Malonylome Analysis Reveals the Involvement of Lysine Malonylation

Subscriber access provided by MacEwan University Libraries

Article

Malonylome Analysis Reveals the Involvement of Lysine Malonylation in Metabolism and Photosynthesis in Cyanobacteria Yanyan Ma, Mingkun Yang, Xiaohuang Lin, Xin Liu, Hui Huang, and Feng Ge J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00017 • Publication Date (Web): 03 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Proteome Research

Malonylome

Analysis

Reveals

the

Involvement

of

Lysine

Malonylation in Metabolism and Photosynthesis in Cyanobacteria

Yanyan Ma1,2#, Mingkun Yang1#, Xiaohuang Lin1,2, Xin Liu1,2, Hui Huang1,2, Feng Ge1*

1

Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences,

Wuhan 430072, China 2

University of Chinese Academy of Sciences, Beijing 100049, China

#

These authors contributed equally to this work.

*To whom correspondence should be addressed: Prof. Feng Ge, Institute of Hydrobiology, Chinese Academy of Sciences, E-mail: [email protected]. Phone/Fax: +86-27-68780500.

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract

As a recently validated reversible post translational modification, lysine malonylation regulates diverse cellular processes from bacteria to mammals, but its existence and function in photosynthetic organisms remain unknown. Cyanobacteria are the most ancient group of photosynthetic prokaryotes and contribute about 50% total primary production on Earth. Previously, we reported the lysine acetylome in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Here, we performed the first proteomic survey of lysine malonylation in Synechocystis using highly accurate tandem mass spectrometry in combination with affinity purification. We identified 598 lysine malonylation sites on 339 proteins with high confidence in total. A bioinformatic analysis suggested that these malonylated proteins may play various functions and were distributed in diverse subcellular compartments. Among them, many malonylated proteins were involved in cellular metabolism. The functional significance of lysine malonylation in the metabolic enzyme activity of phosphoglycerate kinase (PGK) was determined by site-specific mutagenesis and biochemical studies. Interestingly, 27 proteins involved in photosynthesis were found to be malonylated for the first time, suggesting that lysine malonylation may be involved in photosynthesis. Thus, our results provide the first lysine malonylome in a photosynthetic organism and suggest previously unexplored role of lysine malonylation in the regulation of metabolic processes and photosynthesis in Synechocystis, as well as in other photosynthetic organisms.

Keywords: Lysine malonylation, Post-translational modification (PTM), Cyanobacteria, Synechocystis, Photosynthesis, Phosphoglycerate kinase (PGK), metabolism

2


Page 2 of 40

Page 3 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction

Post-translational modifications (PTMs) play critical regulatory roles in diverse cellular pathways and disease processes, and more than 400 various kinds of PTMs have been reported1. Among them, lysine malonylation is a newly identified PTM and the ɛ-amino group of a lysine residue is modified by a malonyl group 2. Because malonyl groups contain a negatively charged carboxyl group, it is conceivable that lysine malonylation has significant effects on protein structure and function3. Lysine malonylated substrates were firstly identified in E. coli and mammals by a high-throughput proteomic analysis in 20112. Since then, comprehensive so-called “malonylome” studies have been performed to identify proteins that contain this PTM in mouse liver4, human fibroblasts4, human HeLa cells5, Saccharopolyspora erythraea6, and Escherichia coli7. These malonylome studies have confirmed that lysine malonylation is a widespread and evolutionarily conserved PTM, which changes dynamically under diverse biological and cellular conditions, such as the stress response and various metabolic processes4, 6-9. In particular, lysine malonylation is abundant in mitochondrial proteins and is enriched in metabolic pathways, particularly in the glycolytic and fatty acid oxidation pathways4, 8. Lysine malonylation affects the activities of metabolic enzymes in E. coli7 and erythromycin biosynthesis in S. erythraea6. Therefore, accumulating evidence demonstrates that lysine malonylation may provide a mechanism for regulating metabolism and cellular physiology in eukaryotic and prokaryotic cells3, 10-11. Cyanobacteria are a large group of prokaryotes and the most ancient group of bacteria capable of photosynthesis12. Cyanobacteria can grow in diverse environments and play critical roles in global O2 production, CO2 assimilation, and N2 fixation13. It is well accepted that cyanobacteria are the progenitors of chloroplasts 14. It is reported that cyanobacteria comprise about 50% of the total primary production on Earth15. Consequently, they are established model organisms for studying photosynthetic mechanisms. Synechocystis sp. PCC 6803 (hereafter Synechocystis) is a model cyanobacterium and the first sequenced phototrophic organism. Synechocystis has been extensively used for studies of photosynthesis and biofuel production16-18. Photosynthesis and carbon metabolism are two well-connected biological processes in photosynthetic organisms19-21. Cyanobacteria have evolved complicated 3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

mechanisms to regulate and coordinate photosynthesis and cellular metabolism21-23. However, many aspects of the regulatory mechanisms of photosynthesis and metabolic processes remain largely unknown. Lysine malonylation has been reported to be a functional PTM that affects central metabolism and the activities of metabolic enzymes in bacteria6-7; therefore, we hypothesized that lysine malonylation may affect diverse metabolic processes in cyanobacteria, including photosynthesis and carbon metabolism. However, no malonylated proteins have been reported previously in any photosynthetic organism, representing a major obstacle for studying their biological functions in cyanobacteria. We previously reported the ﬁrst lysine acetylome in Synechocystis24. In this study, malonylation events in Synechocystis were investigated systematically using high-affinity anti-malonyllysine antibodies in combination with tandem mass spectrometry identification followed by bioinformatic analyses. In total, 598 unique lysine malonylation sites on 339 proteins were identified in Synechocystis. Our study comprise the first extensive dataset regarding malonylation that is available for any photosynthetic organism at present, as well as reveal diverse functions regulated by malonylation in cyanobacteria. The malonylated proteins identified were involved in diverse biological processes, and they were particularly enriched in translational, photosynthetic, and metabolic processes. The functional significance of lysine malonylation sites in phosphoglycerate kinase (PGK) was exmined by mutagenesis and biochemical analyses. Overall, our results revealed the range of functions regulated by malonylation, as well as suggest that lysine malonylation may be a new mechanism involved in Synechocystis metabolic processes and other photosynthetic organisms.

Experimental Procedures

Cyanobacterial Strains, Culture Conditions and Extraction of Protein The Synechocystis was cultivated in medium BG11 bubbled with filtered air under continuous illumination (40 µmol photons m−2 s−1) at 30°C 25. The Synechocystis cultures were collected at the exponential phase ([OD730] ~ 0.8–0.9) and adjusted to OD730 of 0.4 with fresh medium, then immediately illuminated for 2 h at 450 µmol photons m−2 s−1 for the high light condition 4


Page 4 of 40

Page 5 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(HL) treatment. For nitrogen, calcium, potassium and A5 (trace metal elements) deficiency treatments, Synechocystis were grown to exponential phase and collected by centrifugation, cleaned twice with nitrogen deficiency medium BG11 (lacking the specified nutrient), then immediately resuspended in medium BG11 without the specified nutrient: in Ca-free medium BG11 that contained NaCl instead of CaCl2 26; in potassium-free medium BG11 that contained NaCl instead of KH2PO4 and KCl; in nitrogen-free medium without nitrate (NaNO3)

27

or in

A5-free medium BG11 (without the trace elements: CuSO4·5H2O, H3BO3, ZnSO4·7H2O, MnCl2·4H2O, Co(NO3)2·6H2O, Na2MoO4·2H2O). For high salt stress, sodium chloride and sodium malonate were added to the Synechocystis cultures at exponential phase to final concentrations of 1M and 2%, respectively. A protease inhibitor was added to the culture for an additional 30 min to inhibit endogenous protease activities prior to harvesting the cells. After washing twice with PBS buffer, cells were resuspended in ice cold buffer (20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1% Triton X-100, and 1× protease inhibitor cocktail) and sonicated (3 s on and off) for 45 min on ice with an 135 W output (Ningbo Scientz, Guangdong, China). Then, the lysates were cleared by centrifugation at 5,000 × g for 20 min at 4°C. The supernatant was collected, precipitated using ice-cold acetone (five volumes), and dried at R.T. by vacuum centrifuging for further processes. Protein Digestion, Peptide Fractionation and Malonylated Peptide Enrichment The proteins (2 mg) were digested with trypsin according to a method described previously28. Then, 0.1% trifluoroacetic acid (TFA) was used to quench the reaction, and the solution was cleaned by centrifugation at 3,000 × g. Finally, the digested sample was separated into 15 fractions using high pH HPLC by Agilent 300 Extend C18 column (4.6 mm ID, 5 µm particles, 250 mm length) with a 80 min gradient from 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10. The tryptic peptides were dried at room temperature by vacuum centrifugation. The enrichment of malonylated peptides were performed by using preconjugated anti-malonyllysine antibody agarose beads (PTM Biolabs, Chicago, IL, USA). The digested peptides were resuspended in NETN buffer (1 mM ethylenediaminetetraacetic acid, 100 mM sodium chloride, 50 mM Tris-HCl, and 0.5% NP-40, pH 8.0), and then mixed with antibody conjugated protein A agarose beads at 4°C overnight with gentle rotation. After washing three 5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

times using NETN buffer, twice using ETN buffer (1 mM EDTA, 100 mM NaCl, and 50 mM Tris, pH 8.0), and three times using pure water, the malonylated peptides were eluted by 1% TFA for three times. The elutes were dried at room temperature by vacuum centrifuging and desalted by self-packed C18 STAGE tips, prior to nano-high performance liquid chromatography-MS/MS analysis. Mass Spectrometric Analysis The enriched peptides were dissolved in solvent A (0.1% FA/2% ACN), analyzed on an easy nLC-1000 system (Thermo Fisher Scientific, Waltham, MA, USA) connected to the Q-Exactive (Thermo Fisher Scientific) mass spectrometer as described previously24. In brief, peptides were loaded onto a C18-nano capillary LC column (C18 resin with 100 Å pore diameter, 2 µm particle size, 150 mm length × 50 µm inner diameter, Acclaim PepMap RSLC, Thermo Scientific) and eluted using a 40 min gradient from 6% to 90% solvent B (0.1% formic acid/98% ACN, v/v) with 300 nL/min flow rate. The mass spectrometer was operated in a data-dependent mode with an automatic switch between MS and MS/MS acquisition under Xcalibur 3.0. Survey full MS scan (350–1,800 m/z) were collected in the orbitrap using a resolution of 70,000 at m/z = 200. The top 20 most intense ions with charge state 2-5 were allowed for fragmentation by normalized high energy C-trap dissociation (HCD) collision energy of 30%. The max ion times of full MS and each MS/MS were 50 and 200 ms, respectively. A dynamic exclusion of 15 s with a repeat of 1 and ±10 ppm exclusion window was used to collect the MS/MS spectra. Lock mass was enabled for the full MS scans at m/z 445.12003. Raw Data Analysis Raw MS data files from the Q-Exactive mass spectrometer were searched against the Synechocystis

protein

database

(http://genome.microbedb.jp/cyanobase/Synechocystis/;

released 2012, 3672 sequences) using MaxQuant (v. 1.3.0.5)29. Two maximum missed cleavage sites were permitted for trypsin. The mass error of precursor ions and fragment ions was set to 10 ppm and 0.02 Da, respectively. Carbamidomethylation of cysteine was set as a fixed modification and protein N-termini acetylation, deamidation of glutamine/asparagine, oxidation of methionine, and malonylation of lyines (neutral loss of CO2) were included as variable modifications. The minimum peptide length is 6. The false discovery rate (FDR) was 6


Page 6 of 40

Page 7 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


set to < 1% for the protein, peptide, and modification sites. All MS/MS spectra for the malonylatd peptides were manually checked using a method as described previously30-31. Breifly, a spectrum is kept only if the putative malonylated peptides were verified by high coverage of b- or y-ion series. Likewise, at least 5 isotopically resolved and the intensities of matched fragment peaks should be higher than 5% of the maximum intensity. The malonylation sites assigned to C-terminal peptides were discarded to obtain highly confident results. If the localization probability of lysine malonylation sites was higher than 0.75, it was classified as class I malonylation sites, whereas the remaining malonylation sites were reported as ambiguous malonylation sites as described previously 32-33. Bioinformatic Analysis All malonylated proteins were annotated using Gene Ontology (GO) terms (molecular function, biological process and cellular component) using Blast2GO software34. PSORTb 3.0 program35 was used for the prediction of subcellular localization of malonylated proteins. The enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG) and GO were carried out using the DAVID bioinformatic resource36-37. Amino acid sequence motifs were analyzed with the PLogo web tool38 and the heatmap of position-specific was plotted using the log10(ratio of frequencies) as reported previously39. The secondary structures of malonylated proteins were investigated using NetSurfP40. The protein protein interaction (PPI) of malonylated proteins was assessed based on the Synechocystis PPI database (http://bioportal.kobic.kr/SynechoNET) and input into Cytoscape v2.8.341. The PGK structure models were prepared from the available crystalline structure using structural modeling via the SWISS-MODEL Server (https://swissmodel.expasy.org/interactive). The Molecular Graphics System PyMOL ver. 1.7.2 (http://www.pymol.org) was used to show the protein 3D structure. Western Blotting Constant protein quantities from cells under different stress conditions were run on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by polyvinylidene difluoride (PVDF) membrane transfer. TBS buffer (25 mM Tris-HCl, 150 mM NaCl, pH 8.0) with 5% bovine serum albumin (BSA) was used to block the membrane for 1.5 h at R.T. and subsequently mixed with anti-malonyllysine monoclonal antibody (1:2000, 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

PTM Biolabs) overnight at 4°C. TBST buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 8.0) was used to wash the membrane for three times, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5000 dilution) at R.T. for 1 h with gentle shaking. The membrane was washed again and proteins were visualized by the enhanced chemiluminescence (ECL) detection kit (Advansta Inc., Advansta, CA, USA). Densitometry analysis was performed using a fluorescence scanner (GE Healthcare, Piscataway, NJ, USA). Cloning, Mutagenesis, and Purification of PGK The wild-type pgk gene (slr0394) was amplified from Synechocystis genomic DNA using the following primers: slr0394-F: 5’-GGAATTCCATATGTTGTCTAAGCAA TCGATCG-3’ and slr0394-R: 5’-CCGCTCGAGTCGGTCATCTAAAGCGGCA-3’. The polymerase chain reaction (PCR) product was inserted into pMD18-T (Takara Bio, Shiga, Japan). The mutated pgk gene was generated using mutagenic primers (K205E-F: 5’-TCCAGTGAAATCGGT GTGATCGAAA-3’ and K205E-R: 5’-ACCGATTTCACTGGACACTTTGGAA-3’), and mutagenesis was confirmed by sequencing. The plasmids containing the wild-type or mutated pgk genes were digested with NdeI and XhoI. The digested fragments were inserted into the pET21b expression vector (Novagen, Wilmington, DE, USA) to construct the recombinant plasmids. The plasmids were transformed into E. coli to express proteins. The recombinant strains were cultured in Luria–Bertani medium with 25 mg/mL ampicillin to an OD600 of 0.4-0.6. Then, 0.5 mM isopropyl-b-D-thiogalactoside was used to induce the proteins at 18°C for 12 h. The cultures were harvested by centrifugation (5,000 × g for 8 min at 4°C), resuspended in ice-cold PBS and disrupted using a high-pressure homogenizer with 1,500 W output (JNBIO, Guangzhou, China). Debris was discarded by centrifugation at 12,000 g for 10 min at 4°C for two times), and an affinity Ni2+ column (Qiagen Inc., Chatsworth, CA, USA) pre-equilibrated with binding buffer (500 mM NaCl, 20 mM Tris-HCl, pH 8.0) was prepared to purify proteins. Then the lysates were loaded onto the Ni2+ column, washed with washing buffer (20 mM Tris-HCl, 30 or 50 mM imidazole, 500 mM NaCl, pH 8.0), the target protein was eluted twice using elution buffer (100 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl, pH 8.0). The elution was desalted, concentrated with a 30,000 MWCO concentrator (Millipore, Billerica, MA, USA) using storage buffer (50 8


Page 8 of 40

Page 9 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


mM NaCl, 20 mM Tris-HCl, pH 8.0). Protein concentrations were determined by a BCA protein assay (Beyotime, Jiangsu, China), and all purified proteins were examined by SDS-PAGE. In Vitro Enzymatic Activities Assay The activities of purified wild type and mutated PGKs were determined by measuring 3-phosphoglycerate-dependent oxidation of NADH at 340 nm42-43. The purified protein (about 40 ng) was incubated with the reaction buffer (5 mM MgCl2, 0.15 mM NADH, 2.5 mM ATP, 50 mM Tris-HCl, pH 8.0, and 2 U of glyceraldehyde phosphate dehydrogenase). The enzymatic activity assay was started by adding various amounts of 3-phosphoglycerate (3-PGA) at 37°C. The initial velocities were measured at varying substrate concentrations (3-PGA). The Km and kcat values were obtained from the Michaelis-Menten equation. Analysis of PGK Thermal Stability To estimate the structural basis of thermal stability between wild-type and mutant PGK (K205E), we measured the melting temperature by the Nano differential scanning calorimetry (Nano-DSC) instrument (Calorimetry Sciences Corp. Lindon, UT, USA). All protein samples (1.0 mg/ml) were firstly loaded into the sample cells of the Nano-DSC System. The reference cell was filled with the same volume of dialysis buffer (50 mM Tris-HCl, pH 8.0). Samples were then scanned using an increasing temperature from 25 to 100 °C with 1°C/min rate. Baseline value was measured by adding dialysis buffer only. The curve fitting and data analysis were performed in excel.

