Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

Abstract

SUMMARY Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies.

New Zebrafish Models of Neurodegeneration

“…In this review we shall critically assess the most notable and recently published models, disease mechanisms and therapeutic testing utilising zebrafish (Table 1) and endeavour to stimulate further research in this important area. Although overlaps exist between epilepsy and neurodegenerative disease, we will refrain from reviewing zebrafish models of epilepsy in detail here as this disorder has been recently reviewed [1,6], along with zebrafish models of mitochondrial disease [7,8], metabolic disease [9] and neurological disorders [1, 10, 11].…”

Regulation of heme biosynthesis and transport in metazoa

“…Animal FECHs contain iron-sulfur [2Fe-2S] clusters. Both the stability and the enzymatic activity of metazoan FECH proteins are regulated by iron availability as well as the production of iron-sulfur clusters [56,57]. Because of the presence of [2Fe-2S] clusters, animal FECHs are also regulated by mitochondrial pH and redox potential.…”

“…Because of the presence of [2Fe-2S] clusters, animal FECHs are also regulated by mitochondrial pH and redox potential. For example, alkalization of mitochondria due to deficiency of ATPase inhibitory factor 1 reduced the activity of FECH and the production of heme [57].…”

Hematopoietic stem cells develop in the absence of endothelial cadherin 5 expression

“…To further verify the loss-of-function phenotype for cdh5, we injected 2 independently derived cdh5 antisense splice-blocking and translational-blocking morpholinos (MOs) 17 to knockdown cdh5 expression in zebrafish embryos. The cdh5-silenced embryos (morphants) phenocopied mlb's anemia (supplemental Figure 1D-E).…”

“…16,17 The complementary DNA prepared from wild-type (WT) and mlb embryos were sequenced and the mutation in the exon 3 sequence was verified using an allele-specific oligohybridization technique. 18 We performed quantitative reverse transcriptasepolymerase chain reaction (qRT-PCR) using TaqMan gene expression assays (zf-cdh5: Dr03089732_g1 spanning exon 3-4, Dr03089733_m1 spanning exon 4-5, Dr03089737_m1 spanning exon 8-9, Dr03089729_m1 spanning exon 11-12, and zf-hprt: Dr03138604_m1; Applied Biosystems) to measure transcript levels of cdh5 along the length of the gene.…”