Bi-allelic GOT2 Mutations Cause a Treatable Malate-Aspartate Shuttle-Related Encephalopathy.

Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2.

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.

Plasma obtained following murine hindlimb ischemic conditioning protects against oxidative stress in zebrafish models through activation of nrf2a and downregulation of duox.

Ischemia/reperfusion of organ systems in trauma patients with resuscitated hemorrhagic shock (HSR) contributes to tissue injury and organ dysfunction. Previous studies using a murine model of HSR showed that remote ischemic preconditioning (RIC) protected against organ injury and that the plasma was able to prevent neutrophil migration in a zebrafish tailfin-cut inflammation model. In this study, we hypothesized that RIC plasma inhibits neutrophil function through a decrease in reactive oxygen species (ROS) production via the upregulation of the transcription factor Nrf2 and downstream antioxidative genes. Plasma from mice subjected to RIC (4 cycles of 5-min hindlimb ischemia/reperfusion) was microinjected into zebrafish. The results show that RIC plasma caused a reduction of ROS generation in response to tail injury. In addition, RIC plasma protected the fish larvae in the survival studies when exposed to either H2O2 or LPS. Oxidative stress PCR Array showed that RIC plasma treatment led to upregulation of antioxidative related genes including hsp70, hmox1a, nqo1 as well as downregulation of duox, the producer of H2O2. To explore the role of nrf2 in RIC, RIC plasma from Nrf2 KO mice were injected to the zebrafish and showed no inhibitory effect on neutrophil migration. Moreover, knockdown of nrf2a attenuated the anti-inflammatory and protective effect of RIC plasma. The downregulation of duox and upregulation of hmox1a were confirmed to require the activation of nrf2a. Therefore, we show that the protective effect of RIC may be related to the elaboration of humoral factors which counter injury-induced ROS generation in a nrf2-dependent fashion.

Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function.

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.

Substrate share in the suicide inactivation of mushroom tyrosinase.

To address the real cause of the suicide inactivation of mushroom tyrosinase (MT), under in vitro conditions, cresolase and catecholase reactions of this enzyme were investigated in the presence of three different pairs of substrates, which had been selected for their structural specifications. It was showed that the cresolase activity is more vulnerable to the inactivation. Acetylation of the free tyrosyl residues of MT did not cure susceptibility of the cresolase activity, but clearly decreased the inactivation rate of MT in the presence of 4-[(4-methylbenzo)azo]-1,2-benzenediol (MeBACat) as a catecholase substrate. Considering the results of the previous works and this research, some different possible reasons for the suicide inactivation of MT have been discussed. Accordingly, it was proposed that the interruption in the conformational changes in the tertiary and quaternary structures of MT, triggered by the substrate then mediated by the solvent molecules, might be the real reason for the suicide inactivation of the enzyme. However, minor causes like the toxic effect of the ortho-quinones on the protein body of the enzyme or the oxidation of some free tyrosyl residues on the surface of the enzyme by itself, which could boost the inactivation rate, should not be ignored.

Hypoxia in early life is associated with lasting changes in left ventricular structure and function at maturity in the rat.

BACKGROUND: There is a growing population of adults with repaired cyanotic congenital heart disease. These patients have increased risk of impaired cardiac health and premature death. We hypothesized that hypoxia in early life before surgical intervention causes lasting changes in left ventricular structure and function with physiological implications in later life. METHODS: Sprague-Dawley rats reared initially hypoxic conditions (FiO(2)=0.12) for days 1-10 of life were compared to rats reared only in ambient air. Cellular morphology and viability were compared among LV cardiomyocytes and histological analyses were performed on LV myocardium and arterioles. Intracellular calcium transients and cell shortening were measured in freshly-isolated cardiomyocytes, and mitochondrial hexokinase 2 (HK2) expression and activity were determined. Transthoracic echocardiography was used to assess LV function in anesthetized animals. RESULTS: Cardiomyocytes from adult animals following hypoxia in early life had greater cellular volumes but significantly reduced viability. Echocardiographic analyses revealed LV hypertrophy and diastolic dysfunction, and alterations in cardiomyocyte calcium transients and cell shortening suggested impaired diastolic calcium reuptake. Histological analyses revealed significantly greater intima-media thickness and decreased lumen area in LV arterioles from hypoxic animals. Alterations in mitochondrial HK2 protein distribution and activity were also observed which may contribute to cardiomyocyte fragility. CONCLUSIONS: Hypoxia in early life causes lasting changes in left ventricular structure and function that may negatively influence myocardial and vascular responses to physiological stress in later life. These data have implications for the growing population of adults with repaired or palliated cyanotic congenital heart disease.

Dissociation of the octameric enolase from S. pyogenes--one interface stabilizes another.

Most enolases are homodimers. There are a few that are octamers, with the eight subunits arranged as a tetramer of dimers. These dimers have the same basic fold and same subunit interactions as are found in the dimeric enolases. The dissociation of the octameric enolase from S. pyogenes was examined, using NaClO(4), a weak chaotrope, to perturb the quaternary structure. Dissociation was monitored by sedimentation velocity. NaClO(4) dissociated the octamer into inactive monomers. There was no indication that dissociation of the octamer into monomers proceeded via formation of significant amounts of dimer or any other intermediate species. Two mutations at the dimer-dimer interface, F137L and E363G, were introduced in order to destabilize the octameric structure. The double mutant was more easily dissociated than was the wild type. Dissociation could also be produced by other salts, including tetramethylammonium chloride (TMACl) or by increasing pH. In all cases, no significant amounts of dimers or other intermediates were formed. Weakening one interface in this protein weakened the other interface as well. Although enolases from most organisms are dimers, the dimeric form of the S. pyogenes enzyme appears to be unstable.

Calorimetric analysis of thermodynamic stability and aggregation for apo and holo amyotrophic lateral sclerosis-associated Gly-93 mutants of superoxide dismutase.

Differential scanning calorimetry was used to measure changes in thermodynamic stability and aggregation for glycine 93 mutants of human copper, zinc-superoxide dismutase (SOD). Glycine 93 is a conserved residue at position i + 3 of a tight turn and has been found to be a mutational hot spot in familial amyotrophic lateral sclerosis (fALS). The fALS-associated mutations, G93A, G93S, G93R, G93D, and G93V, were made in a pseudo wild-type background containing no free cysteines, which prevented the formation of aberrant disulfide bonds upon thermal unfolding, and enabled quantitative thermodynamic analysis of the effects of the mutations. Thermal unfolding was highly reversible for all the SODs in both the fully metallated (holo) and metal-free (apo) forms. The data for all the holo-SODs and for the apo-pseudo-wild-type SOD were well fit by a 2-state unfolding model for native dimer (N2) to two unfolded monomers (2U), N2 <--> 2U. The holo- and apo-forms of the mutants are significantly destabilized (by 1.5-3.5 kcal mol(-1) monomer) relative to the corresponding forms of pseudo wild-type, with the relative stabilities being correlated with statistical preferences for amino acids in this structural context. Although van't Hoff (DeltaHvH) to calorimetric (DeltaHcal) enthalpy ratios are close to unity for all the holo-SODs and for apo-pseudo-wild-type, consistent with a 2-state transition, DeltaHvH is considerably larger than DeltaHcal for all the apo-mutants. This suggests that the mutations cause apo-SOD to have an increased propensity to misfold or aggregate, which may be linked to increased toxic mutant SOD aggregation in fALS.