Allosteric Transmission along a Loosely Structured Backbone Allows a Cardiac Troponin C Mutant to Function with Only One Ca2+ Ion.

Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyopathies and a major cause of sudden death in young athletes. The Ca2+ sensor of the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating muscle contraction. Although several cardiomyopathy-causing mutations have been identified in cTnC, the limited information about their structural defects has been mapped to the HCM phenotype. Here, we used high-resolution electron-spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG-RD), and affinity measurements of cTnC for the thin filament in reconstituted papillary muscles to provide evidence of an allosteric mechanism in mutant cTnC that may play a role to the HCM phenotype. We showed that the D145E mutation leads to altered dynamics on a µs-ms time scale and deactivates both of the divalent cation-binding sites of the cTnC C-domain. CPMG-RD captured a low populated protein-folding conformation triggered by the Glu-145 replacement of Asp. Paradoxically, although D145E C-domain was unable to bind Ca2+, these changes along its backbone allowed it to attach more firmly to thin filaments than the wild-type isoform, providing evidence for an allosteric response of the Ca2+-binding site II in the N-domain. Our findings explain how the effects of an HCM mutation in the C-domain reflect up into the N-domain to cause an increase of Ca2+ affinity in site II, thus opening up new insights into the HCM phenotype.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

Brazilian Science and Research Integrity: Where are We? What Next?

Building a world-class scientific community requires first-class ingredients at many different levels: funding, training, management, international collaborations, creativity, ethics, and an understanding of research integrity practices. All over the world, addressing these practices has been high on the science policy agenda of major research systems. Universities have a central role in fostering a culture of research integrity, which has posed additional challenges for faculty, students and administrators - but also opportunities. In Brazil, the leading universities and governmental funding agencies are collaborating on this project, but much remains to be done.

Insights into the intramolecular coupling between the N- and C-domains of troponin C derived from high-pressure, fluorescence, nuclear magnetic resonance, and small-angle X-ray scattering studies.

Troponin C (TnC), the Ca(2+)-binding component of the troponin complex of vertebrate skeletal muscle, consists of two structurally homologous domains, the N- and C-domains; these domains are connected by an exposed α-helix. Mutants of full-length TnC and of its isolated domains have been constructed using site-directed mutagenesis to replace different Phe residues with Trp. Previous studies utilizing these mutants and high hydrostatic pressure have shown that the apo form of the C-domain is less stable than the N-domain and that the N-domain has no effect on the stability of the C-domain [Rocha, C. B., Suarez, M. C., Yu, A., Ballard, L., Sorenson, M. M., Foguel, D., and Silva, J. L. (2008) Biochemistry 47, 5047-5058]. Here, we analyzed the stability of full-length F29W TnC using structural approaches under conditions of added urea and hydrostatic pressure denaturation; F29W TnC is a fluorescent mutant, in which Phe 29, located in the N-domain, was replaced with Trp. From these experiments, we calculated the thermodynamic parameters (ΔV and ΔG°(atm)) that govern the folding of the intact F29W TnC in the absence or presence of Ca(2+). We found that the C-domain has only a small effect on the structure of the N-domain in the absence of Ca(2+). However, using fluorescence spectroscopy, we demonstrated a significant decrease in the stability of the N-domain in the Ca(2+)-bound state (i.e., when Ca(2+) was also bound to sites III and IV of the C-domain). An accompanying decrease in the thermodynamic stability of the N-domain generated a reduction in ΔΔG°(atm) in absolute terms, and Ca(2+) binding affects the Ca(2+) affinity of the N-domain in full-length TnC. Cross-talk between the C- and N-domains may be mediated by the central helix, which has a smaller volume and likely greater rigidity and stability following binding of Ca(2+) to the EF-hand sites, as determined by our construction of low-resolution three-dimensional models from the small-angle X-ray scattering data.

Strong cross-bridges potentiate the Ca(2+) affinity changes produced by hypertrophic cardiomyopathy cardiac troponin C mutants in myofilaments: a fast kinetic approach.

