We seek new, medically relevant insights into the biology of red blood cell (RBC) formation (erythropoiesis). In erythroid precursors, the ubiquitin proteasome system (UPS) identifies and eliminates endogenous proteins that become unnecessary or potentially deleterious during progressive maturation. The UPS also functions as a protective mechanism to eliminate toxic proteins that accumulate in RBC disorders, as we and others have demonstrated for ? thalassemia, a common anemia caused by imbalanced hemoglobin synthesis. While the UPS is believed to be critical for erythropoiesis, very little is known regarding the specific molecules involved. Large-scale genome wide association studies (GWAS) of human populations have identified numerous UPS components predicted to regulate erythropoiesis. We combined these GWAS with global transcriptome analyses to identify several potentially important UPS proteins expressed in RBC precursors. One interesting candidate that we have studied in depth is Trim58, a protein that marks other proteins for degradation and has also been implicated by GWAS to regulate the formation of platelets. We showed that Trim58 deficient RBC precursors exhibit faulty maturation, including impaired ability to expel the nucleus, a key step in mammalian erythropoiesis. Preliminary studies indicate that Trim58 facilitates enucleation by eliminating dynein, a molecular motor complex with multiple essential functions in virtually all other cell types. We will perform biochemical studies of purifed proteins and genetic manipulations of cultured RBCs to examine the mechanisms by which Trim58 degrades dynein and how this facilitates RBC precursor enucleation. To investigate potential dynein independent functions of Trim58, we will perform proteomic studies to identify its additional degradation targets (Aim 1). To examine Trim58 functions in vivo, we will ablate the gene in mice and determine the consequences on RBC and platelet formation at baseline and after exposure to various physiological stresses (Aim 2). Finally, we will use short hairpin RNAs to suppress the expression of additional GWAS-identified UPS candidates in cultured primary erythroid precursors and determine how this affects their maturation (Aim 3). Our studies aim to elucidate new pathways that promote erythropoiesis through regulated protein degradation. By altering these pathways through drugs or genetic manipulation, it should be possible to enhance ongoing efforts to generate RBCs in vitro for transfusion therapies and to treat various blood diseases caused by dysregulated erythropoiesis. More generally, our planned investigations synergize with GWAS to better understand how genetic variation influences medically relevant phenotypes.

Public Health Relevance

We are studying the molecular pathways that degrade unnecessary or unstable proteins during red blood cell and platelet development. Defining these pathways and the specific molecules that are involved will elucidate how nascent blood cells streamline their contents during normal maturation and protect themselves against various insults that damage proteins. Once these protein degradation pathways are better understood, it may be possible to manipulate them therapeutically in various anemias or diseases associated with excessive production of red blood cells.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
7R01DK061692-12
Application #
8843634
Study Section
Molecular and Cellular Hematology (MCH)
Program Officer
Bishop, Terry Rogers
Project Start
Project End
Budget Start
Budget End
Support Year
12
Fiscal Year
2014
Total Cost
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
City
Memphis
State
TN
Country
United States
Zip Code
38105
Sankaran, Vijay G; Weiss, Mitchell J (2015) Anemia: progress in molecular mechanisms and therapies. Nat Med 21:221-30
Thom, Christopher S; Traxler, Elizabeth A; Khandros, Eugene et al. (2014) Trim58 degrades Dynein and regulates terminal erythropoiesis. Dev Cell 30:688-700
Strader, Michael Brad; Hicks, Wayne A; Kassa, Tigist et al. (2014) Post-translational transformation of methionine to aspartate is catalyzed by heme iron and driven by peroxide: a novel subunit-specific mechanism in hemoglobin. J Biol Chem 289:22342-57
Thom, Christopher S; Dickson, Claire F; Gell, David A et al. (2013) Hemoglobin variants: biochemical properties and clinical correlates. Cold Spring Harb Perspect Med 3:a011858
Mollan, Todd L; Banerjee, Sambuddha; Wu, Gang et al. (2013) α-Hemoglobin stabilizing protein (AHSP) markedly decreases the redox potential and reactivity of α-subunits of human HbA with hydrogen peroxide. J Biol Chem 288:4288-98
Khandros, Eugene; Thom, Christopher S; D'Souza, Janine et al. (2012) Integrated protein quality-control pathways regulate free α-globin in murine β-thalassemia. Blood 119:5265-75
Mollan, Todd L; Khandros, Eugene; Weiss, Mitchell J et al. (2012) Kinetics of α-globin binding to α-hemoglobin stabilizing protein (AHSP) indicate preferential stabilization of hemichrome folding intermediate. J Biol Chem 287:11338-50
Raess, Philipp W; Paessler, Michelle E; Bagg, Adam et al. (2012) α-Hemoglobin-stabilizing protein is a sensitive and specific marker of erythroid precursors. Am J Surg Pathol 36:1538-47
Khandros, Eugene; Mollan, Todd L; Yu, Xiang et al. (2012) Insights into hemoglobin assembly through in vivo mutagenesis of α-hemoglobin stabilizing protein. J Biol Chem 287:11325-37
Crowley, Moira A; Mollan, Todd L; Abdulmalik, Osheisa Y et al. (2011) A hemoglobin variant associated with neonatal cyanosis and anemia. N Engl J Med 364:1837-43

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