RESUMO
The sarcomeric titin springs influence myocardial distensibility and passive stiffness. Titin isoform composition and protein kinase (PK)A-dependent titin phosphorylation are variables contributing to diastolic heart function. However, diastolic tone, relaxation speed, and left ventricular extensibility are also altered by PKG activation. We used back-phosphorylation assays to determine whether PKG can phosphorylate titin and affect titin-based stiffness in skinned myofibers and isolated myofibrils. PKG in the presence of 8-pCPT-cGMP (cGMP) phosphorylated the 2 main cardiac titin isoforms, N2BA and N2B, in human and canine left ventricles. In human myofibers/myofibrils dephosphorylated before mechanical analysis, passive stiffness dropped 10% to 20% on application of cGMP-PKG. Autoradiography and anti-phosphoserine blotting of recombinant human I-band titin domains established that PKG phosphorylates the N2-B and N2-A domains of titin. Using site-directed mutagenesis, serine residue S469 near the COOH terminus of the cardiac N2-B-unique sequence (N2-Bus) was identified as a PKG and PKA phosphorylation site. To address the mechanism of the PKG effect on titin stiffness, single-molecule atomic force microscopy force-extension experiments were performed on engineered N2-Bus-containing constructs. The presence of cGMP-PKG increased the bending rigidity of the N2-Bus to a degree that explained the overall PKG-mediated decrease in cardiomyofibrillar stiffness. Thus, the mechanically relevant site of PKG-induced titin phosphorylation is most likely in the N2-Bus; phosphorylation of other titin sites could affect protein-protein interactions. The results suggest that reducing titin stiffness by PKG-dependent phosphorylation of the N2-Bus can benefit diastolic function. Failing human hearts revealed a deficit for basal titin phosphorylation compared to donor hearts, which may contribute to diastolic dysfunction in heart failure.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Insuficiência Cardíaca Diastólica/metabolismo , Ventrículos do Coração/metabolismo , Proteínas Musculares/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Conectina , Sequência Consenso , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Cães , Elasticidade , Humanos , Dados de Sequência Molecular , Proteínas Musculares/genética , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/ultraestrutura , Miofibrilas/efeitos dos fármacos , Miofibrilas/ultraestrutura , Óxido Nítrico/fisiologia , Fosforilação , Isoformas de Proteínas/metabolismo , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão/fisiologia , Sarcômeros/metabolismo , Sarcômeros/ultraestrutura , Relação Estrutura-Atividade , Remodelação Ventricular/fisiologiaRESUMO
The giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titin's spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-B(us)), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-B(us) molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-B(us). We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart.
Assuntos
Dissulfetos/química , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Quinases/química , Proteínas Quinases/genética , Sequência de Aminoácidos , Fenômenos Químicos , Simulação por Computador , Conectina , Elasticidade , Escherichia coli , Humanos , Microscopia de Força Atômica , Modelos Químicos , Dados de Sequência Molecular , Proteínas Mutantes/química , Miofibrilas/química , Oxirredução , Estabilidade Proteica , Proteínas Recombinantes/química , Tiorredoxinas/químicaRESUMO
The mammalian class IX myosin Myo9b can move considerable distances along actin filaments before it dissociates. This is remarkable, because it is single headed and because the rate-limiting step in its ATPase cycle is ATP hydrolysis. Thus, it spends most of its cycling time in the ATP-bound state that has a weak affinity for F-actin in other myosins. It has been speculated that the very extended loop 2 in the Myo9b head domain comprises an additional actin-binding site that prevents it from dissociation in the weak binding states. Here we show that two regions in the loop 2 determine the F-actin concentrations needed to maximally activate the steady-state ATPase activity. Together these two regions regulate the amount capable of binding F-actin and the affinity of the nucleotide-free state. The isolated loop 2 behaved like an entropic spring and bound stoichiometrically and with high affinity to F-actin. Subfragment 1 from skeletal muscle myosin II bound to F-actin simultaneously with the isolated loop 2 of Myo9b and could not displace it. Furthermore, the present results imply also a regulatory role for the tail region. Taken together, the results demonstrate that the extended loop 2 in Myo9b binds F-actin and influences the binding of the conventional stereo-specific actin-binding site.
Assuntos
Actinas/química , Trifosfato de Adenosina/química , Miosinas/química , Actinas/genética , Actinas/metabolismo , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/fisiologia , Linhagem Celular , Hidrólise , Miosinas/genética , Miosinas/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , RatosRESUMO
Perturbation of a protein away from its native state by mechanical stress is a physiological process immanent to many cells. The mechanical stability and conformational diversity of proteins under force therefore are important parameters in nature. Molecular-level investigations of "mechanical proteins" have enjoyed major breakthroughs over the last decade, a development to which atomic force microscopy (AFM) force spectroscopy has been instrumental. The giant muscle protein titin continues to be a paradigm model in this field. In this paper, we review how single-molecule mechanical measurements of titin using AFM have served to elucidate key aspects of protein unfolding-refolding and mechanisms by which biomolecular elasticity is attained. We outline recent work combining protein engineering and AFM force spectroscopy to establish the mechanical behavior of titin domains using molecular "fingerprinting." Furthermore, we summarize AFM force-extension data demonstrating different mechanical stabilities of distinct molecular-spring elements in titin, compare AFM force-extension to novel force-ramp/force-clamp studies, and elaborate on exciting new results showing that AFM force clamp captures the unfolding and refolding trajectory of single mechanical proteins. Along the way, we discuss the physiological implications of the findings, not least with respect to muscle mechanics. These studies help us understand how proteins respond to forces in cells and how mechanosensing and mechanosignaling events may proceed in vivo.
Assuntos
Microscopia de Força Atômica , Proteínas Musculares/química , Proteínas Quinases/química , Animais , Fenômenos Biomecânicos , Conectina , Elasticidade , Humanos , Estrutura Molecular , Proteínas Musculares/metabolismo , Proteínas Musculares/ultraestrutura , Dobramento de Proteína , Proteínas Quinases/metabolismo , Proteínas Quinases/ultraestrutura , Sarcômeros/metabolismoRESUMO
Titin is a giant protein responsible for passive-tension generation in muscle sarcomeres. Here, we used single-molecule AFM force spectroscopy to investigate the mechanical characteristics of a recombinant construct from the human cardiac-specific N2B-region, which harbors a 572-residue unique sequence flanked by two immunoglobulin (Ig) domains on either side. Force-extension curves of the N2B-construct revealed mean unfolding forces for the Ig-domains similar to those of a recombinant fragment from the distal Ig-region in titin (I91-98). The mean contour length of the N2B-unique sequence was 120 nm, but there was a bimodal distribution centered at approximately 95 nm (major peak) and 180 nm (minor peak). These values are lower than expected if the N2B-unique sequence were a permanently unfolded entropic spring, but are consistent with the approximately 100 nm maximum extension of that segment measured in isolated stretched cardiomyofibrils. A contour-length below 200 nm would be reasonable, however, if the N2B-unique sequence were stabilized by a disulphide bridge, as suggested by several disulphide connectivity prediction algorithms. Since the N2B-unique sequence can be phosphorylated by protein kinase A (PKA), which lowers titin-based stiffness, we studied whether addition of PKA (+ATP) affects the mechanical properties of the N2B-construct, but found no changes. The softening effect of PKA on N2B-titin may require specific conditions/factors present inside the cardiomyocytes.