This spectroscopic study examined the steady-state and kinetic parameters governing the cross-bridge effect on the increased Ca(2+) affinity of hypertrophic cardiomyopathy-cardiac troponin C (HCM-cTnC) mutants. Previously, we found that incorporation of the A8V and D145E HCM-cTnC mutants, but not E134D into thin filaments (TFs), increased the apparent Ca(2+) affinity relative to TFs containing the WT protein. Here, we show that the addition of myosin subfragment 1 (S1) to TFs reconstituted with these mutants in the absence of MgATP(2-), the condition conducive to rigor cross-bridge formation, further increased the apparent Ca(2+) affinity. Stopped-flow fluorescence techniques were used to determine the kinetics of Ca(2+) dissociation (k(off)) from the cTnC mutants in the presence of TFs and S1. At a high level of complexity (i.e. TF + S1), an increase in the Ca(2+) affinity and decrease in k(off) was achieved for the A8V and D145E mutants when compared with WT. Therefore, it appears that the cTnC Ca(2+) off-rate is most likely to be affected rather than the Ca(2+) on rate. At all levels of TF complexity, the results obtained with the E134D mutant reproduced those seen with the WT protein. We conclude that strong cross-bridges potentiate the Ca(2+)-sensitizing effect of HCM-cTnC mutants on the myofilament. Finally, the slower k(off) from the A8V and D145E mutants can be directly correlated with the diastolic dysfunction seen in these patients.

Redox state of troponin C cysteine in the D/E helix alters the C-domain affinity for the thin filament of vertebrate striated muscle.

BACKGROUND: Despite a broad spectrum of structural studies, it is not yet clear whether the D/E helix of troponin C (TnC) contributes to the interaction of TnC with troponin I (TnI). Redox modifications at Cys 98 in the D/E helix were explored for clues to TnC binding to the thin filament off-state, using recombinant wild-type TnC and an engineered mutant without Cys (Cys98Leu). METHODS: Recombinant proteins and rabbit psoas skinned fibres were reduced with dithiothreitol (DTT) and variously recombined. Changes in affinity of reduced or oxidised TnC for the thin filament were evaluated via TnC binding and dissociation, using a standardized test for maximal force as an index of fibre TnC content. RESULTS: All oxidation and reduction effects observed were reversible and led to changes in TnC content. Oxidation (H(2)O(2)) reduced TnC affinity for the filament; reduction (DTT) increased it. Reducing other fibre proteins had no effect. Binding of the Cys-less TnC mutant was not altered by DTT, nor was dissociation of wild-type TnC from reconstituted hybrids (skeletal TnC in cardiac trabeculae). Thus when Cys 98 in the D/E helix of TnC is fully reduced, its binding affinity for the thin filament of skeletal muscle is enhanced and helps to anchor it to the filament. GENERAL SIGNIFICANCE: Signal transmission between TnC and the other proteins of the regulatory complex is sensitive to the redox state of Cys 98.

Redox balance and mitochondrial glycerol phosphate dehydrogenase activity in trained rats.

Free radical production is increased in many disease states and during exercise, but in the latter the concurrent stimulation of the antioxidant defense system seems to protect the organism from excessive production of reactive oxygen species. Chronic exercise can exert negative effects on the activity of mitochondrial glycerol phosphate dehydrogenase (mGPdH), which may offer some explanation for the antioxidant effects of training, since this enzyme is a relevant producer of free radicals. To test this correlation, we compared mGPdH activity, two antioxidant defense markers and two markers of oxidative stress in sedentary and trained (Tr) rats. Training was through a swimming exercise 3 days a week. After 8 weeks, Tr rats lasted twice as long as controls in an acute swimming test with a 5% load. Forty-eight hours after the last exercise, the animals were killed to collect blood and tissues. Tr animals presented lower body weight and visceral fat mass with lower triglyceride content in visceral fat and plasma (p < 0.05). The specific activity of mGPdH in muscle mitochondria was reduced in Tr rats by 88% (p < 0.05). Total antioxidant capacity, lipid peroxidation and reduced glutathione (GSH) in liver and muscle were unaltered, while plasma GSH increased by 21% (p < 0.05). These data suggest a profile of successful redox equilibrium maintenance in Tr rats, with a potentially significant contribution from the lower level of mGPdH activity in muscle. This training protocol appears to be suitable for use in detailed studies of biochemical adaptations to oxidative stress.

