Facile synthesis of N-6 adenosine modified analogue toward S-adenosyl methionine derived probe for protein arginine methyltransferases

Chemically modified cellular co-factors that provide function,such as immobilization or incorporation of fluorescent dyes,are valuable probes of biological activity.A convenient route to obtain S-adenosyl methionine（AdoMet） analogues modified at N-6 adenosine to feature a linker terminating in azide functionality is described herein.Subsequent decoration of such AdoMet analogues with guanidinium terminated linkers leads to novel potential bisubstrate inhibitors for protein arginine methyltrans-ferases, PRMTs.

Protein arginine methyltransferase 6 is a novel substrate of protein arginine methyltransferase 1

BACKGROUND Post-translational modifications play key roles in various biological processes.Protein arginine methyltransferases(PRMTs)transfer the methyl group to specific arginine residues.Both PRMT1 and PRMT6 have emerges as crucial factors in the development and progression of multiple cancer types.We posit that PRMT1 and PRMT6 might interplay directly or in-directly in multiple ways accounting for shared disease phenotypes.AIM To investigate the mechanism of the interaction between PRMT1 and PRMT6.METHODS Gel electrophoresis autoradiography was performed to test the methyltranferase activity of PRMTs and characterize the kinetics parameters of PRMTs.Liquid chromatography-tandem mass spectrometryanalysis was performed to detect the PRMT6 methylation sites.RESULTS In this study we investigated the interaction between PRMT1 and PRMT6,and PRMT6 was shown to be a novel substrate of PRMT1.We identified specific arginine residues of PRMT6 that are methylated by PRMT1,with R106 being the major methylation site.Combined biochemical and cellular data showed that PRMT1 downregulates the enzymatic activity of PRMT6 in histone H3 methylation.CONCLUSION PRMT6 is methylated by PRMT1 and R106 is a major methylation site induced by PRMT1.PRMT1 methylation suppresses the activity of PRMT6.

Combining protein arginine methyltransferase inhibitor and antiprogrammed death-ligand-1 inhibits pancreatic cancer progression

BACKGROUND Immunotherapy targeting programmed death-1(PD-1)or programmed deathligand-1(PD-L1)has been shown to be effective in a variety of malignancies but has poor efficacy in pancreatic ductal adenocarcinoma(PDAC).Studies have shown that PD-L1 expression in tumors is an important indicator of the efficacy of immunotherapy.Tumor cells usually evade chemotherapy and host immune surveillance by epigenetic changes.Protein arginine methylation is a common posttranslational modification.Protein arginine methyltransferase(PRMT)1 is deregulated in a wide variety of cancer types,whose biological role in tumor immunity is undefined.AIM To investigate the combined effects and underlying mechanisms of anti-PD-L1 and type I PRMT inhibitor in pancreatic cancer in vivo.METHODS PT1001B is a novel type I PRMT inhibitor with strong activity and good selectivity.A mouse model of subcutaneous Panc02-derived tumors was used to evaluate drug efficacy,toxic and side effects,and tumor growth in vivo.By flow cytometry,we determined the expression of key immune checkpoint proteins,detected the apoptosis in tumor tissues,and analyzed the immune cells.Immunohistochemistry staining for cellular proliferation-associated nuclear protein Ki67,TUNEL assay,and PRMT1/PD-L1 immunofluorescence were used to elucidate the underlying molecular mechanism of the antitumor effect.RESULTS Cultured Panc02 cells did not express PD-L1 in vitro,but tumor cells derived from Panc02 transplanted tumors expressed PD-L1.The therapeutic efficacy of anti-PD-L1 mAb was significantly enhanced by the addition of PT1001B as measured by tumor volume(1054.00±61.37 mm3 vs 555.80±74.42 mm3,P<0.01)and tumor weight(0.83±0.06 g vs 0.38±0.02 g,P<0.05).PT1001B improved antitumor immunity by inhibiting PD-L1 expression on tumor cells(32.74%±5.89%vs 17.95%±1.92%,P<0.05).The combination therapy upregulated tumorinfiltrating CD8+T lymphocytes(23.75%±3.20%vs 73.34%±4.35%,P<0.01)and decreased PD-1+leukocytes(35.77%±3.30%vs 6.48%±1.08%,P<0.001)in tumor tissue compared to the control.In addition,PT1001B amplified the inhibitory effect of anti-PD-L1 on tumor cell proliferation and enhanced the induction of tumor cell apoptosis.PRMT1 downregulation was correlated with PD-L1 downregulation.CONCLUSION PT1001B enhances antitumor immunity and combining it with anti-PD-L1 checkpoint inhibitors provides a potential strategy to overcome anti-PD-L1 resistance in PDAC.

