Cooperation between CYB5R3 and NOX4 via coenzyme Q mitigates endothelial inflammation.

NADPH oxidase 4 (NOX4) regulates endothelial inflammation by producing hydrogen peroxide (H2O2) and to a lesser extent O2•-. The ratio of NOX4-derived H2O2 and O2•- can be altered by coenzyme Q (CoQ) mimics. Therefore, we hypothesize that cytochrome b5 reductase 3 (CYB5R3), a CoQ reductase abundant in vascular endothelial cells, regulates inflammatory activation. To examine endothelial CYB5R3 in vivo, we created tamoxifen-inducible endothelium-specific Cyb5r3 knockout mice (R3 KO). Radiotelemetry measurements of systolic blood pressure showed systemic hypotension in lipopolysaccharides (LPS) challenged mice, which was exacerbated in R3 KO mice. Meanwhile, LPS treatment caused greater endothelial dysfunction in R3 KO mice, evaluated by acetylcholine-induced vasodilation in the isolated aorta, accompanied by elevated mRNA expression of vascular adhesion molecule 1 (Vcam-1). Similarly, in cultured human aortic endothelial cells (HAEC), LPS and tumor necrosis factor α (TNF-α) induced VCAM-1 protein expression was enhanced by Cyb5r3 siRNA, which was ablated by silencing the Nox4 gene simultaneously. Moreover, super-resolution confocal microscopy indicated mitochondrial co-localization of CYB5R3 and NOX4 in HAECs. APEX2-based electron microscopy and proximity biotinylation also demonstrated CYB5R3's localization on the mitochondrial outer membrane and its interaction with NOX4, which was further confirmed by the proximity ligation assay. Notably, Cyb5r3 knockdown HAECs showed less total H2O2 but more mitochondrial O2•-. Using inactive or non-membrane bound active CYB5R3, we found that CYB5R3 activity and membrane translocation are needed for optimal generation of H2O2 by NOX4. Lastly, cells lacking the CoQ synthesizing enzyme COQ6 showed decreased NOX4-derived H2O2, indicating a requirement for endogenous CoQ in NOX4 activity. In conclusion, CYB5R3 mitigates endothelial inflammatory activation by assisting in NOX4-dependent H2O2 generation via CoQ.

Decoding the role of SOD2 in sickle cell disease.

Sickle cell disease (SCD) is an inherited hemoglobinopathy caused by a single point mutation in the ß-globin gene. As a consequence, deoxygenated hemoglobin polymerizes triggering red blood cell sickling and hemolysis, vaso-occlusion, and ischemia/reperfusion. Allied to these pathologies is the overproduction of reactive oxygen species driven by hemoglobin Fenton chemistry and peroxidase reactions as well as by secondary activation of vascular oxidases, including NAD(P)H oxidase and xanthine oxidase. In addition, hypoxia, produced by sickle red blood cell occlusion, disrupts mitochondrial metabolism and generates excess superoxide through electron leak from the mitochondrial respiratory chain. Superoxide dismutase 2 (SOD2) is a mitochondrial-specific antioxidant enzyme that dismutates superoxide to hydrogen peroxide, which is then converted to water by catalase and glutathione peroxidase. In SCD, the antioxidant defense system is significantly diminished through decreased expression and activity levels of antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase. From a translational perspective, genetic variants including a missense variant in SOD2 (valine to alanine at position 16) are present in 45% of people with African ancestry and are associated with increased sickle complications. While it is known that there is an imbalance between oxidative species and antioxidant defenses in SCD, much more investigation is warranted. This review summarizes our current understanding of antioxidant defense systems in SCD, particularly focused on SOD2, and provides insight into challenges and opportunities as the field moves forward.

Structure of a highly conserved domain of Rock1 required for Shroom-mediated regulation of cell morphology.

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.