Biochemical Characterization of Protein Complexes from the Helicobacter pylori Protein Interaction Map

• DOI: 10.1074/mcp.m400048-mcp200
• Publication Date: 2004-05-11T00:53:39Z
• Authors: Laurent Terradot; Nathan Durnell; Min Li; Ming Li; Jeremiah Ory; Agnes Labigne; Pierre Legrain; Frederic Colland; Gabriel Waksman
• Keywords: Interactome; Proteome; Secretory protein; Pathogenicity island

We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as source protein-protein complexes for biochemical and structural studies H. interactome; ii) biochemically shown involve components type IV secretion systems. Thus, 17 involving 31 proteins fragments were studied, general strategy was designed produce protein-interacting partners characterization. show that is valid proteome-scale characterization interactome, because 76% tested confirmed biochemically. Of proteins, three could be confirmed. One between apparatus, ComB10 ComB4, which are VirB10 VirB4 homologs, respectively. Another component (HP0525, VirB11 homolog) non-type (HP01451), indicating other than core VirB (1-11)-VirD4 may play role in secretion. Finally, third CagA, virulence factor secreted system encoded Cag pathogenicity island, protein, HP0496. Proteomics aims at studying on scale an entire pathway or whole cell. Proteomic analyses encompass expression profile, identification post-translational modification, detection (see review Ref. 1Pandey A. Mann M. genes genomes..Nature. 2000; 405: 837-846Google Scholar). The has benefited development large-scale (ht) 1The abbreviations used are: ht, high-thoughput; Y2H, system; MBP, maltose-binding protein; SID, selected interacting domain; FC, functional category. methods (2Ito T. Ota K. Kubota Yamaguchi Y. Chiba Sakuraba Yoshida Roles exploration interactome..Mol. Cell. Proteomics. 2002; 1: 561-566Google Scholar) based vitro vivo systems, such systems (Y2H) (3Boulton S.J. Gartner Reboul J. Vaglio P. Dyson N. Hill D.E. Vidal Combined genomic maps C. elegans DNA damage response..Science. 295: 127-131Google Scholar, 4Fromont-Racine Rain J.C. Legrain Toward analysis genome through exhaustive screens..Nat. Genet. 1997; 16: 277-282Google 5Ito Ozawa R. Hattori Sakaki A comprehensive explore interactome..Proc. Natl. Acad. Sci. U. S. 2001; 98: 4569-4574Google 6Uetz Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan Pochart Qureshi-Emili Li Godwin B. Conover Kalbfleisch Vijayadamodar Yang Johnston Fields Rothberg J.M. Saccharomyces cerevisiae..Nature. 403: 623-627Google Scholar), chips (7Zhu Bilgin Bangham Hall Casamayor Bertone Lan Jansen Bidlingmaier Houfek Mitchell Miller Dean R.A. Gerstein Snyder Global activities using proteome chips..Science. 293: 2101-2105Google systematic tandem affinity purification mass spectrometry (8Ho Gruhler Heilbut Bader G.D. Moore Adams S.L. Millar Taylor Bennett Boutilier Wolting Donaldson I. Schandorff Shewnarane Vo Taggart Goudreault Muskat Alfarano Dewar Lin Z. Michalickova Willems A.R. Sassi Nielsen P.A. Rasmussen K.J. Andersen Johansen L.E. Hansen L.H. Jespersen Podtelejnikov E. Crawford Poulsen Sorensen B.D. Matthiesen Hendrickson R.C. Gleeson F. Pawson Moran M.F. Durocher Hogue C.W. Figeys Tyers Systematic cerevisiae spectrometry..Nature. 415: 180-183Google 9Gavin A.C. Bosche Krause Grandi Marzioch Bauer Schultz Rick Michon A.M. Cruciat C.M. Remor Hofert Schelder Brajenovic Ruffner Merino Klein Hudak Dickson Rudi Gnau Bauch Bastuck Huhse Leutwein Heurtier M.A. Copley R.R. Edelmann Querfurth Rybin Drewes Raida Bouwmeester Bork Seraphin Kuster Neubauer Superti-Furga Functional organization complexes..Nature. 141-147Google Information about interactions, molecular complexes, pathways can now classified examined via databases World Wide Web (10Bader Betel BIND: Biomolecular Interaction Network Database..Nucleic Acids Res. 2003; 31: 248-250Google Interpretation these provides framework variety target discovery strategies implemented (11Legrain Wojcik Gauthier Protein-protein maps: lead towards cellular functions..Trends 17: 346-352Google fundamental caveat ht protein-protein-interacting both Y2H tagging/MS generate numbers false-positives -negatives. need developed assess and/or biological significance (12Sprinzak Sattath Margalit How Reliable Experimental Protein-Protein Data?.J. Mol. Biol. 327: 919-923Google 13Deane Salwinski Xenarios Eisenberg Protein interactions: Two assessment reliability high throughput observations..Mol. 349-356Google Depending methodological approach, some false-negatives might arise incorrect folding inadequate subcellular localization under investigation (14Schachter Protein-interaction networks: experiments analysis..Drug Discov. Today. 7: S48-S54Google noteworthy genome-wide projects missed most (as much 90%) already characterized False-positives generated searching many potential increases probability selecting polypeptides no relevance As result, does not guarantee inferred physiological users network must further evaluate each interest. means alleviating least partially this problem increase sampling overlapping preys. Such leads added redundancy data, increasing data accuracy, decreasing -negatives significantly. It additional advantage defining more finely regions involved (selected domains SIDs) 11Legrain first reported human pathogen (15Rain Selig De Reuse Battaglia Reverdy Simon Lenzen Petel Schachter Chemama Labigne pylori..Nature. 409: 211-215Google probably common chronic bacterial infection humans, present almost half population (16Cover T.L. Blaser M.J. infection, paradigm mucosal inflammation: Pathogenesis implications eradication prevention..Adv. Intern. Med. 1996; 41: 85-117Google Gram-negative, spiral-shaped bacterium colonizes gastric mucosa primates known responsible several major diseases B gastritis peptic ulcer disease (17Blaser pylori: Its disease..Clin. Infect. Dis. 1992; 15: 386-391Google risk (18Nomura Stemmermann G.N. Chyou P.H. Kato Perez-Perez G.I. carcinoma among Japanese Americans Hawaii..N. Engl. 1991; 325: 1132-1136Google 19Parsonnet Friedman Vandersteen D.P. Chang Vogelman J.H. Orentreich Sibley R.K. carcinoma..N. 1127-1131Google mucosa-associated lymphoid tissue lymphoma (20Wotherspoon Doglioni Diss T.C. Pan Moschini de Boni Isaacson P.G. Regression primary low-grade B-cell after pylori..Lancet. 1993; 342: 575-577Google part associated with cytotoxin thought interfere normal signaling once injected into duodenal epithelial cells. CagA requires assembly clustered island (21Censini Lange Xiang Crabtree J.E. Ghiara Borodovsky Rappuoli Covacci cag, pylori, encodes I-specific disease-associated factors..Proc. 93: 14648-14653Google Typically, composed 12 termed VirB1-11 VirD4. machinery formed VirB8, 9, 10 (22Krall Wiedemann Unsin Weiss Domke Baron Detergent extraction identifies different subassemblies membranes Agrobacterium tumefaciens..Proc. 99: 11405-11410Google extracellular pilus system: VirB2 subunit itself, while VirB5 minor (23Lai E.-M. Kado C.I. Processed promiscuous tumefaciens..J. Bacteriol. 1998; 180: 2711-2717Google 24Schmidt-Eisenlohr Angerer Wanner Zambryski P.C. Vir stabilize mediate its association T 1999; 181: 7485-7492Google one ATPase ATP-binding appear power machinery: VirB11, VirB4, VirD4, respectively (25Dang Zhou X.R. Graf Christie P.J. Dimerization tumefaciens effect cassette mutations function T-DNA transporter..Mol. Microbiol. 