Characterization of two peptide epitopes on Mdm2 oncoprotein that affect p53 degradation.

Phosphorylation of Mdm2, in response to DNA damage, resulted in prevention of p53 degradation in the cytoplasm as well as reduction of its binding with monoclonal antibody (mAb) 2A10. Using a 15-mer phage-peptide library, we identified two 2A10-epitopes on human Mdm2 (hdm2): at positions 255-266 (LDSEDYSLSEEG) and 389-400 (QESDDYSQPSTS). Synthetic peptides corresponding to the above sites, inhibit the binding of mAb2A10 to Mdm2 with high (4.5 x 10(-9)M) and moderate affinity (1.1 x 10(-7)M), respectively. Phospho-derivatives of these peptides, and of single human Mdm2 mutations S260D or S395D resulted in a considerable reduction in their binding with mAb2A10. These results provide a molecular explanation for the observation that reactivity of Mdm2 with mAb2A10 is inhibited by phosphorylation.

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.

A beta -hairpin structure in a 13-mer peptide that binds alpha -bungarotoxin with high affinity and neutralizes its toxicity.

Snake-venom alpha-bungarotoxin is a member of the alpha-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between alpha-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC(50) of 2 nM, has been solved by (1)H-NMR spectroscopy. The bound peptide folds into a beta-hairpin structure created by two antiparallel beta-strands, which combine with the already existing triple-stranded beta-sheet of the toxin to form a five-stranded intermolecular, antiparallel beta-sheet. Peptide residues Y3(P), E5(P), and L8(P) have the highest intermolecular contact area, indicating their importance in the binding of alpha-bungarotoxin; W1(P), R2(P), and Y4(P) also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole alpha-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds alpha-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of alpha-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs.

ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage.

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major target on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity.

BACKGROUND: Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake venom alpha-neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. We describe the design and synthesis of peptides that bind alpha-BTX with high affinity, and inhibit its interaction with AChR with an IC(50) of 2 nM. The design of these peptides was based on a lead peptide with an IC(50) of 3x10(-7) M, previously identified by us [M. Balass et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6054] using a phage-display peptide library. RESULTS: Employing nuclear magnetic resonance-derived structural information [T. Scherf et al., Proc. Natl. Acad. Sci. USA 94 (1997) 6059] of the complex of alpha-BTX with the lead peptide, as well as structure-function analysis of the ligand-binding site of AChR, a systematic residue replacement of the lead peptide, one position at a time, yielded 45 different 13-mer peptides. Of these, two peptides exhibited a one order of magnitude increase in inhibitory potency in comparison to the lead peptide. The design of additional peptides, with two or three replacements, resulted in peptides that exhibited a further increase in inhibitory potency (IC(50) values of 2 nM), that is more than two orders of magnitude better than that of the original lead peptide, and better than that of any known peptide derived from AChR sequence. The high affinity peptides had a protective effect on mice against alpha-BTX lethality. CONCLUSIONS: Synthetic peptides with high affinity to alpha-BTX may be used as potential lead compounds for developing effective antidotes against alpha-BTX poisoning. Moreover, the procedure employed in this study may serve as a general approach for the design and synthesis of peptides that interact with high affinity with any desired biological target.

A cyclic peptide with high affinity to alpha-bungarotoxin protects mice from the lethal effect of the toxin.

Employing a combinatorial phage-peptide library, we previously identified the peptide MRYYESSLKSYPD (designated, library-peptide) that binds the snake toxin alpha-bungarotoxin (alpha-BTX) with a moderate binding constant of 10(-6)M (Balass et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6054-6058). Under the experimental conditions employed, we found that the library-peptide did not protect mice from alpha-BTX lethality when injected concomitantly with the toxin. In order to improve the affinity of the peptide to alpha-BTX, we designed and synthesized the peptide CRYYESSLKSYCD (Met1 and Pro12 were replaced by cysteines), which following oxidation creates a single disulfide bond and forms a cyclic structure. The design of the cyclic peptide was based on our previous NMR analysis of the library-peptide/alpha-BTX complex (Scherf et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6059-6064). The cyclic peptide binds alpha-BTX with affinity two orders of magnitude higher than that of the linear library selected peptide. Whereas the library peptide was ineffective, the cyclic peptide conferred protection from alpha-BTX lethality in mice, even when given 1h after the toxin injection. The cyclic peptide conferred complete protection from alpha-BTX lethality in mice when administered 40min prior to toxin injection. However, experiments with the whole venom of the snake Bungarus multicinctus showed that protection could be achieved only when the cyclic peptide was administered concomitantly with the venom.

Prevention of passively transferred experimental autoimmune myasthenia gravis by a phage library-derived cyclic peptide.

Many pathogenic antibodies in myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are directed against the main immunogenic region (MIR) of the acetylcholine receptor (AcChoR). These antibodies are highly conformation dependent; hence, linear peptides derived from native receptor sequences are poor candidates for their immunoneutralization. We employed a phage-epitope library to identify peptide-mimotopes capable of preventing the pathogenicity of the anti-MIR mAb 198. We identified a 15-mer peptide (PMTLPENYFSERPYH) that binds specifically to mAb 198 and inhibits its binding to AcChoR. A 10-fold increase in the affinity of this peptide was achieved by incorporating flanking amino acid residues from the coat protein as present in the original phage library. This extended peptide (AEPMTLPENYFSERPYHPPPP) was constrained by the addition of cysteine residues on both ends of the peptide, thus generating a cyclic peptide that inhibited the binding of mAb 198 to AcChoR with a potency that is three orders of magnitude higher when compared with the parent library peptide. This cyclic peptide inhibited the in vitro binding of mAb 198 to AcChoR and prevented the antigenic modulation of AcChoR caused by mAb 198 in human muscle cell cultures. The cyclic peptide also reacted with several other anti-MIR mAbs and the sera of EAMG rats. In addition, this peptide blocked the ability of mAb 198 to passively transfer EAMG in rats. Further derivatization of the cyclic peptide may aid in the design of suitable synthetic mimotopes for modulation of MG.