2,4-Dinitrophenol reduces the reactivity of Lys553 in the lower 50-kDa region of myosin subfragment 1.

2,4-Dinitrophenol (DNP) increases the affinity of myosin for actin and accelerates its Mg(2+)ATPase activity, suggesting that it acts on a region of the myosin head that transmits conformational changes to actin- and ATP-binding sites. The binding site/s for DNP are unknown; however similar hydrophobic compounds bind to the 50-kDa subfragment of the myosin head, near the actin-binding interface. In this region, a helix-loop-helix motif contains Lys553, which is specifically labeled with the fluorescent probe 6-[fluorescein-5(and 6)-carboxamido] hexanoic acid succinimidyl ester (FHS). This reaction is sensitive to conformational changes in the helix-loop-helix and the labeling efficiency was reduced when S1 was bound to actin, DNP or nucleotide analogs. The nucleotide analogs had a range of effects (PPi>ADP·AlF(4)(-)>ADP) irrespective of the open-closed state of switch 2. The greatest reduction in labeling was in the presence of actin or DNP. When we measured the effect of each ligand on the fluorescence of FHS previously attached to S1, only DNP quenched the emission. Together, the results suggest that the helix-loop-helix region is flexible, it is part of the communication pathway between the ATP- and actin-binding sites of myosin and it is proximal to the region of myosin where DNP binds.

Myosin is reversibly inhibited by S-nitrosylation.

Nitric oxide (NO*) is synthesized in skeletal muscle and its production increases during contractile activity. Although myosin is the most abundant protein in muscle, it is not known whether myosin is a target of NO* or NO* derivatives. In the present study, we have shown that exercise increases protein S-nitrosylation in muscle, and, among contractile proteins, myosin is the principal target of exogenous SNOs (S-nitrosothiols) in both skinned skeletal muscle fibres and differentiated myotubes. The reaction of isolated myosin with S-nitrosoglutathione results in S-nitrosylation at multiple cysteine thiols and produces two populations of protein-bound SNOs with different stabilities. The less-stable population inhibits the physiological ATPase activity, without affecting the affinity of myosin for actin. However, myosin is neither inhibited nor S-nitrosylated by the NO* donor diethylamine NONOate, indicating a requirement for transnitrosylation between low-mass SNO and myosin cysteine thiols rather than a direct reaction of myosin with NO* or its auto-oxidation products. Interestingly, alkylation of the most reactive thiols of myosin by N-ethylmaleimide does not inhibit formation of a stable population of protein-SNOs, suggesting that these sites are located in less accessible regions of the protein than those that affect activity. The present study reveals a new link between exercise and S-nitrosylation of skeletal muscle contractile proteins that may be important under (patho)physiological conditions.

Free-energy linkage between folding and calcium binding in EF-hand proteins.

Troponin is the singular Ca(2+)-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca(2+)-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca(2+) was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (DeltaV and DeltaG(atm)). In this study, we propose that part of the affinity for Ca(2+) is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca(2+) binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca(2+) in EF-hand proteins, which is crucial to function.

Betaine protects urea-induced denaturation of myosin subfragment-1.

We have demonstrated previously that urea inhibits the activity and alters the tertiary structure of skeletal muscle myosin in a biphasic manner. This was attributed to differential effects on its globular and filamentous portion. The inhibition of catalytic activity was counteracted by methylamines. With the aim of comprehending the effects of urea on the catalytic (globular) portion of myosin, this study examines the effects of urea and the countereffects of betaine on the catalytic activity and structure of myosin subfragment-1. It is shown that urea inactivates subfragment-1 in parallel with its ability to induce exposure of the enzyme hydrophobic domains, as assessed using intrinsic and extrinsic fluorescence. Both effects are counteracted by betaine, which alone does not significantly affect subfragment-1. Urea also enhances the accessibility of thiol groups, promotes aggregation and decreases the alpha-helix content of S1, effects that are also counteracted by betaine. We conclude that urea-induced inactivation of the enzyme is caused by partial unfolding of the myosin catalytic domain.

