Description: Available online 29 December 2012 Publication year: 2012 Source: FEBS Open Bio A water-soluble selenoxide (DHS ox ) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS ox would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS ox was extensively studied at pH 4–10 and 25°C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS ox (375 mV at 25°C) compared to L-cystine supported the high ability of DHS ox for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the p K a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT red ) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains. Graphical abstract Highlights ▸ DHS ox was applied as a SS-forming reagent for five polythiol peptides and DTT red . ▸ The SS formation velocities depend on the kind of substrate and solvent conditions. ▸ The SS-formation rate constants are proportional to the number of free SH groups. ▸ Folded structures and SH p K a modify the stochastic nature and absolute values of the rate constants. ▸ DHS ox is a useful probe of chemical reactivity and intermediate structures in oxidative protein folding.

Description: Available online 26 December 2012 Publication year: 2012 Source: FEBS Open Bio Human Angiogenin (ANG) is an angiogenic molecule and a ribonucleolytic enzyme with significant amino acid sequence identity to pancreatic RNase A, plays a critical role in the establishment and growth of tumours. An association between ANG and cancer has been observed in more than 25 clinical studies to date. In addition, ANG has now been shown to be implicated in Amyotrophic Lateral Sclerosis and Parkinson's Disease. Structural and biochemical studies so far have showed several distinguishing features of ANG molecule compared to RNase A and provided details of the putative cell binding site, active site, nuclear translocation sequence and the roles of residues in binding and cleaving RNA. A key finding elucidated from the structural study on ANG is the presence of a ‘blocked’ C-terminus (part of the active site apparatus) compared with RNase A. Here we report the crystal structure of ANG with an ‘engineered-loop’ from eosinophil derived neurotoxin (a homologue of ANG) which has resulted with local perturbations (conformational flexibility) at the cell binding site and at the C-terminus of the molecule. This experimental observation will now provide a new avenue to design compounds (potent inhibitors) through a structure guided drug design route. Graphical abstract Highlights ▸ A hybrid form (AEH) of human angiogenin (ANG) was created. ▸ The RI binding loop of ANG was substituted with the RI binding loop of EDN. ▸ Significant conformational changes were observed in the structure of AEH. ▸ Changes include the C-terminal segment and a putative cell binding domain of ANG. ▸ Binding of a chloride ion at the active site was observed

Description: Available online 22 December 2012 Publication year: 2012 Source: FEBS Open Bio As a member of peroxiredoxin (Prx) family, PrxIII is predominantly located in mitochondria and plays an important role as a scavenger of reactive oxygen species (ROS). Since previous reports demonstrated over-expression of PrxIII in cervical cancers, we conducted the present study to investigate the significance of PrxIII in cervical cancer development and/or progression. Cervical cancer cells were cultured from tissues derived from cervical cancer patients. After successful knockdown of PrxIII expression by small interfering RNA, we evaluated ROS level, viable cell number, and apoptosis of cervical cancer cells along with the culture time. The production of ROS was increased in cervical cancer cells as compared with normal cervical epithelia. Knockdown of PrxIII expression induced up-regulation of other Prx members including PrxI, PrxII, and PrxV. ROS level was higher in down-regulated cervical cancer cells than in controls and the difference was increasing with culture time. We also observed increased apoptosis and decreased viable cell number in down-regulated cervical cancer cells. Our results suggest that PrxIII is an indispensable ROS scavenger, which protects tumor cells against oxidative damage and subsequent apoptosis.

Description: Available online 19 December 2012 Publication year: 2012 Source: FEBS Open Bio The green fluorescent protein (GFP) is the most commonly used reporter protein for monitoring gene expression and protein localization in a variety of living and fixed cells, including not only prokaryotes, but also eukaryotes, e.g., yeasts, mammals, plants and fish. In general, it is thought that GFP is nontoxic to cells, although there are some reports on the side effect of GFP. However, details of the molecular mechanism concerning the side effect of GFP remain unclear. Here we show that Ku80, but not XRCC4, plays an important role in the mechanism of the resistance to cytotoxicity induced by enhanced GFP (EGFP). EGFP inhibited both cell proliferation and colony formation, and induced cell death in Ku80-deficient hamster cells, i.e., xrs-6 cells. In addition, Ku80 attenuated EGFP-induced cytotoxicity in the xrs-6 cells. No EGFP-induced cytotoxicity was observed in the NHEJ core protein XRCC4-deficient hamster cells, i.e., XR-1 cells. Furthermore, EGFP markedly enhanced X-ray-induced cytotoxicity in the xrs-6 cells. These results suggest that Ku80 plays a key role in the novel NHEJ-independent defense mechanism against EGFP-induced cytotoxicity. Caution should be taken in considering of the potential influence by the stress response mechanism, namely, the Ku80-dependent elimination mechanism of EGFP-induced cytotoxicity, being activated, even when using EGFP-expressing cells in which Ku80 functions normally.

