MINT MINT, Dpt of Biology, University of Rome Tor Vergata MINT http://mint.bio.uniroma2.it/mint url>http://mint.bio.uniroma2.it/mint/ https://mint.bio.uniroma2.it MINT-[0-9]+ https://mint.bio.uniroma2.it/index.php/results-interactions/?id=${ac} The Molecular INTeraction database (MINT) is a relational database designed to store interactions between biological molecules. http://mint.bio.uniroma2.it Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. (1742-464X) 2012 imex curation Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. Only protein-protein interactions human-hep_g2 Homo sapiens hepatocellular carcinoma (HCC) cells (p53-wt). hep_g2 human hepatocellular carcinoma (HCC) cells (p53-wt). fluorescence imaging fluorescence microscopy fluorescence imaging predetermined predetermined participant predetermined Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. (1742-464X) 2012 imex curation Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. (1742-464X) 2012 imex curation Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. Only protein-protein interactions in vitro In vitro pull down pull down predetermined predetermined participant predetermined Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. (1742-464X) 2012 imex curation Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. Only protein-protein interactions human-hep_g2 Homo sapiens hepatocellular carcinoma (HCC) cells (p53-wt). hep_g2 human hepatocellular carcinoma (HCC) cells (p53-wt). anti tag coip anti tag coimmunoprecipitation anti tag coip predetermined predetermined participant predetermined Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma. FEBS J. (1742-464X) 2012 imex curation Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. Only protein-protein interactions human-mcf7 Homo sapiens mammary carcinoma cells mcf7 mammary carcinoma cells fluorescence imaging fluorescence microscopy fluorescence imaging predetermined predetermined participant predetermined Zhong J., Cao RX., Zu XY., Hong T., Yang J., Liu L., Xiao XH., Ding WJ., Zhao Q., Liu JH., Wen GB. FEBS J. (1742-464X) 2012 Only protein-protein interactions imex curation aldr_human Aldo-keto reductase family 1 member B1 Aldehyde reductase ALR2 Aldose reductase AKR1B1 ALDR1 protein protein human Homo sapiens Human MASRLLLNNGAKMPILGLGTWKSPPGQVTEAVKVAIDVGYRHIDCAHVYQNENEVGVAIQEKLREQVVKREELFIVSKLWCTYHEKGLVKGACQKTLSDLKLDYLDLYLIHWPTGFKPGKEFFPLDESGNVVPSDTNILDTWAAMEELVDEGLVKAIGISNFNHLQVEMILNKPGLKYKPAVNQIECHPYLTQEKLIQYCQSKGIVVTAYSPLGSPDRPWAKPEDPSLLEDPRIKAIAAKHNKTTAQVLIRFPMQRNLVVIPKSVTPERIAENFKVFDFELSSQDMTTLLSYNRNWRVCALLSCTSHKDYPFHEEF 1852E8616B5DCEAE esr1_human Estrogen receptor ESR1 ESR NR3A1 Estradiol receptor ER-alpha Nuclear receptor subfamily 3 group A member 1 protein protein human Homo sapiens Human MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEFNAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPHGQQVPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYCAVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKCYEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV DIP protein P03372 original sequence version: 214 DIP protein P03372 original sequence version: 139 DIP protein P03372 original sequence version: 167 5455C57AB0CCCAA7 anm2_human Protein arginine N-methyltransferase 2 PRMT2 HMT1 Histone-arginine N-methyltransferase PRMT2 HRMT1L1 protein protein human Homo sapiens Human MATSGDCPRSESQGEEPAECSEAGLLQEGVQPEEFVAIADYAATDETQLSFLRGEKILILRQTTADWWWGERAGCCGYIPANHVGKHVDEYDPEDTWQDEEYFGSYGTLKLHLEMLADQPRTTKYHSVILQNKESLTDKVILDVGCGTGIISLFCAHYARPRAVYAVEASEMAQHTGQLVLQNGFADIITVYQQKVEDVVLPEKVDVLVSEWMGTCLLFEFMIESILYARDAWLKEDGVIWPTMAALHLVPCSADKDYRSKVLFWDNAYEFNLSALKSLAVKEFFSKPKYNHILKPEDCLSEPCTILQLDMRTVQISDLETLRGELRFDIRKAGTLHGFTAWFSVHFQSLQEGQPPQVLSTGPFHPTTHWKQTLFMMDDPVPVHTGDVVTGSVVLQRNPVWRRHMSVALSWAVTSRQDPTSQKVGEKVFPIWR 6DEE0350C15ECD4F esr1-prmt2 5 ER-alpha 7 immunostaining immunostaining predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell PRMT2 8 tag fluorescence tag visualisation by fluorescence tag fluorescence predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f2a homomint mint As shown in Fig. 2A, cells expressing wild-type PRMT2, appear to be largely localized to the nucleus excluding the nucleoli