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2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C-40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4. [ABSTRACT FROM AUTHOR]

Introduction: Prion diseases are deadly neurodegenerative disorders in both animals and humans, causing the destruction of neural tissue and inducing behavioral manifestations. Heat shock proteins (Hsps), act as molecular chaperones by supporting the appropriate folding of proteins and eliminating the misfolded proteins as well as playing a vital role in cell signaling transduction, cell cycle, and apoptosis control. SW02 is a potent activator of Hsp 70 kDa (Hsp70). Methods: In the current study, the protective effects of SW02 against prion protein 106-126 (PrP106-126)-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) were investigated. In addition, the therapeutic effects of SW02 in ME7 scrapie-infected mice were evaluated. Results: The results showed that SW02 treatment significantly increased Hsp70 mRNA expression levels and Hsp70 ATPase activity (p < 0.01). SW02 also significantly inhibited cytotoxicity and apoptosis induced by PrP106-126 (p < 0.01) and promoted neurite extension. In vivo, intraperitoneal administration of SW02 did not show a statistically significant difference in survival time (p = 0.16); however, the SW02-treated group exhibited a longer survival time of 223.6 ± 6.0 days compared with the untreated control group survival time of 217.6 ± 5.4 days. In addition, SW02 reduced the PrPSc accumulation in ME7 scrapie-infected mice at 5 months post-injection (p < 0.05). A significant difference was not observed in GFAP expression, an astrocyte marker, between the treated and untreated groups. Conclusion: In conclusion, the potential therapeutic role of the Hsp70 activator SW02 was determined in the present study and may be a novel and effective drug to mitigate the pathologies of prion diseases and other neurodegenerative diseases. Further studies using a combination of two pharmacological activators of Hsp70 are required to maximize the effectiveness of each intervention. [ABSTRACT FROM AUTHOR]

Type VII secretion (T7S) systems, also referred to as ESAT‐6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host–pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX‐5 system is exclusively found in slow‐growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N‐terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX‐3 and ESX‐5 systems have revealed EccCDUF to be an ATPase‐like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide‐free structure with degenerated cis‐acting and trans‐acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX‐like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N‐terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter‐protomer interactions. [ABSTRACT FROM AUTHOR]

Introduction: ATP-binding cassette (ABC) transporters use the hydrolysis of ATP to power the active transport of molecules, but paradoxically the cystic fibrosis transmembrane regulator (CFTR, ABCC7) forms an ion channel. We previously showed that ATP-binding cassette subfamily C member 4 (ABCC4) is the closest mammalian paralog to CFTR, compared to other ABC transporters. In addition, Lamprey CFTR (Lp-CFTR) is the oldest known CFTR ortholog and has unique structural and functional features compared to human CFTR (hCFTR). The availability of these evolutionarily distant orthologs gives us the opportunity to study the changes in ATPase activity that may be related to their disparate functions. Methods: We utilized the baculovirus expression system with Sf9 insect cells and made use of the highly sensitive antimony-phosphomolybdate assay for testing the ATPase activity of human ABCC4 (hABCC4), Lp-CFTR, and hCFTR under similar experimental conditions. This assay measures the production of inorganic phosphate (Pi) in the nanomolar range. Results: Crude plasma membranes were purified, and protein concentration, determined semi-quantitatively, of hABCC4, Lp-CFTR, and hCFTR ranged from 0.01 to 0.36 μg/μL. No significant difference in expression level was found although hABCC4 trended toward the highest level. hABCC4 was activated by ATP with the equilibrium constant (Kd) 0.55 ± 0.28 mM (n = 8). Estimated maximum ATPase rate (Vmax) for hABCC4 was about 0.2 nmol/μg/min when the protein was activated with 1mM ATP at 37°C (n = 7). Estimated maximum ATPase rate for PKA-phosphorylated Lp-CFTR reached about half of hCFTR levels in the same conditions. Vmax for both Lp-CFTR and hCFTR were significantly increased in high PKA conditions compared to low PKA conditions. Maximum intrinsic ATPase rate of hABCC4 in the absence of substrate was twice that of hCFTR when activated in 1mM ATP. Conclusion: The findings here suggest that while both ABCC4 and hCFTR bear one consensus and one degenerate ATPase site, the hCFTR exhibited a reduced intrinsic ATPase activity. In addition, ATPase activity in the CFTR lineage increased from Lp-CFTR to hCFTR. Finally, the studies pave the way to purify hABCC4, Lp-CFTR, and hCFTR from Sf9 cells for their structural investigation, including by cryo-EM, and for studies of evolution in the ABC transporter superfamily. [ABSTRACT FROM AUTHOR]

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C–40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

IntroductionPrion diseases are deadly neurodegenerative disorders in both animals and humans, causing the destruction of neural tissue and inducing behavioral manifestations. Heat shock proteins (Hsps), act as molecular chaperones by supporting the appropriate folding of proteins and eliminating the misfolded proteins as well as playing a vital role in cell signaling transduction, cell cycle, and apoptosis control. SW02 is a potent activator of Hsp 70 kDa (Hsp70).MethodsIn the current study, the protective effects of SW02 against prion protein 106-126 (PrP106-126)-induced neurotoxicity in human neuroblastoma cells (SH-SY5Y) were investigated. In addition, the therapeutic effects of SW02 in ME7 scrapie-infected mice were evaluated.ResultsThe results showed that SW02 treatment significantly increased Hsp70 mRNA expression levels and Hsp70 ATPase activity (p < 0.01). SW02 also significantly inhibited cytotoxicity and apoptosis induced by PrP106-126 (p < 0.01) and promoted neurite extension. In vivo, intraperitoneal administration of SW02 did not show a statistically significant difference in survival time (p = 0.16); however, the SW02-treated group exhibited a longer survival time of 223.6 ± 6.0 days compared with the untreated control group survival time of 217.6 ± 5.4 days. In addition, SW02 reduced the PrPSc accumulation in ME7 scrapie-infected mice at 5 months post-injection (p < 0.05). A significant difference was not observed in GFAP expression, an astrocyte marker, between the treated and untreated groups.ConclusionIn conclusion, the potential therapeutic role of the Hsp70 activator SW02 was determined in the present study and may be a novel and effective drug to mitigate the pathologies of prion diseases and other neurodegenerative diseases. Further studies using a combination of two pharmacological activators of Hsp70 are required to maximize the effectiveness of each intervention.

