ABSTRACT
BACKGROUND: In recent years, the study of creatine supplementation in professional athletes has been of great interest. However, the genetics involved in response to supplementation is unknown. The aim of this study was to analyse, for the first time, the relationship between muscle performance-related genes and the risk of an increased body mass index (BMI) and muscle mass and a decrease in fat mass in professional football players after creatine supplementation. METHODS: For this longitudinal study, one hundred and sixty-one men's professional football players were recruited. The polymorphisms ACE I/D, ACTN3 c.1729C>T, AMPD1 c.34C>T, CKM c.*800A>G, and MLCK (c.49C>T and c.37885C>A) were genotyped using Single-Nucleotide Primer Extension (SNPE). To assess the combined impact of these six polymorphisms, a total genotype score (TGS) was calculated. The creatine supplementation protocol consisted of 20 g/day of creatine monohydrate for 5 days (loading dose) and 3-5 g/day for 7 weeks (maintenance dose). Anthropometric characteristics (body mass index (BMI), fat, and muscle mass) were recorded before and after the creatine supplementation protocol. Characteristics of non-contact muscle injuries during the 2022/2023 season were classified according to a consensus statement for injury recording. The results showed that the allelic frequencies of ACE and AMPD1 differed between responders and non-responders in muscle mass increase (all p < 0.05). Players with a TGS exceeding 54.16 a.u. had an odds ratio (OR) of 2.985 (95%CI: 1.560-5.711; p = 0.001) for muscle mass increase. By contrast, those with a TGS below 54.16 a.u. had an OR of 9.385 (95%CI: 4.535-19.425; p < 0.001) for suffering non-contact muscle injuries during the season. CONCLUSIONS: The increase in BMI and muscle mass in response to creatine supplementation in professional football players was influenced by a TGS derived from the combination of favourable genotypes linked to muscle performance. The CC genotype and C allele of AMPD1 were particularly associated with a higher likelihood of muscle mass increase under creatine supplementation in this group of professional football players.
Subject(s)
AMP Deaminase , Actinin , Body Mass Index , Creatine , Dietary Supplements , Muscle, Skeletal , Polymorphism, Single Nucleotide , Soccer , Humans , Male , Creatine/administration & dosage , Muscle, Skeletal/drug effects , Actinin/genetics , AMP Deaminase/genetics , Adult , Longitudinal Studies , Young Adult , Peptidyl-Dipeptidase A/genetics , Creatine Kinase, MM Form/genetics , Athletes , Athletic Injuries/genetics , Athletic Injuries/prevention & control , GenotypeABSTRACT
Multiple genetic variants are known to influence athletic performance. These include polymorphisms of the muscle-specific creatine kinase (CKM) gene, which have been associated with endurance and/or power phenotypes. However, independent replication is required to support those findings. The aim of the present study was to determine whether the CKM (rs8111989, c.*800A>G) polymorphism is associated with power athlete status in professional Russian and Lithuanian competitors. Genomic DNA was collected from 693 national and international standard athletes from Russia (n = 458) and Lithuania (n = 235), and 500 healthy non-athlete subjects from Russia (n = 291) and Lithuania (n = 209). Genotyping for the CKM rs8111989 (A/G) polymorphism was performed using PCR or micro-array analysis. Genotype and allele frequencies were compared between all athletes and non-athletes, and between non-athletes and athletes, segregated according to population and sporting discipline (from anaerobic-type events). No statistically significant differences in genotype or allele frequencies were observed between non-athletes and power athletes (strength-, sprint- and speed/strength-oriented) athletes. The present study reports the non-association of the CKM rs8111989 with elite status in athletes from sports in which anaerobic energy pathways determine success.
Subject(s)
Athletic Performance , Creatine Kinase, MM Form/genetics , Polymorphism, Single Nucleotide , Sports , Adult , Female , Gene Frequency , Humans , Male , Young AdultABSTRACT
Tenofovir (TFV) is a key component of human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP). TFV is a nucleotide analog reverse-transcriptase inhibitor prodrug that requires two separate phosphorylation reactions by intracellular kinases to form the active metabolite tenofovir-diphosphate (TFV-DP). Muscle-type creatine kinase (CKM) has previously been demonstrated to be the kinase most responsible for the phosphorylation of tenofovir-monophosphate (TFV-MP) to the active metabolite in colon tissue. Because of the importance of CKM in TFV activation, genetic variation in CKM may contribute to interindividual variability in TFV-DP levels. In the present study, we report 10 naturally occurring CKM mutations that reduced TFV-MP phosphorylation in vitro: T35I, R43Q, I92M, H97Y, R130H, R132C, F169L, Y173C, W211R, V280L, and N286I. Interestingly, of these 10, only 4-R130H, R132C, W211R, and N286I-reduced both canonical CKM activities: ADP phosphorylation and ATP dephosphorylation. Although positions 130, 132, and 286 are located in the active site, the other mutations that resulted in decreased TFV-MP phosphorylation occur elsewhere in the protein structure. Four of these eight mutations-T35I, R43Q, I92M, and W211R-were found to decrease the thermal stability of the protein. Additionally, the W211R mutation was found to impact protein structure both locally and at a distance. These data suggest a substrate-specific effect such that certain mutations are tolerated for canonical activities while being deleterious toward the pharmacological activity of TFV activation, which could influence PrEP outcomes. SIGNIFICANCE STATEMENT: Muscle-type creatine kinase (CKM) is important to the activation of tenofovir, a key component of HIV prophylaxis. This study demonstrates that naturally occurring CKM mutations impact enzyme function in a substrate-dependent manner such that some mutations that do not reduce canonical activities lead to reductions in the pharmacologically relevant activity. This finding at the intersection of drug metabolism and energy metabolism is important to the perspective on pharmacology of other drugs acted on by atypical drug-metabolizing enzymes.
Subject(s)
Creatine Kinase, MM Form/chemistry , Mutation , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Binding Sites , Creatine Kinase, MM Form/genetics , Creatine Kinase, MM Form/metabolism , Humans , Molecular Docking Simulation , Phosphorylation , Protein Binding , Tenofovir/chemistry , Tenofovir/pharmacologyABSTRACT
A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.
Subject(s)
Creatine Kinase, MM Form/metabolism , Heart Failure/metabolism , Acetylation , Amino Acid Sequence , Animals , Creatine Kinase, MM Form/chemistry , Creatine Kinase, MM Form/genetics , Disease Models, Animal , Energy Metabolism , Humans , In Vitro Techniques , Male , Mice , Mice, 129 Strain , Models, Molecular , Myocardium/metabolism , Protein Conformation , Protein Multimerization , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sirtuin 2/metabolism , Sirtuin 2/pharmacologyABSTRACT
Muscle-type creatine kinase (CK-MM) is the target protein of ginsenosides in skeletal muscle. 20(S)-protopanaxadiol [20(S)-PPD] is an activator of CK-MM and exerts an anti-fatigue effect. In this study, twelve dammarane-type compounds were used for structure-activity relationship analysis in terms of enzyme activity, intermolecular interaction, and molecular docking. Enzyme activity analysis showed that 20(S)-PPD, 20(R)-PPD, 20(S)-protopanaxatriol [20(S)-PPT], 25-OH-PPD, 24-COOH-PPD, panaxadiol (PD), and ginsenoside Rh2 significantly increased CK-MM activity. Panaxatriol (PT), ocotillol, ginsenoside Rg1, and ginsenoside Rd had no significant influence on CK-MM activity, while jujubogenin inhibited its activity. Biolayer Interferometry (BLI) assay produced the same results as those on enzyme activity. The interaction intensity between dammarane-type compounds and CK-MM was linearly related to the compounds' maximum increment rate of enzyme activity. Molecular docking showed the following sequence of docking scores: Rd > Rg1 > Rh2 > 24-COOH-PPD > 20(S)-PPD > 20(S)-PPT > 25-OH-PPD > 20(R)-PPD > ocotillol > PT > PD > jujubogenin. We demonstrated that 20(S)-PPD was the best activator of CK-MM among the 12 dammarane-type compounds. The cyclization of the dammarane side chain, the hydroxyl group at position C6, and the glycosylation of C3, C6, and C20 reduced the ability to activate CK-MM. These findings can help in the development of enhanced CK-MM activators through structural modification.
Subject(s)
Biological Products/chemistry , Creatine Kinase, MM Form/metabolism , Triterpenes/chemistry , Binding Sites , Biological Products/metabolism , Creatine Kinase, MM Form/chemistry , Creatine Kinase, MM Form/genetics , Ginsenosides/chemistry , Ginsenosides/metabolism , Humans , Molecular Docking Simulation , Protein Structure, Tertiary , Structure-Activity Relationship , Triterpenes/metabolism , DammaranesABSTRACT
Friedreich's ataxia (FA) is due to deficiency of the mitochondrial protein, frataxin, which results in multiple pathologies including a deadly, hypertrophic cardiomyopathy. Frataxin loss leads to deleterious accumulations of redox-active, mitochondrial iron, and suppressed mitochondrial bioenergetics. Hence, there is an urgent need to develop innovative pharmaceuticals. Herein, the activity of the novel compound, 6-methoxy-2-salicylaldehyde nicotinoyl hydrazone (SNH6), was assessed in vivo using the well-characterized muscle creatine kinase (MCK) conditional frataxin knockout (KO) mouse model of FA. The design of SNH6 incorporated a dual-mechanism mediating: (1) NAD+-supplementation to restore cardiac bioenergetics; and (2) iron chelation to remove toxic mitochondrial iron. In these studies, MCK wild-type (WT) and KO mice were treated for 4-weeks from the asymptomatic age of 4.5-weeks to 8.5-weeks of age, where the mouse displays an overt cardiomyopathy. SNH6-treatment significantly elevated NAD+ and markedly increased NAD+ consumption in WT and KO hearts. In SNH6-treated KO mice, nuclear Sirt1 activity was also significantly increased together with the NAD+-metabolic product, nicotinamide (NAM). Therefore, NAD+-supplementation by SNH6 aided mitochondrial function and cardiac bioenergetics. SNH6 also chelated iron in cultured cardiac cells and also removed iron-loading in vivo from the MCK KO heart. Despite its dual beneficial properties of supplementing NAD+ and chelating iron, SNH6 did not mitigate cardiomyopathy development in the MCK KO mouse. Collectively, SNH6 is an innovative therapeutic with marked pharmacological efficacy, which successfully enhanced cardiac NAD+ and nuclear Sirt1 activity and reduced cardiac iron-loading in MCK KO mice. No other pharmaceutical yet designed exhibits both these effective pharmacological properties.
Subject(s)
Aldehydes/therapeutic use , Cardiomyopathies/drug therapy , Friedreich Ataxia/drug therapy , Hydrazones/therapeutic use , Iron Chelating Agents/therapeutic use , NAD/metabolism , Adenosine Triphosphate/metabolism , Aldehydes/pharmacology , Animals , Cardiomyopathies/metabolism , Cell Line , Creatine Kinase, MM Form/genetics , Disease Models, Animal , Friedreich Ataxia/metabolism , Hydrazones/pharmacology , Iron/metabolism , Iron Chelating Agents/pharmacology , Iron-Binding Proteins/genetics , Mice, Inbred C57BL , Mice, Knockout , Mitochondria, Heart/drug effects , Mitochondria, Heart/metabolism , Rats , FrataxinABSTRACT
The creatine/phosphocreatine system is the principal energy buffer in mammals, but is scarcely documented in fish. We measured the gene expression of major enzymes of this system, glycine amidinotransferase (GATM), guanidinoacetate N-methyltransferase (GAMT) and muscle-type creatine kinase (CKM) in kidney, liver, and muscle tissues of fish and mammals. CKM was expressed strongly in the muscles of all examined species. In contrast, GATM and GAMT were strongly expressed in the muscle tissue of fish, but not of mammals. This indicates that creatine synthesis and usage are spatially separated in mammals, but not in fish, which is supported by RNA-Seq data of 25 species. Differences in amino acid metabolism along with methionine adenosyltransferase gene expression in muscle from fishes but not mammals further support a central metabolic role of muscle in fish, and hence different organization of the creatine/phosphocreatine biosynthesis system in higher and lower vertebrates.
Subject(s)
Creatine/biosynthesis , Evolution, Molecular , Muscle, Skeletal/metabolism , Amidinotransferases/genetics , Animals , Creatine Kinase, MM Form/genetics , Fishes , Gene Expression Profiling , Muscle, Skeletal/enzymology , Sequence Analysis, RNAABSTRACT
AIMS: Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. METHODS AND RESULTS: We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. CONCLUSION: Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes' Ca2+ handling but also directly alters the heart's electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.
