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(C) Quantification of individual acetylation sites is displayed as the fold change versus the results for the control. The dot color represents the standard deviation with respect to the unmodified peptide distribution. Acetylated peptides representing SAGA components with changes greater than 1 standard deviation are indicated by colored squares. The standard deviation (SD) of this subset was used to approximate the significance of changes in levels of acetylated peptides. Changes observed in the subset of unmodified peptides were assumed to represent a distribution of measurements unaffected by SIRT1 inhibition. Quantitative measurements of peptide-level changes following acetyllysine IP were normalized to pre-IP total protein measurements. Both unmodified (black) and acetylated (colored) peptides were recovered following antiacetyllysine IP. (B) Dot plot of individual peptides representing the sum of the signal to noise for a given peptide plotted against its fold change versus the results for the control. (A) Outline of the work flow used to analyze acetylation of USP22-associated proteins in response to SIRT1 inhibition. Quantitative mass spectrometry reveals a SIRT1-regulated SAGA acetylome. (E) GCN5-FLAG was purified from HEK 293T cells treated with DMSO or EX527 (10 μM) and immunoblotted with the indicated antibodies. (D) Several FLAG-HA-tagged SAGA components or a FLAG-HA-GFP control were purified from HEK 293T cells treated with DMSO or EX527 (10 μM) and immunoblotted for the indicated proteins. Eluted complex pools were immunoblotted with the indicated antibodies. (C) Whole-cell lysates from HEK 293T cells stably expressing FLAG-HA-USP22 treated with DMSO or EX527 (10 μM) were separated by gel filtration, pooled by size, and further purified by FLAG IP. (B) FLAG-HA-USP22 was purified as described for panel A from HEK 293T cells treated with DMSO, EX527 (10 μM), or FK866 (10 nM). The lower Ac-K 9441 blot is a shorter exposure displaying only the main band found between 50 and 75 kDa. (A) FLAG-HA-USP22 purified from HEK 293T cells treated with DMSO, TSA (400 nM), NAM (20 mM), or EX527 (10 μM) was immunoblotted with the indicated antibodies. SAGA components are acetylated in response to SIRT1 inhibition. FPLC, fast-performance liquid chromatography. (D) (Left) Whole-cell lysates from FLAG-HA-USP22-expressing HEK 293T cells were separated by gel filtration and immunoblotted (right) fractions from gel filtration samples were pooled on the basis of size, purified by IP, and treated with DMSO or 5 μM UbVS. (C) Equal amounts of whole-cell lysates from HEK 293T cells were treated with DMSO or 5 μM UbVS for the indicated times and immunoblotted with the indicated antibodies. (B) Purified intact mononucleosomes were incubated with full-length or catalytic inactive (CS) recombinant human Dubs and immunoblotted with the indicated antibodies. Data are presented in relative fluorescence units (RFU). (Bottom) Kinetic deubiquitinating activity assay of recombinant USP22 or the catalytic core of USP2 (USP2cc) with the substrate Ub-AMC. (Middle) Reactions were performed as described above with various concentrations of DTT. A representative gel stained with Coomassie brilliant blue is shown. (A) (Top) Purified recombinant human USP22 or catalytically inactive USP22-C185S was incubated with either DMSO or 5 μM UbVS. Human USP22 requires complex formation for deubiquitinating activity. Our results indicate an important role of SIRT1-mediated deacetylation in regulating the formation of DUBm subcomplexes within the larger SAGA complex. Furthermore, USP22-mediated recruitment of SIRT1 activity promotes the deacetylation of individual SAGA complex components. Moreover, we show that USP22 is acetylated on multiple lysine residues and that alteration of a single lysine (K129) within the ZnF-UBP domain is sufficient to alter interaction of the DUBm with the core SAGA complex. We found that this interaction is highly specific, requires the ZnF-UBP domain of USP22, and is disrupted by the inactivating H363Y mutation within SIRT1. Employing this method, we identified the deubiquitinating enzyme ubiquitin-specific protease 22 (USP22), a component of the deubiquitinating module (DUBm) of the SAGA transcriptional coactivating complex, as a SIRT1-interacting partner. Using a comparative statistical analysis of binding partners, we have assembled a high-confidence SIRT1 interactome. Although many functions and targets have been attributed to the histone and protein deacetylase SIRT1, a comprehensive analysis of SIRT1 binding proteins yielding a high-confidence interaction map has not been established.