Results Identification of Lysine Malonylation in Synechocystis To detect the existence of malonylated proteins in photosynthetic organisms, we first performed an immunoblot analysis using anti-malonyllysine antibody with the cell lysates from Synechocystis, Synechococcus elongatus PCC 7942, Chlamydomonas reinhardtii, Synechococcus sp. PCC 7002, Chlorella sp. NJ-18, Arabidopsis thaliana, Spinacia oleracea, and Oryza sativa. As shown in Figure 1A, multiple bands were detected, indicating that lysine malonylation is a prevalent PTM in these photosynthetic organisms. Emerging evidence shows that different culture conditions may change protein acylation levels 9


44-45

.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Also, our previous work has confirmed that several protein PTM levels were changed under different stress conditions in cyanobacteria 45. We further assessed the relative abundance of lysine malonylation in whole-cell extracts from Synechocystis under various stresses. Immunoblot analysis demonstrated that malonylation level in several protein bands were changed between 10-55 kDa mass range, suggesting that lysine malonylation may be involved in the Synechocystis stress response (Fig. 1B). Notably, Synechocystis exhibited significant difference of malonylation level in response to HL, suggesting that lysine malonylation may be involved in the HL response. However, further experiments are needed to confirm the speculation and reveal the potential regulatory mechanism in Synechocystis. We used a previously described procedure to map lysine malonylation sites and understand the potential regulatory mechanism of this novel modification in cyanobacteria (Fig. 1C)

4, 6-7, 9

. To ensure confidence with the identifications, all spectra containing

malonylations were manually checked, and only the Class I malonylated peptides were selected for further analysis. In total, we identified 598 lysine malonylation sites (Class I) on 339 Synechocystis proteins with high confidence. Details of the malonylated peptides are presented in Supplemental Table S1. The raw data and annotated spectra of all malonylated peptides have been uploaded onto the PeptideAtlas public database with the identifier PASS00965 (http://www.peptideatlas.org/PASS/PASS00965). Figure 1D represents a MS/MS spectrum of a malonylated peptide from the phycocyanin beta subunit (CpcB) protein. In addition, the high peptide score (mean score, 80.57) and overall absolute peptide mass accuracy (0.94 ppm) indicated the high quality of all identified malonylated peptides (Supplemental Figure S1A). We also checked the number of malonylation sites identified per protein and the result showed that 64% (213) of malonylated proteins contained a single malonylation site, 19% (65) contained two malonylation sites and the remaining proteins contained three or more malonylation sites (Supplemental Figure S1B). Interestingly, multiple lysine malonylation sites were observed on one protein. For example, the ribulose bisphosphate carboxylase large subunit involved in carbon fixation in photosynthetic organisms had 10 lysine malonylation sites and the photosynthetic protein phycobilisome core-membrane linker polypeptide (ApcE) was highly malonylated with nine malonylation sites. These observations show the importance of lysine malonylation in the regulatory role of 10


Page 10 of 40

Page 11 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


protein function, and lysine malonylation may play an important regulatory role in carbon metabolism and photosynthesis. Furthermore, to explore the evolutionary conservation of malonylation in bacteria, we compared the Synechocystis malonylome with two published bacterial malonylomes

6-7

. As shown in Figure 1E, 134 orthologs in Synechocystis were

detected in the malonylome of E. coli and S. erythraea (Supplemental Table S2). Notably, the most conserved malonylated proteins were involved in metabolism including carbon metabolism, citrate cycle and amino acid biosynthesis, suggesting an important role of lysine malonylation in the regulation of metabolic processes in bacteria. Functional Annotation of Malonylated Proteins in Synechocystis We performed a functional analysis based on GO annotation and KEGG pathway to further investigate the functions of malonylated proteins. First, all identified proteins were annotated for molecular functions, biological processes and subcellular localization (Supplemental Table S3). Within the classification of biological processes: most malonylated proteins (38%) were involved in metabolic processes, followed by cellular processes (34%), single-organism processes (18%), localization (4%), biological regulation (3%), stimulus response (2%), or signaling (1%). Consistent with the biological process analysis, a large number of malonylated proteins (246) were assigned to the cytoplasm according to the subcellular localization category, suggesting an important role of malonylation in cellular metabolism. Notably, many identified proteins belong to the cytoplasmic membrane (38), periplasmic (8), outer membrane (2), and extracellular (2) processes, which are linked with the Synechocystis photosynthetic pathway. The molecular function classification revealed that the majority of identified proteins were assigned to catalytic activities, binding to targets, and several other groups, including structural molecule activities, transporter activities, electron carrier activities, antioxidant activities, nucleic acid binding, molecular transducer activities, and receptor activities. To further explore how lysine malonylation might affect cellular processes, DAVID bioinformatics resources were used to analyze the GO and pathway enrichment (Supplemental Table S4). Figure 2B showed that most malonylated proteins were enriched in translation (p = 1.36E-12), generation of precursor metabolites and energy (p = 8.31E-05) or photosynthesis (p = 5.22E-04), based on biological process enrichment. Consistently, from 11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

the enrichment analysis of cellular components, many identified proteins were enriched in intracellular non-membrane-bounded organelles (p = 2.86E-10), ribosomes (p = 2.86E-10), plasma membrane parts (p = 5.52E-06), thylakoids (p = 1.58E-05), and phycobilisomes (p = 1.73E-05). Furthermore, the KEGG metabolic pathway analysis also showed that the malonylated proteins were significantly enriched in ribosomes (p = 4.42E-05), photosynthesis (p = 1.20E-03), and carbon metabolism, including the pentose phosphate pathway, glycolysis/gluconeogenesis, and carbon fixation. Our results inferred the potential role of lysine malonylation in protein translation, carbon metabolism, and photosynthesis in Synechocystis. Analysis of Lysine Malonylation Sites We generated position-specific intensity maps to assess if there are specific biases for neighboring amino acid residues at malonylation sites. As shown in Figure 3A, cysteine (Cys) was highly enriched in the −3, −1, and +3 positions in our Synechocystis dataset. Furthermore, we also compared our site-specific malonylation motifs to the published lysine malonylome of E. coli 46. The malonylation pattern of E. coli was similar with Synechocystis site-specific malonylation motif. A significant preference for Cys at nearly all positions was detected in E. coli. This bias for a specific malonylation site motif suggested a unique preference for substrates in these organisms. However, we also observed quite a different situation in the S. erythraea malonylome6. For example, lysine was most commonly found at −6, −5, +2, +3, and +4 positions, whereas glutamine occurred frequently at the −3 position. The differences in the lysine malonylation motif in these organisms implicate the existence of different acyltransferases or deacylases in prokaryotes. We predicted secondary structure localizations and solvent accessibility of malonylated and non-malonylated lysines of all identified malonylated proteins using NetSurfP software to further investigate the local secondary structures of the amino acid sequences surrounding malonylation sites (Supplemental Table S5). As expected, malonylated lysines were enriched in structured regions of proteins, whereas unmodified lysines were mainly located in coiled structures (56.79% in alpha-helix and beta-strand vs. 43.22% in coil). Interestingly, malonylated lysines had a weak preference for beta-strands compared to non-malonylated lysines. About 14.41% of malonylated lysines were assigned to beta-strand structures 12


Page 12 of 40

Page 13 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compared to 12.7% of non-malonylated lysines (Fig. 3B). Additionally, malonylated lysines had an average relative side chain solvent accessibility of 95.38, which was slightly lower than that of non-malonylated lysines. This result differs from those of protein acetylation sites as described previously24. Moreover, the localizations of malonylated and non-malonylated lysines obtained in the other organisms (E. coli and S. erythraea) were in line with those of Synechocystis (Fig. 3B). Functional Interactions among Malonylated Proteins in Synechocystis Protein-protein interactions were analyzed using STING to obtain a general functional overview of the identified malonylated proteins (Supplemental Table S6). Supplemental Figure S2 shows a vast and densely connected network, in which 289 identified proteins were mapped to the Synechocystis protein interactome database and connected by 7,437 direct physical connections. Owing to the two highly interconnected processes, photosynthesis and carbon metabolism in Synechocystis47-49, we further constructed a sub-network centered on photosynthesis covering 24 identified photosynthetic proteins. These proteins interacted tightly with other function-associated proteins, as shown in Figure 4A, indicating that an intricate regulatory mechanism may exist, and lysine malonylation may play a functional role in photosynthesis in Synechocystis. We further characterized protein complexes that were related with malonylated proteins. The highly connected complexes and cellular functions clusters were extracted from the complete complexes, as assessed by molecular complex detection. Figure 4B shows the top four highly connected interaction clusters. Interestingly, cluster 1 and 4 consisted of ribosome-related proteins, whereas the remaining clusters consisted of many malonylated proteins involved in photosynthesis, including photosystem (PS) I-related proteins and PSII-related proteins. The finding was consistent with our KEGG pathway enrichment analysis that lysine malonylation substrates were significantly enriched in ribosome and photosynthesis-associated events. Taken together, our data suggest that lysine malonylation might play roles in the regulation of photosynthesis in Synechocystis, similar to acetylation24. Lysine Malonylation in Carbon Metabolism and Photosynthesis It has been reported that lysine acetylation and/or succinylation frequently occur in almost all enzymes in the central carbon metabolism, as observed in other organisms 13


