Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.     

Moving calls: a vocal mechanism underlying quorum decisions in cohesive groups

Members of social groups need to coordinate their behaviour when choosing between alternative activities. Consensus decisions enable group members to maintain group cohesion and one way to reach consensus is to rely on quorums. A quorum response is where the probability of an activity change sharply increases with the number of individuals supporting the new activity. Here, we investigated how meerkats (Suricata suricatta) use vocalizations in the context of movement decisions. Moving calls emitted by meerkats increased the speed of the group, with a sharp increase in the probability of changing foraging patch when the number of group members joining the chorus increased from two up to three. These calls had no apparent effect on the group's movement direction. When dominant individuals were involved in the chorus, the group's reaction was not stronger than when only subordinates called. Groups only increased speed in response to playbacks of moving calls from one individual when other group members emitted moving calls as well. The voting mechanism linked to a quorum probably allows meerkat groups to change foraging patches cohesively with increased speed. Such vocal coordination may reflect an aggregation rule linking individual assessment of foraging patch quality to group travel route.     

Synthesis and evaluation of ferrocenoyl pentapeptide (Fc-KLVFF) as an inhibitor of Alzheimer's Aβ₁-₄₂ fibril formation in vitro

Aggregation and fibril formation of β-amyloid peptides (Aβ) is the key event in the pathogenesis of Alzheimer's disease. Many efforts have been made on the development of effective inhibitors to prevent Aβ fibril formation or disassemble the preformed Aβ fibrils. Peptide inhibitors with sequences homologous to the hydrophobic segments of Aβ can alter the aggregation pathway of Aβ, together with decrease of the cell toxicity. In this study, the conjugate of ferrocenoyl (Fc) with pentapeptide KLVFF (Fc-KLVFF), was synthesized by HBTU/HOBt protocol in solution. The inhibitory effect of Fc-KLVFF on Aβ(1-42) fibril formation was evaluated by thioflavin T fluorescence assay, and confirmed by atomic force microscopy (AFM) and transmission electron microscopy (TEM) analyses. Fc-KLVFF shows high inhibitory effect towards the fibril formation of Aβ(1-42). Additionally, the attachment of ferrocenoyl moiety onto peptides allows us to investigate the interaction between the inhibitor and Aβ(1-42) in real-time by electrochemical method. As expected, tethering of ferrocenoyl moiety onto pentapeptide shows improved lipophilicity and significant resistance towards proteolytic degradation compared to its parent peptide.     

Strand 6B deformation and residues exposure towards N-terminal end of helix B during proteinase inhibition by Serpins

Serine Protease inhibitors (Serpins) like antithrombin, antitrypsin, neuroserpin, antichymotrypsin, protein C-inhibitor and plasminogen activator inhibitor is involved in important biological functions like blood coagulation, fibrinolysis, inflammation, cell migration and complement activation. Serpins native state is metastable, which undergoes transformation to a more stable state during the process of protease inhibition. Serpins are prone to conformation defects, however little is known about the factors and mechanisms which promote its conformational change and misfolding. Helix B region in serpins is with several point mutations which result in pathological conditions due to polymerization. Helix B analysis for residue burial and cavity was undertaken to understand its role in serpin structure function. A structural overlap and an accessible surface area analysis showed the deformation of strand 6B and exposure of helix B at N-terminal end in cleaved conformation but not in the native and latent conformation of various inhibitory serpins. A cleaved polymer like conformation of antitrypsin also showed deformation of s6B and helix B exposure. Cavity analysis showed that helix B residues were part of the largest cavity in most of the serpins in the native state which increase in size during the transformation to cleaved and latent states. These data for the first time show the importance of strand 6B deformation and exposure of helix B in smooth insertion of the reactive center loop during serpin inhibition and indicate that helix B exposure due to variants may increase its polymer propensity. ABBREVIATIONS: serpin -serine protease inhibitors RCL -reactive center loop ASA -accessible surface area.     

Effects of arginine on heat-induced aggregation of concentrated protein solutions

Arginine is one of the commonly used additives to enhance refolding yield of proteins, to suppress aggregation of proteins, and to increase solubility of proteins, and yet the molecular interactions that contribute to the role of arginine are unclear. Here, we present experiments, using bovine serum albumin (BSA), lysozyme (LYZ), and β-lactoglobulin (BLG) as model proteins, to show that arginine can enhance heat-induced aggregation of concentrated protein solutions, contrary to the conventional belief that arginine is a universal suppressor of aggregation. Results show that the enhancement in aggregation is caused only for BSA and BLG, but not for LYZ, indicating that arginine's preferential interactions with certain residues over others could determine the effect of the additive on aggregation. We use this previously unrecognized behavior of arginine, in combination with density functional theory calculations, to identify the molecular-level interactions of arginine with various residues that determine arginine's role as an enhancer or suppressor of aggregation of proteins. The experimental and computational results suggest that the guanidinium group of arginine promotes aggregation through the hydrogen-bond-based bridging interactions with the acidic residues of a protein, whereas the binding of the guanidinium group to aromatic residues (aggregation-prone) contributes to the stability and solubilization of the proteins. The approach, we describe here, can be used to select suitable additives to stabilize a protein solution at high concentrations based on an analysis of the amino acid content of the protein.     

Measuring Eco-efficiency of Production with Data Envelopment Analysis

Aggregation of environmental pressures into a single environmental damage index is a major challenge of eco-efficiency measurement. This article examines how the data envelopment analysis (DEA) method can be adapted for this purpose. DEA accounts for substitution possibilities between different natural resources and emissions and does not require subjective judgment about the weights. Although DEA does not require subjective or normative judgment, soft weight restrictions can be incorporated into the framework. The proposed approach is illustrated by an application to assessing ecoefficiency of road transportation in the three largest towns of eastern Finland.     

Modulation of antioxidant enzyme activities, platelet aggregation and serum prostaglandins in rats fed spray-dried milk containing n-3 fatty acid

Spray-dried milk enriched with n-3 fatty acids from linseed oil (LSO) or fish oil (FO) were fed to rats to study its influence on liver lipid peroxides, hepatic antioxidant enzyme activities, serum prostaglandins and platelet aggregation. Significant level of α linolenic acid, eicosapentaenoic acid and docosahexaenoic acid were accumulated at the expense of arachidonic acid in the liver of rats fed n-3 fatty acid enriched formulation. The linseed oil and fish oil enriched formulation fed group had 44 and 112% higher level of lipid peroxides in liver homogenate compared to control rats fed groundnut oil enriched formulation. Catalase activity in liver homogenate was increased by 37 and 183% respectively in linseed oil and fish oil formulation fed rats. The glutathione peroxidase activity decreased to an extent of 25–36% and glutathione transferase activity increased to an extent of 34–39% in rats fed n-3 fatty acids enriched formulation. Feeding n-3 fatty acid enriched formulation significantly elevated the n-3 fatty acids in platelets and increased the lipid peroxide level to an extent of 4.2 to 4.5-fold compared to control. The serum thromboxane B₂ level was decreased by 35 and 42% respectively in linseed oil and fish oil enriched formulation fed rats, whereas 6-keto-prostaglandin F1α level was decreased by 17 and 23% respectively in linseed oil and fish oil enriched formulation fed rats. The extent and rate of platelet aggregation was decreased significantly in n-3 fatty acids enriched formulation fed rats. This indicated that n-3 fatty acids enriched formulation beneficially reduces platelet aggregation and also enhances the activities of hepatic antioxidant enzymes such as catalase and glutathione transferase.