Temporary inhibition of papain by hairpin loop mutants of chicken cystatin Distorted binding of the loops results in cleavage of the Gly9-Ala10 bond

AbstractTemporary inhibition of the cysteine proteinases papain and cathepsin L was observed with several hairpin loop mutants of recombinant chicken cystatin at enzyme concentrations above nanomolar. Kinetic modelling of inhibition data, gel electrophoresis and amino acid sequencing revealed that reappearance of papain activity is due to selective cleavage of the Gly9-Ala10 bond in the N-terminal binding area of the chicken cystatin variants, resulting in truncated inhibitors of lower affinity. Cleavage of the same bond by contaminating papaya proteinase IV was ruled out by previous purification of papain and suitable control experiments. According to the proposed kinetic model, cleavage occurs within the enzyme-inhibitor complex with first order rate constants ktemp of 2.3 × 10−3 up to 5 × 10−1 s−1. A similar ktempKm ratio was found for all mutants (0.7 × 106–2.1 × 106 s−1·M−1); it is almost identical with the kcatKm ratio of the peptide substrate Z-Phe-Arg-NHMec. These results suggest that distorted contacts of one of the hairpin loops affect binding of the N-terminal contact area in a way that covalent interaction of the Gly9-Ala10 bond with the active-site Cys residue of papain can occur and the bond is cleaved in a substrate-like manner

Constraints on Non-Newtonian Gravity from Recent Casimir Force Measurements

Corrections to Newton's gravitational law inspired by extra dimensional physics and by the exchange of light and massless elementary particles between the atoms of two macrobodies are considered. These corrections can be described by the potentials of Yukawa-type and by the power-type potentials with different powers. The strongest up to date constraints on the corrections to Newton's gravitational law are reviewed following from the E\"{o}tvos- and Cavendish-type experiments and from the measurements of the Casimir and van der Waals force. We show that the recent measurements of the Casimir force gave the possibility to strengthen the previously known constraints on the constants of hypothetical interactions up to several thousand times in a wide interaction range. Further strengthening is expected in near future that makes Casimir force measurements a prospective test for the predictions of fundamental physical theories.Comment: 20 pages, crckbked.cls is used, to be published in: Proceedings of the 18th Course of the School on Cosmology and Gravitation: The Gravitational Constant. Generalized Gravitational Theories and Experiments (30 April- 10 May 2003, Erice). Ed. by G. T. Gillies, V. N. Melnikov and V. de Sabbata, 20pp. (Kluwer, in print, 2003

The associations between sedentary behaviour and mental health among adolescents:A systematic review

Background: With technological developments and modernised sedentary lifestyles has come an increase in diseases associated with inactivity such as obesity and other non-communicable diseases. Emerging evidence suggests that time spent sedentary may also interact with mental health. This systematic review examined the associations between sedentary behaviour and mental health problems among adolescents. Methods: This systematic review followed Preferred Reporting Items for Systematic Reviews and Meta-Analyses, and applied a quality assessment tool for quantitative studies to identity best available evidence. Following stringent search strategy of the databases; Cumulative Index to Nursing and Allied Health Literature, Global Health, Health Source: Nursing and Academic Edition, MEDLINE, PsychARTICLES and PsycINFO, we identified 32 articles eligible for review. Results: All studies reported leisure screen time among adolescents, and two thirds of identified studies examined depressive symptomatology. Other mental health measures were; anxiety symptoms, self-esteem, suicide ideation, loneliness, stress, and psychological distress. Strong consistent evidence was found for the relationship between both depressive symptomatology and psychological distress, and time spent using screens for leisure. Moderate evidence supported the relationship between low self-esteem and screen use. Poorer mental health status was found among adolescents using screen time more than 2-3 h per day, and gender differences exist. Essential information was missing for quality of evidence including heterogeneity in mental health and screen time-based measures, and self-report data collection methods. Conclusions: The findings are of particular significance given the global public health concern of lifestyle-attributed diseases and the possibility for novel approaches to mental health. Future research should examine the psychological impact of reducing time spent using screens for leisure among adolescents, whilst accounting for possible confounding factors such as physical activity and dietary behaviours. It is critical that the reciprocal relationship between lifestyle behaviours and mental health is represented in both the psychiatric and public health forum

Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster

Crystallization of the cystine-knot protein Spätzle occurred following serendipitous limited degradation of the pro-Spätzle propeptide during the crystallization experiment

New insights into the membrane-binding and activation mechanism of pyruvate oxidase from Escherichia coli

