Yvette Y. Yien, Ph.D. - US grants

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify and characterize novel proteins that regulate mitochondrial heme metabolism, particularly proteins that play a role in mitochondrial heme/porphyrin transport. This project has implications for identifying genetic modifiers for erythroid porphyria and anemia, and thus has public health significance. Our lab has identified Tmem14c as a novel mitochondrial protein that is required for terminal erythroid differentiation and hemoglobinization in vertebrates. Heme synthesis enzymes are present in Tmem14c-deficient murine erythroleukemia (MEL) cells, and preliminary data from our lab indicate that iron transport in Tmem14c-deficient MEL cells is comparable to wild-type MEL cells. However, Tmem14c-deficient MEL cells synthesize decreased levels of heme and protoporphyrin IX. These data suggest that Tmem14c may facilitate mitochondrial porphyrin transport, thereby playing a key role in heme/porphyrin metabolism. In Aim 1, we will test this hypothesis by identifying the block in porphyrin metabolism in Tmem14c deficient cells, comparing the levels of uroporphyrinogen III (cytoplasmic), coproporphyrinogen III (mitochondrial/cytoplasmic) and protoporphyrin IX (mitochondrial) in the mitochondrial and cytoplasmic fractions of wild-type and Tmem14c-deficient MEL cells. In this manner, we will determine if the block in heme synthesis in Tmem14c-deficient cells is a result of defective porphyrin trafficking. As Tmem14c forms higher-order complexes with other proteins, we hypothesize that Tmem14c's interactions with its partners play a crucial role in regulating its function. In Aim 2, we will identify Tmem14c partner and characterize their role in regulating Tmem14c in the heme synthetic pathway; we will identify interacting proteins that are required for terminal erythroid differentiation, heme synthesis and porphyrin transport, and focus on characterizing proteins that regulate Tmem14c's localization and protein stability. Collectively, our studies will contribute to our lon-term objective of understanding regulatory mechanisms controlling heme metabolism, and the interaction of heme metabolism with erythropoiesis. The specific aims in this proposal are of particular significance because studies of Tmem14c function will shed light on the poorly understood porphyrin trafficking pathways that are central to the regulation of heme metabolism and erythropoiesis. These experimental aims are a logical continuation of my graduate work in hematopoiesis, but provide a framework with which to obtain substantial training in the use of the zebrafish as an animal model and additional techniques that will prove invaluable for further studies in erythroid biology.

? 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.

During terminal erythropoiesis, erythroid cells produce significant quantities of heme and heme intermediates which must be coupled to hemoglobin production and iron uptake. Dysregulation of heme synthesis can cause toxic accumulation of heme intermediates and heme deficiency, leading to diseases such as iron overload, anemia and porphyria. We have demonstrated that mitochondrial CLPX, a member of the ubiquitious AAA+ (ATPases associated with various cellular activities) protein unfoldases family, plays a key role in erythroid differentiation by direct regulation of heme synthesis. ClpX functions as a ring-shaped homo-hexamer and is best understood for its function in a proteasome-like enzyme complex with the peptidase ClpP (the ClpXP ATP-dependent protease). CLPX regulates the heme synthesis pathway by mediating activation and degradation of the heme biosynthetic enzymes ALAS1 and ALAS2, which catalyze the committed step of heme synthesis. Beyond regulation of the ALAS enzymes, our data indicate that CLPX regulates the terminal steps of porphyrin synthesis. This finding is conceptually significant as ALA synthesis has until now been understood to be the regulated step of porphyrin synthesis. The goal of this proposal is to identify the mechanisms by which CLPX regulates erythoid heme synthesis and erythropoiesis. This will be accomplished by Specific Aim 1, which will interrogate the regulation of terminal heme synthesis enzymes by CLPX, and Specific Aim 2, which will examine the effects of CLPX deficiency on erythropoiesis within the context of mouse and zebrafish models.

