Hsp70 (HSPA1) Lysine Methylation Status as a Potential Prognostic Factor in Metastatic High-Grade Serous Carcinoma.
ABSTRACT: Cellular proteins are subject to frequent methylation on lysine residues, introduced by specific methyltransferases, and each lysine residue can receive up to three methyl groups. Histone methylations, which are key determinants of chromatin state and transcriptional status, have been subject to particularly intense studies, but methylations on non-histone protein substrates are also abundant and biologically significant. Numerous studies have addressed lysine methylation in the realm of cancer biology. A recent study used an antibody-based approach to investigate the methylation of Lys-561 of the stress-inducible Hsp70 protein HSPA1, focusing exclusively on dimethylated HSPA1, concluding that it was elevated in cancer [Cho et al. (2012), Nat. Commun.,3, 1072]. In the present study, we have performed a more extensive analysis of HSPA1 methylation status in cancer samples, using protein mass spectrometry. We found that the four methylation states of Lys561 on HSPA1 (un-, mono-, di- and trimethylated) could be measured accurately and reproducibly in samples from carcinomas. We investigated HSPA1 methylation in 70 effusions, representing 53 high-grade serous ovarian carcinomas and 17 breast carcinomas. Notably, we found the trimethylated form of HSPA1 to be predominant in the cancer samples. HSPA1 methylation was studied for association with clinicopathologic parameters, including chemotherapy response and survival. The trimethylated form was more prevalent in breast carcinoma effusions (p = 0.014), whereas the dimethylated (p = 0.025), monomethylated (p = 0.004) and unmethylated (p = 0.021) forms were overrepresented in the ovarian carcinomas. For the ovarian carcinomas, the monomethylated (p = 0.028) and unmethylated (p = 0.007) forms were significantly related to the presence of higher residual disease volume, while the unmethylated form was significantly associated with poor overall (p = 0.015) and progression-free (p = 0.012) survival. In conclusion, lysine methylation of HSPA1 differs between metastatic breast and ovarian carcinoma, and unmethylated HSPA1 shows potential as a prognostic marker in high-grade serous carcinoma.
Project description:The in vitro deuteroacetylation of histones obtained from biological sources has been used previously in bottom-up mass spectrometry analyses to quantitate the percent of endogenous acetylation of specific lysine sites and/or peptides. In this report, derivatization of unmodified lysine residues on histones is used in combination with high performance mass spectrometry, including combined HPLC MS/MS, to distinguish and quantitate endogenously acetylated isoforms occurring within the same tryptic peptide sequence and to extend this derivatization strategy to other post-translational modifications, specifically methylation, dimethylation and trimethylation. The in vitro deuteroacetylation of monomethylated lysine residues is observed, though dimethylated or trimethylated residues are not derivatised. Comparison of the relative intensities ascribed to the deuteroacetylated and monomethylated species with the deuteroacetylated but unmethylated analog, provides an opportunity to estimate the percent of methylation at that site. In addition to the observed fragmentation patterns, the very high mass accuracy available on the Orbitrap mass spectrometer can be used to confirm the structural isoforms, and in particular to distinguish between trimethylated and acetylated species.
Project description:We show that the Saccharomyces cerevisiae ribosomal protein Rpl42ab (the identical product of the RPL42A and RPL42B genes) is monomethylated at Lys-40 and Lys-55. The methylation of Lys-40 is dependent upon the Ybr030w gene product; the methylation of Lys-55 is dependent upon the Set7 gene product. Ybr030w and SET7 genes both encode SET domain containing proteins homologous to known protein lysine methyltransferases, suggesting that their products are the specific enzymes responsible for the monomethylation of the two sites in Rpl42ab. We thus designate Ybr030w as Rkm3 and Set7 as Rkm4. Yeast strains with deletions in both the Ybr030w and SET7 genes produce unmethylated Rpl42ab. A slow growth phenotype was seen for the SET7 deletion strain and the double knock-out when grown in low concentrations of the eukaryotic protein synthesis inhibitor, cycloheximide. These results suggest that modification of Rpl42ab at Lys-55 can fine-tune its structure to avoid inhibition. An intact mass fragmentation approach ("top down mass spectrometry") was used to quantitate the extent of methylation of Rpl42ab. In wild-type strains, it was found that 78% was monomethylated at both Lys-40 and Lys-55 and that 22% was a mixture of species with either Lys-40 or Lys-55 monomethylated. The top down approach was also used to reevaluate the methylation sites of Rpl12ab. We found that the yeast Rpl12ab protein is dimethylated at the N-terminal proline residue, trimethylated at Lys-3 by Rkm2, and monomethylated at Arg-66.
