HMGB1 Interacts with Many Apparently Unrelated Proteins by Recognizing Short Amino Acid Sequences
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m108417200
ISSN1083-351X
AutoresAgnès Dintilhac, Jordi Bernués,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe chromatin high mobility group protein 1 (HMGB1) is a very abundant and conserved protein that is structured into two HMG box domains plus a highly acidic C-terminal domain. From the ability to bind DNA nonspecifically and to interact with various proteins, several functions in DNA-related processes have been assigned to HMGB1. Nevertheless, its functional role remains the subject of controversy. Using a phage display approach we have shown that HMGB1 can recognize several peptide motifs. A computer search of the protein data bases found peptide homologies with proteins already known to interact with HMGB1, like p53, and have allowed us to identify new potential candidates. Among them, transcriptional activators like the heterogeneous nuclear ribonucleoprotein K (hnRNP K), repressors like methyl-CpG binding protein 2 (MeCP2), and co-repressors like the retinoblastoma susceptibility protein (pRb) and Groucho-related gene proteins 1 (Grg1) and 5 (Grg5) can be found. A detailed analysis of the interaction of Grg1 with HMGB1 confirmed that the binding region contained the sequence homologous to one of the peptides identified. Our results have led us to propose that HMGB1 may play a central role in the stabilization and/or assembly of several multifunctional complexes through protein-protein interactions. The chromatin high mobility group protein 1 (HMGB1) is a very abundant and conserved protein that is structured into two HMG box domains plus a highly acidic C-terminal domain. From the ability to bind DNA nonspecifically and to interact with various proteins, several functions in DNA-related processes have been assigned to HMGB1. Nevertheless, its functional role remains the subject of controversy. Using a phage display approach we have shown that HMGB1 can recognize several peptide motifs. A computer search of the protein data bases found peptide homologies with proteins already known to interact with HMGB1, like p53, and have allowed us to identify new potential candidates. Among them, transcriptional activators like the heterogeneous nuclear ribonucleoprotein K (hnRNP K), repressors like methyl-CpG binding protein 2 (MeCP2), and co-repressors like the retinoblastoma susceptibility protein (pRb) and Groucho-related gene proteins 1 (Grg1) and 5 (Grg5) can be found. A detailed analysis of the interaction of Grg1 with HMGB1 confirmed that the binding region contained the sequence homologous to one of the peptides identified. Our results have led us to propose that HMGB1 may play a central role in the stabilization and/or assembly of several multifunctional complexes through protein-protein interactions. In the eukaryotic cell nucleus of all vertebrate cell types, HMGB1 1HMGB1high mobility group protein 1pRbretinoblastoma proteinhnRNP Kheterogeneous nuclear ribonucleoprotein KMeCP2methyl-CpG binding protein 2Grg1Groucho-related gene protein 1Grg5Groucho-related gene protein 5GSTglutathione S-transferase (formerly named HMG1, see Ref. 1Bustin M. Trends Biochem. Sci. 2001; 26: 152-153Abstract Full Text Full Text PDF PubMed Google Scholar for a revised nomenclature) is one of the most abundant non-histone proteins. HMGB1 has been shown to be essential because knock-out mice die 24 h after birth (2Calogero S. Grassi F. Aguzzi A. Voitländer T. Ferrier P. Ferrari S. Bianchi M.E. Nat. Genet. 1999; 22: 276-280Crossref PubMed Scopus (459) Google Scholar). HMGB1 is highly conserved, particularly in mammals and to a lesser extent throughout the animal kingdom. HMGB1 is structured into three domains, two basic HMG boxes (HMG domains A and B) and a highly acidic C-terminal domain, which confer an overall dipolar appearance to this protein (see Refs.3Bustin M. Reeves R. Prog. Nucleic Acids Res. Mol. Biol. 1996; 54: 35-100Crossref PubMed Google Scholar, 4Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (772) Google Scholar, 5Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar for reviews). Each of the HMG boxes is formed by two short and one long α-helix that upon folding produce an L- or V-shaped three-dimensional domain structure (6Read C.M. Cary P.D. Crane-Robinson C. Driscoll P.C. Norman D.G. Nucleic Acids Res. 1993; 21: 3427-3436Crossref PubMed Scopus (249) Google Scholar, 7Weir H.M. Kraulis P.J. Hill C.S. Raine A.R. Laue E.D. Thomas J.O. EMBO J. 1993; 12: 1311-1319Crossref PubMed Scopus (377) Google Scholar, 8Hardman C.H. Broadhurst R.W. Raine A.R.C. Grasser K.D. Thomas J.O. Laue E.D. Biochemistry. 