Khib Sites Research Articles

Identification and Functional Analysis of Lysine 2-Hydroxyisobutyrylation in Cyanobacteria.

Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H2O2 resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

The lysine 2-hydroxyisobutyrylome of Helicobacter pylori: Indicating potential roles of lysine 2-hydroxyisobutyrylation in the bacterial metabolism

Helicobacter pylori (H. pylori) is a pathogen which colonizes the stomach, causing ulcers, chronic gastritis and other related diseases. Protein post-translational modifications (PTMs) in bacteria mainly include glycosylation, ubiquitination, nitrosylation, methylation, phosphorylation and acetylation, all of which have divergent functions in the physiology and pathology of the bacterium. Lysine 2-hydroxyisobutyrylation (Khib) is a newly discovered type of PTM in recent years in some kinds of organisms, and this PTM is involved in the regulation of a variety of metabolic process, such as bacterial glucose metabolism, lipid metabolism and protein synthesis. This study performed the first qualitative lysine 2-hydroxyisobutyrylome in H. pylori, and a total of 4419 Khib sites in 812 proteins were identified. The results show that Khib sites are mainly located in the key functional regions or active domains of proteins involved in nickel-trafficking, energy production, virulence factors, anti-oxidation, metal resistance, and ribosome biosynthesis in H. pylori. The study presented here provides new hints in the metabolism and pathology of H. pylori and the proteins with Khib modification may be potentially promising targets for the further development of antibiotics, especially considering the high occurrence of treatment failure of H. pylori failure due to development of antibiotics-resistance.

Analysis of proteome and post-translational modifications of 2-hydroxyisobutyrylation reveals the glycolysis pathway in oral adenoid cystic carcinoma

PurposeOral adenoid cystic carcinoma (OACC) has high rates of both local–regional recurrence and distant metastasis. The objective of this study is to investigate the impact of Khib on OACC and its potential as a targeted therapeutic intervention.Experimental designWe investigated the DEPs (differentially expressed proteins) and DHMPs between OACC-T and OACC-N using LC–MS/MS-based quantitative proteomics and using several bioinformatics methods, including GO enrichment analysis, KEGG pathway analysis, subcellular localization prediction, MEA (motif enrichment analysis), and PPI (protein–protein interaction networks) to illustrate how Khib modification interfere with OACC evolution.ResultsCompared OACC-tumor samples (OACC-T) with the adjacent normal samples (OACC-N), there were 3243 of the DEPs and 2011 Khib sites were identified on 764 proteins (DHMPs). DEPs and DHMPs were strongly associated to glycolysis pathway. GAPDH of K254, ENO of K228, and PGK1 of K323 were modified by Khib in OACC-T. Khib may increase the catalytic efficiency to promote glycolysis pathway and favor OACC progression.Conclusions and clinical relevanceKhib may play a significant role in the mechanism of OACC progression by influencing the enzyme activity of the glycolysis pathway. These findings may provide new therapeutic options of OACC.

ResNetKhib: a novel cell type-specific tool for predicting lysine 2-hydroxyisobutylation sites via transfer learning.

Lysine 2-hydroxyisobutylation (Khib), which was first reported in 2014, has been shown to play vital roles in a myriad of biological processes including gene transcription, regulation of chromatin functions, purine metabolism, pentose phosphate pathway and glycolysis/gluconeogenesis. Identification of Khib sites in protein substrates represents an initial but crucial step in elucidating the molecular mechanisms underlying protein 2-hydroxyisobutylation. Experimental identification of Khib sites mainly depends on the combination of liquid chromatography and mass spectrometry. However, experimental approaches for identifying Khib sites are often time-consuming and expensive compared with computational approaches. Previous studies have shown that Khib sites may have distinct characteristics for different cell types of the same species. Several tools have been developed to identify Khib sites, which exhibit high diversity in their algorithms, encoding schemes and feature selection techniques. However, to date, there are no tools designed for predicting cell type-specific Khib sites. Therefore, it is highly desirable to develop an effective predictor for cell type-specific Khib site prediction. Inspired by the residual connection of ResNet, we develop a deep learning-based approach, termed ResNetKhib, which leverages both the one-dimensional convolution and transfer learning to enable and improve the prediction of cell type-specific 2-hydroxyisobutylation sites. ResNetKhib is capable of predicting Khib sites for four human cell types, mouse liver cell and three rice cell types. Its performance is benchmarked against the commonly used random forest (RF) predictor on both 10-fold cross-validation and independent tests. The results show that ResNetKhib achieves the area under the receiver operating characteristic curve values ranging from 0.807 to 0.901, depending on the cell type and species, which performs better than RF-based predictors and other currently available Khib site prediction tools. We also implement an online web server of the proposed ResNetKhib algorithm together with all the curated datasets and trained model for the wider research community to use, which is publicly accessible at https://resnetkhib.erc.monash.edu/.

