Functional insights into protein acetylation in the hyperthermophilic archaeon Sulfolobus islandicus
Proteins undergo acetylation at the Nε-amino group of lysine residues and the Nα-amino group of the N-terminus in Archaea as in Bacteria and Eukarya. However, the extent, pattern and roles of the modifications in Archaea remain poorly understood. Here we report the proteomic analyses of a wild-type Sulfolobus islandicus strain and its mutant derivative strains lacking either a homologue of the protein acetyltransferase Pat (SisPat) or a homologue of the Nt-acetyltransferase Ard1 (ΔSisArd1). A total of 1,708 Nε-acetylated lysine residues in 684 proteins (26% of the total proteins), and 158 Nt-acetylated proteins (44% of the identified proteins) were found in S. islandicus. ΔSisArd1 grew more slowly than the parental strain, whereas ΔSisPat showed no significant growth defects. Only 24 out of the 1,503 quantifiable Nε-acetylated lysine residues were differentially acetylated, and all but one of the 24 residues were less acetylated, by >1.3 fold in ΔSisPat than in the parental strain, indicating the narrow substrate specificity of the enzyme. Six acyl-CoA synthetases were the preferred substrates of SisPat in vivo, suggesting that Nε-acetylation by the acetyltransferase is involved in maintaining metabolic balance in the cell. Acetylation of acyl-CoA synthetases by SisPat occurred at a sequence motif conserved among all three domains of life. On the other hand, 92% of the identified N-termini were acetylated by SisArd1 in the cell. The enzyme exhibited broad substrate specificity and was capable of modifying nearly all types of the target N-termini of human NatA-NatF. The deletion of the SisArd1 gene altered the cellular levels of 18% of the quantifiable proteins (1,518) by >1.5 fold. Consistent with the growth phenotype of ΔSisArd1, the cellular levels of proteins involved in cell division and cell cycle control, DNA replication, and purine synthesis were significantly lowered in the mutant than those in the parental strain.
Sample Processing Protocol
Cells were resuspended in lysis buffer [8 M urea, 1% protease inhibitor cocktail (Roche), 3 μM trichostatin A, 50 mM nicotinamide and 2 mM EDTA], and disrupted on ice by sonication. After centrifugation, proteins were precipitated with ice-cold 20% TCA for 2 h at -20°C. After centrifugation, the pellet was washed three times with cold acetone. The proteins were dissolved in 8 M urea and 50 mM NH4HCO3, and the protein concentration was determined by using the BCA kit (Beyotime). The protein solution was reduced with 5 mM DTT for 30 min at 56°C, and subsequently alkylated with 11 mM iodoacetamide (IAA) for 45 min at room temperature in dark. After adding 50 mM NH4HCO3 to lower the urea concentration to < 2 M, trypsin was added at a trypsin-to-protein mass ratio of 1:50. Following digestion for overnight, trypsin was added at a trypsin-to-protein mass ratio of 1:100. The second trypsin digestion was for 4 h. For TMT-based quantitative lysine acetylomic analysis of ΔSisPat and parental strain E233S, a sample of the tryptic peptides (from 4 mg of proteins) was desalted, vacuum-dried and reconstituted in 0.5 M triethylammonium bicarbonate (TEAB). Two replicates of the ΔSisPat sample were labeled with tandem mass tag (TMT) (Thermo Fisher) regent 126-tag and 130-tag, respectively, and two replicates of the E233S sample were labeled with 127-tag and 131-tag, respectively. All peptides were mixed, desalted and vacuum-dried. The peptides were resuspended in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 0.5% Nonidet P-40), and incubated with anti-acetyllysine agarose beads (PTM Biolabs, Hangzhou, China) at 4°C for overnight with gentle shaking. The beads were washed four times with 1 ml of NETN buffer and twice with deionized H2O. The bound peptides were eluted from the beads with 1% trifluoroacetic acid (TFA). The eluted fractions were combined, vacuum-dried, and subjected to LC-MS/MS analysis. For TMT-based quantitative proteomic analyses of ΔSisArd1 and E233S, the tryptic peptides (from 200μg of total proteins for each sample) were labeled with the TMT regents. Two duplicate ΔSisArd1 samples were labeled with TMT regent 129-tag and 130-tag, respectively, and two duplicate E233S samples were labeled with 127-tag and 128-tag, respectively. The labeled samples were mixed at equal amounts and fractionated by high-pH reverse-phase HPLC. The fractions were combined into 10 pools, vacuum-dried and analyzed by LC-MS/MS. For N-terminal acetylome analysis of ΔSisArd1 and E233S using the terminal amine isotopic labeling of substrates (TAIlS) technique, proteins from the two strains were reduced and alkylated as described above. A sample (4 mg) of