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1.  SnapShot: Histone Modifications 
Cell  2014;159(2):458-458.e1.
doi:10.1016/j.cell.2014.09.037
PMCID: PMC4324475  PMID: 25303536
2.  Covalent Modification of a Cysteine Residue in the XPB Subunit of the General Transcription Factor TFIIH Through Single Epoxide Cleavage of the Transcription Inhibitor Triptolide** 
Triptolide is a key component of the traditional Chinese medicinal plant Thunder God Vine and has potent anticancer and immunosuppressive activities. It is an irreversible inhibitor of eukaryotic transcription through covalent modification of XPB, a subunit of the general transcription factor TFIIH. Cys342 of XPB was identified as the residue that undergoes covalent modification by the 12,13-epoxide group of triptolide. Mutation of Cys342 of XPB to threonine conferred resistance to triptolide on the mutant protein. Replacement of the endogenous wild-type XPB with the Cys342Thr mutant in a HEK293T cell line rendered it completely resistant to triptolide, thus validating XPB as the physiologically relevant target of triptolide. Together, these results deepen our understanding of the interaction between triptolide and XPB and have implications for the future development of new analogues of triptolide as leads for anticancer and immunosuppressive drugs.
doi:10.1002/anie.201408817
PMCID: PMC4314353  PMID: 25504624
inhibitors; medicinal chemistry; natural products; target validation; transcription factors
3.  SIRT5-Mediated Lysine Desuccinylation Impacts Diverse Metabolic Pathways 
Molecular cell  2013;50(6):919-930.
SUMMARY
Protein function is regulated by diverse posttranslational modifications. The mitochondrial sirtuin SIRT5 removes malonyl and succinyl moieties from target lysines. The spectrum of protein substrates subject to these modifications is unknown. We report systematic profiling of the mammalian succinylome, identifying 2,565 succinylation sites on 779 proteins. Most of these do not overlap with acetylation sites, suggesting differential regulation of succinylation and acetylation. Our analysis reveals potential impacts of lysine succinylation on enzymes involved in mitochondrial metabolism; e.g., amino acid degradation, the tricarboxylic acid cycle (TCA) cycle, and fatty acid metabolism. Lysine succinylation is also present on cytosolic and nuclear proteins; indeed, we show that a substantial fraction of SIRT5 is extra-mitochondrial. SIRT5 represses biochemical activity of, and cellular respiration through, two protein complexes identified in our analysis, pyruvate dehydrogenase complex and succinate dehydrogenase. Our data reveal widespread roles for lysine succinylation in regulating metabolism and potentially other cellular functions.
doi:10.1016/j.molcel.2013.06.001
PMCID: PMC3769971  PMID: 23806337
4.  Renewable, recombinant antibodies to histone post-translational modifications 
Nature methods  2013;10(10):10.1038/nmeth.2605.
Variability in the quality of antibodies to histone post-translational modifications (PTMs) presents widely recognized hindrance in epigenetics research. Here, by using antibody engineering technologies we produced recombinant antibodies directed to the trimethylated lysine residues of histone H3 with high specificity and affinity and no lot-to-lot variation. These recombinant antibodies performed well in common epigenetics applications, and their high specificity enabled us to identify positive and negative correlations among histone PTMs.
doi:10.1038/nmeth.2605
PMCID: PMC3828030  PMID: 23955773
5.  Brown Remodeling of White Adipose Tissue by SirT1-Dependent Deacetylation of Pparγ 
Cell  2012;150(3):620-632.
