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 i n 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.
The proposed project, which seeks to characterize the role of Tmem14c in heme synthesis and identify novel proteins involved in heme metabolism, will advance our understanding of the genetic mechanisms underlying human blood disorders, particularly anemia and erythroid porphyria. Because a disproportionate number of anemic patients are women and children, the success of this project will have particular impact on the management of their health. The intellectual environment and scientific infrastructure available to Dr. Yien will facilitate the completion of her proposed aims, laying the foundation for development of her independent research program during a mentored transition to independence.
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|Grillo, Anthony S; SantaMaria, Anna M; Kafina, Martin D et al. (2017) Restored iron transport by a small molecule promotes absorption and hemoglobinization in animals. Science 356:608-616|
|Yien, Yvette Y; Ducamp, Sarah; van der Vorm, Lisa N et al. (2017) Mutation in human CLPX elevates levels of ?-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria. Proc Natl Acad Sci U S A 114:E8045-E8052|
|Yien, Yvette Y; Paw, Barry H (2016) A role for iron deficiency in dopaminergic neurodegeneration. Proc Natl Acad Sci U S A 113:3417-8|
|Chung, Jacky; Bauer, Daniel E; Ghamari, Alireza et al. (2015) The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability. Sci Signal 8:ra34|
|Kardon, Julia R; Yien, Yvette Y; Huston, Nicholas C et al. (2015) Mitochondrial ClpX Activates a Key Enzyme for Heme Biosynthesis and Erythropoiesis. Cell 161:858-67|
|Yien, Yvette Y; Gnanapragasam, Merlin Nithya; Gupta, Ritama et al. (2015) Alternative splicing of EKLF/KLF1 in murine primary erythroid tissues. Exp Hematol 43:65-70|
|Yien, Yvette Y; Robledo, Raymond F; Schultz, Iman J et al. (2014) TMEM14C is required for erythroid mitochondrial heme metabolism. J Clin Invest 124:4294-304|
|Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek et al. (2014) Characterization of genomic deletion efficiency mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J Biol Chem 289:21312-24|
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