Subject(s)
Action Potentials , Arrhythmias, Cardiac/metabolism , Calcium Signaling , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Heart Rate , Myocardium/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/physiopathology , Clozapine/analogs & derivatives , Clozapine/pharmacology , Connexin 43/metabolism , Creatine Kinase, MM Form/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Isolated Heart Preparation , Male , Mice, Inbred C57BL , Mice, Transgenic , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C/metabolism , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/geneticsABSTRACT
Tenofovir (TFV), a nucleotide reverse transcriptase inhibitor, requires two phosphorylation steps to form a competitive inhibitor of HIV reverse transcriptase. Adenylate kinase 2 (AK2) has been previously demonstrated to phosphorylate tenofovir to tenofovir-monophosphate, while creatine kinase, muscle (CKM), pyruvate kinase, muscle (PKM) and pyruvate kinase, liver and red blood cell (PKLR) each have been found to phosphorylate tenofovir-monophosphate to the pharmacologically active tenofovir-diphosphate. In the present study, genomic DNA isolated from dried blood spots collected from 505 participants from Bangkok, Thailand; Cape Town, South Africa; and New York City, USA were examined for variants in AK2, CKM, PKM, and PKLR using next-generation sequencing. The bioinformatics tools SIFT and PolyPhen predicted that 19 of the 505 individuals (3.7% frequency) carried variants in at least one kinase that would result in a decrease or loss of enzymatic activity. To functionally test these predictions, AK2 and AK2 variants were expressed in and purified from E. coli, followed by investigation of their activities towards tenofovir. Interestingly, we found that purified AK2 had the ability to phosphorylate tenofovir-monophosphate to tenofovir-diphosphate in addition to phosphorylating tenofovir to tenofovir-monophosphate. Further, four of the six AK2 variants predicted to result in a loss or decrease of enzyme function exhibited a ≥30% decrease in activity towards tenofovir in our in vitro assays. Of note, an AK2 K28R variant resulted in a 72% and 81% decrease in the formation of tenofovir-monophosphate and tenofovir-diphosphate, respectively. These data suggest that there are naturally occurring genetic variants that could potentially impact TFV activation.
Subject(s)
Adenylate Kinase/genetics , Creatine Kinase, MM Form/genetics , Genetic Variation , HIV Infections/drug therapy , HIV-1/drug effects , Pyruvate Kinase/genetics , Tenofovir/pharmacology , Anti-HIV Agents/pharmacology , HIV Infections/epidemiology , HIV Infections/genetics , HIV Infections/virology , HIV-1/enzymology , HIV-1/genetics , High-Throughput Nucleotide Sequencing , Humans , South Africa/epidemiology , Thailand/epidemiology , United States/epidemiologyABSTRACT
PURPOSE: Exertional rhabdomyolysis can occur in individuals performing various types of exercise but it is unclear why some individuals develop this condition while others do not. Previous investigations have determined the role of several single nucleotide polymorphisms (SNPs) to explain inter-individual variability of serum creatine kinase (CK) concentrations after exertional muscle damage. However, there has been no research about the interrelationship among these SNPs. The purpose of this investigation was to analyze seven SNPs that are candidates for explaining individual variations of CK response after a marathon competition (ACE = 287bp Ins/Del, ACTN3 = p.R577X, CKMM = NcoI, IGF2 = C13790G, IL6 = 174G>C, MLCK = C37885A, TNFα = 308G>A). METHODS: Using Williams and Folland's model, we determined the total genotype score from the accumulated combination of these seven SNPs for marathoners with a low CK response (n = 36; serum CK <400 U·L-1) vs. marathoners with a high CK response (n = 31; serum CK ≥400 U·L-1). RESULTS: At the end of the race, low CK responders had lower serum CK (290±65 vs. 733±405 U·L-1; P<0.01) and myoglobin concentrations (443±328 vs. 1009±971 ng·mL-1, P<0.01) than high CK responders. Although the groups were similar in age, anthropometric characteristics, running experience and training habits, total genotype score was higher in low CK responders than in high CK responders (5.2±1.4 vs. 4.4±1.7 point, P = 0.02). CONCLUSION: Marathoners with a lower CK response after the race had a more favorable polygenic profile than runners with high serum CK concentrations. This might suggest a significant role of genetic polymorphisms in the levels of exertional muscle damage and rhabdomyolysis. Yet other SNPs, in addition to exercise training, might also play a role in the values of CK after damaging exercise.
