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• W4312085004 abstract "•Global lysine crotonylation and autophagy levels are enhanced by leucine deprivation•Crotonylome profiling identifies many proteins regulated by leucine deprivation•14-3-3ε crotonylation affects its binding to PPM1B, which is a phosphatase for ULK1 Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy. Lysine crotonylation as a protein post-translational modification regulates diverse cellular processes and functions. However, the role of crotonylation in nutrient signaling pathways remains unclear. Here, we find a positive correlation between global crotonylation levels and leucine-deprivation-induced autophagy. Crotonylome profiling identifies many crotonylated proteins regulated by leucine deprivation. Bioinformatics analysis dominates 14-3-3 proteins in leucine-mediated crotonylome. Expression of 14-3-3ε crotonylation-deficient mutant significantly inhibits leucine-deprivation-induced autophagy. Molecular dynamics analysis shows that crotonylation increases molecular instability and disrupts the 14-3-3ε amphipathic pocket through which 14-3-3ε interacts with binding partners. Leucine-deprivation-induced 14-3-3ε crotonylation leads to the release of protein phosphatase 1B (PPM1B) from 14-3-3ε interaction. Active PPM1B dephosphorylates ULK1 and subsequently initiates autophagy. We further find that 14-3-3ε crotonylation is regulated by HDAC7. Taken together, our findings demonstrate that the 14-3-3ε-PPM1B axis regulated by crotonylation may play a vital role in leucine-deprivation-induced autophagy. 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Rajagopal N. et al.Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028https://doi.org/10.1016/j.cell.2011.08.008Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar Lysine crotonylation was initially identified and verified as a novel PTM in histone26Tan M. Luo H. Lee S. Jin F. Yang J.S. Montellier E. Buchou T. Cheng Z. Rousseaux S. Rajagopal N. et al.Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification.Cell. 2011; 146: 1016-1028https://doi.org/10.1016/j.cell.2011.08.008Abstract Full Text Full Text PDF PubMed Scopus (1140) Google Scholar and plays a vital role in epigenetically marking sex chromosomes in the post-meiotic stages of spermatogenesis. Histone crotonylation catalyzed by the histone acetyltransferase p300/CBP has been shown to directly stimulate transcriptional process.27Sabari B.R. Tang Z. Huang H. Yong-Gonzalez V. Molina H. Kong H.E. Dai L. Shimada M. Cross J.R. Zhao Y. et al.Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation.Mol. Cell. 2015; 58: 203-215https://doi.org/10.1016/j.molcel.2015.02.029Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar In addition to “writers” (p300/CBP), “erasers” for histone crotonylation were identified to be class Ⅰ histone deacetylases (HDACs) in mammalian cells.28Wei W. Liu X. Chen J. Gao S. Lu L. Zhang H. Ding G. Wang Z. Chen Z. Shi T. et al.Class I histone deacetylases are major histone decrotonylases: evidence for critical and broad function of histone crotonylation in transcription.Cell Res. 2017; 27: 898-915https://doi.org/10.1038/cr.2017.68Crossref PubMed Scopus (134) Google Scholar Furthermore, the YEATS domain was demonstrated to be the “reader” of histone crotonylation.29Zhao D. Guan H. Zhao S. Mi W. Wen H. Li Y. Zhao Y. Allis C.D. Shi X. Li H. YEATS2 is a selective histone crotonylation reader.Cell Res. 2016; 26: 629-632https://doi.org/10.1038/cr.2016.49Crossref PubMed Scopus (113) Google Scholar,30Zhang Q. Zeng L. Zhao C. Ju Y. Konuma T. Zhou M.-M. Structural insights into histone crotonyl-lysine recognition by the AF9 YEATS domain.Structure. 