Identification of VCP/p97, Carboxyl Terminus of Hsp70-interacting Protein (CHIP), and Amphiphysin II Interaction Partners Using Membrane-based Human Proteome Arrays
2005; Elsevier BV; Volume: 5; Issue: 2 Linguagem: Inglês
10.1074/mcp.m500198-mcp200
ISSN1535-9484
AutoresGerlinde Grelle, Susanne Kostka, Albrecht Otto, Birgit Kersten, K. Genser, Eva‐Christina Müller, Stephanie Wälter, Annett Böddrich, Ulrich Stelzl, Christian Hänig, Rudolf Volkmer, Christiane Landgraf, Simon Alberti, Jörg Höhfeld, Martin Strödicke, Erich E. Wanker,
Tópico(s)vaccines and immunoinformatics approaches
ResumoProteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles. Proteins mediate their biological function through interactions with other proteins. Therefore, the systematic identification and characterization of protein-protein interactions have become a powerful proteomic strategy to understand protein function and comprehensive cellular regulatory networks. For the screening of valosin-containing protein, carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II interaction partners, we utilized a membrane-based array technology that allows the identification of human protein-protein interactions with crude bacterial cell extracts. Many novel interaction pairs such as valosin-containing protein/autocrine motility factor receptor, CHIP/caytaxin, or amphiphysin II/DLP4 were identified and subsequently confirmed by pull-down, two-hybrid and co-immunoprecipitation experiments. In addition, assays were performed to validate the interactions functionally. CHIP e.g. was found to efficiently polyubiquitinate caytaxin in vitro, suggesting that it might influence caytaxin degradation in vivo. Using peptide arrays, we also identified the binding motifs in the proteins DLP4, XRCC4, and fructose-1,6-bisphosphatase, which are crucial for the association with the Src homology 3 domain of amphiphysin II. Together these studies indicate that our human proteome array technology permits the identification of protein-protein interactions that are functionally involved in neurodegenerative disease processes, the degradation of protein substrates, and the transport of membrane vesicles. As protein-protein interactions (PPIs) 1The abbreviations used are: PPI, protein-protein interaction; HA, hemagglutinin; SH3, Src homology 3; CHIP, carboxyl terminus of Hsp70-interacting protein; VCP, valosin-containing protein; AMFR, autocrine motility factor receptor; FBP, fructose-1,6-bisphosphatase; aa, amino acid(s); HRP, horseradish peroxidase; DLP4, Discs large-associated protein 4; Y2H, yeast two-hybrid; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; E4, ubiquitin-conjugating enzyme; E2Q, ubiquitin-conjugating enzyme 1The abbreviations used are: PPI, protein-protein interaction; HA, hemagglutinin; SH3, Src homology 3; CHIP, carboxyl terminus of Hsp70-interacting protein; VCP, valosin-containing protein; AMFR, autocrine motility factor receptor; FBP, fructose-1,6-bisphosphatase; aa, amino acid(s); HRP, horseradish peroxidase; DLP4, Discs large-associated protein 4; Y2H, yeast two-hybrid; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; E4, ubiquitin-conjugating enzyme; E2Q, ubiquitin-conjugating enzyme are central to most biological processes, their systematic identification is considered a key strategy for uncovering the complex organization principles of functional cellular networks (1Legrain P. 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Identification of barley CK2α targets by using the protein microarray technology.Phytochemistry. 2004; 65: 1777-1784Google Scholar, 26Feilner T. Hultschig C. Lee J. Meyer S. Immink R.G.H. Koenig A. Possling A. Seitz H. Beveridge A. Scheel D. Cahill D.J. Lehrach H. Kreutzberger J. Kersten B. High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates.Mol. Cell. Proteomics. 2005; 4: 1558-1568Google Scholar). In principle, the production of protein arrays and their utilization for systematic large scale interaction and activity screens has proven viable. One major drawback, however, has been the necessity to use purified proteins. Protein purification is usually difficult, time-consuming, and expensive. For array-based studies, especially high throughput systematic screening (19Ramachandran N. Hainsworth E. Bhullar B. Eisenstein S. Rosen B. Lau A.Y. Walter J.C. LaBaer J. Self-assembling protein microarrays.Science. 