Molecular Forces Governing the Biological Function of Per-Arnt-Sim-B (PAS-B) Domains: A Comparative Computational Study

\ua9 2021 by the authors.Per-Arnt-Sim (PAS) domains are evolutionarily-conserved regions found in proteins in all living systems, involved in transcriptional regulation and the response to hypoxic and xenobiotic stress. Despite having low primary sequence similarity, they show an impressively high structural conservation. Nonetheless, understanding the underlying mechanisms that drive the biological function of the PAS domains remains elusive. In this work, we used molecular dynamics simulations and bioinformatics tools in order the investigate the molecular characteristics that govern the intrinsic dynamics of five PAS-B domains (human AhR receptor, NCOA1, HIF1α, and HIF2α transcription factors, and Drosophila Suzukii (D. Suzukii) juvenile hormone receptor JHR). First, we investigated the effects of different length of N and C terminal regions of the AhR PAS-B domain, showing that truncation of those segments directly affects structural stability and aggregation propensity of the domain. Secondly, using the recently annotated PAS-B located in the methoprene-tolerant protein/juvenile hormone receptor (JHR) from D. Suzukii, we have shown that the mutation of the highly conserved “gatekeeper” tyrosine to phenylalanine (Y322F) does not affect the stability of the domain. Finally, we investigated possible redox-regulation of the AhR PAS-B domain by focusing on the cysteinome residues within PAS-B domains. The cysteines in AhR PAS-B are directly regulating the dynamics of the small molecule ligand-gating loop (residues 305 to 326). In conclusion, we comprehensibly described several molecular features governing the behaviour of PAS-B domains in solution, which may lead to a better understanding of the forces driving their biological functions

Force-Sensitive Autoinhibition of the von Willebrand Factor ls Mediated by Interdomain Interactions

Von Willebrand factor (VWF) plays a central role in hemostasis. Triggered by shear-stress, it adheres to platelets at sites of vascular injury. Inactivation of VWF has been associated to the shielding of its adhesion sites and proteolytic cleavage. However, the molecular nature of this shielding and its coupling to cleavage under shear-forces in flowing blood remain unknown. In this study, we describe, to our knowledge, a new force-sensory mechanism for VWF-platelet binding, which addresses these questions, based on a combination of molecular dynamics (MD) simulations, atomic force microscopy (AFM), and microfluidic experiments. Our MD simulations demonstrate that the VWF A2 domain targets a specific region at the VWF A1 domain, corresponding to the binding site of the platelet glycoprotein Ibα (GPIbα) receptor, thereby causing its blockage. This implies autoinhibition of the VWF for the binding of platelets mediated by the A1-A2 protein-protein interaction. During force-probe MD simulations, a stretching force dissociated the A1A2 complex, thereby unblocking the GPIbα binding site. Dissociation was found to be coupled to the unfolding of the A2 domain, with dissociation predominantly occurring before exposure of the cleavage site in A2, an observation that is supported by our AFM experiments. This suggests that the A2 domain prevents platelet binding in a force-dependent manner, ensuring that VWF initiates hemostasis before inactivation by proteolytic cleavage. Microfluidic experiments with an A2-deletion VWF mutant resulted in increased platelet binding, corroborating the key autoinhibitory role of the A2 domain within VWF multimers. Overall, autoinhibition of VWF mediated by force-dependent interdomain interactions offers the molecular basis for the shear-sensitive growth of VWF-platelet aggregates, and might be similarly involved in shear-induced VWF self-aggregation and other force-sensing functions in hemostasis

Whole Earth Telescope observations of the pulsating subdwarf B star PG 0014+067

PG 0014+067 is one of the most promising pulsating subdwarf B stars for seismic analysis, as it has a rich pulsation spectrum. The richness of its pulsations, however, poses a fundamental challenge to understanding the pulsations of these stars, as the mode density is too complex to be explained only with radial and nonradial low degree (l < 3) p-modes without rotational splittings. One proposed solution, for the case of PG 0014+067 in particular, assigns some modes with high degree (l=3). On the other hand, theoretical models of sdB stars suggest that they may retain rapidly rotating cores, and so the high mode density may result from the presence of a few rotationally-split triplet (l=1), quintuplet (l=2) modes, along with radial (l=0) p-modes. To examine alternative theoretical models for these stars, we need better frequency resolution and denser longitude coverage. Therefore, we observed this star with the Whole Earth Telescope for two weeks in October 2004. In this paper we report the results of Whole Earth Telescope observations of the pulsating subdwarf B star PG 0014+067. We find that the frequencies seen in PG 0014+067 do not appear to fit any theoretical model currently available; however, we find a simple empirical relation that is able to match all of the well-determined frequencies in this star.Comment: 19 pages, preprint of paper accepted for publication in The Astrophysical Journa

Oxidation of SQSTM1/p62 mediates the link between redox state and protein homeostasis.