Project description:Histone lysine methylations have primarily been linked to selective recruitment of reader or effector proteins that subsequently modify chromatin regions and mediate genome functions. Here, we describe a divergent role for histone H4 lysine 20 mono-methylation (H4K20me1) and demonstrate that it directly facilitates chromatin openness and accessibility by disrupting chromatin folding. Thus, accumulation of H4K20me1 demarcates highly accessible chromatin at genes, and this is maintained throughout the cell cycle. In vitro, H4K20me1-containing nucleosomal arrays with nucleosome repeat lengths (NRL) of 187 and 197 are less compact than unmethylated (H4K20me0) or trimethylated (H4K20me3) arrays. Concordantly, and in contrast to trimethylated and unmethylated tails, solid-state NMR data shows that H4K20 mono-methylation changes the H4 conformational state and leads to more dynamic histone H4-tails. Notably, the increased chromatin accessibility mediated by H4K20me1 facilitates gene expression, particularly of housekeeping genes. Altogether, we show how the methylation state of a single histone H4 residue operates as a focal point in chromatin structure control. While H4K20me1 directly promotes chromatin openness at highly transcribed genes, it also serves as a stepping-stone for H4K20me3-dependent chromatin compaction.
Project description:The human methyltransferases (MTases) METTL21A and VCP-KMT (METTL21D) were recently shown to methylate single lysine residues in Hsp70 proteins and in VCP, respectively. The yet uncharacterized MTase encoded by the YNL024C gene in Saccharomyces cerevisiae shows high sequence similarity to METTL21A and VCP-KMT, as well as to their uncharacterized paralogues METTL21B and METTL21C. Despite being most similar to METTL21A, the Ynl024c protein does not methylate yeast Hsp70 proteins, which were found to be unmethylated on the relevant lysine residue. Eukaryotic translation elongation factor eEF1A in yeast has been reported to contain four methylated lysine residues (Lys30, Lys79, Lys318 and Lys390), and we here show that the YNL024C gene is required for methylation of eEF1A at Lys390, the only of these methylations for which the responsible MTase has not yet been identified. Lys390 was found in a partially monomethylated state in wild-type yeast cells but was exclusively unmethylated in a ynl024c? strain, and over-expression of Ynl024c caused a dramatic increase in Lys390 methylation, with trimethylation becoming the predominant state. Our results demonstrate that Ynl024c is the enzyme responsible for methylation of eEF1A at Lys390, and in accordance with prior naming of similar enzymes, we suggest that Ynl024c is renamed to Efm6 (Elongation factor MTase 6).
Project description:Arginine methylation is a post-translational protein modification that is catalyzed by proteins known as arginine methyl transferases (RMTs). Recently, arginine methylation was postulated as an important modification in modulating biomolecular interactions. RMTs largely target nuclear proteins, so it is highly likely that they aid in modulating protein...DNA interactions. In this study, we probe the influence that sequential guanidinium methylation has on the energetics of the guanidinium...guanine and guanidinium...guanine...cytosine complexes using ab initio and double-hybrid density functional theory (DFT) methods. Structures of guanidinium...guanine complexes derived at the MP2/6-31+G** level of theory show that monomethylated, symmetrically dimethylated, and unsymmetrical dimethylated guanidiniums are all capable of forming guanidinium...guanine complexes. However, when cytosine is involved in a base pair to guanine, only the monomethylated and symmetrically dimethylated guanidinium groups are capable of forming hydrogen bond complexes with guanine. At the B2-PLYP/6-311++G** level of theory, we found that methylation of the guanidinium group stabilizes the formation of the guanidinium... guanine complex relative to the unmethylated guanidinium...guanine complex by approximately 2.5 kcal mol(-1). The biological implication of these findings are discussed.
Project description:We employ a stable isotope strategy wherein both histones and their methylations are labeled in synchronized human cells. This allows us to differentiate between old and new methylations on pre-existing versus newly synthesized histones. The strategy is implemented on K79 methylation in an isoform-specific manner for histones H3.1, H3.2, and H3.3. Although levels of H3.3K79 monomethylation are higher than that of H3.2/H3.1, the rate of establishing the K79 methylation is the same for all three isoforms. Surprisingly, we find that pre-existing "old" histones continue to be K79-monomethylated and -dimethylated at a rate equal to the newly synthesized histones. These observations imply that some degree of positional "scrambling" of K79 methylation occurs through the cell cycle.