SUMMARY
Brown adipose tissue (BAT) can disperse stored energy as heat. Promoting BAT-like features in white adipose (WAT) is an attractive, if elusive therapeutic approach to staunch the current obesity epidemic. Here we report that gain-of-function of the NAD-dependent deacetylase SirT1 or loss-of-function of its endogenous inhibitor Deleted in breast cancer-1 (Dbc1) promote “browning” of WAT by deacetylating peroxisome proliferator-activated receptor (Ppar)-γ on Lys268 and Lys293. SirT1-dependent deacetylation of Lys268 and Lys293 is required to recruit the BAT program coactivator Prdm16 to Pparγ, leading to selective induction of BAT genes and repression of visceral WAT genes associated with insulin resistance. An acetylation-defective Pparγ mutant induces a brown phenotype in white adipocytes, while an acetylated mimetic fails to induce “brown” genes, but retains the ability to activate “white” genes. We propose that SirT1-dependent Pparγ deacetylation is a form of selective Pparγ modulation of potential therapeutic import.
doi:10.1016/j.cell.2012.06.027
PMCID: PMC3413172  PMID: 22863012
6.  Tumor suppression in the absence of p53-mediated cell cycle arrest, apoptosis, and senescence 
Cell  2012;149(6):1269-1283.
Summary
Cell-cycle arrest, apoptosis, and senescence are widely accepted as major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53K117R) or three (p533KR; K117R+K161R+K162R) of the critical p53 acetylation sites. While p53K117R/K117R cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p533KR/3KR cells. Surprisingly, unlike p53-null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53K117R/K117R or p533KR/3KR animals. Notably, p533KR retains the ability to modulate energy metabolism and reactive oxygen species (ROS) production by regulating metabolic p53 target genes. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.
doi:10.1016/j.cell.2012.04.026
PMCID: PMC3688046  PMID: 22682249
p53; Tip60; CBP/p300; K120; k164; Mdm2; Mdmx; acetylation; deacetylation; Growth arrest; apoptosis; p21; PUMA; DNA binding domain
7.  Catheter-Based Intramyocardial Delivery (NavX) of Adenovirus Achieves Safe and Accurate Gene Transfer in Pigs 
PLoS ONE  2013;8(1):e53007.
Background
Hepatocyte growth factor (HGF) is one of the major angiogenic factors being studied for the treatment of ischemic heart diseases. Our previous study demonstrated adenovirus-HGF was effective in myocardial ischemia models. The first clinical safety study showed a positive effect in patients with severe and diffused triple coronary disease.
Methods
12 Pigs were randomized (1∶1) to receive HGF, which was administered as five injections into the infarcted myocardium, or saline (control group). The injections were guided by EnSite NavX left ventricular electroanatomical mapping.
Results
The catheter-based injections caused no pericardial effusion, malignant arrhythmia or death. During mapping and injection, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, serum creatinine and creatine kinase-MB levels have no significant increase as compared to those before and after the injection in HGF group(P>0.05). HGF group has high HGF expression with Western blot, less myocardial infarct sizes by electroanatomical mapping (HGF group versus after saline group, 5.28±0.55 cm2 versus 9.06±1.06 cm2, P<0.01), better cardiac function with Gated-Single Photon Emission Computed Tomography compared with those in saline group. Histological, strongly increased lectin–positive microvessels and microvessel density were found in the myocardial ischemic regions in HGF group.
Conclusion
Intramyocardial injection guided by NavX system provides a method of feasible and safe percutaneous gene transfer to myocardial infarct regions.
doi:10.1371/journal.pone.0053007
PMCID: PMC3536803  PMID: 23301013
8.  Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification 
Cell  2011;146(6):1016-1028.
Summary
Using an integrated approach, we here report the identification of 67 novel histone marks, a discovery that increases the current number of known histone marks by about 70%. We verified one of the newly-identified marks, lysine crotonylation (Kcr), as a novel, evolutionarily-conserved histone post-translational modification. The unique structure and genomic localization of Kcr suggest that it is mechanistically and functionally different from lysine acetylation (Kac), a previously-described post-translational modification. Specifically, in both human somatic and mouse male germ cell genomes, histone Kcr marks either active promoters or potential enhancers. In male germinal cells immediately following meiosis, Kcr is enriched on sex chromosomes and specifically marks testis-specific genes, including a significant proportion of X-linked genes that escape sex chromosome inactivation in haploid cells. These results therefore dramatically extend the repertoire of histone PTM sites and designate Kcr as a specific mark of active sex chromosome-linked genes in post-meiotic male germ cells.