1995; 34: 16596-16607Crossref PubMed Scopus (168) Google Scholar). Whereas the acidic C-terminal domain is presumably involved in the modulation of HMGB1 activity, the HMG box domains allow the protein to bind to linear DNA with moderate affinity and to highly structured (3- and 4-way junction DNA, cruciform DNA) or distorted DNA (bent or kink DNA, bulged DNA, cisplatin-modified DNA) with higher affinity, but always without sequence specificity. The concave surface of the L- or V-shaped HMG box domain contacts the DNA in the minor groove in two slightly different ways introducing important modifications in the structure of DNA, in particular a strong bend (reviewed in Ref. 5Thomas J.O. Travers A.A. Trends Biochem. Sci. 2001; 26: 167-174Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). Presumably, these features will be of relevance for the biological functions in which HMGB1 has been involved (DNA repair, recombination, replication, and transcription). high mobility group protein 1 retinoblastoma protein heterogeneous nuclear ribonucleoprotein K methyl-CpG binding protein 2 Groucho-related gene protein 1 Groucho-related gene protein 5 glutathione S-transferase The activity of HMGB1 is not solely mediated by its ability to bind to DNA. Indeed, HMGB1 and the related HMGB2 protein can interact through their HMG box domains with a broad range of proteins ranging from nuclear cell proteins to viral proteins. Interactions of HMGB1 have been described with the recombination activation gene protein RAG1 (9Aidinis V. Bonaldi T. Beltrame M. Santgata S. Bianchi M.E. Spanopoulou E. Mol. Cell. Biol. 1999; 19: 6532-6542Crossref PubMed Scopus (111) Google Scholar), several transcription factors including the cellular tumor suppressor p53 (10Jayaraman L. Moorthy N.C. Murthy K.G.K. Manley J.L. Bustin M. Prives C. Genes Dev. 1998; 12: 462-472Crossref PubMed Scopus (286) Google Scholar), the octamer transcription factors Oct1, Oct2, Oct4, and Oct6 (11Zwilling S. König H. Wirth T. EMBO J. 1995; 14: 1198-1208Crossref PubMed Scopus (218) Google Scholar, 12Butteroni C., De Felici M. Scholer H.R. Pesce M. J. Mol. Biol. 2000; 304: 529-540Crossref PubMed Scopus (58) Google Scholar), some homeotic HOX proteins (13Zappavigna V. Faciola L. Citterich M.H. Mavilio F. Bianchi M.E. EMBO J. 1996; 15: 4981-4991Crossref PubMed Scopus (217) Google Scholar), the steroid receptors (progesterone (PR), glucocorticoid (GR), estrogen (ER), and androgen (AR)) (14Oñate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijhon D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar, 15Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E.A. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (306) Google Scholar), the general initiation factor human TATA-binding protein (hTBP) (16Ge H. Roeder R.G. J. Biol. Chem. 1994; 269: 17136-17140Abstract Full Text PDF PubMed Google Scholar, 17Sutrias-Grau M. Bianchi M.E. Bernues J. J. Biol. Chem. 1999; 274: 1628-1634Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 18Lu W. Peterson R. Dasgupta A. Scovell W.M. J. Biol. Chem. 2000; 275: 35006-35012Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), and the viral replication proteins Rep78 and Rep68 (19Costello E. Saudan P. Winocour E. Pizer L. Beard P. EMBO J. 1997; 16: 5943-5954Crossref PubMed Scopus (87) Google Scholar). The consequences of these interactions are multiple. HMGB1 in general increases the DNA binding affinity of those factors and depending on the context and the assay conditions HMGB1 has been shown to have a positive or a negative effect on transcription (14Oñate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijhon D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar, 15Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E.A. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (306) Google Scholar, 16Ge H. Roeder R.G. J. Biol. Chem. 1994; 269: 17136-17140Abstract Full Text PDF PubMed Google Scholar, 17Sutrias-Grau M. Bianchi M.E. Bernues J. J. Biol. Chem. 1999; 274: 1628-1634Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20Shykind B.M. Kim J. Sharp P.A. Genes Dev. 1995; 9: 1354-1365Crossref PubMed Scopus (130) Google Scholar). In the case of RAG1 and Rep68/78 HMGB1 enhances the rate of the sequence-specific DNA cleavage reaction (19Costello E. Saudan P. Winocour E. Pizer L. Beard P. EMBO J. 1997; 16: 5943-5954Crossref PubMed Scopus (87) Google Scholar, 21Van Gent D.C. Hiom K. Paull T.T. Gellert M. EMBO J. 1997; 16: 2665-2670Crossref PubMed Scopus (221) Google Scholar). Interestingly, HMGB1 can also stimulate the ATPase activity of Rep78 (19Costello E. Saudan P. Winocour E. Pizer L. Beard P. EMBO J. 1997; 16: 5943-5954Crossref PubMed Scopus (87) Google Scholar). The two HMG box domains of HMGB1 appear to have a similar but not identical behavior with respect to their protein-interacting features. Thus, HMG box A is important for binding to hTBP and p53, whereas the binding to Oct factors, HOX factors, and hormone receptors can take place through boxes A or B (11Zwilling S. König H. Wirth T. EMBO J. 1995; 14: 1198-1208Crossref PubMed Scopus (218) Google Scholar, 13Zappavigna V. Faciola L. Citterich M.H. Mavilio F. Bianchi M.E. EMBO J. 1996; 15: 4981-4991Crossref PubMed Scopus (217) Google Scholar, 17Sutrias-Grau M. Bianchi M.E. Bernues J. J. Biol. Chem. 1999; 274: 1628-1634Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 22Imamura T. Izumi H. Nagatani G. Ise T. Nomoto M. Iwamoto Y. Kohno K. J. Biol. Chem. 2001; 276: 7534-7540Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). However, the interaction with RAG requires both HMG box domains (9Aidinis V. Bonaldi T. Beltrame M. Santgata S. Bianchi M.E. Spanopoulou E. Mol. Cell. Biol. 1999; 19: 6532-6542Crossref PubMed Scopus (111) Google Scholar). To date, neither the HMG box surface that is involved in the interaction with other proteins nor the required amino acids of HMGB1 are known. On the other hand, sequence analysis of the factors interacting with HMGB1 does not suggest any apparent homology or similarity. For instance, the interaction with RAG1, Oct, and HOX factors occurs at the homeodomain. In the case of hTBP, it is the H2′ α-helix of the core and with Rep78, two different regions are recognized. From these data, no consensus can be defined. The fast progress of genomics and proteomics has made it obvious that an important focus in understanding biological processes is to characterize how proteins interact in macromolecular complexes. Attempts to define general rules for predicting specific recognition between proteins have been unsuccessful because each protein-protein interaction has its own properties. Nevertheless, some indications are emerging. The development of powerful tools has led to the discovery that one type of recognition involves asymmetric interactions that occur between a particular domain and a short region, often less than 10 amino acids in length, within the other protein. A recent review (23Kay B.K. Kasanov J. Knight S. Kurakin A. FEBS Lett. 2000; 480: 55-62Crossref PubMed Scopus (55) Google Scholar) recapitulated several examples of protein domains involved in these kinds of interactions like SH3 (Src homology 3), phosphotyrosine-binding WW, EH (Eps15 homology), PDZ modules (PSD-95/dlg/ZO1), as well as pRb and the ER. Some of them, like the ER, could interact in several modes with different peptides, in a manner that depends on the bound ligand. In the present study we have explored the molecular recognition properties of HMGB1 by ligand selection from a large library of heptapeptides displayed on phages. Our results do not give support to one unique strong consensus sequence but rather to a few different kinds of peptide sequences. A BLAST search enabled us to predict new proteins that may interact with HMGB1. We have tested and confirmed the interactions with a few of these and have shown that HMGB1 can interact not only with transcriptional activators