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

• 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
• 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

 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（由申请人提供）：本提案的长期目标是鉴定促进血红素中间体转运进入线粒体和在线粒体内转运的线粒体蛋白，并概述需要血红素合成中间体特异性转运蛋白功能的生理过程。我以前已经表明，Tmem14c是必需的终端红细胞生成和进口的原卟啉原IX到线粒体基质血红素合成。在我们的遗传模型中，Tmem14c缺乏导致贫血和卟啉积累。 然而，TMEM14 C功能和原卟啉原IX转运的几个方面仍然不清楚，并导致在我的具体目标中解决的两个主要假设。首先，TMEM14 C促进原卟啉原IX转运的机制仍不清楚。由于TMEM14 C的紧密结构表明其作为跨膜通道起作用，因此我假设TMEM14 C直接将原卟啉原IX转运到线粒体基质中。我将在具体目标1中通过定量TMEM14C对血红素和四吡咯血红素中间体的相对亲和力来测试这一点。我还将测量在体外血红素合成率在野生型和Tmem14c缺陷的线粒体在外源血红素中间体的存在下。 其次，缺乏TMEM14C的红系细胞具有与野生型细胞相似的存活率、线粒体电位和线粒体质量。由于线粒体和细胞呼吸是维持生命的基本过程，需要血红素蛋白（具有血红素辅因子的蛋白质），因此细胞可能具有维持管家血红素合成的其他原卟啉原IX转运蛋白。由于TMEM14蛋白的结构非常相似，我推测TMEM14超家族的其他成员作为原卟啉原IX转运蛋白发挥功能，以维持管家血红素合成和细胞生理学。在具体目标2中，我将通过敲低脊椎动物细胞系中的TMEM 14基因并量化敲低对血红素合成的影响来测试这一假设。参与血红素合成的候选TMEM 14基因将在原代肝细胞和原代造血细胞中被敲低，以检查它们对线粒体生理学和血红蛋白化的影响。我将在斑马鱼中通过敲低研究来测试红系和肝脏发育中对tmem14基因的体内需求。 我的项目的完成将阐明血红素合成途径的遗传学和生物化学，并将有助于我们对当血红素中间体的运输受到干扰时发生的病理后果的基本理解。本建议书中所述的具体目标和职业发展计划是我先前培训的合理延续，但将提供一个框架，使我能够科学地将自己与我的顾问区分开来，最终为成功过渡到独立铺平道路。