学地与我的顾问区分开来，最终为成功过渡到独立性铺平了道路。