Proteomic analysis of protein lysine 2-hydroxyisobutyrylation (Khib) in soybean leaves

BackgroundProtein lysine 2-hydroxyisobutyrylation (Khib) is a novel post-translational modification (PTM) discovered in cells or tissues of animals, microorganisms and plants in recent years. Proteome-wide identification of Khib-modified proteins has been performed in several plant species, suggesting that Khib-modified proteins are involved in a variety of biological processes and metabolic pathways. However, the protein Khib modification in soybean, a globally important legume crop that provides the rich source of plant protein and oil, remains unclear.ResultsIn this study, the Khib-modified proteins in soybean leaves were identified for the first time using affinity enrichment and high-resolution mass spectrometry-based proteomic techniques, and a systematic bioinformatics analysis of these Khib-modified proteins was performed. Our results showed that a total of 4251 Khib sites in 1532 proteins were identified as overlapping in three replicates (the raw mass spectrometry data are available via ProteomeXchange with the identifier of PXD03650). These Khib-modified proteins are involved in a wide range of cellular processes, particularly enriched in biosynthesis, central carbon metabolism and photosynthesis, and are widely distributed in subcellular locations, mainly in chloroplasts, cytoplasm and nucleus. In addition, a total of 12 sequence motifs were extracted from all identified Khib peptides, and a basic amino acid residue (K), an acidic amino acid residue (E) and three aliphatic amino acid residues with small side chains (G/A/V) were found to be more preferred around the Khib site. Furthermore, 16 highly-connected clusters of Khib proteins were retrieved from the global PPI network, which suggest that Khib modifications tend to occur in proteins associated with specific functional clusters.ConclusionsThese findings suggest that Khib modification is an abundant and conserved PTM in soybean and that this modification may play an important role in regulating physiological processes in soybean leaves. The Khib proteomic data obtained in this study will help to further elucidate the regulatory mechanisms of Khib modification in soybean in the future.

Global landscape of 2-hydroxyisobutyrylation in human pancreatic cancer.

As a new type of post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) was firstly identified in histones and functioned as a regulator of transactivation in mammals. However, the role of Khib proteins remains to be investigated. Here, we firstly identified 10,367 Khib sites on 2,325 modified proteins in seven patients with pancreatic cancer by applying liquid chromatography with tandem mass spectrometry (LC-MS/MS) qualitative proteomics techniques. Among them, 27 Khib-modified sites were identified in histones. Bioinformatics analysis revealed that the Khib-modified proteins were mainly distributed in the cytoplasm and enhanced in metabolic pathways, including glycolysis/gluconeogenesis, the tricarboxylic acid cycle (TCA cycle), and fatty acid degradation. In an overlapping comparison of lysine 2-hydroxyisobutyrylation, succinylation, and acetylation in humans, 105 proteins with 80 sites were modified by all three PTMs, suggesting there may be a complex network among the different modified proteins and sites. Furthermore, MG149, which was identified as a Tip60 inhibitor, significantly decreased the total Khib modification level in pancreatic cancer (PC) and strongly suppressed PC’s proliferation, migration, and invasion ability. Overall, our study is the first profiling of lysine 2-hydroxyisobutyrylome and provides a new database for better investigating Khib in PC.

Global analysis of lysine 2-hydroxyisobutyrylation during Fusarium graminearum infection in maize.