Subject(s)
Creatine Kinase, MM Form/genetics , Physical Exertion , Polymorphism, Single Nucleotide , Rhabdomyolysis/diagnosis , Rhabdomyolysis/genetics , Actinin/blood , Actinin/genetics , Adolescent , Adult , Aged , Creatine Kinase, MM Form/blood , Female , Gene Expression Profiling , Gene Expression Regulation , Humans , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Interleukin-6/blood , Interleukin-6/genetics , Male , Middle Aged , Myoglobin/blood , Myosin-Light-Chain Kinase/blood , Myosin-Light-Chain Kinase/genetics , Peptidyl-Dipeptidase A/blood , Peptidyl-Dipeptidase A/genetics , Prognosis , Rhabdomyolysis/blood , Rhabdomyolysis/pathology , Running , Tumor Necrosis Factor-alpha/blood , Tumor Necrosis Factor-alpha/geneticsABSTRACT
Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate, which are critical fuel metabolites of skeletal muscle particularly during exercise. However, the physiological relevance of LDH remains poorly understood. Here we show that Ldhb expression is induced by exercise in human muscle and negatively correlated with changes in intramuscular pH levels, a marker of lactate production, during isometric exercise. We found that the expression of Ldhb is regulated by exercise-induced peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). Ldhb gene promoter reporter studies demonstrated that PGC-1α activates Ldhb gene expression through multiple conserved estrogen-related receptor (ERR) and myocyte enhancer factor 2 (MEF2) binding sites. Transgenic mice overexpressing Ldhb in muscle (muscle creatine kinase (MCK)-Ldhb) exhibited increased exercise performance and enhanced oxygen consumption during exercise. MCK-Ldhb muscle was shown to have enhanced mitochondrial enzyme activity and increased mitochondrial gene expression, suggesting an adaptive oxidative muscle transformation. In addition, mitochondrial respiration capacity was increased and lactate production decreased in MCK-Ldhb skeletal myotubes in culture. Together, these results identified a previously unrecognized Ldhb-driven alteration in muscle mitochondrial function and suggested a mechanism for the adaptive metabolic response induced by exercise training.
Subject(s)
Gene Expression Regulation, Enzymologic/physiology , L-Lactate Dehydrogenase/biosynthesis , Mitochondria, Muscle/enzymology , Muscle, Skeletal/enzymology , Physical Conditioning, Animal , Animals , Creatine Kinase, MM Form/genetics , Creatine Kinase, MM Form/metabolism , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , L-Lactate Dehydrogenase/genetics , Mice , Mice, Transgenic , Mitochondria, Muscle/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/genetics , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolismABSTRACT
OBJECTIVE: Discusses the distributive characters of the Creatine Kinase MM (CKMM) gene A/G Polymorphism in XinjiangUyghur, One hundred and fourtheen athletes and 441 general population of Uyghur were involved in the study. METHODS: Polymerase chain reaction-restriction fragment length polymorphism was used. RESULTS: (1) The CKMM gene A/G frequency in Uyghur general population was(AA, AG and GG) 0.497, 0.392 and 0.111, the result test by Hardy-Weinberg (H-W) equilibrium and x² = 2.72, P = 0.1, df = 2, indicated that the control group had representative. (2) AA, AG and GG genotype frequency of power-oriented athlete respectively was 0.442,0.302 and 0.256, frequency of GG genotype and G allele was higher than the control group, there were significant differences compared to thecontrol( P < 0.05, df = 2); (3) A/G genotype frequency of Endurance-oriented athletere spectively was 0.571, 0.400 and 0.029, there were nosignificant differences compared to the controls ( P > 0. 05, df = 2). (4) A/G genotype frequency of Uyghur soccer athletes respectively was0.472, 0.361 and 0.167, G allele was higher than the Endurance-oriented athlete and lower than the power-oriented athletes. and no significant differences compared to the controls( P > 0.05, df = 2). CONCLUSION: The results indicate that the CKMM gene GG genotype and G alleleare represented in power-oriented athletes, but don't find A/G polymorphism correlation with endurance and the football sport performance.
