Hydroxylation activity at the 6β-position of steroid hormones (testosterone, progesterone, and cortisol) by human cytochromes P450 (CYP) 3A4, polymorphic CYP3A5, and fetal CYP3A7 were compared to understand the catalytic properties of the major forms of human CYP3A subfamily. Testosterone, progesterone, and cortisol 6β-hydroxylation activities of recombinant CYP3A4, CYP3A5, and CYP3A7 were determined by liquid chromatography. Michaelis constants (Km) for CYP3A7-mediated 6β-hydroxylation of testosterone, progesterone, and cortisol were similar to those of CYP3A4 and CYP3A5. The maximal velocity (kcat) and kcat/Km values for CYP3A4 were the highest, followed by CYP3A5 and those for CYP3A7 were the lowest among three CYP3A subfamily members. A decrease in Km values for for progesterone 6β-hydroxylation by CYP3A4, CYP3A5, and CYP3A7 in the presence of testosterone was observed, and the kcat values for CYP3A5 gradually increased with increasing testosterone. This indicated that testosterone stimulated progesterone 6β-hydroxylation by all three CYP3A subfamily members. However, progesterone inhibited testosterone 6β-hydroxylation mediated by CYP3A4, CYP3A5, and CYP3A7. In conclusion, the kcat values, rather than Km values, for 6β-hydroxylation of three steroid hormones mediated by CYP3A7 were different from those for CYP3A4 and CYP3A5. In addition, the inhibitory/stimulatory pattern of steroid-steroid interactions would be different among CYP3A subfamily members.

Upregulation of nerve growth factor (NGF) in parenchymal hepatocytes has been shown to exert hepatoprotective function during cholestatic liver injury. However, the modulatory role of NGF in regulation of liver autophagy remains unclear. This study aimed to scrutinize the regulatory role of NGF in hepatic expression of farnesoid X receptor (FXR), a bile acid (BA)-activated nuclear receptor, and to determine its cytoprotective effect on BA-induced autophagy and cytotoxicity. Livers of human hepatolithiasis and bile duct ligation (BDL)-induced mouse cholestasis were used for histopathological and molecular detection. The regulatory roles of NGF in autophagy flux and FXR expression, as well as its hepatoprotection against BA cytotoxicity were examined in cultured hepatocytes. FXR downregulation in human hepatolithiasis livers showed positive correlation with hepatic NGF levels. NGF administration upregulated hepatic FXR levels, while neutralization of NGF decreased FXR expression in BDL-induced cholestatic mouse livers. In vitro studies demonstrated that NGF upregulated FXR expression, increased cellular LC3 levels, and exerted hepatoprotective effect in cultured primary rat hepatocytes. Conversely, autophagy inhibition abrogated NGF-driven cytoprotection under BA exposure, suggesting involvement of NGF-modulated auophagy flux. Although FXR agonistic GW4064 stimulation did not affect auophagic LC3 levels, FXR activity inhibition significantly potentiated BA-induced cytotoxicity and increased cellular p62/SQSTM1 and Rab7 protein in SK-Hep1 hepatocytes. Moreover, FXR gene silencing abolished the protective effect of NGF under BA exposure. These findings support that NGF modulates autophagy flux via FXR upregulation and protects hepatocytes against BA-induced cytotoxicity. NGF/FXR axis is a novel therapeutic target for treatment of cholestatic liver diseases.

Tropomyosin and cofilin are involved in the regulation of actin filament dynamic polymerization and depolymerization. Binding of cofilin changes actin filaments structure, leading to their severing and depolymerization. Non-muscle tropomyosin isoforms were shown before to differentially regulate the activity of cofilin 1; products of TPM1 gene stabilized actin filaments, but products of TPM3 gene promoted cofilin-dependent severing and depolymerization. Here, conformational changes at the longitudinal and lateral interface between actin subunits resulting from tropomyosin and cofilin 1 binding were studied using skeletal actin and yeast wild type and mutant Q41C and S265C actins. Cross-linking of F-actin and fluorescence changes in F-actin labeled with acrylodan at Cys41 (in D-loop) or Cys265 (in H-loop) showed that tropomyosin isoforms differentially regulated cofilin-induced conformational rearrangements at longitudinal and lateral filament interfaces. Tryptic digestion of F–Mg-actin confirmed the differences between tropomyosin isoforms in their regulation of cofilin-dependent changes at actin-actin interfaces. Changes in the fluorescence of AEDANS attached to C-terminal Cys of actin, as well as FRET between Trp residues in actin subdomain 1 and AEDANS, did not show differences in the conformation of the C-terminal segment of F-actin in the presence of different tropomyosins ± cofilin 1. Therefore, actin's D- and H-loop are the sites involved in regulation of cofilin activity by tropomyosin isoforms.

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorentz Mie scattering theory yields the bead position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables (LUT). Here, we introduce an alternative 3D phasor algorithm, that provides robust bead tracking with nanometric localization accuracy in a z-range of over 10 μm under non-optimal imaging conditions. The algorithm is based on a 2D cross-correlation using Fast Fourier Transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real-time on a generic CPU. The accuracy of 3D Phasor tracking was extensively tested and compared to a LUT approach using Lorentz Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead tracking applications, especially where high throughput is required and image artefacts are difficult to avoid.

Ribosomes are essential machines for protein synthesis in cells. Their structures are very complex but conserved in different species. Since most parts of a ribosome are composed of negatively charged RNAs, its electrostatics should play a fundamental role in the realization of its functions. However, a complete picture of the electrostatics of ribosomes is still absent at present. Here, assisted by the latest version of DelPhi (Version: 8.4), we illustrate a picture of the electrostatics of a prokaryotic ribosome as well as its molecular chaperones. The revealed electrostatics features are well consistent with available experimental data as well as the functions of the ribosome and its molecular chaperones and provides a basis for further studying the mechanism underlying these functions.

