Polyphosphate and tyrosine phosphorylation in the N-terminal domain of the human mitochondrial Lon protease disrupts its functions.

Phosphorylation plays a crucial role in the regulation of many fundamental cellular processes. Phosphorylation levels are increased in many cancer cells where they may promote changes in mitochondrial homeostasis. Proteomic studies on various types of cancer identified 17 phosphorylation sites within the human ATP-dependent protease Lon, which degrades misfolded, unassembled and oxidatively damaged proteins in mitochondria. Most of these sites were found in Lon's N-terminal (NTD) and ATPase domains, though little is known about the effects on their function. By combining the biochemical and cryo-electron microscopy studies, we show the effect of Tyr186 and Tyr394 phosphorylations in Lon's NTD, which greatly reduce all Lon activities without affecting its ability to bind substrates or perturbing its tertiary structure. A substantial reduction in Lon's activities is also observed in the presence of polyphosphate, whose amount significantly increases in cancer cells. Our study thus provides an insight into the possible fine-tuning of Lon activities in human diseases, which highlights Lon's importance in maintaining proteostasis in mitochondria.

Eu3+ detects two functionally distinct luminal Ca2+ binding sites in ryanodine receptors.

Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type. RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane's electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate.

Characterization of a newly discovered putative DNA replication initiator from Paenibacillus polymyxa phage phiBP.

The bacteriophage phiBP contains a newly discovered putative replisome organizer, a helicase loader, and a beta clamp, which together may serve to replicate its DNA. Bioinformatics analysis of the phiBP replisome organizer sequence showed that it belongs to a recently identified family of putative initiator proteins. We prepared and isolated a wild type-like recombinant protein, gpRO-HC, and a mutant protein gpRO-HCK8A, containing a lysine to alanine substitution at position 8. gpRO-HC had low ATPase activity regardless of the presence of DNA, while the ATPase activity of the mutant was significantly higher. gpRO-HC bound to both single- and double-stranded DNA substrates. Different methods showed that gpRO-HC forms higher oligomers containing about 12 subunits. This work provides the first information about another group of phage initiator proteins, which trigger DNA replication in phages infecting low GC Gram-positive bacteria.

Yeast Sec14-like lipid transfer proteins Pdr16 and Pdr17 bind and transfer the ergosterol precursor lanosterol in addition to phosphatidylinositol.

Yeast Sec14-like phosphatidylinositol transfer proteins (PITPs) contain a hydrophobic cavity capable of accepting a single molecule of phosphatidylinositol (PI) or another molecule in a mutually exclusive manner. We report here that two yeast Sec14 family PITPs, Pdr16p (Sfh3p) and Pdr17p (Sfh4p), possess high-affinity binding and transfer towards lanosterol. To our knowledge, this is the first identification of lanosterol transfer proteins. In addition, a pdr16Δpdr17Δ double mutant had a significantly increased level of cellular lanosterol compared with the corresponding wild-type. Based on the lipid profiles of wild-type and pdr16Δpdr17Δ cells grown in aerobic and anaerobic conditions, we suggest that PI-lanosterol transfer proteins are important predominantly for the optimal functioning of the post-lanosterol part of sterol biosynthesis.

Synthesis and Optical Characterization of a Rhodamine B Spirolactam Dimer.

Amide derivatives of xanthene dyes such as rhodamine B are useful in a variety of sensing applications due to their colorimetric responses to stimuli such as acidity changes and UV light. The optical properties of these molecules can be influenced by intermolecular associations into dimeric structures, but the exact impact can be hard to predict. We have designed a covalently linked intramolecular dimer of the dye rhodamine B utilizing p-phenylenediamine to link the two dyes via amide bonds. The doubly closed spirolactam version of this dimer, RSL2, is isolated as a colorless solid. Under acidic conditions or UV exposure, RSL2 solutions develop a pink color that is expected for the ring-opened form of the molecule. However, nuclear magnetic resonance (NMR) and single-crystal diffraction data show that the equilibrium still prefers the closed dimer state. Interestingly, the emission profile of RSL2 shows solvatochromic blue fluorescence. Control studies of model compounds with similar structural motifs do not display similar blue fluorescence, indicating that this optical behavior is unique to the dimeric form. This behavior may lend itself to applications of such xanthene dimers to more sophisticated sensors beyond those with traditional binary on/off fluorescence profiles.