Background and Aim: Pollution and lifestyle changes expose mankind to a number of toxicants such as polycyclic aromatic hydrocarbons that may cause life-threatening diseases. The present study was undertaken to explicate the protective role of Acetyl-11-Keto-Beta Boswellic Acid (AKBA), if any, in containing benzo(a)pyrene (BaP) induced alterations on hepatic membrane dynamics and lipidiome in female rats. Experimental procedure: The animals were divided into five groups viz. Normal control, Vehicle treated, BaP treated, AKBA treated and BaP + AKBA treated. To induce hepatotoxicity, BaP was administered orally at a dose level of 50 mg/kg b.wt. dissolved in olive oil twice a week for 4 weeks. AKBA was supplemented to animals four weeks prior to BaP treatment and continued for 8 weeks at a dose level of 50 mg/kg b.wt. thrice a week. Certain key indices that included oxidative stress biomarkers, lipid profile of membranes, membrane fluidity parameters and activities of ATPases were studied. Results and Conclusion: The results showed that benzo(a)pyrene treatment resulted in a significant increase in the levels of lipid peroxidation (LPO) and ROS but caused a significant decrease in the levels of total lipids, phospholipids, cholesterol, glycolipids and activities of ATPases. Hepatic membrane fluidity as assessed by 1,6-Diphenyl-1,3,5-hexatriene (DPH) and Pyrene fluorescence probes was significantly increased in rats intoxicated with BaP. Interestingly, AKBA supplementation to BaP treated rats appreciably contained altered membrane dynamics and lipidiome as well as modulated hepatotoxicity by skirmishing activities of oxidative stress markers and also improved hepatic histoarchitecture. Our study thus concludes that AKBA can be used as a prophylactic intervention in providing protection to hepatocytes as it maintains membrane integrity in conditions of BaP induced toxicity.

Proper plant nutrition can be an effective strategy to alleviate the phytotoxicity and damaging effects of Cd stress on plants and to avoid its entry into the food chain. This study aimed to investigate effects of phosphorus concentrations, and pH levels in the nutrient solution on Cd uptake, growth, photosynthetic characters, total phytochelatins (PCs), and plasma membrane ATPase activity of sugar beet (Beta vulgaris L.) plants grown in sand culture. Two successive experimental studies were carried out under controlled conditions. In the first study, 10-day-old sugar beet seedlings were irrigated with a half-strength Hoagland and Arnon nutrient solution, adjusted to different pH (3.5, 5.0, 6.0, 7.0, or 8.0), and containing 0, 0.15, or 0.30 mg Cd L−1 as CdCl2. In the second study, 10-day-old sugar beet seedlings were irrigated with the same nutrient solution (pH 6), but combined with different phosphorus concentrations (0, 10, 25 and 50 µg P ml−1 as KH2PO4) and Cd levels (0, 0.15, and 0.30 mg Cd L−1 as CdCl2). The first study revealed that pH had a strong influence on Cd uptake by sugar beet roots. Cd stress significantly decreased sugar beet growth and Mg2+-ATPase activity, whereas it increased total phytochelatins (PCs). Second study indicated an antagonistic effect between P and Cd. Furthermore, phosphorus had the potential to stimulate Mg2+-ATPase activity and synthesis of photosynthetic pigments and to promote growth of sugar beet seedlings. Cadmium induced phytotoxicity in sugar beet seedlings can be alleviated by a proper pH and phosphorus nutrition. [ABSTRACT FROM AUTHOR]

In Vibrio cholerae, the master regulator FlrA controls transcription of downstream flagellar genes in a σ54‐dependent manner. However, the molecular basis of regulation by VcFlrA, which contains a phosphorylation‐deficient N‐terminal FleQ domain, has remained elusive. Our studies on VcFlrA, four of its constructs, and a mutant showed that the AAA+ domain of VcFlrA, with or without the linker 'L', remains in ATPase‐deficient monomeric states. By contrast, the FleQ domain plays a pivotal role in promoting higher‐order functional oligomers, providing the required conformation to 'L' for ATP/cyclic di‐GMP (c‐di‐GMP) binding. The crystal structure of VcFlrA‐FleQ at 2.0 Å suggests that distinct structural features of VcFlrA‐FleQ presumably assist in inter‐domain packing. VcFlrA at a high concentration forms ATPase‐efficient oligomers when the intracellular c‐di‐GMP level is low. Conversely, excess c‐di‐GMP locks VcFlrA in a non‐functional lower oligomeric state, causing repression of flagellar biosynthesis. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum is a unicellular protozoan parasite and causative agent of a severe form of malaria in humans, accounting for very high worldwide fatality rates. At the molecular level, survival of the parasite within the human host is mediated by P. falciparum heat shock proteins (PfHsps) that provide protection during febrile episodes. The ATP-dependent chaperone activity of Hsp70 relies on the co-chaperone J domain protein (JDP), with which it forms a chaperone-co-chaperone complex. The exported P. falciparum JDP (PfJDP), PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots, highly mobile structures found in the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full-length exported PfJDP, PFE0055c. Recombinant PFE0055c was successfully expressed and purified and found to stimulate the basal ATPase activity of PfHsp70-x to a greater extent than PFA0660w but, like PFA0660w, did not significantly stimulate the basal ATPase activity of human Hsp70. Small-molecule inhibition assays were conducted to determine the effect of known inhibitors of JDPs (chalcone, C86) and Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) on the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. In this study, JG231 and JG98 were found to inhibit both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future antimalarial drug discovery studies.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

The surimi-like material made from duck (DSLM) was investigated on the freeze-thaw stability during 4 months of frozen storage using different low-sweetness cryoprotectants such as palatinose (PAL), trehalose (TRE), polydextrose (PLD), maltodextrin (MAL), sucrose and sorbitol (SS), and lactitol (LAC) at a 6% concentration. As compared to the CON, the inclusion of cryoprotectants significantly improved the water-holding capacity of all treated samples. Particularly, as compared to all of the cryoprotectants evaluated, the PLD and PAL samples displayed greater stability concerning Ca2+-ATPase activity, sulfhydryl concentration, and protein solubility. All samples' endothermic peaks declined over time, showing changes in thermal characteristics during storage. A scanning electron microscope (SEM) observation of the CON and LAC-treated samples after 4 months of frozen storage revealed an increase in the number of pores. These findings imply that low-sweetness PLD and PAL outperformed the usual cryoprotectant combination of sucrose and sorbitol (SS) and that they could be used in frozen DSLM. They provide greater water-holding capacity, anti-freezing characteristics, lowered sulfhydryl oxidation, increased protein solubility, and structural integrity, which renders them useful components for innovation in the frozen surimi-like material industry. [ABSTRACT FROM AUTHOR]

