Self-assembly and Self-replication of Short Amphiphilic β-sheet Peptides

Most self-replicating peptide systems are made of α-helix forming sequences. However, it has been postulated that shorter and simpler peptides may also serve as templates for replication when arranged into well-defined structures. We describe here the design and characterization of new peptides that form soluble β-sheet aggregates that serve to significantly accelerate their ligation and self-replication. We then discuss the relevance of these phenomena to early molecular evolution, in light of additional functionality associated with β-sheet assemblies.     

The Ras Antagonist,Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy^2J/dy^2J mouse model of merosin deficient congenital muscular dystrophy. The dy^2J/dy^2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy^2J/dy^2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy^2J/dy^2J mouse model of congenital muscular dystrophy.     

Cardinium in Plagiomerus diaspidis (Hymenoptera: Encyrtidae)

The bacterial symbiont Cardinium (Bacteroidetes) was previously implicated in the thelytokous reproduction of the parasitoid Plagiomerus diaspidis Crawford (Hymenoptera: Encyrtidae). Horizontal transmission of the symbiont among the cactus scale Diaspis echinocacti Bouché (Homoptera: Diaspididae) and its hymenopteran parasitoids has been suggested. In this study, the bacteria associated with D. echinocacti, its parasitoids P. diaspidis and Aphytis sp. (Hymenoptera: Aphelinidae), and the hyperparasitoid Marietta leopardina Motschulsky (Hymenoptera: Aphelinidae) were characterized using molecular fingerprinting techniques, and the localization of Cardinium in P. diaspidis was studied using fluorescence in situ hybridizations (FISH). Cardinium was the only bacterium found in P. diaspidis, but it could not be detected in any of the other insects tested. The symbiont was specifically located in the reproductive tissues of its P. diaspidis host.     

A conservation and rigidity based method for detecting critical protein residues

Background

Certain amino acids in proteins play a critical role in determining their structural stability and function. Examples include flexible regions such as hinges which allow domain motion, and highly conserved residues on functional interfaces which allow interactions with other proteins. Detecting these regions can aid in the analysis and simulation of protein rigidity and conformational changes, and helps characterizing protein binding and docking. We present an analysis of critical residues in proteins using a combination of two complementary techniques. One method performs in-silico mutations and analyzes the protein's rigidity to infer the role of a point substitution to Glycine or Alanine. The other method uses evolutionary conservation to find functional interfaces in proteins.

Results

We applied the two methods to a dataset of proteins, including biomolecules with experimentally known critical residues as determined by the free energy of unfolding. Our results show that the combination of the two methods can detect the vast majority of critical residues in tested proteins.

Conclusions

Our results show that the combination of the two methods has the potential to detect more information than each method separately. Future work will provide a confidence level for the criticalness of a residue to improve the accuracy of our method and eliminate false positives. Once the combined methods are integrated into one scoring function, it can be applied to other domains such as estimating functional interfaces.

     

Phytochrome involvement in stomatal movement in Pisum sativum, Vicia faba and Pelargonium sp.

Red and blue light triggered the opening of isolated stomata of Pisum sativum L. cv. Peleg Alvador, Vicia faba L. (unknown cultivar) and Pelargonium sp. The stimulatory effect of short irradiation with red or blue light was reversed by a subsequent short irradiation with far-red light. In Pisum the stimulatory effect of a continuous irradiation with red or blue light was also abolished by a concomitant far-red light. In leaf pieces of P. sativum blue light was more effective than red, but not in isolated guard cells. In the presence of mesophyll, DCMU inhibited stomatal opening in red light more than in blue, and thus increased the relative response to blue light. This was less evident in isolated guard cells.     

The Ras antagonist, farnesylthiosalicylic acid (FTS), decreases fibrosis and improves muscle strength in dy/dy mouse model of muscular dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy(2J)/dy(2J) mouse model of merosin deficient congenital muscular dystrophy. The dy(2J)/dy(2J) mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy(2J)/dy(2J) mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy(2J)/dy(2J) mouse model of congenital muscular dystrophy.     

A stress-responsive system for mitochondrial protein degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.     

Cytosolic phospholipase A2alpha is targeted to the p47phox-PX domain of the assembled NADPH oxidase via a novel binding site in its C2 domain

We have previously demonstrated a physical interaction between cytosolic phospholipase A2alpha (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, F?rster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain.