Project description:To identify acetylation-dependent posttranslational modifications (PTMs) of G6PD, site-specifically acetylated and Flag-tagged G6PD was expressed in HEK293T cells by genetically encoding the incorporation of acetylated lysine in response to an in-frame TAG stop codon. K403-acetylated G6PD (sample) and K414-acetylated G6PD (control) were co-expressed with WT Fyn kinase and a catalytically inactive mutant of Fyn (FynDN). G6PD was immunoprecipitated using anti-Flag beads before MS analysis.
Project description:Peroxisome proliferator-activated receptorg (PPARg), which is expressed in a variety of malignancies, governs biological functions through transcriptional programs. Defining the molecular mechanisms governing selection of canonical versus non-canonical PPARg binding sequences may provide the opportunity to design regulators with distinct functions and side effects. PPARg is acetylated at multiple residues including K268/293 in mouse Pparg2 and the conserved lysine residues (K154/155) in mouse Pparg1. Herein, the PPARg acetylated residues K154/155 were shown to be essential for growth control, and the induction of transcriptional modules governing growth factor signaling, cellular apoptosis and autophagy. The K154/155 residues determined the selection of genome wide DNA binding sites, altering the selection from canonical to non-canonical (C/EBP) DNA sequence specific binding. The gene signature reflecting the acetylation-dependent genomic occupancy provided predictive value in patient survival outcome. Pparg1 participates in ErbB2-induced tumor growth and inflammation and represents a relevant target for therapeutic coextinction.
Project description:cAMP receptor protein (CRP, also known as the catabolite activator protein [CAP]) is arguably the best-studied of the global transcription factors of E coli. CRP alone is responsible for regulating at least 283 operons. Upon binding cAMP, the CRP dimer binds DNA and directly interacts with RNA polymerase (RNAP). At Class II promoters, CRP binds near position -41,5 relative to the transcription start site and contacts the amino-terminal domain of the RNAP α subunit (RNAPα-NTD). This interaction requires AR2, a patch of primarily positively charged residues (H19, H21, E96, and K101) that interact with negatively charged residues on RNAPα-NTD. Acetylome analyses consistently detect lysine 100 (K100) of CRP as acetylated. Since K100 is adjacent to the positively charged AR2, we hypothesized that the K100 positive charge may also play a role in CRP function. We further hypothesized that acetylation of K100 would neutralize this positive charge, leading to a potential regulatory mechanism
Project description:Previously, analysis of EGF-induced EGFR ubiquitination by mass spectrometry revealed that ubiquitinated lysine residues were located in the TKD of EGFR. However, the specific lysine residues for the CBL-mediated polyubiquitination have not been identified. To investigate the differences in EGFR ubiquitination by ZNRF1 and CBL, we sought to identify the acceptor residues for polyubiquitin chains mediated by these two E3 ubiquitin ligases. We co-transfected HEK293T cells with EGFR and ZNRF1 or CBL, and immunoprecipitated EGFR for liquid chromatography-tandem mass spectrometry analysis.
Project description:Protein lysine acetylation is a reversible and dynamic post-translational modification. It plays an important role in regulating diverse cellular processes including chromatin dynamic, metabolic pathways and transcription in both prokaryotes and eukaryotes. Although studies of lysine acetylome on plants have been reported, the throughput was not high enough, hindering the deep understanding of lysine acetylation in plant physiology and pathology. In this study, taking advantages of anti-acetyllysine-based enrichment and high-sensitive-mass spectrometer, we applied an integrated proteomic approach to comprehensively investigate lysine acetylome in strawberry. In total, we identified 1392 acetylation sites in 684 proteins, representing the largest dataset of acetylome in plant to date. To reveal the functional impacts of lysine acetylation in strawberry, intensive bioinformatic analysis was performed. The results significantly expanded our current understanding of plant acetylome and demonstrated that lysine acetylation is involved in multiple cellular metabolism and cellular processes. More interestingly, nearly 50% of all acetylated proteins identified in this work were localized in chloroplast and the vital role of lysine acetylation in photosynthesis was also revealed. Taken together, this study not only established the most extensive lysine acetylome in plants to date, but also systematically suggests the significant and unique roles of lysine acetylation in plants.
Project description:We assessed the genomic distribution of H4-acetylated chromatin by ChIP using SNP chips and an antibody specific to four acetylated lysine residues of H4. Keywords: Comparative genomic hybridization
Project description:To better examine the molecular mechanisms behind the virus infection, we conducted a correlation analysis of RNA-Seq and quantitative iTRAQ-LC-MS/MS in TuMV-infected and in healthy Chinese cabbage leaves.
Project description:We immunoprecipitated exogengusly expressed TaRACK1B-GFP,TaSGT1-GFP and TaHSP90-GFP fused protein in wheat protoplasts.A high-throughput technology, LC-MS/MS, was applied to identify the candidate interacting proteins of TaRACK1B-GFP,TaSGT1-GFP and TaHSP90-GFP
Project description:Histone lysine methylation is an important post-translational modifications (PTMs) that plays critical roles in numerous biological processes with abnormal histone lysine methylation having been associated with developmental defects and human diseases. The primary amine of all lysine residues can be mono-, di-, or trimethylated and the extent of methylation at a single site is shown to inspire unique protein function. Moreover, histone lysine methylation can activate or repress transcription depending on which sites are modified and to what degree. Therefore, a comprehensive stoichiometric analysis of histone lysine methylation is necessary to fully elucidate its function. As histones are lysine-rich and highly-hydrophilic proteins (Figure S1), trypsin digestion of histones results in small, hydrophilic peptides which often suffer from substantial losses during sample preparation, and are difficult to detect in conventional reversed phase LC-MS. Additionally, trypsin fails to cleave histone proteins when lysine residues are methylated, resulting in inaccurate estimations of site occupancy and methylation extent. Previously, lysine propionylation labeling has been developed to overcome this drawback and facilitate quantitative analysis of PTMs that frequently occur on the lysine residue of histones. However, propionylation labeling of unmodified and monomethylated lysine residues causes a mismatch in hydrophobicity and charge state between labeled and unlabeled di-/trimethylated peptides. This mismatch forces retention time and signal intensity differences that inspire inaccurate quantitation and miscalculation of site occupancy. In this work, we developed a method that facilitates blocking of free lysine groups and discrimination of native lysine methylation, enables accurate calculation of the histone lysine methylation stoichiometry.
Project description:Nucleoid-associated proteins (NAPs) are critical during the process of chromatin compaction in Streptomyces soil bacteria. HupS is one of the two NAPs encoded in the Streptomyces genome. Its unique C-terminal domain, rich in lysine repeats (LR domain), is alike to the H2B histone found in eukaryotic cells or the HupB protein found in Mycobacterium. Project study aim was to identify post-translationally acetylated lysine residues of HupS via bottom-up LC-MS. Two approaches were uptaken. One was based on HupS enrichment from a strain expressing a recombinant HupS with a C-terminal FLAG tag (TM015) via an antiFLAG pull-down, and the other relied on a proteome-wide search of acetylated peptides in a WT strain lysate. Controls for spectral false-positives were also carried out in parallel, which were an antiFLAG pull-down with a WT strain, and a proteome-wide search of acetylated peptides in a HupS deletion mutant strain (ΔhupS) lysate. 5 lysine acetylation sites were confidently determined for the HupS protein within this study, them being Lys51, Lys85, Lys104, Lys119, and either Lys193 or Lys194.