The human ABCB1 (P-glycoprotein, Pgp) protein is an active exporter expressed in the plasma membrane of cells forming biological barriers. In accordance with its broad substrate spectrum and tissue expression pattern, it affects the pharmacokinetics of numerous chemotherapeutic drugs and it is involved in unwanted drug–drug interactions leading to side effects or toxicities. When expressed in tumor tissues, it contributes to the development of chemotherapy resistance in malignancies. Therefore, the understanding of the molecular details of the ligand–ABCB1 interactions is of crucial importance. In a previous study, we found that quercetin (QUR) hampers both the transport and ATPase activity of ABCB1, while cyandin-3O-sophroside (C3S) stimulates the ATPase activity and causes only a weak inhibition of substrate transport. In the current study, when QUR and C3S were applied together, both a stronger ATPase inhibition and a robust decrease in substrate transport were observed, supporting their synergistic ABCB1 inhibitory effect. Similar to cyclosporine A, a potent ABCB1 inhibitor, co-treatment with QUR and C3S shifted the conformational equilibrium to the "inward-facing" conformer of ABCB1, as it was detected by the conformation-selective UIC2 mAb. To gain deeper insight into the molecular details of ligand–ABCB1 interactions, molecular docking experiments and MD simulations were also carried out. Our in silico studies support that QUR and C3S can bind simultaneously to ABCB1. The most favourable ligand–ABCB1 interaction is obtained when C3S binds to the central substrate binding site and QUR occupies the "access tunnel". Our results also highlight that the strong ABCB1 inhibitory effect of the combined treatment with QUR and C3S may be exploited in chemotherapy protocols for the treatment of multidrug-resistant tumors or for improving drug delivery through pharmacological barriers. [ABSTRACT FROM AUTHOR]

This research is aimed at the investigation the electrosurface and biochemical parameters of bacterial cells B. cereus B4368, L. plantarum, E. coli K-A, P. fluorescens B5040 under the influence of copper in ionic form and as nanoparticles in order to determine the nature and level of their toxic effect on bacteria. Copper nanoparticles synthesized in aqueous solution with NaBH4 and stabilized with dextran were used. Changes in membrane transport parameters were assessed by the value of ATPase activity; changes in transmembrane potential were assessed by the method of penetrating tetraphenylphosphonium cations (TPP+); and bacterial integrity was assessed by UV spectroscopy of cellular metabolites. A concentration-dependent inhibition of the membrane ATPase reaction and dissipation of the transmembrane potential under the action of both forms of copper was found, and the inhibitory effect in the case of the nanoparticles was on average 20 % higher than in the ionic form. As a result of heterocoagulation of dextran-stabilized copper nanoparticles and bacteria, a decrease in the negative ξ - potential of bacteria was observed, which was 40 % more effective under the action of copper nanoparticles compared to Cu2+ ions. The most significant changes in membrane parameters were observed in the range 10–60 μM of copper concentrations. With B. cereus B4368 cells taken as an example, we found a violation of the barrier function of their cell membrane under the influence of both copper preparations. In the case of copper nanoparticles, nucleic acid leakage from the bacterial cytoplasm was detected, which was confirmed by the absorption band at 260 nm. The results obtained indicate a high level of sensitivity of the studied electrosurface and biochemical parameters of bacterial cells to the effects of ionic and nanoparticle copper, which allows them to be used as indicators of the toxicity of metal nanoparticles in the development of metal-containing probiotic preparations. [ABSTRACT FROM AUTHOR]

The binding of calcium and magnesium ions to proteins is crucial for regulating heart contraction. However, other divalent cations, including xenobiotics, can accumulate in the myocardium and enter cardiomyocytes, where they can bind to proteins. In this article, we summarized the impact of these cations on myosin ATPase activity and EF-hand proteins, with special attention given to toxic cations. Optimal binding to EF-hand proteins occurs at an ionic radius close to that of Mg2+ and Ca2+. In skeletal Troponin C, Cd2+, Sr2+, Pb2+, Mn2+, Co2+, Ni2+, Ba2+, Mg2+, Zn2+, and trivalent lanthanides can substitute for Ca2+. As myosin ATPase is not a specific MgATPase, Ca2+, Fe2+, Mn2+, Ni2+, and Sr2+ could support myosin ATPase activity. On the other hand, Zn2+ and Cu2 significantly inhibit ATPase activity. The affinity to various divalent cations depends on certain proteins or their isoforms and can alter with amino acid substitution and post-translational modification. Cardiac EF-hand proteins and the myosin ATP-binding pocket are potential molecular targets for toxic cations, which could significantly alter the mechanical characteristics of the heart muscle at the molecular level. [ABSTRACT FROM AUTHOR]

Introduction: Protein p97 is an extensively investigated AAA ATPase with various cellular activities, including cell cycle control, ubiquitin-proteasome system, autophagy, and NF-κB activation. Method: In this study, we designed, synthesized and evaluated eight novel DBeQanalogs as potential p97 inhibitors in vivo and in vitro. Results: In the p97 ATPase inhibition assay, compounds 6 and 7 showed higher potency than the known p97 inhibitors, DBeQ and CB-5083. Compounds 4-6 dramatically induced G0/G1 phase arrest in the HCT116 cells, and compound 7 arrested the cells in both G0/G1 and S phases. Western blots showed elevated levels of SQSTM/p62, ATF-4, and NF-κB in HCT116 cells with the treatment of compounds 4-7, confirming their role in inhibiting the p97 signaling pathway in cells. In addition, the IC50 of compounds 4-6 against HCT116, RPMI-8226, and s180 proliferation were 0.24-6.9 µM with comparable potency as DBeQ. However, compounds 4-6 displayed low toxicity against the normal human colon cell line. Thus, compounds 6 and 7 were proved to be potential p97 inhibitors with less cytotoxicity. In vivo studies using the s180 xenograft model have demonstrated that compound 6 inhibited tumor growth, led to a significant reduction of p97 concentration in the serum and tumor, and indicated non-toxicity on the body weight and organ-to-brain weight ratios except for the spleen at the dose of 90 μmol/kg/day for 10 days. Furthermore, the present study indicated that compound 6 may not induce s180 mice myelosuppression often observed in the p97 inhibitors. Conclusion: Compound 6 displayed high binding affinity to p97, great p97 ATPase inhibition, selective cytotoxicity, remarkable anti-tumor effect, and upregulated safety, which improved the clinical potential of p97 inhibitors. [ABSTRACT FROM AUTHOR]

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell. Many ATP-binding cassette (ABC) transporters display a basal ATPase activity in the absence of transported substrates. Multidrug ABC exporters function according to an alternating access mechanism between inward- and outward-facing conformations. The basal ATPase activity of microbial multidrug ABC exporters is often either not or poorly stimulated by the presence of drugs. The sole binding of drugs to multidrug ABC exporters does not produce a major conformational change. When drugs stimulate the ATPase activity, they likely shift the equilibrium toward the outward-facing conformation of multidrug ABC exporters, a conformation prone to drug release. [ABSTRACT FROM AUTHOR]

Much is known about the structure, function, and stability of the SecA motor ATPase that powers the secretion of periplasmic proteins across the inner membrane of Escherichia coli. Most studies of SecA are carried out in buffered sodium or potassium chloride salt solutions. However, the principal intracellular salt of E. coli is potassium glutamate (KGlu), which is known to stabilize folded proteins and protein-nucleic acid complexes. Here we report that KGlu stabilizes SecA, including its dimeric state, and increases its ATPase activity, suggesting that SecA is likely fully folded, stable, and active in vivo at 37°C. Furthermore, KGlu also stabilizes a precursor form of the secreted maltose-binding protein.

