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Proteome and Crotonylome of normal and leucine deprivation treated AML12 cells

ABSTRACT: Leucine, as a branched amino acid, is not only a substrate for protein synthesis, but also a signaling molecule that affects many biological processes. Lysine crotonylation is a novel post-translational modification in both histone and non-histone proteins. What effect amino acids have on crotonylation remains unclear. Here, we identified a large number of crotonylated proteins and sites affected by leucine deprivation for 2 hours in AML12 cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Zheng Zilong

LAB HEAD: Zilong Zheng

PROVIDER: PXD018118 | Pride | 2023-05-10

REPOSITORIES: Pride

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Publications

KAT7-mediated CANX (calnexin) crotonylation regulates leucine-stimulated MTORC1 activity.

Yan Guokai G Li Xiuzhi X Zheng Zilong Z Gao Weihua W Chen Changqing C Wang Xinkai X Cheng Zhongyi Z Yu Jie J Zou Geng G Farooq Muhammad Zahid MZ Zhu Xiaoyan X Zhu Weiyun W Zhong Qing Q Yan Xianghua X

Autophagy 20220310 12

Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully understood. Here, we establish a cell-free system to mimic the activation of MTORC1, by which we identify CANX (calnexin) as an essential regulator for leucine-stimulated MTORC1 pathway. CANX translocates to lysosomes after leucine deprivation, and its loss of function renders either the MTORC1 activity or the lysosomal translocation of ...[more]

PMID: 35266843

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SIRT7-induced PHF5A de-crotonylation regulates ageing progress through alternative splicing-mediated downregulation of CDK2

Project description:Dysregulation of protein post-translational modification (PTM) can lead to a variety of pathological process, such as abnormal sperm development, malignant tumorigenesis, depression, and ageing process. SIRT7 is a NAD + dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other de-acylation in ageing is still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan- lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5149 Kcr sites across 1541 protein in senescent fibroblasts, providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified protein, we found SIRT7 de-crotonylated PHF5A, an alternative splicing factor, at K25. De-crotonylation of PHF5A K25 contributed to decreased CDK2 expression by Retained Intron (RI) induced abnormal alternative splicing, thereby accelerating fibroblasts senescence, supporting a key role of PHF5A K25 de-crotonylation in ageing. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging, and provide new ideas and molecular targets for drug intervention in cellular ageing and the treatment of aging-related diseases, indicating that protein crotonylation has important implication in the regulation of ageing progress.

2022-02-17 | PXD027232 | Pride

Global Lysine Crotonylation Alterations of Host Cell Proteins Caused by Brucella GNAT Containing Effector BspF

Project description:In Brucella spp., the type IV secretion system (T4SS) is essential for bacterial intracellular survival and inhibition of the host innate immune response. The Brucella T4SS secretes 15 different effectors to escape host immunity and promote intracellular replication. Among them, BspF has a GNAT-family acetyltransferase domain, implying its acetyltransferase activity. To test this, in this study, we first tested and confirmed that BspF has de-histone crotonyltransferase activity in vitro. Unexpectedly, we also found that BspF enhances histone crotonylation of host cell proteins. We then conducted enrichment of crotonylated proteins and used LC-MS to study the crotonylation of proteins in HEK-293T cells caused BspF overexpression. A total of 5,559 crotonylation sites were identified on 1,525 different proteins, of which 331 sites on 265 proteins were significantly changed 30 h after transfection. Among these differentially modified proteins, a significant proportions were involved in immune response and defense mechanism. We propose that BspF may influence the function of these host proteins by changing the level of crotonylation, thereby promoting the intracellular propagation of Brucella.