but also with repressors and co-repressors. Taken together the data suggest a complex network of protein-protein interactions that will clarify the biological function(s) of HMGB1. The plasmids pT7-HMGB1bA, pT7-HMGB1bB, and pET14b-HMGB1 used for the expression of rat HMGB1 box A, box B, and full-length HMGB1, respectively, have already been described. The procedure for the expression and purification of the three recombinant proteins was as described previously (17Sutrias-Grau M. Bianchi M.E. Bernues J. J. Biol. Chem. 1999; 274: 1628-1634Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Native calf thymus HMGB1 was purified as described previously (24Bernués J. Espel E. Querol E. Biochim. Biophys. Acta. 1986; 866: 242-251Crossref PubMed Scopus (30) Google Scholar). A phage display heptapeptide library kit (New England Biolabs, Beverly, MA) was used to screen for peptides binding to HMGB1 box domains A and B. The kit contained a random combinatorial collection of heptapeptides fused via a flexible linker sequence to the N terminus of protein pIII of bacteriophage M13. Each phage expressed at the tip of the cover 3–5 copies of the unique peptide it encoded. The library complexity contained all the possible combinations of the 20 natural amino acids taken as 7-mer sequences. For the phage biopanning process, we followed the kit instructions as indicated by the manufacturer. Four independent experiments were run in which 30–40 μg of HMGB1 box A or B were immobilized overnight at 4 °C on 96-well microtiter plates (Costar 3690). Wells were then blocked for 1 h at 4 °C with Tris-buffered saline, 0.1% Tween 20. 2 × 1011plaque-forming units of the phage library were added per well and incubated for an additional hour at room temperature. Wells were washed ten times with Tris-buffered saline, 0.1% Tween 20 for the first round. For subsequent rounds of washing, Tris-buffered saline, 0.5% Tween 20 was used to increase stringency. Finally, phages were either eluted at room temperature by incubation with 0.2 mglycine-HCl, pH 2.2 for 10 min or affinity eluted by incubation for 1 h with 30–40 μg of the respective HMGB1 box A or B. Phage amplification, titration, and purification were carried out according to the manufacturer's protocol. Automatic phage DNA sequencing used the −96gIII primer (5′-CCCCTCATAGTTAGCGTAACG-3′) and was performed by the Serveis Cientı́fico-Tècnics of the Universitat de Barcelona. The data base search for homology with the sequences selected in the phage display experiments was performed using the BLAST (Basic Local Alignment Search Tool) program (NCBI, National Center for Biotechnology Information) (25Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (61065) Google Scholar). The alignments were obtained using the MultAlin program (Multiple Sequence Alignment, INRA, Institut National de Recherche Agronomique, Toulouse, France) (26Corpet F. Nucleic Acids Res. 1988; 16: 10881-10890Crossref PubMed Scopus (4415) Google Scholar). All GST constructs were prepared using the pGEX-4T3 plasmid (Amersham Biosciences, Inc.). The twelve peptides selected to be further analyzed as GST fusions were amplified from the phages with PCR using the following primers 5′-TGGTACCTTTGAATTCTCACTC-3′ and 5′-TCAACAGTGTCGACCGAACC-3′, which introducedEcoRI and SalI sites, respectively (underlined). The PCR products were inserted between these sites in the pGEX-4T3 plasmid. pGEX-Grg1 and pGEX-Grg5 were constructed by inserting theNaeI/XhoI fragments obtained from the pBS-Grg1 and pBS-Grg5 (kindly provided by Dr. C. Lobe) into pGEX-4T3 digested with SmaI and XhoI. pGEX-Grg Q-GP-CcN was produced by inserting a NaeI/SmaI fragment from pBS-Grg1 into a SmaI site of the pGEX vector. pGEX-Grg SP-WD was generated by inserting a SmaI/XhoI fragment of pBS-Grg1 into the same sites of the pGEX vector. pGEX-Grg Q was obtained by digesting pGEX-Grg1 with Bpu1102I andXhoI. The vector was blunt-ended and then self-ligated. pGEX-Grg GP-CcN was prepared by digestion of pGEX-Grg1 Q-GP-CcN withBamHI and Bpu1102I. The vector was blunt-ended and then self-ligated. pGEX-Grg ΔGP-CcN was obtained by inserting aBamHI/XhoI fragment obtained by PCR from the pGEX-Grg1 construct by using the primers 