Simple Summary: In comparison to other neoplasias, Multiple Myeloma is extremely sensitive to changes in protein homeostasis. Therefore, proteasome inhibitors are highly efficient and widely used for this disease. Genetic alterations of the PSMC genes, such as the ones shown in this manuscript, affect this Multiple Myeloma vulnerability and, therefore, play a key role in the physiopathogenesis and resistance to proteasome inhibitors. By different modes of action, those alterations are likely to increase the proteolytic capacity of cancer cells. Even though they occur in low frequencies, they can serve as biomarkers to predict and monitor a patient's response to proteasome inhibitors over time and might be useful to guide therapy. For the treatment of Multiple Myeloma, proteasome inhibitors are highly efficient and widely used, but resistance is a major obstacle to successful therapy. Several underlying mechanisms have been proposed but were only reported for a minority of resistant patients. The proteasome is a large and complex machinery. Here, we focus on the AAA ATPases of the 19S proteasome regulator (PSMC1-6) and their implication in PI resistance. As an example of cancer evolution and the acquisition of resistance, we conducted an in-depth analysis of an index patient by applying FISH, WES, and immunoglobulin-rearrangement sequencing in serial samples, starting from MGUS to newly diagnosed Multiple Myeloma to a PI-resistant relapse. The WES analysis uncovered an acquired PSMC2 Y429S mutation at the relapse after intensive bortezomib-containing therapy, which was functionally confirmed to mediate PI resistance. A meta-analysis comprising 1499 newly diagnosed and 447 progressed patients revealed a total of 36 SNVs over all six PSMC genes that were structurally accumulated in regulatory sites for activity such as the ADP/ATP binding pocket. Other alterations impact the interaction between different PSMC subunits or the intrinsic conformation of an individual subunit, consequently affecting the folding and function of the complex. Interestingly, several mutations were clustered in the central channel of the ATPase ring, where the unfolded substrates enter the 20S core. Our results indicate that PSMC SNVs play a role in PI resistance in MM. [ABSTRACT FROM AUTHOR]

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C‐terminal α‐helical lid subdomain of substrate‐binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β‐sandwich substrate‐binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C‐terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C‐terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s. [ABSTRACT FROM AUTHOR]

In order to improve the quality of frozen bream and use it to produce high-quality surimi, the effect of phosphate ratio in compound cryoprotectant on frozen Acanthopagrus latus was studied. The freezing point and latent heat of fish were measured by differential scanning calorimeter. The ratio of phosphates in compound cryoprotectant was optimized by orthogonal experiment based on the results of single-factor experiment. The results of orthogonal experiment showed that when 3% sodium pyrophosphate, 3% sodium tripolyphosphate and 1% sodium hexametaphosphate were added into the compound cryoprotectant, the freezing point and latent heat of fish decreased to −2.3 ℃ and 147.2 J/g respectively. Acanthopagrus latus were treated with the optimized compound cryoprotectant and frozenat −18 ℃ for 30 days, the protein solubility of the fish increased by 8.1%, and the activities of Ca2+-ATPase, Mg2+-ATPase and Mg2+-Ca2+-ATPase increased by 75.6%, 26.9% and 113.7% respectively, compared with the unsoaking group. The activities of Ca2+-ATPase and Mg2+-Ca2+-ATPase could reach 98.6% and 84.5% of those in fresh fish. The breaking force and breaking distance of the surimi prepared by fish of the soaking group were 84.9% and 96.3% of those in surimi prepared by fresh fish respectively, and the gel strength was more similar to the surimi prepared by fresh fish compared with the unsoaking group. In conclusion, the optimized compound cryoprotectant could effectively reduce the denaturation of Acanthopagrus latus proteins during frozen storage.

Cohesin is key to eukaryotic genome organization and acts throughout the cell cycle in an ATP-dependent manner. The mechanisms underlying cohesin ATPase activity are poorly understood. Here, we characterize distinct steps of the human cohesin ATPase cycle and show that the SMC1A and SMC3 ATPase domains undergo specific but concerted structural rearrangements along this cycle. Specifically, whereas the proximal coiled coil of the SMC1A ATPase domain remains conformationally stable, that of the SMC3 displays an intrinsic flexibility. The ATP-dependent formation of the heterodimeric SMC1A/SMC3 ATPase module (engaged state) favors this flexibility, which is counteracted by NIPBL and DNA binding (clamped state). Opening of the SMC3/RAD21 interface (open-engaged state) stiffens the SMC3 proximal coiled coil, thus constricting together with that of SMC1A the ATPase module DNA-binding chamber. The plasticity of the ATP-dependent interface between the SMC1A and SMC3 ATPase domains enables these structural rearrangements while keeping the ATP gate shut. [Display omitted] [Display omitted] • SMC1A and SMC3 ATPase domains show specific structural dynamics • SMC3 ATPase domain proximal coiled coil has a specific intrinsic flexibility • SMC3/RAD21 interface dissociation constricts SMC1A/SMC3 ATPase DNA-binding chamber • SMC1A/SMC3 ATP interface plasticity allows dynamic moves but keeps the ATP gate shut Vitoria Gomes et al. show that SMC1A and SMC3 ATPase domains have distinct, specific, but concerted conformational dynamics during the human cohesin ATPase cycle. Whereas the SMC1A proximal coiled coil is stable, that of the SMC3 has an intrinsic flexibility that modifies the size of the SMC1A/SMC3 ATPase module DNA-binding chamber. [ABSTRACT FROM AUTHOR]

The universal N6-threonylcarbamoyladenosine (t6A) modification occurs at position 37 of tRNAs that decipher codons starting with adenosine. Mechanistically, t6A stabilizes structural configurations of the anticodon stem loop, promotes anticodon–codon pairing and safeguards the translational fidelity. The biosynthesis of tRNA t6A is co-catalyzed by two universally conserved protein families of TsaC/Sua5 (COG0009) and TsaD/Kae1/Qri7 (COG0533). Enzymatically, TsaC/Sua5 protein utilizes the substrates of L-threonine, HCO3−/CO2 and ATP to synthesize an intermediate L-threonylcarbamoyladenylate, of which the threonylcarbamoyl-moiety is subsequently transferred onto the A37 of substrate tRNAs by the TsaD–TsaB –TsaE complex in bacteria or by the KEOPS complex in archaea and eukaryotic cytoplasm, whereas Qri7/OSGEPL1 protein functions on its own in mitochondria. Depletion of tRNA t6A interferes with protein homeostasis and gravely affects the life of unicellular organisms and the fitness of higher eukaryotes. Pathogenic mutations of YRDC, OSGEPL1 and KEOPS are implicated in a number of human mitochondrial and neurological diseases, including autosomal recessive Galloway–Mowat syndrome. The molecular mechanisms underscoring both the biosynthesis and cellular roles of tRNA t6A are presently not well elucidated. This review summarizes current mechanistic understandings of the catalysis, regulation and disease implications of tRNA t6A-biosynthetic machineries of three kingdoms of life, with a special focus on delineating the structure–function relationship from perspectives of conservation and diversity. [ABSTRACT FROM AUTHOR]