Subject(s)
Athletic Performance , Creatine Kinase, MM Form/genetics , Physical Endurance/genetics , Polymorphism, Genetic , Alleles , Asian People/genetics , Athletes , China , Gene Frequency , Genotype , Humans , Polymorphism, Restriction Fragment LengthABSTRACT
Creatine kinase (CK) helps maintain homeostasis of intracellular ATP level by catalyzing the reversible phosphotransfer between ATP and phosphocreatine. In humans, there are two cytosolic CK isoforms, the muscle-type (M) and the brain-type (B), which frequently function as homodimers (hMMCK and hBBCK). Interestingly, these isoenzymes exhibit significantly different thermostabilities, despite high similarity in amino acid sequences and tertiary structures. In order to investigate the mechanism of this phenomenon, in this work, we first used domain swapping and site-directed mutagenesis to search for the key residues responsible for the isoenzyme-specific thermostability. Strikingly, the difference in thermostability was found to principally arise from one single residue substitution at position 36 (Pro in hBBCK vs. Leu in hMMCK). We then engaged the molecular dynamics simulations to study the molecular mechanism. The calculations imply that the P36L substitution introduces additional local interactions around residue 36 and thus further stabilizes the dimer interface through a complex interaction network, which rationalizes the observation that hMMCK is more resistant to thermal inactivation than hBBCK. We finally confirmed this molecular explanation through thermal inactivation assays on Asp36 mutants that were proposed to devastate the local interactions and thus the dimer associations in both isoenzymes.
Subject(s)
Amino Acid Substitution , Creatine Kinase, BB Form/chemistry , Creatine Kinase, BB Form/genetics , Creatine Kinase, MM Form/chemistry , Creatine Kinase, MM Form/genetics , Protein Stability , Humans , Isoenzymes , Models, Molecular , Mutation , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , TemperatureABSTRACT
Creatine kinase (CK) and lactate dehydrogenase (LDH) are widely used markers of tissue damage. To search for sequence variants influencing serum levels of CK and LDH, 28.3 million sequence variants identified through whole-genome sequencing of 2,636 Icelanders were imputed into 63,159 and 98,585 people with CK and LDH measurements, respectively. Here we describe 13 variants associating with serum CK and 16 with LDH levels, including four that associate with both. Among those, 15 are non-synonymous variants and 12 have a minor allele frequency below 5%. We report sequence variants in genes encoding the enzymes being measured (CKM and LDHA), as well as in genes linked to muscular (ANO5) and immune/inflammatory function (CD163/CD163L1, CSF1, CFH, HLA-DQB1, LILRB5, NINJ1 and STAB1). A number of the genes are linked to the mononuclear/phagocyte system and clearance of enzymes from the serum. This highlights the variety in the sources of normal diversity in serum levels of enzymes.
Subject(s)
Creatine Kinase, MM Form/genetics , Creatine Kinase/blood , L-Lactate Dehydrogenase/blood , Anoctamins , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Biomarkers/blood , Cell Adhesion Molecules, Neuronal/genetics , Chloride Channels/genetics , Complement Factor H/genetics , Female , Gene Frequency , Genetic Variation , HLA-DQ beta-Chains/genetics , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Iceland , Isoenzymes/genetics , L-Lactate Dehydrogenase/genetics , Lactate Dehydrogenase 5 , Macrophage Colony-Stimulating Factor/genetics , Male , Membrane Glycoproteins , Nerve Growth Factors/genetics , Polymorphism, Single Nucleotide , Receptors, Cell Surface/genetics , Receptors, Immunologic/genetics , Receptors, Lymphocyte Homing/genetics , Receptors, Scavenger , Regression AnalysisABSTRACT
Protein glutathionylation is an important post-translational modification that regulates many cellular processes, including energy metabolism, signal transduction, and protein homeostasis. Global profiling of glutathionylated proteins (denoted as glutathionylome) is crucial for understanding redox-regulated signal transduction. Here, we developed a novel method based on click reaction and proteomics to enrich and identify the glutathionylated peptides in Escherichia coli and Drosophila lysates, in which 937 and 1,930 potential glutathionylated peptides were identified, respectively. Bioinformatics analysis showed that the cysteine residue next to negatively charged amino acid residues has a higher frequency of glutathionylation. Importantly, we found that most proteins associated with metabolic pathways were glutathionylated and that the glutathionylation sites of metabolic enzymes were highly conserved among different species. Our results indicate that the glutathione analog is a useful tool to characterize protein glutathionylation, and glutathionylation of metabolic enzymes, which play important roles in regulating cellular metabolism, is conserved.
