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.This work was supported by National Institutes of Health (NIH) R01 awards [R01 HL 133864 (A.C.S), R01 HL 128304 (A.C.S), R01 HL 149825 (A.C.S), R01 HL 153532 (A.C.S), R01 GM 125944 (F.J.S.), R01 DK 112854 (F.J.S.), 1S10OD021540-01 (Center for Biologic Imaging, University of Pittsburgh), 1S10RR019003-01 (Simon Watkins (S.W.)), 1S10RR025488-01 (S.W.), 1S10RR016236-01 (S.W)]. American Heart Association (AHA) [Established Investigator Award 19EIA34770095 (A.C.S.)], Post-doctoral Fellowship 19POST34410028 (S.Y.)]. American Society of Hematology (ASH) Minority Hematology Graduate Award (A.M.D-O.). Junta de Andalucía grant BIO-177 (P.N.), the FEDER Funding Program from the European Union and Spanish Ministry of Science, Innovation and Universities grant RED2018-102576-T (P.N.)

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. © 2013 Mohan et al

The Role of Superoxide Dismutase 2 in Modulating Sickle Cell Disease Pathology

Sickle cell disease (SCD) is an inherited hemoglobinopathy resulting from a point mutation in the β-globin gene. This substitution leads to the polymerization of sickle hemoglobin (HbS) under deoxygenated conditions and sickling of red blood cells (RBCs). Sickled RBCs can block the microvasculature causing vaso-occlusions and hemolyze releasing free hemoglobin and heme into the intravascular space. These processes cause an increase in the production of reactive oxygen species (ROS). Despite there being an increase in ROS production, there is a decrease in the antioxidant defense system. Superoxide dismutase 2 (SOD2) is an enzymatic antioxidant found only within the mitochondria which functions to dismutate superoxide to hydrogen peroxide. Given the decrease in antioxidants in SCD and that SOD2 is a known regulator of vascular function, this thesis project sought to identify whether SOD2 could modify SCD pathology. In aim 1, we identified that a common polymorphism of SOD2, SOD2 V16A, is strongly associated with clinical indicators of pulmonary hyper`tension in SCD patients. In human pulmonary arterial endothelial cells (HPAECs), we found that this polymorphism pathologically vii associates with complex IV of the respiratory chain leading to a decrease in complex activity and mitochondrial respiration. HPAECs transduced with SOD2 V16A also had increased ROS production which was exacerbated with inhibition of complex III of the respiratory chain. In aim 2, we found that SOD2 protein expression is decreased in the pulmonary endothelium of SCD patients. In addition to this, we discovered that SOD2 protein expression was decreased in whole lung tissue of transgenic sickle mice. Given its significant decrease in sickle conditions, we went on to further define the function of SOD2 in the pulmonary endothelium using human pulmonary microvascular endothelial cells (HPMVECs). In HPMVECs, SOD2 deficiency led to a disruption in barrier function as well as a decrease in migration and proliferation. We found that these reduced functions were linked to a reduction in adhesion stemming from dysregulation of fibronectin processing. Altogether, we are the first to thoroughly investigate the role of SOD2 within the endothelium, and our findings can aide in the development of new therapeutics in the treatment of SCD patients

Rock1 SBD variants show decreased interaction with Shrm.

<p>Fluorescence anisotropy experiments monitored 50-Green labeled human Shrm2 SD2 domain with increasing concentrations of Rock1 (707–946) containing the indicated amino acid substitutions. The change in anisotropy was fit to Equation 1 to determine binding affinities (<i>K_d</i>) as indicated.</p

Crystallographic Data collection and refinement statistics for human Rock1 SBD^SER.

<p>Values in parentheses correspond to those in the outer resolution shell.</p><p>R_merge = (|(ΣI−<i>)|)/(ΣI), where <i> is the average intensity of multiple measurements.</i></i></p><p><i><i>R_work = Σ_hkl∥F_obs(hkl)∥−F_calc (hkl)∥/Σ_hkl|F_obs(hkl)|.</i></i></p><p><i><i>R_free = crossvalidation R factor for 7.3% of the reflections against which the model was not refined.</i></i></p

A conserved region on the SBD surface mediates Shrm binding.