2004; 305: 86-90Google Scholar), technologies are needed that permit the use of crude protein extracts.Here we describe the design and application of an array-based technology that allows the identification of PPIs with crude bacterial cell extracts containing recombinant human proteins. Using this approach, novel partners for the proteins valosin-containing protein (VCP), carboxyl terminus of Hsp70-interacting protein (CHIP), and amphiphysin II were detected and confirmed with different independent binding assays, validating our strategy.EXPERIMENTAL PROCEDURESClones and Constructs—For the production of protein arrays, clones from the hEx1 library (27Büssow K. Cahill D. Nietfeld W. Bancroft D. Scherzinger E. Lehrach H. Walter G. A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.Nucleic Acids Res. 1998; 26: 5007-5008Google Scholar) expressing His-tagged human fusion proteins were used. For the expression of GST-tagged fusions, cDNA fragments encoding CHIP (aa 2–303) and amphiphysin II (aa 497–593) were PCR-amplified from the brain cDNA pool number 588 (RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH), Berlin, Germany) and subcloned into the plasmids pGEX-6P-1 and −2 (Amersham Biosciences), respectively. The cDNA encoding VCP was obtained from ATCC (American Type Culture Collection), amplified, and subcloned into pGEX-6P-1 (Amersham Biosciences) and pTL-1 (28Sittler A. Walter S. Wedemeyer N. Hasenbank R. Scherzinger E. Eickhoff H. Bates G.P. Lehrach H. Wanker E.E. SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polyGln-containing protein aggregates.Mol. Cell. 1998; 2: 427-436Google Scholar), respectively. Dynamin1-encoding cDNA (aa 634–864) was amplified from full-length cDNA obtained from RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH) and subcloned into pQE30-NST. For co-localization studies, a cDNA fragment encoding autocrine motility factor receptor (AMFR) (aa 396–643) was subcloned into pTL-HA-2 (28Sittler A. Walter S. Wedemeyer N. Hasenbank R. Scherzinger E. Eickhoff H. Bates G.P. Lehrach H. Wanker E.E. SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polyGln-containing protein aggregates.Mol. Cell. 1998; 2: 427-436Google Scholar). For confirmation of interactions with the two-hybrid system, the plasmids pBTM117c (29Wanker E.E. Rovira C. Scherzinger E. Hasenbank R. Walter S. Tait D. Colicelli J. Lehrach H. HIP-I: a huntingtin interacting protein isolated by the yeast two-hybrid system.Hum. Mol. Genet. 1997; 6: 487-495Google Scholar) and pACT4 (derivative of pACT2 from Clontech) were used.Sequence Analysis—Clones were subjected to DNA tag sequencing, starting from the 5′-ends of the cDNA inserts. After base calling and quality clipping of sequencer trace data (PHRED, trim_cutoff = 0.05), 13,964 sequences were translated into protein sequences. BLASTP searches in the NCBI and TrEMBL (Swiss-Prot) protein databases were performed.Overexpression of His-tagged Proteins for Array Production—For expression of His-tagged human fusion proteins, 384-well microtiter plates were filled with 40 μl of TB medium/well (100 mm glucose, 100 μg/ml ampicillin, and 15 μg/ml kanamycin) and inoculated with 13,824 Escherichia coli clones from the hEx1 cDNA library. Cells were grown for 3 h at 37 °C followed by induction of protein expression by addition of 40 μl of 1 mm isopropyl thiogalactoside and overnight incubation at 30 °C. Then cells were pelleted by centrifugation for 10 min at 4,000 rpm at 4 °C and stored at −80 °C.Native Cell Lysis and Generation of Protein Arrays—Cell pellets were resuspended in 50 μl of lysis buffer (1 mg/ml lysozyme, 0.5% Nonidet P-40, 1% Triton X-100, 1 mm PMSF, 25 units/ml benzonase, and 150 mm NaCl in PBS) and incubated for 90 min on ice. During the incubation, cell suspensions were mixed carefully every 10 min using a 384-pin replicator. Then the plates were centrifuged at 4,000 rpm at 4 °C for 1 h to pellet cell debris. Supernatants were spotted onto 225 × 225-mm nitrocellulose membranes (Protran BA 83, Schleicher & Schuell) using the K2-003 Gridder (KBiosystems). Each extract was spotted 20 times on each position in a 5 × 5 duplicate gridding pattern.Overlay Assay with GST-tagged Fusion Proteins—GST fusion proteins were expressed in E. coli as described previously (30Wanker E.E. Scherzinger E. Heiser V. Sittler A. Eickhoff H. Lehrach H. Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates.Methods Enzymol. 1999; 309: 375-386Google Scholar). Crude E. coli cell extracts were used for overlay screens. Membranes with high density spotted His-tagged fusion proteins were blocked for 4 h at 4 °C with PBS-T (PBS supplemented with 0.05% Tween 20) containing 0.5 mm DTT and 5% nonfat dry milk powder. Then they were rinsed with PBS-T and incubated overnight at 4 °C in 100 ml of overlay buffer (PBS-T supplemented with 5% fetal calf serum, 100 μm ATP, and 100 μm GTP) to which crude bacterial cell extract containing the GST fusion protein (∼0.5 mg) had been added prior to use. After incubation, membranes were washed once for 10 min with PBS-TT (TT = 0.05% Tween 20 + 0.2% Triton X-100) and three times for 10 min each with PBS-T. Subsequently filters were incubated for 1 h at room temperature in 100 ml of PBS-T containing anti-GST HRP-conjugated antibody (5,000-fold diluted, Amersham Biosciences). Membranes were rinsed twice with PBS-T, washed once with PBS-T for 15 min, and then washed three times for 5 min. Subsequently filters were incubated for 3 min in CHEMIGLOW solution (Alphainnotech) according to the manufacturer's instructions. Signals were detected using a Fuji luminescent image analyzer (LAS 1000 CH).The resulting images were analyzed with Aida Image Analyzer version 3.21.001 (Raytest GmbH). The intensity of spots was determined using a spot diameter of 2,280 μm if both spots of a respective duplicate gave a distinct signal in the image. Background correction of the average signal intensities of duplicates was performed blockwise. Interactions were considered positive when the relative signal of respective duplicates was greater than 3 times the standard deviation of average background signal.In Vitro Protein-Protein Binding Assays—For pull-down experiments, ∼20 μg of GST fusion protein was immobilized on 100 μl of glutathione-agarose beads (Sigma) and incubated with bacterial protein extracts containing 20 μg of His-tagged fusion protein in IP buffer (50 mm Hepes, pH 7.4, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, 20 mm NaF, 10% glycerol, 1% Nonidet P-40, and protease inhibitors) at 7 °C for 4 h. Then beads were washed four times with IP buffer and incubated with 100 μl of 4× SDS gel loading buffer at 95 °C for 5 min. Proteins were analyzed by SDS-PAGE and Western blotting using anti-GST (HRP conjugate, Amersham Biosciences) and anti-His (penta-His HRP conjugate, Qiagen) antibodies.Mass Spectrometry—For identification, proteins of interest were separated by SDS-PAGE. Protein bands were excised from the gels and digested with trypsin. The peptide mixture was analyzed by peptide mass fingerprinting using a MALDI TofSpec2E mass spectrometer (Micromass). In case of equivocal results, the identification was performed with MS/MS sequencing. The MS/MS measurement was achieved with a nanoelectrospray hybrid quadrupole mass spectrometer (Q-TOF, Micromass). The Mascot software package (Matrix Science) was used for protein identification.Yeast Two-hybrid (Y2H) Analysis—L40ccU MATa and L40ccα MATα yeast strains were transformed with plasmids encoding bait and prey proteins, respectively (10Goehler H. Lalowski M. Stelzl U. Waelter S. Stroedicke M. Worm U. Droege A. Lindenberg K.S. Knoblich M. Haenig C. Herbst M. Suopanki J. Scherzinger E. Abraham C. Bauer B. Hasenbank R. Fritzsche A. Ludewig A.H. Buessow K. Coleman S.H. Gutekunst C.A. Landwehrmeyer B.G. Lehrach H. Wanker E.E. A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.Mol. Cell. 2004; 15: 853-865Google Scholar). For interaction mating, the MATα and MATa strains were individually mixed in 96-well microtiter plates, transferred onto YPD agar plates using the K2-003 