Oral microbes are a contributing factor to hyperglycemia by inducing an increase in insulin resistance resulting in uncontrolled blood glucose levels. However, the relationship between the distribution of oral flora and hyperglycemia is still controversial. Combining the power of MALDI-Biotyper with anaerobic bacterial culture, this study explores the correlation between anaerobic bacteria in the oral cavity and blood glucose levels. The results demonstrated that altered blood glucose levels contributed to a varied bacterial distribution in the oral cavity. Specifically, Veillonella spp. and Prevotella spp. were identified in a higher proportion in people with elevated blood glucose levels. Six bacterial species identified in this study (Prevotella melaninogenica, Campylobacter rectus, Streptococcus gordonii, Streptococcus mitis, Streptococcus salivarius, and Veillonella parvula) not only demonstrated a positive association with higher blood glucose levels, but also likely contribute to the development of the condition. The data demonstrated MALDI-TOF MS to be a simpler, faster, and more economical clinical identification tool that provides clarity and depth to the research on blood glucose and oral microbiota.

Precise structural differentiation of often isomeric glycans is important given their roles in numerous biological processes. Mass spectrometry (MS) (and tandem MS) is one of the analytical techniques at the forefront of glycan analysis given its speed, sensitivity in producing structural information as well as the fact it can be coupled to other orthogonal analytical techniques such as liquid chromatography (LC) and ion mobility spectrometry (IMS). This review describes another family of techniques that are more commonly being hybridized to MS(/MS) namely gas-phase infrared (IR) spectroscopy, whose rise is in part due to the development and improved accessibility of tunable IR lasers. Gas-phase IR can often differentiate fine isomeric differences ubiquitous within carbohydrates that MS may be ‘blind’ to. There are also examples of cryogenic gas-phase IR spectroscopy with much greater spectral resolution as well as hybridizing with separative methods (LC, IMS). Furthermore, collision-induced dissociation (CID) product ions can also be probed by IR, which may be beneficial to deconvolute spectra, aid analysis and build spectral libraries, thus generating novel opportunities for fragment-based approaches to analyze glycans.

Advances in imaging methods such as electron microscopy, tomography, and other modalities are enabling high-resolution reconstructions of cellular and organelle geometries. Such advances pave the way for using these geometries for biophysical and mathematical modeling once these data can be represented as a geometric mesh, which, when carefully conditioned, enables the discretization and solution of partial differential equations. In this study, we outline the steps for a naïve user to approach GAMer 2, a mesh generation code written in C++ designed to convert structural datasets to realistic geometric meshes, while preserving the underlying shapes. We present two example cases, 1) mesh generation at the subcellular scale as informed by electron tomography, and 2) meshing a protein with structure from x-ray crystallography. We further demonstrate that the meshes generated by GAMer are suitable for use with numerical methods. Together, this collection of libraries and tools simplifies the process of constructing realistic geometric meshes from structural biology data.

Excessive accumulation of acylcarnitines (ACs), often caused by metabolic disorders, has been associated with obesity, arrhythmias, cardiac ischemia, insulin resistance, etc. Mechanisms whereby elevated ACs might contribute to pathophysiological effects remain largely unexplored. We have aimed to gain insight into AC interactions with the mitochondrial inner membrane. To model its outer leaflet, Langmuir monolayers and cushioned supported bilayers were employed. Their interactions with ACs were monitored with epifluorescence microscopy, which revealed a local leaflet expansion upon exposure to elevated concentrations of a long-chain AC, plausibly caused by its insertion. To assess the AC insertion parameters, constant pressure insertion assays were performed. A value of 21 ± 3 Å2 was obtained for the AC insertion area, which is roughly the same as the cross-sectional area of an acyl chain. By contrast, the carnitine moiety was found to require an area of 37 ± 3 Å2. The AC insertion has thus been concluded to involve solely the AC acyl chain. This mode of insertion implies that the carnitine moiety with its nontitratable positive charge is left dangling at the membrane surface, which is likely to alter the surface electrostatics of the outer leaflet. The extrapolation of these findings has enabled us to hypothesize that, by altering the morphology and surface electrostatics of the outer leaflet, the insertion of ACs, in particular their long-chain counterparts, may trigger a nonspecific activation of signaling pathways in the inner mitochondrial membrane thereby modulating its function and potentially leading to pathophysiological responses.

All medications have adverse effects. Among the most serious of these are cardiac arrhythmias. Current paradigms for drug safety evaluation are costly, lengthy, conservative, and impede efficient drug development. Here we combine multiscale experiment and simulation, high-performance computing, and machine learning to create a risk estimator to stratify new and existing drugs according to their pro-arrhythmic potential. We capitalize on recent developments in machine learning and integrate information across ten orders of magnitude in space and time to provide a holistic picture of the effects of drugs, either individually or in combination with other drugs. We show, both experimentally and computationally, that drug-induced arrhythmias are dominated by the interplay between two currents with opposing effects: the rapid delayed rectifier potassium current and the L-type calcium current. Using Gaussian process classification, we create a classifier that stratifies drugs into safe and arrhythmic domains for any combinations of these two currents. We demonstrate that our classifier correctly identifies the risk categories of 22 common drugs, exclusively on the basis of their concentrations at 50% current block. Our new risk assessment tool explains under which conditions blocking the L-type calcium current can delay or even entirely suppress arrhythmogenic events. Using machine learning in drug safety evaluation can provide a more accurate and comprehensive mechanistic assessment of the pro-arrhythmic potential of new drugs. Our study paves the way towards establishing science-based criteria to accelerate drug development, design safer drugs, and reduce heart rhythm disorders.

