Gain of function in an ERV/ALR sulfhydryl oxidase by molecular engineering of the shuttle disulfide

The ERV/ALR sulfhydryl oxidase domain is a versatile module adapted for catalysis of disulfide bond formation in various organelles and biological settings. Its four-helix bundle structure juxtaposes a Cys-X-X-Cys dithiol/disulfide motif with a bound flavin adenine dinucleotide (FAD) cofactor, enabling transfer of electrons from thiol substrates to non-thiol electron acceptors. ERV/ALR family members contain an additional di-cysteine motif outside the four-helix-bundle core. Although the location and context of this "shuttle" disulfide differs among family members, it is proposed to perform the same basic function of mediating electron transfer from substrate to the enzyme active site. We have determined by X-ray crystallography the structure of AtErv1, an ERV/ALR enzyme that contains a Cys-X4-Cys shuttle disulfide and oxidizes thioredoxin in vitro, and compared it to ScErv2, which has a Cys-X-Cys shuttle and does not oxidize thioredoxin at an appreciable rate. The AtErv1 shuttle disulfide is in a region of the structure that is disordered and thus apparently mobile and exposed. This feature may facilitate access of protein substrates to the shuttle disulfide. To test whether the shuttle disulfide region is modular and can confer on other enzymes oxidase activity toward new substrates, we generated chimeric enzyme variants combining shuttle disulfide and core elements from AtErv1 and ScErv2 and monitored oxidation of thioredoxin by the chimeras. We found that the AtErv1 shuttle disulfide region could indeed confer thioredoxin oxidase activity on the ScErv2 core. Remarkably, various chimeras containing the ScErv2 Cys-X-Cys shuttle disulfide were found to function efficiently as well. Since neither the ScErv2 core nor the Cys-X-Cys motif is therefore incapable of participating in oxidation of thioredoxin, we conclude that wild-type ScErv2 has evolved to repress activity on substrates of this type, perhaps in favor of a different, as yet unknown, substrate.     

Mild caloric restriction up-regulates the expression of prohibitin: a proteome study

The eukaryotic initiation factor 4E (eIF4E) serves as a master switch that controls mRNA translation through the promotive binding to eIF4G and the regulative binding with the endogenous inhibitor 4E-BP. Although the bindings of eIF4G and 4E-BP to eIF4E proceed through the common eIF4E recognition Y(X)₄Lφ motif (X: variable, φ: hydrophobic) (first binding site), the relationship between their eIF4E binding mode and the functional difference is hardly known. Recently, we have clarified the existence and function of the second eIF4E binding site in 4E-BP. Surface plasmon resonance (SPR) analysis based on the sequential comparison between 4E-BP and eIF4GI clarified that eIF4G has the second binding site at the periphery of the ⁵⁹⁷SDVVL⁶⁰¹ sequence and that it plays an auxiliary but indispensable function in stabilizing the binding of the first binding sequence ⁵⁷²YDREFLL⁵⁷⁸. The kinetic parameters of the interactions of the eIF4GI and 4E-BP2 fragment peptides with eIF4E showed that the association (ka) and dissociation (kd) rates of the former peptide are about three and two orders of magnitude lower than those of the latter peptide, respectively. This means that eIF4G has a potent resistive property for release from eIF4E, although its rate of binding to eIF4E is not as high as that of 4E-BP, that is, 4E-BP is apt to bind to and be released from eIF4E, as compared with eIF4G. Isothermal titration calorimetry (ITC) showed the opposite behavior between the second binding sites of eIF4GI and 4E-BP for the interaction with eIF4E. This clearly indicates the importance of the second binding region for the difference in function between eIF4G and 4E-BP for eIF4E translation.     

