Potential role of cardiac calsequestrin in the lethal arrhythmic effects of cocaine.

BACKGROUND: Cocaine-related deaths are continuously rising and its overdose is often associated with lethal cardiotoxic effects. METHODS AND RESULTS: Our approach, employing isothermal titration calorimetry (ITC) and light scattering in parallel, has confirmed the significant affinity of human cardiac calsequestrin (CASQ2) for cocaine. Calsequestrin (CASQ) is a major Ca(2+)-storage protein within the sarcoplasmic reticulum (SR) of both cardiac and skeletal muscles. CASQ acts as a Ca(2+) buffer and Ca(2+)-channel regulator through its unique Ca(2+)-dependent oligomerization. Equilibrium dialysis and atomic absorption spectroscopy experiments illustrated the perturbational effect of cocaine on CASQ2 polymerization, resulting in substantial reduction of its Ca(2+)-binding capacity. We also confirmed the accumulation of cocaine in rat heart tissue and the substantial effects cocaine has on cultured C2C12 cells. The same experiments were performed with methamphetamine as a control, which displayed neither affinity for CASQ2 nor any significant effects on its function. Since cocaine did not have any direct effect on the Ca(2+)-release channel judging from our single channel recordings, these studies provide new insights into how cocaine may interfere with the normal E-C coupling mechanism with lethal arrhythmogenic consequences. CONCLUSION: We propose that cocaine accumulates in SR through its affinity for CASQ2 and affects both SR Ca(2+) storage and release by altering the normal CASQ2 Ca(2+)-dependent polymerization. By this mechanism, cocaine use could produce serious cardiac problems, especially in people who have genetically-impaired CASQ2, defects in other E-C coupling components, or compromised cocaine metabolism and clearance.

Structural and catalytic differences between two FADH(2)-dependent monooxygenases: 2,4,5-TCP 4-monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-monooxygenase (TcpA) from Cupriavidus necator JMP134.

2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH(2))-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.

Furfural reduction mechanism of a zinc-dependent alcohol dehydrogenase from Cupriavidus necator JMP134.

FurX is a tetrameric Zn-dependent alcohol dehydrogenase (ADH) from Cupriavidus necator JMP134. The enzyme rapidly reduces furfural with NADH as the reducing power. For the first time among characterized ADHs, the high-resolution structures of all reaction steps were obtained in a time-resolved manner, thereby illustrating the complete catalytic events of NADH-dependent reduction of furfural and the dynamic Zn(2+) coordination among Glu66, water, substrate and product. In the fully closed conformation of the NADH complex, the catalytic turnover proved faster than observed for the partially closed conformation due to an effective proton transfer network. The domain motion triggered by NAD(H) association/dissociation appeared to facilitate dynamic interchanges in Zn(2+) coordination with substrate and product molecules, ultimately increasing the enzymatic turnover rate. NAD(+) dissociation appeared to be a slow process, involving multiple steps in concert with a domain opening and reconfiguration of Glu66. This agrees with the report that the cofactor is not dissociated from FurX during ethanol-dependent reduction of furfural, in which ethanol reduces NAD(+) to NADH that is subsequently used for furfural reduction.

A soluble α-synuclein construct forms a dynamic tetramer.

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.

Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:FAD oxidoreductase (TftC) of Burkholderia cepacia AC1100.

Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH:FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 A resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide:isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.

Reduction of axial kinetic energy induced perturbations on observed cyclotron frequency.

With Fourier transform ion cyclotron resonance (FTICR) mass spectrometry one determines the mass-to-charge ratio of an ion by measuring its cyclotron frequency. However, the need to confine ions to the trapping region of the ion cyclotron resonance (ICR) cell with electric fields induces deviations from the unperturbed cyclotron frequency. Additional perturbations to the observed cyclotron frequency are often attributed to changes in space charge conditions. This study presents a detailed investigation of the observed ion cyclotron frequency as a function of ion z-axis kinetic energy. In a perfect three-dimensional quadrupolar field, cyclotron frequency is independent of position within the trap. However, in most ICR cell designs, this ideality is approximated only near the trap center and deviations arise from this ideal quadrupolar field as the ion moves both radially and axially from the center of the trap. To allow differentiation between deviations in observed cyclotron frequency caused from changes in space charge conditions or differences in oscillation amplitude, ions with identical molecular weights but different axial kinetic energy, and thus amplitude of z-axis motion, were simultaneously trapped within the ICR cell. This allows one to attribute deviations in observed cyclotron frequency to differences in the average force from the radial electric field experienced by ions of different axial amplitude. Experimentally derived magnetron frequency is compared with the magnetron frequency calculated using SIMION 7.0 for ions of different axial amplitude. Electron promoted ion coherence, or EPIC, is used to reduce the differences in radial electric fields at different axial positions. Thus with the application of EPIC, the differences in observed cyclotron frequencies are minimized for ions of different axial oscillation amplitudes.