Schilling - Wolfe_KAT_2018

Schilling - Wolfe_KAT_2018
Identification of novel protein lysine acetyltransferases in Escherichia coli

  • Organism: Escherichia coli
  • Instrument: TripleTOF 6600
  • SpikeIn: No
Post-translational modifications, such as Nε -lysine acetylation, regulate protein function. Nε-lysine acetylation can occur either non-enzymatically or enzymatically. The non-enzymatic mechanism uses acetyl phosphate (AcP) or acetyl coenzyme A (AcCoA) as acetyl donors to modify an Nε-lysine residue of a protein. The enzymatic mechanism uses Nε-lysine acetyltransferases (KATs) to specifically transfer an acetyl group from AcCoA to Nε-lysine residues on proteins. To date, only one KAT (YfiQ, also known as Pka and PatZ) has been identified in E. coli. Here, we demonstrate the existence of 4 additional E. coli KATs: RimI, YiaC, YjaB, and PhnO. In a genetic background devoid of all known acetylation mechanisms (most notably AcP and YfiQ) and one deacetylase (CobB), overexpression of these putative KATs elicited unique patterns of protein acetylation. We mutated key active site residues and found that most of them eliminated enzymatic acetylation activity. We used mass spectrometry to identify and quantify the specificity of YfiQ and the four novel KATs. Surprisingly, our analysis revealed a high degree of substrate specificity. The overlap between KAT-dependent and AcP-dependent acetylation was extremely limited, supporting the hypothesis that these two acetylation mechanisms play distinct roles in the post-translational modification of bacterial proteins. We further showed that these novel KATs are conserved across broad swaths of bacterial phylogeny. Finally, we determined that one of the novel KATs (YiaC) and the known KAT (YfiQ) can negatively regulate bacterial migration in soft agar. Together, these results emphasize distinct and specific non-enzymatic and enzymatic protein acetylation mechanisms present in bacteria.
Experiment Description
We processed isolated frozen bacterial pellets from the gutted strains carrying vector control (AJW5426) or one of the 5 KAT candidates (i) YjgM (AJW5496), (ii) RimI (AJW5499), (iii) YiaC (AJW5501), (iv) YjaB (AJW5504), (v) PhnO (AJW5513), as well as the known KAT YfiQ (AJW5497). Each of the strains was processed as 3 biological replicates.
Sample Description
Cell pellets of the indicated strains were suspended in 6 mL of PBS and centrifuged at 4°C, 15,000 g for 20 min. The firm cell pellet was collected and re-suspended and denatured in a final solution of 6 M urea, 100 mM Tris, 75 mM NaCl, and the deacetylase inhibitors tricostatin A (1 mM) and nicotinamide (3 mM). Samples were sonicated on ice (5× each for 15 sec), cellular debris removed, and the supernatants were processed for proteolytic digestion. Lysate containing 1.5 mg of protein was reduced with 20 mM DTT (37°C for 1 h), and subsequently alkylated with 40 mM iodoacetamide (30 min at RT in the dark). Samples were diluted 10-fold with 100 mM Tris (pH 8.0) and incubated overnight at 37°C with sequencing grade trypsin (Promega) added at a 1:50 enzyme:substrate ratio (wt/wt). In parallel, separate 1.5 mg protein aliquots were digested using endoproteinase Glu-C (Roche, Indianapolis, IN) by adding Glu-C at a 1:50 protease to substrate protein ratio (wt:wt), and incubating overnight at 37 °C. Subsequently, samples were acidified with formic acid and desalted using HLB Oasis SPE cartridges (Waters) (Keshishian et al., 2007). Proteolytic peptides were eluted, concentrated to near dryness by vacuum centrifugation, and re-suspended in NET buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). A small aliquot of each protein digestion (∼ 10 μg) was saved for protein-level identification and quantification. The remaining proteolytic peptide samples were used for affinity purification of acetylated peptides (Kac). Acetylated peptides were enriched using 1/4 tube of anti-acetyl lysine antibody-bead conjugated ‘PTMScan Acetyl-Lysine Motif [Ac-K]’ Kit (Cell Signaling Technologies) for each of the 1 mg protein lysate samples according to the manufacturer’s instructions. Prior to mass spectrometric analysis, the acetylated peptide enrichment samples were concentrated and desalted using C-18 zip-tips (Millipore, Billerica, MA).
Created on 7/5/18, 9:17 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates 15:20:4490961922,76490 22:10:23131402165 21:25:59264762373 21:25:5911921636 21:25:582,77924,31330,00390,00933
Sky0 ACE2 TMPRSS2 cell 13:38:456252919732 16:24:3290961922,76493 15:34:1311261 15:34:13112668 15:34:1311262 15:34:1311268 15:34:13112663 15:34:13112672 15:34:131121069 15:34:13112101 15:34:13112102 15:34:13112460 15:34:13112412 15:34:13112669 15:34:1311262
Sky1 Synthetic SARS-CoV2 proteome 13:21:56121431351,2121
Sky3 52 patients nano PRM-MS2 13:20:236345734055
Sky4 5 positive patients nano PRM-MS2 13:20:22634492945
Sky6 11 positive patients micro PRM-MS2 13:11:206345527311
Sky5 91 patients micro PRM-MS2 13:11:206345527391
Sky2 Dilution series SARS Cov2 nano and 13:04:536345737118 20:16:492828562164 20:16:492627542064 20:16:492424481824 05:16:2773154154616102
Intra-day 05:14:01731541546166
Inter-day 05:13:11731541546165
Instrumental set-up 05:11:30731541546165 01:26:17217715461713 01:17:00217815662512 20:17:53751481622,58918 20:17:272141254 20:17:272141263 20:17:272151639 20:17:272151631 20:17:272141235 20:17:272141238 20:17:272141265 20:17:272141211 20:17:262141264 20:17:262141213 20:17:262041284
Batch 20:17:262141265 20:17:262141219 20:16:45417201228 20:16:44299668 20:16:10299668 15:59:1690961921,02896 19:31:421,6914,7464,87926,1230 17:38:291,5075,0745,10425,3220 10:58:1317494921860 05:51:586253,2053,20513,12660 07:21:051,2225,9545,95422,91166 16:35:09112434 16:35:09112644 16:35:09112638 16:35:09112428 16:35:09112644 16:35:09112632 16:35:09112643 16:35:09112442 16:35:09112442 16:35:09112436 16:35:09112433 16:35:09112442 16:35:09112643 16:35:09112444 16:35:09112444 16:35:09112441 16:23:121616328224 16:23:121616328423 16:23:121616328229 16:23:121616328027 16:23:121616328425 16:23:121616328632 16:23:121616328225 16:23:121616328223 16:23:121616328218 16:23:121616328244 16:23:121616328230 16:23:121616328415 16:23:121616328021 16:23:121616328430 16:23:121616328424 16:23:121616328227 16:23:121616328028 16:23:121616328229 16:23:121616328224 16:23:121616328425 16:23:121616328422 16:23:111616328428 16:23:111616328428 16:23:111616328224 16:23:111616328235 16:23:111616328422