Zeolites are effective ROS-scavengers in vitro

We report on the use of zeolites to limit the effects of reactive oxygen species (ROS) on human albumin under in vitro conditions. Zeolites of different structure type, channel size, channel polarity, and charge-compensating cation were screened for the elimination of ROS, notably HO, resulting from the Fenton reaction. A test based on ischemia-modified albumin (IMA) was used as a marker to monitor the activity of HO after co-exposure of human serum to these zeolites. Two commercial zeolites, faujasite (FAU 13×, channel opening 0.74 × 0.74 nm with Na⁺ as charge-compensating cation) and ferrierite (FER, channel opening 0.54 × 0.42 nm with H⁺ as charge-compensating cation), were found to reduce IMA formation by more than 65% due to removal of HO relative to reference values. It was established that partial ion exchange of the zeolites’ respective charge-compensating cation vs. Fe³⁺ implicated in the Fenton reaction plays a major role in HO deactivation process. Moreover, our results show that no saturation of the respective zeolite active sites occurred. This is possible only when ROS are actively converted to water molecules within the zeolite void system, which generates H⁺ ion transport.Because zeolites cannot be administered in blood, their use in medicine should be limited to extra corporeal circuits. Zeolites could be of use during cardiopulmonary bypass or hemodialysis procedures.     

Interactions of the DNA polymerase X from African Swine Fever Virus with the ssDNA. Properties of the total DNA-binding site and the strong DNA-binding subsite

Interactions of the polymerase X from the African Swine Fever Virus with the ssDNA have been studied, using quantitative fluorescence titration and fluorescence resonance energy transfer techniques. The primary DNA-binding subsite of the enzyme, independent of the DNA conformation, is located on the C-terminal domain. Association of the bound DNA with the catalytic N-terminal domain finalizes the engagement of the total DNA-binding site of the enzyme and induces a large topological change in the structure of the bound ssDNA. The free energy of binding includes a conformational transition of the protein. Large positive enthalpy changes accompanying the ASFV pol X-ssDNA association indicate that conformational changes of the complex are induced by the engagement of the N-terminal domain. The enthalpy changes are offset by large entropy changes accompanying the DNA binding to the C-terminal domain and the total DNA-binding site, predominantly resulting from the release of water molecules.     

Dark-mediated dormancy release in stratified Lolium rigidum seeds is associated with higher activities of cell wall-modifying enzymes and an apparent increase in gibberellin sensitivity

Dormancy release in freshly matured, imbibed annual ryegrass (Lolium rigidum) seeds is inhibited by light and involves a decrease in seed sensitivity to abscisic acid. Other processes involved in dormancy release in the dark were investigated by measuring seed storage compound mobilisation and the activity of cell wall-degrading enzymes. Activities of endo-β-mannanase and total peroxidase were higher in dark-stratified compared to light-stratified seeds, indicating that weakening of the structures constraining the embryo was accelerated in the dark. A dramatic degradation of storage proteins in light-stratified seeds, accompanied by induction of a high molecular mass protease, suggests that maintenance of storage(-like) proteins is also important in dark-mediated dormancy release. α-Amylase activity was induced in dark-stratified seeds at least 48 h prior to radicle emergence upon transfer to conditions permitting germination, or in light-stratified seeds supplied with exogenous gibberellin A₄. This suggests that (a) α-amylase is involved in stimulation of germination of non-dormant L. rigidum seeds, and (b) dark-stratified seeds have an increased sensitivity to gibberellins which permits the rapid induction of α-amylase activity upon exposure to germination conditions. Overall, it appears that a number of processes, although possibly minor in themselves, occur in concert during dark-stratification to contribute to dormancy release.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

Enzymatic activity of Lecithin:retinol acyltransferase: A thermostable and highly active enzyme with a likely mode of interfacial activation

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions. A much larger tLRAT activity has been obtained compared to previous reports and to an enzyme with a similar activity. In addition, tLRAT is able to hydrolyze phospholipids bearing different chain lengths with a preference for micellar aggregated substrates. It therefore presents an interfacial activation property, which is typical of classical phospholipases. Furthermore, given that stability is a very important quality of an enzyme, the influence of different parameters on the activity and stability of tLRAT has thus been studied in detail. For example, storage buffer has a strong effect on tLRAT activity and high enzyme stability has been observed at room temperature. The thermostability of tLRAT has also been investigated using circular dichroism and infrared spectroscopy. A decrease in the activity of tLRAT was observed beyond 70 °C, accompanied by a modification of its secondary structure, i.e. a decrease of its α-helical content and the appearance of unordered structures and aggregated β-sheets. Nevertheless, residual activity could still be observed after heating tLRAT up to 100 °C. The results of this study highly improved our understanding of this enzyme.     

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.