Ionic interventions that alter the association of troponin C C-domain with the thin filaments of vertebrate striated muscle.

The regulatory complex of vertebrate skeletal muscle integrates information about cross-bridge binding, divalent cations and other intracellular ionic conditions to control activation of muscle contraction. Relatively little is known about the role of the troponin C (TnC) C-domain in the absence of Ca2+. Here, we use a standardized condition for measuring isometric tension in rabbit psoas skinned fibers to track TnC attachment and detachment in the absence of Ca2+ under different conditions of ionic strength, pH and MgATP. In the presence of MgATP and Mg2+, TnC detaches more readily and has a 1.5- to 2-fold lower affinity for the intact thin filament at pH 8 and 250 mM K+ than at pH 6 or in 30 mM K+; changes in affinity are fully reversible. The response to ionic strength is lost when Mg2+ and MgATP are absent, whereas the response to pH persists, suggesting that weaker electrostatic TnC-TnI-TnT interactions can be overridden by strongly bound cross-bridges. In solution, titration of a fluorescent C-domain mutant (F154W TnC) with Mg2+ reveals no significant changes in Mg2+ affinity with pH or ionic strength, suggesting that these parameters influence TnC binding by acting directly on electrostatic forces between TnC and TnI rather than by changing Mg2+ binding to C-domain sites III and IV.

Amide hydrogens reveal a temperature-dependent structural transition that enhances site-II Ca2+-binding affinity in a C-domain mutant of cardiac troponin C.

The hypertrophic cardiomyopathy-associated mutant D145E, in cardiac troponin C (cTnC) C-domain, causes generalised instability at multiple sites in the isolated protein. As a result, structure and function of the mutant are more susceptible to higher temperatures. Above 25 °C there are large, progressive increases in N-domain Ca2+-binding affinity for D145E but only small changes for the wild-type protein. NMR-derived backbone amide temperature coefficients for many residues show a sharp transition above 30-40 °C, indicating a temperature-dependent conformational change that is most prominent around the mutated EF-hand IV, as well as throughout the C-domain. Smaller, isolated changes occur in the N-domain. Cardiac skinned fibres reconstituted with D145E are more sensitive to Ca2+ than fibres reconstituted with wild-type, and this defect is amplified near body-temperature. We speculate that the D145E mutation destabilises the native conformation of EF-hand IV, leading to a transient unfolding and dissociation of helix H that becomes more prominent at higher temperatures. This creates exposed hydrophobic surfaces that may be capable of binding unnaturally to a variety of targets, possibly including the N-domain of cTnC when it is in its open Ca2+-saturated state. This would constitute a potential route for propagating signals from one end of TnC to the other.

Ca(2+) and Mg(2+) binding to weak sites of TnC C-domain induces exposure of a large hydrophobic surface that leads to loss of TnC from the thin filament.

The C-domain of troponin C, the Ca(2+)-binding subunit of the troponin complex, has two high-affinity sites for Ca(2+) that also bind Mg(2+) (Ca(2+)/Mg(2+) sites), whereas the N-domain has two low-affinity sites for Ca(2+). Two more sites that bind Mg(2+) with very low affinity (K(a)<10(3)M(-1)) have been detected by several laboratories but have not been localized or studied in any detail. Here we investigated the effects of Ca(2+) and Mg(2+) binding to isolated C-domain, focusing primarily on low-affinity sites. Since TnC has no Trp residues, we utilized a mutant with Phe 154 replaced by Trp (F154W/C-domain). As expected from previous reports, the changes in Trp fluorescence revealed different conformations induced by the addition of Ca(2+) or Mg(2+) (Ca(2+)/Mg(2+) sites). Exposure of hydrophobic surfaces of F154W/C-domain was monitored using the fluorescence intensity of bis-anilino naphthalene sulfonic acid. Unlike the changes reported by Trp, the increments in bis-ANS fluorescence were much greater (4.2-fold) when Ca(2+)+Mg(2+) were both present or when Ca(2+) was present at high concentration. Bis-ANS fluorescence increased as a function of [Ca(2+)] in two well-defined steps: one at low [Ca(2+)], consistent with the Ca(2+)/Mg(2+) sites (K(a) approximately 1.5 x 10(6)M(-1)), and one of much lower affinity (K(a) approximately 52.3M(-1)). Controls were performed to rule out artifacts due to aggregation, high ionic strength and formation of the bis-ANS-TnC complex itself. With a low concentration of Ca(2+) (0.6mM) to occupy the Ca(2+)/Mg(2+) sites, a large increase in bis-ANS binding also occurred as Mg(2+) occupied a class of low-affinity sites (K(a) approximately 59 M(-1)). In skinned fibers, a high concentration of Mg(2+) (10-44 mM) caused TnC to dissociate from the thin filament. These data provide new evidence for a class of weak binding sites for divalent cations. They are located in the C-domain, lead to exposure of a large hydrophobic surface, and destabilize the binding of TnC to the regulatory complex even when sites III and IV are occupied.

