Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.     

Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1

Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross‐linker in mSOD1‐linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo‐form of the wild‐type SOD1 proteins in a calcium‐dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non‐cell‐autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2‐mediated soluble SOD1 oligomers induced tumor necrosis factor‐α, interleukin‐1β, and nitric oxide in microglial BV2 cells. TG2 was up‐regulated in the spinal cord of pre‐symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer‐mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.

     

To dare or not to dare? Risk management by owls in a predator–prey foraging game

In a foraging game, predators must catch elusive prey while avoiding injury. Predators manage their hunting success with behavioral tools such as habitat selection, time allocation, and perhaps daring—the willingness to risk injury to increase hunting success. A predator’s level of daring should be state dependent: the hungrier it is, the more it should be willing to risk injury to better capture prey. We ask, in a foraging game, will a hungry predator be more willing to risk injury while hunting? We performed an experiment in an outdoor vivarium in which barn owls (Tyto alba) were allowed to hunt Allenby’s gerbils (Gerbillus andersoni allenbyi) from a choice of safe and risky patches. Owls were either well fed or hungry, representing the high and low state, respectively. We quantified the owls’ patch use behavior. We predicted that hungry owls would be more daring and allocate more time to the risky patches. Owls preferred to hunt in the safe patches. This indicates that owls manage risk of injury by avoiding the risky patches. Hungry owls doubled their attacks on gerbils, but directed the added effort mostly toward the safe patch and the safer, open areas in the risky patch. Thus, owls dared by performing a risky action—the attack maneuver—more times, but only in the safest places—the open areas. We conclude that daring can be used to manage risk of injury and owls implement it strategically, in ways we did not foresee, to minimize risk of injury while maximizing hunting success.     

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.     

Clinical chemistry of serotonin and metabolites

Analyses of serotonin and other 5-hydroxyindoles, such as its precursor 5-hydroxytryptophan and major metabolite 5-hydroxyindoleacetic acid (5-HIAA), are indispensable for the elucidation of their (patho)physiological roles. In clinical chemistry attention is mainly focused on the diagnosis and follow-up of carcinoid tumours. For this most laboratories routinely measure urinary 5-HIAA. More recently, measurements of serotonin in platelets and urine have been advocated. Platelet serotonin may be the most sensitive indole marker for the detection of carcinoid tumours that secrete only small amounts of serotonin and/or its precursor 5-hydroxytryptophan. Although several chromatographic techniques have emerged for the analysis of tryptophan-related indoles, HPLC with either electrochemical or fluorometric detection have become the methods of choice for their quantification. HPLC-based methods combine selectivity, sensitivity and high precision, and enable the simultaneous investigation of several metabolically related indoles. This review aims to place the analysis of indoles in biological matrices in a biochemical, physiological and clinical perspective and highlights several important steps in their chromatographic analysis and quantification.     

Structural insights into the role of the WW2 domain on tandem WW–PPxY motif interactions of oxidoreductase WWOX

Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW–peptide interactions is not always intuitive. The WW domain–containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1 domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here, we combined NMR, isothermal calorimetry, and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. Isothermal calorimetry and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases 10-fold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1–WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.     

Restoration of gene function by homologous recombination: from PCR to gene expression in one step

We have developed a simple method for single-step cloning of any PCR product into a plasmid. A novel selection principle has been applied, in which activation of a drug selection marker is achieved following homologous recombination. In this method a DNA fragment is amplified by PCR with standard oligonucleotides that contain flanking tails derived from the host plasmid and the complete lambdaPR or rrnA1 promoter regions. The resulting PCR product is then electroporated into an Escherichia coli strain harboring both the phage lambda Red functions and the host plasmid. Upon homologous recombination of the PCR fragment into the plasmid, expression of a drug selection marker is fully induced due to restoration of its truncated promoter, thus allowing appropriate selection. Recombinant plasmid vectors encoding beta-galactosidase and neomycin phosphotransferase were constructed by using this method in two well-known Red systems. This cloning strategy significantly reduces both the time and costs associated with cloning procedures.     

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na₂SeO₃, Na₂SeO₄, Na₂Se, seleno-dl-cystine, seleno-l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na₂SeO₃ was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na₂SeO₃ was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na₂SeO₃, may be the critical enzyme.     

Use of In Vitro and Predictive In Silico Models to Study the Inhibition of Cytochrome P4503A by Stilbenes

CYP3A4 is recognized as the main enzyme involved in the metabolism of drugs and xenobiotics in the human body and its inhibition may lead to undesirable consequences. Stilbenes, including resveratrol, belong to a group of dietary health-promoting compounds that also act as inhibitors of CYP3A4. The aim of this study was to examine the use of computer modeling of enzyme-ligand interactions to analyze and predict the inhibition of structurally related compounds. To this end, an aldehyde group was attached to resveratrol and the interactions of CYP3A4 with resveratrol, its aldehyde analogue (RA) and a known synthetic inhibitor were studied and compared in two biological models. Specifically, the metabolism of testosterone was examined in a human intestine cell line (Caco-2/TC7) and in rat liver microsomes (RLM). The results demonstrated a weak inhibitory effect of RA on CYP3A4, as compared to resveratrol itself, in both biological models. Human CYP3A4 was more susceptible to inhibition than the commonly used model isozyme from rat. Modeling of the binding site of CYP3A4 revealed a combination of three types of interactions: hydrophobic interactions, electrostatic interactions and hydrogen bonds. A docking simulation revealed that the RA lacked an important binding feature, as compared to resveratrol, and that that difference may be responsible for its lower level of affinity for CYP3A4. Software analysis of binding affinity may serve as a predictive tool for designing new therapeutic compounds in terms of inhibition of CYP3A4 and help to reveal the biochemical nature of the interactions of dietary compounds, herbal compounds and drugs whose metabolism is mediated by this enzyme.     

Purification and physicochemical and immunological analysis of chicken alpha-fetoprotein

Chicken alpha-fetoprotein was isolated from 12 to 13-day-old embryonic chicken serum by column chromatography on CM-Sephadex C-50. Hydroxyapatite and DEAE-Sephadex A-25. The purified protein was homogeneous based on polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The purified protein had the following physicochemical and immunological properties. (1) It was a glycoprotein with a single polypeptide chain. (2) The molecular weight of the protein was estimated at 71,000 by SDS-polyacrylamide gel electrophoresis. (3) The isoelectric point of the protein was 4.90. (4) The amino acid composition of the protein was similar to those of mammalian alpha-fetoproteins. (5) The protein showed no steroid-binding capacity. (6) It was immunologically distinct from mammalian alpha-fetoprotein. (7) No immunological cross-reaction was observed between the protein and chicken albumin.