Maximal ATPase Activity Research Articles

The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation

Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.

Structural insights into the transient closed conformation and pH dependent ATPase activity of S.Typhi GyraseB N- terminal domain

DNA Gyrase is a type II topoisomerase that utilizes the energy of ATP hydrolysis for introducing negative supercoils in DNA. The protein comprises two subunits GyrA and GyrB that form a GyrA2GyrB2 heterotetramer. GyrB subunit contains the N-terminal domain (GBNTD) for ATPase activity and the C-terminal domain (GBCTD) for interaction with GyrA and DNA. Earlier structural studies have revealed three different conformational states for GBNTD during ATP hydrolysis defined as open, semi-open, and closed. Here we report, the three-dimensional structure of a new transient closed conformation of GBNTD from Salmonella Typhi (StGBNTD) at 1.94 Å resolution. Based on the structural analysis of this transient closed conformation, we propose the role of protein in the mechanism of ATP hydrolysis. We further explored the effect of pH on ATPase activity and structural stability of the GBNTD using CD and fluorescence spectroscopy at varying pH environment. Kinetic parameters obtained from the ATPase assay were correlated with its secondary and tertiary structure at their respective pH environment. The protein possessed maximum ATPase activity and structural stability at optimum pH 8. At acidic pH, a remarkable decrease in both enzymatic activity and structural stability was observed whereas at alkaline pH there was no significant change. The structural analysis of StGBNTD reveals the role of polar interactions in stabilizing the overall dimeric conformation of the protein.

The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP

The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria.

Impact of Dilated Cardiomyopathy Mutation and Small Molecule Regulator on Human Beta-Cardiac Myosin

Missense mutations in human β-cardiac myosin are associated with inherited cardiomyopathies. The mechanisms for how mutations alter myosin's motor performance are still unclear. We expressed and purified human beta-cardiac myosin subfragment 1 (M2β-S1) containing a point mutation in the converter domain (F764L) that is associated with dilated cardiomyopathy (DCM) and compared it to wild-type (WT) M2β-S1. We demonstrate that the F764L mutation slows down maximum actin-activated ATPase activity (20-30%). Direct measurements of ADP release in the presence of actin using mant labeled ADP demonstrate the F764L mutant has a 20% slower ADP release rate constant. The in vitro motility assay demonstrates a 10-15% slower actin sliding velocity for F764L. Monitoring ATP binding and hydrolysis with intrinsic tryptophan fluorescence demonstrates that the F764L mutation causes a 2-fold increase in K0.5 without changing the maximal rate. We also examined the impact of the heart failure drug Omecamtiv Mecarbil (OM) on the F764L mutant. The steady-state actin-activated ATPase results suggest that the F764L mutant shares a similar OM binding affinity with WT. Interestingly, the drug does not reduce the maximum ATPase activity and actin sliding velocity as much as it does with WT. We observed no difference in the ionic strength dependence or density dependence of in vitro motility in WT and F764L in the presence and absence of OM. Our previous results suggested OM dramatically slows down the ADP isomerization step in WT M2β-S1, making it the rate-limiting step. Our results demonstrate OM does not slow the ADP isomerization step as much in F764L, but this step is still rate-limiting. Therefore, we propose that OM will increase the myosin duty ratio in the F764L mutant enough to enhance isomeric force while only moderately slowing shortening velocity.

Biochemical properties of the sensitivity to GABAAergic ligands, Cl-/HCO3--ATPase isolated from fish (Cyprinus carpio) olfactory mucosa and brain.

This paper presents a comparative study of the roles of Cl- and HCO3- in the functioning of the GABAAR-associated Cl-/HCO3--ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl-(15-30mmoll-1), whereas the enzyme from MBNs was not significantly affected by Cl-. By contrast, HCO3-(15-30mmoll-1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl-(15mmoll-1)/HCO3-(27mmoll-1) and these activities were inhibited in the presence of picrotoxin (100μmoll-1), bumetanide (150μmoll-1), and DIDS (1000μmoll-1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~ 56kDa that binds [3H]muscimol and [3H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-32P and Mg2+, as well as Cl-/HCO3- sensitive dephosphorylation, is also associated with this 56kDa peptide. Both preparations also showed one subunit with molecular mass 56kDa that was immunoreactive with GABAAR β3 subunit. The use of a fluorescent dye for Cl- demonstrated that HCO3-(27mmoll-1) causes a twofold increase in Cl- influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO3- had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl- and HCO3- in the regulation of the Cl-/HCO3--ATPases functioning in neurons with different specializations.

Protein Biochemistry and Expression Regulation of Cadmium/Zinc Pumping ATPases in the Hyperaccumulator Plants Arabidopsis halleri and Noccaea caerulescens.

P1B-ATPases are decisive for metal accumulation phenotypes, but mechanisms of their regulation are only partially understood. Here, we studied the Cd/Zn transporting ATPases NcHMA3 and NcHMA4 from Noccaea caerulescens as well as AhHMA3 and AhHMA4 from Arabidopsis halleri. Protein biochemistry was analyzed on HMA4 purified from roots of N. caerulescens in active state. Metal titration of NcHMA4 protein with an electrochromic dye as charge indicator suggested that HMA4 reaches maximal ATPase activity when all internal high-affinity Cd2+ binding sites are occupied. Although HMA4 was reported to be mainly responsible for xylem loading of heavy metals for root to shoot transport, the current study revealed high expression of NcHMA4 in shoots as well. Further, there were additional 20 and 40 kD fragments at replete Zn2+ and toxic Cd2+, but not at deficient Zn2+ concentrations. Altogether, the protein level expression analysis suggested a more multifunctional role of NcHMA4 than previously assumed. Organ-level transcription analysis through quantitative PCR of mRNA in N. caerulescens and A. halleri confirmed the strong shoot expression of both NcHMA4 and AhHMA4. Further, in shoots NcHMA4 was more abundant in 10 μM Zn2+ and AhHMA4 in Zn2+ deficiency. In roots, NcHMA4 was up-regulated in response to deficient Zn2+ when compared to replete Zn2+ and toxic Cd2+ treatment. In both species, HMA3 was much more expressed in shoots than in roots, and HMA3 transcript levels remained rather constant regardless of Zn2+ supply, but were up-regulated by 10 μM Cd2+. Analysis of cellular expression by quantitative mRNA in situ hybridisation showed that in A. halleri, both HMA3 and HMA4 mRNA levels were highest in the mesophyll, while in N. caerulescens they were highest in the bundle sheath of the vein. This is likely related to the different final storage sites for hyperaccumulated metals in both species: epidermis in N. caerulescens, mesophyll in A. halleri.

Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial Hypertrophic Cardiomyopathy Phenotype

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by ventricular hypertrophy, myofibrillar disarray and often caused by mutations in genes encoding for major sarcomeric proteins. This study is focused on Arginine to Glutamine substitution (R58Q) in the ventricular myosin regulatory light chain (RLC) resulting in a malignant hypertrophic phenotype. We previously showed a mutation-induced decrease in the endogenous level of cardiac RLC phosphorylation in R58Q mice, resulting in compromised communication between the functional regions of the RLC and affecting the interaction of myosin and actin and cardiac muscle contraction. In this report we explored the therapeutic potential of pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphorylatable Serine-15 (S15D) in the R58Q background. In vitro actin-activated myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal ATPase activity, Vmax (∼30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten constant (in μM) for R58Q (Km=4.04±0.45) was significantly increased compared to WT (Km=1.68±0.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a small but significant increase in Km (2.56±0.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled actin to quantify the dissociation constants (Kd) and stoichiometry of binding (n) between myosin and actin in the absence of nucleotide. Binding of WT-RLC to actin was strong (Kd value =5.5 nM, n=0.89). A large decrease in binding (∼67-fold increase in Kd) was observed for R58Q-RLC. Kd of S15D-R58Q-RLC was only slightly increased (∼3-fold) compared with WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC mutant. Our findings on S15D-R58Q support the idea that myosin phosphorylation may have important translational applications for the treatment of RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 (DSC).

ЗМІНА АКТИВНОСТІ Na+/K+–АТФ-ази В ЕМБРІОНАХ КОРОПОВИХ РИБ ЗА ДІЇ РІЗНОГО ТЕМПЕРАТУРНОГО ТА КИСНЕВОГО РЕЖИМУ ВОДОЙМ

Due to the increase in global average annual temperatures considerable changes of living conditions of aquatic organisms. Recently, many reservoirs an increase in water temperature 4–5 °C. Since fish are poikilothermic animals then these changes will greatly affect their life. That is why the purpose of our research was to determine the influence of environmental factors aquatic environment and their fluctuations in the early stages of embryos of fish, assess the extent of action by anatomical, morphological, biochemical and genetic parameters. In this paper, lit only part of the total of the results, namely a change of activity Na + /K + –ATP-ase embryos for carp, grass carp and silver carp white. The noted inter specific differences in activity of the enzyme. With the deteriorating conditions of temperature and decrease with the concentration of dissolved oxygen in the white carp ATP-ase activity increases, that is reinforced exchange between external and internal environment of the eggs that we saw in temperatures of 30.4 and 30.6 °C, which was the highest enzyme activity. Also while there was accelerated growth of embryos was observed and a significant percentage of anomalies of development. The most optimal, in our opinion, was the temperature during incubation of silver carp within 25–28 ºC. For grass carp found the maximum ATPase activity at temperatures 25.6 ºC (41.1 mg P / h. × mg protein). The most optimal in terms of enzyme activity investigated, we believe the temperature at 25.0 ºC as for white carp. In carp on stage pigmentation eye we observed the highest activity of Na + /K + –ATP-ase in temperatures 22.0–24.0 ºC, who was 22.8–59.0 % higher than 25.0–28.0 ºC. It 22.0–24.0 ºC is the optimal temperature conditions for this species in natural waters. As the temperature at this stage, the reduced activity of the enzyme that caused fetal compensatory response to the growing influence of temperature factor and reduction of dissolved oxygen.

Protective mechanisms of resveratrol against methotrexate-induced renal damage may involve BCRP/ABCG2.

Resveratrol (RES) is a well-known polyphenol antioxidant. We have previously shown that testicular protective effect of RES against the anticancer drug methotrexate (MTX)-induced toxicity involves transporter-mediated mechanisms. Here, we investigated the effect of RES on MTX-induced nephrotoxicity. Rats were administered RES (10 mg/kg/day) for 8 days, with or without a single MTX dose (20 mg/kg i.p.) at day 4 of the experiment. MTX induced nephrotoxicity, as evidenced by a significant increase in serum blood urea nitrogen and creatinine compared to the control, as well as distortion of kidney microscopic structure. MTX also significantly increased renal nitric oxide level along with inducible nitric oxide synthase, fas ligand, and caspase 3 expressions. Administering RES prior to MTX significantly improved kidney function and microscopic picture and also significantly decreased nitrosative and apoptotic markers compared to MTX alone. RES, but not MTX, caused a significant increase in expression of breast cancer resistance protein (BCRP), an apical efflux renal transporter that participates in urinary elimination of both MTX and RES. Interestingly, concomitant MTX and RES caused further upregulation of renal BCRP compared to RES alone. Using Human BCRP ATPase assay, both RES and MTX exhibited a dose-dependent increase in ATPase activity, with Km values of 0.52 ± 0.03 and 30.9 ± 4.2 μm, respectively. Furthermore, combined RES and MTX caused ATPase activity which was significantly less than maximum ATPase activity attained by the positive control, sulfasalazine (12.5 μm). In conclusion, RES exerted nephroprotection against MTX-induced toxicity through antinitrosative and anti-apoptotic effects, as well as via upregulation of renal BCRP.

Application of a Euryarchaeota-Specific Helicase from Thermococcus kodakarensis for Noise Reduction in PCR.

DNA/RNA helicases, which are enzymes for eliminating hydrogen bonds between bases of DNA/DNA, DNA/RNA, and RNA/RNA using the energy of ATP hydrolysis, contribute to various biological activities. In the present study, the Euryarchaeota-specific helicase EshA (TK0566) from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-EshA) was obtained as a recombinant form, and its enzymatic properties were examined. Tk-EshA exhibited maximal ATPase activity in the presence of RNA at 80°C. Unwinding activity was evaluated with various double-stranded DNAs (forked, 5' overhung, 3' overhung, and blunt end) at 50°C. Tk-EshA unwound forked and 3' overhung DNAs. These activities were expected to unwind the structured template and to peel off misannealed primers when Tk-EshA was added to a PCR mixture. To examine the effect of Tk-EshA on PCR, various target DNAs were selected, and DNA synthesis was investigated. When 16S rRNA genes were used as a template, several misamplified products (noise DNAs) were detected in the absence of Tk-EshA. In contrast, noise DNAs were eliminated in the presence of Tk-EshA. Noise reduction by Tk-EshA was also confirmed when Taq DNA polymerase (a family A DNA polymerase, PolI type) and KOD DNA polymerase (a family B DNA polymerase, α type) were used for PCR. Misamplified bands were also eliminated during toxA gene amplification from Pseudomonas aeruginosa DNA, which possesses a high GC content (69%). Tk-EshA addition was more effective than increasing the annealing temperature to reduce misamplified DNAs during toxA amplification. Tk-EshA is a useful tool to reduce noise DNAs for accurate PCR. PCR is a technique that is useful for genetic diagnosis, genetic engineering, and detection of pathogenic microorganisms. However, troubles with nonspecific DNA amplification often occur from primer misannealing. In order to achieve a specific DNA amplification by eliminating noise DNAs derived from primer misannealing, a thermostable Euryarchaeota-specific helicase (Tk-EshA) was included in the PCR mixture. The addition of Tk-EshA has reduced noise DNAs in PCR.

PTS regulation domain-containing transcriptional activator CelR and sigma factor σ(54) control cellobiose utilization in Clostridium acetobutylicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity.

Phospholamban overexpression in mice causes a centronuclear myopathy-like phenotype.

ABSTRACTCentronuclear myopathy (CNM) is a congenital myopathy that is histopathologically characterized by centrally located nuclei, central aggregation of oxidative activity, and type I fiber predominance and hypotrophy. Here, we obtained commercially available mice overexpressing phospholamban (PlnOE), a well-known inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs), in their slow-twitch type I skeletal muscle fibers to determine the effects on SERCA function. As expected with a 6- to 7-fold overexpression of phospholamban, SERCA dysfunction was evident in PlnOE muscles, with marked reductions in rates of Ca2+ uptake, maximal ATPase activity and the apparent affinity of SERCA for Ca2+. However, our most significant discovery was that the soleus and gluteus minimus muscles from the PlnOE mice displayed overt signs of myopathy: they histopathologically resembled human CNM, with centrally located nuclei, central aggregation of oxidative activity, type I fiber predominance and hypotrophy, progressive fibrosis and muscle weakness. This phenotype is associated with significant upregulation of muscle sarcolipin and dynamin 2, increased Ca2+-activated proteolysis, oxidative stress and protein nitrosylation. Moreover, in our assessment of muscle biopsies from three human CNM patients, we found a significant 53% reduction in SERCA activity and increases in both total and monomeric PLN content compared with five healthy subjects, thereby justifying future studies with more CNM patients. Altogether, our results suggest that the commercially available PlnOE mouse phenotypically resembles human CNM and could be used as a model to test potential mechanisms and therapeutic strategies. To date, there is no cure for CNM and our results suggest that targeting SERCA function, which has already been shown to be an effective therapeutic target for murine muscular dystrophy and human cardiomyopathy, might represent a novel therapeutic strategy to combat CNM.

Functional Effects of the H1-Helix of Rat Cardiac Troponin T on Crossbridge Detachment Rate is Differently Modulated by α- and β-Myosin Heavy Chain Isoforms

The primary structure of the H1-helix of troponin T (TnT) varies among different types of striated muscles. Moreover, these muscles also express different myosin heavy chain (MHC) isoforms. Recently, we demonstrated that pseudo-phosphorylation of residue 204 (near the H1-helix) of cardiac TnT affected the functional state of the thin filament differently in fibers that expressed either α- or β-MHC isoforms (Michael et al., Basic Res Cardiol, 109:442, 2014). In this follow-up study, we investigated how the isoform-specific function of the H1-helix of cardiac TnT was influenced by α- and β-MHC isoforms. We generated a mutant rat cardiac TnT (RfsH1) in which the cardiac H1-helix was replaced by the fast skeletal H1-helix. Recombinant RfsH1 was reconstituted into detergent-skinned cardiac muscle fibers from either normal rats (expressing α-MHC) or propylthiouracil treated rats (expressing β-MHC). Steady-state and dynamic measurements were carried out at sarcomere length 2.3 µm. Our results demonstrated that RfsH1 decreased Ca2+-activated maximal ATPase activity differently in α-MHC (∼33%) and β-MHC (∼17%) fibers. Furthermore, RfsH1 decreased tension cost (∼31%) and crossbridge (XB) distortion dynamics (∼25%) in α-MHC but not in β-MHC fibers. Because the above mentioned parameters are indices of the rate of XB detachment, our results suggest that the interplay between the RfsH1- and α-MHC-mediated effects on the thin filament modulates XB detachment kinetics. Our findings suggest that the conformational changes in the H1-helix of TnT are sensitive to MHC isoform-mediated changes in the thin filament.

Activity of Na+/K+-activated Mg2+-dependent ATP-hydrolase in the cell-free extracts of the sulfate-reducing bacteria Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9

The aim of our work was to study Na+/K+-activated Mg2+-dependent ATPase activity in cell-free extracts of the sulfate-reducing bacteriaDesulfovibrio pigerVib-7 andDesulfomicrobiumsp. Rod-9 isolated from the human large intestine, and to carry out the kinetic analysis of the enzyme reaction. The maximum ATPase activity for both bacterial strains at +35 ºC was determined. The highest activities of the studied enzyme in the cell-free extracts ofD. pigerVib-7 at pH 7.0 andDesulfomicrobiumsp. Rod-9 at pH 6.5 were measured. Based on experimental data, the analysis of kinetic properties of the ATP-hydrolase reaction by the studied bacteria was carried out. The enzyme activity, initial (instantaneous) reaction rate (V0) and maximum rate of the ATPase reaction (Vmax) was significantly higher inD. pigerVib-7 cells than inDesulfomicrobiumsp. Rod-9. Michaelis constants (Km) of the enzyme reaction for both bacterial strains were determined.

Phosphorylation of protein kinase C sites Ser42/44 decreases Ca2+-sensitivity and blunts enhanced length-dependent activation in response to protein kinase A in human cardiomyocytes

Protein kinase C (PKC)-mediated phosphorylation of troponin I (cTnI) at Ser42/44 is increased in heart failure. While studies in rodents demonstrated that PKC-mediated Ser42/44 phosphorylation decreases maximal force and ATPase activity, PKC incubation of human cardiomyocytes did not affect maximal force. We investigated whether Ser42/44 pseudo-phosphorylation affects force development and ATPase activity using troponin exchange in human myocardium. Additionally, we studied if pseudo-phosphorylated Ser42/44 modulates length-dependent activation of force, which is regulated by protein kinase A (PKA)-mediated cTnI-Ser23/24 phosphorylation. Isometric force was measured in membrane-permeabilized cardiomyocytes exchanged with human recombinant wild-type troponin or troponin mutated at Ser42/44 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after PKA incubation. ATPase activity was measured in troponin-exchanged cardiac muscle strips. Compared to wild-type, 42D/44D decreased Ca2+-sensitivity without affecting maximal force in failing and donor cardiomyocytes. In donor myocardium, 42D/44D did not affect maximal ATPase activity or tension cost. Interestingly, 42D/44D blunted the length-dependent increase in Ca2+-sensitivity induced upon PKA-mediated phosphorylation. Since the drop in Ca2+-sensitivity at physiological Ca2+-concentrations is relatively large phosphorylation of Ser42/44 may result in a decrease of force and associated ATP utilization in the human heart.

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load.

Myosin-10 is an actin-based molecular motor that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration, and mitotic spindle maintenance. To study this motor protein's mechano-chemical properties, we used a recombinant, truncated form of myosin-10 consisting of the first 936 amino acids, followed by a GCN4 leucine zipper motif, to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of ∼310 nm/s. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s(-1)) is similar to the maximum ATPase activity (∼12-14 s(-1)) and therefore contributes to rate limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN), myosin-10 interacts intermittently with actin and produces a power stroke of ∼17 nm, composed of an initial 15-nm and subsequent 2-nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six ∼35-nm steps per binding interaction. We discuss the implications of this load-dependent processivity of myosin-10 as a filopodial transport motor.

Biochemical characterization of sporadic/familial hemiplegic migraine mutations

Sporadic hemiplegic migraine type 2 (SHM2) and familial hemiplegic migraine type 2 (FHM2) are rare forms of hemiplegic migraine caused by mutations in the Na+,K+-ATPase α2 gene. Today, more than 70 different mutations have been linked to SHM2/FHM2, randomly dispersed over the gene. For many of these mutations, functional studies have not been performed. Here, we report the functional characterization of nine SHM2/FHM2 linked mutants that were produced in Spodoptera frugiperda (Sf)9 insect cells. We determined ouabain binding characteristics, apparent Na+ and K+ affinities, and maximum ATPase activity. Whereas membranes containing T345A, R834Q or R879W possessed ATPase activity significantly higher than control membranes, P796S, M829R, R834X, del 935–940 ins Ile, R937P and D999H membranes showed significant loss of ATPase activity compared to wild type enzyme. Further analysis revealed that T345A and R879W showed no changes for any of the parameters tested, whereas mutant R834Q possessed significantly decreased Na+ and increased K+ apparent affinities as well as decreased ATPase activity and ouabain binding. We hypothesize that the majority of the mutations studied here influence interdomain interactions by affecting formation of hydrogen bond networks or interference with the C-terminal ion pathway necessary for catalytic activity of Na+,K+-ATPase, resulting in decreased functionality of astrocytes at the synaptic cleft expressing these mutants.

Elucidating the Role of Myosin Pseudo-Phosphorylation in a Novel Rescue Mouse Model of Cardiomyopathy

This study focuses on a novel rescue mouse model generated to test the effect of regulatory light chain (RLC) phosphorylation on a diseased cardiac phenotype. Pseudo-phosphorylation of the D166V (aspartic acid to valine) mutation shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) was examined. Transgenic (Tg) mice carrying the D166V mutation demonstrated severe functional and physiological abnormalities and a significant decrease in endogenous RLC phosphorylation compared with Tg-WT mice. These detrimental phenotypes were partially rescued by either myosin light chain kinase-induced phosphorylation or by using phosphomimic (S15D) D166V-RLC exchanged in porcine cardiac muscle preparations. Transgenic rescue mice carrying the S15D-D166V mutation have been generated and subjected to functional measurements. Myofibrillar ATPase assays performed on Tg-S15D-D166V vs. Tg-D166V and Tg-WT mice cardiac samples showed a pseudo-phosphorylation rescue of an abnormal increase in calcium sensitivity (hallmark of FHC) caused by the D166V mutation. A D166V-induced decrease in maximal ATPase activity was also reversed in Tg-S15D-D166V mice. Similarly, maximal force and myofilament Ca2+-sensitivity, that were largely compromised in Tg-D166V skinned muscle fibers, were improved in the rescue mice and brought to the level observed in Tg-WT. Histopathological analyses of heart sections from age and gender matched mice from all groups demonstrated an approximately four-fold decrease in the extent of fibrosis in the rescue mice compared to Tg-D166V. The beneficial effects of S15D on the function of the D166V hearts were confirmed in vivo by invasive hemodynamics. Results from this study emphasize the significance of RLC phosphorylation in alleviating an FHC induced phenotype and could be used in the development of therapeutic interventions to restore the normal cardiac function.Supported by NIH-HL108343 and HL071778.

Myosin-10 Produces its Power-Stroke in Two Phases and Moves Processively along a Single Actin Filament under Low-Load

Myosin-10 is an actin-activated ATPase that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration and mitotic spindle maintenance. The myosin-10 duty-cycle ratio - i.e. the fraction of time the motor remains tightly bound to actin during its total ATPase cycle - from previous biochemical studies are inconclusive, thus whether this myosin displays intermediate or high duty ratio is still under debate. To study this motor protein's mechano-chemical properties we have used a recombinant, truncated form of myosin-10 consisting of the first 940 amino acids, followed by a GCN4 leucine zipper motif to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules (∼87%) are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of 150 - 400 nm s−1. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12 - 14 s−1) and, therefore, contributes to rate-limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN) myosin-10 interacts intermittently with actin and produces a working stroke of ∼17 nm, composed of an initial 15 nm and subsequent 2 nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six, ∼35 nm, steps per binding interaction. Here we describe the kinetics and mechanics of myosin-10, interrogating bulk and single molecule biophysical/biochemical properties to further our understanding of its ATP-driven, motor mechanism and how this relates to its cellular functions.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60Å2 to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (Vmax) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller Kapp) than that of the wild-type actin, with the Vmax being almost unchanged. The Kapp and Vmax of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/Kapp reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.