Mutant Ras proteins are important drivers of human cancers, yet no approved drugs act directly on this difficult target. Over the last decade, the idea has emerged that oncogenic signaling can be diminished by molecules that drive Ras into orientations in which effector binding interfaces are occluded by the cell membrane. To support this approach to drug discovery, we characterize the orientational preferences of membrane-bound K-Ras4B in 1.45 milliseconds aggregate time of atomistic molecular dynamics simulations. Individual simulations probe active or inactive states of Ras on membranes with or without anionic lipids. We find that the membrane orientation of Ras is relatively insensitive to its bound guanine nucleotide and activation state but depends strongly on interactions with anionic phosphatidylserine lipids. These lipids slow Ras’ translational and orientational diffusion and promote a discrete population in which small changes in orientation control Ras’ competence to bind multiple regulator and effector proteins. Our results suggest that compound-directed conversion of constitutively active mutant Ras into functionally inactive forms may be accessible via subtle perturbations of Ras’ orientational preferences at the membrane surface.

Low molecular weight protein tyrosine phosphatases (LMW-PTP) are ubiquitous enzymes found across a spectrum of genera from prokaryotes to higher eukaryotes. LMW-PTP belong to the Cys-based PTP class II protein family. Here, we show that LMW-PTP can be categorized into two different groups, referred as class II subdivision I (class II.I) and subdivision II (class II.II). Using to BPtpA from the opportunistic pathogen Burkholderia cenocepacia, as a representative member of the LMW-PTP class II.I, we demonstrated that four conserved residues (W47, H48, D80, and F81) are required for enzyme function. Guided by an in silico model of BPtpA, we show that the conserved residues at α3-helix (D80 and F81) contribute to protein stability, while the other conserved residues in the W-loop (W47 and H48) likely play a role in substrate recognition. Overall, our results provide new information on LMW-PTP protein family and establish B. cenocepacia as a suitable model to investigate how substrates are recognized and sorted by these proteins.

Rieske iron-sulfur protein (RISP) is a catalytic subunit of the complex III in the mitochondrial electron transport chain. Studies for years have revealed that RISP is essential for the generation of intracellular reactive oxygen species (ROS) via delicate signaling pathways associated with many important molecules such as protein kinase C-ε, NADPH oxidase, and ryanodine receptors. More significantly, mitochondrial RISP-mediated ROS production has been implicated in the development of hypoxic pulmonary vasoconstriction, leading to pulmonary hypertension, right heart failure, and death. Investigations have also shown the involvement of RISP in ROS-dependent cardiac ischemic/reperfusion injuries. Further research may provide novel and valuable information that can not only enhance our understanding of the functional roles of RISP and the underlying molecular mechanisms in the pulmonary vasculature and other systems, but also elucidate whether RISP targeting can act as preventative and restorative therapies against pulmonary hypertension, cardiac diseases, and other disorders.

Background Current experimental models using either human or mouse cell lines, are not representative of the complex features of GBM. In particular, there is no model to study patient-derived iPSCs to generate a GBM model. Overexpression of c-met gene is one of the molecular features of GBM leading to increased signaling via STAT3 phosphorylation. We generated an iPSC line from a patient with c-met mutation and we asked whether we could use it to generate neuronal-like organoids mimicking features of GBM. Methods We have generated iPSC-aggregates differentiating towards organoids. We analyzed them by gene expression profiling, immunostaining and transmission electronic microscopy analyses (TEM). Results Herein we describe that c-met-mutated iPSC aggregates spontaneously differentiate into dopaminergic neurons more rapidly than control iPSC aggregates in culture. Gene expression profiling of c-met-mutated iPSC aggregates at day +90 showed neuronal- and GBM-related genes, reproducing a genomic network described in primary human GBM. Comparative TEM analyses confirmed the enrichment of these structures in intermediate filaments and abnormal cilia, a feature described in human GBM. The c-met-mutated iPSC-derived organoids, as compared to controls expressed high levels of glial fibrillary acidic protein (GFAP), which is a typical marker of human GBM, as well as high levels of phospho-MET and phospho-STAT3. The use of temozolomide (TMZ) showed a preferential cytotoxicity of this drug in c-met-mutated neuronal-like organoids. General significance This study shows the feasibility of generating “off-the shelf” neuronal-like organoid model mimicking GBM using c-met-mutated iPSC aggregates and its potential future use in research.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N- and C-) with fluorescent probes, and used intramolecular TR-FRET to assess inter-lobe distances when CaM is bound to either RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an inter-lobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+ the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: (1) The two conformations are resolved and more ordered. (2) The open state is the major component. (3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

In cell membranes, the functional constituents, such as peptides, proteins, and polysaccharides, diffuse in a sea of lipids as single molecules and molecular aggregates. Thus, the fluidity of the heterogeneous multicomponent membrane is important to understand the roles of the membrane in cell functionality. Recently, Henle and Levine described the hydrodynamics of molecular diffusion in a spherical membrane. A tangential point force at the north pole induces a pair of vortices whose centers lie on a line perpendicular to the point force and are symmetrical with respect to the point force. The position of the vortex center depends on ηm /Rηw where R is the radius of the spherical membrane, and ηm and ηw are the viscosities of the membrane and the surrounding medium, respectively. Based on this theoretical prediction, we applied a point force to a phase separated spherical vesicle composed of DPPC/DOPC/Cholesterol by means of a microinjection technique. The path lines were visualized by trajectories of microdomains. We determined the position of the vortex center and estimated the membrane viscosity using the dependence of the position of the vortex center on ηm/Rηw. The obtained apparent membrane viscosities for various compositions are mapped on the phase diagram. The membrane viscosity is almost constant in the range of 0 < ϕLo ≤ 0.5 (ϕLo: area fraction of the liquid ordered phase), whereas that in the range of 0.5 ≤ ϕLo < 1.0 exponentially increases with increasing of ϕLo. The obtained viscosity landscape provides a basic understanding of the fluidity of heterogeneous multicomponent membranes.

