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 Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. FEBS Lett. (0014-5793) 2012 Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk in vitro In vitro enzymatic study enzymatic study predetermined predetermined participant predetermined Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk FEBS Lett. (0014-5793) 2012 Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. FEBS Lett. (0014-5793) 2012 Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk in vitro In vitro pull down pull down predetermined predetermined participant predetermined Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk FEBS Lett. (0014-5793) 2012 Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. Histaminylation of glutamine residues is a novel posttranslational modification implicated in G-protein signaling. FEBS Lett. (0014-5793) 2012 Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk in vitro In vitro solid phase assay solid phase assay predetermined predetermined participant predetermined Vowinckel J., Stahlberg S., Paulmann N., Bluemlein K., Grohmann M., Ralser M., Walther DJ. jv330@cam.ac.uk FEBS Lett. (0014-5793) 2012 pak3_mouse Serine/threonine-protein kinase PAK 3 p21-activated kinase 3 Beta-PAK CDC42/RAC effector kinase PAK-B Pak3 Pak-3 Pakb Stk4 protein protein mouse Mus musculus Mouse MSDSLDNEEKPPAPPLRMNSNNRDSSALNHSSKPLPMAPEEKNKKARLRSIFPGGGDKTNKKKEKERPEISLPSDFEHTIHVGFDAVTGEFTPDLYGSQMCPGKLPEGIPEQWARLLQTSNITKLEQKKNPQAVLDVLKFYDSKETVNNQKYMSFTSGDKSAHGYIAAHQSNTKTASEPPLAPPVSEEEDEEEEEEEDDNEPPPVIAPRPEHTKSIYTRSVVESIASPAAPNKEDIPPSAENANSTTLYRNTDRQRKKSKMTDEEILEKLRSIVSVGDPKKKYTRFEKIGQGASGTVYTALDIATGQEVAIKQMNLQQQPKKELIINEILVMRENKNPNIVNYLDSYLVGDELWVVMEYLAGGSLTDVVTETCMDEGQIAAVCRECLQALDFLHSNQVIHRDIKSDNILLGMDGSVKLTDFGFCAQITPEQSKRSTMVGTPYWMAPEVVTRKAYGPKVDIWSLGIMAIEMVEGEPPYLNENPLRALYLIATNGTPELQNPERLSAVFRDFLNRCLEMDVDRRGSAKELLQHPFLKLAKPLSSLTPLIIAAKEAIKNSSR 9AD07B0328F0B08C gnaq_mouse Guanine nucleotide-binding protein G(q) subunit alpha Guanine nucleotide-binding protein alpha-q Gnaq protein protein mouse Mus musculus Mouse MTLESIMACCLSEEAKEARRINDEIERQLRRDKRDARRELKLLLLGTGESGKSTFIKQMRIIHGSGYSDEDKRGFTKLVYQNIFTAMQAMIRAMDTLKIPYKYEHNKAHAQLVREVDVEKVSAFENPYVDAIKSLWNDPGIQECYDRRREYQLSDSTKYYLNDLDRVADPSYLPTQQDVLRVRVPTTGIIEYPFDLQSVIFRMVDVGGQRSERRKWIHCFENVTSIMFLVALSEYDQVLVESDNENRMEESKALFRTIITYPWFQNSSVILFLNKKDLLEEKIMYSHLVDYFPEYDGPQRDAQAAREFILKMFVDLNPDSDKIIYSHFTCATDTENIRFVFAAVKDTILQLNLKEYNLV DIP protein P21279 original sequence version: 157 DIP protein P21279 original sequence version: 127 DIP protein P21279 original sequence version: 141 DIP protein P21279 original sequence version: 170 3BCBA4EE7DADADBF tgm2_cavcu Protein-glutamine gamma-glutamyltransferase 2 Tissue transglutaminase Transglutaminase-2 TGM2 Guinea pig liver transglutaminase Isopeptidase TGM2 Protein-glutamine deamidase TGM2 Protein-glutamine dopaminyltransferase TGM2 Protein-glutamine histaminyltransferase TGM2 Protein-glutamine noradrenalinyltransferase TGM2 Protein-glutamine serotonyltransferase TGM2 protein protein cavcu Cavia cutleri (Guinea pig) MAEDLILERCDLQLEVNGRDHRTADLCRERLVLRRGQPFWLTLHFEGRGYEAGVDTLTFNAVTGPDPSEEAGTMARFSLSSAVEGGTWSASAVDQQDSTVSLLLSTPADAPIGLYRLSLEASTGYQGSSFVLGHFILLYNPRCPADAVYMDSDQERQEYVLTQQGFIYQGSAKFINGIPWNFGQFEDGILDICLMLLDTNPKFLKNAGQDCSRRSRPVYVGRVVSAMVNCNDDQGVLQGRWDNNYSDGVSPMSWIGSVDILRRWKDYGCQRVKYGQCWVFAAVACTVLRCLGIPTRVVTNFNSAHDQNSNLLIEYFRNESGEIEGNKSEMIWNFHCWVESWMTRPDLEPGYEGWQALDPTPQEKSEGTYCCGPVPVRAIKEGHLNVKYDAPFVFAEVNADVVNWIRQKDGSLRKSINHLVVGLKISTKSVGRDEREDITHTYKYPEGSEEEREAFVRANHLNKLATKEEAQEETGVAMRIRVGQNMTMGSDFDIFAYITNGTAESHECQLLLCARIVSYNGVLGPVCSTNDLLNLTLDPFSENSIPLHILYEKYGDYLTESNLIKVRGLLIEPAANSYVLAERDIYLENPEIKIRVLGEPKQNRKLIAEVSLKNPLPVPLLGCIFTVEGAGLTKDQKSVEVPDPVEAGEQAKVRVDLLPTEVGLHKLVVNFECDKLKAVKGYRNVIIGPA 047223D38DBEA4A4 rgs4_rat Regulator of G-protein signaling 4 Rgs4 protein protein rat Rattus norvegicus (Rat) Rat MCKGLAGLPASCLRSAKDMKHRLGFLLQKSDSCEHSSSHSKKDKVVTCQRVSQEEVKKWAESLENLINHECGLAAFKAFLKSEYSEENIDFWISCEEYKKIKSPSKLSPKAKKIYNEFISVQATKEVNLDSCTREETSRNMLEPTITCFDEAQKKIFNLMEKDSYRRFLKSRFYLDLTNPSSCGAEKQKGAKSSADCTSLVPQCA 9647C0EC909D0F6F cdc42_human Cell division control protein 42 homolog CDC42 G25K GTP-binding protein protein protein human Homo sapiens Human MQTIKCVVVGDGAVGKTCLLISYTTNKFPSEYVPTVFDNYAVTVMIGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHHCPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQKGLKNVFDEAILAALEPPEPKKSRRCVLL 51A437E22A4D8FFF gnao_rat Guanine nucleotide-binding protein G(o) subunit alpha Gnao1 Gna0 Gnao protein protein rat Rattus norvegicus (Rat) Rat MGCTLSAEERAALERSKAIEKNLKEDGISAAKDVKLLLLGAGESGKSTIVKQMKIIHEDGFSGEDVKQYKPVVYSNTIQSLAAIVRAMDTLGVEYGDKERKADSKMVCDVVSRMEDTEPFSAELLSAMMRLWGDSGIQECFNRSREYQLNDSAKYYLDSLDRIGAADYQPTEQDILRTRVKTTGIVETHFTFKNLHFRLFDVGGQRSERKKWIHCFEDVTAIIFCVALSGYDQVLHEDETTNRMHESLMLFDSICNNKFFIDTSIILFLNKKDLFGEKIKKSPLTICFPEYPGSNTYEDAAAYIQTQFESKNRSPNKEIYCHMTCATDTNNIQVVFDAVTDIIIANNLRGCGLY DIP protein P59215 original sequence version: 119 DIP protein P59215 original sequence version: 86 mint Guanine nucleotide-binding proteins (G proteins) are involved as modulators or transducers in various transmembrane signaling systems. The G(o) protein function is not clear homomint 577024F61B179C89 cdc42-tgm2 1 CDC42 8 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate neutral component neutral component 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 TGM2 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction f1c mint TGM2 catalyzed a transfer of histamine to the G proteins Go1, Gq and Cdc42. gnao1-tgm2 1 Galpha1 9 ms participant Identification by mass spectrometry ms participant predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate neutral component neutral component 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 certain certain sequence position certain certain certain sequence position certain other modification other modification uncategorized protein modification uncategorized protein modification modified residue ms mass detection of residue modification modified residue ms certain certain sequence position certain certain certain sequence position certain resulting-ptm TGM2 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction mint TGM2 catalyzed a transfer of histamine to the G proteins Go1, Gq and Cdc42. To determine the stoichiometry of the histaminylation, we incubated GST-Gαo1 and TGM2 for 60 min with different [3H]-HA concentrations. After correction for unspecific binding, the HA/GST-Gαo1 ratio approached the saturation closely to 1, indicating histaminylation of a single residue (Fig 1d). We next used liquid chromatography/tandem mass spectrometry (LCMS/ MS) for localizing the modification. Using a nano LC- (nanoLC 2D Ultra, Eksigent) coupled hybrid triple quadrupole/ion trap mass spectrometer (QTRAP5500, AB/Sciex), we established selective reaction monitoring (SRM) assays [30,33] for tryptic digests of the G proteins Go1, Cdc42 and Gq, taking into account a mass shift (+94) caused by potential histaminylations (Fig 2e). Peptides corresponding to the histaminylated catalytic center of Go1 (aa 199-206, Fig 2a) and Gq (aa 203-210, Fig 2b) were detected by three SRM transitions each (upper panels). f1c f1d f2a f2d gnaq-tgm2 1 G alpha q 5 ms participant Identification by mass spectrometry ms participant predetermined predetermined participant predetermined enzyme target enzyme target substrate substrate neutral component neutral component 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 other modification other modification uncategorized protein modification uncategorized protein modification modified residue ms mass detection of residue modification modified residue ms certain certain sequence position certain certain certain sequence position certain resulting-ptm binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain TGM2 6 predetermined predetermined participant predetermined predetermined predetermined participant predetermined enzyme enzyme neutral component neutral component purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined enzymatic reaction enzymatic reaction Biochemical reaction Biochemical reaction mint f1c f2b TGM2 catalyzed a transfer of histamine to the G proteins Go1, Gq and Cdc42. To determine the stoichiometry of the histaminylation, we incubated GST-Gαo1 and TGM2 for 60 min with different [3H]-HA concentrations. After correction for unspecific binding, the HA/GST-Gαo1 ratio approached the saturation closely to 1, indicating histaminylation of a single residue (Fig 1d). We next used liquid chromatography/tandem mass spectrometry (LCMS/ MS) for localizing the modification. Using a nano LC- (nanoLC 2D Ultra, Eksigent) coupled hybrid triple quadrupole/ion trap mass spectrometer (QTRAP5500, AB/Sciex), we established selective reaction monitoring (SRM) assays [30,33] for tryptic digests of the G proteins Go1, Cdc42 and Gq, taking into account a mass shift (+94) caused by potential histaminylations (Fig 2e). Peptides corresponding to the histaminylated catalytic center of Go1 (aa 199-206, Fig 2a) and Gq (aa 203-210, Fig 2b) were detected by three SRM transitions each (upper panels). gnao1-rgs4 2 Gnao1 9 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Rgs4 7 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey 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 f3b homomint mint Go1 was pre-loaded with GTP, GDP or GMP-PNP, a non-hydrolysable GTP analog leading to constitutive G protein activation. Then, the protein was incubated with recombinant RGS4, and the resulting protein complex was immunoprecipitated. Analyzed by western-blotting and densitometry, Go1 strongly bound RGS4 in the presence of GMP-PNP, whereas GDP- and GTP-preloaded Go1/RGS4-binding was significantly weaker (Fig 3b). cdc42-pak3-8 2 Pak3 4 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified binding site binding region binding-associated region binding region certain certain sequence position certain certain certain sequence position certain 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 CDC42 8 western blot western blot Immuno blot Immuno blot predetermined predetermined participant predetermined unspecified role unspecified role bait bait purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association f3e homomint mint This result was additionally confirmed by pulldown experiments, where a 6xHis-Cdc42/GST-Pak3BD complex was purified using a Ni-NTA matrix and analyzed by western-blotting. When Cdc42 was histaminylated, or pre-loaded with the non-hydrolysable GTP analogue GMP-PNP, a significantly higher amount of Pak3BD was co-precipitated with Cdc42 (Fig 3e). pak3-cdc42 3 CDC42 8 predetermined predetermined participant predetermined predetermined predetermined participant predetermined unspecified role unspecified role prey prey purified purified his tag his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined Pak3 4 predetermined predetermined participant predetermined 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 certain certain sequence position certain certain certain sequence position certain physical association physical association f3d homomint mint To provide additional evidence if histaminylation leads to activation of Cdc42, we thus studied the ability of Cdc42 to form a complex with the G protein binding domain of Pak3 (Pak3BD) [37,38] using a multiwellbased binding assay as described in the Materials and Methods section, and co-precipitation. Histaminylation of Cdc42 significantly increased its binding to Pak3BD in the multiwellbased binding assay