While many aspects of archaeal cell biology remain relatively unexplored, systems biology approaches like mass spectrometry (MS) based proteomics offer an opportunity for rapid advances. Unfortunately, the enormous amount of MS data generated often remains incompletely analyzed due to a lack of sophisticated bioinformatic tools and field-specific biological expertise for data interpretation. Here we present the initiation of the Archaeal Proteome Project (ArcPP), a community-based effort to comprehensively analyze archaeal proteomes. Starting with the model archaeon Haloferax volcanii, we reanalyze MS datasets from various strains and culture conditions. Optimized peptide spectrum matching, with strict control of false discovery rates, facilitates identifying > 72% of the reference proteome, with a median protein sequence coverage of 51%. These analyses, together with expert knowledge in diverse aspects of cell biology, provide meaningful insights into processes such as Nterminal protein maturation, N-glycosylation, and metabolism. Altogether, ArcPP serves as an invaluable blueprint for comprehensive prokaryotic proteomics.
Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine-S-sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant.
Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1′s deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.
Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A chase assay is described to monitor the impact of proteasome function on the stability of Ubl-modified proteins in the cell. A method to reconstitute Ubl ligation using a purified E1-like enzyme (UbaA), Ubl (SAMP2), methionine sulfoxide reductase A (MsrA), and cell lysate of an ΔmsrA ΔubaA Δsamp1-3 mutant is also described. MsrA is found to have the surprising ability to stimulate the formation of Ubl bonds. Haloferax volcanii, a halophilic archaeon originally isolated from the Dead Sea, serves as the model organism for these protocols.
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