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 Meprins process matrix metalloproteinase-9 (MMP-9)/gelatinase B and enhance the activation kinetics by MMP-3. Meprins process matrix metalloproteinase-9 (MMP-9)/gelatinase B and enhance the activation kinetics by MMP-3. FEBS Lett. (0014-5793) 2012 Geurts N., Becker-Pauly C., Martens E., Proost P., Van den Steen PE., Stöcker W., Opdenakker G. in vitro In vitro enzymatic study enzymatic study experimental particp experimental participant identification experimental particp Geurts N., Becker-Pauly C., Martens E., Proost P., Van den Steen PE., Stöcker W., Opdenakker G. FEBS Lett. (0014-5793) 2012 Meprins process matrix metalloproteinase-9 (MMP-9)/gelatinase B and enhance the activation kinetics by MMP-3. Meprins process matrix metalloproteinase-9 (MMP-9)/gelatinase B and enhance the activation kinetics by MMP-3. FEBS Lett. (0014-5793) 2012 Geurts N., Becker-Pauly C., Martens E., Proost P., Van den Steen PE., Stöcker W., Opdenakker G. in vitro In vitro zymography zymography weight by comassie molecular weight estimation by coomasie staining weight by comassie Geurts N., Becker-Pauly C., Martens E., Proost P., Van den Steen PE., Stöcker W., Opdenakker G. FEBS Lett. (0014-5793) 2012 mep1b_human Meprin A subunit beta MEP1B Endopeptidase-2 N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit beta PABA peptide hydrolase PPH beta Meprin B protein protein human Homo sapiens Human MDLWNLSWFLFLDALLVISGLATPENFDVDGGMDQDIFDINEGLGLDLFEGDIRLDRAQIRNSIIGEKYRWPHTIPYVLEDSLEMNAKGVILNAFERYRLKTCIDFKPWAGETNYISVFKGSGCWSSVGNRRVGKQELSIGANCDRIATVQHEFLHALGFWHEQSRSDRDDYVRIMWDRILSGREHNFNTYSDDISDSLNVPYDYTSVMHYSKTAFQNGTEPTIVTRISDFEDVIGQRMDFSDSDLLKLNQLYNCSSSLSFMDSCSFELENVCGMIQSSGDNADWQRVSQVPRGPESDHSNMGQCQGSGFFMHFDSSSVNVGATAVLESRTLYPKRGFQCLQFYLYNSGSESDQLNIYIREYSADNVDGNLTLVEEIKEIPTGSWQLYHVTLKVTKKFRVVFEGRKGSGASLGGLSIDDINLSETRCPHHIWHIRNFTQFIGSPNGTLYSPPFYSSKGYAFQIYLNLAHVTNAGIYFHLISGANDDQLQWPCPWQQATMTLLDQNPDIRQRMSNQRSITTDPFMTTDNGNYFWDRPSKVGTVALFSNGTQFRRGGGYGTSAFITHERLKSRDFIKGDDVYILLTVEDISHLNSTQIQLTPAPSVQDLCSKTTCKNDGVCTVRDGKAECRCQSGEDWWYMGERCEKRGSTRDTIVIAVSSTVAVFALMLIITLVSVYCTRKKYRERMSSNRPNLTPQNQHAF DIP protein Q16820 original sequence version: 124 4733DBF018A4F3CD mep1a_human Meprin A subunit alpha MEP1A Endopeptidase-2 N-benzoyl-L-tyrosyl-P-amino-benzoic acid hydrolase subunit alpha PABA peptide hydrolase PPH alpha protein protein human Homo sapiens Human MAWIRSTCILFFTLLFAHIAAVPIKYLPEENVHDADFGEQKDISEINLAAGLDLFQGDILLQKSRNGLRDPNTRWTFPIPYILADNLGLNAKGAILYAFEMFRLKSCVDFKPYEGESSYIIFQQFDGCWSEVGDQHVGQNISIGQGCAYKAIIEHEILHALGFYHEQSRTDRDDYVNIWWDQILSGYQHNFDTYDDSLITDLNTPYDYESLMHYQPFSFNKNASVPTITAKIPEFNSIIGQRLDFSAIDLERLNRMYNCTTTHTLLDHCTFEKANICGMIQGTRDDTDWAHQDSAQAGEVDHTLLGQCTGAGYFMQFSTSSGSAEEAALLESRILYPKRKQQCLQFFYKMTGSPSDRLVVWVRRDDSTGNVRKLVKVQTFQGDDDHNWKIAHVVLKEEQKFRYLFQGTKGDPQNSTGGIYLDDITLTETPCPTGVWTVRNFSQVLENTSKGDKLQSPRFYNSEGYGFGVTLYPNSRESSGYLRLAFHVCSGENDAILEWPVENRQVIITILDQEPDVRNRMSSSMVFTTSKSHTSPAINDTVIWDRPSRVGTYHTDCNCFRSIDLGWSGFISHQMLKRRSFLKNDDLIIFVDFEDITHLSQTEVPTKGKRLSPQGLILQGQEQQVSEEGSGKAMLEEALPVSLSQGQPSRQKRSVENTGPLEDHNWPQYFRDPCDPNPCQNDGICVNVKGMASCRCISGHAFFYTGERCQAVQVHGSVLGMVIGGTAGVIFLTFSIIAILSQRPRK mint homomint B5F0812F2850F958 mmp9_human Matrix metalloproteinase-9 MMP9 CLG4B 92 kDa type IV collagenase 92 kDa gelatinase Gelatinase B protein protein human Homo sapiens Human MSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGESKSLGPALLLLQKQLSLPETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVYSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRSYSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTQDGNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANYDRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYSTCTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAAHEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIRHLYGPRPEPEPRPPTTTTPQPTAPPTVCPTGPPTVHPSERPTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNIFDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKWPALPRKLDSVFEERLSKKLFFFSGRQVWVYTGASVLGPRRLDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYWRVSSRSELNQVDQVGYVTYDILQCPED DIP protein P14780 original sequence version: 114 DIP protein P14780 original sequence version: 120 2165AC8CA1466209 mep1a-mmp9 2 Meprin alpha 4 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme enzyme neutral component neutral component purified purified MMP-9 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme target enzyme target substrate substrate neutral component neutral component purified purified cleavage cleavage reaction cleavage homomint f1a f1c mint To analyze whether proMMP-9 processing is triggered by meprins, in vitro experiments were performed in which wild type human proMMP-9 and active meprin α or meprin β were incubated at different physiological E/S ratios for one hour at 37°C. As shown on gelatin zymography, incubation with meprin α (fig. 1A) or β (fig. 1B) resulted in a concentration-dependent processing of proMMP-9 into lower molecular weight zymolytic bands and this was not observed in the condition without meprin α or β. In addition, processing of MMP-9 multimers also occurred in the presence of meprin α or β. To investigate the gelatinolytic activity of both meprin α or β itself, different concentrations of the enzyme were analyzed by zymography. As shown in panel C of figure 1, no enzymatic activity towards gelatin was detected in the samples containing different concentrations of only meprin α or β. Hence, the fragments observed following zymography analysis of MMP-9/meprin incubation samples (panel A and B and lane 5 panel C of figure 1) represent meprin-mediated cleavage products derived from proMMP-9. mep1b-mmp9 2 Meprin beta 3 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme enzyme neutral component neutral component purified purified MMP-9 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme target enzyme target substrate substrate neutral component neutral component purified purified cleavage cleavage reaction cleavage f1b f1c homomint mint To analyze whether proMMP-9 processing is triggered by meprins, in vitro experiments were performed in which wild type human proMMP-9 and active meprin α or meprin β were incubated at different physiological E/S ratios for one hour at 37°C. As shown on gelatin zymography, incubation with meprin α (fig. 1A) or β (fig. 1B) resulted in a concentration-dependent processing of proMMP-9 into lower molecular weight zymolytic bands and this was not observed in the condition without meprin α or β. In addition, processing of MMP-9 multimers also occurred in the presence of meprin α or β. To investigate the gelatinolytic activity of both meprin α or β itself, different concentrations of the enzyme were analyzed by zymography. As shown in panel C of figure 1, no enzymatic activity towards gelatin was detected in the samples containing different concentrations of only meprin α or β. Hence, the fragments observed following zymography analysis of MMP-9/meprin incubation samples (panel A and B and lane 5 panel C of figure 1) represent meprin-mediated cleavage products derived from proMMP-9. mep1a-mmp9-1 1 Meprin alpha 4 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp enzyme enzyme neutral component neutral component purified purified MMP-9 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme target enzyme target substrate substrate neutral component neutral component purified purified cleavage cleavage reaction cleavage f2 homomint mint As a second approach, inactive rh proMMP-9 mutant, in which the catalytic Glu402 is mutated into Ala, was incubated with meprin α or β (E/S: 1/16) at 37°C. The use of an inactive mutant was relevant to exclude autocatalysis of MMP-9. Panel A of figure 2 shows the CBB-stained MMP-9 fragments derived from 1 hour incubation with meprin α or β. A similar pattern was observed after 10 hours of incubation, although the stability of meprin α was reduced (data not shown). At different time points after incubation, sample mixtures were separated by SDS-PAGE (12,5%) and after electroblotting, the CBB-stained protein bands were excised and identified by automated Edman degradation (fig. 2B). mep1b-mmp9-1 1 MMP-9 5 weight by comassie molecular weight estimation by coomasie staining weight by comassie experimental particp experimental participant identification experimental particp enzyme target enzyme target substrate substrate neutral component neutral component purified purified Meprin beta 3 predetermined predetermined participant predetermined experimental particp experimental participant identification experimental particp enzyme enzyme neutral component neutral component purified purified cleavage cleavage reaction cleavage f2 homomint mint As a second approach, inactive rh proMMP-9 mutant, in which the catalytic Glu402 is mutated into Ala, was incubated with meprin α or β (E/S: 1/16) at 37°C. The use of an inactive mutant was relevant to exclude autocatalysis of MMP-9. Panel A of figure 2 shows the CBB-stained MMP-9 fragments derived from 1 hour incubation with meprin α or β. A similar pattern was observed after 10 hours of incubation, although the stability of meprin α was reduced (data not shown). At different time points after incubation, sample mixtures were separated by SDS-PAGE (12,5%) and after electroblotting, the CBB-stained protein bands were excised and identified by automated Edman degradation (fig. 2B).