Master of Science

thesisChediak-Higashi Syndrome (CHS) is a rare, autosomal recessive genetic disorder caused by mutations in the lysosomal trafficking regulator gene (CHS1) that affects vesicle morphology. The CHS1 gene encodes a protein, Lyst, which belongs to a family of proteins containing a conserved BEACH (BEige And Chediak-Higashi) domain and WD40-repeat domain. The beigej mouse has been the best-studied animal model for CHS. Factor associated with neutral sphingomyelinase activation or FAN is the smallest member of the BEACH family. FAN interacts with the Tumor Necrosis Factor Receptor (TNFR1) to activate Neutral SphingoMyelinase2 (NSMase2). The absence of Lyst or FAN gives rise to enlarged lysosomes. Loss of both proteins results in an additive effect, as demonstrated by increased lysosome size in FAN-/- /beigej mouse. An additive phenotype suggests that there are at least two pathways to regulate lysosome size. Overexpression of Lyst or FAN results in smaller than normal lysosomes. Overexpression of FAN in the absence of Lyst does not give rise to small lysosomes. NSMases are present on lysosomes and inhibition of NSMases, using GW4869 or 3-OMethylsphingomyelin, results in enlarged lysosomes. Further, the decrease in lysosome size seen in FAN-overexpressing cells is blocked by GW4869. These results suggest that FAN activation of NSMase at the lysosome is a crucial step in regulating lysosome size

The mitochondrial metal transporters mitoferrin1 and mitoferrin2 are required for liver regeneration and cell proliferation in mice

International audienceMitochondrial iron import is essential for iron-sulfur cluster formation and heme biosynthesis. Two nuclear-encoded vertebrate mitochondrial high-affinity iron importers, mitoferrin1 (Mfrn1) and Mfrn2, have been identified in mammals. In mice, the gene encoding Mfrn1, solute carrier family 25 member 37 (Slc25a37), is highly expressed in sites of erythropoiesis, and whole-body Slc25a37 deletion leads to lethality. Here, we report that mice with a deletion of Slc25a28 (encoding Mfrn2) are born at expected Mendelian ratios, but show decreased male fertility due to reduced sperm numbers and sperm motility. Mfrn2 -/- mice placed on a low-iron diet exhibited reduced mitochondrial manganese, cobalt, and zinc levels, but not reduced iron. Hepatocyte-specific loss of Slc25a37 (encoding Mfrn1) in Mfrn2 -/- mice did not affect animal viability, but resulted in a 40% reduction in mitochondrial iron and reduced levels of oxidative phosphorylation proteins. Placing animals on a low-iron diet exaggerated the reduction in mitochondrial iron observed in liver-specific Mfrn1/2-knockout animals. Mfrn1 -/-/Mfrn2 -/- bone marrow-derived macrophages or skin fibroblasts in vitro were unable to proliferate, and overexpression of Mfrn1-GFP or Mfrn2-GFP prevented this proliferation defect. Loss of both mitoferrins in hepatocytes dramatically reduced regeneration in the adult mouse liver, further supporting the notion that both mitoferrins transport iron and that their absence limits proliferative capacity of mammalian cells. We conclude that Mfrn1 and Mfrn2 contribute to mitochondrial iron homeostasis and are required for high-affinity iron import during active proliferation of mammalian cells

FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism