Reactive oxygen species (ROS) are cytotoxic. oxidations. Many PTMs targeted a

Reactive oxygen species (ROS) are cytotoxic. oxidations. Many PTMs targeted a single reactive SOD1 cysteine Cys111. An intriguing observation was that unlike native SOD1 cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may safeguard SOD1 from oxidation cysteine altered SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly Rabbit Polyclonal to B-RAF. total protection from peroxide-induced oxidation of SOD1. Moreover Apremilast SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue. Introduction Reactive oxygen species (ROS) are by-products of aerobic metabolism and are also the primary products of certain oxidoreductases. For example the incomplete reduction of oxygen to water during mitochondrial respiration can create both hydrogen peroxide (H2O2) and superoxide anion (O2?·). These by-products are harmful to cells because they can alter protein conformation disrupt enzyme function and mutate DNA amongst other things 1-3. A testament to the toxicity Apremilast of ROS is the monocyte-resident oxidoreductase NADPH oxidase (NOX) which generates superoxide enzymatically to kill targeted cells including microorganisms. Cells combat harmful ROS species with a multi-faceted anti-oxidant defense mechanism that includes the metalloenzyme Cu/Zn superoxide dismutase (SOD1). SOD1 catalyzes the disproportionation of the superoxide anion as follows 4: showed inhibits fast axonal transport in a similar fashion to SOD1 familial Amyotrophic Lateral Sclerosis (FALS) variants 13. Here we characterize post-translational modifications (PTMs) of SOD1 in situ including peroxide- and cysteine-related modifications and provide in vitro evidence that cysteinylation protects SOD1 from oxidative damage. Methods SOD1 Purification from Human Tissue Two purification protocols using unique elution buffers and antibodies were used to investigate PTMs and their relative amounts in human tissue. The first purification protocol was previously described and used polyclonal rabbit antibodies raised in house against a mixture of native and altered (by both oxygen and sulfur adducts on Cys111) SOD1 purified from human erythrocytes and elution with 5 % acetic acid 14. This protocol provides protein that can be directly infused into a mass spectrometer avoiding lengthy liquid chromatography. In the second purification SOD1 was isolated from human nervous tissue as previously explained 13 using a sheep polyclonal antibody raised against SOD1 from human erythrocytes and Gentle Elution Buffer? (reportedly 3M MgCl2 at roughly neutral pH). Frozen human nervous tissue was homogenized in lysis buffer (25 mM Tris pH 7.8 supplemented Apremilast with protease inhibitor cocktail (Roche)) at 4 °C followed by centrifugation at 14 0 RPM and this supernatant was applied to an individual immunoaffinity column. Columns were washed four occasions with 600 μl (~20 column volumes total) wash buffer (25 mM Tris 100 mM NaCl pH 7.8). SOD1 proteins were eluted with 2 × 500 μl of either 5 % acetic acid (purification 1) or gentle antibody elution buffer (GEB) pH 6.6 (Pierce 21027 (purification 2). To ensure that the purified Apremilast samples contained a representative sampling of native and altered SOD1 we verified that SOD1 was immunodepleted from your homogenates. Following the first immunopurification the column was re-equilibrated in lysis buffer the depleted homogenates (flow-through) were re-applied and the purifications were repeated in this way a total of 3 times. If protein was detected in a repeat purification (using MALDI-TOF MS only the second purification occasionally contained minor amounts of SOD1) that purification was pooled with the first. Proteins eluted with GEB were buffer exchanged into 25 mM HEPES pH 7.4 and concentrated to ~100 μl and the concentrations were determined by western blot and densitometry (ImageJ) analyses with recombinant wild-type SOD1 requirements. Proteins eluted with 5 % acetic acid were used as purified. In addition SOD1 was purified anaerobically in the presence or absence of.