Project description:Human L3MBTL1, which contains three malignant brain tumor (MBT) repeats, binds monomethylated and dimethylated lysines, but not trimethylated lysines, in several histone sequence contexts. In crystal structures of L3MBTL1 complexes, the monomethyl- and dimethyllysines insert into a narrow and deep cavity of aromatic residue-lined pocket 2, while a proline ring inserts into shallower pocket 1. We have also engineered a single Y to E substitution within the aromatic cage of the BPTF PHD finger, resulting in a reversal of binding preference from trimethyl- to dimethyllysine in an H3K4 sequence context. In both the "cavity insertion" (L3MBTL1) and "surface groove" (PHD finger) modes of methyllysine recognition, a carboxylate group both hydrogen bonds and ion pairs to the methylammonium proton. Our structural and binding studies of these two modules provide insights into the molecular principles governing the decoding of lysine methylation states, thereby highlighting a methylation state-specific layer of histone mark readout impacting on epigenetic regulation.
Project description:SET domain protein lysine methyltransferases (PKMTs) regulate transcription and other cellular functions through site-specific methylation of histones and other substrates. PKMTs catalyze the formation of monomethylated, dimethylated, or trimethylated products, establishing an additional hierarchy with respect to methyllysine recognition in signaling. Biochemical studies of PKMTs have identified a conserved position within their active sites, the Phe/Tyr switch, that governs their respective product specificities. To elucidate the mechanism underlying this switch, we have characterized a Phe/Tyr switch mutant of the histone H4 Lys-20 (H4K20) methyltransferase SET8, which alters its specificity from a monomethyltransferase to a dimethyltransferase. The crystal structures of the SET8 Y334F mutant bound to histone H4 peptides bearing unmodified, monomethyl, and dimethyl Lys-20 reveal that the phenylalanine substitution attenuates hydrogen bonding to a structurally conserved water molecule adjacent to the Phe/Tyr switch, facilitating its dissociation. The additional space generated by the solvent's dissociation enables the monomethyllysyl side chain to adopt a conformation that is catalytically competent for dimethylation and furnishes sufficient volume to accommodate the dimethyl epsilon-ammonium product. Collectively, these results indicate that the Phe/Tyr switch regulates product specificity through altering the affinity of an active-site water molecule whose dissociation is required for lysine multiple methylation.
Project description:Polycomb group proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by modifying chromatin states. One Polycomb group complex, the PRC2 complex, is composed of several proteins, including the histone H3 lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 and the WD-repeat protein embryonic ectoderm development (EED). Histone H3K27 can be monomethylated (H3K27me1), dimethylated (H3K27me2), or trimethylated (H3K27me3). However, it remains unclear what regulates the number of methyl groups added to H3K27 in a particular nucleosome. In mammalian cells, EED is present as four distinct isoforms, which are believed to be produced by utilizing four distinct, in-frame translation start sites in a common Eed mRNA. A mutation that disables all four EED isoforms produces defects in H3K27 methylation [Montgomery, N.D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S.J., Otte, A.P. & Magnuson, T. (2005). The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr. Biol., 15, 942-947]. To assess the roles of individual EED isoforms in H3K27 methylation, we first characterized three of the four EED isoform start sites and then demonstrated that individual isoforms are not necessary for H3K27me1, H3K27me2, or H3K27me3. Instead, we show that the core WD-40 motifs and the histone-binding region of EED alone are sufficient for the generation of all three marks, demonstrating that EED isoforms do not control the number of methyl groups added to H3K27.
Project description:Arsenic is the most ubiquitous environmental toxin and carcinogen and consequently ranks first on the Environmental Protection Agency's Superfund Priority List of Hazardous Substances. It is introduced primarily from geochemical sources and is acted on biologically, creating an arsenic biogeocycle. A common biotransformation is methylation to monomethylated, dimethylated and trimethylated species. Methylation is catalyzed by the ArsM (or AS3MT) arsenic(III) S-adenosylmethionine methyltransferase, an enzyme (EC 184.108.40.206) that is found in members of every kingdom from bacteria to humans. ArsM from the thermophilic alga Cyanidioschyzon sp. 5508 was expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapor-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=84.85, b=46.89, c=100.35 A, beta=114.25 degrees and one molecule in the asymmetric unit. Diffraction data were collected at the Advanced Light Source and were processed to a resolution of 1.76 A.