Dose-dependent oxidative stress by the anthracycline doxorubicin (Dox) and other chemotherapeutic agents causes irreversible cardiac damage, restricting their clinical effectiveness. We hypothesized that the resultant protein oxidation could be monitored and correlated with physiological functional impairment. We focused on protein carbonylation as an indicator of severe oxidative damage because it is irreversible and results in proteasomal degradation. We identified and investigated a specific high-molecular weight cardiac protein that showed a significant increase in carbonylation under Doxinduced cardiotoxic conditions in a spontaneously hypertensive rat model. We confirmed carbonylation and degradation of this protein under oxidative stress and prevention of such effect in the presence of the iron chelator dexrazoxane. Using MS, the Dox-induced carbonylated protein was identified as the 140-kDa cardiac myosin binding protein C (MyBPC). We confirmed the carbonylation and degradation of MyBPC using HL-1 cardiomyocytes and a purified recombinant untagged cardiac MyBPC under metal-catalyzed oxidative stress conditions. The carbonylation and degradation of MyBPC were time-and drug concentration-dependent. We demonstrated that carbonylated MyBPC undergoes proteasome-mediated degradation under Dox-induced oxidative stress. Cosedimentation, immunoprecipitation, and actin binding assays were used to study the functional consequences of carbonylated MyBPC. Carbonylation of MyBPC showed significant functional impairment associated with its actin binding properties. The dissociation constant of carbonylated recombinant MyBPC for actin was 7.35 ± 1.9 μM compared with 2.7 ± 0.6 μM for native MyBPC. Overall, our findings indicate that MyBPC carbonylation serves as a critical determinant of cardiotoxicity and could serve as a mechanistic indicator for Doxinduced cardiotoxicity.cancer | ROS | cardioprotection

Iron regulatory protein 2 coordinates cellular regulation of iron metabolism by binding to iron responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. This iron-dependent degradation requires the presence of a 73-residue degradation domain, but its functions have not yet been established. We now show that the domain can act as an iron sensor, mediating its own covalent modification. The domain forms an ironbinding site with three cysteine residues located in the middle of the domain. It then reacts with molecular oxygen to generate a reactive oxidizing species at the iron-binding site. One cysteine residue is oxidized to dehydrocysteine and other products. This covalent modification may thus mark the protein molecule for degradation by the proteasome system.Iron metabolism is exquisitely regulated by all organisms, from bacteria to humans. In mammals, the iron regulatory proteins (IRPs) 1 mediate the coordinate expression of proteins that participate in iron metabolism (1, 2). When iron stores are low, the IRPs bind to an RNA stem-loop structure known as an iron-responsive element (IRE) located in either the 5Ј-or 3Ј-untranslated region of mRNA. If the IRE is close to the cap site, binding of the IRP blocks initiation of translation, causing a decrease in the level of the protein encoded by that mRNA. Conversely, when the IRE is located in the 3Ј-untranslated region of the transferrin receptor transcript, binding of the IRP stabilizes the mRNA by decreasing susceptibility to nuclease attack, causing an increase in the level of the protein encoded by the mRNA (3).Mammals have two known IRPs, IRP1 and IRP2, with the tissue roles of each still being defined. IRP1 contains an ironsulfur center, whereas IRP2 does not. The general mechanism by which each IRP is regulated is distinct (1). When cellular iron stores are low, IRP1 lacks a functional iron-sulfur center and binds to its IRE targets. When iron stores are sufficient, IRP1 regains its full iron-sulfur center, loses the ability to bind to IRE, and functions as a cytosolic aconitase. The cellular levels of IRP1 are unaffected by iron status in most cell types.In contrast, IRP2 protein and IRE binding activity are readily detected when iron stores are limited but are low or absent when iron stores are sufficient (4, 5). The decrease in IRP2 protein occurs as a consequence of rapid degradation by the proteasome; synthesis of the protein is constitutive and does not vary substantially with iron status. The sequences of the two IRPs are similar except that IRP2 contains one domain not present in IRP1, and this domain is both required and sufficient to confer susceptibility to iron-dependent degradation. Deletion of the degradation domain produces an IRP2 whose levels no longer vary with iron status. Conversely, insertion of the degradation domain into IRP1 renders it susceptible to iron-triggered degradation (6).We showed previously that IRP2 is oxidatively modified and ubiquitinylated in vivo, ...

Iron regulatory protein 2 coordinates the cellular regulation of iron metabolism by binding to iron-responsive elements in mRNA. The protein is synthesized constitutively but is rapidly degraded when iron stores are replete. The mechanisms that prevent degradation during iron deficiency or promote degradation during iron sufficiency are not delineated. Iron regulatory protein 2 contains a domain not present in the closely related iron regulatory protein 1, and we found that this domain binds heme with high affinity. A cysteine within the domain is axially liganded to the heme, as occurs in cytochrome P450. The protein-bound heme reacts with molecular oxygen to mediate the oxidation of cysteine, including ␤-elimination of the sulfur to yield alanine. This covalent modification may thus mark the protein molecule for degradation by the proteasome system, providing another mechanism by which heme can regulate the level of iron regulatory protein 2.