Metastasis of mesenchymal tumor cells is traditionally considered as a single cell process. Here we report an emergent collective phenomenon where the dissemination rate of mesenchymal breast cancer cells from 3D tumors depends on the tumor geometry. Combining experimental measurements and computational modeling we demonstrate that the collective dynamics is coordinated by the mechanical feedback between individual cells and their surrounding extracellular matrix (ECM). We find the tissue-like fibrous ECM supports long-range physical interactions between cells, which turn geometric cues into regulated cell dissemination dynamics. Our results suggest that migrating cells in 3D ECM represent a distinct class of active particle system where the collective dynamics is governed by remodeling of the environment rather than direct particle-particle interactions.

Stroke is one of the world's most deadly pathologies, and the rate of stroke recurrence is high. However, due to the complex nature of ischemia and reperfusion injury, there is presently no reliable treatment. The main factors driving brain damage from ischemic stroke are neuronal cell death resulting from oxidative stress, inflammation, and failure of the blood brain barrier. While under normal conditions, the blood brain barrier acts as a selectively permeable membrane allowing solutes and other substances to pass into the tissues of the central nervous system, ischemia and reperfusion alter the expression of tight junction proteins such as occludin, which leads to unmitigated perfusion and loss of homeostasis. Phoenixin-14 is a 14-amino acid neuropeptide that has been shown to play a role in regulating reproduction, blood sugar metabolism, pain, anxiety, and more recently, certain aspects of ischemic cardiac injury. In the present study, we found that phoenixin-14 confers protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R) injury in bEnd.3 brain endothelial cells. Phoenixin-14 attenuated oxidative stress via downregulation of ROS and NOX1 and inhibited HMGB1 expression. Additionally, phoenixin-14 increased the expression of eNOS and NO, which play a protective role. Phoenixin-14 reduced endothelial monolayer permeability by increasing the expression of occludin. Finally, we found that the effects of phoenixin-14 on the expression of eNOS and occludin are dependent on the KLF2 transcriptional pathway, as evidenced by the results of our KLF2 knockdown experiment. Thus, phoenixin-14 may serve as a novel therapeutic agent for ischemic stroke.

Molecular solutes are known to have a strong effect on the structural and dynamical properties of the surrounding water. In our recent study (PNAS, 114, 322 (2017)) we have identified the presence of strengthened water hydrogen bonds near hydrophobic solutes by using both IR spectroscopy and ab-initio molecular dynamics simulations. The water molecules involved in the enhanced hydrogen bonding have been shown to display extensive structural ordering and restricted mobility. We observed that an individual pair of water molecules can make stronger hydrogen bond to each other if it is not surrounded by intercalating water molecules. Here we present compelling simulation results which unravel a simple mechanistic picture of the emergence of the hydrogen bond (HB) strengthening around solvated methane. We show explicitly that actual absence of water molecules within the excluded volume due to the hydrophobic molecule assures smaller residual torque on neighboring water molecules enabling the formation of stronger HBs between them.

Background Cancer is one of the major threats to human health and current cancer therapies have been unsuccessful in eradicating it. Ferroptosis is characterized by iron-dependence and lipid hydroperoxides accumulation, and its primary mechanism involves the suppression of system Xc−-GSH (glutathione)-GPX4 (glutathione peroxidase 4) axis. Co-incidentally, cancer cells are also metabolically characterized by iron addiction and ROS tolerance, which makes them vulnerable to ferroptosis. This may provide a new tactic for cancer therapy. Scope of review The general features and mechanisms of ferroptosis, and the basis that makes cancer cells vulnerable to ferroptosis are described. Further, we emphatically discussed that disrupting GSH may not be ideal for triggering ferroptosis of cancer cells in vivo, but directly inhibiting GPX4 and its compensatory members could be more effective. Finally, the various approaches to directly inhibit GPX4 without disturbing GSH were described. Major conclusions Targeting system Xc− or GSH may not effectively trigger cancer cells' ferroptosis in vivo the existence of other compensatory pathways. However, directly targeting GPX4 and its compensatory members without disrupting GSH may be more effective to induce ferroptosis in cancer cells in vivo, as GPX4 is essential in preventing ferroptosis. General significance Cancer is a severe threat to human health. Ferroptosis-based cancer therapy strategies are promising, but how to effectively induce ferroptosis in cancer cells in vivo is still a question without clear answers. Thus, the viewpoints raised in this review may provide some references and different perspectives for researchers working on ferroptosis-based cancer therapy.

The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pre-transition at 72 °C with an unfolding enthalpy of ΔHcal ∼ 200-300 kcal/mol and a main transition at 85 °C with an enthalpy of ∼900 - 1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analysed with the multi-state Zimm-Bragg theory. For the investigated mAb, unfolding is characterised by a cooperativity parameter σ ∼6x10−5 and a Gibbs free energy of unfolding of gnu ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220±50 amino acids are involved in the pre-transition and ν∼850±30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔHcal and lowers the midpoint temperature Tm. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25 °C are predicted to be 2-3 M for the pre-transition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCL molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pre-transition and 0.55 for the main transition. The pre-transition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.

Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin’s spectrin repeat 1, and related the changes in the protein’s structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically-induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein’s small protrusions in structurally ordered waters that are characteristic of the so-called “small-molecule regime”, which allow water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically-induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.

We consider a model lipid plasma membrane, one that describes the outer leaf as consisting of sphingomyelin, phosphatidylcholine, and cholesterol, and the inner leaf of phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine, and cholesterol. Their relative compositions are taken from experiment; the cholesterol freely interchanges between leaves. Fluctuations in local composition are coupled to fluctuations in the local membrane curvature, as in the Leibler-Andelman mechanism. Structure factors of components in both leaves display a peak at non-zero wavevector. This indicates that the disordered fluid membrane is characterized by structure of the corresponding wavelength. The scale is given by membrane properties: its bending modulus and its surface tension, that arises from the membrane’s connections to the cytoskeleton. From measurements on the plasma membrane, this scale is on the order of 100 nm. We find that the membrane can be divided into two different kinds of domains that differ not only in their composition, but also in their curvature. The first domain in the outer, exoplasmic, leaf is rich in cholesterol and sphingomyelin, while the inner, cytoplasmic, leaf is rich in phosphatidylserine and phosphatidylcholine. The second kind of domain is rich in phosphatidylcholine in the outer leaf, and in cholesterol and phosphatidylethanolamine in the inner leaf. The theory provides a tenable basis for the origin of structure in the plasma membrane, and an illuminating picture of the organization of lipids therein.

Intercellular bridges are plasma continuities formed at the end of the cytokinesis process that facilitate intercellular mass transport between the two daughter cells. However, it remains largely unknown how the intercellular bridge mediates Ca2+ communication between post-mitotic cells. In the present work, we utilize BV-2 microglial cells planted on dumbbell-shaped micropatterned assemblies to resolve spatiotemporal characteristics of Ca2+ signal transfer over the intercellular bridges. With use of such micropatterns considerably longer and more regular intercellular bridges can be obtained than in conventional cell cultures. The initial Ca2+ signal is evoked by mechanical stimulation of one of the daughter cells. A considerable time delay is observed between the arrivals of passive Ca2+ diffusion and endogenous Ca2+ response in the intercellular bridge-connected cell, indicating two different pathways of the Ca2+ communication. Extracellular Ca2+ and the paracrine pathway have practically no effect on the endogenous Ca2+ response demonstrated by application of Ca2+-free medium, exogenous ATP and P2Y13 receptor antagonist. In contrast, endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin and inositol trisphosphate (IP3) receptor blocker 2-aminoethyl diphenylborate (2-APB) significantly inhibit the endogenous Ca2+ increase, which signifies involvement of IP3-sensitive calcium store release. Notably, passive Ca2+ diffusion into the connected cell can clearly be detected when IP3-sensitive calcium store release is abolished by 2-APB. Those observations prove that both, passive Ca2+ diffusion and IP3-mediated endogenous Ca2+ response contribute to the Ca2+ increase in intercellular bridge-connected cells. Moreover, a simulation model agreed well with the experimental observations.

Transcription factor (TF) proteins bind to DNA to regulate gene expression. Normally, accessibility to DNA is required for their function. However, in the nucleus the DNA is often inaccessible, wrapped around histone proteins in nucleosomes forming the chromatin. Pioneer TFs are thought to induce chromatin opening by recognizing their DNA binding sites on nucleosomes. For example, Oct4, a master regulator and inducer of stem cell pluripotency, binds to DNA in nucleosomes in a sequence specific manner. Here we reveal the structural dynamics of nucleosomes that mediate Oct4 binding from molecular dynamics simulations. Nucleosome flexibility and the amplitude of nucleosome motions such as breathing and twisting are enhanced in nucleosomes with multiple TF binding sites. Moreover, the regions around the binding sites display higher local structural flexibility. Probing different structures of Oct4-nucleosome complexes, we show that alternative configurations in which Oct4 recognizes partial binding sites display stable TF-DNA interactions similar to those observed in complexes with free DNA and compatible with the DNA curvature and DNA-histone interactions. Therefore, we propose a structural basis for nucleosome recognition by a pioneer TF which is essential for understanding how chromatin is unraveled during cell fate conversions.

The rigidity and flexibility of a protein is reflected in its structural dynamics. Studies on protein dynamics often focus on flexibility and softness; this review focuses on protein structural rigidity. The extent of rigidity can be assessed experimentally with incoherent neutron scattering; a method that is complementary to molecular dynamics simulation. This experimental technique can provide information about protein dynamics in timescales of pico- to nanoseconds and at spatial scales of nanometers; these dynamics can help quantify the rigidity of a protein by indices such as force constant, Boson peak, dynamical transition, and dynamical heterogeneity. These indicators also reflect the rigidity of a protein's secondary and tertiary structures. In addition, the indices reveal how rigidity is influenced by different environmental parameters, such as hydration, temperature, pressure, and protein-protein interactions. Hydration affects both rigidity and softness more than other environmental factors. Interestingly, hydration affects harmonic and anharmonic motions in opposite ways. This difference is probably due to the protein's dynamic coupling with water molecules via hydrogen bonding.