Cellular homoeostatic pathways such as macroautophagy (hereinafter autophagy) are regulated by basic mechanisms that are conserved throughout the eukaryotic kingdom. However, it remains poorly understood how these mechanisms further evolved in higher organisms. Here we describe a modification in the autophagy pathway in vertebrates, which promotes its activity in response to oxidative stress. We have identified two oxidation-sensitive cysteine residues in a prototypic autophagy receptor SQSTM1/p62, which allow activation of pro-survival autophagy in stress conditions. The Drosophila p62 homologue, Ref(2)P, lacks these oxidation-sensitive cysteine residues and their introduction into the protein increases protein turnover and stress resistance of flies, whereas perturbation of p62 oxidation in humans may result in age-related pathology. We propose that the redox-sensitivity of p62 may have evolved in vertebrates as a mechanism that allows activation of autophagy in response to oxidative stress to maintain cellular homoeostasis and increase cell survival

Autoregulation of von Willebrand factor function by a disulfide bond switch

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this “mechanopresentation” remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.: This study was supported by grants from the National Health and Medical Research Council of Australia (P.J.H.), Royal College of Pathologists Foundation Kanematsu/Novo Nordisk Research Award (F.P. and L.J.), Diabetes Australia Research Trust grant G179720 and Sydney Medical School Early-Career Researcher Kickstart Grant (L.J.), National Heart Foundation of Australia Postdoctoral Fellowship (101285) (L.J.) and British Heart Foundation Intermediate Basic Science Research Fellowship (FS/11/51/28920) (B.M.L.), Deutsche Forschungsgemeinschaft (research unit FOR 1543 to C.A.-S., C.B., and F.G.), the Center for Modelling and Simulation in the Biosciences postdoctoral program of the Heidelberg University (A.B.), and the Klaus Tschira Foundation (F.G.). B.L. was supported by the Dutch Thrombosis Foundation through grant number 2016-03.

Estimation of the acute cesium toxicity by the microbial assay for risk assessment (MARA) test

The microbial assay for risk assessment (MARA) test was used for acute cesium toxicity evaluation in water solutions. The test contained 11 different microorganisms with a wide spectrum of sensitivity. The resistance of microorganisms to cesium was characterized as follows: microbial toxic concentration (MTC), half maximal inhibitory concentration (IC50), maximal inhibitory concentration (IC100). The sensitivity to cesium was characterized by the lowest observed effect level (LOEL). High levels of sensitivity in the range 3.1–6.3 mM were shown by the following microorganisms: Serratia rubidaea > Pseudomonas aurantiaca, Delftia acidovorans, Citrobacter freundii, Staphylococcus warneri. Lower levels of sensitivity (up to 16 mM) were noted for Comamonas testosteroni, Microbacterium species, Kurthia gibsonii, Pichia anomala, whereas that in the range 24–31 mM for Brevundimonas diminuta > Enterococcus casseliflavus. High resistance to Cs+ was found for E. casseliflavus (MTC 86.9 g/l) > the yeast – P. anomala (MTC 19.3 g/l) > K. gibsoni (MTC 17.4 g/l) > B. diminuta (MTC 13.4 g/l). The phenomenon of resistance of enterococcus and yeast strains was discussed

Reserch on the speed of cyclist for various infrastructure solutions

Artykuł przedstawia wyniki badań prędkości rowerzystów dla różnych rozwiązań infrastrukturalnych. Badania przeprowadzono w jedenastu lokalizacjach o nawierzchni asfaltowej, na pasach ruchu na jezdni, ciągach pieszo rowerowych, kontrapasach, wydzielonych ścieżkach rowerowych oraz na pasie ruchu dla autobusów. Przeanalizowano także prędkość rowerzystów na ścieżce rowerowej o nawierzchni z kostki brukowej oraz prędkości na pasie rowerowym o dużym pochyleniu wzdłużnym. Szczegółowe wyniki pomiarów przedstawiono w podziale na kategorie wiekowe, płeć i rodzaj roweru.The article presents the results of research on cycling speed on various infrastructure solutions. The measurements were taken in 13 different locations: on road lanes, bicycle counterflow lanes, isolated paths and bus lanes. Additional research were made on bike paths with paving surface and on bicycle lane where a 6.3% gradient exists Results are presented as average speed and quantile to of the order of 0.15 and are separated by age group and bike type