Human antiquitin: structural and functional studies.
ABSTRACT Antiquitin (ALDH7) is a member of the aldehyde dehydrogenase superfamily which oxidizes various aldehydes to form the corresponding carboxylic acids. Human antiquitin (ALDH7A1) is believed to play a role in detoxification, osmoregulation and more specifically, in lysine metabolism in which alpha-aminoadipic semialdehyde is identified as the specific, physiological substrate of the enzyme. In the present study, the structural basis for the substrate specificity was studied by site-directed mutagenesis. Kinetic analysis on wild-type human antiquitin and its mutants E121A and R301A demonstrated the importance of Glu121 and Arg301 in the binding as well as the turnover of alpha-aminoadipic semialdehyde. On the functional aspect, in addition to the already diversified physiological functions of antiquitin, the recent demonstration of its presence in the nucleus suggests that it may also play a role in cell growth and cell cycle progression. In this investigation, the expression level of antiquitin was monitored in synchronized WRL68 and HEK293 cell culture systems. It was found that the protein was up-regulated during G(1)-S phase transition. Immunofluorescence staining of the synchronized cells demonstrated an increased expression and accumulation of antiquitin in the nucleus during the G(1)-S phase transition. Knockdown of antiquitin using shRNA transfection also resulted in changes in the levels of several key cell cycle-regulating proteins.
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ABSTRACT: Uveal coloboma is a potentially blinding congenital ocular malformation caused by failure of the optic fissure to close during development. Although mutations in numerous genes have been described, these account for a minority of cases, complicating molecular diagnosis and genetic counseling. Here we describe a key role of aldh7a1 as a gene necessary for normal eye development. We show that morpholino knockdown of aldh7a1 in zebrafish causes uveal coloboma and misregulation of nlz1, another known contributor to the coloboma phenotype, as well as skeletal abnormalities. Knockdown of aldh7a1 leads to reduced cell proliferation in the optic cup of zebrafish, delaying the approximation of the edges of the optic fissure. The aldh7a1 morphant phenotype is partially rescued by co-injection of nlz1 mRNA suggesting that nlz1 is functionally downstream of aldh7a1 in regulating cell proliferation in the optic cup. These results support a role of aldh7a1 in ocular development and skeletal abnormalities in zebrafish.PLoS ONE 07/2014; 9(7):e101782. · 3.53 Impact Factor
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ABSTRACT: The lysine catabolism pathway differs in adult mammalian brain from that in extracerebral tissues. The saccharopine pathway is the predominant lysine degradative pathway in extracerebral tissues, whereas the pipecolate pathway predominates in adult brain. The two pathways converge at the level of ∆(1)-piperideine-6-carboxylate (P6C), which is in equilibrium with its open-chain aldehyde form, namely, α-aminoadipate δ-semialdehyde (AAS). A unique feature of the pipecolate pathway is the formation of the cyclic ketimine intermediate ∆(1)-piperideine-2-carboxylate (P2C) and its reduced metabolite L-pipecolate. A cerebral ketimine reductase (KR) has recently been identified that catalyzes the reduction of P2C to L-pipecolate. The discovery that this KR, which is capable of reducing not only P2C but also other cyclic imines, is identical to a previously well-described thyroid hormone-binding protein [μ-crystallin (CRYM)], may hold the key to understanding the biological relevance of the pipecolate pathway and its importance in the brain. The finding that the KR activity of CRYM is strongly inhibited by the thyroid hormone 3,5,3'-triiodothyronine (T3) has far-reaching biomedical and clinical implications. The inter-relationship between tryptophan and lysine catabolic pathways is discussed in the context of shared degradative enzymes and also potential regulation by thyroid hormones. This review traces the discoveries of enzymes involved in lysine metabolism in mammalian brain. However, there still remain unanswered questions as regards the importance of the pipecolate pathway in normal or diseased brain, including the nature of the first step in the pathway and the relationship of the pipecolate pathway to the tryptophan degradation pathway.Amino Acids 09/2013; 45(6). · 3.65 Impact Factor
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ABSTRACT: Objective: A high incidence of structural brain abnormalities has been reported in individuals with pyridoxine-dependent epilepsy (PDE). PDE is caused by mutations in ALDH7A1, also known as antiquitin. How antiquitin dysfunction leads to cerebral dysgenesis is unknown. In this study, we analyzed tissue from a child with PDE as well as control human and murine brain to determine the normal distribution of antiquitin, its distribution in PDE, and associated brain malformations. Methods: Formalin-fixed human brain sections were subjected to histopathology and fluorescence immunohistochemistry studies. Frozen brain tissue was utilized for measurement of PDE-associated metabolites and Western blot analysis. Comparative studies of antiquitin distribution were performed in developing mouse brain sections. Results: Histologic analysis of PDE cortex revealed areas of abnormal radial neuronal organization consistent with type Ia focal cortical dysplasia. Heterotopic neurons were identified in subcortical white matter, as was cortical astrogliosis, hippocampal sclerosis, and status marmoratus of the basal ganglia. Highly elevated levels of lysine metabolites were present in postmortem PDE cortex. In control human and developing mouse brain, antiquitin immunofluorescence was identified in radial glia, mature astrocytes, ependyma, and choroid plexus epithelium, but not in neurons. In PDE cortex, antiquitin immunofluorescence was greatly attenuated with evidence of perinuclear accumulation in astrocytes. Interpretation: Antiquitin is expressed within glial cells in the brain, and its dysfunction in PDE is associated with neuronal migration abnormalities and other structural brain defects. These malformations persist despite postnatal pyridoxine supplementation and likely contribute to neurodevelopmental impairments. ANN NEUROL 2013. © 2013 American Neurological Association.Annals of Neurology 10/2013; 75(1). · 11.91 Impact Factor