ABSTRACT
Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.
Subject(s)
Cell Differentiation , Dual-Specificity Phosphatases , Megakaryocytes , Mitogen-Activated Protein Kinase Phosphatases , Adult , Animals , Child , Female , Humans , Male , Middle Aged , Young Adult , Arginine/metabolism , Cell Line , Dual-Specificity Phosphatases/metabolism , Enzyme Stability , HEK293 Cells , MAP Kinase Signaling System , Megakaryocytes/cytology , Megakaryocytes/enzymology , Methylation , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase Phosphatases/metabolism , Myelodysplastic Syndromes/enzymology , Myelodysplastic Syndromes/pathology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolism , Polyubiquitin/metabolism , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/metabolism , Proteolysis , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , UbiquitinationABSTRACT
Protein arginine methyltransferases (PRMTs) are essential epigenetic and post-translational regulators in eukaryotic organisms. Dysregulation of PRMTs is intimately related to multiple types of human diseases, particularly cancer. Based on the previously reported PRMT1 inhibitors bearing the diamidine pharmacophore, we performed virtual screening to identify additional amidine-associated structural analogs. Subsequent enzymatic tests and characterization led to the discovery of a top lead K313 (2-(4-((4-carbamimidoylphenyl)amino)phenyl)-1H-indole-6-carboximidamide), which possessed low-micromolar potency with biochemical IC50 of 2.6 µM for human PRMT1. Limited selectivity was observed over some other PRMT isoforms such as CARM1 and PRMT7. Molecular modeling and inhibition pattern studies suggest that K313 is a nonclassic noncompetitive inhibitor to PRMT1. K313 significantly inhibited cell proliferation and reduced the arginine asymmetric dimethylation level in the leukaemia cancer cells.
ABSTRACT
Dynamic regulation of histone modification enzymes such as PRMT1 (protein arginine methyltransferase 1) determines the ordered epigenetic transitions in hematopoiesis. Sorting cells according to the expression levels of histone modification enzymes may further define subpopulations in hematopoietic lineages with unique differentiation potentials that are presently defined by surface markers. We discovered a vital near infrared dye, E84, that fluoresces brightly following binding to PRMT1 and excitation with a red laser. The staining intensity as measured by flow cytometry is correlated with the PRMT1 expression level. Importantly, E84 staining has no apparent negative effect on the proliferation of the labeled cells. Given that long-term hematopoietic stem cells (LT-HSCs) produce low levels of PRMT1, we used E84 to sort LT-HSCs from mouse bone marrow. We found that SLAM (the signalling lymphocyte activation molecule family) marker-positive LT-HSCs were enriched in the E84low cell fraction. We then performed bone marrow transplantations with E84high or E84low Lin-Sca1+Kit+ (LSK) cells and showed that whole blood cell lineages were successfully reconstituted 16 weeks after transplanting 200 E84low LSK cells. Thus, E84 is a useful new tool to probe the role of PRMT1 in hematopoiesis and leukemogenesis. Developing E84 and other small molecules to label histone modification enzymes provides a convenient approach without modifying gene loci to study the interaction between hematopoietic stem/progenitor cell epigenetic status and differentiation state.
Subject(s)
Blood Cells/metabolism , Carbocyanines/chemistry , Epigenesis, Genetic , Fluorescent Dyes/chemistry , Protein-Arginine N-Methyltransferases/genetics , Animals , Ataxin-1/metabolism , Blood Cells/pathology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Bone Marrow Transplantation , Cell Lineage , Flow Cytometry/methods , HEK293 Cells , Hematopoietic Stem Cells/metabolism , Hematopoietic Stem Cells/pathology , Humans , Leukocyte Common Antigens/metabolism , Mice , Protein-Arginine N-Methyltransferases/metabolism , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of ß-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.
Subject(s)
Alternative Splicing/genetics , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Erythroid Cells/cytology , Mutation , Phosphoproteins/metabolism , Proto-Oncogene Proteins/genetics , RNA Splicing Factors/metabolism , Humans , K562 Cells , Protein Isoforms/genetics , Protein-Arginine N-Methyltransferases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , T-Cell Acute Lymphocytic Leukemia Protein 1ABSTRACT
Antisense RNAs regulate the transcription and translation of the corresponding sense genes. Here, we report that an antisense RNA, AS-RBM15, is transcribed in the opposite direction within exon 1 of RBM15 RBM15 is a regulator of megakaryocyte (MK) differentiation and is also involved in a chromosome translocation t(1;22) in acute megakaryocytic leukemia. MK terminal differentiation is enhanced by up-regulation of AS-RBM15 expression and attenuated by AS-RBM15 knockdown. At the molecular level, AS-RBM15 enhances RBM15 protein translation in a CAP-dependent manner. The region of the antisense AS-RBM15 RNA, which overlaps with the 5'UTR of RBM15, is sufficient for the up-regulation of RBM15 protein translation. In addition, we find that transcription of both RBM15 and AS-RBM15 is activated by the transcription factor RUNX1 and repressed by RUNX1-ETO, a leukemic fusion protein. Therefore, AS-RBM15 is a regulator of megakaryocyte differentiation and may play a regulatory role in leukemogenesis.
