Quick facts about this article:
Protein post-translational modifications regulate protein function and are of great significance for biology and disease research. Researchers have been exploring the field of protein post-translational modifications, including phosphorylation, ubiquitination, acetylation, and other types. In previous tweets, Xiaoyuan has introduced you to some common types of protein post-translational modifications, including phosphorylation and ubiquitination. If you are interested in these types of modifications, you may wish to click here for more information!"Protein Post-Translational Modifications - Ubiquitination, Protein Post-Translational Modifications - Ubiquitination (II), Protein Post-Translational Modifications - Phosphorylation (1), Protein Post-Translational Modifications - Phosphorylation (II)". Today, Xiaoyuan will bring you another high-profile "guest" - acetylation modification. Let's follow in Xiaoyuan's footsteps and explore the mystery of acetylation together!
1. Background of protein acetylation modification
1. Acetylation
Protein acetylation is a type of post-translational modification that is widespread in both eukaryotes and prokaryotes, and it is also the most common type of acylation modification. Acetylation refers to the process by which acetyl groups are transferred and added to the N-terminus or lysine residues of proteins catalyzed by acetyltransferases or abiotic catalysts, which plays an important role in regulating protein function.
Most of the N-terminal acetylation modifications of proteins occur in eukaryotes and are catalyzed by N-terminal acetyltransferases (NATS), and no N-terminal deacetylase has been found, so it is considered that N-terminal acetylation is irreversible (Wang Xuchu et al., 2022). KATs) and lysine deacetylases (KDACs) are a reversible process.
Fig.1 Schematic diagram of the reversible reaction of lysine acetylation (Wang et al., 2020).
2. N-terminal acetyltransferase
N-terminal acetyltransferase belongs to the GNAT (Gcn5-related N-acetyltransferase) protein family, which can be divided into multiple isoforms such as NatA, NatB, NatC, NatD, NatE, NatF and NatG according to their substrate properties and subunit composition, among which NatA, NatB and NatC mainly act on the N-terminus of the protein, usually composed of a catalytic subunit and one or two auxiliary subunits.
图2 真核生物中不同类型的NATs(Aksnes et al., 2016)。
3. Lysine acetyltransferase
Lysine acetyltransferases can be divided into three protein families: the GNAT family, which includes KAT2A and KAT2B, the MYST family, which includes MOZ, Ybf2/Sas3, Sas2, and Tip60, and the CBP/p300 family. KAT2A and KAT2B are highly homologous acetyltransferases in the GNAT family, and they have been shown to repel each other in chromatin modification complexes (Nagy et al., 2010; Foumier et al., 2016)。 As the most studied KAT in the MYST family, Tip60 plays an important role not only in transcriptional regulation, but also in DNA damage repair (Zhang et al., 2015). CBP/p300 acetyltransferase is thought to be a transcriptional coactivator that plays an important role in transcriptional regulation (Robert G. Roeder, 2019).
4. Lysine deacetylase
Lysine deacetylase can be divided into two families according to the different cofactors that exert their functions: a family of Zn2+-dependent proteins for deacetylation and a NAD+-dependent family for deacetylation (Lü et al., 2015). The Zn2+-dependent protein family is similar in secondary structure to the Hda1/Rpd3 protein, which exhibits only deacetylase activity, while the NAD+-dependent protein family is similar to the Sir2 protein, which exhibits ADP ribosyltransferase activity in addition to lysine deacetylase (Barneda-Zahonero and Parra, 2012).
2. Protein acetylation detection method
Acetylation detection is an important technical means to identify protein acetylation, acetylation sites and acetylation levels, and the commonly used methods include mass spectrometry, immunoblotting and chromatin immunoprecipitation detection.
1. Mass spectrometry detection method
Analysis of protein samples using mass spectrometry allows for the identification of acetylated proteins and acetylation sites. This method has high sensitivity and specificity, and enables comprehensive analysis of acetylation modifications, but it is relatively difficult to quantify acetylation levels. The general process can be divided into five steps: total protein extraction, proteolysis, acetylated peptide enrichment, mass spectrometry and data analysis.
In September 2022, Wenwu Guo's research group at Huazhong Agricultural University published a research paper titled "Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus" in the journal Plant Physiology. The authors used mass spectrometry detection techniques to analyze citrus heterotetraploid hybrids and their biparent pulp tissues. A large number of non-histone proteins were identified as acetylated modifications, with more than 4000 acetylation sites for more than 1600 proteins identified.