Description: 2013 Publication year: 2013 Source: FEBS Open Bio, Volume 3 Some ligand–receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A 2A receptors (A 2A R), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A 2A R remains to be established. We examined the situation using patients over-expressing lymphocyte A 2A R and an agonist-like mAb to A 2A R. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 μM, respectively). Lymphocyte A 2A R expression was correlated to plasma adenosine level and A 2A R occupation but not to A 2A R response. These results are consistent with a lack of a reserve of functional A 2A R on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A 2A R determine the maximal level of the lymphocyte response to adenosine.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Adaptor proteins play an important role in signaling pathways by providing a platform on which many other proteins can interact. Malfunction or mislocalization of these proteins may play a role in the development of disease. Lipoma preferred partner (LPP) is a nucleocytoplasmic shuttling adaptor protein. Previous work shows that LPP plays a role in the function of smooth muscle cells and in atherosclerosis. In this study we wanted to determine whether LPP has a role in the myocardium. LPP expression increased by 56% in hearts from pressure overload aortic-banded rats ( p 〈 0.05 n = 4), but not after myocardial infarction, suggesting hemodynamic load regulates its expression. In vitro, LPP expression was 87% higher in cardiac fibroblasts than myocytes ( p 〈 0.05 n = 3). LPP expression was downregulated in the absence of the actin cytoskeleton but not when microtubules were disassembled. We mechanically stretched cardiac fibroblasts using the Flexcell 4000 for 48 h (1 Hz, 5% maximum strain), which decreased total LPP total expression and membrane localization in subcellular fractions ( p 〈 0.05, n = 5). However, L-NAME, an inhibitor of nitric oxide synthase (NOS), significantly upregulated LPP expression. These findings suggest that LPP is regulated by a complex interplay between NO and mechanical cues and may play a role in heart failure induced by increased hemodynamic load. Highlights ► Lipoma preferred partner (LPP) is highly expressed in the heart, especially in cardiac fibroblasts. ► LPP increases in pressure overload hypertrophy but not in myocardial infarction. ► LPP may be used as a novel marker for heart failure due to hemodynamic overload. ► Inhibition of nitric oxide upregulates LPP, but not other fibroblast proteins. ► LPP may play an important role in cardiac homeostasis and heart disease.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 For successful mitochondrial transgene expression, an optimal packaging exogenous DNA is an important issue. We report herein on the effects of DNA packaged with mitochondrial transcription factor A (TFAM), which packages mitochondrial DNA (mtDNA), on the transcription process. Our initial findings indicated that the transcription of the TFAM/DNA complex was activated, when the complex was formed at an optimal ratio. We also found that TFAM has a significant advantage over protamine, a nuclear DNA packaging protein, from the viewpoint of transcription efficiency. This result indicates that TFAM can be useful packaging protein for exogenous DNA to achieve mitochondrial transgene expression. Graphical abstract Highlights ► The base-pair interval at TFAM binds to DNA is critical for transcription. ► TFAM/DNA complex was transcriptionally active only when DNA was optimally packaged. ► The transcription inhibition appears to result from the overpackaging of DNA by TFAM. ► The manner in which DNA is packaged with TFAM has an impact on transcription.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 The Arabidopsis thaliana genome contains a small group of bipartite F-box proteins, consisting of an N-terminal F-box domain and a C-terminal domain sharing sequence similarity with Nictaba, the jasmonate-induced glycan-binding protein (lectin) from tobacco. Based on the high sequence similarity between the C-terminal domain of these proteins and Nictaba, the hypothesis was put forward that the so-called F-box-Nictaba proteins possess carbohydrate-binding activity and accordingly can be considered functional homologs of the mammalian sugar-binding F-box or Fbs proteins which are involved in proteasomal degradation of glycoproteins. To obtain experimental evidence for the carbohydrate-binding activity and specificity of the A. thaliana F-box-Nictaba proteins, both the complete F-box-Nictaba sequence of one selected Arabidopsis F-box protein (in casu At2g02360) as well as the Nictaba-like domain only were expressed in Pichia pastoris and analyzed by affinity chromatography, agglutination assays and glycan micro-array binding assays. These results demonstrated that the C-terminal Nictaba-like domain provides the F-box-protein with a carbohydrate-binding activity that is specifically directed against N - and O -glycans containing N -acetyllactosamine (Galβ1-3GlcNAc and Galβ1-4GlcNAc) and poly- N- acetyllactosamine ([Galβ1-4GlcNAc] n ) as well as Lewis A (Galβ1-3(Fucα1-4)GlcNAc), Lewis X (Galβ1-4(Fucα1-3)GlcNAc, Lewis Y (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc) and blood type B (Galα1-3(Fucα1-2)Galβ1-3GlcNAc) motifs. Based on these findings one can reasonably conclude that at least the A. thaliana F-box-Nictaba protein encoded by At2g02360 can act as a carbohydrate-binding protein. The results from the glycan array assays revealed differences in sugar-binding specificity between the F-box protein and Nictaba, indicating that the same carbohydrate-binding motif can accommodate unrelated oligosaccharides. Highlights ► Recombinant expression of a new type of Arabidopsis F-box protein. ► Arabidopsis F-box-Nictaba protein is a functional lectin. ► Arabidopsis F-box-Nictaba recognizes N-acetyllactosamine and Lewis structures.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P) + -dependent decarboxylation of isocitrate into α-ketoglutarate and CO 2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH ( Ct IDH), a native open apo Ct IDH to 2.35 Å and a quaternary complex of Ct IDH with NADP + , isocitrate and Mg 2+ to 2.5 Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH ( Dp IDH) was also resolved to 1.93 Å. Ct IDH and Dp IDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9 °C, respectively. Ct IDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. Ct IDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to Dp IDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in Dp IDH, were absent in Ct IDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251′ and Arg255 ( Ct IDH). These interactions lock the large domain to the small domain and direct NADP + into the correct orientation, which together are important for NADP + selectivity. Graphical abstract Highlights New insights into the locking mechanism with open, closing, closed and locked structures of two isocitrate dehydrogenases. ► Focus is on a thermophilic ( Ct IDH) and cold active ( Dp IDH) isocitrate dehydrogenase. ► Biochemical characterization and three new IDH crystal structures are presented. ► Ct IDH has many ionic interactions and hydrogen bonds, which explain the T m of 68 °C. ► Prokaryotic IDH subfamily II seems to have a unique locking mechanism. ► The large domain is locked to the small domain and NADP + is also directed correctly.

