Triadins are not triad-specific proteins: two new skeletal muscle triadins possibly involved in the architecture of sarcoplasmic reticulum

We have cloned two new triadin isoforms from rat skeletal muscle, Trisk 49 and Trisk 32, which were named according to their theoretical molecular masses (49 and 32 kDa, respectively). Specific antibodies directed against each protein were produced to characterize both new triadins. Both are expressed in adult rat skeletal muscle, and their expression in slow twitch muscle is lower than that in fast twitch muscle. Using double immunofluorescent labeling, the localization of these two triadins was studied in comparison to well-characterized proteins such as ryanodine receptor, calsequestrin, desmin, Ca(2+)-ATPase, and titin. None of these two triadins are localized within the rat skeletal muscle triad. Both are instead found in different parts of the longitudinal sarcoplasmic reticulum. We attempted to identify partners for each isoform: neither is associated with ryanodine receptor; Trisk 49 could be associated with titin or another sarcomeric protein; and Trisk 32 could be associated with IP(3) receptor. These results open further fields of research concerning the functions of these two proteins; in particular, they could be involved in the set up and maintenance of a precise sarcoplasmic reticulum structure.     

Reduced Verbal Fluency following Subthalamic Deep Brain Stimulation: A Frontal-Related Cognitive Deficit?

Objective

The decrease in verbal fluency in patients with Parkinson’s disease (PD) undergoing subthalamic nucleus deep brain stimulation (STN-DBS) is usually assumed to reflect a frontal lobe-related cognitive dysfunction, although evidence for this is lacking.

Methods

To explore its underlying mechanisms, we combined neuropsychological, psychiatric and motor assessments with an examination of brain metabolism using F-18 fluorodeoxyglucose positron emission tomography, in 26 patients with PD, 3 months before and after surgery. We divided these patients into two groups, depending on whether or not they exhibited a postoperative deterioration in either phonemic (10 patients) or semantic (8 patients) fluency. We then compared the STN-DBS groups with and without verbal deterioration on changes in clinical measures and brain metabolism.

Results

We did not find any neuropsychological change supporting the presence of an executive dysfunction in patients with a deficit in either phonemic or semantic fluency. Similarly, a comparison of patients with or without impaired fluency on brain metabolism failed to highlight any frontal areas involved in cognitive functions. However, greater changes in cognitive slowdown and apathy were observed in patients with a postoperative decrease in verbal fluency.

Conclusions

These results suggest that frontal lobe-related cognitive dysfunction could play only a minor role in the postoperative impairment of phonemic or semantic fluency, and that cognitive slowdown and apathy could have a more decisive influence. Furthermore, the phonemic and semantic impairments appeared to result from the disturbance of distinct mechanisms.     

The anti-apoptotic Bcl-x(L) protein, a new piece in the puzzle of cytochrome c interactome

A structural model of the adduct between human cytochrome c and the human anti-apoptotic protein Bcl-x(L), which defines the protein-protein interaction surface, was obtained from solution NMR chemical shift perturbation data. The atomic level information reveals key intermolecular contacts identifying new potentially druggable areas on cytochrome c and Bcl-x(L). Involvement of residues on cytochrome c other than those in its complexes with electron transfer partners is apparent. Key differences in the contact area also exist between the Bcl-x(L) adduct with the Bak peptide and that with cytochrome c. The present model provides insights to the mechanism by which cytochrome c translocated to cytosol can be intercepted, so that the apoptosome is not assembled.     

NMR of molecules interacting with lipids in small unilamellar vesicles

Detailed characterization of protein, peptide or drug interactions with natural membrane is still a challenge. This review focuses on the use of nuclear magnetic resonance (NMR) for the analysis of interaction of molecules with small unilamellar vesicles (SUV). These phospholipid vesicles are often used as model membranes for fluorescence or circular dichroism experiments. The various NMR approaches for studying molecule-lipid association are reviewed. After a brief survey of the SUV characterization, the use of heteronuclear NMR (phosphorous, carbon, fluorine) is described. Applications of proton NMR through transferred nuclear Overhauser effect to perform structural determination of peptide are presented. Special care is finally given to the influence of the kinetic of the interactions for the proton NMR of bound molecules in SUV, which can constitute a good model for the study of dynamical processes at the membrane surface. Presented at the joint biannual meeting of the SFB-GEIMM-GRIP, Anglet France, 14–19 October, 2006.     

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.     

Effect of ryanodine on cardiac calcium current and calcium channel gating current.

The effects of 100 microM ryanodine on the L-type calcium channel were studied using the pacth-clamp technique in isolated guinea pig ventricular myocytes. The inactivation kinetics of the calcium current were slowed down in the presence of ryanodine in agreement with the blockade of the release of calcium from the sarcoplasmic reticulum by the drug. The I-V and steady-state inactivation curves of the calcium current were shifted to negative values by ryanodine. A similar shift was observed in the activation and inactivation curves of the intramembrane charge movement associated with the calcium channel. Due to this shift, ryanodine slightly reduced the maximal amount of displaced charge although it did not modify the transition from the inactivated to the activated state (i.e., charge movement repriming). This result is in notable contrast with that obtained in skeletal muscle, where it has been found that ryanodine interferes with charge movement repriming. These results provide additional evidence of the postulated differences between the architecture of the excitation-contraction coupling system in cardiac and skeletal muscle.     

Identification of a Cd2+- and Zn2+-binding site in cytochrome c using FTIR coupled to an ATR microdialysis setup and NMR spectroscopy

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.