Contents were transferred to a 15-ml conical tube and were centrifuged at room temperature

Contents were transferred to a 15-ml conical tube and were centrifuged at room temperature. the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival. revealed that only a small fraction, representing about 1% of total SOD1, is required for protection against oxidative stress (16). This suggests that SOD1 may have additional functions beyond its traditional role in reactive oxygen species (ROS) scavenging. Indeed, studies suggest that SOD1 plays roles in zinc and copper buffering (11, 17) and in regulating gene transcription (18, 19). In addition, a recent study by Reddi and Culotta found that yeast SOD1 suppressed mitochondrial respiration (20). However, despite this emerging complexity in SOD1 biology and clear roles for SOD1 in human disease, we have a limited understanding of SOD1 regulation at a posttranslational level. Here we uncover a novel regulatory mechanism by which a sirtuin-governed acylation within the electrostatic loop of SOD1, at K122, suppresses SOD1-mediated inhibition of mitochondrial metabolism in mammalian cells. This observation provided genetic tools to help us understand the relationship between SOD1 mitochondrial localization and metabolic regulation, as well as the potential contribution of this metabolic function of SOD1 to its role in promoting cell survival. Our data suggest a model in which sirtuin-mediated deacylation of SOD1 promotes its inhibition of respiration, which in turn, elevates levels of mitochondrial SOD1 and contributes to the prosurvival function of SOD1. RESULTS As a starting point, with the goal of identifying PTMs on cell survival signaling nodes, we used several PTM-specific antibody resins to compare PTMs across multiple mouse tissues (brain, liver, and embryo homogenates). The experimental layout, shown in Fig. 1A, included several phosphomotif, ubiquitin, and acetyl-lysine affinity resins. A complete set of database search results from this experiment is publicly available as a Scaffold file (Proteome Software Inc.) at In an effort to zoom in on PTMs on cell survival signaling nodes, we applied Gene Ontology analysis, as well as manual sorting by protein function. Two proteins of interest, 14-3-3 and SOD1, are shown in Fig. Cefadroxil 1B. 14-3-3 is a phospho-serine/threonine binding protein that is overexpressed in a variety of cancers and promotes cell survival by directly modulating a network of phosphoproteins. In combining our PTM data sets, we identified PTMs of unknown function, including phosphorylation of Y149 (phospho-Y149) and ubiquitination of K139 (Ub-K139), on 14-3-3, in addition to well-described PTMs, such as acetylation of K49 (Ac-K49) (21,C23). In particular, acetylation of K49 is known to disrupt 14-3-3 interactions, and our previous work identified histone deacetylase 6 (HDAC6) as the K49-targeted lysine deacylase (KDAC) (23). Open in a separate window FIG 1 Identification of PTMs on 14-3-3 and SOD1. (A) Brain, liver, and whole-embryo mouse tissues were homogenized and digested with trypsin. Peptides were subjected to affinity purification by the indicated antibody resin. Peptides were eluted and analyzed by LCCMS-MS. Proteomics data were analyzed with Scaffold software. IP, immunoprecipitation. (B) Crystal structures of human 14-3-3 (PDB accession no. 4IHL) and mouse SOD1 (PDB accession no. 3GTT) with PTMs identified in Cefadroxil the proteomics data. SAPH-ire identifies PTMs with high function potential in the SOD domain family. Our attention was also drawn to SOD1, Rabbit polyclonal to ADAMTS18 which acts as one of the main modes of defense against oxidative stress by catalyzing the disproportionation of superoxide radicals (O2?) to molecular oxygen (O2) and hydrogen peroxide (H2O2). The lower panel of Fig. 1B shows the crystal structure of the SOD1 dimer and PTMs identified from our proteomics data. In an effort to prioritize PTMs on SOD1, we utilized SAPH-ire FPx, a machine learning-based PTM hot spot finder that examines experimentally identified PTMs and prioritizes them for the likelihood of biological function based on a number of parameters as Cefadroxil described previously (1, 2, 24). By use of human SOD1 (UniProt ID “type”:”entrez-protein”,”attrs”:”text”:”P00441″,”term_id”:”134611″,”term_text”:”P00441″P00441) as an anchor, PTMs were evaluated in the context of the entire eukaryotic SOD domain family (InterPro ID IPR001424). Interestingly, PTMs.

Comments are closed.