2021-09-09 | PXD021423 | Pride

Crotonylation versus acetylation in PaACL-silenced corollas in petunia

Project description:Protein acetylation and crotonylation are important post-translational modifications (PTMs) of lysine. In animal cells, histone lysines are crotonylated or acetylated depends on the relative intracellular concentrations of crotonyl-CoA and acetyl-CoA, and adding crotonate to cell cultures, or reducing the intracellular levels of acetyl-CoA, leads to an increase in histone crotonylation through the direct production of crotonyl-CoA. However, in plant the relationship of acetylation and crotonylation and the effect of the concentration of acetyl-CoA on protein crotonylation were not well known. Our previous study showed that PhACL silencing changed the content of acetyl CoA in petunia corollas. In this study, we performed a global crotonylation proteome analysis of petunia (Petunia hybrida) and found that protein crotonylation have close relationship with protein acetylation and the protein with more crotonylation sites often had more acetylation sites. Crotonylated proteins and acetylated proteins enriched in many common KEGG pathway. However, PhACL silencing resulted in different KEGG pathway enrichment of proteins with different level of crotonylated sites and acetylated sites. Cytoplasm non-histone lysine crotonylation may not depend on the concentrations of acetyl-CoA in petunia corollas. There was a positive correlation between crotonylome and acetylome expression levels in corollas of PhACL-silenced plants and control.

2022-10-12 | PXD023270 | Pride

Label-free quantitative crotonylproteomics of mouse ovaries-Chronic resistant stress (CRS) and control group, two repeats each

Project description:We found that in Chronic Resistant Stress (CRS)-treated mice, the fertility and oocyte quality significantly decreased, and plenty of biochemical indexes significantly changed, so we guessed that the overall physical health of CRS mice was severely impacted. As one major strategy to investigate the mechanism how CRS impact the physical health, We chose label-free quantitative ovarian crotonylproteomics service from Hangzhou PTM Bio Co. We sent two repeats of two-month-old CRS CD1/ICR mouse ovaries and two repeats of control ovaries for characterization of differentially crotonylated proteins. Each repeat contained about 60 ovaries from 30 female mice, about 300 mg. Gross protein levels were also examined and used to normalize the protein crotonylation level.

2024-05-22 | PXD028961 | Pride

The Role of CDYL-Regulated Crotonylation of RPA1 in Homologous Recombination-Mediated DNA Repair

Project description:Lysine crotonylation (Kcr) is a recently-identified protein short-chain acylation. We have previously reported that chromodomain Y-like transcription corepressor CDYL acts as a crotonyl-CoA hydratase and negatively regulates histone Kcr. However, the global crotonylome of CDYL-regulated Kcr on non-histone substrates remains unclear. Using proteome-wide quantitative Kcr analysis, we identified 14,311 Kcr sites across 3,734 proteins in HeLa cells, providing by far the largest crotonylome data set from a single study. Upon depletion of CDYL, 1,141 Kcr sites from 759 proteins were increased by more than 1.5 fold, and 933 Kcr sites from 528 proteins were decreased by more than 0.67 fold. Upregulated crotonylome alterations upon CDYL depletion include components from diverse cellular pathways such as RNA splicing, DNA replication, and amino acid metabolism. Specifically, CDYL regulates K88 and K379 of crotonylation on RPA1, which affects its binding to other DNA repair factors including BLM, DNA2L, RAD50 and WRN. We showed evidence that CDYL-mediated RPA1 crotonylation is critical for the homologous recombination (HR) repair of camptothecin (CPT)-induced DNA damage. Together, our results provide a broad lysine crotonylome in response to CDYL and shed new light on the role of RPA1 Kcr in DNA repair, implicating functional importance of Kcr on non-histone substrates in diverse cellular processes.

2020-04-02 | PXD013616 | Pride

Global Profiling of the Lysine Crotonylome in Different Pluripotent States

Project description:Understanding the mechanisms of pluripotency maintenance and exit are important for developmental biology and regenerative medicine. However, studies of pluripotency and post-translational modifications of proteins are scarce. To systematically profile protein crotonylation in mouse pluripotent stem cells (PSCs) in different culture conditions, we used affinity purification of crotonylated peptides, TMT labeling, and LC-MS/MS. Our study included PSCs in ground, metastable, and primed state, as well as metastable PSCs undergoing early pluripotency exit. We have successfully identified 8,102 crotonylation sites in 2,578 proteins, among which 3,628 sites in 1,426 proteins were with high-confidence. These high-confidence crotonylated proteins are enriched for factors involved in functions related to pluripotency such as RNA biogenesis, central carbon metabolism, and proteasome function. Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of crotonylation in other cell fate transitions.