Selecting peptides that bind strongly to the major histocompatibility complex (MHC) for inclusion in a vaccine has therapeutic potential for infections and tumors. Machine learning models trained on sequence data exist for peptide:MHC (p:MHC) binding predictions. Here, we train support vector machine classifier (SVMC) models on physicochemical sequence-based and structure-based descriptor sets to predict peptide binding to a well-studied model mouse MHC I allele, H-2Db. Recursive feature elimination and two-way forward feature selection were also performed. Although low on sensitivity compared to the current state-of-the-art algorithms, models based on physicochemical descriptor sets achieve specificity and precision comparable to the most popular sequence-based algorithms. The best-performing model is a hybrid descriptor set containing both sequence-based and structure-based descriptors. Interestingly, close to half of the physicochemical sequence-based descriptors remaining in the hybrid model were properties of the anchor positions, residues 5 and 9 in the peptide sequence. In contrast, residues flanking position 5 make little to no residue-specific contribution to the binding affinity prediction. The results suggest that machine-learned models incorporating both sequence-based descriptors and structural data may provide information on specific physicochemical properties determining binding affinities.

Background: We re-evaluate our RNA-As-Graphs clustering approach, using our expanded graph library and new RNA structures, to identify potential RNA-like topologies for design. Our coarse-grained approach represents RNA secondary structures as tree and dual graphs, with vertices and edges corresponding to RNA helices and loops. The graph theoretical framework facilitates graph enumeration, partitioning, and clustering approaches to study RNA structure and its applications. Methods: Clustering graph topologies based on features derived from graph Laplacian matrices and known RNA structures allows us to classify topologies into ‘existing’ or hypothetical, and the latter into, ‘RNA-like’ or ‘non RNA-like’ topologies. Here we update our list of existing tree graph topologies and RAG-3D database of atomic fragments to include newly determined RNA structures. We then use linear and quadratic regression, optionally with dimensionality reduction, to derive graph features and apply several clustering algorithms on our tree-graph library and recently expanded dual-graph library to classify them into the three groups. Results: The unsupervised PAM and K-means clustering approaches correctly classify 72–77% of all existing graph topologies and 75–82% of newly added ones as RNA-like. For supervised k-NN clustering, the cross-validation accuracy ranges from 57 to 81%. Conclusions: Using linear regression with unsupervised clustering, or quadratic regression with supervised clustering, provides better accuracies than supervised/linear clustering. All accuracies are better than random, especially for newly added existing topologies, thus lending credibility to our approach. General Significance: Our updated RAG-3D database and motif classification by clustering present new RNA substructures and RNA-like motifs as novel design candidates.

Highly charged, single α-helical (SAH) domains contain a high percentage of Arg, Lys, and Glu residues. Their dynamic salt bridge pairing creates exceptional stiffness of these helical rods, with a persistence length of more than 200 Å for the myosin VI SAH domain. With the aim of modulating the stiffness of the helical structure, we investigated by NMR spectroscopy the effect of substituting key charged Arg, Lys, Glu and Asp residues by Gly or His. Results indicate that such mutations result in transient breaking of the helix at the site of mutation, but with noticeable impact on amide hydrogen exchange rates extending as far as ±2 helical turns, pointing to a substantial degree of cooperativity in SAH helical stability. Whereas a single Gly substitution caused transient breaks for ca 20% of the time, two consecutive Gly substitutions break the helix for ca 65% of the time. NMR relaxation measurements indicate that the exchange rate between intact and broken helix is fast (>300,000 s-1) and that for the wild-type sequence the finite persistence length is dominated by thermal fluctuations of backbone torsion angles and H-bond lengths, and not by transient helix breaking. The double mutation D27H/E28H causes a pH-dependent fraction of helix disruption, where the helix breakage increases from 26% at pH 7.5 to 53% at pH 5.5. The ability to modulate helical integrity by pH may enable incorporation of externally tunable dynamic components in the design of molecular machines.

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme’s intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, non-cooperative variants.

We describe here a simple strategy to characterize transport specificity of NADH:quinone oxidoreductases, using Na+-translocating (NQR) and H+-translocating (NDH-1) enzymes of the soil bacterium Azotobacter vinelandii as the models. Submillimolar concentrations of Na+ and Li+ increased the rate of deaminoNADH oxidation by the inverted membrane vesicles prepared from the NDH-1-deficient strain. The vesicles generated carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-resistant electric potential difference and CCCP-stimulated pH difference (alkalinization inside) in the presence of Na+. These findings testified a primary Na+-pump function of A. vinelandii NQR. Furthermore, ΔpH measurements with fluorescent probes (acridine orange and pyranine) demonstrated that A. vinelandii NQR cannot transport H+ under various conditions. The opposite results obtained in similar measurements with the vesicles prepared from the NQR-deficient strain indicated a primary H+-pump function of NDH-1. Based on our findings, we propose a package of simple experiments that are necessary and sufficient to unequivocally identify the pumping specificity of a bacterial Na+ or H+ transporter. The NQR-deficient strain, but not the NDH-1-deficient one, exhibited impaired growth characteristics under diazotrophic condition, suggesting a role for the Na+ transport in nitrogen fixation by A. vinelandii.

Human peroxidasin 1 (hsPxd01) is a homotrimeric multidomain heme peroxidase embedded in the extracellular matrix. It catalyses the two-electron oxidation of bromide by hydrogen peroxide to hypobromous acid which mediates the formation of essential sulfilimine cross-links between methionine and hydroxylysine residues in collagen IV. This confers critical structural reinforcement to the extracellular matrix. This study presents for the first time transient kinetic measurements of the reactivity of hsPxd01 compound I and compound II with the endogenous one-electron donors nitrite, ascorbate, urate, tyrosine and serotonin using the sequential stopped-flow technique. At pH 7.4 and 25 °C compound I of hsPxd01 is reduced to compound II with apparent second-order rate constants ranging from (1.9 ± 0.1) × 104 M−1 s−1 (urate) to (4.8 ± 0.1) × 105 M−1 s−1 (serotonin). Reduction of compound II to the ferric state occurs with apparent second-order rate constants ranging from (4.3 ± 0.2) × 102 M−1 s−1 (tyrosine) to (7.7 ± 0.1) × 103 M−1 s−1 (serotonin). The relatively fast rates of compound I reduction suggest that these reactions may take place in vivo and modulate bromide oxidation due to formation of compound II. Urate is shown to inhibit the bromination activity of hsPxd01, whereas nitrite stimulates the formation of hypobromous acid. The results are discussed with respect to known kinetic data of homologous mammalian peroxidases and to the physiological role of human peroxidasin 1.