Controversial information about the role of chaperonins in the amyloid transformation of proteins and, in particular, α-synuclein, requires a more detailed study of the observed effects due to the structure and functional state of various chaperonins. In this work, two types of phage chaperonins, the double-ring EL and the single-ring OBP, were shown to stimulate α-synuclein fibrillation in an ATP-dependent manner. Chaperonin morphology does not affect the stimulation of α-synuclein amyloid transformation. However, the ATP-dependent effect of single- and double-ring chaperonins on this process differs, which can lead to different morphology of resulting fibrils. Fibril formation seems to proceed without substrate encapsulation in the internal cavity of chaperonin, because of the structural features of phage chaperonins and their ability to function without co-chaperonins. In the absence of ATP, both chaperonins, on the contrary, completely prevent α-synuclein amyloid transformation, which provides the possibility of their use as anti-amyloid agents, in the form of incomplete molecules or mutants with suppressed ATPase activity. • Phage chaperonins EL and OBP passively prevent α-synuclein fibrillation without ATP. • Chaperonins EL and OBP stimulate α-synuclein fibrillation in an ATP-dependent manner. • Chaperonin morphology does not affect the stimulation of α-synuclein fibrillation. [ABSTRACT FROM AUTHOR]

2C is a highly conserved picornaviral non-structural protein with ATPase activity and plays a multifunctional role in the viral life cycle as a promising target for anti-picornavirus drug development. While the structure-function of enteroviral 2Cs have been well studied, cardioviral 2Cs remain largely uncharacterized. Here, an endogenous ATP molecule was identified in the crystal structure of 2C from encephalomyocarditis virus (EMCV, Cardiovirus A). The ATP is bound into the ATPase active site with a unique compact conformation. Notably, the γ-phosphate of ATP directly interacts with Arg311 (conserved in cardioviral 2Cs), and its mutation significantly inhibits the ATPase activity. Unexpectedly, this mutation remarkably promotes 2C self-oligomerization and viral replication efficiency. Molecular dynamic simulations showed that the Arg311 side chain is highly dynamic, indicating it may function as a switch between the activation state and the inhibition state of ATPase activity. A hexameric ring model of EMCV 2C full length indicated that the C-terminal helix may get close to the N-terminal amphipathic helices to form a continuous positive region for RNA binding. The RNA-binding studies of EMCV 2C revealed that the RNA length is closely associated with the RNA-binding affinities and indicated that the substrate may wrap around the outer surface of the hexamer. Our studies provide a biochemical framework to guide the characterization of EMCV 2C and the essential role of arginine in cardioviral 2C functions., Importance: Encephalomyocarditis virus (Cardiovirus A) is the causative agent of the homonymous disease, which may induce myocarditis, encephalitis, and reproductive disorders in various mammals. 2C protein is functionally indispensable and a promising target for drug development involving broad-spectrum picornaviral inhibitors. Here, an endogenous ATP molecule with a unique conformation was discovered by a combination of protein crystallography and high-performance liquid chromatography in the encephalomyocarditis virus (EMCV) 2C structure. Biochemical and structural characterization analysis of EMCV 2C revealed the critical role of conserved Arg311 in ATPase activity and self-oligomerization of EMCV 2C. The viral replication kinetics and infectivity study suggested that the residue negatively regulated the infectivity titer and virus encapsulation efficiency of EMCV and is, therefore, crucial for 2C protein to promote viral replication. Our systemic structure-function analysis provides unique insights into the function and regulation mechanism of cardioviral 2C protein., Competing Interests: The authors declare no conflict of interest.

ABCA4 is an ATP-binding cassette (ABC) transporter that prevents the buildup of toxic retinoid compounds by facilitating the transport of N-retinylidene-phosphatidylethanolamine across membranes of rod and cone photoreceptor cells. Over 1500 missense mutations in ABCA4, many in the nucleotide-binding domains (NBDs), have been genetically linked to Stargardt disease. Here, we show by cryo-EM that ABCA4 is converted from an open outward conformation to a closed conformation upon the binding of adenylyl-imidodiphosphate. Structural information and biochemical studies were used to further define the role of the NBDs in the functional properties of ABCA4 and the mechanisms by which mutations lead to the loss in activity. We show that ATPase activity in both NBDs is required for the functional activity of ABCA4. Mutations in Walker A asparagine residues cause a severe reduction in substrate-activated ATPase activity due to the loss in polar interactions with residues within the D-loops of the opposing NBD. The structural basis for how disease mutations in other NBD residues, including the R1108C, R2077W, R2107H, and L2027F, affect the structure and function of ABCA4 is described. Collectively, our studies provide insight into the structure and function of ABCA4 and mechanisms underlying Stargardt disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cypermethrin contamination was a potential threat to soil organisms. In the present work, reproductive damage in earthworms (Amynthas corticis) exposed to cypermethrin was investigated. It was found that earthworms could absorb and accumulate residual cypermethrin in soil, and also earthworm activities helped accelerate the degradation of cypermethrin in soil. The accumulation of cypermethrin in earthworms induced sperm damage, and cypermethrin not only caused the imbalance of calcium homeostasis in earthworm sperm cells by inhibiting earthworm sperm Ca2+-ATP and Ca2+-Mg2+-ATP enzyme activities but also caused barriers in acrosome reaction. It also affected sperm energy supply of earthworms by inhibiting the activity of Na+-K+-ATPase and Mg2+-ATPase of earthworm sperm. Meanwhile, the inhibition of acrosome enzyme activity of earthworm sperm by cypermethrin led to hinder fertilization and reduced cocoon production of earthworms, and the damage of cypermethrin to sperm of earthworm was a significant cause of its reproductive toxicity. The results of the evaluation of IBR index showed that reproductive toxicity of cypermethrin to earthworms reduced with the increasing time. The decreased reproductive toxicity of cypermethrin to earthworms at the later stage of exposure (42–56 d) might be due to a combination of reduced absorption of cypermethrin in soil by earthworms, decreased accumulation of cypermethrin in the body, and improved sperm capacitation.