Proteins post-translational modification (PTMs) is necessary in the whole life process of organisms. Among them, lysine 2-hydroxyisobutyrylation (Khib) plays an important role in protein synthesis, transcriptional regulation, and cell metabolism. Khib is a newly identified PTM in several plant species. However, the function of Khib in maize was unclear. In this study, western blotting results showed that Khib modification level increased significantly after Fusarium graminearum infection, and 2,066 Khib modified sites on 728 proteins were identified in maize, among which 24 Khib sites occurred on core histones. Subcellular localization results showed that these Khib modified proteins were localized in cytoplasm, chloroplast, and nucleus. Then, comparative proteomic analysis of the defense response to F. graminearum infection showed that Khib modification participated in plant resistance to pathogen infection by regulating glycolysis, TCA cycle, protein synthesis, peroxisome, and secondary metabolic processes, such as benzoxazinoid biosynthesis, phenylpropanoid biosynthesis, jasmonic acid synthesis, and tyrosine and tryptophan biosynthesis. In addition, we also demonstrated that lysine 2-hydroxyisobutyrylation sites on histones were involved in the gene expression of pathogenesis-related proteins. Our results provide a new perspective for the study of plant disease resistance, and had directive significance of maize disease resistance for molecular breeding.

Proteome-wide analysis of lysine 2-hydroxyisobutyrylation in Frankliniella occidentalis

BackgroundLysine 2-hydroxyisobutyrylation (Khib) is a novel and conserved post-translational modification (PTM). Frankliniella occidentalis are economically important agricultural pests globally and also notorious for vectoring destructive plant viruses. To better study the disease transmission mechanism of F. occidentalis, it is necessary to conduct in-depth analysis of it. So far, no Khib modification of insects has been reported.ResultsIn this study, a proteome-wide analysis of Khib modifications in F. occidentalis was analyzed for the first time through the combination of high performance liquid chromatography fractionation technology and 2-hydroxyisobutyrylated peptide enrichment and other advanced technologies, 4093 Khib sites were identified on 1125 modified proteins. Bioinformatics and functional enrichment analyses showed that Khib-modified proteins were significantly enriched in many cell compartments and pathways, especially related to various cellular components and biological processes, and were more concentrated in ribosomes and proteasome subunits, involved in energy metabolism, protein synthesis and degradation, compared to the other nine species including Japonica rice, Homo sapiens, P. patens, Botrytis, Ustilaginoidea virens, Saccharomyces cerevisiae, T. gondii, C. albicans, and F. oxysporum. And Khib sites on virus-interacting insect proteins were discovered for the first time, such as cyclophilin and endoCP-GN.ConclusionsAfter three repeated experiments, we found a total of 4093 Khib sites on 1125 proteins. These modified proteins are mainly concentrated in ribosomes and proteasome subunits, and are widely involved in a variety of critical biological activities and metabolic processes of F. occidentalis. In addition, for the first time, Khib modification sites are found on the proteome of F. occidentalis, and these sites could be acted as for the virus interaction, including cyclophilin and endoCP-GN. The global map of 2-hydroxyisobutyrylation in thrips is an invaluable resource to better understand the biological processes of thrips and provide new means for disease control and mitigation of pest damage to crops.

Lysine 2‐hydroxyisobutyrylation orchestrates cell development and aflatoxin biosynthesis in Aspergillus flavus

Lysine 2-hydroxyisobutyrylation (Khib ) is a recently identified post-translational modification (PTM) that regulates numerous cellular metabolic processes. In pathogenic microorganism, although glycolysis and fungal virulence are regulated by Khib , its potential roles in fungi remain to be elusive. Our preliminary results showed that levels of Khib fluctuate over time in Aspergillus flavus, which frequently contaminates crops and produces carcinogenic aflatoxins. However, the perception of Khib function in A. flavus is limited, especially in mycotoxin-producing strains. Here, we performed a comprehensive analysis of Khib in A. flavus, and 7156 Khib sites were identified in 1473 proteins. Notably, we demonstrated that Khib of AflM, a key enzyme in aflatoxin biosynthesis, affected conidia production and sclerotia formation. Furthermore, aflM deletion impaired aflatoxin biosynthesis, and more importantly, strains in which Khib was mimicked by K to T mutation at K49, K179 and K180 sites showed reduced aflatoxin production compared with wild type and ΔaflM complementation strains. These results indicate that Khib at these sites of AflM negatively regulates aflatoxin biosynthesis in A. flavus. In summary, our study revealed the potential roles of Khib in A. flavus, and particularly shed light on a new way to regulate aflatoxin production via Khib .