doi:10.1016/j.cell.2011.08.008
PMCID: PMC3176443  PMID: 21925322
Crotonyl-CoA; histones; lysine acetylation; lysine crotonylation; mass spectrometry; protein post-translational modifications
9.  Acetylation of yeast AMP-Activated Protein Kinase Controls Intrinsic Aging Independently of Caloric Restriction 
Cell  2011;146(6):969-979.
SUMMARY
(De)acetylation of histone and non-histone proteins is an important post-translational modification affecting many cellular processes. Here we report that NuA4 acetylation of Sip2, one of three regulatory β subunits of Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. We used mutations at four acetylation sites, K12, 16, 17 and 256, to study acetyl-Sip2 function. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), ultimately leads to slower growth but extends replicative lifespan. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a novel protein acetylation- phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging and extends replicative lifespan in yeast.
doi:10.1016/j.cell.2011.07.044
PMCID: PMC3176974  PMID: 21906795
10.  Acetylation of EGF Receptor Contributes to Tumor Cell Resistance to Histone Deacetylase Inhibitors 
Alteration of epidermal growth factor receptor (EGFR) is involved in various human cancers and has been intensively investigated. A plethora of evidence demonstrates that posttranslational modifications of EGFR play a pivotal role in controlling its function and metabolism. Here, we show that EGFR can be acetylated by CREB binding protein (CBP) acetyltransferase. Interestingly, EGFR acetylation affects its tyrosine phosphorylation, which may contribute to cancer cell resistance to histone deacetylase inhibitors (HDACIs). Since there is an increasing interest in using HDACIs to treat various cancers in the clinic, our current study provides insights and rationale for selecting effective therapeutic regimen. Consistent with the previous reports, we also show that HDACI combined with EGFR inhibitors achieves better therapeutic outcomes and provides a molecular rationale for the enhanced effect of combination therapy. Our results unveil a critical role of EGFR acetylation that regulates EGFR function, which may have an important clinical implication.
doi:10.1016/j.bbrc.2010.11.064
PMCID: PMC3049249  PMID: 21094134
11.  Strategic Plan for Lung Vascular Research 
The Division of Lung Diseases of the National Heart, Lung, and Blood Institute, with the Office of Rare Diseases Research, held a workshop to identify priority areas and strategic goals to enhance and accelerate research that will result in improved understanding of the lung vasculature, translational research needs, and ultimately the care of patients with pulmonary vascular diseases. Multidisciplinary experts with diverse experience in laboratory, translational, and clinical studies identified seven priority areas and discussed limitations in our current knowledge, technologies, and approaches. The focus for future research efforts include the following: (1) better characterizing vascular genotype–phenotype relationships and incorporating systems biology approaches when appropriate; (2) advancing our understanding of pulmonary vascular metabolic regulatory signaling in health and disease; (3) expanding our knowledge of the biologic relationships between the lung circulation and circulating elements, systemic vascular function, and right heart function and disease; (4) improving translational research for identifying disease-modifying therapies for the pulmonary hypertensive diseases; (5) establishing an appropriate and effective platform for advancing translational findings into clinical studies testing; and (6) developing the specific technologies and tools that will be enabling for these goals, such as question-guided imaging techniques and lung vascular investigator training programs. Recommendations from this workshop will be used within the Lung Vascular Biology and Disease Extramural Research Program for planning and strategic implementation purposes.
doi:10.1164/rccm.201006-0869WS
PMCID: PMC3029941  PMID: 20833821
right ventricle; pulmonary hypertension; metabolism; genomics; phenotyping
12.  Bioinformatic Analysis and Post-Translational Modification Crosstalk Prediction of Lysine Acetylation 
PLoS ONE  2011;6(12):e28228.
Recent proteomics studies suggest high abundance and a much wider role for lysine acetylation (K-Ac) in cellular functions. Nevertheless, cross influence between K-Ac and other post-translational modifications (PTMs) has not been carefully examined. Here, we used a variety of bioinformatics tools to analyze several available K-Ac datasets. Using gene ontology databases, we demonstrate that K-Ac sites are found in all cellular compartments. KEGG analysis indicates that the K-Ac sites are found on proteins responsible for a diverse and wide array of vital cellular functions. Domain structure prediction shows that K-Ac sites are found throughout a wide variety of protein domains, including those in heat shock proteins and those involved in cell cycle functions and DNA repair. Secondary structure prediction proves that K-Ac sites are preferentially found in ordered structures such as alpha helices and beta sheets. Finally, by mutating K-Ac sites in silico and predicting the effect on nearby phosphorylation sites, we demonstrate that the majority of lysine acetylation sites have the potential to impact protein phosphorylation, methylation, and ubiquitination status. Our work validates earlier smaller-scale studies on the acetylome and demonstrates the importance of PTM crosstalk for regulation of cellular function.
doi:10.1371/journal.pone.0028228
PMCID: PMC3229533  PMID: 22164248
13.  The first global screening of protein substrates bearing protein-bound 3,4-dihydroxyphenylalanine in E. coli and human mitochondria 
Journal of proteome research  2010;9(11):5705-5714.
Protein hydroxylation at proline and lysine residues is known to have important effects on cellular functions, such as the response to hypoxia. However, for protein hydroxylation at tyrosine residues (called protein-bound 3,4-dihydroxy-phenylalanine (PB-DOPA) has not been carefully examined. Here we report the first proteomics screening of the PB-DOPA protein substrates and their sites in E. coli and human mitochondria by nano-LC/MS/MS and protein sequence alignment using the PTMap algorithm. Our study identified 67 novel PB-DOPA sites in 43 E. coli proteins, and 9 novel PB-DOPA sites in 7 proteins from HeLa mitochondria. Bioinformatics analysis indicates that the structured region is more favored than the unstructured regions of proteins for the PB-DOPA modification. The PB-DOPA substrates in E. coli were dominantly enriched in proteins associated with carbohydrate metabolism. Our study showed that PB-DOPA may be involved in regulation of the specific activity of certain evolutionarily conserved proteins such as superoxide dismutase and glyceraldehyde 3-phosphate dehydrogenase, suggesting the conserved nature of the modification among distant biological species. The substrate proteins identified in this study offer a rich source for hunting their regulatory enzymes, and for further characterization of the possible contributions of this modification to cellular physiology and human diseases.
doi:10.1021/pr1005179
PMCID: PMC2974785  PMID: 20818827
Protein hydroxylation; protein-bound 3,4-dihydroxy-phenylalanine; PB-DOPA; E. coli; human mitochondria; tyrosine oxidation
14.  Correction: Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease 
PLoS ONE  2011;6(7):10.1371/annotation/e66e7e90-9503-46b8-91f9-abe4d5056ba1.
doi:10.1371/annotation/e66e7e90-9503-46b8-91f9-abe4d5056ba1
PMCID: PMC3144976
15.  An Improved Transplantation Strategy for Mouse Mesenchymal Stem Cells in an Acute Myocardial Infarction Model 
PLoS ONE  2011;6(6):e21005.