2016; 24: 1606-1612https://doi.org/10.1016/j.str.2016.05.023Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar Lysine crotonylation exists not only on histone proteins but also non-histone proteins.31Xu W. Wan J. Zhan J. Li X. He H. Shi Z. Zhang H. Global profiling of crotonylation on non-histone proteins.Cell Res. 2017; 27: 946-949https://doi.org/10.1038/cr.2017.60Crossref PubMed Scopus (75) Google Scholar,32Wu Q. Li W. Wang C. Fan P. Cao L. Wu Z. Wang F. Ultradeep lysine crotonylome reveals the crotonylation enhancement on both histones and nonhistone proteins by SAHA treatment.J. Proteome Res. 2017; 16: 3664-3671https://doi.org/10.1021/acs.jproteome.7b00380Crossref PubMed Scopus (39) Google Scholar,33Wei W. Mao A. Tang B. Zeng Q. Gao S. Liu X. Lu L. Li W. Du J.X. Li J. et al.Large-scale identification of protein crotonylation reveals its role in multiple cellular functions.J. Proteome Res. 2017; 16: 1743-1752https://doi.org/10.1021/acs.jproteome.7b00012Crossref PubMed Scopus (72) Google Scholar Although many crotonylated non-histone proteins have been identified, their functional roles remain largely unknown. Here, the link between autophagy and lysine crotonylation was found. Lysine crotonylation correlated well with leucine-mediated autophagy both in vitro and in vivo. Quantitative crotonylomic profiling identified a series of differentially crotonylated proteins and sites regulated by leucine deprivation. Crotonylation 14-3-3ε, K73 and K78, regulated leucine-deprivation-induced autophagy. We further revealed the mechanism by which 14-3-3ε crotonylation controls leucine-mediated autophagy. To investigate the underlying link between autophagy and PTMs, we assessed the autophagy and global PTMs levels in AML12 cells. Leucine starvation and rapamycin increased the abundance of LC3-Ⅱ, the well-established maker for autophagosome, and decreased the p62 level, an autophagic substrate (Figure 1A ). Impressively, we found that lysine crotonylation levels were increased significantly upon leucine deprivation (Figure 1A), but the levels of lysine acetylation, lysine succinylation, and ubiquitination showed no change or a limited change (Figure S1A). Consistently, immunofluorescence using pan-lysine crotonylation (pan-Kcr) antibody displayed stronger signals after leucine deprivation for 2 h both in AML12 (Figures 1H and 1I) and HEK293T cells (Figures S1B and S1C), which suggests that the enhanced crotonylation levels coupled with increased autophagy were not limited in hepatocytes. We found more cytosolic immunostaining by pan-Kcr antibody in AML12 cells compared with HEK293T cells, suggesting different distributions of crotonylated proteins in different types of cell lines (Figures 1H and S1B). To evaluate the relationship of protein crotonylation and leucine-deprivation-induced autophagy in vivo, 8-week-old C57BL/6J mice were fed with amino acid-completed diets (+Leu) or leucine-free diets (−Leu) for 7 days. The crotonylation and LC3-Ⅱ levels increased in the liver of mice fed with −Leu diets (Figure 1B), consistent with more autophagic vehicles observed in the liver by transmission electron microscopy (TEM) (Figures 1C and 1D). Immunohistochemical results confirmed the enhanced crotonylation levels in the liver of mice fed with −Leu diets (Figures 1E and 1F). Furthermore, we observed more autophagic vehicles in the liver of mice with overnight starvation compared with the control group (Figures S1D and S1E). Immunohistochemical assay showed increased crotonylation in the liver of mice starved for 12 h (Figures S1F and S1G). Thus, these results demonstrated that nutrient limitation (starvation or Leu deprivation) could induce autophagy and upregulate global crotonylation levels. Histone and non-histone protein crotonylation could be enhanced by sodium crotonate (NaCr; pH 7.4) in cells such as HeLa, PC3, and LNCap.27Sabari B.R. Tang Z. Huang H. Yong-Gonzalez V. Molina H. Kong H.E. Dai L. Shimada M. Cross J.R. Zhao Y. et al.Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation.Mol. Cell. 2015; 58: 203-215https://doi.org/10.1016/j.molcel.2015.02.029Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar,33Wei W. Mao A. Tang B. Zeng Q. Gao S. Liu X. Lu L. Li W. Du J.X. Li J. et al.Large-scale identification of protein crotonylation reveals its role in multiple cellular functions.J. Proteome Res. 2017; 16: 1743-1752https://doi.org/10.1021/acs.jproteome.7b00012Crossref PubMed Scopus (72) Google Scholar Consistently, NaCr treatment for 12 h enhanced global crotonylation levels in AML12 cells (Figure 1G). Immunofluorescence using pan-Kcr antibody confirmed that NaCr can be used as a crotonylation activator in AML12 (Figures 1H and 1I) and HEK293T cells (Figures S1B and S1C). To further explore the relationship between crotonylation modification and autophagy, we measured the autophagy level of AML12 cells culturing in medium with NaCr treatment. Interestingly, we found that autophagy was induced by NaCr in a concentration-dependent manner (Figure 1J). The average number of GFP-LC3 puncta per cell, which represents the magnitude of autophagy activity, was significantly augmented in GFP-LC3 stable HeLa cells (Figures 1K and 1L). Autophagic flux was increased significantly by NaCr treatment (Figure S1H). These results thus suggested that lysine crotonylation correlated with the autophagy level both in vitro and in vivo. To identify the crotonylated proteins and sites regulated by Leu deprivation, crotonylome profiling was conducted in the cells treated with or without Leu. The protein extracts were prepared and digested with trypsin, and the resulting peptides were labeled and then fractionated through basic reversed-phase high-performance liquid chromatography (HPLC), followed by antibody affinity enrichment from each fraction. The eluted peptides were analyzed by high-resolution tandem mass spectrometry (MS/MS) (Figure 2A ). Global proteomics data were also collected for normalization. We totally identified 1,898 crotonylated sites on 654 proteins, in which 1,501 sites on 536 proteins were quantifiable (n = 3) (Table S1; Figure S2A). The distribution of most of the peptide length is from 7 to 19 aa, which was consistent with the length of tryptic peptides (Figure 2B). About 46.94% (307/654) proteins contained one crotonylation site, 19.88% (130/654) contained two sites, and 10.40% (68/654) contained three sites (Figure 2C). Relative standard deviation (RSD) (Figure 2D) and Pearson correlation coefficient (Figure S2B) indicated that these data have high repeatability. To explore the evolutionary conservation of this kind of modification, we searched the orthologs of crotonylated proteins in Mus musculus (this study) against the following previously reported crotonylomes: Homo sapiens,32Wu Q. Li W. Wang C. Fan P. Cao L. Wu Z. Wang F. Ultradeep lysine crotonylome reveals the crotonylation enhancement on both histones and nonhistone proteins by SAHA treatment.J. Proteome Res. 2017; 16: 3664-3671https://doi.org/10.1021/acs.jproteome.7b00380Crossref PubMed Scopus (39) Google Scholar Danio rerio,34Kwon O.K. Kim S.J. Lee S. First profiling of lysine crotonylation of myofilament proteins and ribosomal proteins in zebrafish embryos.Sci. Rep. 2018; 8: 3652https://doi.org/10.1038/s41598-018-22069-3Crossref PubMed Scopus (32) Google Scholar Carica papaya,35Liu K. Yuan C. Li H. Chen K. Lu L. Shen C. Zheng X. A qualitative proteome-wide lysine crotonylation