In all domains of life, transmembrane proteins from the ATP‐binding cassette (ABC) transporter family drive the translocation of diverse substances across lipid bilayers. In pathogenic fungi, the ABC transporters of the pleiotropic drug resistance (PDR) subfamily confer antibiotic resistance and so are of interest as therapeutic targets. They also drive the quest for understanding how ABC transporters can generally accommodate such a wide range of substrates. The Pdr5 transporter from baker's yeast is representative of the PDR group and, ever since its discovery more than 30 years ago, has been the subject of extensive functional analyses. A new perspective of these studies has been recently provided in the framework of the first electron cryo‐microscopy structures of Pdr5, as well as emergent applications of machine learning in the field. Taken together, the old and the new developments have been used to propose a mechanism for the transport process in PDR proteins. This mechanism involves a "flippase" step that moves the substrates from one leaflet of the bilayer to the other, as a central element of cellular efflux. [ABSTRACT FROM AUTHOR]

Na+/K+-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na+/K+-ATPase activity in vitro and silico. The inhibitory assays showed that calotropin and C3OG decreased the Na+/K+-ATPase activity with IC50 values of 0.27 and 0.87 mM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na+/K+-ATPase activity, with Ki values of 0.2 mMto calotropin and 0.5 mMto C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr797 and Gln111 residues, considered essential to the interaction with the Na+/K+-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe783, Leu125, and Ala323, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata, proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule. [ABSTRACT FROM AUTHOR]

Na+/K+-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na+/K+-ATPase activity in vitro and silico. The inhibitory assays showed that calotropin and C3OG decreased the Na+/K+-ATPase activity with IC50 values of 0.27 and 0.87 μM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na+/K+-ATPase activity, with Ki values of 0.2 μM to calotropin and 0.5 μM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr797 and Gln111 residues, considered essential to the interaction with the Na+/K+-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe783, Leu125, and Ala323, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata, proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process. • RecN increases E. coli recombination rate. • E. coli RecN has intrinsic and ssDNA-stimulated ATPase activity. • E. coli RecN binds preferentially to ssDNA. • RecN does not associate with nucleoid in the absence of SOS response. [ABSTRACT FROM AUTHOR]

Hereditary cancer syndromes account for approximately 5%–10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N‐terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in‐cell fluorescent‐based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign. [ABSTRACT FROM AUTHOR]

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The tea aphid, Aphis aurantii (Boyer de Fonscolombe), is a serious pest that can infest many economically important plants. Tea aphids damage plants by directly sucking phloem sap, transmitting viruses, and secreting honeydew to cause sooty mold. At present, tea aphids has become one of the most important pests in tropical and subtropical tea plants. The heat shock protein 70 (Hsp70) is a key protein involved in heat stress tolerance. In this study, we cloned four Hsp70 genes that are highly expressed in tea aphids after heat shock. Bioinformatic analysis of the deduced amino acid sequences showed that these four AaHsp70s had a close genetic relationship to Hsp70 in Hemiptera insects and shared a conserved ATPase domain. After incubation at low (14 °C) or high (36 °C) temperature, the expression of four AaHsp70s was significantly up-regulated compared to the control (25 °C); however, the up-regulation of the AaHsp70s in the low-temperature treatment was far less than that of the high-temperature treatment. The ATPase activity of the four purified recombinant AaHsp70 proteins after high-temperature treatment was significantly increased compared to the control. In addition, these proteins effectively improved the heat tolerance of Escherichia coli in vivo. Our data indicate that AaHsp701, AaHsp702, AaHsp703, AaHsp704 play important roles in response to the high-temperature tolerance in tea aphids. [ABSTRACT FROM AUTHOR]

We report the steady state ATPase activities of the ATP Binding Cassette (ABC) exporter NaAtm1 in the absence and presence of a transported substrate, oxidized glutathione (GSSG), in detergent, nanodiscs, and proteoliposomes. The steady state kinetic data were fit to the "nonessential activator model" where the basal ATPase rate of the transporter is stimulated by GSSG. The detailed kinetic parameters varied between the different reconstitution conditions, highlighting the importance of the lipid environment for NaAtm1 function. The increased ATPase rates in the presence of GSSG more than compensate for the modest negative cooperativity observed between MgATP and GSSG in lipid environments. These studies highlight the central role of the elusive ternary complex in accelerating the ATPase rate that is at the heart of coupling mechanism between substrate transport and ATP hydrolysis. [ABSTRACT FROM AUTHOR]

Abstract Hereditary cancer syndromes account for approximately 5%–10% of all diagnosed cancer cases. Lynch syndrome (LS) is an autosomal dominant hereditary cancer condition that predisposes individuals to an elevated lifetime risk for developing colorectal, endometrial, and other cancers. LS results from a pathogenic mutation in one of four mismatch repair (MMR) genes (MSH2, MSH6, MLH1, and PMS2). The diagnosis of LS is often challenged by the identification of missense mutations, termed variants of uncertain significance, whose functional effect on the protein is not known. Of the eight PMS2 variants initially selected for this study, we identified a variant within the N‐terminal domain where asparagine 335 is mutated to serine, p.Asn335Ser, which lacked ATPase activity, yet appears to be proficient in MMR. To expand our understanding of this functional dichotomy, we performed biophysical and structural studies, and noted that p.Asn335Ser binds to ATP but is unable to hydrolyze it to ADP. To examine the impact of p.Asn335Ser on MMR, we developed a novel in‐cell fluorescent‐based microsatellite instability reporter that revealed p.Asn335Ser maintained genomic stability. We conclude that in the absence of gross structural changes, PMS2 ATP hydrolysis is not necessary for proficient MMR and that the ATPase deficient p.Asn335Ser variant is likely benign.

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process. [ABSTRACT FROM AUTHOR]