spotting robot (KBiosystems) and incubated for 36 h at 30 °C. After mating, the clones were transferred onto SDII (Leu−Trp−) agar plates and grown for 50 h at 30 °C to select for diploid yeast strains. For selection of interactions, diploid yeasts were spotted onto SDIV (Leu−Trp−Ura−His− where Ura is uracil) agar plates as well as on nylon membranes placed on SDIV agar plates. After 7 days of incubation at 30 °C, the agar plates and nylon membranes were assessed for growth and β-galactosidase activity, respectively (10Goehler H. Lalowski M. Stelzl U. Waelter S. Stroedicke M. Worm U. Droege A. Lindenberg K.S. Knoblich M. Haenig C. Herbst M. Suopanki J. Scherzinger E. Abraham C. Bauer B. Hasenbank R. Fritzsche A. Ludewig A.H. Buessow K. Coleman S.H. Gutekunst C.A. Landwehrmeyer B.G. Lehrach H. Wanker E.E. A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.Mol. Cell. 2004; 15: 853-865Google Scholar).Co-immunoprecipitation and Co-localization Studies—Co-immunoprecipitation experiments were performed as described previously (28Sittler A. Walter S. Wedemeyer N. Hasenbank R. Scherzinger E. Eickhoff H. Bates G.P. Lehrach H. Wanker E.E. SH3GL3 associates with the Huntingtin exon 1 protein and promotes the formation of polyGln-containing protein aggregates.Mol. Cell. 1998; 2: 427-436Google Scholar). For immunofluorescence microscopy, COS-1 cells were grown on coverslips and co-transfected with pTL-VCP and pTL-HA-AMFR encoding full-length VCP and C-terminal hemagglutinin (HA)-tagged AMFR (aa 396–643). 40 h post-transfection, cells were treated with 2% paraformaldehyde. Immunolabeling was performed with rabbit anti-VCP (1:500) coupled to CY3-conjugated antibody (red) (1:100, Dianova) and with mouse anti-HA antibody (1:200, Babco) coupled to Alexa 488-conjugated antibody (green) (1:100, Molecular Probes). Nuclei were counterstained with Hoechst (Sigma). Samples were observed with the fluorescence microscope Axioplan-2 (Zeiss).Ubiquitination Assays—Ubiquitination of caytaxin in vitro was performed essentially as described previously for Raf-1 (31Demand J. Alberti S. Patterson C. Hohfeld J. Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.Curr. Biol. 2001; 11: 1569-1577Google Scholar). All reactions contained 5% of protein extract from bacteria expressing His-tagged caytaxin and 1.25 mg/ml of purified ubiquitin (Sigma). When indicated 0.1 μm E1, 4 μm UbcH5b, 3 μm CHIP, 0.3 μm Hsp40, and 3 μm Hsc70 were added. Samples were incubated for 2 h at 30 °C and subsequently analyzed by SDS-PAGE and immunoblotting using an anti-His antibody (hexa-His, Roche Applied Science).Peptide Arrays—Two identical arrays were synthesized on a cellulose-(3-amino-2-hydroxy-propyl)-ether (CAPE) membrane using the Spot technology as described previously (32Landgraf C. Panni S. Montecchi-Palazzi L. Castagnoli L. Schneider-Mergener J. Volkmer-Engert R. Cesareni G. Protein interaction networks by proteome peptide scanning.PLoS Biol. 2004; 2: E14Google Scholar). Each array represents four scans of overlapping 15-mer peptides with a shift of three amino acids representing the interaction partners (Table I). Probing the array toward binding to amphiphysin II was performed as described previously (32Landgraf C. Panni S. Montecchi-Palazzi L. Castagnoli L. Schneider-Mergener J. Volkmer-Engert R. Cesareni G. Protein interaction networks by proteome peptide scanning.PLoS Biol. 2004; 2: E14Google Scholar) except that the peroxidase-labeled anti-GST antibody (RPN1236, Amersham Biosciences) was used for detection. To exclude false positive results in the binding experiment, one array was pre-examined with GST/anti-GST antibody. All spot signal intensities (Boehringer light unit) were measured on a Lumi-Imager™ (Roche Diagnostics) and evaluated by using the software Genespotter® (Microdiscovery).Table IProtein interaction partners of VCP, CHIP, and amphiphysin II identified by overlay experiments with protein arraysGene nameGene IDProtein nameAccession no.Over layPull-downY2HSequence (aa)MSVCP7415VCPP550721–47915NSFL1C55968NSFL1 cofactor p47 (p47)Q9UNZ2+++1–3708AMFR267AMFR, isoform 2Q9UKV5+++396–6438STUB110273CHIPQ9UNE72–303MAPT4137Microtubule-associated