5′-TTCAGCCTCCTGGATCCCCG-3′ and 5′-GTTTTCTCGAGGTGAGTGTG-3′ (restriction sites underlined) into pGEX 4T3 digested with the same enzymes. pGEX-Grg SP was generated by inserting a SmaI/XhoI fragment obtained as above by PCR with primers 5′-ACTCACCCCGGGAAAACG-3′ and 5′-GTTGATCTCTCGAGCATGTCG-3′ into pGEX 4T3 digested with the same enzymes. All constructions were verified by manual or automated DNA sequencing. pGEX-MeCP2-(207–492) was obtained from Dr. A. Bird. pGEX-KG-hn RNP K was a gift of Drs. S. K. Jong and J. H. Kim. pGEX-pRb-(379–928) and pGEX-p53 were obtained from Dr. M. A. Martı́nez-Balbás. All the GST fusions were expressed in Escherichia coliBL21(DE3) and purified according to standard methods as suggested by the manufacturer (Amersham Biosciences, Inc.). GST-peptide fusions were separated by SDS-PAGE and electroblotted in transfer buffer (25 mm Tris, 40 mm glycine, 0.05% SDS, 20% methanol) to nitrocellulose membranes (Optitran BA-85, Schleicher & Schuell). After blocking in phosphate-buffered saline, 0.1% Tween 20) containing 5% nonfat dry milk for 1–2 h at room temperature, membranes were incubated overnight in 10 ml of D buffer without glycerol (20 mm HEPES, pH 7.9, 0.2 mm EDTA, 100 mm NaCl, 0.5 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride) containing 10–20 μg of either HMGB1 or its derivatives, HMGB1 box A or B. After several washes in phosphate-buffered saline/Tween, membranes were incubated with a primary chicken anti-HMGB1 antibody raised in our laboratory against recombinant HMGB1 deleted of the C-terminal domain and subsequently with a secondary anti-chicken IgY-HRP antibody (Jackson Laboratories) and detected using ECL reagents. Glutathione-Sepharose beads (Amersham Biosciences, Inc.) were loaded with the different GST fusion proteins as suggested by the manufacturer and washed five times with 450 μl of D buffer containing 20% glycerol. Then, they were incubated with HMGB1 box A or B for 1 h at 4 °C in the same buffer and washed six times more with the same buffer. Beads were finally boiled in protein-loading buffer, and proteins were separated by SDS-PAGE and detected by Western blotting with specific antibodies. In an attempt to identify targets that could be potentially recognized by HMGB1, and because other general approaches like yeast two-hybrid analysis were not possible (likely due to toxic effects of the expression of HMGB1 boxes in yeast, results not shown) a peptide library screening approach was carried out. A highly complex library containing the whole collection of natural heptapeptide sequences displayed on phage M13 was used. Four rounds of selection were carried out for each of the four independent experiments that were performed using highly purified recombinant HMGB1 boxes A or B as bait proteins. During the biopanning process stringency was increased by using higher detergent concentrations in the washing buffer. Bound phages were recovered by either a nonspecific acid elution (experiment 1) or by affinity competition using HMGB1 boxes A or B free in solution (experiments 2–4). Several peptides were selected as potentially interacting with HMGB1 with some specificity (TableI). From these results, it became clear from their sequences that they were not related to a single strong consensus sequence, suggesting that either the interactions between HMGB1 and the peptides were weak or that HMGB1 boxes could interact with several unrelated motifs. We noted that the frequency of appearance of some amino acids in the selected peptides clearly deviated from the random theoretical level indicating that the selection process was successful. That is, if interactions of HMGB1 were specific they should be independent of the particular growth features of the phages and their statistics should clearly differ from those of the unselected phages in the biopanning assays and be due to their ligand-binding requirements. For example, positively and negatively charged amino acids usually presented frequencies lower than expected in the selected peptides, suggesting that the interactions did not mainly rely on electrostatic forces despite the highly basic character of the HMGB1 boxes. Also, a high rate of aromatic residues was observed, in particular a