Mechanism and Function of TMEM14 proteins in vertebrate heme synthesis

 描述(申请人提供)：这项提案的长期目标是确定促进血红素中间体进入线粒体和在线粒体内运输的线粒体蛋白质，并概述需要特定转运体功能的血红素合成中间体的生理过程。我以前已经证明，Tem14c是终末红细胞生成所必需的，并将原卟啉原IX输入线粒体基质中以合成血红素。在我们的遗传模型中，Tem14c缺乏会导致贫血和卟啉蓄积。然而，TMEM14C功能和原卟啉原IX转运的几个方面仍然不清楚，并导致了两个主要假设，在我的特定目标中解决了这两个假设。首先，TMEM14C促进原卟啉原IX转运的机制尚不清楚。由于TMEM14C的紧密结构表明它具有跨膜通道的功能，我推测TMEM14C直接将原卟啉原IX运输到线粒体基质中。我将在特定目标1中测试这一点，方法是量化TMEM14C与亚铁血红素和四吡咯亚铁血红素中间体的相对亲和力。我还将测量在外源血红素中间体存在的情况下，野生型和Tem14c缺陷线粒体的体外血红素合成速率。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(含血红素辅因子的蛋白质)，因此细胞可能拥有其他原卟啉原IX转运体，维持内务血红素的合成。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转运蛋白来维持家务、血红素合成和细胞生理。在特定的目标2中，我将通过敲除脊椎动物细胞系中的TMEM14基因并量化敲除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基因TMEM14将在原代肝细胞和原代造血细胞中被敲除，以检测它们对线粒体生理学和血红素化的影响。我将通过在斑马鱼中进行基因敲除研究来测试在红系和肝脏发育中对tem14基因的体内需求。我的项目的完成将有助于揭示血红素合成途径的遗传学和生物化学，并将有助于我们从根本上理解当该途径受到血红素中间体运输中断时发生的病理后果。这份建议中描述的具体目标和职业发展计划是我之前培训的合乎逻辑的延续，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。

 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.