AADC deficiency is a rare genetic disease caused by mutations in the gene of aromatic amino acid decarboxylase, the pyridoxal 5′-phosphate dependent enzyme responsible for the synthesis of dopamine and serotonin. Here, following a biochemical approach together with an in silico bioinformatic analysis, we present a structural and functional characterization of 13 new variants of AADC. The amino acid substitutions are spread over the entire protein from the N-terminal (V60A), to its loop1 (H70Y and F77L), to the large domain (G96R) and its various motifs, i.e. loop2 (A110E), or a core β-barrel either on the surface (P210L, F251S and E283A) or in a more hydrophobic milieu (L222P, F237S and W267R) or loop3 (L353P), and to the C-terminal domain (R453C). Results show that the β-barrel variants exhibit a low solubility and those belonging to the surface tend to aggregate in their apo form, leading to the identification of a new enzymatic phenotype for AADC deficiency. Moreover, five variants of residues belonging to the large interface of AADC (V60A, G96R, A110E, L353P and R453C) are characterized by a decreased catalytic efficiency. The remaining ones (H70Y and F77L) present features typical of apo-to-holo impaired transition. Thus, defects in catalysis or in the acquirement of the correct holo structure are due not only to specific local domain effects but also to long-range effects at either the protein surface or the subunit interface. Altogether, the new characterized enzymatic phenotypes represent a further step in the elucidation of the molecular basis for the disease.

Background Bruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies. Methods Covalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC50 studies) or following a time course where inactivation kinetics were measured. Results Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M−1 s−1 for BTK with selectivity against important off-targets. Conclusions For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. General significance This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.

Background Hybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP. Methods Two repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1–8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated. Results Hybrid peptides KFSP1–4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression. Conclusion Hybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.

Background Epidemiologic studies suggest that diabetes is associated with an increased risk of cancer. Concurrently, clinical trials have shown that metformin, which is a first-line antidiabetic drug, displays anticancer activity. The underlying mechanisms for these effects are, however, still not well recognized. Methods Methods based on atomic force microscopy (AFM) were used to directly evaluate the influence of metformin on the nanomechanical and adhesive properties of endothelial and cancer cells in chronic hyperglycemia. AFM single-cell force spectroscopy (SCFS) was used to measure the total adhesion force and the work of detachment between EA.hy926 endothelial cells and A549 lung carcinoma cells. Nanoindentation with a spherical AFM probe provided information about the nanomechanical properties of cells, particularly the length and grafting density of the glycocalyx layer. Fluorescence imaging was used for glycocalyx visualization and monitoring of E-selectin and ICAM-1 expression. Results SCFS demonstrated that metformin attenuates adhesive interactions between EA.hy926 endothelial cells and A549 lung carcinoma cells in chronic hyperglycemia. Nanoindentation experiments, confirmed by confocal microscopy imaging, revealed metformin-induced recovery of endothelial glycocalyx length and density. The recovery of endothelial glycocalyx was correlated with a decrease in the surface expression of E-selectin and ICAM-1. Conclusion Our results identify metformin-induced endothelial glycocalyx restoration as a key factor responsible for the attenuation of adhesion between EA.hy926 endothelial cells and A549 lung carcinoma cells. General significance Metformin-induced glycocalyx restoration and the resulting attenuation of adhesive interactions between the endothelium and cancer cells may account for the antimetastatic properties of this drug.

In a contracting muscle, myosin cross-bridges extending from thick filaments pull the interdigitating thin (actin-containing) filaments, during cyclical ATP-driven interactions, toward the center of the sarcomere, the structural unit of striated muscle. Cross-bridge attachments in the sarcomere have been reported to exhibit a similar stiffness under both positive and negative forces. However, in vitro measurements on filaments with a sparse complement of heads detected a decrease of the cross-bridge stiffness at negative forces attributed to the buckling of the subfragment 2 tail portion. Here we review some old and new data which confirm that cross-bridge stiffness is nearly linear in the muscle filament lattice. Implications of high myosin stiffness at positive and negative strains are considered in muscle fibers and in non-muscle intracellular cargo transport.

A ubiquitously expressed transcription factor, specificity protein 1 (Sp1), interacts with the amyloid precursor protein (APP) promoter and likely mediates APP expression. Promoter-interaction strengths variably regulate the level of APP expression. Here, we examined the interactions of finger 3 of Sp1 (Sp1–f3) with a DNA fragment containing the APP promoter in different ionic solutions using atomic force microscope (AFM) spectroscopy. Sp1–f3 molecules immobilized on an Si substrate were bound to the APP promoter, which was linked to the AFM tips via covalent bonds. The interactions were strongly influenced by Pb2+, considering that substituting Zn2+ with Pb2+ increased the binding affinity of Sp1 for the APP promoter. The results revealed that the enhanced interaction force facilitated APP expression and that APP overexpression could confer a high-risk for disease incidence. An increased interaction force between Sp1–f3 and the APP promoter in Pb2+ solutions was consistent with a lower binding free energy, as determined by computer-assisted analysis. The impact of Pb2+ on cell morphology and related mechanical properties were also detected by AFM. The overexpression of APP caused by the enhanced interaction force triggered actin reorganization and further resulted in an increased Young's modulus and viscosity. The correlation with single-force measurements revealed that altered cellular activities could result from alternation of Sp1–APP promoter interaction. Our AFM findings offer a new approach in understanding Pb2+ associated neurodegeneration.