<p>A) Surface view of the SBD dimer colored by sequence conservation. Residues colored light blue are identical in >95% of the Rock sequences in our alignment, while residues that are invariant across all 14 sequences are color darker blue. Residues that were altered in our mutational analysis are labeled. B) Surface of the SBD dimer colored by Surface Triplet Propensity. Scoring is colored as a heat map with lowest scores in dark blue and the highest scores in red. A prominent patch containing residues Y851 and F852 is indicated. C) Residues within the conserved patch contribute to Shrm binding. Human Shrm2 SD2 was mixed with wild-type Rock1 SBD or the indicated mutant and the formation of a Rock-Shrm complex was detected by native gel electrophoresis.</p

A Central region within the coiled-coil domain interacts with Shrm SD2.

<p>A) Diagram of Rock1 domain structure. Domains and their boundaries within Rock1 are indicated. N- and C-terminal extensions on the Rock1 kinase domain are shown in red. Sequence conservation from a multiple sequence alignment of 22 Rock sequences is shown with sequence positions containing 90% identity indicated in blue. B) Identification of a minimal Shrm SD2 binding domain within Rock1. Purified untagged human Shrm2 SD2 was mixed with beads pre-bound to the indicated his-tagged fragment of Rock1. Complexes were precipitated by spinning down the beads and the resulting samples were resolved on SDS-PAGE. P, pelleted beads; S supernatant. C) Rock fragments were assayed for binding to Shrm SD2. Increasing concentrations of Rock1 (707–946) or (834–913) were added to a reaction mixture containing 50 nM Oregon-Green labeled human Shrm2 SD2 domain in a fluorescence spectrophotometer. The binding isotherm was fit to Equation 1 using a non-linear regression to determine binding affinity (<i>K_d</i>).</p

Opposing Shrm binding sites within the SBD.

<p>A) Surface representation of the Rock SBD colored by the effect of substitutions in cell based and in vitro assays. Included are positions which altered Shrm colocalization (magenta), positions which affected SD2 binding in vitro (red), residues which did not affect SD2 binding in vitro (green), and residues 900–902 which had a subtle affect on SD2 binding (pale green). B) Cutaway view of the Shrm binding region. Ribbon diagram and positions of side chains with a demonstrated affect (sticks) are colored as above. Black represents the Rock surface which has been cut away to reveal the backbone and side chains underneath. A hydrophobic patch comprised of residues Y851, F852, and L855 is indicated for each binding site.</p

The Rock1 SBD is required for localization with Shrm3.

<p>(A–F) Myc-tagged wild type or SBD variants of hRock1 (681–942) were co-expressed with wildtype Shrm3 or a Shrm3 variant lacking the SD2 in Cos7 cells and stained to detect Shrm3 (green) or the myc tag (red). The right-hand panels in A, C–F depict the results of pulldown assays to detect the interaction of the indicated SBD variant and the Shrm3 SD2. Binding of the Rock SBD variants was tested by using immobilized GST-Shrm3 SD2 and lysates from HEK293 cells expressing the indicated SBD protein, followed by western blotting to detect the myc-tagged SBD proteins. Input = total cell lysate, GST = pulldown using GST bound to beads, GST-SD2 = GST-Shrm3-SD2 bound to beads. Arrowhead denotes the myc-tagged Rock protein. (G–I) T23 MDCK epithelial cells were transfected with expression vectors for EndoShrm3 and Rock1 SBD (G), EndoShrm3 and Rock1-SBD ⁸⁵⁵LYKTQ⁸⁵⁹ to ⁸⁵⁵AAAAA⁸⁵⁹ (H) or EndoShrm3ΔSD2 and Rock1-SBD (I), grown on transwell filters overnight to form confluent monolayers, and stained to detect EndoShrm3 (green) and Rock-SBD (red). Dashed lines indicate the position of the Z-projections that are shown in the lower panels. Ap, apical surface; Bsl, basal surface.</p