To develop an effective therapeutic strategy for cardiac regeneration using bone marrow mesenchymal stem cells (BM-MSCs), the primary mouse BM-MSCs (1st BM-MSCs) and 5th passage BM-MSCs from β-galactosidase transgenic mice were respectively intramyocardially transplanted into the acute myocardial infarction (AMI) model of wild type mice. At the 6th week, animals/tissues from the 1st BM-MSCs group, the 5th passage BM-MSCs group, control group were examined. Our results revealed that, compared to the 5th passage BM-MSCs, the 1st BM-MSCs had better therapeutic effects in the mouse MI model. The 1st BM-MSCs maintained greater differentiation potentials towards cardiomocytes or vascular endothelial cells in vitro. This is indicated by higher expressions of cardiomyocyte and vascular endothelial cell mature markers in vitro. Furthermore, we identified that 24 proteins were down-regulated and 3 proteins were up-regulated in the 5th BM-MSCs in comparison to the 1st BM-MSCs, using mass spectrometry following two-dimensional electrophoresis. Our data suggest that transplantation of the 1st BM-MSCs may be an effective therapeutic strategy for cardiac tissue regeneration following AMI, and altered protein expression profiles between the 1st BM-MSCs and 5th passage BM-MSCs may account for the difference in their maintenance of stemness and their therapeutic effects following AMI.
doi:10.1371/journal.pone.0021005
PMCID: PMC3117862  PMID: 21698117
16.  A novel acetylation of β-tubulin by San modulates microtubule polymerization via down-regulating tubulin incorporation 
Molecular Biology of the Cell  2011;22(4):448-456.
We report that San, an acetyltransferase required for sister chromatid cohesion, also acetylates β-tubulin at lysine 252. The acetylation happens only on free tubulin heterodimers, and it delays the incorporation of modified tubulins into microtubules in vivo.
Dynamic instability is a critical property of microtubules (MTs). By regulating the rate of tubulin polymerization and depolymerization, cells organize the MT cytoskeleton to accommodate their specific functions. Among many processes, posttranslational modifications of tubulin are implicated in regulating MT functions. Here we report a novel tubulin acetylation catalyzed by acetyltransferase San at lysine 252 (K252) of β-tubulin. This acetylation, which is also detected in vivo, is added to soluble tubulin heterodimers but not tubulins in MTs. The acetylation-mimicking K252A/Q mutants were incorporated into the MT cytoskeleton in HeLa cells without causing any obvious MT defect. However, after cold-induced catastrophe, MT regrowth is accelerated in San-siRNA cells while the incorporation of acetylation-mimicking mutant tubulins is severely impeded. K252 of β-tubulin localizes at the interface of α-/β-tubulins and interacts with the phosphate group of the α-tubulin-bound GTP. We propose that the acetylation slows down tubulin incorporation into MTs by neutralizing the positive charge on K252 and allowing tubulin heterodimers to adopt a conformation that disfavors tubulin incorporation.
doi:10.1091/mbc.E10-03-0203
PMCID: PMC3038643  PMID: 21177827
17.  Phosphorylation-Dependent 14-3-3 Binding to LRRK2 Is Impaired by Common Mutations of Familial Parkinson's Disease 
PLoS ONE  2011;6(3):e17153.
Background
Recent studies show that mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are the cause of the most common inherited and some sporadic forms of Parkinson's disease (PD). The molecular mechanism underlying the pathogenic role of LRRK2 mutations in PD remains unknown.
Methodology/Principal Findings
Using affinity purification and mass spectrometric analysis, we investigated phosphorylation sites and binding proteins of LRRK2 purified from mouse brain. We identified multiple phosphorylation sites at N-terminus of LRRK2 including S910, S912, S935 and S973. Focusing on the high stoichiometry S935 phosphorylation site, we developed an anti-pS935 specific antibody and showed that LRRK2 is constitutively phosphorylated at S935 in various tissues (including brain) and at different ages in mice. We find that 14-3-3 proteins (especially isoforms γ and η) bind LRRK2 and this binding depends on phosphorylation of S935. The binding of 14-3-3, with little effect on dimer formation of LRRK2, confers protection of the phosphorylation status of S935. Furthermore, we show that protein kinase A (PKA), but not LRRK2 kinase itself, can cause the phosphorylation of LRRK2 at S935 in vitro and in cell culture, suggesting that PKA is a potential upstream kinase that regulates LRRK2 function. Finally, our study indicates that the common PD-related mutations of LRRK2, R1441G, Y1699C and G2019S, decrease homeostatic phosphorylation levels of S935 and impair 14-3-3 binding of LRRK2.