Methohexital for procedural sedation of cardioversions in the emergency department.

BACKGROUND: Procedural sedation for electrical cardioversion is a common practice in the emergency department (ED). Ideal sedative properties for this procedure are a short half-life and minimal hemodynamic effects. There is limited literature examining methohexital for this use. OBJECTIVE: To compare the use of methohexital to propofol and etomidate for procedural sedation for electrical cardioversions in the ED. METHODS: This was a single-center, retrospective study of adult patients who underwent procedural sedation for electrical cardioversion in the ED between February 1, 2015 and July 31, 2020. Included patients received methohexital, propofol, or etomidate as an initial sedative agent in the ED. The primary outcome was time from initial dose of sedative to goal Aldrete score. The main secondary outcome was time from sedative agent to ED discharge. The safety outcome was the occurrence of a critical hemodynamic change requiring intervention. Outcomes were assessed using a single-factor ANOVA analysis. RESULTS: One-hundred and fifty cardioversion encounters were included with 50 encounters per cohort. The median (IQR) time (minutes) to goal Aldrete score was 10.5 (7-18.5) for methohexital, 12.0 (9-16.8) for propofol, and 11.0 (8-15) for etomidate (p = 0.863). Mean (SD) time (minutes) to discharge from the ED (n = 105) was 90.4 ± 40.4 for methohexital, 89.0 ± 57.4 for propofol, and 94.0 ± 42.5 for etomidate (p = 0.897). No difference was seen between the groups regarding hemodynamic changes requiring intervention. CONCLUSION: Methohexital was found to have a similar efficacy and safety profile when compared to propofol and etomidate when used as procedural sedation for cardioversions in the ED.

Extracting the Dynamic Motion of Proteins Using Normal Mode Analysis.

Normal mode analysis (NMA) is a technique for describing the conformational states accessible to a protein in a minimum energy conformation. NMA gives results similar to those produced by principal components analysis of a molecular dynamics simulation, but with only a fraction of the computational effort. Here, we provide a brief overview of the theory and describe three methods for carrying out NMA, including the use of one of the on-line services, the use of off-line software for calculating the projection of the modes calculated from one conformation onto another, and an all-atom NMA calculated using GROMACS. For all three methods, we will use the E1·2Ca2+ form of the Ca2+-ATPase as a concrete example.

Glucose Oxidase, an Enzyme "Ferrari": Its Structure, Function, Production and Properties in the Light of Various Industrial and Biotechnological Applications.

Glucose oxidase (GOx) is an important oxidoreductase enzyme with many important roles in biological processes. It is considered an "ideal enzyme" and is often called an oxidase "Ferrari" because of its fast mechanism of action, high stability and specificity. Glucose oxidase catalyzes the oxidation of ß-d-glucose to d-glucono-δ-lactone and hydrogen peroxide in the presence of molecular oxygen. d-glucono-δ-lactone is sequentially hydrolyzed by lactonase to d-gluconic acid, and the resulting hydrogen peroxide is hydrolyzed by catalase to oxygen and water. GOx is presently known to be produced only by fungi and insects. The current main industrial producers of glucose oxidase are Aspergillus and Penicillium. An important property of GOx is its antimicrobial effect against various pathogens and its use in many industrial and medical areas. The aim of this review is to summarize the structure, function, production strains and biophysical and biochemical properties of GOx in light of its various industrial, biotechnological and medical applications.

Mitochondrial Processing Peptidases-Structure, Function and the Role in Human Diseases.

