Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS−/−

Abstract

In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS•) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 kDa and 75 kDa subunits was detected under the conditions of •O2− production, and mass spectrometry confirmed that formation of Complex I PrS• mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS• (Kang et al Free Radical Biol. Med. 2012; 52: 962–73). In this investigation, we tested our hypothesis in the murine heart of eNOS−/−. The eNOS−/− mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS−/− myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in •O2− generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS−/− murine heart. When Complex I from the mitochondria of the eNOS−/− murine heart was analyzed by immuno-spin trapping and probed with anti-GSH antibody, both PrS• and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS•, supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS• is a unique pathway for the redox regulation of mitochondrial function in vivo.

Differential protein acetylation assists import of excess SOD2 into mitochondria and mediates SOD2 aggregation associated with cardiac hypertrophy in the murine SOD2-tg heart

“…Mitochondria were prepared from mouse hearts by differential centrifugation according to published methods [9, 11]. Mitochondria were precipitated by centrifugation at 20,000 × g for 10 min in the final step and resuspended in a medium (M-buffer) containing the following agents: in mM, mannitol 230, sucrose 70, EDTA 1, Trizma 1; pH 7.4.…”

“…Mitochondria were precipitated by centrifugation at 20,000 × g for 10 min in the final step and resuspended in a medium (M-buffer) containing the following agents: in mM, mannitol 230, sucrose 70, EDTA 1, Trizma 1; pH 7.4. The cytosolic compartment was collected from the supernatant after centrifugation at 20,000 × g. The mitochondrial and cytosolic fractions were evaluated by immunoblotting using a monoclonal antibody (1:500) against glyceraldehyde 3-phosphate dehydrogenase (GAPDH, a housekeeping cytosolic protein, Santa Cruz Biotechnology, Inc., Dallas, TX; catalog number: sc-32233 (6C5), blotting of cytosolic preparations served as a positive control) and a monoclonal antibody (1:1000, Santa Cruz Biotechnology, Inc. catalog number: sc-65237 (20E9),) against the ND1 subunit of complex I (a mitochondrial DNA-encoded protein) to ensure no cross contamination between mitochondria and cytosol [9, 11]. …”

“…The reaction mixture was mixed with the Laemmli sample buffer at a ratio of 4:1 (v/v) under the reducing conditions in the presence of dithiothretol (40 mM), incubated at 70 °C for 10 min, and then immediately loaded onto a 4–12% Bis-Tris polyacrylamide gradient gel according to previous published methods [9, 11]. Samples were run in the running buffer (in mM, MES 50, Tris Base 50, SDS 1.39, EDTA 1, DTT 0.31) at room temperature for 55 min at 190 V. Protein bands were electrophoretically transferred to a nitrocellulose membrane in 25 mM Bis-Tris, 25 mM Bicine, 0.029% (w/v) EDTA, and 10% methanol.…”

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

“…In combination with MS, immuno-spin trapping has been used to define the role of protein thiol radicals in mediating S -glutathionylation of mitochondrial complex I at the level of isolated enzymes, myocytes, and hearts of eNOS −/− mice. 13,30,222 In endothelial cells, the same approach has been used to examine a similar mechanism in regulating protein S -glutathionylation coupling, and function of eNOS. 223 Anti-DMPO antibodies can also be covalently bound to an albumin-gadolinium-diethylenetriaminepentaacetic acid-biotin magnetic resonance imaging contrast agent and used for molecular magnetic resonance imaging.…”

Thiyl radicals and induction of protein degradation

“…Physiologically, peptide and protein thiyl radicals can form through electron/hydrogen transfer between Cys and tyrosyl radicals [28, 29] or carbon-centered radicals [30]. Chemically, thiyl radicals can be generated by reaction of Cys with tryptophan radicals/radical cations [31], peroxyl radicals [32] (including superoxide [33, 34], where superoxide-induced protein thiyl radical formation has been implicated in S-glutathionylation of mitochondrial complex I [35, 36] and endothelial nitric oxide synthase [37, 38]), carbon-centered radicals [30], nitrogen dioxide ( • NO 2 ) [39, 40], carbonate radical (CO 3 •− ) [40] and the hydroxyl radical (HO • ). Protein thiyl radicals have been involved in mechanisms leading to S-nitrosation, and specifically in mechanisms of nucleotide exchange of various GTPases [41–47].…”