24, 44, 50-66

. To


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

explore if this new acyl modification could also be found in these important pathways and determine whether they have a potential functional effect on enzymatic activities and/or flux of metabolism, we mapped all identified proteins to the KEGG pathway (Supplemental Table S7). Notably, many malonylation sites were found on 27 photosynthetic proteins including a large proportion of allophycocyanin (ApcA, ApcD, ApcB, ApcE, and ApcF) and phycocyanin subunits (CpcA, CpcC, CpcB and CpcG), PSII components (PsbB, PsbC, PsbO, and PsbV), PSI components (PsaA, PsaB, PsaC, PsaF, and PsaL), cytochrome b6/f complex (PetD and PetH), and ATPase complex (AtpA, AtpB, AtpD, AtpF, and AtpG) (Fig. 5A). It is likely that lysine malonylation is involved in the processes of assembly/disassembly of the photosynthetic complex and energy down-transfer, similar to lysine acetylation 24. However, there are no direct evidences demonstrating the effects of malonylation on the functions of these proteins and the photosynthesis pathway. Further studies are needed to reveal whether and how these identified malonylation sites have impacts on the functions of these proteins and the photosynthesis process. To our knowledge, most of major metabolic enzymes are conserved and subjected to acetylation in eukaryotes and prokaryotes. In the present study, we found that many enzymes involved in glycolysis/gluconeogenesis, TCA cycle, the pentose phosphate pathway, and the Calvin–Bassham–Benson (CBB) cycle were malonylated (Fig. 5B). Among these pathways, the CBB cycle is the primary carbon fixation pathway, and 12 enzymes in the CBB cycle were found to be malonylated in this study. For example, the key enzymes of ribulose-1,5-bi-sphosphate carboxylase and fructose-1,6-/sedoheptulose -1,7-bisphosphatase, which are important for controlling CO2 fixation rate67, were subjected to malonylation at ten and eight lysine residues, respectively. Our observations strongly suggest that many malonylation sites on these enzymes may have a potential role in regulating carbon metabolism in Synechocystis. Effect of Lysine Malonylation on PGK Activity Among these identified enzymes, one cellular metabolic protein of interest is PGK, encoded by slr0394. PGK was identified as catalytic enzyme in the conversion of 3-phosphoglycerate (3-PG) to 1,3-diphosphoglycerate (Fig. 6A), which is the second CBB step in photosynthetic organisms. In this study, six reliable malonylation sites (K48, K69, K201, K205, K246, and 14


Page 14 of 40

Page 15 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


K251) on PGK were identified. Notably, at least two malonylation sites (K201 and K205) appeared to be highly conserved in other organisms according to the multiple sequence alignment analysis, suggesting that these lysine residues may be important for PGK functions (Fig. 6B). To examine the possible effects of malonylation on PGK activity, we mapped malonylation sites using the crystalline structures of PGK and the structure revealed the functional importance of lysine malonylation on PGK. As shown in Figure 6C, PGK consisted of two well-conserved domains, namely the N- and C-terminal domains. The highly conserved K201, which was positively charged before malonylation, was located in the α-helix region and adjacent to the near α-helix of the C-terminal domain. Similarly, K205 was located in the functional α-helix of the C-terminal domain. Due to the additional malonyl group and the change in lysine charge status (from +1 to -1) on PGK, it is conceivable that malonylation at these residues would affect the interaction of these conserved residues with the near structure, such as the helix and coil regions, resulting in partial perturbation of these regions and altered domain conformation. We further explore the potential regulatory mechanisms of malonylation at these conserved residues by constructing site-specific mutants to mimic the -1 charge state of the malonyl group (K205E). We purified and examined enzymatic activity of wild-type PGK and its mutants. We showed that the K205E mutant gave rise to an evident decrease in enzymatic activity (p < 0.01) (Fig. 6D-F). In addition, because thermal stability can reflect the intrinsic stability of a protein in dilute solution

68

, we assessed the thermal stability of PGK using a

Nano-DSC instrument. Tm is related to the Gibbs free energy change to unfold the protein structure and the calorimetric enthalpy of a protein reflects its ability to absorb heat

69

. As

shown in Figure 6G, the PGK Tm value (50.05°C) was higher than that of the mutant (49.63°C) and molar heat capacity of PGK was also much higher than that of the mutant, suggesting that the wild-type was more stable than the mutant and that malonylation could be a potential way to regulate PGK activity, further leading to the regulation of cellular metabolism.

DISCUSSION We recently reported the Synechocystis lysine acetylome and revealed the functional roles of 15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

lysine acetylation in photosynthesis24. In the current study, we report the first lysine malonylome in a model cyanobacterium. This is the first global survey of lysine malonylation in a photosynthetic organism. The malonylated proteins identified are assigned to diverse molecular functions and biological processes. Notably, proteins in major metabolic and photosynthetic pathways are modified by malonylation. Because lysine malonylation has been demonstrated as one of the PTMs that is involved in metabolism, our dataset provides a list of potential functional modification sites in Synechocystis. An immunoblot analysis using an anti-malonyllysine antibody revealed that malonylation occurs in diverse photosynthetic organisms, suggesting that malonylation is a prevalent PTM in photosynthetic organisms. Thus, understanding the physiological functions of modifying these sites would certainly help to elucidate the regulation of the photosynthesis and metabolic networks in cyanobacteria, as well as in other photosynthetic organisms. In this study, many proteins involved in carbon metabolism were malonylated. For example, Slr0394, which encodes PGK, was malonylated. In the Calvin–Bassham–Benson (CBB) cycle, PGK uses ATP to catalyze the phosphorylation of 3-PGA to 1,3-bis-phosphoglycerate 70. PGK is highly conserved and found in all kingdoms71 where it catalyzes the step following carbon fixation in the Calvin cycle of photosynthetic organisms72-73. PGK is a thioredoxin or S-thiolation target in both eukaryotic and prokaryotic cells74-77, suggesting that glutathionylation and oxidation could play a role regulating PGK activity. Tsukamoto et al. reported that PGK activity is regulated by the redox PTM in Synechocystis78. Because lysine malonylation induces significant structural changes by producing a two-charge shift on substrate residues, it is conceivable that malonylation could affect the protein structure and function. Consistent with this notion, several groups have reported the important role of lysine malonylation in the regulation of the activities of the central metabolic enzymes in various eukaryotes and bacteria4, 6-8. Our functional results also show that malonylation could affect PGK activity significantly. Therefore, lysine malonylation may be one of the PTMs which can regulate the PGK activity in Synechocystis. Because cyanobacteria are some of Earth’s oldest organisms, we propose that malonylation plays an evolutionarily conserved role in regulating metabolic process of photosynthetic organisms. Increasing studies showed that diverse PTM events play an important role in regulating 16


Page 16 of 40

Page 17 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


photosynthetic processes in cyanobacteria

24, 79-82

. In this study, many proteins related to

photosynthesis were malonylated, ranging from light harvesting, PSI and PSII, and the ATPase complex. We found that representative proteins from the full photosynthetic pathway harbored this PTM. Thus, understanding the physiological functions of modifications of these sites would certainly help to elucidate regulation of the photosynthesis pathway in cyanobacteria, as well as in other photosynthetic organisms. The roles of lysine malonylation in the control of photosynthesis are largely unknown. However, their important roles in the regulation of metabolic pathways in prokaryotes and mammalian cells, as well as conservation of these metabolic enzymes in photosynthetic organisms, suggest that malonylation helps regulate photosynthetic pathways because photosynthesis and metabolism are highly interconnected to cell energy status24,