the proteins was precipitated by the addition of eight sample volumes of cold acetone and subsequent incubation for 2 h at -20°C. After centrifugation, the precipitate was dissolved in 6M GuHCl, 20 mM HEPES-KOH, pH 8.0, 40 mM deuterated formaldehyde (12CD2O) and 20 mM NaBH3CN, and incubated at 37°C for overnight. The reaction was quenched by the addition of 1 M NH4HCO3 to 100 mM. After 4 h at 37°C, proteins were precipitated by the addition of cold acetone. After 2 h at -20°C, the sample was centrifuged, and the precipitate was dissolved in 8 M urea and 50 mM NH4HCO3. The proteins were digested with trypsin as described above. The sample was fractionated into 60 fractions by high-pH reverse-phase HPLC. These fractions were combined into 6 pools and dried. The peptides were dissolved in PBS buffer, and incubated with pre-washed NHS-activated agarose beads (Lot number 26196, Thermo) at 4°C for overnight with gentle shaking. Unbound peptides were collected after removing the beads by centrifugation, vacuum-dried and subjected to LC-MS/MS. LC-MS/MS analysis was performed on an EASY-nLC 1000 UPLC system (Thermo Fisher) coupled online to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher). Peptides were desalted online by an in-house packed trap column (C18, 5 μm particles, 100 μm ID, 3 mm length, Dr. Maisch GmbH). The trapped peptides were loaded onto an in-house packed reversed-phase C18 column (3 μm particles, 75 μm ID, 150 mm length, Dr. Maisch GmbH) and eluted at a flow rate of 300 nl/min with a gradient from solvent A (0.1% formic acid in water) to solvent B (0.1% formic acid in acetonitrile): 4% to 22% B over 65 min, 22% to 35% B over 15 min, 35% to 80% B over 5 min and 80% B over 5min. The eluted peptides were subjected to analysis on an Orbitrap Fusion Tribrid mass spectrometer with an MS survey scan (m/z range 350-1800, 70,000 resolution, 3×106 AGC target, and 50 ms maximal ion time) and an MS/MS scan (m/z range 100-2000, 17,500 resolution, high-energy collision dissociation, 2 m/z isolation window, 30 normalized collision energy, 5×104 AGC, 200 ms maximal ion time, 30 s dynamic exclusion, and top number 20), respectively.
Data Processing Protocol
The RAW mass spectrometry files were processed using MaxQuant (v.188.8.131.52) with an integrated Andromeda search engine. Tandem mass spectra were searched against the Uniprot S. islandicus REY15A database (UP000002664, 2,631 sequences) concatenated with a reverse decoy database. The mass tolerance for precursor ions was set at 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set at 0.02 Da. Trypsin/P was specified as the cleavage enzyme, allowing up to 4 missing cleavages. The minimum peptide length was set at 7. For RAW mass spectrometry files of the TMT-based quantitative lysine acetylomic analysis, carbamidomethyl (C) and TMT-6plex (N-terminus, K) were specified as fixed modifications, whereas oxidation (M) and acetylation (K) as variable modifications. False discovery rate (FDR) was adjusted to <1% and the minimum score for modified peptides was set at >40. Quantitative information for each peptide or lysine site was calculated according to TMT ratios. A differentially acetylated lysine peptide was identified using a 1.3 or 2 fold cutoff. For RAW mass spectrometry files of the TMT-based quantitative proteomic analyses, carbamidomethyl (C), TMT-6plex (N-terminus, K) were specified as fixed modifications, whereas oxidation (M) as a variable modification. At least one unique peptide was required for the identification and quantification of a protein. A maximum FDR of 1% was employed for the identification of a protein. A differentially regulated protein was identified using a 1.5 fold cutoff. For RAW mass spectrometry files of the TAIlS-based N-terminal acetylomic analysis, Carbamidomethyl (C) was set as a fixed modification, whereas acetylation (N-terminus), tetradeutero-dimethyl (C2H2D4, 32.0564 Da) (K, N-terminus) and oxidation (M) were set as variable modifications. FDR was adjusted to <1% and the minimum score for modified peptides was set at >40. Representative MS2 spectra of modified peptides (i.e., ones with the highest MASCOT score for a given modified peptide) were manually inspected. MS2 spectra, in which fewer than four sequential matched fragment ions were present or most of the matched ions were at the level of background noise, were deleted. The extent of Nt-acetylation for each protein was calculated as the ratio of the intensity of Nt-acetylated peptides to that of the identified N-terminal peptides from that protein.
Cao J, Wang T, Wang Q, Zheng X, Huang L. Functional insights into protein acetylation in the hyperthermophilic archaeon Sulfolobus islandicus. Mol Cell Proteomics. 2019 PubMed: 31182439