The RVB proteins, composed of the conservative paralogs, RVB1 and RVB2, belong to the AAA+ (A TPases A ssociated with various cellular A ctivities) protein superfamily and are present in archaea and eukaryotes. The most distinct structural features are their ability to interact with each other forming the RVB1/2 complex and their participation in several macromolecular protein complexes leading them to be involved in many biological processes. We report here the biochemical and biophysical characterization of the Neurospora crassa RVB-1/RVB-2 complex. Chromatographic analyses revealed that the complex (APO) predominantly exists as a dimer in solution although hexamers were also observed. Nucleotides influence the oligomerization state, while ATP favors hexamers formation, ADP favors the formation of multimeric states, likely dodecamers, and the Molecular Dynamics (MD) simulations revealed the contribution of certain amino acid residues in the nucleotide stabilization. The complex binds to dsDNA fragments and exhibits ATPase activity, which is strongly enhanced in the presence of DNA. In addition, both GFP-fused proteins are predominantly nuclear, and their nuclear localization signals (NLS) interact with importin-α (NcIMPα). Our findings show that some properties are specific of the fungus proteins despite of their high identity to orthologous proteins. They are essential proteins in N. crassa, and the phenotypic defects exhibited by the heterokaryotic strains, mainly related to growth and development, indicate N. crassa as a promising organism to investigate additional biological and structural aspects of these proteins. • The essential RVB-1 and RVB-2 proteins are the RuvBL/RVB homologues in Neurospora crassa. • The proteins are functionally related to growth and development. • The RVB-1/RVB-2 complex predominantly exists as a dimer in solution and nucleotides strongly influence the oligomerization state. • Binding of nucleotides leads to conformational changes that may influence the interaction with DNA. • The RVB-1/RVB-2 complex exhibits ATPase activity, binds to dsDNA fragments and DNA strongly increase the ATPase activity. • The GFP-fused RVB-1 and RVB-2 are nuclear proteins and their NLSs interact with the N. crassa importin-α (NcIMPα). [ABSTRACT FROM AUTHOR]

This study compared two histochemical staining methods of muscle fibres and evaluated their relationship with the meat quality traits of two high-value porcine muscles. Immunohistochemical (IHC) and adenosine triphosphatase (ATPase) staining was used to assess the cross-sectional area and proportion of fibre-types I, IIa, IIx and IIb in the samples of longissimus lumborum (LL) and psoas major (PM) muscles collected one-hour post-mortem from 25 crossbred pigs [Large WhiteSire × (Landrace × Large WhiteDam)] at an average age of 152 days. Muscles differed in all fibre parameters, except the proportion and relative area of type IIx fibres. The LL muscle exhibited greater fibre cross-sectional areas of all fibre types, higher proportions of type IIb/IIB, and lower proportions of I and IIa fibres than the PM muscle in both staining techniques. These two muscles also differed marginally in moisture, crude protein and intramuscular fat content. The PM muscle showed a low correlation between fibre types and chemical composition, but the LL muscle showed moderate correlations between fibre CSA and area composition for moisture and ash content. After IHC staining, an increase in LL eye muscle area and drip loss were correlated with lower proportions of type I fibres, while a greater proportion of type IIx fibres resulted in increased LL eye muscle area and moisture content. Furthermore, a higher CSA of all fibre types in the LL decreased redness (a*) and moisture content of the muscle. Results showed that IHC is more appropriate than ATPase staining for the assessment of relationships between muscle fibre parameters and meat quality traits in pigs.

Abstract Background Bacterial gas vesicles, composed of two major gas vesicle proteins and filled with gas, are a unique class of intracellular bubble-like nanostructures. They provide buoyancy for cells, and thus play an essential role in the growth and survival of aquatic and soil microbes. Moreover, the gas vesicle could be applied to multimodal and noninvasive biological imaging as a potential nanoscale contrast agent. To date, cylinder-shaped gas vesicles have been found in several strains of cyanobacteria. However, whether the functional gas vesicles could be produced in the model filamentous cyanobacteria Anabaena sp. PCC 7120 remains controversial. Results In this study, we found that an intact gvp gene cluster indeed exists in the model filamentous cyanobacteria Anabaena sp. PCC 7120. Real-time PCR assays showed that the gvpA gene is constitutively transcribed in vivo, and its expression level is upregulated at low light intensity and/or high growth temperature. Functional expression of this intact gvp gene cluster enables the recombinant Escherichia coli to gain the capability of floatation in the liquid medium, thanks to the assembly of irregular gas vesicles. Furthermore, crystal structure of GvpF in combination with enzymatic activity assays of GvpN suggested that these two auxiliary proteins of gas vesicle are structurally and enzymatically conserved, respectively. Conclusions Our findings show that the laboratory strain of model filamentous cyanobacteria Anabaena sp. PCC 7120 possesses an intact but partially degenerated gas vesicle gene cluster, indicating that the natural isolate might be able to produce gas vesicles under some given environmental stimuli for better floatation.

The antimicrobial peptide (AMP) ARV-1502 was designed based on naturally occurring short proline-rich AMPs, including pyrrhocoricin and drosocin. Identification of chaperone DnaK as a therapeutic target in Escherichia coli triggered intense research on the ligand-DnaK-interactions using fluorescence polarization and X-ray crystallography to reveal the binding motif and characterize the influence of the chaperone on protein refolding activity, especially in stress situations. In continuation of this research, 182 analogs of ARV-1502 were designed by substituting residues involved in antimicrobial activity against Gram-negative pathogens. The peptides synthesized on solid-phase were examined for their binding to E. coli and S. aureus DnaK providing 15 analogs with improved binding characteristics for at least one DnaK. These 15 analogs were distinguished from the original sequence by their increased hydrophobicity parameters. Additionally, the influence of the entire DnaK chaperone system, including co-chaperones DnaJ and GrpE on refolding and ATPase activity, was investigated. The increasingly hydrophobic peptides showed a stronger inhibitory effect on the refolding activity of E. coli chaperones, reducing protein refolding by up to 64%. However, these more hydrophobic peptides had only a minor effect on the ATPase activity. The most dramatic changes on the ATPase activity involved peptides with aspartate substitutions. Interestingly, these peptides resulted in a 59% reduction of the ATPase activity in the E. coli chaperone system whereas they stimulated the ATPase activity in the S. aureus system up to 220%. Of particular note is the improvement of the antimicrobial activity against S. aureus from originally >128 µg/mL to as low as 16 µg/mL. Only a single analog exhibited improved activity over the original value of 8 µg/mL against E. coli. Overall, the various moderate-throughput screenings established here allowed identifying (un)favored substitutions on 1) DnaK binding, 2) the ATPase activity of DnaK, 3) the refolding activity of DnaK alone or together with co-chaperones, and 4) the antimicrobial activity against both E. coli and S. aureus.

The toxin hemolysin A was first identified in uropathogenic E. coli strains and shown to be secreted in a one-step mechanism by a dedicated secretion machinery. This machinery, which belongs to the Type I secretion system family of the Gram-negative bacteria, is composed of the outer membrane protein TolC, the membrane fusion protein HlyD and the ABC transporter HlyB. The N-terminal domain of HlyA represents the toxin which is followed by a RTX (Repeats in Toxins) domain harboring nonapeptide repeat sequences and the secretion signal at the extreme C-terminus. This secretion signal, which is necessary and sufficient for secretion, does not appear to require a defined sequence, and the nature of the encoded signal remains unknown. Here, we have combined structure prediction based on the AlphaFold algorithm together with functional and in silico data to examine the role of secondary structure in secretion. Based on the presented data, a C-terminal, amphipathic helix is proposed between residues 975 and 987 that plays an essential role in the early steps of the secretion process.