Subject(s)
Drosophila Proteins/chemistry , Drosophila/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli/metabolism , Glutathione/analogs & derivatives , Molecular Probes/chemistry , Amino Acid Sequence , Animals , Citric Acid Cycle , Click Chemistry , Creatine Kinase, MM Form/chemistry , Creatine Kinase, MM Form/genetics , Creatine Kinase, MM Form/metabolism , Cysteine/chemistry , Cysteine/metabolism , Drosophila Proteins/metabolism , Escherichia coli Proteins/metabolism , Glutathione/chemical synthesis , Humans , Malate Dehydrogenase/antagonists & inhibitors , Malate Dehydrogenase/metabolism , Molecular Sequence Data , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Protein Processing, Post-Translational , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
OBJECTIVE: Muscle-specific creatine kinase is thought to play an integral role in maintaining energy homeostasis by providing a supply of creatine phosphate. The genetic variant, rs8111989, contributes to individual differences in physical performance, and thus the purpose of this study was to determine if rs8111989 variant is predictive of Physical Fitness Test (PFT) scores in male, military infantry recruits. METHODS: DNA was extracted from whole blood, and genotyping was performed in 176 Marines. Relationships between PFT measures (run, sit-ups, and pull-ups) and genotype were determined. RESULTS: Participants with 2 copies of the T allele for rs8111989 variant had higher PFT scores for run time, pull-ups, and total PFT score. Specifically, participants with 2 copies of the TT allele (variant) (n = 97) demonstrated an overall higher total PFT score as compared with those with one copy of the C allele (n = 79) (TT: 250 ± 31 vs. CC/CT: 238 ± 31; p = 0.02), run score (TT: 82 ± 10 vs. CC/CT: 78 ± 11; p = 0.04) and pull-up score (TT: 78 ± 11 vs. CC/CT: 65 ± 21; p = 0.04) or those with the CC/CT genotype. CONCLUSION: These results demonstrate an association between physical performance measures and genetic variation in the muscle-specific creatine kinase gene (rs8111989).
Subject(s)
Creatine Kinase, MM Form/genetics , Military Personnel , Physical Fitness , Adolescent , Exercise Test , Genotype , Humans , Male , Polymorphism, Single Nucleotide , United States , Young AdultABSTRACT
Cyclin D3 is important for muscle development and regeneration, and is involved in post-mitotic arrest of muscle cells. Cyclin D3 also has cell-cycle-independent functions such as regulation of specific genes in other tissues. Ectopic expression of cyclin D3 in myoblasts, where it is normally undetectable, promotes muscle gene expression and faster differentiation kinetics upon serum depletion. In the present study, we investigated the mechanistic role of cyclin D3 in muscle gene regulation. We initially showed by mutational analysis that a stable and functional cyclin D3 was required for promoting muscle differentiation. Using chromatin immunoprecipitation assays, we demonstrated that expression of cyclin D3 in undifferentiated myoblasts altered histone epigenetic marks at promoters of muscle-specific genes like MyoD, Pax7, myogenin and muscle creatine kinase but not non-muscle genes. Cyclin D3 expression also reduced the mRNA levels of certain epigenetic modifier genes. Our data suggest that epigenetic modulation of muscle-specific genes in cyclin-D3-expressing myoblasts may be responsible for faster differentiation kinetics upon serum depletion. Our results have implications for a regulatory role for cyclin D3 in muscle-specific gene activation.