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators.

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.

Exogenous ß-amyloid peptide interferes with GLUT4 localization in neurons.

Aging represents a major risk factor for numerous illnesses that are of increasing importance to society, including two of the most prevalent: diabetes and Alzheimer's disease. Studies have shown that diabetes is a risk factor for spontaneous Alzheimer's disease. While these studies suggest that diabetes can contribute to Alzheimer's disease, the implications of AD on diabetes are practically unexplored. The major mediator of the pathophysiological effects, the Aß42 peptide, has been shown to enter neurons and lead to an alteration of the intracellular distribution of the molecular motor myosin Vb. Myosin Vb functions in memory and learning by participating in the strengthening of the long-term potentiation (LTP) of synaptic transmissions. It has also been implicated in the translocation of the glucose transporter, GLUT4, to the plasma membrane in response to insulin, a process that is defective in diabetes. Here, the effect on GLUT4 upon entry of the Aß42 peptide into cultured chick retinal neurons was explored. The results suggest an alteration in distribution and a reduced level at the cell surface, as well as an increased colocalization with myosin Vb, which can partially explain the changes in glucose metabolism associated with AD. It is also shown that the presence of the Aß40 peptide inhibits the internalization of the Aß42 peptide in cultured cells. Together, the results provide additional targets for the development of therapeutics against the progression and effects of Alzheimer's disease.

S-Nitrosylation of Sarcomeric Proteins Depresses Myofilament Ca2+)Sensitivity in Intact Cardiomyocytes.

AIMS: The heart responds to physiological and pathophysiological stress factors by increasing its production of nitric oxide (NO), which reacts with intracellular glutathione to form S-nitrosoglutathione (GSNO), a protein S-nitrosylating agent. Although S-nitrosylation protects some cardiac proteins against oxidative stress, direct effects on myofilament performance are unknown. We hypothesize that S-nitrosylation of sarcomeric proteins will modulate the performance of cardiac myofilaments. RESULTS: Incubation of intact mouse cardiomyocytes with S-nitrosocysteine (CysNO, a cell-permeable low-molecular-weight nitrosothiol) significantly decreased myofilament Ca(2+) sensitivity. In demembranated (skinned) fibers, S-nitrosylation with 1 µM GSNO also decreased Ca(2+) sensitivity of contraction and 10 µM reduced maximal isometric force, while inhibition of relaxation and myofibrillar ATPase required higher concentrations (≥ 100 µM). Reducing S-nitrosylation with ascorbate partially reversed the effects on Ca(2+) sensitivity and ATPase activity. In live cardiomyocytes treated with CysNO, resin-assisted capture of S-nitrosylated protein thiols was combined with label-free liquid chromatography-tandem mass spectrometry to quantify S-nitrosylation and determine the susceptible cysteine sites on myosin, actin, myosin-binding protein C, troponin C and I, tropomyosin, and titin. The ability of sarcomere proteins to form S-NO from 10-500 µM CysNO in intact cardiomyocytes was further determined by immunoblot, with actin, myosin, myosin-binding protein C, and troponin C being the more susceptible sarcomeric proteins. INNOVATION AND CONCLUSIONS: Thus, specific physiological effects are associated with S-nitrosylation of a limited number of cysteine residues in sarcomeric proteins, which also offer potential targets for interventions in pathophysiological situations.