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC 5 , a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC 5 DUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC DUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC 5 DUF , supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC 5 DUF with ATP/Mg 2+ , supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC 5 DUF , it is hypothesized that MtbEccC 5 DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions., (open access.)

The EccC enzyme of ESX-1 system contains (i) a membrane bound Rv3870 with single ATPase domain and (ii) a cytoplasmic Rv3871 with two ATPase domains and involved in secretion of ESAT6/CFP10 factor out of the cell. In current study, we have structurally and biochemically characterized the ATPase domain (442–747 residues) of Rv3870 enzyme. The ΔRv3870 eluted as oligomer (~813 kDa) from Superdex 200 (16/60) column, as identified based on molecular mass standard and dynamics light scattering. The SAXS analysis yielded a tetrameric ring envelope of ΔRv3870, quite consistent to dynamic light scattering data. The ΔRv3870 exhibited ATPase activity having kinetic parameters, K m ~ 100 ± 40 μM, k cat ~ 1.81 ± 0.27 min−1 and V max ~ 54.41 μM/min/mg. ATPase activity using nine ΔRv3870 mutants showed 70–91% decrease in catalytic efficiency of the enzyme. ΔRv3870 binds Rv3871 with K D ~ 484.0 ± 10.3 nM and its catalytic efficiency is enhanced ~6.7-fold in presence of Rv3871. CD data revealed the high T M ~ 82.2 ± 0.5 °C for ΔRv3870 and enhanced in presence of ATP + Mg2+, as observed in dynamics simulation on ΔRv3870 hexameric models. Overall, our structural and biochemical studies on ΔRv3870 have explained the mechanism, which will contribute in development of antivirulence inhibitors against M. tuberculosis. [ABSTRACT FROM AUTHOR]

Mitotic kinesin Eg5 remains a validated target in antimitotic therapy because of its essential role in the formation and maintenance of bipolar mitotic spindles. Although numerous Eg5 inhibitors of synthetic origin are known, only a few inhibitors derived from natural products have been reported. In our study, we focused on identifying novel Eg5 inhibitors from medicinal plants, particularly Garcinia species. Herein, we report the inhibitory effect of kolaflavanone (KLF), a Garcinia biflavonoid, on the ATPase and microtubule-gliding activities of mitotic kinesin Eg5. Additionally, we showed the interaction mechanism between Eg5 and KLF via in vitro and in silico analyses. The results revealed that KLF inhibited both the basal and microtubule-activated ATPase activities of Eg5. The inhibitory mechanism is allosteric, without a direct competition with adenosine-5′-diphosphate for the nucleotide-binding site. KLF also suppressed the microtubule gliding of Eg5 in vitro. The Eg5–KLF model obtained from molecular docking showed that the biflavonoid exists within the α2/α3/L5 (α2: Lys111–Glu116 and Ile135–Asp149, α3: Asn206–Thr226; L5: Gly117–Gly134) pocket, with a binding pose comparable to known Eg5 inhibitors. Overall, our data suggest that KLF is a novel allosteric inhibitor of mitotic kinesin Eg5. [ABSTRACT FROM AUTHOR]

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35–50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2–3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25–40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity. [ABSTRACT FROM AUTHOR]

[Display omitted] • Taurine and COQ-10 increased spermatogenesis in CPZ-treated rats. • Taurine and COQ-10 enhance CPZ-induced decreased Na+K+-, Ca2+-, Mg2+- and H+-ATPase biosynthesis. • Taurine and COQ-10 increase CPZ-induced decreased 3ß-HSD, 17ß –HSD, G6PDH and LDH-X enzymes. • Taurine and COQ-10 increased sperm capacitation and acrosomal reaction rats tests. Evidences have shown that alterations in testicular dehydrogenase and ionic-ATPase activities have important implications in spermatogenesis and sperm capacitation, a penultimate biochemical change required for fertilization. Previous studies have revealed that taurine and coenzyme-Q10 (COQ-10), which are synergistic testicle-active bioflavonoids, with proven gonadotropin-enhancing properties reduce testicular damage in rats. Hence, this study investigated the effects of taurine and COQ-10 or their combination alone, and in the preventive and reversal of chlorpromazine-induced inhibition of testicular dehydrogenase enzymes, electrogenic pumps, sperm capacitation and acrosomal-reaction in male Wister rats. In the drug-treatment alone or preventive-protocol, rats received oral treatment of saline (10 mL/kg), taurine (150 mg/kg/day), COQ-10 (10 mg/kg/day) or both alone repeatedly for 56 days, or in combination with chlorpromazine (30 mg/kg/p.o./day) from days 29−56. In the reversal-protocol, the animals received chlorpromazine for 56 days prior to saline, taurine, COQ-10 or the combination from days 29−56. Thereafter, spermatogenesis (sperm count, viability, motility and morphology), testicular dehydrogenase [3beta-hydroxysteroid dehydrogenase (3ß-HSD), 17beta-hydroxysteroid dehydrogenase (17ß-HSD), glucose-6-phosphate dehydrogenase (G6PDH), lactate dehydrogenase-X (LDH-X)], ATPase (Na+/K+, Ca2+, Mg2+, H+) activities, sperm capacitation and acrosomal reaction were evaluated. Taurine and COQ-10 or their combination increased spermatogenesis, testicular 3ß-HSD, 17ß-HSD, G6PDH and LDH-X enzymes of naïve and chlorpromazine-treated rats. Both taurine and COQ-10 increased Na+/K+, Ca2+, Mg2+ and H+-ATPase activities. Also, taurine and COQ-10 or their combination prevented and reversed chlorpromazine-induced inhibition of sperm capacitation and acrosomal-reaction. The study showed that taurine and COQ-10 prevent and reverse chlorpromazine-induced inhibition of spermatogenesis, epididymal sperm capacitation and acrosomal reaction in rats through increased testicular dehydrogenases and electrogenic pump activities. [ABSTRACT FROM AUTHOR]

ATP‐binding cassette (ABC) transporter proteins include efflux pumps that confer multixenobiotic resistance to zebrafish embryos, a valuable toxico/pharmacological model. Here, we established an automated microscopy‐based rhodamine B dye accumulation assay in which enhanced dye accumulation in live zebrafish embryos indicates inhibition of multixenobiotic efflux transporter activity. Twenty structurally divergent known substrates and/or inhibitors of human ABC transporters and environmentally relevant compounds were examined using this assay and the ATPase activity of recombinant zebrafish Abcb4 as readouts. These two assays confirmed that Abcb4 functions as an efflux transporter in zebrafish, whereas they gave discordant results for some of the tested substances. The dye accumulation assay in zebrafish embryos could be useful to screen environmental pollutants and other chemicals for efflux transporter interaction in a medium‐throughput fashion. [ABSTRACT FROM AUTHOR]