83-84

. Plants may also have special

mechanisms related to the control of metabolic and photosynthetic pathways via lysine malonylation because much of the energy status of plants is related to the chloroplasts via photosynthesis. Thus, to elucidate the role of lysine malonylation in photosynthesis, it is critical to understand the photosynthetic organism malonylomes and demonstrate the extent to which malonylation is important in the control of photosynthesis. This will involve several key approaches including: (i) determining how environmental and growth conditions can affect the status of protein malonylation; (ii) investigating the consequences of malonylation on the functions of proteins; and (iii) identifying the involvement of lysine malonyltransferase and demalonylase in the control of the malonylation status of proteins involved in photosynthesis, as well as understanding their regulation. Protein malonylation is only one of many types of PTM, which compete with other forms of acylation, such as acetylation, succinylation, crotonylation, and butyrylation for the same lysine residues85. Thus, a complicated interaction may exist between malonylation and other PTMs, which ultimately determines protein functions. Unraveling the combined and hierarchical patterns of malonylation and other PTMs will be essential for understanding metabolic and photosynthetic pathways in cyanobacteria. Here, we compared the malonylome with the published Synechocystis acetylome86. Specifically, we compared 776 acetylation sites in 513 proteins with 598 malonylation sites in 339 proteins (Supplemental Table S8). We found that 199 malonylated proteins (58.7% of all malonylated proteins) were also acetylated 17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in Synechocystis (Fig. 7A). These overlapping proteins were mainly involved in photosynthesis and carbon metabolism, suggesting the similar regulation functions of the two modifications on these proteins (Fig. 7B). We also mapped the remaining proteins (314 acetylated proteins and 140 malonylated proteins) to KEGG pathways and the results demonstrated that 23 acetylated proteins involved in 14 specific pathways, while 59 malonylated proteins involved in 9 specific pathways (Supplemental Table S9). Therefore, these proteins may be involved in different pathways and regulated by different modifications. Malonylation and acetylation use malonyl-CoA and acetyl-CoA as cofactors, and malonyl-CoA is more reactive than acetyl-CoA; thus, it is likely that these two modifications could also have different functions in cellular metabolism. Consistent with this notion, we found that only 17.6% (105/598) of the malonylation sites and 14.7% (105/713) of the acetylation sites overlapped (Fig. 7C). This limited overlap suggests that these two types of acylations may be regulated by different enzymes and involved in different pathways in vivo. We also compared the sequence motifs of lysine malonylation and acetylation in Synechocystis. The frequency of distribution of acidic amino acids near malonylation or acetylation sites was almost the same (Fig. 7D). Aspartic acid or methionine/lysine was often located at the −1 position of acetylation or malonylation site, respectively. The distribution of basic amino acids near malonylation or acetylation sites was different. These differences suggest that these two PTMs might be catalyzed by different acetyl/malonyl-transferases and deacetylases/demalonylases, similar to Sir family deacylase in eukaryotes87. Malonylation chemically adds a bulkier three carbon acyl group to lysine compared to two carbons for acetylation. Malonylation changes the charge on lysine from +1 to −1, while acetylation is from +1 to 03. Thus, it is conceivable that these two PTMs may have a different effect on protein structure and function. Consistent with this notion, it has been reported that Tip60 can induce different p53 activities when using different acyl donors (acetyl-CoA or malonyl-CoA) 88. Sirtuins have different catalytic activities toward thirteen different types of acylated lysine 89. Interestingly, SIRT5 is a dedicated enzyme that removes malonylation and succinylation modifications in mice, suggesting that these two PTMs are different from acetylation90. In fact, we found two potential deacylases (slr0245; slr0168) based on the analysis of the complete Synechocystis genome. We speculate that these two predicted 18


Page 18 of 40

Page 19 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


deacylases may have different activities toward lysine malonylation and acetylation in Synechocystis. Our future studies will test this hypothesis.

Conclusion

A lysine malonylome analysis was performed on a model cyanobacterium, which led to the identification of 598 malonylation sites in 339 corresponding Synechocystis proteins. Our dataset represents the first and largest malonylome dataset in a photosynthetic organism to date. Bioinformatics analyses suggested the potential roles of lysine malonylation in multiple cyanobacterial cellular pathways. Our comparative analysis suggested that lysine malonylation and acetylation could possibly play distinct roles in cellular physiology. In conclusion, our results indicate that lysine malonylation is widespread in cyanobacteria and that it is probably one of the mechanisms that regulates photosynthesis and carbon metabolism in both plants and cyanobacteria, although further studies are needed to provide direct evidences on the functions of malonylation in photosynthesis. This study therefore provides a start point for further functional study of lysine malonylation in cyanobacteria and other photosynthetic organisms.

Acknowledgements

This work was supported by the National Key Research and Development Program (2016YFA0501304), the Chinese Academy of Sciences Grant QYZDY-SSW-SMC004, the Strategic Priority Research Program of the Chinese Academy of Sciences (grant no. XDB14030202).

Supporting Information The following additional data are available with the online version of this paper. Table S1: Details of the identified malonylated peptides in Synechocystis. Table S2: List of Synechocystis malonylome and other bacterial malonylomes. 19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table S3: GO classification of malonylated proteins in Synechocystis. Table S4: GO and pathway enrichment analysis of identified malonylated proteins in Synechocystis. Table S5: Comparison of lysine malonylation sites in Synechocystis with those in other two organisms. Table S6: List of malonylated proteins and the interactiong proteins. Table S7: List of KEGG pathways represented by the identified malonylated proteins. Table S8: Comparison of lysine malonylome with acetylome in Synechocystis. Table S9: List of KEGG pathways represented by the acetylated and malonylated proteins in Synechocystis. Figure S1: Evaluation of the quality of proteomic data. (A) Peptide score and mass error statistics for malonylated peptides. (B) Histogram illustrating the number of malonylation sites identified per protein. Figure S2: Protein-protein interaction (PPI) network of all identified malonylated proteins.

20


Page 20 of 40

Page 21 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References 1.

Xu, Y.; Ding, Y. X.; Ding, J.; Wu, L. Y.; Xue, Y., Mal-Lys: prediction of lysine

malonylation sites in proteins integrated sequence-based features with mRMR feature selection. Sci Rep 2016, 6, 38318. 2. Peng, C.; Lu, Z.; Xie, Z.; Cheng, Z.; Chen, Y.; Tan, M.; Luo, H.; Zhang, Y.; He, W.; Yang, K.; Zwaans, B. M.; Tishkoff, D.; Ho, L.; Lombard, D.; He, T. C.; Dai, J.; Verdin, E.; Ye, Y.; Zhao, Y., The first identification of lysine malonylation substrates and its regulatory enzyme. Molecular & cellular proteomics 2011, 10, (12), M111 012658. 3.

Hirschey, M. D.; Zhao, Y., Metabolic Regulation by Lysine Malonylation, Succinylation,

and Glutarylation. Molecular & cellular proteomics 2015, 14, (9), 2308-15. 4.

Colak, G.; Pougovkina, O.; Dai, L.; Tan, M.; Te Brinke, H.; Huang, H.; Cheng, Z.; Park,

J.; Wan, X.; Liu, X.; Yue, W. W.; Wanders, R. J.; Locasale, J. W.; Lombard, D. B.; de Boer, V. C.; Zhao, Y., Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation. Molecular & cellular proteomics 2015, 14, (11), 3056-71. 5.

Bao, X.; Zhao, Q.; Yang, T.; Fung, Y. M.; Li, X. D., A chemical probe for lysine

malonylation. Angew Chem Int Ed Engl 2013, 52, (18), 4883-6. 6.

Xu, J. Y.; Xu, Z.; Zhou, Y.; Ye, B. C., Lysine Malonylome May Affect the Central

Metabolism and Erythromycin Biosynthesis Pathway in Saccharopolyspora erythraea. J Proteome Res 2016, 15, (5), 1685-701. 7.

Qian, L.; Nie, L.; Chen, M.; Liu, P.; Zhu, J.; Zhai, L.; Tao, S. C.; Cheng, Z.; Zhao, Y.;

Tan, M., Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism. J Proteome Res 2016, 15, (6), 2060-71. 8.

Nishida, Y.; Rardin, M. J.; Carrico, C.; He, W.; Sahu, A. K.; Gut, P.; Najjar, R.; Fitch, M.;

Hellerstein, M.; Gibson, B. W.; Verdin, E., SIRT5 Regulates both Cytosolic and Mitochondrial Protein Malonylation with Glycolysis as a Major Target. Mol Cell 2015, 59, (2), 321-32. 9.

