Muscle wasting: A review of exercise, classical and non-classical RAS axes.

This review identifies how the classical/non-classical renin-angiotensin system (RAS) and exercise influence muscle wasting. The classical RAS axis enhances muscle loss through the interaction with NADPH oxidase (NOX), ubiquitin proteasome system (UPS), protein synthesis and fibrosis pathways. The mainstream hypothesis identifies reactive oxygen species (ROS) as the key pathway in muscle, this review recognizes alternative pathways that lead to an increase in muscle wasting through the classical RAS axis. In addition, pathways in which the non-classical RAS axis and exercise inhibit the classical RAS axis are also explored. The non-classical RAS axis and exercise have a significant negative impact on ROS production and protein synthesis. The non-classical RAS axis has been identified in this review to directly affect protein synthesis pathways not by altering the pre-existing intracellular ROS level, further supporting the idea that muscle wasting caused by the classical RAS system is not entirely due to ROS production. Exercise has been identified to modify the RAS axes making it a therapeutic option.

Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold.

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydF(EG)), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydF(EG). The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.

HydF as a scaffold protein in [FeFe] hydrogenase H-cluster biosynthesis.

In an effort to determine the specific protein component(s) responsible for in vitro activation of the [FeFe] hydrogenase (HydA), the individual maturation proteins HydE, HydF, and HydG from Clostridium acetobutylicum were purified from heterologous expressions in Escherichia coli. Our results demonstrate that HydF isolated from a strain expressing all three maturation proteins is sufficient to confer hydrogenase activity to purified inactive heterologously expressed HydA (expressed in the absence of HydE, HydF, and HydG). These results represent the first in vitro maturation of [FeFe] hydrogenase with purified proteins, and suggest that HydF functions as a scaffold upon which an H-cluster intermediate is synthesized.

On the accuracy of density functional theory for iron-sulfur clusters.

A simple, yet powerful wave function manipulation method was introduced utilizing a generalized ionic fragment approach that allows for systematic mapping of the wave function space for multispin systems with antiferromagnetic coupling. The use of this method was demonstrated for developing ground state electronic wave function for [2Fe-2S] and [Mo-3Fe-4S] clusters. Using well-defined ionic wave functions for ferrous and ferric irons, sulfide, and thiolate fragments, the accuracy of various density functionals and basis sets including effective core potentials were evaluated on a [4Fe-4S] cluster by comparing the calculated geometric and electronic structures with crystallographic data and experimental atomic spin densities from X-ray absorption spectroscopy, respectively. We found that the most reasonable agreement for both geometry and atomic spin densities is obtained by a hybrid functional with 5% HF exchange and 95% density functional exchange supplemented with Perdew's 1986 correlation functional. The basis set seems to saturate only at the triple-zeta level with polarization and diffuse functions. Reasonably preoptimized structures can be obtained by employing computationally less expensive effective core potentials, such as the Stuttgart-Dresden potential with a triple-zeta valence basis set. The extension of the described calibration methodology to other biologically important and more complex iron-sulfur clusters, such as hydrogenase H-cluster and nitrogenase FeMo-co will follow.