Mitochondrial proteins are encoded by both nuclear and mitochondrial DNA. While some of the essential subunits of the oxidative phosphorylation (OXPHOS) complexes responsible for cellular ATP production are synthesized directly in the mitochondria, most mitochondrial proteins are first translated in the cytosol and then imported into the organelle using a sophisticated transport system. These proteins are directed mainly by targeting presequences at their N-termini. These presequences need to be cleaved to allow the proper folding and assembly of the pre-proteins into functional protein complexes. In the mitochondria, the presequences are removed by several processing peptidases, including the mitochondrial processing peptidase (MPP), the inner membrane processing peptidase (IMP), the inter-membrane processing peptidase (MIP), and the mitochondrial rhomboid protease (Pcp1/PARL). Their proper functioning is essential for mitochondrial homeostasis as the disruption of any of them is lethal in yeast and severely impacts the lifespan and survival in humans. In this review, we focus on characterizing the structure, function, and substrate specificities of mitochondrial processing peptidases, as well as the connection of their malfunctions to severe human diseases.

Interpretation of Single-Molecule Force Experiments on Proteins Using Normal Mode Analysis.

Single-molecule force spectroscopy experiments allow protein folding and unfolding to be explored using mechanical force. Probably the most informative technique for interpreting the results of these experiments at the structural level makes use of steered molecular dynamics (MD) simulations, which can explicitly model the protein under load. Unfortunately, this technique is computationally expensive for many of the most interesting biological molecules. Here, we find that normal mode analysis (NMA), a significantly cheaper technique from a computational perspective, allows at least some of the insights provided by MD simulation to be gathered. We apply this technique to three non-homologous proteins that were previously studied by force spectroscopy: T4 lysozyme (T4L), Hsp70 and the glucocorticoid receptor domain (GCR). The NMA results for T4L and Hsp70 are compared with steered MD simulations conducted previously, and we find that we can recover the main results. For the GCR, which did not undergo MD simulation, our approach identifies substructures that correlate with experimentally identified unfolding intermediates. Overall, we find that NMA can make a valuable addition to the analysis toolkit for the structural analysis of single-molecule force experiments on proteins.

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids.

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.

Mitochondrial HSP70 Chaperone System-The Influence of Post-Translational Modifications and Involvement in Human Diseases.

Since their discovery, heat shock proteins (HSPs) have been identified in all domains of life, which demonstrates their importance and conserved functional role in maintaining protein homeostasis. Mitochondria possess several members of the major HSP sub-families that perform essential tasks for keeping the organelle in a fully functional and healthy state. In humans, the mitochondrial HSP70 chaperone system comprises a central molecular chaperone, mtHSP70 or mortalin (HSPA9), which is actively involved in stabilizing and importing nuclear gene products and in refolding mitochondrial precursor proteins, and three co-chaperones (HSP70-escort protein 1-HEP1, tumorous imaginal disc protein 1-TID-1, and Gro-P like protein E-GRPE), which regulate and accelerate its protein folding functions. In this review, we summarize the roles of mitochondrial molecular chaperones with particular focus on the human mtHsp70 and its co-chaperones, whose deregulated expression, mutations, and post-translational modifications are often considered to be the main cause of neurological disorders, genetic diseases, and malignant growth.

Mitochondrial Kinases and the Role of Mitochondrial Protein Phosphorylation in Health and Disease.

The major role of mitochondria is to provide cells with energy, but no less important are their roles in responding to various stress factors and the metabolic changes and pathological processes that might occur inside and outside the cells. The post-translational modification of proteins is a fast and efficient way for cells to adapt to ever changing conditions. Phosphorylation is a post-translational modification that signals these changes and propagates these signals throughout the whole cell, but it also changes the structure, function and interaction of individual proteins. In this review, we summarize the influence of kinases, the proteins responsible for phosphorylation, on mitochondrial biogenesis under various cellular conditions. We focus on their role in keeping mitochondria fully functional in healthy cells and also on the changes in mitochondrial structure and function that occur in pathological processes arising from the phosphorylation of mitochondrial proteins.

The vascular occlusion test using multispectral imaging: a validation study : The VASOIMAGE study.