2021-02-26 | PXD017121 | Pride

A proteomic analysis of lysine crotonylation in systemic lupus erythematosus patients

Project description:Posttranslational modification (PTM) refers to the process of the covalent processing of translated proteins, as well as the activities and functions of proteins, and it is involved in almost all cellular pathways and processes. Lysine crotonylation (Kcr) is a recently identified PTM and plays a key role in regulating various biological processes. In the present study, we performed a crotonylation proteome analysis in both Systemic lupus erythematosus(SLE) and normal control (NC) subjects using high resolution LC-MS/MS. We identified a total of 1109 lysine modification sites distributed on 347 proteins, including 417 crotonylated sites that were upregulated and 275 that were downregulated. By intensive bioinformatic analysis, our data proved that subcellular locations of crotonylated proteins include the cytoplasm, mitochondria, nucleus and extracellular regions. Kcr was related to multiple metabolism pathways, such as the focal adhesion, PI3K-Akt signaling, salmonella infection, cell cycle, hypertrophic cardiomyopathy (HCM), neurotrophin signaling, natural killer cell-mediated cytotoxicity, malaria, Hippo signaling and legionellosis pathways, while downregulated Kcr proteins were related to carbon metabolism, biosynthesis of amino acids, glutathione metabolism, complement and coagulation cascades and the pentose phosphate pathway. Several Kcr proteins related to focal adhesion have also been discussed. Enrichment analysis using Gene Ontology (GO) revealed that the Kcr proteins were enriched in the platelet alpha granule lumen, actin filament binding and platelet degranulation classes. Protein domain enrichment analysis revealed that the 14-3-3, immunoglobulin-like fold, EF-hand and Ca-insensitive domains were enriched in Kcr proteins.

2023-03-10 | PXD012966 | Pride

Qualitative Proteome-wide Analysis Reveal the Diverse Functions of Lysine Crotonylation in Dendrobium huoshanense C. Z. Tang et S. J. Cheng

Project description:Lysine crotonylation of histone proteins is a newly identified post-translational modification with diversified cellular functions. However, there’s few reports on lysine crotonylation of non-histone proteins in fruit plant cells. By using high-resolution LC-MS coupled with highly sensitive specific immune-affinity antibody analysis, a whole crotonylation proteome analysis of Dendrobium huoshanese was performed. In total, 1,591 proteins with 4,725 lysine crotonylation sites were found, among them eleven conserved motifs have been identified. Bioinformatic analysis linked crotonylated proteins to multiple metabolic pathways, including secondary metabolites biosynthesis, transport and catabolism; energy production and conversion; carbohydrate transport and metabolism; and translation, ribosomal structure and biogenesis.

2021-11-29 | PXD028293 | Pride

Global Proteomic Analysis of Lysine crotonylation in The Plant Pathogen Botrytis cinerea

Project description:Lysine crotonylation (Kcr), a recently discovered post-translational modification, plays a key role in the regulation of diverse cellular processes. Botrytis cinerea is a destructive necrotrophic fungal pathogen distributed worldwide with broad ranging hosts. However, the functions of Kcr are unknown in B. cinerea or any other plant fungal pathogens. Here, we comprehensively evaluated the crotonylation proteome of B. cinerea and identified 3967 Kcr sites in 1041 proteins, of which contained in 9 types of modification motifs. Our results show that although the crotonylation were largely conserved, different organisms contained distinct crotonylated proteins with unique functions. Bioinformatic analysis demonstrated that the majority of crotonylated proteins were distributed in cytoplasm (35%), mitochondria (26%) and nucleus (22%). The identified proteins were found to be involved in various metabolic and cellular processes, such as cytoplasmic translation and structural constituent of ribosome. Particularly, 26 crotonylated proteins participated in the pathogenicity of B. cinerea, suggesting a significant role for Kcr in this process. Protein interaction network analysis demonstrated that a mass of protein interactions are regulated by crotonylation. These data represent the ﬁrst report of the crotonylome of B. cinerea and provide a good foundation for further explorations of the role of Kcr in plant fungal pathogens.

2020-09-07 | PXD019358 | Pride

Global involvement of lysine crotonylation in protein modification and transcription regulation in rice

Project description:Lysine crotonylation (Kcr) is a newly discovered post-translational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice (Oryza sativa L. japonica) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome, and might mark other cis-regulatory DNA elements which are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants.

2018-09-20 | PXD010376 | Pride