Conclusions/Significance
LRRK2 is extensively phosphorylated in vivo, and the phosphorylation of specific sites (e.g. S935) determines 14-3-3 binding of LRRK2. We propose that 14-3-3 is an important regulator of LRRK2-mediated cellular functions. Our study suggests that PKA, a cAMP-dependent kinase involved in regulating dopamine physiology, is a potential upstream kinase that phosphorylates LRRK2 at S935. Furthermore, the reduction of phosphorylation/14-3-3 binding of LRRK2 due to the common familial PD-related mutations provides novel insight into the pathogenic mechanism of LRRK2-linked PD.
doi:10.1371/journal.pone.0017153
PMCID: PMC3046972  PMID: 21390248
18.  MS/MS/MS reveals false positive identification of histone serine methylation 
Journal of proteome research  2010;9(1):585-594.
Methylation of lysine and arginine residues is known to play a key role in regulating histone structure and function. However, methylation of other amino acid residues in histones has not been previously described. Using exhaustive nano-HPLC/MS/MS and blind protein sequence database searches, we tentatively assigned methylation to serine 28 of histone H3 from calf thymus. The assignment was in agreement with our stringent manual verification rules, co-elution in HPLC/MS/MS with its corresponding synthetic peptide, the dynamic nature of such methylation in distinct cell lines, and isotopic labeling. However, careful inspection of the MS/MS and MS/MS/MS spectra of a series of synthetic peptides confirmed that methylation actually occurs on K27 rather than on S28. The misassignment was caused by the fact that the (y9 + 14) of the putative S28-methylated peptide and (b9 + 18) ions of the K27 methylated peptide share the same m/z value (m/z 801). This MS/MS peak was used as the major evidence to assign methylation to S28 (consecutive y8 and (y9 + 14) ions). MS/MS/MS analysis revealed the false positive nature of serine methylation: the ambiguous ion at m/z 801 is indeed (b9 + 18), an ion resulting from an in vitro reaction in the gas phase during collisionally activated dissociation (CAD). When lysine (K27) was acetylated, the degree of such in vitro reactions was greatly reduced, and such reactions were completely eliminated when the C-terminus was blocked by carboxylic group derivatization. Moreover, such side-chain assisted C-terminal rearrangement was found to be charge dependent. In aggregate, these results suggest that extra caution should be taken in interpretation of post-translational modification (PTM) data and that MS/MS as well as MS/MS/MS of synthetic peptides are needed for verifying the identity of peptides bearing a novel PTM.
doi:10.1021/pr900864s
PMCID: PMC2801770  PMID: 19877717
MS/MS/MS analysis; false positive identification; protein methylation; charge dependence; side-chain assisted C-terminal rearrangement; C-terminal elimination; loss of C-terminal; histone modifications
19.  CHD7 cooperates with PBAF to control multipotent neural crest formation 
Nature  2010;463(7283):958-962.
Summary
Heterozygous mutations in the gene encoding CHD7, an ATP-dependent chromatin remodeler result in a complex constellation of congenital anomalies called CHARGE syndrome. Here we show that in humans and in Xenopus, CHD7 is essential for the formation of multipotent migratory neural crest cells, a transient cell population that is ectodermal in origin, but undergoes a major gene expression reprogramming to acquire a remarkably broad differentiation potential and ability to migrate throughout the body to give rise to bones, cartilages, nerves, and cardiac structures. We demonstrate that CHD7 function is essential for activation of core components of neural crest transcriptional circuitry, including Sox9, Twist and Slug. Moreover, the major features of CHARGE are recapitulated in Xenopus embryo by the downregulation of CHD7 levels or overexpression of its catalytically inactive ATP-ase mutant. We further show that in human multipotent neural crest cells, CHD7 associates with a BRG1-containing complex PBAF, and both factors co-occupy a neural crest-specific distal SOX9 enhancer, as well as a novel genomic element located upstream from TWIST1 gene and marked by H3K4me1. Furthermore, in the embryo CHD7 and PBAF act synergistically to promote neural crest gene expression and cell migration. Our work identifies an evolutionary conserved role for CHD7 in orchestrating neural crest gene expression programs, provides insights into the synergistic regulation of distal genomic elements by two distinct chromatin remodelers, and illuminates the patho-embryology of CHARGE syndrome.