Description: 2012 Publication year: 2012 Source: FEBS Open Bio, Volume 2 GTPases are important regulatory proteins that hydrolyze GTP to GDP. A novel GTP-hydrolysis mechanism is employed by MnmE, YqeH and FeoB, where a potassium ion plays a role analogous to the Arginine finger of the Ras-RasGAP system, to accelerate otherwise slow GTP hydrolysis rates. In these proteins, two conserved asparagines and a ‘K-loop’ present in switch-I, were suggested as attributes of GTPases employing a K + -mediated mechanism. Based on their conservation, a similar mechanism was suggested for TEES family GTPases. Recently, in Dynamin, Fzo1 and RbgA, which also conserve these attributes, a similar mechanism was shown to be operative. Here, we probe K + -activated GTP hydrolysis in TEES (TrmE-Era-EngA-YihA-Septin) GTPases – Era, EngB and the two contiguous G-domains, GD1 and GD2 of YphC (EngA homologue) – and also in HflX, another GTPase that also conserves the same attributes. While GD1-YphC and Era exhibit a K + -mediated activation of GTP hydrolysis, surprisingly GD2-YphC, EngB and HflX do not. Therefore, the attributes identified thus far, do not necessarily predict a K + -mechanism in GTPases and hence warrant extensive structural investigations. Highlights ▸ An emerging alternative mechanism of GTP hydrolysis is mediated by K + ions. ▸ Features characteristic of the K + -mediated mechanism were suggested. ▸ As TEES family GTPases possess these, they were suggested to employ this mechanism. ▸ Not all GTPases that possess these features utilize the K + -mediated mechanism. ▸ Unambiguously identifying determinants of K + mechanism requires extensive studies.