Du, Y.; Cai, T.; Li, T.; Xue, P.; Zhou, B.; He, X.; Wei, P.; Liu, P.; Yang, F.; Wei, T., Lysine

malonylation is elevated in type 2 diabetic mouse models and enriched in metabolic associated proteins. Molecular & cellular proteomics 2015, 14, (1), 227-36. 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10. Papanicolaou, K. N.; O'Rourke, B.; Foster, D. B., Metabolism leaves its mark on the powerhouse: recent progress in post-translational modifications of lysine in mitochondria. Front Physiol 2014, 5, 301. 11. Choudhary, C.; Weinert, B. T.; Nishida, Y.; Verdin, E.; Mann, M., The growing landscape of lysine acetylation links metabolism and cell signalling. Nature reviews. Molecular cell biology 2014, 15, (8), 536-50. 12. Abed, R. M. M.; Dobretsov, S.; Sudesh, K., Applications of cyanobacteria in biotechnology. J Appl Microbiol 2009, 106, (1), 1-12. 13. Nozzi, N. E.; Oliver, J. W.; Atsumi, S., Cyanobacteria as a Platform for Biofuel Production. Front Bioeng Biotechnol 2013, 1, 7. 14. Chellamuthu, V. R.; Alva, V.; Forchhammer, K., From cyanobacteria to plants: conservation of PII functions during plastid evolution. Planta 2013, 237, (2), 451-62. 15. Johnson, Z. I.; Zinser, E. R.; Coe, A.; McNulty, N. P.; Woodward, E. M.; Chisholm, S. W., Niche partitioning among Prochlorococcus ecotypes along ocean-scale environmental gradients. Science 2006, 311, (5768), 1737-40. 16. Kaneko, T.; Sato, S.; Kotani, H.; Tanaka, A.; Asamizu, E.; Nakamura, Y.; Miyajima, N.; Hirosawa, M.; Sugiura, M.; Sasamoto, S.; Kimura, T.; Hosouchi, T.; Matsuno, A.; Muraki, A.; Nakazaki, N.; Naruo, K.; Okumura, S.; Shimpo, S.; Takeuchi, C.; Wada, T.; Watanabe, A.; Yamada, M.; Yasuda, M.; Tabata, S., Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Research 1996, 3, (3), 109-36. 17. Williams, J. G. K., Construction of Specific Mutations in Photosystem-Ii Photosynthetic Reaction Center by Genetic-Engineering Methods in Synechocystis 6803. Method Enzymol 1988, 167, 766-778. 18. Gao, L.; Wang, J.; Ge, H.; Fang, L.; Zhang, Y.; Huang, X.; Wang, Y., Toward the complete proteome of Synechocystis sp. PCC 6803. Photosynth Res 2015, 126, (2-3), 203-19. 19. Noctor, G.; Dutilleul, C.; De Paepe, R.; Foyer, C. H., Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism. J Exp Bot 2004, 55, (394), 49-57. 22


Page 22 of 40

Page 23 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. Padmasree, K.; Padmavathi, L.; Raghavendra, A. S., Essentiality of mitochondrial oxidative metabolism for photosynthesis: optimization of carbon assimilation and protection against photoinhibition. Crit Rev Biochem Mol Biol 2002, 37, (2), 71-119. 21. Tan, X.; Yao, L.; Gao, Q.; Wang, W.; Qi, F.; Lu, X., Photosynthesis driven conversion of carbon dioxide to fatty alcohols and hydrocarbons in cyanobacteria. Metab Eng 2011, 13, (2), 169-76. 22. Singh, A. K.; Elvitigala, T.; Bhattacharyya-Pakrasi, M.; Aurora, R.; Ghosh, B.; Pakrasi, H. B., Integration of carbon and nitrogen metabolism with energy production is crucial to light acclimation in the cyanobacterium Synechocystis. Plant Physiol 2008, 148, (1), 467-78. 23. Wegener, K. M.; Singh, A. K.; Jacobs, J. M.; Elvitigala, T.; Welsh, E. A.; Keren, N.; Gritsenko, M. A.; Ghosh, B. K.; Camp, D. G., 2nd; Smith, R. D.; Pakrasi, H. B., Global proteomics reveal an atypical strategy for carbon/nitrogen assimilation by a cyanobacterium under diverse environmental perturbations. Molecular & cellular proteomics 2010, 9, (12), 2678-89. 24. Mo, R.; Yang, M.; Chen, Z.; Cheng, Z.; Yi, X.; Li, C.; He, C.; Xiong, Q.; Chen, H.; Wang, Q.; Ge, F., Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803. J Proteome Res 2015, 14, (2), 1275-86. 25. Takahashi, H.; Uchimiya, H.; Hihara, Y., Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry. Journal of Experimental Botany 2008, 59, (11), 3009-3018. 26. Berry, S.; Esper, B.; Karandashova, I.; Teuber, M.; Elanskaya, I.; Rogner, M.; Hagemann, M., Potassium uptake in the unicellular cyanobacterium Synechocystis sp strain PCC 6803 mainly depends on a Ktr-like system encoded by slr1509 (ntpJ). Febs Lett 2003, 548, (1-3), 53-58. 27. Ogawa, T.; Sonoike, K., Effects of Bleaching by Nitrogen Deficiency on the Quantum Yield of Photosystem II in Synechocystis sp PCC 6803 Revealed by Chl Fluorescence Measurements. Plant Cell Physiol 2016, 57, (3), 558-567. 28. Chen, Z.; Yang, M. K.; Li, C. Y.; Wang, Y.; Zhang, J.; Wang, D. B.; Zhang, X. E.; Ge, F., 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Phosphoproteomic analysis provides novel insights into stress responses in Phaeodactylum tricornutum, a model diatom. J Proteome Res 2014, 13, (5), 2511-23. 29. Cox, J.; Mann, M., MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 2008, 26, (12), 1367-1372. 30. Chen, Y.; Kwon, S. W.; Kim, S. C.; Zhao, Y. M., Integrated approach for manual evaluation of peptides identified by searching protein sequence databases with tandem mass spectra. J Proteome Res 2005, 4, (3), 998-1005. 31. Macek, B.; Gnad, F.; Soufi, B.; Kumar, C.; Olsen, J. V.; Mijakovic, I.; Mann, M., Phosphoproteome analysis of E-coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Molecular & Cellular Proteomics 2008, 7, (2), 299-307. 32. Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M., Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127, (3), 635-648. 33. Olsen, J. V.; Mann, M., Improved peptide identification in proteomics by two consecutive stages of mass spectrometric fragmentation. P Natl Acad Sci USA 2004, 101, (37), 13417-13422. 34. Conesa, A.; Gotz, S.; Garcia-Gomez, J. M.; Terol, J.; Talon, M.; Robles, M., Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 2005, 21, (18), 3674-3676. 35. Yu, N. Y.; Wagner, J. R.; Laird, M. R.; Melli, G.; Rey, S.; Lo, R.; Dao, P.; Sahinalp, S. C.; Ester, M.; Foster, L. J.; Brinkman, F. S. L., PSORTb 3.0: improved protein subcellular localization prediction with refined localization subcategories and predictive capabilities for all prokaryotes. Bioinformatics 2010, 26, (13), 1608-1615. 36. Jiao, X. L.; Sherman, B. T.; Huang, D. W.; Stephens, R.; Baseler, M. W.; Lane, H. C.; Lempicki, R. A., DAVID-WS: a stateful web service to facilitate gene/protein list analysis. Bioinformatics 2012, 28, (13), 1805-1806. 37. Huang, D. W.; Sherman, B. T.; Lempicki, R. A., Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009, 37, (1), 1-13. 24


Page 24 of 40

Page 25 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


38. O'Shea, J. P.; Chou, M. F.; Quader, S. A.; Ryan, J. K.; Church, G. M.; Schwartz, D., pLogo: a probabilistic approach to visualizing sequence motifs. Nat Methods 2013, 10, (12), 1211-1212. 39. Yang, M. K.; Qiao, Z. X.; Zhang, W. Y.; Xiong, Q.; Zhang, J.; Li, T.; Ge, F.; Zhao, J. D., Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp Strain PCC 7002. J Proteome Res 2013, 12, (4), 1909-1923. 40. Petersen, B.; Petersen, T. N.; Andersen, P.; Nielsen, M.; Lundegaard, C., A generic method for assignment of reliability scores applied to solvent accessibility predictions. Bmc Struct Biol 2009, 9. 41. Shannon, P.; Markiel, A.; Ozier, O.; Baliga, N. S.; Wang, J. T.; Ramage, D.; Amin, N.; Schwikowski, B.; Ideker, T., Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Res 2003, 13, (11), 2498-2504. 42. Meijer,

W.