Quantitative proteomics profiling of lysine 2‐hydroxyisobutyrylation in right atrial appendage from rheumatic heart valve disease patients

ObjectivesLysine 2‐hydroxyisobutyrylation (Khib) is a novel acylation that regulates glycolysis. Little has been done in human diseases regarding the role of Khib. The present study was designed to reveal the role of Khib in the development of atrial fibrillation (AF) associated with heart valve disease.MethodsDiscarded right atrial appendage tissues during the cannulation procedure were collected from AF and sinus rhythm (SR) patients with rheumatic heart valve disease. The protein profiles and Khib sites were identified and qualified by Tandem Mass Tag labeling and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) technology. Bioinformatic tools, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein‐protein interaction, were also used to explore the biological function between AF and SR.ResultsA total of 6,523 matched spectrum, 3,234 peptides, 2,867 modified peptides, corresponding to 579 proteins and 2,900 sites from LC‐MS/MS analysis were identified. Of these identified proteins and sites, 480 proteins and 2,275 sites were quantifiable. The length of most peptides distributed between 7 and 22, which was consistent with the length of tryptic peptides. Differentially expressed Khib sites analysis showed that there were 35 and 48 Khib sites related to 124 and 67 proteins exhibited upregulation and downregulation in AF compared to SR. Subcellular location analysis of Khib ‐related proteins revealed that cytoplasm, nucleus, and extracellular accounted for the most proportion. Bioinformatic analysis demonstrated that proteins with Khib were significantly enriched in different biological approach, such as cysteine and methionine metabolism, cGMP‐PKG signaling pathway, and glucose‐responsive energy metabolism.ConclusionsThe present study for the first time revealed larger number of differentially expressed Khib sites between AF and SR in patients with rheumatic valve disease. The proteins related to these sites are involved in diverse cellular processes including energy metabolism. These results indicate that Khib modification might be a key factor in occurrence of AF in patients with rheumatic heart valve disease.

Quantitative multiplexed proteomics analysis reveals reshaping of the lysine 2-hydroxyisobutyrylome in Fusarium graminearum by tebuconazole

BackgroundsLysine 2-hydroxyisobutyrylation (Khib) is a newly discovered posttranslational modification (PTM) and has been identified in several prokaryotic and eukaryotic organisms. Fusarium graminearum, a major pathogen of Fusarium head blight (FHB) in cereal crops, can cause considerable yield loss and produce various mycotoxins that threaten human health. The application of chemical fungicides such as tebuconazole (TEC) remains the major method to control this pathogen. However, the distribution of Khib in F. graminearum and whether Khib is remodified in response to fungicide stress remain unknown.ResultsHere, we carried out a proteome-wide analysis of Khib in F. graminearum, identifying the reshaping of the lysine 2-hydroxyisobutyrylome by tebuconazole, using the most recently developed high-resolution LC–MS/MS technique in combination with high-specific affinity enrichment. Specifically, 3501 Khib sites on 1049 proteins were identified, and 1083 Khib sites on 556 modified proteins normalized to the total protein content were changed significantly after TEC treatment. Bioinformatics analysis showed that Khib proteins are involved in a wide range of biological processes and may be involved in virulence and deoxynivalenol (DON) production, as well as sterol biosynthesis, in F. graminearum.ConclusionsHere, we provided a wealth of resources for further study of the roles of Khib in the fungicide resistance of F. graminearum. The results enhanced our understanding of this PTM in filamentous ascomycete fungi and provided insight into the remodification of Khib sites during azole fungicide challenge in F. graminearum.

Quantitative Proteomics Reveals the Role of Lysine 2-Hydroxyisobutyrylation Pathway Mediated by Tip60.

