ABSTRACT
Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d0-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.
Subject(s)
Histones/metabolism , Acetylation , Cell Line, Tumor , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Humans , Mass Spectrometry , MethylationABSTRACT
Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Purine-Nucleoside Phosphorylase/deficiency , Alternative Splicing , Antineoplastic Agents/chemistry , Biomarkers , Cell Line, Tumor , Drug Synergism , Enzyme Inhibitors/chemistry , Humans , Methylation , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Protein-Arginine N-Methyltransferases/chemistry , Substrate SpecificityABSTRACT
Evasion of the potent tumour suppressor activity of p53 is one of the hurdles that must be overcome for cancer cells to escape normal regulation of cellular proliferation and survival. In addition to frequent loss of function mutations, p53 wild-type activity can also be suppressed post-translationally through several mechanisms, including the activity of PRMT5. Here we describe broad anti-proliferative activity of potent, selective, reversible inhibitors of protein arginine methyltransferase 5 (PRMT5) including GSK3326595 in human cancer cell lines representing both hematologic and solid malignancies. Interestingly, PRMT5 inhibition activates the p53 pathway via the induction of alternative splicing of MDM4. The MDM4 isoform switch and subsequent p53 activation are critical determinants of the response to PRMT5 inhibition suggesting that the integrity of the p53-MDM4 regulatory axis defines a subset of patients that could benefit from treatment with GSK3326595.
Subject(s)
Nuclear Proteins/metabolism , Protein-Arginine N-Methyltransferases/metabolism , Proto-Oncogene Proteins/metabolism , RNA Splicing/genetics , Tumor Suppressor Protein p53/metabolism , Alternative Splicing/genetics , Antineoplastic Agents , Arginine/analogs & derivatives , Arginine/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Cycle Proteins , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Enzyme Inhibitors/pharmacology , Humans , Nuclear Proteins/genetics , Protein Isoforms/genetics , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Proto-Oncogene Proteins/genetics , Tumor Suppressor Protein p53/genetics , snRNP Core Proteins/metabolismABSTRACT
Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin, cx40.8, is co-expressed with cx43 in the population of proliferating cells during fin regeneration. Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.
Subject(s)
Animal Fins/chemistry , Animal Fins/physiology , Connexins/metabolism , Regeneration , Zebrafish Proteins/metabolism , Zebrafish/metabolism , Animals , Cell Proliferation , Connexin 43 , Gap Junction alpha-5 ProteinABSTRACT
In addition to having a Cx43 ortholog, the zebrafish genome also contains a Cx43-like gene, Cx40.8. Here, we investigate the expression of cx40.8 in zebrafish fins and the function of Cx40.8 in HeLa cells. We find that cx40.8 is present in the same population of dividing cells as cx43. Unlike Cx43, dye coupling assays suggest that Cx40.8 only inefficiently forms functional gap junction channels. However, co-transfection reveals that Cx40.8 can co-localize with Cx43 in gap junction plaques, and that the resulting plaques contain functional gap junction channels. Together, these data suggest the possibility that Cx40.8 may functionally interact with Cx43 to regulate cell proliferation in vivo.