Screening of Substrates of Protein Arginine Methyltransferase 1 in Glioma

Objective To screen the asymmetric dimethyl arginines (ADMA)-containing proteins which could combine with protein arginine methyltransferase 1 (PRMT1). Methods Western blot was adopted to identify the expression of PRMT1 and the proteins with ADMA in glioma cell lines and normal brain tissues, and then to detect the changes of ADMA level after knock-down of PRMT1 with RNAi transfection in U87MG cells. Co-Immunoprecipitation (Co-IP), western blot, and sliver staining were employed to screen the candidate binding proteins of PRMT1. Then liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify the binding proteins of PRMT1. Results The expression of PRMT1 and some levels of ADMA were higher in glioma cell lines than in normal brain tissues. After knocking down PRMT1, some ADMA levels were found declined. After screening the binding proteins of PRMT1 with Co-IP and LC-MS/MS, 26 candidate binding proteins were identified. Among them, 6 candidate proteins had higher ions scores (>38) and bioinformation analysis predicted that SEC23-IP, ANKHD1-EIF4EBP3 protein, and 1-phosphatidylinositol-3-phosphate 5-kinase isoform 2 had possible methylated aginine sites. Conclusions The high expression of PRMT1 in glioma may induce the change of ADMA levels. Altogether 26 candidate proteins were identified, which contain ADMA and specifically bind with PRMT1.

Inhibition of histone methyltransferase PRMT5 attenuates cisplatininduced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatininduced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

Inhibition of Nonsmall Cell Lung Cancer Cell Migration by Protein Arginine Methyltransferase 1-small Hairpin RNA Through Inhibiting Epithelial-mesenchymal Transition,Extracellular Matrix Degradation, and Src Phosphorylation In Vitro

Background：Protein arginine methyltransferases 1 （PRMT1） is over-expressed in a variety of cancers,including lung cancer,and is correlated with a poor prognosis of tumor development.This study aimed to investigate the role of PRMT1 in nonsmall cell lung cancer （NSCLC） migration in vitro.Methods：In this study,PRMT1 expression in the NSCLC cell line A549 was silenced using lentiviral vector-mediated short hairpin RNAs.Cell migration was measured using both scratch wound healing and transwell cell migration assays.The mRNA expression levels of matrix metalloproteinase 2 （MMP-2） and tissue inhibitor ofmetalloproteinase 1,2 （TIMP l,2） were measured using quantitative real-time reverse transcription-polymerase chain reaction.The expression levels of protein markers for epithelial-mesenchymal transition （EMT） （E-cadherin,N-cadherin）,focal adhesion kinase （FAK）,Src,AKT,and their corresponding phosphorylated states were detected by Western blot.Results：Cell migration was significantly inhibited in the PRMT1 silenced group compared to the control group.The mRNA expression of MMP-2 decreased while TIMP 1 and TIMP2 increased significantly.E-cadherin mRNA expression also increased while N-cadherin decreased.Only phosphorylated Src levels decreased in the silenced group while FAK or AKT remained unchanged.Conclusions：PRMT1-small hairpin RNA inhibits the migration abilities of NSCLC A549 cells by inhibiting EMT,extracellular matrix degradation,and Src phosphorylation in vitro.

The enzymatic activity of Arabidopsis protein arginine methyltransferase 10 is essential for flowering time regulation

Arabidopsis AtPRMT10 is a plant-specific type I protein arginine methyltransferase that can asymmetrically dimethylate arginine 3 of histone H4 with auto-methylation activity.Mutations of AtPRMT10 derepress FLOWERING LOCUS C(FLC)expression resulting in a late-flowering phenotype.Here,to further investigate the biochemical characteristics of AtPRMT10,we analyzed a series of mutated forms of the AtPRMT10 protein.We demon-strate that the conserved“VLD”residues and“double-E loop”are essential for enzymatic activity of AtPRMT10.In addition,we show that Arg54 and Cys259 of AtPRMT10,two residues unreported in animals,are also important for its enzymatic activity.We find that Arg13 of AtPRMT10 is the auto-methylation site.However,substitution of Arg13 to Lys13 does not affect its enzymatic activity.In vivo complementation assays reveal that plants expressing AtPRMT10 with VLD-AAA,E143Q or E152Q mutations retain high levels of FLC expression and fail to rescue the late-flowering phenotype of atprmt10 plants.Taken together,we conclude that the methyltransferase activity of AtPRMT10 is essential for repressing FLC expression and promoting flowering in Arabidopsis.

Discovery of a potent and dual-selective bisubstrate inhibitor for protein arginine methyltransferase 4/5

Protein arginine methyltransferases(PRMTs)have been implicated in the progression of many diseases.Understanding substrate recognition and specificity of individual PRMT would facilitate the discovery of selective inhibitors towards future drug discovery.Herein,we reported the design and synthesis of bisubstrate analogues for PRMTs that incorporate a S-adenosylmethionine(SAM)analogue moiety and a tripeptide through an alkyl substituted guanidino group.Compound AH237 is a potent and selective inhibitor for PRMT4 and PRMT5 with a half-maximal inhibition concentration(IC_(50)) of 2.8 and0.42 nmol/L,respectively.Computational studies provided a plausible explanation for the high potency and selectivity of AH237 for PRMT4/5 over other 40 methyltransferases.This proof-of-principle study outlines an applicable strategy to develop potent and selective bisubstrate inhibitors for PRMTs,providing valuable probes for future structural studies.