Subject(s)
Creatine Kinase, MM Form/genetics , Cyclin D3/genetics , Muscle Cells/metabolism , MyoD Protein/genetics , Myogenin/genetics , PAX7 Transcription Factor/genetics , Promoter Regions, Genetic/genetics , Animals , Cells, Cultured , Chromatin Immunoprecipitation , Cyclin D3/metabolism , Gene Expression Regulation/genetics , Mice , Muscle Cells/cytology , RNA, Messenger/genetics , Regeneration/geneticsABSTRACT
Muscle-specific creatine kinase (CKMM) plays a vital role in the energy homeostasis of muscle cells. The A/G variation (rs8111989) located in the 3'-untranslated region of the CKM gene has been found to be the most relevant in terms of genetic testing in sport. The aim of the presented study was to test the hypothesis that the G allele might represent a genetic element that contributes to the improvement of endurance performance in Polish and Russian rowers. The distribution of the CKM genotypes was examined in a group of Polish and Russian athletes in comparison with non-athlete controls. There were no statistical differences between the rowers and the control groups across the CKM genotypes when Polish or Russian participants were analyzed. Based on the obtained results, it may be speculated that the CKM A/G polymorphism is not an important determinant of endurance performance level in Polish and Russian rowers. However, these results should be interpreted with caution as they can be limited by many factors.
Subject(s)
3' Untranslated Regions , Creatine Kinase, MM Form/genetics , Physical Endurance/physiology , Polymorphism, Genetic , Adult , Athletes , Humans , Male , Poland , RussiaABSTRACT
Myoblast fusion is critical for proper muscle growth and regeneration. During myoblast fusion, the localization of some molecules is spatially restricted; however, the exact reason for such localization is unknown. Creatine kinase B (CKB), which replenishes local ATP pools, localizes near the ends of cultured primary mouse myotubes. To gain insights into the function of CKB, we performed a yeast two-hybrid screen to identify CKB-interacting proteins. We identified molecules with a broad diversity of roles, including actin polymerization, intracellular protein trafficking, and alternative splicing, as well as sarcomeric components. In-depth studies of α-skeletal actin and α-cardiac actin, two predominant muscle actin isoforms, demonstrated their biochemical interaction and partial colocalization with CKB near the ends of myotubes in vitro. In contrast to other cell types, specific knockdown of CKB did not grossly affect actin polymerization in myotubes, suggesting other muscle-specific roles for CKB. Interestingly, knockdown of CKB resulted in significantly increased myoblast fusion and myotube size in vitro, whereas knockdown of creatine kinase M had no effect on these myogenic parameters. Our results suggest that localized CKB plays a key role in myotube formation by limiting myoblast fusion during myogenesis.
Subject(s)
Creatine Kinase, BB Form/genetics , Muscle Development/genetics , Muscle Fibers, Skeletal/enzymology , Myoblasts/enzymology , Actins/genetics , Actins/metabolism , Alternative Splicing , Animals , Cell Fusion , Creatine Kinase, BB Form/antagonists & inhibitors , Creatine Kinase, BB Form/metabolism , Creatine Kinase, MM Form/genetics , Creatine Kinase, MM Form/metabolism , Gene Expression Regulation, Developmental , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Muscle Fibers, Skeletal/cytology , Myoblasts/cytology , Polymerization , Primary Cell Culture , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction , Two-Hybrid System TechniquesABSTRACT
Maintenance of skeletal muscle structure and function requires a precise stoichiometry of sarcomeric proteins for proper assembly of the contractile apparatus. Absence of components of the sarcomeric thin filaments causes nemaline myopathy, a lethal congenital muscle disorder associated with aberrant myofiber structure and contractility. Previously, we reported that deficiency of the kelch-like family member 40 (KLHL40) in mice results in nemaline myopathy and destabilization of leiomodin-3 (LMOD3). LMOD3 belongs to a family of tropomodulin-related proteins that promote actin nucleation. Here, we show that deficiency of LMOD3 in mice causes nemaline myopathy. In skeletal muscle, transcription of Lmod3 was controlled by the transcription factors SRF and MEF2. Myocardin-related transcription factors (MRTFs), which function as SRF coactivators, serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. Conversely, conditions that favor actin polymerization de-repress MRTFs and activate SRF-dependent genes. We demonstrated that the actin nucleator LMOD3, together with its stabilizing partner KLHL40, enhances MRTF-SRF activity. In turn, SRF cooperated with MEF2 to sustain the expression of LMOD3 and other components of the contractile apparatus, thereby establishing a regulatory circuit to maintain skeletal muscle function. These findings provide insight into the molecular basis of the sarcomere assembly and muscle dysfunction associated with nemaline myopathy.