doi:10.1038/nature08733
PMCID: PMC2890258  PMID: 20130577
20.  Identification and Validation of Eukaryotic Aspartate and Glutamate Methylation in Proteins 
Journal of proteome research  2008;7(3):1001-1006.
Methylation of lysine and arginine is known to be critical in cellular processes. However, methylation of other amino acidic residues has been largely overlooked. Here, we report a systematic screening for methylation of side chains of aspartate and glutamate (D/E-methylation), involving exhaustive nano-HPLC/MS/MS, a protein sequence database search, and manual verification. The putative D/E-methylated peptides were confirmed by MS/MS of synthetic peptides. Our analysis identified several D/E-methylation substrate proteins and their modification sites in human and yeast cells. To our knowledge, this is the first report conclusively identifying in vivo D/E-methylation substrates and their modification sites in eukaryotic cells, demonstrating that D/E-methylations are abundant protein modifications. The substrate proteins identified here provide a stepping stone for future biochemical characterization of protein methylation pathways.
doi:10.1021/pr0705338
PMCID: PMC2921173  PMID: 18220335
methylation; methylation of aspartic acid; methylation of glutamic acid; mass spectrometry; proteomics
21.  Identification and Verification of Lysine Propionylation and Butyrylation in Yeast Core Histones Using PTMap Software 
Journal of proteome research  2009;8(2):900-906.
Ten types of post-translational modifications (PTMs) known to be critical to diverse cellular functions have been described in core histone proteins. However, it remains unclear whether additional PTMs exist in histones, and if so, what roles these undiscovered signals play in epigenetic phenomena. Here, we report a systematic analysis of yeast histone PTMs by mass spectrometry in combination with protein sequence alignment using PTMap, a computer program we recently developed. We have identified, for the first time, multiple sites of lysine propionylation and butyrylation in yeast histones H2B, H3, and H4. We confirmed these modifications by Western blotting using modification-specific antibodies, MS/MS of synthetic peptides, and coelution of synthetic and in vivo-derived peptides from an HPLC column. The presence of multiple modification sites in several yeast histones suggests that these two PTMs are histone marks that are evolutionarily conserved among eukaryotes. In addition, we identified 14 novel mass shifts that do not match any known PTM, suggesting the presence of previously undescribed histone modifications. The chemical natures of these modifications remain to be determined. Our studies therefore expand current knowledge of the “histone code”.
doi:10.1021/pr8005155
PMCID: PMC2921183  PMID: 19113941
protein post-translational modifications; PTMap; histones; lysine propionylation; lysine butyrylation
22.  Complications in the Assignment of 14 and 28 Da Mass Shift Detected by Mass Spectrometry as in Vivo Methylation from Endogenous Proteins 
Analytical chemistry  2008;80(5):1721-1729.
Identification of protein methylation sites typically starts with database searching of MS/MS spectra of proteolytic digest of the target protein by allowing addition of 14 and 28 Da in the selected amino acid residues that can be methylated. Despite the progress in our understanding of lysine and arginine methylation, substrates and functions of protein methylation at other amino acid residues remain unknown. Here we report the analysis of protein methylation for p53, SMC3, iNOS, and MeCP2. We found that a large number of peptides can be modified on the lysine, arginine, histidine, and glutamic acid residues with a mass increase of 14 or 28 Da, consistent with methylation. Surprisingly, a majority of which did not demonstrate a corresponding mass shift when cells were cultured with isotope-labeled methionine, a precursor for the synthesis of S-adenosyl-L-methionine (SAM), which is the most commonly used methyl donor for protein methylation. These results suggest the possibility of either exogenous protein methylation during sample handling and processing for mass spectrometry or the existence of SAM-independent pathways for protein methylation. Our study found a high occurrence of protein methylation from SDS-PAGE isolated endogenous proteins and identified complications for assigning such modifications as in vivo methylation. This study provides a cautionary note for solely relying on mass shift for mass spectrometric identification of protein methylation and highlights the importance of in vivo isotope labeling as a necessary validation method.