high level of tryptophan in experiment number 4, which contrasts with the observation that this amino acid tends to decrease naturally without selection. A remarkable level of proline was also obtained in experiment number 3. Hydrophilic amino acids, which tend to be involved in hydrogen-bond recognition, showed some decrease as well. These data indicate that selection had in fact occurred in the presence of the HMGB1 boxes although no clear-cut consensus could be easily drawn.Table IAmino acid sequences of the heptapeptides bound to HMG boxes A and B of HMGB1Exp. 1 box A1-aPeptides eluted unspecifically by low pH.Exp. 2 box A1-bPeptides eluted by affinity with the same HMG-box used for the biopanning.Exp. 3 box B1-bPeptides eluted by affinity with the same HMG-box used for the biopanning.Exp. 4 box B1-bPeptides eluted by affinity with the same HMG-box used for the biopanning.71-cNumber of peptides sequenced per experiment.311-cNumber of peptides sequenced per experiment.291-cNumber of peptides sequenced per experiment.201-cNumber of peptides sequenced per experiment.HWGMWSYSSPHNHS (6)1-dThe number of clones encoding the same peptide for each experiment is indicated in parentheses.IQSPHFF (8)HWGMWSY (5)NWGMWSYHAIYPRH (6)LPLTPLP (7)HSWLWWP (5)PHWTWVLQISFMAN (4)NQDVPLF (2)LAMPQYE (2)HMSKPVQTLTTPIL (2)WPKLASH (2)KLWVIPQSSGTHAKHWGMWSYSPAHAAKSHWFWSWYNINIRPISIPRTMASMSVAIHAIYPRHNYTQTVPTPAHNDYAWLPWAKSRPHTSDFHMGQPFMPNRTANHYWWWPRAPTPVKLNLPAYTSSSSSHPTHMALNxVCSSVETHPGAQLTKMHSLSYRGTPTLxSSTTLRYFWHWWPxLVMPWVHKGETRAPLYAPRLRSVQASNSNAPPTRNQGNSLRWDSAPQILLDTDPPGLHeptapeptide amino acid sequences deduced from phage sequencing are recapitulated here for each independent experiment.1-a Peptides eluted unspecifically by low pH.1-b Peptides eluted by affinity with the same HMG-box used for the biopanning.1-c Number of peptides sequenced per experiment.1-d The number of clones encoding the same peptide for each experiment is indicated in parentheses. Open table in a new tab Heptapeptide amino acid sequences deduced from phage sequencing are recapitulated here for each independent experiment. Despite the fact that the sequences selected were very variable (as shown in Table I), the appearance of several copies of the same peptide in each experiment indicated a high enrichment and specificity in the screening. Note also that some peptides (e.g. HWGMWSY, HAIYPRH) were selected in independent experiments with both HMGB1 domains. Despite this variability in the peptide sequences it was also clear that amino acid distribution in the peptides was not random (Table I). Thus, peptides could be grouped into at least two classes: proline-rich and tryptophan-rich. Two peptides enriched in tryptophan, HWGMWSY and HSWLWWP, accounted for 50% of the clones in experiment 4, and HWGMWSY appeared also in experiments 1 and 2. A minimal consensus WXXW motif could be a potential site for interaction. In the case of proline-enriched peptides it was more difficult to define a consensus given the diversity of these sequences. Because of the high complexity in the peptide sequences retrieved in the phage display experiments we were concerned about the potential existence of false positives in the selected set of peptides. As a second approach to confirm the bona fide association of HMGB1 with those peptides, we performed in vitro assays in which peptide sequences were fused to GST. The twelve peptides were used among those selected with HMGB1 boxes A and B and were representative of the different kinds of peptides obtained. They were fused to the C terminus of GST to facilitate their expression and purification in E. coli. As a control a GST with an extended, unrelated, and never selected peptide was included (GST-KG). Because direct GST pull-down assays did not work, likely because of steric hindrance of the GST moiety to the very small peptide (not shown), Far-Western experiments were performed (Fig.1). In contrast to the negative control, all the other peptides showed interaction with HMGB1 box B (Fig.1 B). The relative intensity of each band varied from experiment to experiment indicating that the results were not quantitative. Nevertheless, these interactions were always detectable, whereas interaction with