Multispectral imaging (MSI) is a new, non-invasive method to continuously measure oxygenation and microcirculatory perfusion, but has limitedly been validated in healthy volunteers. The present study aimed to validate the potential of multispectral imaging in the detection of microcirculatory perfusion disturbances during a vascular occlusion test (VOT). Two consecutive VOT's were performed on healthy volunteers and tissue oxygenation was measured with MSI and near-infrared spectroscopy (NIRS). Correlations between the rate of desaturation, recovery and the hyperemic area under the curve (AUC) measured by MSI and NIRS were calculated. Fifty-eight volunteers were included. The MSI oxygenation curves showed identifiable components of the VOT, including a desaturation and recovery slope and hyperemic area under the curve, similar to those measured with NIRS. The correlation between the rate of desaturation measured by MSI and NIRS was moderate: r = 0.42 (p = 0.001) for the first and r = 0.41 (p = 0.002) for the second test. Our results suggest that non-contact multispectral imaging is able to measure changes in regional oxygenation and deoxygenation during a vascular occlusion test in healthy volunteers. When compared to measurements with NIRS, correlation of results was moderate to weak, most likely reflecting differences in physiology of the regions of interest and measurement technique.

Structure and Function of the Human Ryanodine Receptors and Their Association with Myopathies-Present State, Challenges, and Perspectives.

Cardiac arrhythmias are serious, life-threatening diseases associated with the dysregulation of Ca2+ influx into the cytoplasm of cardiomyocytes. This dysregulation often arises from dysfunction of ryanodine receptor 2 (RyR2), the principal Ca2+ release channel. Dysfunction of RyR1, the skeletal muscle isoform, also results in less severe, but also potentially life-threatening syndromes. The RYR2 and RYR1 genes have been found to harbor three main mutation "hot spots", where mutations change the channel structure, its interdomain interface properties, its interactions with its binding partners, or its dynamics. In all cases, the result is a defective release of Ca2+ ions from the sarcoplasmic reticulum into the myocyte cytoplasm. Here, we provide an overview of the most frequent diseases resulting from mutations to RyR1 and RyR2, briefly review some of the recent experimental structural work on these two molecules, detail some of the computational work describing their dynamics, and summarize the known changes to the structure and function of these receptors with particular emphasis on their N-terminal, central, and channel domains.

Mitochondrial HMG-Box Containing Proteins: From Biochemical Properties to the Roles in Human Diseases.

Mitochondrial DNA (mtDNA) molecules are packaged into compact nucleo-protein structures called mitochondrial nucleoids (mt-nucleoids). Their compaction is mediated in part by high-mobility group (HMG)-box containing proteins (mtHMG proteins), whose additional roles include the protection of mtDNA against damage, the regulation of gene expression and the segregation of mtDNA into daughter organelles. The molecular mechanisms underlying these functions have been identified through extensive biochemical, genetic, and structural studies, particularly on yeast (Abf2) and mammalian mitochondrial transcription factor A (TFAM) mtHMG proteins. The aim of this paper is to provide a comprehensive overview of the biochemical properties of mtHMG proteins, the structural basis of their interaction with DNA, their roles in various mtDNA transactions, and the evolutionary trajectories leading to their rapid diversification. We also describe how defects in the maintenance of mtDNA in cells with dysfunctional mtHMG proteins lead to different pathologies at the cellular and organismal level.

Disease-associated mutations alter the dynamic motion of the N-terminal domain of the human cardiac ryanodine receptor.