PROJECT SUMMARY The overall objective of this proposal is to identify and characterize novel proteins involved in erythroid iron/heme metabolism. Erythropoiesis is a massive exercise in cellular proliferation and synthesis of a single protein, hemoglobin. As a consequence, there is a tremendous demand for iron and heme to be efficiently trafficked within the developing erythron. Despite advances in our understanding of extra-cellular iron trafficking and proto-porphyrin biosynthesis, significant gaps remain, especially with respect to components involving the egress of iron from the endosomes to the mitochondria, the trafficking of iron/heme within the mitochondria, the transporters required for proto-porphyrin genesis, the cofactors that facilitate the intracellular trafficking of iron/heme, and the eventual export of heme from the mitochondria for its incorporation in hemoglobin. Using complementary approaches of genetics and bioinformatics from transcriptional profiling, we previously identified several proteins, such as Mitoferrin1 (Mfrn1), Sorting Nexin3 (Snx3), Tmem14c, Lat3, and Clpx1, as new components in the intracellular trafficking of iron, heme and nutrients crucial to red cell development. Although transcriptional profiling as provided insights, we showed that post-translational mechanisms play equally critical roles in the expression and function of proteins involved in iron and heme metabolism. Using quantitative mass spectrometry, we examined changes in the mitochondrial proteome as erythroid cells undergo maturation. We identified several solute carriers and transmembrane proteins, whose function in erythropoiesis have not been previously ascribed, that were induced with hemoglobinization. We propose to study the expression and loss-of-function phenotype of these 7 candidate genes (Aim 1). In particular, we plan to focus previously identified gene, Fam210b (c20orf108), and its interacting partners in red cell development (Aim 2). Functional elucidation of these structural genes will expand our knowledge into the unknown additional steps in intracellular solute, iron and heme trafficking crucial for erythropoiesis. The results of our proposal will provide us with new genetic tools to explore human disorders of anemias.

ABSTRACT The long-term goal of this project is to identify the mechanisms that regulate ?housekeeping? mitochondrial heme metabolism. Heme plays a central role in the redox reactions of life-essential processes such as mitochondrial respiration. My research program is particularly interested in 1. identifying proteins that regulate mitochondrial heme/porphyrin transport and 2. proteins that are part of the housekeeping mitochondrial homeostasis machinery that structurally or functionally interact with the core enzymes of the heme synthesis pathway. While the enzymes of the heme synthesis are well characterized and identical in all tissues, the regulatory mechanisms that couple heme synthesis to cellular requirements are very poorly understood. The importance of these regulatory mechanisms is underscored by the existence of disorders of heme synthesis and iron metabolism that are caused by dysregulation of proteins that are commonly associated with ?housekeeping? homeostatic processes or mitochondrial respiration. Although all tissues require heme, most of our studies on regulatory aspects of heme synthesis have focused on erythroid cells to the exclusion of understanding heme synthesis in other cell types. This project uses our knowledge of erythroid heme synthesis as a springboard to identify heme regulatory mechanisms that are required for housekeeping heme synthesis. Project 1 aims to identify proteins that are required for mitochondrial porphyrin transport in non-erythroid cells. Heme intermediates are photosensitive and cytotoxic, requiring mechanisms for cells to quickly and efficiently transport heme intermediates across cell membranes, to the next enzyme in the heme synthetic pathway. When efficient transport does not occur, cytotoxic porphyrins accumulate in cells, potentially causing porphyria and heme deficiency. While TMEM14C is essential for porphyrin transport in erythroid cells, TMEM14 proteins are not required for heme synthesis in non-erythroid cells. Using a combination of proteomics and whole genome sequencing/RNAseq analysis of TMEM14C suppressor mutants, we will identify novel mitochondrial proteins that regulate porphyrin transport in non-erythroid cells. The long-term goal of Project 2 is to understand the regulation of the heme synthesis complex by proteins that regulate housekeeping mitochondrial homeostasis. During this project period, we focus on the ubiquitous mitochondrial unfoldase, CLPX, which plays an essential role in regulating the activity and protein stability of heme synthesis enzymes. We show that the function of CLPX is highly tissue-specific, and propose to understand its role in the systemic regulation of heme metabolism in the setting of ?housekeeping? heme synthesis. These studies will provide essential datasets that will be invaluable to the mitochondrial protein unfoldase community, but will also be essential for determining how CLPX globally regulates mitochondrial homeostasis via tight regulation of heme.