protein Tau (Tau)P10636++−685–7562HSPA83312Heat shock cognate 71-kDa protein (Hsc70)P11142+++417–64612HSPA1B3304Heat shock 70-kDa protein 1 (Hsp70)P08107+++70–64121HSP90A30591Heat shock protein HSP90-α (Hsp90)P07900+++227–3787UBE2Q55585Ubiquitin-conjugating enzyme E2Q (E2Q)Q7Z7E8+++107–42210ATCAY85300CaytaxinQ86WG3+++78–3707BIN1274Myc box-dependent interacting protein 1 (amphiphysin II)O00499497–5934DNM11759Dynamin 1 (DNM1)Q05193+NDND634–8642DLGAP422839DLP4Q9Y2H0+++726–98916XRCC47518DNA repair protein (XRCC4)Q13426+++1–3367FBP12203FBPP09467+++1–33712 Open table in a new tab RESULTSProtein Arrays and Overlay Assays—To produce protein arrays for interaction studies, a human fetal brain cDNA library (hEx1) allowing the expression of His-tagged fusion proteins was used (27Büssow K. Cahill D. Nietfeld W. Bancroft D. Scherzinger E. Lehrach H. Walter G. A method for global protein expression and antibody screening on high-density filters of an arrayed cDNA library.Nucleic Acids Res. 1998; 26: 5007-5008Google Scholar). cDNA inserts of 13,824 expression plasmids were sequenced at their 5′-ends, and the sequence information was stored in a database. BLASTP searches in protein databases (33Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Google Scholar) and bioinformatic analysis of sequence data revealed that about 64% of the cDNA fragments encoded human proteins in the correct reading frame, 19% of which were full-length.For production of protein arrays, the E. coli clones were expressed in 384-well microtiter plates by addition of isopropyl thiogalactoside to the culture medium, and cells were lysed under non-denaturing conditions using lysozyme. After removal of insoluble cell debris by centrifugation, crude bacterial protein extracts were double gridded onto membrane filters using a robot. The resulting high density spotted membranes (225 × 225 mm in size) containing 13,824 protein extracts were dried and stored at 4 °C prior to protein interaction screens.To determine how many of the protein samples on each membrane contained a recombinant His-tagged human fusion protein, the filter membranes were first probed with an anti-His antibody. Image analysis revealed that ∼22% of the expression clones produced a detectable fusion protein. This is in agreement with previous studies demonstrating that ∼20% of the hEx1 library clones express a soluble human protein (34Bussow K. Quedenau C. Sievert V. Tischer J. Scheich C. Seitz H. Hieke B. Niesen F.H. Gotz F. Harttig U. Lehrach H. A catalog of human cDNA expression clones and its application to structural genomics.Genome Biol. 2004; 5: R71Google Scholar). In total, about 3,000 His-tagged recombinant proteins were accessible for interaction studies on each high density spotted filter membrane.To identify PPIs, the protein arrays were used for proof-of-principle overlay experiments with the human proteins VCP, CHIP, and amphiphysin II (Table I). GST-tagged fusions were expressed in E. coli, and after native lysis with lysozyme, crude protein extracts were prepared and incubated overnight with the high density spotted filter membranes (Fig. 1). After extensive washing, PPIs were detected by ELISA using an anti-GST antibody. Specific double spots that gave signals above background were detected with the relevant GST fusion protein (Supplemental Figs. 1–3) but not with the control protein GST (data not shown). For each GST fusion protein three independent overlay experiments were performed, all of which essentially gave the same results (data not shown). The identity of the His-tagged proteins detected by overlay screening was determined using image analysis tools directly linked to our sequence database. In addition, interacting His-tagged proteins expressed in E. coli were affinity-purified with nickel-nitrilotriacetic acid-agarose beads and analyzed by MS (Table I).Identification and Characterization of VCP Protein-Protein Interactions—VCP, a member of the AAA ATPase family, is involved in a variety of cellular processes including membrane fusion (35Kondo H. Rabouille C. Newman R. Levine T.P. Papp
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