Summary. Two brothers with X-linked ataxia (XLA) were found to have hypochromic red cells and increased erythrocyte protoporphyrin despite normal iron stores. The mother was unaffected by ataxia and had normal iron stores but showed evidence of some red cell hypochromia with heavy basophilic stippling that stained positive for iron. Bone marrow biopsy confirmed the presence of ring sideroblasts in one of the brothers. The absence of mutations in the ALAS2 gene and the predominance of zinc over free protoporphyrin led to a search using a combination of DNA and cDNA analysis for the presence of mutations in the ABC7 gene. ABC7 encodes a mitochondrial half-type ATP Binding Cassette transporter involved in iron homeostasis. The published cDNA sequence was used to search databases for the genomic sequence of which 12 exons spanning 23´4 kb were mapped leaving the most 5 H nucleotides unaccounted for. The identified exons and their exon±intron boundaries were amplified from DNA while the most 5 H sequence including the initiation codon was amplified from cDNA of peripheral blood cells. Direct sequencing revealed hemizygosity in the brothers and heterozygosity in the mother for a G3C transversion at position 1299 of the published cDNA. This predicts a V411L substitution at the beginning of the last of six putative transmembrane regions of the protein. Restriction enzyme digestion confirmed the presence of this mutation in the three family members but could not detect it in 200 normal alleles. An uncle affected by ataxia also carried this mutation. This study supports the recently hypothesized involvement of the ABC7 gene in XLSA/A and highlights a protein structure region of importance to this syndrome.
Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis. Missense mutations within the acetyltransferase domain of these proteins correlate with diseases, including endometrial cancers and Roberts syndrome. Despite their biological importance, the mechanisms underlying acetylation by the ESCO proteins are not understood. Here, we report the X-ray crystal structure of the highly conserved zinc finger-acetyltransferase moiety of ESCO1 with accompanying structure-based mutagenesis and biochemical characterization. We find that the ESCO1 acetyltransferase core is structurally homologous to the Gcn5 HAT, but contains unique additional features including a zinc finger and an ∼40-residue loop region that appear to play roles in protein stability and SMC3 substrate binding. We identify key residues that play roles in substrate binding and catalysis, and rationalize the functional consequences of disease-associated mutations. Together, these studies reveal the molecular basis for SMC3 acetylation by ESCO1 and have broader implications for understanding the structure/function of non-histone acetyltransferases.
In the past, necrosis, which is associated with many diseases was considered to be an uncontrolled process; however, recent data support the notion that necrosis is a regulated process, yet, the only available treatment for necrosis is applying high oxygen pressure. Humanin (HN) is a 24-amino acid peptide, known for its anti-apoptotic activity, especially against neuronal cell death by Alzheimer insults. Recent studies showed that HN has other protective actions such as in myocardial ischemia, atherosclerosis and more; most of the activities were related to anti-apoptotic action. HN was also shown to increase cellular ATP levels. Our study reveals that, in addition to their anti-apoptotic activities, HN and some of its peptide derivatives exhibits a protective effect against necrotic insults in neuronal cell lines (PC-12 and NSC-34). Additionally, we found that HN affects ATP levels in cells undergoing necrosis, and interacts directly with mitochondrial ATP synthase enhancing its activity. Results obtained by fluorescence lifetime imaging microscopy (FLIM) and super-resolution microscopy with fluorescein-labeled HN, support the aforementioned findings. Furthermore, in vivo studies in traumatic brain injury on C57BL/6J mice, show a protective effect of HN, as demonstrated by improved motor performance and by MRI study done in a 1 Tesla small animals device. Thus, the present study may reveal a novel anti-necrotic mechanism of action of HN and its derivatives, and provide a new strategy for potential therapeutic treatment of ischemic states.
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