32: 1239-1253Google 26Krause Barcena Pansegrau W. Lurz Carazo Lanka Sequence-related export NTPases conjugative transfer region RP4 cag share similar hexameric ring structures..Proc. 97: 3067-3072Google 27Sagulenko Sagulenko Chen Role T-pilus substrate selection..J. 183: 5813-5825Google 28Kumar R.B. Das Polar location VirD4..Mol. 43: 1523-1532Google In homologs HPXXXX (where XXXX denotes ORF number defined 29). With exception possessing significant homology members little process assembly. Note only pylori. gene clusters encoding been recently characterized: ComB system, serves uptake machinery, Tfs3 conjugation (30Hofreuter Odenbreit Haas Natural transformation competence mediated basic system..Mol. 379-391Google 31Kersulyte Velapatino Mukhopadhyay A.K. Cahuayme Bussalleu Combe Gilman R.H. Berg Cluster pylori’s plasticity zone..J. 185: 3764-3772Google obtained displays 1,524 285 baits initial prey library 2 × 106 independent Surprisingly, island-encoded detected, found connect them cell unknown functions. examples HP0525, HP1451, HP0527, homolog, HP0149, (HP0547) HP0496 functions any result suggests Cag-encoded depend unidentified proteins. Interactions also discovered, notably described (or HP0042), ComB4 (HP0017), homolog None means. Given abysmal record published studies, recorded et al. trusted if deployed confirm them. report, we describe complex full-length SIDs form binary complexes. goal research whether htY2H-derived reliable enough make initiation proteome-wide interactome important worthwhile. second characterize formation Here starting point time HP0525 HP547 (CagA) HP0496, ComB4. panel targets, i.e. 19 SIDs, within (Table I). (HP0522) (HP1414 HP0819) (between HP1231 HP1247) studied twice either corresponding SID. Also, HP1338, self-interacting. total targets engaged studied. These belong categories (FC) pyloriGen database (genolist.pasteur.fr/pyloriGene/genome.cgi), including FC2 (purines, pyrimidines, nucleosides, nucleotides), FC7 (transport binding proteins), FC8 (DNA metabolism), FC9 (transcription), FC11 (protein fate), FC14 (cellular processes), FC17 (hypothetical, conserved function). Five directly biology HP0547, HP527, HP0017, HP0042). size targeted ranged 3 69 kDa, five predicted transmembrane domain HMMTOP server (32Tusnady G.E. topology prediction server..Bioinformatics. 849-850Google Proteins named according annotation (29Tomb J.F. White O. Kerlavage Clayton Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill Dougherty B.A. Nelson Quackenbush Kirkness E.F. Peterson Loftus Richardson Dodson Khalak H.G. Glodek McKenney Fitzegerald L.M. Lee M.D. Venter complete sequence pylori.Nature. 388: 539-547Google score (PBS) above) Table I putative brief, PBS reflects uniquely basis results.Table IProtein targetsProteinLengthTMMrFCFunctionPBSProteinLengthTMMrFCFunctionHP1414113No12.817Unknown◂A▸HP0522481Yes54.914cag3, UnknownHP0819216No247ProV, import system◂A▸HP0522””””UnknownHP0525330No37.414ATPase, virB11◂A▸HP1451 (92–264)173No3017UnknownHP0149194Yes2217Unknown◂A▸HP0527 (1496–1870)375Yes40.514cag7, homologueHP0261132No15.617Unknown◂A▸HP0537 (39–365)327No3814CagM, UnknownHP1489510Yes56.817Unknown◂B▸HP0540 (126–381)256No28.614cag19, UnknownHP0496133No15.417Unknown◂A▸HP0547 (392–733)342No38.514CagA, CytotoxinHP1032255No29.39RNA pol. Sigma factor, FliA◂A▸HP1122 (48–76)29No3.317Anti sigma FlgMHP1293344No38.39DNA-dependent RNA polymerase, rpoA◂A▸HP1198 (659–1272)614No68.59DNA-dependent rpoBHP1231218No25.28DNA polymerase III, holB◂A▸HP1247 (199–340)142No16.417UnknownHP1231 (136–218)83”9.7””◂A▸HP1247 (199–340)”””””HP0650196No22.117Unknown◂A▸HP1245179No19.98DNA