Crystallographic Study of Mutant Lysl20Leu Xenopus laevis Cu,Zn Superoxide Dismutase

Theoretical calculations and experimental measurements on the Xenopus laevis Cu,Zn superoxide dismutase (XSODB) wild-type protein and on some of its engineered mutants showed that the electrostatic arrangement around the active site channel plays a fundamental role in determining the catalytic properties of the en-zyme. Lysl20, which lies on the lip of the active site channel, about 11 Å from the catalytic copper ion, influences the enzyme electrostatic environment and binding selectivity. Neutralization of this residue has the effect of decreasing the activity of the enzyme versus the negatively charged substrate. In order to get precise information about the mutated residue and its effects on the structure of the engineered protein, the crystal structure of single site Lysl20Leu mutant XSODB was determined at 2.0 Å resolution, and refined to an R-factor value of 0.181. The structure of Lysl20Leu mutant XSODB is little affected by the amino-acid substitution, suggesting that the main effect of the mutation is perturbation of the electrostatic properties of the SOD catalytic center

Protective properties of the cultured stem cell proteome studied in an animal model of acetaminophen-induced acute liver failure

Chronic overuse of common pharmaceuticals, e.g. acetaminophen (paracetamol), often leads to the development of acute liver failure (ALF). This study aimed to elucidate the effect of cultured mesenchymal stem cells (MSCs) proteome on the onset of liver damage and regeneration dynamics in animals with ALF induced by acetaminophen, to test the liver protective efficacy of MSCs proteome depending on the oxygen tension in cell culture, and to blueprint protein components responsible for the effect. Protein compositions prepared from MSCs cultured in mild hypoxic (5% and 10% O2) and normal (21% O2) conditions were used to treat ALF induced in mice by injection of acetaminophen. To test the effect of reduced oxygen tension in cell culture on resulting MSCs proteome content we applied a combination of high performance liquid chromatography and mass-spectrometry (LC–MS/MS) for the identification of proteins in lysates of MSCs cultured at different O2 levels. The treatment of acetaminophen-administered animals with proteins released from cultured MSCs resulted in the inhibition of inflammatory reactions in damaged liver; the area of hepatocyte necrosis being reduced in the first 24 h. Compositions obtained from MSCs cultured at lower O2 level were shown to be more potent than a composition prepared from normoxic cells. A comparative characterization of protein pattern and identification of individual components done by a cytokine assay and proteomics analysis of protein compositions revealed that even moderate hypoxia produces discrete changes in the expression of various subsets of proteins responsible for intracellular respiration and cell signaling. The application of proteins prepared from MSCs grown in vitro at reduced oxygen tension significantly accelerates healing process in damaged liver tissue. The proteomics data obtained for different preparations offer new information about the potential candidates in the MSCs protein repertoire sensitive to oxygen tension in culture medium, which can be involved in the generalized mechanisms the cells use to respond to acute liver failure

Evolutionarily conserved human targets of adenosine to inosine RNA editing

A-to-I RNA editing by ADARs is a post-transcriptional mechanism for expanding the proteomic repertoire. Genetic recoding by editing was so far observed for only a few mammalian RNAs that are predominantly expressed in nervous tissues. However, as these editing targets fail to explain the broad and severe phenotypes of ADAR1 knockout mice, additional targets for editing by ADARs were always expected. Using comparative genomics and expressed sequence analysis, we identified and experimentally verified four additional candidate human substrates for ADAR-mediated editing: FLNA, BLCAP, CYFIP2 and IGFBP7. Additionally, editing of three of these substrates was verified in the mouse while two of them were validated in chicken. Interestingly, none of these substrates encodes a receptor protein but two of them are strongly expressed in the CNS and seem important for proper nervous system function. The editing pattern observed suggests that some of the affected proteins might have altered physiological properties leaving the possibility that they can be related to the phenotypes of ADAR1 knockout mice

PKAN neurodegeneration and residual PANK2 activities in patient erythrocytes

Objective: Pantothenate kinase 2-associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. Methods: Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry-Albright Dystonia Scale (BAD-Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. Results: Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12-56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. Interpretation: Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient-derived cell system with still underestimated diagnostic potential

Dimeric chlorite dismutase from the nitrogen-fixing cyanobacterium Cyanothece sp. PCC7425

It is demonstrated that cyanobacteria (both azotrophic and non-azotrophic) may 34 contain heme b oxidoreductases that can convert chlorite to chloride and molecular oxygen (incorrectly denominated chlorite “dismutase”, Cld). Beside the water-splitting manganese complex of photosystem II this metalloenzyme is the second known enzyme that catalyzes the formation of a covalent oxygen-oxygen bond. All cyanobacterial Clds have a truncated N-terminus and are dimeric (i.e. clade 2) proteins. As model protein, Cld from Cyanothece sp. PCC7425 (CCld) was recombinantly produced in E. coli and shown to efficiently degrade chlorite with an activity optimum at pH 5 (kcat 1144 ± 23.8 s-1, KM 162 ± 10.0 μM, catalytic efficiency (7.1 ± 0.6) × 106 M-1 s-1). The resting ferric high-spin axially symmetric heme enzyme has a standard reduction potential of the Fe(III)/Fe(II) couple of -126 ± 1.9 mV at pH 7. Cyanide mediates the formation of a low-spin complex with kon = (1.6 ± 0.1) × 105 M-1 s-1 and koff = 1.4 ± 2.9 s-1 (KD ~ 8.6 μM). Both, thermal and chemical unfolding follows a non-two state unfolding pathway with the first transition being related to the release of the prosthetic group. The obtained data are discussed with respect to known structure-function relationships of Clds. We ask for the physiological substrate and putative function of these O2-producing proteins in (nitrogen-fixing) cyanobacteria

Roles of distal aspartate and arginine of B-class dye-decolorizing peroxidase in heterolytic hydrogen peroxide cleavage

Dye-decolorizing peroxidases (DyPs) represent the most recently classified hydrogen peroxide dependent heme peroxidase family. Although widely distributed with more than 5000 annotated genes and hailed for their biotechnological potential detailed biochemical characterization of their reaction mechanism remains limited. Here, we present the high resolution crystal structures of wild-type B-class DyP from the pathogenic bacterium Klebsiella pneumoniae (KpDyP) (1.6 \uc5) and the variants D143A (1.3 \uc5), R232A (1.9 \uc5), and D143A/R232A (1.1 \uc5). We demonstrate the impact of elimination of the DyP-typical, distal residues Asp 143 and Arg 232 on (i) the spectral and redox properties, (ii) the kinetics of heterolytic cleavage of hydrogen peroxide, (iii) the formation of the low-spin (LS) cyanide complex as well as on (iv) the stability and reactivity of an oxoiron(IV)porphyrin \u3c0-cation radical (Compound I). Structural and functional studies reveal that the distal aspartate is responsible for deprotonation of H2O2 and for the poor oxidation capacity of Compound I. Elimination of the distal arginine promotes a collapse of the distal heme cavity including blocking of one access channel and a conformational change of the catalytic aspartate. We also provide evidence of formation of an oxoiron(IV)-type Compound II in KpDyP with absorbance maxima at 418, 527 and 553 nm. In summary, a reaction mechanism of the peroxidase cycle of B-class DyPs is proposed. Our observations challenge the idea that peroxidase activity toward conventional aromatic substrates is related to the physiological roles of B-class DyPs

Molecular mechanism of enzymatic chlorite detoxification: insights from structural and kinetic studies

The heme enzyme chlorite dismutase (Cld) degrades chlorite to chloride and dioxygen. Although the structure and steady-state kinetics of pentameric Clds have been elucidated, many questions remain, such as the mechanism of chlorite cleavage and the pH dependence of the reaction. Here, we present high resolution X-ray crystal structures of a dimeric Cld at pH 6.5 and 8.5, its fluoride and isothiocyanate complexes and the neutron structure at pH 9.0 together with the pH dependence of the Fe(III)/Fe(II) couple and the UV-vis and resonance Raman spectral features. We demonstrate that the distal Arg127 cannot act as proton acceptor and is fully ionized even at pH 9.0 ruling out its proposed role in dictating the pH dependence of chlorite degradation. Stopped-flow studies show that (i) Compound I and hypochlorite cannot recombine and (ii) Compound II is the immediately formed redox intermediate that dominates during reaction. Homolytic cleavage of chlorite is propose

Insights into the Role of a Cardiomyopathy-Causing Genetic Variant in ACTN2

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hyper-trophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocar-diography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Reso-lution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell cycle defects and mitochondrial dys-function. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteosomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteosomal system is activated; a mechanism which has been implicated in cardiomyopathies previously. In parallel, lack of functional al-pha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell cycle defects, the likely cause of death of the embryos. The defects also have wide-ranging morphological consequences

PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is a regulatory hub for transcription and RNA processing. Here, we identify PHD-finger protein 3 (PHF3) as a regulator of transcription and mRNA stability that docks onto Pol II CTD through its SPOC domain. We characterize SPOC as a CTD reader domain that preferentially binds two phosphorylated Serine-2 marks in adjacent CTD repeats. PHF3 drives liquid-liquid phase separation of phosphorylated Pol II, colocalizes with Pol II clusters and tracks with Pol II across the length of genes. PHF3 knock-out or SPOC deletion in human cells results in increased Pol II stalling, reduced elongation rate and an increase in mRNA stability, with marked derepression of neuronal genes. Key neuronal genes are aberrantly expressed in Phf3 knock-out mouse embryonic stem cells, resulting in impaired neuronal differentiation. Our data suggest that PHF3 acts as a prominent effector of neuronal gene regulation by bridging transcription with mRNA decay