[Show abstract][Hide abstract] ABSTRACT: Microcephaly affects approximately 1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2(an/an) mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2(an/an) neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation.
[Show abstract][Hide abstract] ABSTRACT: Proteins with iron–sulfur (Fe–S) clusters participate in multiple metabolic pathways throughout the cell. The mitochondrial
ABC half-transporter Abcb7, which is mutated in X-linked sideroblastic anemia with ataxia in humans, is a functional ortholog
of yeast Atm1p and is predicted to export a mitochondrially derived metabolite required for cytosolic Fe–S cluster assembly.
Using an inducible Cre/loxP system to delete exons 9 and 10 of the Abcb7 gene, we examined the phenotype of mice deficient in Abcb7. We found that Abcb7 was essential in extra-embryonic tissues early in gestation and that the mutant allele exhibits an X-linked parent-of-origin
lethality effect. Furthermore, using X-chromosome inactivation assays and tissue-specific deletions, Abcb7 was found to be essential for the development and function of numerous other cell types and tissues. A notable exception
to this was liver, where loss of Abcb7 impaired cytosolic Fe–S cluster assembly but was not lethal. In this situation, control of iron regulatory protein 1, a key
cytosolic modulator of iron metabolism, which is responsive to the availability of cytosolic Fe–S clusters, was impaired and
contributed to the dysregulation of hepatocyte iron metabolism. Altogether, these studies demonstrate the essential nature
of Abcb7 in mammals and further substantiate a central role for mitochondria in the biogenesis of cytosolic Fe–S proteins.
Full-text · Article · Apr 2006 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Heme-regulated eIF2alpha kinase (HRI) controls protein synthesis by phosphorylating the alpha-subunit of eukaryotic translational initiation factor 2 (eIF2alpha). In heme deficiency, HRI is essential for translational regulation of alpha- and beta-globins and for the survival of erythroid progenitors. HRI is also activated by a number of cytoplasmic stresses other than heme deficiency, including oxidative stress and heat shock. However, to date, HRI has not been implicated in the pathogenesis of any known human disease or mouse phenotype. Here we report the essential role of HRI in 2 mouse models of human rbc disorders, namely erythropoietic protoporphyria (EPP) and beta-thalassemia. In both cases, lack of HRI adversely modifies the phenotype: HRI deficiency exacerbates EPP and renders beta-thalassemia embryonically lethal. This study establishes the protective function of HRI in inherited rbc diseases in mice and suggests that HRI may be a significant modifier of many rbc disorders in humans. Our findings also demonstrate that translational regulation could play a critical role in the clinical manifestation of rbc diseases.
[Show abstract][Hide abstract] ABSTRACT: In heme deficiency, protein synthesis is inhibited by the activation of the heme-regulated eIF2alpha kinase (HRI) through its multiple autophosphorylation. Autophosphorylation sites in HRI were identified in order to investigate their functions. We found that there were eight major tryptic phosphopeptides of HRI activated in heme deficiency. In this report we focused on the role of autophosphorylation at Thr483 and Thr485 in the activation loop of HRI. Disruption of the autophosphorylation of Thr485, but not Thr483, resulted in a lower autokinase activity and locked Thr485Ala HRI in a hypophosphorylated state. Most importantly, autophosphorylation of Thr485, but not Thr483, was essential for attaining eIF2alpha kinase activity of HRI. In addition, autophosphorylation of Thr485 was necessary for arsenite-induced activation of the eIF2alpha kinase activity of HRI, while autophosphorylation at Thr483 was not required for activation by arsenite. The function of Thr490, another conserved Thr residue in the activation loop of HRI, was also investigated. Mutations of Thr490 to either Ala or Asp resulted in reduced autokinase activity and loss of eIF2alpha kinase activity in heme deficiency or upon arsenite treatment. Since Thr490 was not identified as an autophosphorylated site, it is likely that Thr490 itself might be critical for the catalytic activity of HRI. Importantly, Thr485 was very poorly phosphorylated in Thr490 mutant HRI. Collectively, our results demonstrate that autophosphorylation of Thr485 is essential for the hyperphosphorylation and activation of HRI and is required for the acquisition of the eIF2alpha kinase activity.