Lysine 2-hydroxyisobutyrylation (Khib) is a new type of posttranslational modifications (PTMs) extensively reported on eukaryotic cell histones. It is evolutionarily conserved and participates in diverse important biological processes, such as transcription and cell metabolism. Recently, it has been demonstrated that Khib can be regulated by p300 and Tip60. Although the specific Khib substrates mediated by p300 have been revealed, how Tip60 regulates diverse cellular processes through the Khib pathway and the different roles between Tip60 and p300 in regulating Khib remain largely unknown, which prevents us from understanding how this modification executes its biological functions. In this study, we report the first Khib proteome mediated by Tip60. In total, 3502 unique Khib sites from 1050 proteins were identified. Among them, 536 Khib sites from 406 proteins were present only in Tip60 overexpressing cells and 13 Khib sites increased more than 2-fold in response to Tip60 overexpression, indicating that Tip60 significantly affected global Khib. Notably, only 5 of the 549 Tip60-targeted Khib sites overlapped with the 149 known Khib sites targeted by p300, indicating the different Khib substrate preferences of Tip60 and p300. In addition, the Khib substrates regulated by Tip60 are deeply involved in processes such as nucleic acid metabolism and translation, and some are associated with Parkinson's and Prion diseases. In summary, our research reveals the Khib substrates targeted by Tip60, which elucidates the effect of Tip60 in regulating various cellular processes through the Khib pathway, and proposes novel views into the functional mechanism of Tip60.

Deep 2-Hydroxyisobutyrylome in mouse liver expands the roles of lysine 2-hydroxyisobutyrylation pathway

Lysine 2-hydroxyisobutyrylation (Khib), a newly characterized post-translational modification, is conserved in both eukaryotic and prokaryotic cells. At present, only about 6500 Khib sites have been identified in mammalian cells, which is insufficient compared with the well-known acetylation and thus hinders the understanding of its roles in diverse cellular processes. Here, utilizing immunoaffinity enrichment coupled with LC-MS/MS approach, we carried out a deep proteomics analysis of Khib in mouse liver. A total of 20861 Khib sites in 3768 proteins were identified, which expands the known Khib sites by two folds and represents the deepest Khib proteome in mammalian cells currently. Bioinformatics analysis showed that the 2-hydroxyisobutyrylated proteins have different subcellular localizations and participate in a wide range of molecular functions and cellular processes, such as metabolic processes and disease-related pathways. In addition, RNA-Seq analysis revealed that 1470 genes up-regulated and 790 genes down-regulated in response to elevated Khib levels in HeLa cells. The 1470 up-regulated genes were mainly associated with human papillomavirus infection, ECM-receptor interaction, as well as protein digestion and absorption, while the 790 down-regulated genes were mainly enriched in the multiple diseases and Glycolysis/Gluconeogenesis processes. Taken together, our research largely expands the known Khib sites, which helps delineate the biological functions of the Khib pathway and provides mechanistic insights into how Khib exerts its functions in specific cellular pathways.

Salt stress downregulates 2-hydroxybutyrylation in Arabidopsis siliques

Lysine 2-hydroxyisobutyrylation (Khib) is one of the newly discovered post-translational modifications (PTMs) through protein acylation. It has been reported to be widely distributed in both eukaryotes and prokaryotes, and plays an important role in chromatin conformation change, gene transcription, protein subcellular localization, protein-protein interaction, signal transduction, and cellular proliferation. In this study, the Khib modification proteome of siliques from A. thaliana under salt stress (Ss) and those in the control (Cs) were compared. The results showed that Khib modification was abundant in siliques. Totally 3810 normalized Khib sites on 1254 proteins were identified, and the Khib modification showed a downregulation trend dramatically: it was down-regulated at 282 sites on 205 proteins while was up-regulated at 96 sites on 78 proteins in Ss siliques (Data are available via ProteomeXchange with identifier PXD028116 and PXD026643). Among them, 13 proteins, including F4IVN6, Q9M1P5, and Q9LF33, had sites with the most significant regulation of Khib modification. Bioinformatics analysis suggested that the differentially Khib-regulated proteins mainly participated in glycolysis/gluconeogenesis and endocytosis. In particular, there were differentially117 Khib-regulated proteins that were mapped to the protein-protein interaction database. In the KEGG pathway enrichment analysis, Khib-modified proteins were enriched in several pathways related to energy metabolism, including gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism. Overall, our work reveals the first systematic analysis of Khib proteome in Arabidopsis siliques under salt stress, and sheds a light on the future studies on the regulatory mechanisms of Khib during the salt stress response of plants. SignificanceIn this study, we found the Khib-modified proteins in silique under salt stress and described the enrichment of Khib-modified proteins involved in the biological processes and cellular localization. Proteins undergoing 2-hydroxyisobutylation were mainly involved in the gluconeogenesis pathway, pentose phosphate pathway, and pyruvate metabolism, suggesting that 2-hydroxyisobutylation affects the energy metabolic pathway, and thus the development of the plant. In addition, specific candidate proteins that may affect plant development under salt stress were selected. This study will provide a theoretical basis for revealing the function and mechanism of these proteins and their 2-hydroxyisobutyryl modifications during the development of silique under salt stress.