helicase, SSBHP1230180No20.817Unknown◂A▸HP1529457No51.58Chromosome replication initiation, DnaAHP1338148No1717Unknown◂A▸HP1338148No1717UnknownHP0042233Yes25.614ComB3, Trbl, virB10 homologue◂A▸HP0017 (34–336)303No35.214VirB4 homologueHP0862223No24.517Unknown◂C▸HP1474 (100–191)92No10.32Thymidylate kinase, tmkHP0608160No17.817Unknown◂A▸HP0175 (43–299)257No29.611Peptidyl-prolyl cis-trans isomerase Open table new tab Unless otherwise stated, PCR amplification specific primers inserted restriction sites BamHI (5′ end) EcoRI (3′ end). For HP0547 BamHI-PstI EcoRI-HindIII used, Most coding sequences digesting bait clones PstI enzymes HP0522 HP0862, BamH1 EcoRI. digested visualized agarose gel, correctly sized bands excised purified gel kit (Concert Life Technologies, Inc., Grand Island, NY). cloned pPROEXHT (-b -c) pMAL (-BE -G) vectors definition below), generating His6- (MBP)-tagged version His6 MBP tags add 6 44 kDa To fusions, designed. pMAL-c2x vector (New England Biolabs, Beverly, MA) expresses N-terminal fusion modified insertion PreScission protease cleavage site XmnI (gift Joe St. Gemes, Washington University, St Louis, MO). then inverted create pMAL-BE. From pMAL-BE, G front pMAL-G. All SID appropriate all pPROEXHTb (for pPROEXHTc proteins) used. HP1451 (a SID), pPROEXHTa. finally transformed BL21(DE3)pLysS competent cells (Invitrogen, San Diego, CA). co-expression subcloned pAlter-Ex2 (Promega, Madison, WI) NcoI-PstI sites. contains tetracycline-resistant well origin (p15a), makes it suitable co-expressing pPROEXHT-cloned partners. Freshly recombinants inoculated 5 ml Luria-Bertani broth containing 100 μg/ml ampicillin 40 chloramphenicol, grown overnight 37 °C. preculture inoculate 500 fresh same antibiotics. °C OD600 0.6 induced 1 mm isopropyl β-d-thiogalactoside h. Solubility fragment assessed harvesting cells, resuspending lysis buffer (20 Tris-HCl, pH 7.6, 150 NaCl, 0.2% (v/v) Triton X-100, 5% glycerol), lyzing sonication pelleting debris centrifugation, collecting supernatant, analyzing supernatant SDS-PAGE presence band visually Coomassie blue staining fusions (all strongly soluble, see “Results”) anti-His6 antibody below) detect His6-tagged fusions. that, our hands, mixing interactive prior did detectable changes solubility individual Nor induction 20 Several protocols devised depending soluble). Cells soluble mixed together harvested. pellet resuspended glycerol) frozen (−80 °C). inhibitor mixture (1 PMSF, 1% aprotinin, β-mercaptoethanol) unfrozen sonication. disrupted sonication, separated centrifugation 45,000 g min. resulting loaded onto amylose column (fast flow resin, New 10-ml volume) equilibrated NaCl). eluted applying short linear gradient (3 volumes) (buffer maltose). usually around 10–15% partner detected followed below). When insoluble (i.e. inclusion bodies centrifugation), washed R (8 m urea, Tris, NaCl), centrifuged. dissolved h 4 constant stirring. discarded (40,000 g, 15 min). diluted times U guanidine, solution gently (0.5 ml/min) 300 F l-arginine, NaCl) stirring centrifuged min) remove precipitated protein. TALON resin (10-ml volume, Clontech, Palo Alto, CA) NaCl 8. imidazole. formation, having expressed fusion. Complex monitored elution column. solubilized described. unfolded 200 8 urea. imidazole buffer. fractions pooled (6–8 total) (final volume 30 ml). dialysed against liter filtered, previously). procedure section “Protein Expression” except tetracycline media concentration μg/ml. carried out 12% polyacrylamide gels Mini Protean III following manufacturer’s protocol. made per complex, stained Simple Blue (Invitrogen) Western blot. Separated blotted PVDF liquid