A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 °C and micro-injected with Seleno-L-Methionine to reach 8 µg g⁻¹ selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P < 0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65 kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.     

Macrocyclization of Organo‐Peptide Hybrids through a Dual Bio‐orthogonal Ligation: Insights from Structure–Reactivity Studies

Macrocycles constitute an attractive structural class of molecules for targeting biomolecular interfaces with high affinity and specificity. Here, we report systematic studies aimed at exploring the scope and mechanism of a novel chemo‐biosynthetic strategy for generating macrocyclic organo‐peptide hybrids (MOrPHs) through a dual oxime‐/intein‐mediated ligation reaction between a recombinant precursor protein and bifunctional, oxyamino/1,3‐amino‐thiol compounds. An efficient synthetic route was developed to access structurally different synthetic precursors incorporating a 2‐amino‐ mercaptomethyl‐aryl (AMA) moiety previously found to be important for macrocyclization. With these compounds, the impact of the synthetic precursor scaffold and of designed mutations within the genetically encoded precursor peptide sequence on macrocyclization efficiency was investigated. Importantly, the desired MOrPHs were obtained as the only product from all the different synthetic precursors probed in this study and across peptide sequences comprising four to 15 amino acids. Systematic mutagenesis of the “i?1” site at the junction between the target peptide sequence and the intein moiety revealed that the majority of the 20 amino acids are compatible with MOrPH formation; this enables the identification of the most and the least favorable residues for this critical position. Furthermore, interesting trends with respect to the positional effect of conformationally constrained (Pro) and flexible (Gly) residues on the reactivity of randomized hexamer peptide sequences were observed. Finally, mechanistic investigations enabled the relative contributions of the two distinct pathways (side‐chain→C‐end ligation versus C‐end→side‐chain ligation) to the macrocyclization process to be dissected. Altogether, these studies demonstrate the versatility and robustness of the methodology to enable the synthesis and diversification of a new class of organo‐peptide macrocycles and provide valuable structure–reactivity insights to inform the construction of macrocycle libraries through this chemo‐biosynthetic strategy.     

Formation versus Hydrolysis of the Peptide Bond from a Quantum-mechanical Viewpoint: The Role of Mineral Surfaces and Implications for the Origin of Life

The condensation (polymerization by water elimination) of molecular building blocks to yield the first active biopolymers (e.g. of amino acids to form peptides) during primitive Earth is an intriguing question that nowadays still remains open since these processes are thermodynamically disfavoured in highly dilute water solutions. In the present contribution, formation and hydrolysis of glycine oligopeptides occurring on a cluster model of sanidine feldspar (001) surface have been simulated by quantum mechanical methods. Results indicate that the catalytic interplay between Lewis and Brønsted sites both present at the sanidine surface, in cooperation with the London forces acting between the biomolecules and the inorganic surface, plays a crucial role to: i) favour the condensation of glycine to yield oligopeptides as reaction products; ii) inhibit the hydrolysis of the newly formed oligopeptides. Both facts suggest that mineral surfaces may have helped in catalyzing, stabilizing and protecting from hydration the oligopeptides formed in the prebiotic era.     

Effects of trypsin‐hydrolyzed wheat gluten peptide on wheat flour dough

BACKGROUND: Gluten peptide was prepared by trypsin hydrolysis and characterized by high‐performance liquid chromatographic analysis. The effects on non‐frozen and frozen doughs of trypsin‐hydrolyzed gluten peptide (THGP) and its combination with ascorbic acid or KBrO₃ were investigated. RESULTS: Molecular analysis of THGP showed a decrease in the high‐molecular‐weight and an increase in the low‐molecular‐weight sodium dodecyl sulfate‐soluble fractions, compared with those of control wheat gluten. The addition of 8% THGP decreased the mixing time and tolerance of the dough, both with and without ascorbic acid or KBrO₃. However, the maximum resistance and extensibility of the rested dough containing 8% THGP, with and without ascorbic acid or KBrO₃, were not significantly different from those of the control dough. The addition of 8% THGP significantly increased the loaf volume of bread baked from non‐frozen dough when combined with 60 ppm ascorbic acid or 30 ppm KBrO₃, but it had a significant effect both with and without ascorbic acid or KBrO₃ on frozen‐dough bread. A large difference in volume was observed between breads made with and without THGP at the oven‐spring, rather than at proofing. CONCLUSION: The addition of 8% THGP increased the loaf volume of bread made from freeze‐damaged dough and this effect increased when THGP was combined with 60 ppm ascorbic acid. Copyright © 2008 Society of Chemical Industry     

Fimbriae of Bacteroides nodosus: protein engineering of the structural subunit for the production of an exogenous peptide

The pattern of sequence variation between Bacteroides nodosusfimbrial subuits of different serotypes suggests a degree offlexibility, which might be exploited for protein engineeringapproaches for the expression of other peptides. We have testedthis using the well-characterized peptide epitope from VP1 offoot-and-mouth disease virus (FMDV), residues 144–159:LRGDLQVLAQKVARTL (strain 01-BFS). Using bacterial codon usage,several oligonucleotides were designed for the substitutionof this sequence internally at hypervariable regions of thefimbrial subunit (aligned for maximum homology), and for itsaddition at the carboxy-terminus with a diglycine spacer asa flexible hinge. Following site-directed mutagenesis in phageM13, the modified genes were placed under P_L promoter controland placed in a broad host range vector. Analysis of the variantproteins expressed in E.coli showed that these substitutionsaffected, to varying extents, recognition by both anti-fimbrialand anti-FMDV antibodies, indicating that hypervariable region2 is a major antigenic determinant of the fimbrial subunit andthat local stereochemical effects can influence antibody bindingto the FMDV peptide antigenic determinant. In Pseudomonas aeruginosa,viable transformants could only be obtained with the mutantgene encoding the carboxy-terminal graft. These cells providedfimbrial preparations comprised of the modified subunit. Thisthen constitutes the prototype for the development of a generalexpression system for the production of vaccine epitopes andother bioactive peptides. Furthermore, there is considerablescope for further modification of the system, for example byengineering specific chemical or protease cleavage sites forrelease of the grafted peptide. Alternatively, the fimbriaethemselves may serve as a useful supramoleuclar carrier or adjuvantfor immune provocation.