Lysine 2-hydroxyisobutyrylation proteomics reveals protein modification alteration in the actin cytoskeleton pathway of oral squamous cell carcinoma

As the most commonplace malignant carcinoma in the oral cavity, oral squamous cell carcinoma (OSCC) is highly invasive and prone to recurrence. The nosogenesis of OSCC are affected by epigenetics. Recently, a newly-found post-translational modification of lysine, 2-hydroxyisobutylation (Khib), has been proved to play a critical role in biological regulation. However, no research has evaluated the mechanism of Khib in oral cancer. Here, we performed liquid chromatography-mass spectrometry-based quantitative proteomics combined with bioinformatics analysis to reveal and evaluate Khib protein alterations in OSCC. Numerous proteins in OSCC undergo up-regulated modification of Khib. We quantified and identified 967 proteins with differential expression levels, and 617 2-hydroxyisobutylated proteins with 938 Khib sites. Among them, 125 proteins both differentially expressed and accompanied by obvious Khib modification were further identified and analyzed through KEGG-based and ingenuity pathway analysis (IPA). These proteins are enriched in the actin cytoskeleton regulatory pathway, and IPA predicted that they alter the state of actin aggregation and stability, hence impacting and regulating the actin cytoskeleton in OSCC. This is the first 2-hydroxyisobutylated modification proteomics performed for OSCC. Khib protein is significantly concentrated in the actin cytoskeleton regulatory pathway, indicating that this pathway may mediate the tumorigenesis or exacerbation of OSCC. SignificanceThis is the first study that revealed the alterations of Khib protein in oral squamous cell carcinoma through LC-MS/MS-based modified proteomic. Our data showed that the protein in the actin cytoskeleton regulatory pathway was underwent significant Khib modification and abundance changes. We applied predictive function in IPA software to analyze and clarify that the aggregation of actin and the regulation of actin stability that mediated by the actin cytoskeleton regulatory pathway may be the potential mechanism of the occurrence and development of oral squamous cell carcinoma. Our research broadens the understanding of the pathogenesis of oral squamous cell carcinoma and provides new insights for future research.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus niger in Peanuts.

Aspergillus niger is a very destructive pathogen causing severe peanut root rot, especially in the seeding stage of peanuts (Arachis hypogaea), and often leading to the death of the plant. Protein lysine 2-hydroxyisobutyrylation (Khib) is a newly detected post-translational modification identified in several species. In this study, we identified 5041 Khib sites on 1,453 modified proteins in A. niger. Compared with five other species, A. niger has conserved and novel proteins. Bioinformatics analysis showed that Khib proteins are widely distributed in A. niger and are involved in many biological processes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that Khib proteins were significantly enriched in many cellular compartments and pathways, such as ribosomes and proteasome subunits. A total of 223 Khib proteins were part of the PPI network, thus, suggesting that Khib proteins are associated with a large range of protein interactions and diverse pathways in the life processes of A. niger. Several identified proteins are involved in pathogenesis regulation. Our research provides the first comprehensive report of Khib and an extensive database for potential functional studies on Khib proteins in this economically important fungus.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection.

The post-translational modification lysine 2-hydroxyisobutyrylation (Khib ) plays an important role in gene transcription, metabolism, and enzymatic activity. Khib sites have been identified in rice (Oryza sativa). However, the Khib status of proteins in rice flowers during pathogen infection remains unclear. Here, we report a comprehensive identification of Khib -modified proteins in rice flowers, and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens. By using a tandem mass tag-based quantitative proteomics approach, we identified 2,891 Khib sites on 964 proteins in rice flowers. Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Khib levels were substantially reduced upon infection with U. virens. Chromatin immunoprecipitation polymerase chain reaction (PCR) and reverse transcription quantitative PCR analyses revealed that histone Khib is involved in the expression of disease-resistance genes. More importantly, most quantified sites on core histones H3 were downregulated upon U. virens infection. In addition, the histone deacetylases HDA705, HDA716, SRT1, and SRT2 are involved in the removal of Khib marks in rice. HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U. virens, Magnaporthe oryzae, and Xanthomonas oryzae pv. oryzae (Xoo). Our data suggest that U. virens could modulate Khib in rice flowers during infection.