Subject(s)
Cell Differentiation/genetics , Megakaryocytes/cytology , Megakaryocytes/metabolism , RNA, Antisense , RNA, Long Noncoding/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Cell Line, Tumor , Core Binding Factor Alpha 2 Subunit/metabolism , Gene Expression , Gene Expression Regulation , Hematopoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Protein Biosynthesis , Protein Transport , Sequence Deletion , Transcription, GeneticABSTRACT
RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578, leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-messenger RNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte differentiation for acute megakaryocytic leukemia.
Subject(s)
Protein Processing, Post-Translational , Protein-Arginine N-Methyltransferases/metabolism , RNA Splicing , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Cell Line , Humans , Methylation , Proteolysis , UbiquitinationABSTRACT
Protein arginine methyltransferase 1 (PRMT1) is involved in many biological activities, such as gene transcription, signal transduction, and RNA processing. Overexpression of PRMT1 is related to cardiovascular diseases, kidney diseases, and cancers; therefore, selective PRMT1 inhibitors serve as chemical probes to investigate the biological function of PRMT1 and drug candidates for disease treatment. Our previous work found trimethine cyanine compounds that effectively inhibit PRMT1 activity. In our present study, we systematically investigated the structure-activity relationship of cyanine structures. A pentamethine compound, E-84 (compound 50), showed inhibition on PRMT1 at the micromolar level and 6- to 25-fold selectivity over CARM1, PRMT5, and PRMT8. The cellular activity suggests that compound 50 permeated the cellular membrane, inhibited cellular PRMT1 activity, and blocked leukemia cell proliferation. Additionally, our molecular docking study suggested compound 50 might act by occupying the cofactor binding site, which provided a roadmap to guide further optimization of this lead compound.
Subject(s)
Carbocyanines/pharmacology , Enzyme Inhibitors/pharmacology , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Repressor Proteins/antagonists & inhibitors , Carbocyanines/chemical synthesis , Carbocyanines/chemistry , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Docking Simulation , Molecular Structure , Protein-Arginine N-Methyltransferases/metabolism , Repressor Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Protein arginine methylation is a posttranslational modification critical for a variety of biological processes. Misregulation of protein arginine methyltransferases (PRMTs) has been linked to many pathological conditions. Most current PRMT inhibitors display limited specificity and selectivity, indiscriminately targeting many methyltransferase enzymes that use S-adenosyl-l-methionine as a cofactor. Here we report diamidine compounds for specific inhibition of PRMT1, the primary type I enzyme. Docking, molecular dynamics, and MM/PBSA analysis together with biochemical assays were conducted to understand the binding modes of these inhibitors and the molecular basis of selective inhibition for PRMT1. Our data suggest that 2,5-bis(4-amidinophenyl)furan (1, furamidine, DB75), one leading inhibitor, targets the enzyme active site and is primarily competitive with the substrate and noncompetitive toward the cofactor. Furthermore, cellular studies revealed that 1 is cell membrane permeable and effectively inhibits intracellular PRMT1 activity and blocks cell proliferation in leukemia cell lines with different genetic lesions.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pentamidine/analogs & derivatives , Pentamidine/pharmacology , Protein-Arginine N-Methyltransferases/antagonists & inhibitors , Repressor Proteins/antagonists & inhibitors , Binding, Competitive/drug effects , Catalytic Domain/drug effects , Cell Line, Tumor , Cell Membrane Permeability/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Computer Simulation , Fluorescence Polarization , Humans , Immunoprecipitation , Kinetics , Leukemia/drug therapy , Leukemia/metabolism , Models, Molecular , Pentamidine/chemical synthesis , Protein Binding , Structure-Activity RelationshipABSTRACT
Praziquantel (PZQ), which is used to treat all forms of schistosomiasis, has been shown to induce strong T cell activities and decrease T regulatory cell levels. In our study, we investigated whether PZQ may be used as an adjuvant for a hepatitis B surface antigen (HBsAg) DNA vaccine (pcD-S2) in eliciting strong humoral and cellular responses. Our data demonstrate that PZQ as an adjuvant increased T cell proliferation and an HBsAg-specific antibody response that was characterized by a higher ratio of IgG2a/IgG1. Moreover, a higher level of IFN-gamma in CD4(+) and CD8(+) T cells were elicited. In addition, a significantly antigen-specific cytotoxic T lymphocyte response was also observed. The expression of TGF-beta can be induced by HBsAg, while PZQ as an adjuvant can inhibit the expression of TGF-beta and TGF-beta/Smad2,3 signaling. The frequency of CD4(+)CD25(+)Foxp3(+) Treg cells was reduced. Importantly, the regulatory function of CD4(+)CD25(+) Treg cells was correspondingly impaired. Together, these results suggest that PZQ can enhance humoral and cellular responses to HBsAg DNA vaccination through inhibition TGF-beta/Smad2,3 signaling.