Pyruvate oxidase from Escherichia coli (EcPOX) is a thiamin diphosphate- (ThDP) and FAD-dependent peripheral membrane protein that carries out the irreversible oxidative decarboxylation of pyruvate to acetate and carbon dioxide. Concomitant two-electron reduction of the flavin cofactor was suggested to induce a Structural rearrangement of the C-terminus triggering recruitment of the protein from the cytosol to the cell membrane, where the electrons are eventually transferred to final electron acceptor ubiquinone 8. Binding to the membrane, or alternatively, mild proteolytic digestion leads to a multifold enhancement of both the catalytic activity and substrate affinity. Recent X-ray crystallographic studies on EcPOX in the resting state and on a C-terminal truncation variant mimicking the membrane-bound activated form have fueled our understanding of the membrane-binding mechanism and concomitant catalytic activation. In the resting state, the auto-inhibitory C-terminal membrane anchor adopts a half-barrel/helix fold that occludes the active site. Upon activation, the C-terminus is expelled and becomes structurally flexible thereby freeing the active site. Circular dichroism spectroscopic analysis revealed the isolated C-terminus to be disordered, however, formation of a helical structure was observed in the presence of micelles. Limited proteolysis experiments indicate that activation of EcPOX involves at least two sequential structural transitions: the first occurring after binding of pyruvate to ThDP and the second after two-electron reduction of the flavin. (C) 2009 Elsevier B.V. All rights reserved

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

The thiamin- and flavin-dependent peripheral membrane enzyme pyruvate oxidase from E. coli catalyzes the oxidative decarboxylation of the central metabolite pyruvate to CO2 and acetate. Concomitant reduction of the enzyme-bound flavin triggers membrane binding of the C terminus and shuttling of 2 electrons to ubiquinone 8, a membrane-bound mobile carrier of the electron transport chain. Binding to the membrane in vivo or limited proteolysis in vitro stimulate the catalytic proficiency by 2 orders of magnitude. The molecular mechanisms by which membrane binding and activation are governed have remained enigmatic. Here, we present the X-ray crystal structures of the full-length enzyme and a proteolytically activated truncation variant lacking the last 23 C-terminal residues inferred as important in membrane binding. In conjunction with spectroscopic results, the structural data pinpoint a conformational rearrangement upon activation that exposes the autoinhibitory C terminus, thereby freeing the active site. In the activated enzyme, Phe-465 swings into the active site and wires both cofactors for efficient electron transfer. The isolated C terminus, which has no intrinsic helix propensity, folds into a helical structure in the presence of micelles

Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases

The synthesis of the catecholic siderophore bacillibactin is accomplished by the nonribosomal peptide synthetase (NRPS) encoded by the dhb operon. DhbE is responsible for the initial step in bacillibactin synthesis, the activation of the aryl acid 2,3-dihydroxybenzoate (DHB). The stand-alone adenylation (A) domain DhbE, the structure of which is presented here, exhibits greatest homology to other NRPS A-domains, acyl-CoA ligases and luciferases. It's structure is solved in three different states, without the ligands ATP and DHB (native state), with the product DHB-AMP (adenylate state) and with the hydrolyzed product AMP and DHB (hydrolyzed state). The 59.9-kDa protein folds into two domains, with the active site at the interface between them. In contrast to previous proposals of a major reorientation of the large and small domains on substrate binding, we observe only local structural rearrangements. The structure of the phosphate binding loop could be determined, a motif common to many adenylate-forming enzymes, as well as with bound DHB-adenylate and the hydrolyzed product DHB*AMP. Based on the structure and amino acid sequence alignments, an adapted specificity conferring code for aryl acid activating domains is proposed, allowing assignment of substrate specificity to gene products of previously unknown function

Crystallization and preliminary X-ray diffraction analysis of full-length and proteolytically activated pyruvate oxidase from Escherichia coli

The peripheral membrane flavoprotein pyruvate oxidase from E. coli has been crystallized in the full-length form and as a proteolytically activated truncation variant lacking the last 23 amino acids at the C-terminus

Mutagenesis and crystallographic studies of Zymomonas mobilis tRNA-guanine transglycosylase to elucidate the role of serine 103 for enzymatic activity

AbstractThe tRNA modifying enzyme tRNA-guanine transglycosylase (TGT) is involved in the exchange of guanine in the first position of the anticodon with preQ1 as part of the biosynthesis of the hypermodified base queuine (Q). Mutation of Ser90 to an alanine in Escherichia coli TGT leads to a dramatic reduction of enzymatic activity (Reuter, K. et al. (1994) Biochemistry 33, 7041–7046). To further clarify the role of this residue in the catalytic center, we have mutated the corresponding Ser103 of the crystallizable Zymomonas mobilis TGT into alanine. The crystal structure of a TGT(S103A)/preQ1 complex combined with biochemical data presented in this paper suggest that Ser103 is essential for substrate orientation in the TGT reaction