profiling of papaya (Carica papaya L.).Sci. Rep. 2018; 8: 8230https://doi.org/10.1038/s41598-018-26676-yCrossref PubMed Scopus (39) Google Scholar and Nicotiana tabacum.36Sun H. Liu X. Li F. Li W. Zhang J. Xiao Z. Shen L. Li Y. Wang F. Yang J. First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum.Sci. Rep. 2017; 7: 3013https://doi.org/10.1038/s41598-017-03369-6Crossref PubMed Scopus (47) Google Scholar The lysine-conserved percentage in these four orthologs was determined for uncrotonylated and crotonylated Mus musculus lysine residues, respectively (Figures 2E and S2C). The calculated p values in Homo sapiens, Danio rerio, Carica papaya, and Nicotiana tabacum were 2.58 × 10−4, 3.32 × 10−4, 9.61 × 10−9, and 1.64 × 10−3, respectively, indicating that lysine crotonylation was an evolutionarily conserved modification among these species. To evaluate the sequence preferences of crotonylation, we analyzed the surrounding sequences (−10 to +10 amino acids) of crotonylated lysine. We found that the negatively charged amino acids aspartic acid (D) and glutamic acid (E) were drastically enriched at positions +1 and −1, which displayed high similarity to a previous study,31Xu W. Wan J. Zhan J. Li X. He H. Shi Z. Zhang H. Global profiling of crotonylation on non-histone proteins.Cell Res. 2017; 27: 946-949https://doi.org/10.1038/cr.2017.60Crossref PubMed Scopus (75) Google Scholar indicating surrounding negatively charged amino acids might facilitate lysine crotonylation (Figure 2F). In addition, alanine (A) and lysine (K) were preferred at positions −1 and +1, respectively, and cysteine (C), proline (P), and serine (S) seemed to be negatively enriched at the surrounding position. Further motif analysis using Motif-x37Chou M.F. Schwartz D. Biological sequence motif discovery using motif-x. Curr Protoc Bioinformatics.Curr. Protoc. Bioinformatics. 2011; Chapter 13:Unit 13.15-24https://doi.org/10.1002/0471250953.bi1315s35Crossref Scopus (294) Google Scholar showed that several motifs were significantly enriched, including KXXXEKcr, KKcr, KcrE, and KcrD (Figure S2D). Principal-component analysis (PCA) analysis indicated that the modification patterns were different between +Leu and −Leu groups (Figure 2G). The −Leu group possessed less low-modified peptides, more high-modified peptides, and higher average modification intensity compared with +Leu group (Figure 2H). These results suggest that Leu deprivation can significantly change the lysine crotonylome. To screen the differentially crotonylated sites regulated by Leu, the modification sites with a quantification ratio (−Leu/+Leu) >1.5 or <0.67 and a p <0.05 were identified as significantly up- or down-regulated sites, respectively. Among the dataset, 298 Kcr sites on 189 proteins were up-regulated, 28 Kcr sites on 19 proteins were down-regulated (Figure 3A ; Table S2), and the heatmap results are shown in Figure 3B. Further domain analysis suggested that a serial of enriched conserved functional domains were enriched, including the 14-3-3 domain, the aminoacyl-tRNA class Ⅱ(G/P/S/T) domain, the ubiquitin-related domain, and other domains (Figure 3C). 14-3-3 family proteins consist of seven isoforms (α/β, γ, ε, η, σ, ζ/δ, and τ/θ) encoded by seven distinct genes (YWHAB, YWHAG, YWHAE, YWHAH, SFN, YWHAZ, and YWHAQ, respectively) in mammalian cells and modulated a wide range of cellular function via protein-protein interactions (PPIs).38Stevers L.M. Sijbesma E. Botta M. MacKintosh C. Obsil T. Landrieu I. Cau Y. Wilson A.J. Karawajczyk A. Eickhoff J. et al.Modulators of 14-3-3 protein–protein interactions.J. Med. Chem. 2018; 61: 3755-3778https://doi.org/10.1021/acs.jmedchem.7b00574Crossref PubMed Scopus (135) Google Scholar,39Aghazadeh Y. Papadopoulos V. The role of the 14-3-3 protein family in health, disease, and drug development.Drug Discov. Today. 