and a weak fluorescence was detected in the cytosol as described previously [15, 32, 37]. Whereas, cells expressing GFP-PRMT2β, showed an even distribution of the GFP fusion proteins between the nucleus including the nucleoli and the cytoplasm. Strikingly, the GFP-PRMT2α and GFP-PRMT2γ fusion protein expression were predominantly localized to the nuclear compartment excluding the nucleoli, as well as that of the wild type PRMT2. In addition, the fluorescence signals related to ERα and PRMT2 variants overlap, indicating colocalization of PRMT2 variants and ERα inside MCF7 cells (Fig. 2 Merge). akr1b1-prmt2 1 AR 6 immunostaining immunostaining predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component physiological level physiological level endogenous level endogenous endogenous endogenous level fixed cell fixed cell PRMT2 8 tag fluorescence tag visualisation by fluorescence tag fluorescence predetermined predetermined participant predetermined unspecified role unspecified role neutral component neutral component over-expressed over expressed level over-expressed fixed cell fixed cell gfp tag gfp tag green fluorescent protein tag GFP green fluorescent protein GFP green fluorescent protein gfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined colocalization colocalization f3 homomint mint To examine whether the subcellular localization of PRMT2 and its variants is MCF7 cellspecific, this study was conducted in HepG2 cells, and similar results were observed (Fig. 3).Furthermore, the fluorescence signals related to AR and PRMT2 variants also overlap, indicating colocalization of PRMT2 variants and AR inside HepG2 cells (Fig. 3 Merge). prmt2-esr1 3 ER-alpha 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified PRMT2 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined direct interaction direct interaction f6a homomint mint As shown in Fig. 6A,PRMT2, PRMT2α and PRMT2γ demonstrated similar binding affinity to ERα, but PRMT2β was weaker. prmt2-esr1-1 3 ER-alpha 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey cell lysate cell lysate physiological level physiological level endogenous level endogenous endogenous endogenous level PRMT2 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified gst tag gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined binding site binding region binding-associated region binding region deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain physical association physical association f7b homomint mint A protein-protein interaction assay confirmed this functional result. As shown in Fig. 7B, mutants PRMT2 (1-218) and PRMT2 (1-277) displayed binding activity to ERα, but PRMT2β (△1-218) and PRMT2 (278-433) did not. These data are consistent with the binding assays of PRMT2 and variants, suggesting the N-terminus of PRMT2 and its variants is responsible for their binding and regulatory activity. prmt2-esr1-2 2 PRMT2 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ER-alpha 7 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level physical association physical association f6b homomint mint This binding activity of PRMT2 and its variants to ERα was confirmed inside the MCF7 cells. As shown in Fig. 6B, A-V5 tagged PRMT2 or its variants was coimmunoprecipitated by anti-a-V5 antibody, and like in vitro study, the affinity of PRMT2β was weaker. prmt2-akr1b1 4 AR 6 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey living cell living cell physiological level physiological level endogenous level endogenous endogenous endogenous level PRMT2 8 anti tag western anti tag western blot anti tag western predetermined predetermined participant predetermined unspecified role unspecified role bait bait over-expressed over expressed level over-expressed living cell living cell v5 tag v5 tag v5 tag GKPIPNPLLGLDST epitope tag GKPIPNPLLGLDST epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f6c homomint mint To examine the binding activity of PRMT2 and its variants to androgen receptor (AR), this study was conducted in HepG2 cells. After treatment of 10 nM DHT for 10 h, as shown in Fig. 6C, A-V5 tagged PRMT2 or its variants was co-immunoprecipitated by anti-a-V5 antibody, and the affinity of them was similar