a Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. Molecular

Reversible lysine acetylation is one of the most important protein posttranslational modifications that plays essential roles in both prokaryotes and eukaryotes. However, only a few lysine deacetylases (KDACs) have been identified in prokaryotes, perhaps in part due to their limited sequence homology. Herein, we developed a ‘clip-chip’ strategy to enable unbiased, activity-based discovery of novel KDACs in the Escherichia coli proteome. In-depth biochemical characterization confirmed that YcgC is a serine hydrolase involving Ser200 as the catalytic nucleophile for lysine deacetylation and does not use NAD+ or Zn2+ like other established KDACs. Further, in vivo characterization demonstrated that YcgC regulates transcription by catalyzing deacetylation of Lys52 and Lys62 of a transcriptional repressor RutR. Importantly, YcgC targets a distinct set of substrates from the only known E. coli KDAC CobB. Analysis of YcgC’s bacterial homologs confirmed that they also exhibit KDAC activity. YcgC thus represents a novel family of prokaryotic KDACs.DOI: http://dx.doi.org/10.7554/eLife.05322.001

CobB is a bacterial NAD+‐dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N‐hydroxyarylamine O‐acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site‐specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O‐acetyltransferase and N‐acetyltransferase activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role in the activity of Escherichia coli NhoA.