the control was never detected (Fig.1 B, lane GST-KG). Moreover, the same assays were also done using either HMGB1 box A or HMGB1 full-length and the results were the same (not shown). We noted that none of the peptides selected was represented in the sequence of HMGB1. Because HMGB1 can only very inefficiently interact with itself forming a homodimer (27Ranatuga W. Lebowitz J. Axe B. Pavlik P. Kar S.R. Scovell W.M. Biochim. Biophys. Acta. 1999; 1432: 1-12Crossref PubMed Scopus (8) Google Scholar) and HMG box domains do not interact with each other inside the HMGB1 molecule (28Ramstein J. Locker D. Bianchi M.E. Leng M. Eur. J. Biochem. 1999; 260: 692-700Crossref PubMed Scopus (34) Google Scholar), this suggested that the peptides selected were representative of reasonable rather than very weak interactions. Once our results were confirmed it was of interest to look for proteins containing regions of homology to the peptides selected in the phage display assay in order to perform a survey. This point was addressed by searching the available data bases with the BLAST program for nuclear proteins. HWGMWSY was a peptide that generated interesting candidates and among them appeared ER, a factor previously identified to interact with HMGB1 (15Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M.E. Taraseviciene L. Nordeen S.K. Allegretto E.A. Edwards D.P. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (306) Google Scholar). We noted a potential WXXW consensus motif (TableIIB) for the factors belonging to this group. These factors were not studied in detail because in comparison another peptide, LPLTPLP, generated the most interesting homologies and allowed the identification of a new set of nuclear proteins that could potentially interact with HMGB1. These putative factors are summarized in Table IIA. Once again, two other factors, p53 and PR, already described to interact with HMGB1 appeared (10Jayaraman L. Moorthy N.C. Murthy K.G.K. Manley J.L. Bustin M. Prives C. Genes Dev. 1998; 12: 462-472Crossref PubMed Scopus (286) Google Scholar, 14Oñate S.A. Prendergast P. Wagner J.P. Nissen M. Reeves R. Pettijhon D.E. Edwards D.P. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar) and interestingly, the region homologous to our peptide in p53 was highly conserved in mammals. Remarkably, all the proteins listed presented a potential PXXPXP consensus motif, and among them components for many transcription factor complexes can be found. In this list we had only included the proteins containing the sequence motifs homologous to the peptide that were conserved among mammalian species. Note that in some cases two conserved motifs could be found (e.g. Grg1, p53). Also, the two orientations of the motif were considered because similar proline-rich motifs were reported to be recognized by SH3 domains independently of their orientation (Ref. 29Kay B.K. Williamson M.P. Sudol M. FASEB J. 2000; 14: 231-241Crossref PubMed Scopus (1057) Google Scholarand references therein).Table IISome potential candidates for interaction with HMGB1APutative interactorsMotifs of homology LPLTPLPAccession numberGrg5140LPLTPLP146Q06195Grg1/TL1141VPLTPLP147Q62440459VPFPPMP465TLE2160VPLTPRP166NP 062699p5376GPVAPAP82P0234083APATPWP89hnRNP K307LPLPPPP313Q60577MeCP2380MPLLPSP386NP 034918pRb780RPIHPIP774A33718P107956PPLSPFP962Q64701P1301028PPLSPYP1034Q64700DP219NPYTPAP25Q64163RelB75GPAAPPP81Q04863PR415FPLAPAP421Q00175AR280LPACPTP274P19091HAIRLESS179WPLAPNP185Q61645CDP913QPTTPLP919P53564RBP-Jκ375APVTPVP381P31266ERF243EPLSPFP249P70459IRF2315APVTPTP321P23906MNT511LPLYPQP517O08789MCMT1327LPLFPEP1333P13864RFX180QPATPAP86P48377Sox18288NPLSPPP294P43680TTP69APLAPRP75P22893Six5535LPVGPSP541P70178FREAC 365GPYTPQP72CAA11239TFE3312LPVPPNP318Q64092SREBP-193APLSPPP99Q9WTN3TGIF154RPVSPKP160AAH05724CTCF648QPVAPAP654NP 031820AF-41103SPLSPMP1109AAB82427HNF-3G26SPVNPVP32CAA52892C-EBPβ12LPLPPPP18P17676TAF2501640GPYTPQP1646P21675HES1187PPLVPIP193P35428MATH129PPLTPQP35P48985MATH2258GPLSPPP265P48986ConsensusXPLTPXPVSAAYPILTHGYFGNVCBPutative interactorsMotifs of homology HWGMWSY HSWLWWPAccession numberER α200HYGVWSC206P19785ER β160HYGVWSC166O08537TIAR309QWGQWSQ315P70318ETS179
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