doi:10.1021/ac7021025
PMCID: PMC2920732  PMID: 18247584
23.  Unbiased proteomic screen for binding proteins to modified lysines on histone H3 
Proteomics  2009;9(9):2343-2354.
We report a sensitive peptide pull-down approach in combination with protein identification by LC-MS/MS and qualitative abundance measurements by spectrum counting to identify proteins binding to histone H3 tail containing dimethyl lysine 4 (H3K4me2), dimethyl lysine 9 (H3K9me2), or acetyl lysine 9 (H3K9ac). Our study identified 86 nuclear proteins that associate with the histone H3 tail peptides examined, including seven known direct binders and 16 putative direct binders with conserved PHD finger, bromodomain, and WD40 domains. The reliability of our proteomic screen is supported by the fact that more than one-third of the proteins identified were previously described to associate with histone H3 tail directly or indirectly. To our knowledge, the results presented here are the most comprehensive analysis of H3K4me2, H3K9me2, and H3K9ac associated proteins and will provide a useful resource for researchers studying the mechanisms of histone code effector proteins.
doi:10.1002/pmic.200800600
PMCID: PMC2914554  PMID: 19337993
Epigenetics; Histone code effectors; Histone modifications; Proteomics
24.  Acetylation Is Indispensable for p53 Activation 
Cell  2008;133(4):612-626.
SUMMARY
The activation of the tumor suppressor p53 facilitates the cellular response to genotoxic stress; however, the p53 response can only be executed if its interaction with its inhibitor Mdm2 is abolished. There have been conflicting reports on the question of whether p53 posttranslational modifications, such as phosphorylation or acetylation, are essential or only play a subtle, fine-tuning role in the p53 response. Thus, it remains unclear whether p53 modification is absolutely required for its activation. We have now identified all major acetylation sites of p53. Although unacetylated p53 retains its ability to induce the p53-Mdm2 feedback loop, loss of acetylation completely abolishes p53-dependent growth arrest and apoptosis. Notably, acetylation of p53 abrogates Mdm2-mediated repression by blocking the recruitment of Mdm2 to p53-responsive promoters, which leads to p53 activation independent of its phosphorylation status. Our study identifies p53 acetylation as an indispensable event that destabilizes the p53-Mdm2 interaction and enables the p53-mediated stress response.
doi:10.1016/j.cell.2008.03.025
PMCID: PMC2914560  PMID: 18485870
25.  Structure-Specific Recognition Protein 1 Facilitates Microtubule Growth and Bundling Required for Mitosis▿†  
Molecular and Cellular Biology  2009;30(4):935-947.
Tight regulation of microtubule (MT) dynamics is essential for proper chromosome movement during mitosis. Here we show, using mammalian cells, that structure-specific recognition protein 1 (SSRP1) is a novel regulator of MT dynamics. SSRP1 colocalizes with the spindle and midbody MTs, and associates with MTs both in vitro and in vivo. Purified SSRP1 facilitates tubulin polymerization and MT bundling in vitro. Knockdown of SSRP1 inhibits the growth of MTs and leads to disorganized spindle structures, reduction of K-fibers and midbody fibers, disrupted chromosome movement, and attenuated cytokinesis in vivo. These results demonstrate that SSRP1 is crucial for MT growth and spindle assembly during mitosis.
doi:10.1128/MCB.01379-09
PMCID: PMC2815565  PMID: 19995907

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