One of the essential steps in every viral ‘life’ cycle is entry into the host cell. Membrane-enveloped viruses carry dedicated proteins to catalyse the fusion of the viral and cellular membrane. Herpesviruses feature a set of essential, structurally diverse glycoproteins on the viral surface that form a multicomponent fusion machinery, necessary for the entry mechanism. For Herpes simplex virus 1, these essential glycoproteins are gD, gH, gL and gB. In this review we describe the functions of the individual components, the potential interactions between them as well as the influence of post-translational modifications on the fusion mechanism.

Background Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastasis at the moment of diagnosis. Methods Osteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. Results Our results suggest that metastatic OS cells posses a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. Conclusions All this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. General significance Our work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Background Wolfram syndrome (WS), caused by mutations in WFS1 gene, is a multi-targeting disease affecting multiple organ systems. Wolframin is localized in the membrane of the endoplasmic reticulum (ER), influencing Ca2+ metabolism and ER interaction with mitochondria, but the exact role of the protein remains unclear. In this study we aimed to characterize alterations in energy metabolism in the cardiac and in the oxidative and glycolytic skeletal muscles in Wfs1-deficiency. Methods Alterations in the bioenergetic profiles in the cardiac and skeletal muscles of Wfs1-knock-out (KO) male mice and their wild type male littermates were determined using high resolution respirometry, quantitative RT-PCR, NMR spectroscopy, and immunofluorescence confocal microscopy. Results Oxygen consumption without ATP synthase activation (leak) was significantly higher in the glycolytic muscles of Wfs1 KO mice compared to wild types. ADP-stimulated respiration with glutamate and malate was reduced in the Wfs1-deficient cardiac as well as oxidative and glycolytic skeletal muscles. Conclusions Wfs1-deficiency in both cardiac and skeletal muscles results in functional alterations of energy transport from mitochondria to ATP-ases. There was a substrate-dependent decrease in the maximal Complex I –linked respiratory capacity of the electron transport system in muscles of Wfs1 KO mice. Moreover, in cardiac and gastrocnemius white muscles a decrease in the function of one pathway were balanced by the increase in the activity of the parallel pathway. General significance This work provides new insights to the muscle involvement at early stages of metabolic syndrome like WS as well as developing glucose intolerance.

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Single molecule photobleaching (smPB) is a powerful technique to measure the number of fluorescent units in sub-resolution molecular complexes, such as in toxic protein oligomers associated with amyloid diseases. However, photo-bleaching can occur before the sample is appropriately placed and focused. Such ‘pre-bleaching’ can introduce a strong systematic bias towards smaller oligomers. Quantitative correction of pre-bleaching is known to be an ill-posed problem, limiting the utility of the technique. Here we provide an experimental solution to improve its reliability. We chemically construct multimeric standards to estimate the pre-bleaching probability, B. We show that B can be used as a constraint to reliably correct the statistics obtained from a known distribution of standard oligomers. Finally, we apply this method to the data obtained from a heterogeneous oligomeric solution of human Islet Amyloid Polypeptide (IAPP). Our results show that photobleaching can critically skew the estimation of oligomeric distributions, so that low abundance monomers display a much higher apparent abundance. In summary, any inference from photobleaching experiments with B > 0.1 is likely to be unreliable, but our method can be used to quantitatively correct possible errors.

Voltage-gated proton channels (HV1) are essential for various physiological tasks but are strongly inhibited by Zn2+ cations. Some determinants of Zn2+ binding have been elucidated, experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn2+ binds to monomeric HV1 despite evidence that HV1 expresses as a dimer, that the dimer has a higher affinity for zinc than the monomer, and experimental data that suggests coordination in the dimer interface. The results of former studies are also controversial, e.g. supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed series of molecular dynamics simulations to address different structures of the human proton channel (hHV1), the monomer and two plausible dimer conformations to compare their respective potential to interact with and bind Zn2+ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29,9 μs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric hHV1. The dimer interface is more efficient to attract and bind Zn2+ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations towards the binding sites.

In the peripheral nervous system (PNS), Schwann cells (SCs) are required for the myelination of axons. Periaxin (PRX), one of the myelination proteins expressed in SCs, is critical for the normal development and maintenance of PNS. As a member of the ERM (ezrin-radxin-moesin) protein family, ezrin holds our attention since their link to the formation of the nodes of Ranvier. Furthermore, PRX and ezrin are co-expressed in cytoskeletal complexes with periplakin and desmoyokin in lens fiber cells. In the present study, we observed that L-periaxin and ezrin interacted in a “head to head and tail to tail” mode in SC RSC96 through NLS3 region of L-periaxin with F3 subdomain of ezrin interaction, and the region of L-periaxin (residues 1368–1461) with ezrin (residues 475–557) interaction. A phosphorylation-mimicking mutation of ezrin resulted in L-periaxin accumulation on SC RSC96 membrane. Ezrin could inhibit the self-association of L-periaxin, and ezrin overexpression in sciatic nerve injury rats could facilitate the repair of impaired myelin sheath. Therefore, the interaction between L-periaxin and ezrin may adopt a close form to complete protein accumulation and to participate in myelin sheath maintenance.