Zhaohua Peng

Global Analysis of Lysine Acetylation Suggests the Involvement of Protein Acetylation in Diverse Biological Processes in Rice (Oryza sativa)

Lysine acetylation is a reversible, dynamic protein modification regulated by lysine acetyltransferases and deacetylases. Recent advances in high-throughput proteomics have greatly contributed to the success of global analysis of lysine acetylation. A large number of proteins of diverse biological functions have been shown to be acetylated in several reports in human cells, E.coli, and dicot plants. However, the extent of lysine acetylation in non-histone proteins remains largely unknown in monocots, particularly in the cereal crops. Here we report the mass spectrometric examination of lysine acetylation in rice (Oryza sativa). We identified 60 lysine acetylated sites on 44 proteins of diverse biological functions. Immunoblot studies further validated the presence of a large number of acetylated non-histone proteins. Examination of the amino acid composition revealed substantial amino acid bias around the acetylation sites and the amino acid preference is conserved among different organisms. Gene ontology analysis demonstrates that lysine acetylation occurs in diverse cytoplasmic, chloroplast and mitochondrial proteins in addition to the histone modifications. Our results suggest that lysine acetylation might constitute a regulatory mechanism for many proteins, including both histones and non-histone proteins of diverse biological functions.

Proteome Profile of Starch Granules Purified from Rice (Oryza sativa) Endosperm

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I2 staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins.

Malonylome analysis in developing rice (Oryza sativa) seeds suggesting that protein lysine malonylation is well-conserved and overlaps with acetylation and succinylation substantially

In recent years, lysine malonylation has garnered wide spread interest due to its potential regulatory roles. While studies have been performed in bacteria, mouse, and human, the involvement and the biological function of this modification in plant are still largely unknown. We examined the global proteome profile of lysine malonylation in developing rice seeds using affinity enrichment followed by LC-MS/MS analysis. We identified 421 malonylated lysine sites across 247 proteins. Functional analyses showed predominant presence of malonylated proteins in metabolic processes, including carbon metabolism, glycolysis/gluconeogenesis, TCA cycle, as well as photosynthesis. Malonylation was also detected on enzymes in starch biosynthesis pathway in developing rice seeds. In addition, we found a remarkable overlap among the malonylated, succinylated and acetylated sites identified in rice. Furthermore, malonylation at conserved sites of homologous proteins was observed across organisms of different kingdoms, including mouse, human, and bacteria. Finally, distinct motifs were identified when the rice malonylation sites were analyzed and conserved motifs were observed from bacterium to human and rice. Our results provide an initial understanding of the lysine malonylome in plants. The study has critical reference value for future understanding of the biological function of protein lysine malonylation in plants. BIOLOGICAL SIGNIFICANCE Lysine malonylation is a newly discovered acylation with functional potential in regulating cellular metabolisms and activities. However, the malonylation status has not been reported in plants. Grain yield and quality, mainly determined during cereal seed development, are closely related to food security, human health and economic value. To evaluate malonylation level in plants and the possible regulatory functions of malonylation in seed development, we conducted comprehensive analyses of malonylome in developing rice seeds. A total of 421 malonylated lysine sites from 247 proteins were identified, which involved in multiple critical metabolic processes, including central carbon metabolism, lipid metabolism, photosynthesis, and starch biosynthesis. We found that charged amino acids, lysine and arginine, were the preferred residues in positions flanking the modified lysines. Highly conserved modification sites on both histone and non-histone proteins were observed among different organisms through sequence alignment analysis. More interestingly, a large number of modification sites shared by malonylation, acetylation and succinylation were identified in rice. The study presents a comprehensive understanding of malonylome in plants, which will serve as an initial platform for further investigation of the functions of lysine malonylation, especially in cereal seeds development.

Proteome-wide lysine acetylation identification in developing rice (Oryza sativa) seeds and protein co-modification by acetylation, succinylation, ubiquitination, and phosphorylation

Protein lysine acetylation is a highly conserved post-translational modification with various biological functions. However, only a limited number of acetylation sites have been reported in plants, especially in cereals, and the function of non-histone protein acetylation is still largely unknown. In this report, we identified 1003 lysine acetylation sites in 692 proteins of developing rice seeds, which greatly extended the number of known acetylated sites in plants. Seven distinguished motifs were detected flanking acetylated lysines. Functional annotation analyses indicated diverse biological processes and pathways engaged in lysine acetylation. Remarkably, we found that several key enzymes in storage starch synthesis pathway and the main storage proteins were heavily acetylated. A comprehensive comparison of the rice acetylome, succinylome, ubiquitome and phosphorylome with available published data was conducted. A large number of proteins carrying multiple kinds of modifications were identified and many of these proteins are known to be key enzymes of vital metabolic pathways. Our study provides extending knowledge of protein acetylation. It will have critical reference value for understanding the mechanisms underlying PTM mediated multiple signal integration in the regulation of metabolism and development in plants.

Proteome-wide Analysis of Lysine 2-hydroxyisobutyrylation in Developing Rice ( Oryza sativa ) Seeds

Lysine 2-hydroxyisobutyrylation is a recently identified protein post-translational modification that is known to affect the association between histone and DNA. However, non-histone protein lysine 2-hydroxyisobutyrylation remains largely unexplored. Utilizing antibody-based affinity enrichment and nano-HPLC/MS/MS analyses of 2-hydroxyisobutyrylation peptides, we efficaciously identified 9,916 2-hydroxyisobutyryl lysine sites on 2,512 proteins in developing rice seeds, representing the first lysine 2-hydroxyisobutyrylome dataset in plants. Functional annotation analyses indicated that a wide variety of vital biological processes were preferably targeted by lysine 2-hydroxyisobutyrylation, including glycolysis/gluconeogenesis, TCA cycle, starch biosynthesis, lipid metabolism, protein biosynthesis and processing. Our finding showed that 2-hydroxyisobutyrylated histone sites were conserved across plants, human, and mouse. A number of 2-hydroxyisobutyryl sites were shared with other lysine acylations in both histone and non-histone proteins. Comprehensive analysis of the lysine 2-hydroxyisobutyrylation sites illustrated that the modification sites were highly sequence specific with distinct motifs, and they had less surface accessibility than other lysine residues in the protein. Overall, our study provides the first systematic analysis of lysine 2-hydroxyisobutyrylation proteome in plants, and it serves as an important resource for future investigations of the regulatory mechanisms and functions of lysine 2-hydroxyisobutyrylation.