G.,

The

Calvin

Cycle

Enzyme

Phosphoglycerate

Kinase

of

Xanthobacter-Flavus Required for Autotrophic Co2 Fixation Is Not Encoded by the Cbb Operon. J Bacteriol 1994, 176, (19), 6120-6126. 43. Pal, B.; Pybus, B.; Muccio, D. D.; Chattopadhyay, D., Biochemical characterization and crystallization of recombinant 3-phosphoglycerate kinase of Plasmodium falciparum. BBA-Proteins Proteom 2004, 1699, (1-2), 277-280. 44. Colak, G.; Xie, Z. Y.; Zhu, A. Y.; Dai, L. Z.; Lu, Z. K.; Zhang, Y.; Wan, X. L.; Chen, Y.; Cha, Y. H.; Lin, H. N.; Zhao, Y. M.; Tan, M. J., Identification of Lysine Succinylation Substrates and the Succinylation Regulatory Enzyme CobB in Escherichia coli. Molecular & Cellular Proteomics 2013, 12, (12), 3509-3520. 45. Yang, M. K.; Yang, Y. H.; Chen, Z.; Zhang, J.; Lin, Y.; Wang, Y.; Xiong, Q.; Li, T.; Ge, F.; Bryant, D. A.; Zhao, J. D., Proteogenomic analysis and global discovery of posttranslational modifications in prokaryotes. P Natl Acad Sci USA 2014, 111, (52), E5633-E5642. 46. Qian, L. L.; Nie, L. T.; Chen, M.; Liu, P.; Zhu, J.; Zhai, L. H.; Tao, S. C.; Cheng, Z. Y.; Zhao, Y. M.; Tan, M. J., Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism. J Proteome Res 2016, 15, (6), 2060-2071. 47. Singh, A. K.; Elvitigala, T.; Bhattacharyya-Pakrasi, M.; Aurora, R.; Ghosh, B.; Pakrasi, 25



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

H. B., Integration of carbon and nitrogen metabolism with energy production is crucial to light acclimation in the cyanobacterium Synechocystis. Plant Physiol 2008, 148, (1), 467-478. 48. Grundel, M.; Scheunemann, R.; Lockau, W.; Zilliges, Y., Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp PCC 6803. Microbiol-Sgm 2012, 158, 3032-3043. 49. Steuer, R.; Knoop, H.; Machne, R., Modelling cyanobacteria: from metabolism to integrative models of phototrophic growth. Journal of Experimental Botany 2012, 63, (6), 2259-2274. 50. Choudhary, C.; Kumar, C.; Gnad, F.; Nielsen, M. L.; Rehman, M.; Walther, T. C.; Olsen, J. V.; Mann, M., Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, (5942), 834-40. 51. Henriksen, P.; Wagner, S. A.; Weinert, B. T.; Sharma, S.; Bacinskaja, G.; Rehman, M.; Juffer, A. H.; Walther, T. C.; Lisby, M.; Choudhary, C., Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae. Molecular & cellular proteomics 2012, 11, (11), 1510-22. 52. Kim, S. C.; Sprung, R.; Chen, Y.; Xu, Y.; Ball, H.; Pei, J.; Cheng, T.; Kho, Y.; Xiao, H.; Xiao, L.; Grishin, N. V.; White, M.; Yang, X. J.; Zhao, Y., Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 2006, 23, (4), 607-18. 53. Liu, F.; Yang, M.; Wang, X.; Yang, S.; Gu, J.; Zhou, J.; Zhang, X. E.; Deng, J.; Ge, F., Acetylome analysis reveals diverse functions of lysine acetylation in Mycobacterium tuberculosis. Molecular & cellular proteomics 2014, 13, (12), 3352-66. 54. Lundby, A.; Lage, K.; Weinert, B. T.; Bekker-Jensen, D. B.; Secher, A.; Skovgaard, T.; Kelstrup, C. D.; Dmytriyev, A.; Choudhary, C.; Lundby, C.; Olsen, J. V., Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns. Cell reports 2012, 2, (2), 419-31. 55. Okanishi, H.; Kim, K.; Masui, R.; Kuramitsu, S., Acetylome with structural mapping reveals the significance of lysine acetylation in Thermus thermophilus. J Proteome Res 2013, 12, (9), 3952-68. 56. Weinert, B. T.; Wagner, S. A.; Horn, H.; Henriksen, P.; Liu, W. R.; Olsen, J. V.; Jensen, L. 26


Page 26 of 40

Page 27 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


J.; Choudhary, C., Proteome-wide mapping of the Drosophila acetylome demonstrates a high degree of conservation of lysine acetylation. Science signaling 2011, 4, (183), ra48. 57. Kim, G. W.; Yang, X. J., Comprehensive lysine acetylomes emerging from bacteria to humans. Trends Biochem Sci 2011, 36, (4), 211-218. 58. Zhang, J. M.; Sprung, R.; Pei, J. M.; Tan, X. H.; Kim, S.; Zhu, H.; Liu, C. F.; Grishin, N. V.; Zhao, Y. M., Lysine Acetylation Is a Highly Abundant and Evolutionarily Conserved Modification in Escherichia Coli. Molecular & Cellular Proteomics 2009, 8, (2), 215-225. 59. Park, J.; Chen, Y.; Tishkoff, D. X.; Peng, C.; Tan, M. J.; Dai, L. Z.; Xie, Z. Y.; Zhang, Y.; Zwaans, B. M. M.; Skinner, M. E.; Lombard, D. B.; Zhao, Y. M., SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways. Molecular Cell 2013, 50, (6), 919-930. 60. Rardin, M. J.; He, W. J.; Nishida, Y.; Newman, J. C.; Carrico, C.; Danielson, S. R.; Guo, A.; Gut, P.; Sahu, A. K.; Li, B.; Uppala, R.; Fitch, M.; Riiff, T.; Zhu, L.; Zhou, J.; Mulhern, D.; Stevens, R. D.; Ilkayeva, O. R.; Newgard, C. B.; Jacobson, M. P.; Hellerstein, M.; Goetzman, E. S.; Gibson, B. W.; Verdin, E., SIRT5 Regulates the Mitochondrial Lysine Succinylome and Metabolic Networks. Cell Metab 2013, 18, (6), 920-933. 61. Weinert, B. T.; Scholz, C.; Wagner, S. A.; Iesmantavicius, V.; Su, D.; Daniel, J. A.; Choudhary, C., Lysine Succinylation Is a Frequently Occurring Modification in Prokaryotes and Eukaryotes and Extensively Overlaps with Acetylation. Cell reports 2013, 4, (4), 842-851. 62. Kosono, S.; Tamura, M.; Suzuki, S.; Kawamura, Y.; Yoshida, A.; Nishiyama, M.; Yoshida, M., Changes in the Acetylome and Succinylome of Bacillus subtilis in Response to Carbon Source. Plos One 2015, 10, (6). 63. Pan, J. Y.; Chen, R.; Li, C. C.; Li, W. Y.; Ye, Z. C., Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus. J Proteome Res 2015, 14, (10), 4309-4318. 64. Yang, M. K.; Wang, Y.; Chen, Y.; Cheng, Z. Y.; Gu, J.; Deng, J. Y.; Bi, L. J.; Chen, C. B.; Mo, R.; Wang, X. D.; Ge, F., Succinylome Analysis Reveals the Involvement of Lysine Succinylation in Metabolism in Pathogenic Mycobacterium tuberculosis. Molecular & Cellular Proteomics 2015, 14, (4), 796-811. 65. He, D. L.; Wang, Q.; Li, M.; Damaris, R. N.; Yi, X. L.; Cheng, Z. Y.; Yang, P. F., Global 27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed. J Proteome Res 2016, 15, (3), 879-890. 66. Mizuno, Y.; Nagano-Shoji, M.; Kubo, S.; Kawamura, Y.; Yoshida, A.; Kawasaki, H.; Nishiyama, M.; Yoshida, M.; Kosono, S., Altered acetylation and succinylation profiles in Corynebacterium glutamicum in response to conditions inducing glutamate overproduction. Microbiology open 2016, 5, (1), 152-173. 67. Raines, C. A., The Calvin cycle revisited. Photosynthesis Research 2003, 75, (1), 1-10. 68. Ausar, S. F.; Foubert, T. R.; Hudson, M. H.; Vedvick, T. S.; Middaugh, C. R., Conformational stability and disassembly of Norwalk virus-like particles. Effect of pH and temperature. J Biol Chem 2006, 281, (28), 19478-88. 69. Bryan, M. A.; Cheng, H.; Brodsky, B., Sequence environment of mutation affects stability and folding in collagen model peptides of osteogenesis imperfecta. Biopolymers 2011, 96, (1), 4-13. 70. Smith, C. D.; Chattopadhyay, D.; Pal, B., Crystal structure of Plasmodium falciparum phosphoglycerate kinase: Evidence for anion binding in the basic patch. Biochem Bioph Res Co 2011, 412, (2), 203-206. 71. Joao, H. C.; Williams, R. J., The anatomy of a kinase and the control of phosphate transfer. Eur J Biochem 1993, 216, (1), 1-18. 72. Canback, B.; Andersson, S. G. E.; Kurland, C. G., The global phylogeny of glycolytic enzymes. P Natl Acad Sci USA 2002, 99, (9), 6097-6102. 73. Wolf, M.; Muller, T.; Dandekar, T.; Pollack, J. D., Phylogeny of Firmicutes with special reference to Mycoplasma (Mollicutes) as inferred from phosphoglycerate kinase amino acid sequence data. Int J Syst Evol Micr 2004, 54, 871-875. 74. Balmer, Y.; Koller, A.; Val, G. D.; Schurmann, P.; Buchanan, B. B., Proteomics uncovers proteins interacting electrostatically with thioredoxin in chloroplasts. Photosynth Res 2004, 79, (3), 275-80. 75. Michelet, L.; Zaffagnini, M.; Vanacker, H.; Le Marechal, P.; Marchand, C.; Schroda, M.; Lemaire, S. D.; Decottignies, P., In vivo targets of S-thiolation in Chlamydomonas reinhardtii. J Biol Chem 2008, 283, (31), 21571-8. 28