Advances in Research of PRMT7,DVL3 and Gastric Cancer

Protein arginine methyltransferase 7(PRMT7)is closely related to the formation of lung cancer,breast cancer and other malignant tumors,and it may be a potential target gene for malignant tumor therapy.Dishevelleds(DVLs)are key factors involved in cell proliferation,invasion and metastasis.Among the dishevelleds,the dishevelled segment polarity protein 3(DVL3)is a key protein in the Wnt signaling pathway,and its abnormal expression plays an important role in the occurrence and development of malignant tumors.This paper reviewed the advances in research of PRMT7 and DVL3 in gastric cancer.

A systematic survey of PRMT interactomes reveals the key roles of arginine methylation in the global control of RNA splicing and translation

Thousands of proteins undergo arginine methylation,a widespread post-translational modification catalyzed by several protein arginine methyltransferases(PRMTs).However,global understanding of their biological functions is limited due to the lack of a complete picture of the catalytic network for each PRMT.Here,we systematically identified interacting proteins for all human PRMTs and demonstrated their functional importance in mRNA splicing and translation.We demonstrated significant overlapping of interactomes of human PRMTs with the known methylarginine-containing proteins.Different PRMTs are functionally redundant with a high degree of overlap in their substrates and high similarities between their putative methylation motifs.Importantly,RNA-binding proteins involved in regulating RNA splicing and translation contain highly enriched arginine methylation regions.Moreover,inhibition of PRMTs globally alternates alternative splicing(AS)and suppresses translation.In particular,ribosomal proteins are extensively modified with methylarginine,and mutations in their methylation sites suppress ribosome assembly,translation,and eventually cell growth.Collectively,our study provides a global view of different PRMT networks and uncovers critical functions of arginine methylation in regulating mRNA splicing and translation.

Ep,gallocatechin-3-gallate ameliorates erectile function in aged rats via regulation of PRMT1/ DDAH/ADMA/NOS metabolism pathway

Aging-related ED is predominantly attributed to neurovascular dysfunction mediated by NO suppression and increased oxidative stress in penis. The alterations of protein arginine methyltransferases 1 （PRMT1）/dimethylarginine dimethylaminohydrolase （DDAH）/ asymmetrical dimethylarginine （ADMA）/NO synthase （NOS） pathway regulate NO production in the vascular endothelium. Epigallocatechin-3-gallate （EGCG） is one of the most abundant and antioxidative ingredients isolated from green tea. In the present study, 40 Sprague-Dawley rats were randomly distributed into four groups： one young rat group and three aged rat groups treated with daily gavage feedings of EGCG at doses of O, 10 mg kg-1, and 100 mg kg-1 for 12 weeks, respectively. Erectile function was assessed by electrical stimulation of the cavernous nerves with intracavernous pressure （ICP） measurement. After euthanasia, penile tissue was investigated using Western blot and ELISA to assess the PRMTI/DDAH/ADMA/NOS metabolism pathway. Superoxide dismutase （SOD） and malondialdehyde （MDA） levels were detected by colorimetry. We also evaluated smooth muscle contents. The ratio of maximal ICP and mean systemic arterial pressure （MAP） was markedly higher in EGCG-treated aged rats than in untreated aged rats. We found that DDAH 1 and DDAH2 were expressed in cavernosal tissue, and they were downregulated in corpora of aged rats. The administration of EGCG upregulated the expression and activity of DDAH. In contrast, EGCG treatment downregulated the expression of PRMT1 and ADMA content. Moreover, EGCG-treated rats showed an improvement in smooth muscle expression, the ratio of smooth muscle cell/collagen fibril, SOD activity, and MDA levels when compared with untreated aged rats.

PRMT6 physically associates with nuclear factor Y to regulate photoperiodic flowering in Arabidopsis

The timing of floral transition is critical for reproductive success in flowering plants.In long-day(LD)plant Arabidopsis,the floral regulator gene FLOWERING LOCUS T(FT)is a major component of the mobile florigen.FT expression is rhythmically activated by CONSTANS(CO),and specifically accumu-lated at dusk of LDs.However;the underlying mechanism of adequate regulation of FT transcription in response to day-length cues to warrant flowering time still remains to be investigated.Here,we identify a homolog of human protein arginine methyltransferases 6(HsPRMT6)in Arabidopsis,and confirm AtPRMT6 physically interacts with three positive regulators of flowering Nuclear Factors YC3(NF-YC3),NF-YC9,and NF-YB3.Further investigations find that AtPRMT6 and its encoding protein accumulate at dusk of LDs.PRMT6-mediated H3 R2me2a modification enhances the promotion of NF-YCs on FT transcription in response to inductive LD signals.Moreover,AtPRMT6 and its homologues proteins AtPRMT4a and AtPRMT4b coordinately inhibit the expression of FLOWERING LOCUS C,a suppressor of FT.Taken together,our study reveals the role of arginine methylation in photoperiodic pathway and how the PRMT6-mediating H3R2me2a system interacts with NF-CO module to dynamically control FT expression and facilitate flowering time.