Fig.3 Characterization of lysine acetylase in citrus fruit (Zhang et al., 2022). (A) Citrus pulp (juice sac) for acetylation analysis, scale bar = 1 cm, (B) Western blot analysis of lysine acetylated protein in fruit pulp with anti-acetyl-lysine (anti-KAc) antibody (left), Coomassie brilliant blue staining as control (right) ;(C) lysine acetylation (KAc) sites and acetylated protein number in 5-week-old leaves of citrus and Arabidopsis thaliana and 14-day-old rice seedlings. Data from Hartl et al., 2017 and Zhou et al., 2018, respectively, were obtained from Arabidopsis thaliana and rice. (D) Comparison of acetylation sites for each acetylated protein between citrus and Arabidopsis thaliana and rice, (E) Comparison of acetylation sites for each acetylated protein in different metabolic pathways of citrus and Arabidopsis, (F) predicted subcellular localization distribution of proteins containing acetylated lysine residues in citrus fruits, (G) Venn plots showing overlap between homologous acetylated proteins from citrus, Arabidopsis thaliana, and rice, and (H) KEGG enrichment analysis of citrus-specific acetylated proteins.
2. Immunoimprinting
Western blot (WB), also known as protein immunotechnology, uses the principle of specific binding of antigens and antibodies to detect acetylated proteins, first separates the protein samples by polyacrylamide gel electrophoresis, and then transfers the separated products to a solid support to bind to the primary antibody, and then reacts with a specific labeled secondary antibody for detection by autoradiography and other methods.
In November 2023, Wenxing Liang's group at Qingdao Agricultural University published a research paper titled "Sir2-mediated cytoplasmic deacetylation facilitates pathogenic fungi infection in host plants" in the journal New Phytologist. In this study, we identified a cytoplasmic deacetylase FolSir2 in Fusarium oxysporum f. sp. lycopersici (Fol) by subcellular localization and acetylation modification immunoblotting, and found that the pathogenicity of the knockout body ΔFolSir2 was significantly reduced.
Fig.4 FolSir2 is essential for the deacetylation and pathogenicity of Fol fungi (Zhang et al., 2023). (a) Phylogenetic tree was established between FolSir2 and homologous human Sirtuin isoforms (SIRT1-7); Western blot analysis of the knockout body ΔFolSir2 and the complement strain ΔFolSir2-C lysine acetylation, Coomassie brilliant blue staining as a control to ensure equal protein load on each lane, triplicate experiments were performed for each gel presented, (c) western blot analysis of histone H3 and H4 acetylation, and (d) assessment of the pathogenicity of the Fol strain on tomato 14 days after infection.
3. Chromatin immunoprecipitation detection method
Chromatin immunoprecipitation (ChIP) is a method for studying the interaction of DNA and proteins in vivo. Specific antibodies can be used to selectively enrich specific DNA-binding proteins and their target sites, which can be used to study histone modifications and/or protein binding at known target sites in the genome. The whole experimental process can be roughly divided into cross-linking, chromatin fragmentation, immunoprecipitation, de-cross-linking, DNA extraction, and data analysis.
In May 2022, Junbin Huang's research group at Huazhong Agricultural University published a research paper titled "A secreted fungal effector suppresses rice immunity through host histone hypoacetylation" in the journal Nucleic Acids Research. This study revealed a novel mechanism of Aspergillus oryzae effector protein manipulation of rice histone deacetylation and inhibition of host immunity at the epigenetic level. In our study, the authors used ChIP-seq to identify and found that the number of target genes regulated by H3K9 acetylation in 35S-UvSec117 transgenic rice was significantly reduced, and the expression of disease-related genes regulated by H3K9 acetylation in 35S-UvSec117 transgenic rice was significantly reduced by RT-qPCR and ChIP-qPCR.
Fig.5 Aspergillus oryzae effector protein UvSec117 manipulates rice histone deacetylase OsHDA701 to inhibit rice immunity (Chen et al., 2022). (a) Left: Resistance determination of 35S-EV and 35S-UvSec117 transgenic lines 25 days after inoculation with B. oryzae HWD-2. Right: Average number of blast bacteria measured in resistance assays, (b) Left: Disease symptoms on leaves 11 days after infection with blast strains of 35S-EV and 35S-UvSec117 transgenic lines. (c) Left: Leaf symptoms and length of 35S-EV and 35S-UvSec117 transgenic lines inoculated with P. alba P. alba PXO99A 21 days after inoculation (right);(d)Histone H3K9ac, H3K27ac, H3K36ac, H3K56ac, Expression levels of H4K5ac, H4K8ac, H4K12ac, and H4K16ac, (e) H3K9ac peak identified in transcription start site (TSS), statistical overlap distribution of reads in the DNA region 2 kb upstream of TSS and 2 kb downstream of transcription termination site (TES), and (f) for inoculation with B. oryzae HWD-2 RT-qPCR analysis of defense-related genes in 35S-EV and 35S-UvSec117 transgenic lines 1 day later, and (g) ChIP-qPCR analysis of defense-related genes in 35S-EV and 35S-UvSec117 transgenic lines 1 day after inoculation with the pathogen.
As an important protein modification modality, protein acetylation plays a key role in cell function and signal transduction. In order to study the function and mechanism of protein acetylation more comprehensively, we can use a variety of methods to investigate. In the above method introduction, Xiaoyuan only highlights the techniques to be described, but throughout the article, the author will use a variety of inquiry methods at the same time, and interested partners can learn on their own.
The above three methods are widely used at present, but Xiaoyuan also heard that the fields of high-throughput proteomics, chemical biology, and bioinformatics are constantly developing, which is expected to provide great help for the analysis of protein acetylation modifications, and the expectations have been full.
3. Function of protein acetylation modification in plants
Protein acetylation modifications are involved in key processes related to the physiology and disease of organisms, such as gene transcription, DNA damage repair, cell division, signal transduction, protein folding, autophagy, and metabolism. What is the function of protein acetylation at the plant level?
1. Participate in plant immune response
病原菌效应蛋白与植物之间的相互作用机制一直是植物领域的研究热点，然而，对于它们如何激活植物免疫的机制仍存在一些待解的谜题。 2021年11月，韩国浦项科技大学Kee Hoon Soh课题组在Molecular Plant杂志上发表了一篇题为“Direct acetylation of a conserved threonine of RIN4 by the bacterial effector HopZ5 or AvrBsT activates RPM1-dependent immunity in Arabidopsis”的研究论文。 揭示了病原菌效应蛋白HopZ5和AvrBsT通过乙酰化修饰RIN4（RPM1-INTERACTING PROTEIN4）来激活植物免疫的新机制。
Previous studies have shown that Arabidopsis thaliana deacetylase SOBER1 can inhibit plant immune responses stimulated by the effector proteins HopZ5 and AvrBsT. In this study, the authors found that in the absence of SOBER1, the immune response activated by these two pathogen effector proteins is dependent on RIN4 and the intracellular anti-disease protein RPM1. The authors selected the Arabidopsis SOBER1 loss-of-function mutant sober1-3, abbreviated as sober1, to verify the dependence of acetyltransferase effector protein-triggered hypersensitivity reaction (HR). The growth of Pto DC3000 pathogens carrying the effector proteins HopZ5 or AvrBsT in the sober1 rpm1 and sober1 rps2 rin4 mutants is comparable to that of the unloaded Pto DC3000 strains, but differs from those in the sober1 rps2 mutants, suggesting that RPM1 and RIN4 are required for HopZ5 or AvrBsT-triggered immunity in Arabidopsis thaliana, rather than RPS2. In summary, the bacterial acetyltransferases HopZ5 and AvrBsT may target RIN4 and activate RPM1-dependent immunity.
Fig.6 The bacterial acetyltransferase effector proteins HopZ5 and AvrBsT trigger RPM1 and RIN4-dependent immunity in Arabidopsis thaliana (Choi et al., 2021).
Other studies have shown that two other effector proteins, AvrB and AvrRpm1, induce phosphorylation of RIN4 threonine at position 166 (T166) and activate the RPM1-dependent immune response via the cytosolic receptor kinase RIPK. Therefore, the authors further studied the interaction between HopZ5 and AvrBsT and RIN4.
Through co-immunoprecipitation experiments, the researchers found an interaction between HopZ5 and AvrBsT and RIN4 (Figure 7A). Interestingly, through in vitro reactions and mass spectrometry identification, the researchers found that HopZ5 and AvrBsT, which are acetylases, were able to acetylate the conserved site T166 of RIN4 (Fig. 7B, C). This acetylation modification is present not only in in vitro reactions, but also in plants and during pathogen infection (Fig. 7D, E). Eventually, acetylation modification at the RIN4 T166 site will activate an RPM1-dependent immune response, allowing plants to resist pathogenic bacteria and demonstrate disease resistance.
Fig. 7 HopZ5 and AvrBsT interact with RIN4 and acetylate RIN4 (Choi et al., 2021). (A) HopZ5 and AvrBsT interact with RIN4 regardless of their catalytic activity;(B) MS/MS profiles of acetylated RIN4 fragment peptide (FGDWDENPSSADGYTHIFNK) incubated with HopZ5-FLAG protein;(C) In vitro study of acetylated RIN4 by HopZ5 and AvrBsT;(D) Acetylated N. (E) In Arabidopsis thaliana, HopZ5 acetylates T166 RIN4.
The results of this study suggest that HopZ5 and AvrBsT activate an RPM1-dependent immune response by directly acetylating the RIN4 protein. This reveals a novel mechanism that regulates plant immune responses through acetylation modifications. The findings of this study are of great significance for understanding the interaction between plants and pathogenic bacteria, revealing the mechanism of immune signal transduction, and developing disease resistance technologies.
