Triazavirine supramolecular complexes as modifiers of the peptide oligomeric structure

In this study, we present molecular dynamics simulations of the antiviral drug triazavirine, that affects formation of amyloid-like fibrils of the model peptide (SI). According to our simulations, triazavirine is able to form linear supramolecular structures which can act as shields and prevent interactions between SI monomers. This model, as validated by simulations, provides an adequate explanation of triazavirine’s mechanism of action as it pertains to SI peptide fibril formation.     

Priming of microbial microcystin degradation in biomass-fed gravity driven membrane filtration biofilms

Gravity-driven membrane (GDM) filtration is a promising tool for low-cost decentralized drinking water production. The biofilms in GDM systems are able of removing harmful chemical components, particularly toxic cyanobacterial metabolites such as microcystins (MCs). This is relevant for the application of GDM filtration because anthropogenic nutrient input and climate change have led to an increase of toxic cyanobacterial blooms. However, removal of MCs in newly developing GDM biofilms is only established after a prolonged period of time. Since cyanobacterial blooms are transient phenomena, it is important to understand MC removal in mature biofilms with or without prior toxin exposure. In this study, the microbial community composition of GDM biofilms was investigated in systems fed with water from a lake with periodic blooms of MC-producing cyanobacteria. Two out of three experimental treatments were supplemented with dead biomass of a MC-containing cyanobacterial strain, or of a non-toxic mutant, respectively. Analysis of bacterial rRNA genes revealed that both biomass-amended treatments were significantly more similar to each other than to a non-supplemented control. Therefore, it was hypothesized that biofilms could potentially be ‘primed’ for rapid MC removal by prior addition of non-toxic biomass. A subsequent experiment showed that MC removal developed significantly faster in mature biofilms that were pre-fed with biomass from the mutant strain than in unamended controls, indicating that MC degradation was a facultative trait of bacterial populations in GDM biofilms. The significant enrichment of bacteria related to both aerobic and anaerobic MC degraders suggested that this process might have occurred in parallel in different microniches.     

New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order

Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and ¹H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.     

Characteristics of fatty acid composition of <Emphasis Type="Italic">Gammarus lacustris</Emphasis> inhabiting lakes with and without fish

The effect of a biotic factor—the presence of predatory fish in water—on the composition and content of fatty acids in crustaceans was studied in the populations of the lake amphipod Gammarus lacustris from two lakes with fish and three lakes without fish. It was found that, at an overall increase in the quantity and quality of food resources (namely, increase in the content of eicosapentaenoic acid and docosahexaenoic acid (DHA) in the biomass), the relative rate of DHA accumulation in gammarids in the lakes without fish is higher than in the lake with fish.     

Membrane integration of a mitochondrial signal-anchored protein does not require additional proteinaceous factors

The MOM (mitochondrial outer membrane) contains SA (signal-anchored) proteins that bear at their N-terminus a single hydrophobic segment that serves as both a mitochondrial targeting signal and an anchor at the membrane. These proteins, like the vast majority of mitochondrial proteins, are encoded in the nucleus and have to be imported into the organelle. Currently, the mechanisms by which they are targeted to and inserted into the OM (outer membrane) are unclear. To shed light on these issues, we employed a recombinant version of the SA protein OM45 and a synthetic peptide corresponding to its signal-anchor segment. Both forms are associated with isolated mitochondria independently of cytosolic factors. Interaction with mitochondria was diminished when a mutated form of the signal-anchor was employed. We demonstrate that the signal-anchor peptide acquires an α-helical structure in a lipid environment and adopted a TM (transmembrane) topology within artificial lipid bilayers. Moreover, the peptide's affinity to artificial membranes with OM-like lipid composition was much higher than that of membranes with ER (endoplasmic reticulum)-like lipid composition. Collectively, our results suggest that SA proteins are specifically inserted into the MOM by a process that is not dependent on additional proteins, but is rather facilitated by the distinct lipid composition of this membrane.     

Critical role of vimentin phosphorylation at Ser-56 by p21-activated kinase in vimentin cytoskeleton signaling

Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and mitosis. In this study, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of p21-activated kinase (PAK) activation). Treatment with PAK led to disassembly of wild-type (but not mutant S56A) vimentin filaments as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS; an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of mutant S56A vimentin in cells inhibited the increase in phosphorylation at Ser-56 and in the ratios of soluble to insoluble vimentin (an index of vimentin disassembly) and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation compared with cells expressing wild-type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing the S56A mutant. Expression of mutant S56A vimentin depressed PAK phosphorylation at Thr-423 induced by 5-HT. Expression of the S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.     

Negative regulation of osteoclastogenesis by ectodomain shedding of receptor activator of NF-kappaB ligand

Receptor activator of NF-kappaB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis.     

Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase

Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).