Primary structure of copper-zinc superoxide dismutase from Neurospora crassa

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.     

Structural and spectroscopic studies of a model for catechol oxidase

A binuclear copper complex, [Cu₂(BPMP)(OAc)₂][ClO₄]·H₂O, has been prepared using the binucleating ligand 2,6-bis[bis(pyridin-2-ylmethylamino)methyl]-4-methylphenol (H-BPMP). The X-ray crystal structure reveals the copper centers to have a five-coordinate square pyramidal geometry, with the acetate ligands bound terminally. The bridging phenolate occupies the apical position of the square-based pyramids and magnetic susceptibility, electron paramagnetic resonance (EPR) and variable-temperature variable-field magnetic circular dichroism (MCD) measurements indicate that the two centers are very weakly antiferromagnetically coupled (J = −0.6 cm⁻¹). Simulation of the dipole–dipole-coupled EPR spectrum showed that in solution the Cu–O–Cu angle was increased from 126° to 160° and that the internuclear distance was larger than that observed crystallographically. The high-resolution spectroscopic information obtained has been correlated with a detailed ligand-field analysis to gain insight into the electronic structure of the complex. Symmetry arguments have been used to demonstrate that the sign of the MCD is characteristic of the tetragonally elongated environment. The complex also displays catecholase activity (k _cat = 15 ± 1.5 min⁻¹, K _M = 6.4 ± 1.8 mM), which is compared with other dicopper catechol oxidase models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Knock-Down of IL-1Ra in Obese Mice Decreases Liver Inflammation and Improves Insulin Sensitivity

Interleukin 1 Receptor antagonist (IL-1Ra) is highly elevated in obesity and is widely recognized as an anti-inflammatory cytokine. While the anti-inflammatory role of IL-1Ra in the pancreas is well established, the role of IL-1Ra in other insulin target tissues and the contribution of systemic IL-1Ra levels to the development of insulin resistance remains to be defined. Using antisense knock down of IL-1Ra in vivo, we show that normalization of IL-1Ra improved insulin sensitivity due to decreased inflammation in the liver and improved hepatic insulin sensitivity and these effects were independent of changes in body weight. A similar effect was observed in IL1-R1 KO mice, suggesting that at high concentrations of IL-1Ra typically observed in obesity, IL-1Ra can contribute to the development of insulin resistance in a mechanism independent of IL-1Ra binding to IL-1R1. These results demonstrate that normalization of plasma IL-1Ra concentration improves insulin sensitivity in diet- induced obese mice.     

Genetic effects on information processing speed are moderated by age – converging results from three samples

Information processing is a cognitive trait forming the basis of complex abilities like executive function. The Trail Making Test (TMT) is a well‐established test of information processing with moderate to high heritability. Age of the individual also plays an important role. A number of genetic association studies with the TMT have been performed, which, however, did not consider age as a moderating factor. We report the results of genome‐wide association studies (GWASs) on age‐independent and age‐dependent TMT performance in two population‐representative community samples (Munich Antidepressant Response Signature, MARS: N₁ = 540; Ludwig Maximilians University, LMU: N₂ = 350). Age‐dependent genome‐wide findings were then evaluated in a third sample of healthy elderly subjects (Sydney Memory and Ageing Study, Sydney MAS: N₃ = 448). While a meta‐analysis on the GWAS findings did not reveal age‐independent TMT associations withstanding correction for multiple testing, we found a genome‐wide significant age‐moderated effect between variants in the DSG1 gene region and TMT‐A performance predominantly reflecting visual processing speed (rs2199301, P_{meta‐analysis} = 1.3 × 10^?7). The direction of the interaction suggests for the minor allele a beneficial effect in younger adults turning into a detrimental effect in older adults. The detrimental effect of the missense single nucleotide polymorphism rs1426310 within the same DSG1 gene region could be replicated in Sydney MAS participants aged 70–79, but not in those aged 80 years and older, presumably a result of survivor bias. Our findings demonstrate opposing effects of DSG1 variants on information processing speed depending on age, which might be related to the complex processes that DSG1 is involved with, including cell adhesion and apoptosis.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

The LG3 module of laminin-5 harbors a binding site for integrin alpha3beta1 that promotes cell adhesion, spreading, and migration

Laminins are a family of extracellular matrix glycoproteins involved in cell adhesion and migration. A major obstacle to understanding their structure-function relationships is the lack of small laminin domains capable of replicating integrin-binding, cell-adhesive, and migratory functions of the intact molecule. Here, we show that the recombinant LG3 (rLG3) module (26 kDa) of laminin-5 (Ln-5) alpha(3) chain replicated key Ln-5 activities. rLG3 but not rLG1 or rLG2 supported cell adhesion and migration of at least two distinct cell lines, in an integrin alpha(3)beta(1)-dependent manner. Cell adhesion to rLG3 was regulated by divalent cations and accompanied by cell spreading and tyrosine phosphorylation of FAK focal adhesion kinase. The integrin binding activity of rLG3 was confirmed by rLG3 affinity chromatography of detergent cell lysates, which resulted in specific purification of integrin alpha(3)beta(1). To our knowledge, this is the first report directly demonstrating that a recombinant laminin LG module is an active domain capable of supporting integrin-dependent cell adhesion and migration.     

(Na+,K+)-ATPase: a new assay of Na+-ATPase reveals covert anti-pump antibodies

A new assay is described for rat (Na⁺,K⁺)-ATPase [EC 3.6.1.3] prepared from renal medullary or crude liver membranes. With ATP at 1 μm, initial rates of ouabain-sensitive decreases in substrate concentrations are followed by measuring diminished ATP-driven luciferin-luciferase light production. Under these conditions, using highly purified enzyme preparations, Na⁺ and K⁺ ions stimulate and inhibit initial ATP hydrolysis rates, respectively. Therefore, it is likely that the assay measures Na⁺-ATPase partial reactions of the pump. A monospecific polyclonal rabbit anti-rat pump antiserum blocks Na⁺-dependent ATPase measured with the luciferase-linked ATPase assay, whereas conventional assays of purified pump activity at 3.0 mm ATP fail to reveal immunochemical blockade.