2016; 21: 278-287https://doi.org/10.1016/j.drudis.2015.09.012Crossref PubMed Scopus (156) Google Scholar,40Falcicchio M. Ward J.A. Macip S. Doveston R.G. Regulation of p53 by the 14-3-3 protein interaction network: new opportunities for drug discovery in cancer.Cell Death Discov. 2020; 6: 126https://doi.org/10.1038/s41420-020-00362-3Crossref PubMed Scopus (23) Google Scholar In total, we found 29 crotonylated sites in 6 kinds of 14-3-3 isoforms, indicating that 14-3-3 proteins were widely crotonylated. Among them, 7 crotonylated sites were up-regulated by Leu deprivation (Figure 3D). The ratios (−Leu/+Leu) of two lysine crotonylation sites of 14-3-3ε, K73 and K78, are 10.684 (p = 1.42 × 10−3) and 5.475 (p = 3.13 × 10−7), respectively. Mass spectrogram confirmed that K73 (Figure 3E) and K78 (Figure 3F) were crotonylated. Consistently, we found that the crotonylation level of 14-3-3ε was upregulated after Leu deprivation either using anti-14-3-3ε (Figure 3G) or pan-Kcr (Figure 3H) in immunoprecipitation assays. Together, these observations suggest that 14-3-3ε crotonylation may have a functional role in Leu-deprivation-mediated autophagy. We then evaluated the effect of 14-3-3ε K73 and K78 crotonylation on Leu-deprivation-induced autophagy. These two crotonylated sites, K73 and K78, were evolutionarily conservative (Figure 4A ). We converted the modified residues at these two lysine crotonylation sites to arginine (K73R and K78R), which locked these two sites into a constitutively decrotonylated state. Two mutants significantly decreased the crotonylated level of 14-3-3ε both under +Leu and −Leu conditions, and K73R led to a greater decrease than K78R (Figure 4B). The crotonylation level was barely increased when the two sites were both mutated under Leu-deprivation conditions (Figure 4B). In addition, the subcellular localization and protein stability were not changed by K73R and/or K78R (Figures S3A and S3B). To evaluate the effect of 14-3-3ε itself on autophagy, we knocked down 14-3-3ε expression, which led to increased LC3-Ⅱ expression and decreased p62 expression, under conditions with or without Leu (Figure 4C). Overexpression of FLAG-tagged wild-type (WT) 14-3-3ε decreased LC3-Ⅱ levels (Figure 4E, lanes 2 and 3), which suggested that 14-3-3ε was a repressor for Leu-deprivation-induced autophagy. Next, we constructed and overexpressed the crotonylation-deficient mutants. The autophagy level was decreased dramatically when overexpressing mutants (Figures 4D and 4E). Furthermore, we expressed FLAG-14-3-3ε for WT or mutant in GFP-LC3-expressing cells and found that K73R/K78R mutation significantly inhibited LC3 puncta formation (Figures 4F and 4G). Together, these data demonstrate that Leu-deprivation-induced autophagy is regulated by crotonylation of 14-3-3ε. To directly determine whether lysine crotonylation affect the structure of 14-3-3ε, we performed molecular dynamics (MD) simulations using GROMACS.41Pronk S. Páll S. Schulz R. Larsson P. Bjelkmar P. Apostolov R. Shirts M.R. Smith J.C. Kasson P.M. van der Spoel D. et al.Gromacs 4.5: a high-throughput and highly parallel open source molecular simulation toolkit.Bioinformatics. 2013; 29: 845-854https://doi.org/10.1093/bioinformatics/btt055Crossref Pu" @default.
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• W4312085004 date "2022-12-01" @default.
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• W4312085004 title "Lysine crotonylation regulates leucine-deprivation-induced autophagy by a 14-3-3ε-PPM1B axis" @default.
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