The human cardiac ryanodine receptor (hRyR2), the ion channel responsible for the release of Ca2+ ions from the sarcoplasmic reticulum into the cytosol, plays an important role in cardiac muscle contraction. Mutations to this channel are associated with inherited cardiac arrhythmias. These mutations appear to cluster in distinct parts of the N-terminal, central and C-terminal areas of the channel. Here, we used molecular dynamics simulation to examine the effects three disease-associated mutations to the N-terminal region, R414L, I419F and R420W, have on the dynamics of a model of residues 1-655 of hRyR2. We find that the R414L and I419F mutations diminish the overall amplitude of motion without greatly changing the direction of motion of the individual domains, whereas R420W both enhances the amplitude and changes the direction of motion. Based on these results, we hypothesize that R414L and I419F hinder channel closing, whereas R420W may enhance channel opening. Overall, it appears that the wild-type protein possesses a moderate level of flexibility which allows the gate to close and not easily open without an opening signal. These mutations, however, disrupt this balance by making the gate either too rigid or too loose, causing closing to become difficult or less effective. Small-angle X-ray scattering studies of the same 1-655 residue fragment are in agreement with the molecular dynamics results and also suggest that the rest of the protein is needed to keep the entire domain properly folded.Communicated by Ramaswamy H. Sarma.

An Evaluation Framework for Spectral Filter Array Cameras to Optimize Skin Diagnosis.

Comparing and selecting an adequate spectral filter array (SFA) camera is application-specific and usually requires extensive prior measurements. An evaluation framework for SFA cameras is proposed and three cameras are tested in the context of skin analysis. The proposed framework does not require application-specific measurements and spectral sensitivities together with the number of bands are the main focus. An optical model of skin is used to generate a specialized training set to improve spectral reconstruction. The quantitative comparison of the cameras is based on reconstruction of measured skin spectra, colorimetric accuracy, and oxygenation level estimation differences. Specific spectral sensitivity shapes influence the results directly and a 9-channel camera performed best regarding the spectral reconstruction metrics. Sensitivities at key wavelengths influence the performance of oxygenation level estimation the strongest. The proposed framework allows to compare spectral filter array cameras and can guide their application-specific development.

Normal Mode Analysis as a Routine Part of a Structural Investigation.

Normal mode analysis (NMA) is a technique that can be used to describe the flexible states accessible to a protein about an equilibrium position. These states have been shown repeatedly to have functional significance. NMA is probably the least computationally expensive method for studying the dynamics of macromolecules, and advances in computer technology and algorithms for calculating normal modes over the last 20 years have made it nearly trivial for all but the largest systems. Despite this, it is still uncommon for NMA to be used as a component of the analysis of a structural study. In this review, we will describe NMA, outline its advantages and limitations, explain what can and cannot be learned from it, and address some criticisms and concerns that have been voiced about it. We will then review the most commonly used techniques for reducing the computational cost of this method and identify the web services making use of these methods. We will illustrate several of their possible uses with recent examples from the literature. We conclude by recommending that NMA become one of the standard tools employed in any structural study.

Residue Mutations in Murine Herpesvirus 68 Immunomodulatory Protein M3 Reveal Specific Modulation of Chemokine Binding.

The M3 protein (M3) encoded by murine gammaherpesvirus 68 (MHV-68) is a unique viral immunomodulator with a high-affinity for a broad spectrum of chemokines, key mediators responsible for the migration of immune cells to sites of inflammation. M3 is currently being studied as a very attractive and desirable tool for blocking the chemokine signaling involved in some inflammatory diseases and cancers. In this study, we elucidated the role of M3 residues E70 and T272 in binding to chemokines by examining the effects of the E70A and T272G mutations on the ability of recombinant M3, prepared in Escherichia coli cells, to bind the human chemokines CCL5 and CXCL8. We found that the E70A mutation enhanced binding of M3 to CCL5 two-fold but had little effect on its binding to CXCL8. In contrast, the T272G mutation was found to be important for the thermal stability of M3 and significantly decreased M3's binding to both CCL5 (by about 4×) and CXCL8 (by about 5×). We also constructed in silico models of the wild-type M3-CCL5 and M3-CCL8 complexes and found substantial differences in their physical and chemical properties. M3 models with single mutation E70A and T272G suggested the role of E70 and T272 in binding M3 protein to chemokines. In sum, we have confirmed that site-directed mutagenesis could be an effective tool for modulating the blockade of particular chemokines by M3, as desired in therapeutic treatments for severe inflammatory illnesses arising from chemokine network dysregulation.