2. Participate in plant stress response
Protein acetylation plays an important regulatory role in plant stress stress, which helps plants adapt to the stress environment and improve their ability to resist stress by regulating the activity, stability and gene expression of proteins. In-depth study of the relationship between protein acetylation and plant stress will help us understand the mechanism of plant stress adaptation and provide a theoretical basis for cultivating stress-tolerant crops.
2022年1月，中国科学院分子植物科学卓越创新中心张蘅课题组在Stress Biology杂志上发表了一篇文章题为“Acetylproteomics analyses reveal critical features of lysine-ε-acetylation in Arabidopsis and a role of 14-3-3 protein acetylation in alkaline response”的研究论文，解析了拟南芥主要器官的蛋白质乙酰化，分析了植物赖氨酸乙酰化的关键特征，并揭示了14-3-3蛋白乙酰化在植物碱胁迫应答中的重要作用。
In this study, the authors used high-resolution tandem mass spectrometry to perform lysine acetylation analysis of five representative plant organs, including leaves, roots, stems, flowers, and seeds of Arabidopsis, and identified a total of 2887 Lysine-ε-acetylation (Kac) proteins and 5929 Kac sites. Evolutionary analysis has found that Kac preferentially targets evolutionarily conserved proteins and conserved lysine.
The authors observed that many Kac proteins are also regulated by other post-translational modifications (e.g., phosphorylation, ubiquitination, and SUMO)ation, and although acetylation, ubiquitination, and SUMO can modify lysine residues, they rarely occur at the same site. Proteins that are co-targeted by acetylation and phosphorylation are often rich in many bromodomains and 14-3-3 protein domains. Structural analysis revealed that lysine acetylation at position 56 (K56ac) at the binding site of the 14-3-3 protein binding phosphorylated substrate may block its binding to the phosphorylated substrate (Figure 8A). Previous studies have shown that phosphorylated recognition protein 14-3-3s is involved in the regulation of plasma membrane H+-ATPase AHA2 activity, and the interaction between 14-3-3s and AHA2 is regulated by the post-translational modification of the 14-3-3s protein itself.
Fig.8 Acetylation of Lys56 in GRF6 negatively regulates alkali tolerance in Arabidopsis thaliana (Guo et al., 2022). (A) The prediction model based on Cp14-3-3 shows the GRF6 structure. The phosphoserine (pSer) binding pocket containing 3 positively charged residues (K56, R63, and R136) is indicated in magnification (top), and the side chain (bottom) of the identified acetylated lysine is shown in red ;(B) immunoprecipitation of GRF6 protein and acetylation levels for western blot analysis, and (C) yeast assay shows that both AtGRF6 and AtAHA2 are required for yeast growth when glucose is the only carbon source. When galactose was used as the sole carbon source, yeast growth was independent of AtGRF6 and AHA2 (left;(D) methyl violet staining showed the effect of AtAHA2 and different AtGRF6 expressions on the pH of the culture medium, (E) quantification of pH changes in (D), (F) growth phenotypes of plant roots on normal pH 5.8 and high pH 8.2 plates, and (G) quantification of root length in (F).
To further explore the biological function of acetylation of 14-3-3 protein, the authors developed a yeast exogenous expression system for validation. In yeast with the deletion of endogenous 14-3-3 and H+-ATPase proteins, Arabidopsis AHA2 and 14-3-3 protein GRF6 were transferred to construct acetylated (K56Q) and deacetylated (K56R) mutants (Figure 8C). By detecting the pH change of yeast extracellular solution, the results showed that the acetylation of Lys56 in GRF6 had a negative regulatory effect on the activity of AHA2. Transgenic experiments in Arabidopsis thaliana also verified the negative regulatory effect of acetylation of GRF6 Lys56 in the response to alkaloid stress (Fig. 8D, E). In the context of grf6 and grf8 double mutations, transfer of GRF6 K56Q did not restore normal plant growth at high pH, and the acetylation level of GRF6 was reduced at high pH (Fig. 8F, G). Therefore, this study revealed a novel mechanism by which protein acetylation regulates the phytoalkal stress response by regulating the interaction of 14-3-3 protein with AHA2.
Xiaoyuan chattered
This article introduces the post-translational modification of acetylated proteins, first introduces the background knowledge of acetylation modification, then introduces three methods for identifying acetylation of proteins, and finally focuses on describing the biological functions of acetylation modification in plants. This article is the last article of the 2023 Year of the Lunar New Year, I would like to thank you all for your companionship and support in 2023, and I would like to take this opportunity to wish you all the best in the 2024 Jiachen Year!
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