Functional interplay of histone lysine 2-hydroxyisobutyrylation and acetylation in Arabidopsis under dark-induced starvation.

Lysine 2-hydroxyisobutyrylation (Khib) is a novel type of histone acylation whose prevalence and function in plants remain unclear. Here, we identified 41 Khib sites on histones in Arabidopsis thaliana, which did not overlap with frequently modified N-tail lysines (e.g. H3K4, H3K9 and H4K8). Chromatin immunoprecipitation-sequencing (ChIP-seq) assays revealed histone Khib in 35% of protein-coding genes. Most Khib peaks were located in genic regions, and they were highly enriched at the transcription start sites. Histone Khib is highly correlated with acetylation (ac), particularly H3K23ac, which it largely resembles in its genomic and genic distribution. Notably, co-enrichment of histone Khib and H3K23ac correlates with high gene expression levels. Metabolic profiling, transcriptome analyses, and ChIP-qPCR revealed that histone Khib and H3K23ac are co-enriched on genes involved in starch and sucrose metabolism, pentose and glucuronate interconversions, and phenylpropanoid biosynthesis, and help fine-tune plant response to dark-induced starvation. These findings suggest that Khib and H3K23ac may act in concert to promote high levels of gene transcription and regulate cellular metabolism to facilitate plant adaption to stress. Finally, HDA6 and HDA9 are involved in removing histone Khib. Our findings reveal Khib as a conserved yet unique plant histone mark acting with lysine acetylation in transcription-associated epigenomic processes.

Systematic analysis of lysine 2-hydroxyisobutyrylation posttranslational modification in wheat leaves.

Lysine 2-hydroxyisobutyrylation (Khib) is a recently discovered post-translational modification (PTM) showing diverse biological functions and effects in living organisms. However, the study of Khib in plant species is still relatively limited. Wheat (Triticum aestivum L.) is a global important cereal plant. In this study, the systematic Khib analysis was performed in wheat leave tissues. A total of 3004 Khib sites in 1104 proteins were repeatedly identified. Structure characterization of these Khib peptides revealed 12 conserved sequence motifs. Function classification and enrichment analysis indicated these Khib proteins showed a wide function and pathway distribution, of which ribosome activity, protein biosynthesis and photosynthesis were the preferred biological processes. Subcellular location predication indicated chloroplast was the dominant subcellular compartment where Khib was distributed. There may be some crosstalks among Khib, lysine acetylation and lysine succinylation modification because some proteins and sites were modified by all these three acylations. The present study demonstrated the critical role of Khib in wheat biological and physiology, which has expanded the scope of Khib in plant species. Our study is an available resource and reference of Khib function demonstration and structure characterization in cereal plant, as well as in plant kingdom.

Global analysis of lysine 2-hydroxyisobutyrylation in wheat root

Lysine 2-hydroxyisobutyrylation (Khib) is a novel naturally occurring post-translational modification. The system Khib identification at proteomics level has been performed in various species and tissues to characterize the role of Khib in biological activities. However, the study of Khib in plant species is relatively less. In the present study, the first plant root tissues lysine 2-hydroxyisobutyrylome analysis was performed in wheat with antibody immunoprecipitation affinity, high resolution mass spectrometry-based proteomics and bioinformatics analysis. In total, 6328 Khib sites in 2186 proteins were repeatedly identified in three replicates. These Khib proteins showed a wide subcellular location distribution. Function and pathways characterization of these Khib proteins indicated that many cellular functions and metabolism pathways were potentially affected by this modification. Protein and amino acid metabolism related process may be regulated by Khib, especially ribosome activities and proteins biosynthesis process. Carbohydrate metabolism and energy production related processes including glycolysis/gluconeogenesis, TCA cycle and oxidative phosphorylation pathways were also affected by Khib modification. Besides, root sulfur assimilation and transformation related enzymes exhibited Khib modification. Our work illustrated the potential regulation role of Khib in wheat root physiology and biology, which could be used as a useful reference for Khib study in plant root.