Page 28 of 40

Page 29 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


76. Rouhier, N.; Villarejo, A.; Srivastava, M.; Gelhaye, E.; Keech, O.; Droux, M.; Finkemeier, I.; Samuelsson, G.; Dietz, K. J.; Jacquot, J. P.; Wingsle, G., Identification of plant glutaredoxin targets. Antioxid Redox Signal 2005, 7, (7-8), 919-29. 77. Fratelli, M.; Demol, H.; Puype, M.; Casagrande, S.; Eberini, I.; Salmona, M.; Bonetto, V.; Mengozzi, M.; Duffieux, F.; Miclet, E.; Bachi, A.; Vandekerckhove, J.; Gianazza, E.; Ghezzi, P., Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci U S A 2002, 99, (6), 3505-10. 78. Tsukamoto, Y.; Fukushima, Y.; Hara, S.; Hisabori, T., Redox control of the activity of phosphoglycerate kinase in Synechocystis sp. PCC6803. Plant Cell Physiol 2013, 54, (4), 484-91. 79. Xiong, Q.; Chen, Z.; Ge, F., Proteomic analysis of post translational modifications in cyanobacteria. J Proteomics 2015, 134, 57-64. 80. Yang, M. K.; Qiao, Z. X.; Zhang, W. Y.; Xiong, Q.; Zhang, J.; Li, T.; Ge, F.; Zhao, J. D., Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp. Strain PCC 7002. J Proteome Res 2013, 12, (4), 1909-23. 81. Chen, Z.; Zhan, J.; Chen, Y.; Yang, M.; He, C.; Ge, F.; Wang, Q., Effects of Phosphorylation of beta Subunits of Phycocyanins on State Transition in the Model Cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 2015, 56, (10), 1997-2013. 82. Zhang, C. C.; Jang, J.; Sakr, S.; Wang, L., Protein phosphorylation on Ser, Thr and Tyr residues in cyanobacteria. Journal of molecular microbiology and biotechnology 2005, 9, (3-4), 154-66. 83. Mettler, T.; Muhlhaus, T.; Hemme, D.; Schottler, M. A.; Rupprecht, J.; Idoine, A.; Veyel, D.; Pal, S. K.; Yaneva-Roder, L.; Winck, F. V.; Sommer, F.; Vosloh, D.; Seiwert, B.; Erban, A.; Burgos, A.; Arvidsson, S.; Schonfelder, S.; Arnold, A.; Gunther, M.; Krause, U.; Lohse, M.; Kopka, J.; Nikoloski, Z.; Mueller-Roeber, B.; Willmitzer, L.; Bock, R.; Schroda, M.; Stitt, M., Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii. Plant Cell 2014, 26, (6), 2310-2350. 84. Wulff-Zottele, C.; Gatzke, N.; Kopka, J.; Orellana, A.; Hoefgen, R.; Fisahn, J.; Hesse, H., 29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Photosynthesis and metabolism interact during acclimation of Arabidopsis thaliana to high irradiance and sulphur depletion. Plant Cell Environ 2010, 33, (11), 1974-88. 85. Lin, H.; Su, X.; He, B., Protein lysine acylation and cysteine succination by intermediates of energy metabolism. ACS Chem Biol 2012, 7, (6), 947-60. 86. Mo, R.; Yang, M. K.; Chen, Z.; Cheng, Z. Y.; Yi, X. L.; Li, C. Y.; He, C. L.; Xiong, Q.; Chen, H.; Wang, Q.; Ge, F., Acetylome Analysis Reveals the Involvement of Lysine Acetylation in Photosynthesis and Carbon Metabolism in the Model Cyanobacterium Synechocystis sp PCC 6803. J Proteome Res 2015, 14, (2), 1275-1286. 87. Wagner, G. R.; Hirschey, M. D., Nonenzymatic Protein Acylation as a Carbon Stress Regulated by Sirtuin Deacylases. Molecular Cell 2014, 54, (1), 5-16. 88. Cheng, Z. Y.; Tang, Y.; Chen, Y.; Kim, S. C.; Liu, H. D.; Shawn, S. C.; Gu, W.; Zhao, Y. M., Molecular Characterization of Propionyllysines in Non-histone Proteins. Molecular & Cellular Proteomics 2009, 8, (1), 45-52. 89. Ooga, T.; Ohashi, Y.; Kuramitsu, S.; Koyama, Y.; Tomita, M.; Soga, T.; Masui, R., Degradation of ppGpp by Nudix Pyrophosphatase Modulates the Transition of Growth Phase in the Bacterium Thermus thermophilus. Journal of Biological Chemistry 2009, 284, (23), 15549-15556. 90. Du, J.; Zhou, Y.; Su, X.; Yu, J. J.; Khan, S.; Jiang, H.; Kim, J.; Woo, J.; Kim, J. H.; Choi, B. H.; He, B.; Chen, W.; Zhang, S.; Cerione, R. A.; Auwerx, J.; Hao, Q.; Lin, H., Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 2011, 334, (6057), 806-9.

30


Page 30 of 40

Page 31 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Legends Figure 1. Synopsis of proteome wide analysis of lysine malonylation in cyanobacteria. (A) Identification of lysine malonylation presented in photosynthetic organisms. The SDS-PAGE gel was stained with Coomassie Brilliant Blue or transferred to a polyvinylidene fluoride membrane and incubated with anti-malonyllysine antibody. (B) Identification of lysine malonylation presented in Synechocystis under various stressors. Protein (20 µg) was extracted from cells cultured under the normal condition (NC), high light condition (HL), nitrogen deficiency (-N), sodium malonate (+Mal), sodium chloride (+NaCl), calcium deficiency (-Ca), potassium deficiency (-K), or A5 deficiency (-A5). (C) Flowchart showing the experimental procedure for the malonylation proteomics study. (D) A representative MS/MS spectrum of a malonylated peptide sequence from the phycocyanin beta subunit (CpcB) protein. (E) Venn diagram showing the number of evolutionarily conserved malonylation proteins between Synechocystis and two other bacteria (E. coli and S. erythraea). Figure 2. Functional annotaion of the malonylated proteins. (A) Pie charts representation of the distribution of all the identified malonylated proteinsaccording to their biological processes, molecular function and subcellular localization. (B) Gene ontology and KEGG pathway enrichment analysis of all the identified malonylated proteins. Figure 3. Analysis of lysine malonylation sites. (A) Heatmap depicting the sequence motifs of malonylation sites across three bacteria (Synechocystis, E. coli and S. erythraea). Colors were plotted by using intensity map and represent the log10 of the ratio of frequencies within malonyl-13-mers versus non-malonyl-13-mers (red shows enrichment, blue shows depletion). (B) Proportion of malonylated and non-malonylated lysines localized in different secondary structures (α-helix, β-strand, coil and solvent accessibility) in Synechocystis, E. coli and S. erythraea. Figure 4. Protein-protein interaction networks of malonylated proteins. (A) Interaction network of malonylated proteins involved in photosynthesis. Identified proteins are highlighted in yellow and the interacted proteins are highlighted in light blue. (B) The top four highly connected interaction clusters. The clusters were generated by MCODE plugin in cytoscape tool. 31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Synopsis of lysine malonylation events involved in photosynthesis and carbon metabolism. (A) Schematic of photosynthesis process in Synechocystis. Malonylated proteins are highlighted in red. (B) Schematic illustration of malonylated enzymes involved in major metabolic pathways. Malonylated enzymes are highlighted in red. Figure 6. Effects of lysine malonylation on phosphoglycerate kinase (PGK) activity. (A) Schematic illustration of the metabolic pathway catalyzed by PGK. (B) Multiple sequence alignment of PGK from different species. The conserved malonylation sites are marked by the red arrow. (C) Crystal structure of PGK in Synechocystis. Six reliable malonylation sites (K48, K69, K201, K205, K246, and K251) are highlighted. (D) Coomassie blue staining of purified PGK and the K205E mutant. Enzyme activity of PGK (E) and K205E mutant (F). (G) Thermal stability of PGK and K205E mutant. Figure 7. Comparative analysis of lysine malonylome with acetylome in Synechocystis. (A) Overlapping of acetylated and malonylated proteins identified in Synechocystis. (B) Cellular component and KEGG Pathway analysis of 199 both acetylated and malonylated proteins. (C) Comparison of malonylation and acetylation sites in Synechocystis. (D) The frequency of amino acid residues around acetylated and malonylated lysines (p < 0.05).

32


Page 32 of 40

Page 33 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2

34


Page 34 of 40

Page 35 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4

36


Page 36 of 40

Page 37 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5

37



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 6

38


Page 38 of 40

Page 39 of 40

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7

39



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FOR TOC ONLY

40


Page 40 of 40

Malonylome Analysis Reveals the Involvement of Lysine Malonylation

Recommend Documents