Biology subjects

Danoff, A.

Publications and source records attributed to Danoff, A..

3 recordsLinked to original sources

bioRxiv2022

Regulation of iron transport is required for terminal erythroid differentiation even under iron-replete conditions that are sufficient for hemoglobinization

Iron metabolism drives key erythropoietic processes, including hemoglobinization, survival, and proliferation. Here, we developed in vivo methods to interrogate how iron regulates erythropoiesis and report that mitochondrial iron transport via MFRN1 is essential for erythroid cell cycle progression. mfrn1 embryos had severely decreased erythroid cell number caused by cell cycle arrest at G2/M. They had enlarged nuclei, suggesting a mitotic defect. Iron supplementation rescued the cell cycle defect, implicating mitochondrial iron deficiency as its cause. In contrast, fpn1 mutants, anemic from systemic iron deficiency, had less severe decreases in erythroid mitochondrial iron than mfrn1 mutants and no proliferative defects. scRNAseq and FACS analyses for cd41 (thrombocytic) and gata1 reporters indicated that developmental defects in mfrn1 mutants were largely erythroid restricted. mfrn1 mutant gata1+ erythroid progenitors were severely decreased at 3 dpf, and a further decrease in globin- expressing terminally differentiating erythroid cells. While wild-type erythroid cells mostly lost expression of the gata1 progenitor marker by 3 dpf, mfrn1 mutant erythroid cells retained gata1 expression. These data are consistent with a model where mitochondrial iron transport facilitates development of gata1+ erythroid progenitors and is required for the completion of erythropoiesis by facilitating mitosis in the terminal cell cycles. Key pointsO_LIMitochondrial iron import through MFRN1 is required for progress through the G2/M checkpoint in the terminal cell cycles leading to terminal erythroid differentiation. C_LIO_LIMitochondrial iron supply through MFRN1 is required for maintenance/proliferation of erythroid progenitors and terminal maturation into globin expressing erythroid cells. C_LI

biochemistry↗

bioRxiv2020

CLPX regulates erythroid heme synthesis by control of mitochondrial heme synthesis enzymes and iron utilization

Heme is a prosthetic group that plays a critical role in catalyzing life-essential redox reactions in all cells, including critical metabolic processes. Heme synthesis must be tightly co-regulated with cellular requirements in order to maximize utilization and minimize toxicity. Terminally differentiating erythroid cells have an extremely high demand for heme for hemoglobin synthesis. While the enzymatic reactions of heme synthesis are extremely well studied, the mechanisms by which the mitochondrial homeostatic machinery interacts with and regulates heme synthesis are poorly understood. Knowledge of these regulatory mechanisms are key to understanding how red cells couple heme production with heme demand. Heme synthesis is tightly regulated by the mitochondrial AAA+ unfoldase CLPX, which has been reported to promote heme synthesis by activation of yeast {delta}-aminolevulinate synthase (ALAS/Hem1). CLPX was also reported to mediate heme-induced turnover of ALAS1 in human cells. However, a mutation in the ATP binding domain of CLPX that abrogated ATP binding caused an increase in ALAS activity, contrary to previous predictions that CLPX activated ALAS. Using loss-of-function assays in murine cells and zebrafish, we interrogated the mechanisms by which CLPX regulates erythroid heme synthesis. We found that consistent with previous studies, CLPX is required for erythroid heme synthesis. We show that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover. However, we also showed that CLPX is required for PPOX activity and maintenance of FECH levels, likely accounting for the heme deficiency in the absence of CLPX. Lastly, CLPX is required for iron metabolism during erythroid terminal differentiation. Our results show that the role of CLPX in heme synthesis is not conserved across eukaryotes. Our studies reveal a potential mechanism for the role of CLPX in anemia and porphyria, and reveal multiple nodes at which heme synthesis is regulated by the mitochondrial housekeeping machinery.

biochemistry↗

bioRxiv2019

A missense mutation dissociates triglyceride and phospholipid transfer activities in zebrafish and human microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) transfers triglycerides and phospholipids and is essential for the assembly of Apolipoprotein B (ApoB)-containing lipoproteins in the endoplasmic reticulum. We have discovered a zebrafish mutant (mttpc655) expressing a C-terminal missense mutation (G863V) in Mttp, one of the two subunits of MTP, that is defective at transferring triglycerides, but retains phospholipid transfer activity. Mutagenesis of the conserved glycine in the human MTTP protein (G865V) also eliminates triglyceride but not phospholipid transfer activity. The G863V mutation reduces the production and size of ApoB-containing lipoproteins in zebrafish embryos and results in the accumulation of cytoplasmic lipid droplets in the yolk syncytial layer. However, mttpc655 mutants exhibit only mild intestinal lipid malabsorption and normal growth as adults. In contrast, zebrafish mutants bearing the previously identified mttpstl mutation (L475P) are deficient in transferring both triglycerides and phospholipids and exhibit gross intestinal lipid accumulation and defective growth. Thus, the G863V point mutation provides the first evidence that the triglyceride and phospholipid transfer functions of a vertebrate MTP protein can be separated, arguing that selective inhibition of the triglyceride transfer activity of MTP may be a feasible therapeutic approach for dyslipidemia.

physiology↗