High affinity binding of monoclonal antibodies to the sequential epitope EFRH of beta-amyloid peptide is essential for modulation of fibrillar aggregation.

Monoclonal antibodies raised against the N-terminal of Alzheimer's beta-amyloid peptide (betaAP) were found to modulate its fibrillar aggregation. While mAbs 6C6 and 10D5 inhibit the formation of beta-amyloid fibrils, trigger disaggregation and reversal to its non-toxic form, mAb 2H3 is devoid of these properties. MAb 2H3 binds the sequence DAEFRHD, corresponding to position 1-7 of the betaAP with high affinity (2 x 10(-9) M) similar to its binding with the whole betaAP. The EFRH peptide strongly inhibits binding of mAbs 6C6 and 10D5 to betaAP, whereas it inhibits weakly the interaction of 2H3 with betaAP. Low affinity binding of mAb 2H3 to EFRH might explain its failure in prevention of beta-amyloid formation.

N-terminal EFRH sequence of Alzheimer's beta-amyloid peptide represents the epitope of its anti-aggregating antibodies.

Monoclonal antibodies 6C6 and 10D5 raised against the N-terminal of beta-amyloid peptide interfere with the formation of beta-amyloid and trigger reversal to its non-toxic components. The epitopes of these antibodies were localized employing a library composed of filamentous phage displaying random combinatorial hexapeptides. Among 44 positive phage-clones, selected from the library by both antibodies, 40 clones carried the consensus sequence EFRH. These EFRH phage-clones bind specifically mAbs 6C6 or 10D5 with an apparent binding constant of approximately 10(-9) M. The peptide EFRH inhibits binding of mAbs 6C6 or 10D5 to beta-amyloid peptide in affinities identical to those obtained. with the peptides corresponding to positions 1-9, 1-16 and 1-40 of beta-peptide. These findings confirm that the peptide EFRH which is located at positions 3-6 within beta-amyloid peptide represents the sequential epitope of mAbs 6C6 and 10D5.

The alpha-bungarotoxin binding site on the nicotinic acetylcholine receptor: analysis using a phage-epitope library.

The nicotinic acetylcholine receptor (AcChoR) is a ligand-gated ion channel that is activated upon binding of acetylcholine. alpha-Neurotoxins, in particular alpha-bungarotoxin (alpha-BTX), bind specifically and with high affinity to the AcChoR and compete with binding of the natural ligand. We employed a 15-mer phage-display peptide library to select epitopes reacting with alpha-BTX. Phages bearing the motif YYXSSL as a consensus sequence were found to bind with high affinity to alpha-BTX. The library-derived peptide (MRYYESSLKSYPD) bears amino acid sequence similarities to a region of the alpha-subunit of the Torpedo muscle AcChoR, as well as of other muscle and neuronal AcChoRs that bind alpha-BTX. The library-derived peptide and the corresponding peptides containing residues 187-199 of the Torpedo AcChoR alpha-subunit (WVYYTCCPDTPYL), as well as peptides analogous to the above region in the neuronal AcChoR (e.g., human alpha7; ERFYECCKEPYPD) that binds alpha-BTX, inhibit the binding of alpha-BTX to the intact Torpedo AcChoR with IC50 values of 10(-6) M. A synthetic peptide from a neuronal AcChoR that does not bind alpha-BTX (e.g., human alpha2; ERKYECCKEPYPD) which differs by just one amino acid from the homologous peptide from the alpha-BTX-binding protein (alpha7)-i.e., Lys in alpha2 and Tyr in alpha7-does not inhibit the binding of alpha-BTX to Torpedo AcChoR. These results indicate the requirement for two adjacent aromatic amino acid residues for binding to alpha-BTX.

Three-dimensional solution structure of the complex of alpha-bungarotoxin with a library-derived peptide.

The solution structure of the complex between alpha-bungarotoxin (alpha-BTX) and a 13-residue library-derived peptide (MRYYESSLKSYPD) has been solved using two-dimensional proton-NMR spectroscopy. The bound peptide adopts an almost-globular conformation resulting from three turns that surround a hydrophobic core formed by Tyr-11 of the peptide. The peptide fills an alpha-BTX pocket made of residues located at fingers I and II, as well as at the C-terminal region. Of the peptide residues, the largest contact area is formed by Tyr-3 and Tyr-4. These findings are in accord with the previous data in which it had been shown that substitution of these aromatic residues by aliphatic amino acids leads to loss of binding of the modified peptide with alpha-BTX. Glu-5 and Leu-8, which also remarkably contribute to the contact area with the toxin, are present in all the library-derived peptides that bind strongly to alpha-BTX. The structure of the complex may explain the fact that the library-derived peptide binds alpha-BTX with a 15-fold higher affinity than that shown by the acetylcholine receptor peptide (alpha185-196). Although both peptides bind to similar sites on alpha-BTX, the latter adopts an extended conformation when bound to the toxin [Basus, V., Song, G. & Hawrot, E. (1993) Biochemistry 32, 12290-12298], whereas the library peptide is nearly globular and occupies a larger surface area of alpha-BTX binding site.

Recovery of high-affinity phage from a nitrostreptavidin matrix in phage-display technology.

A novel approach for the selection of high-affinity phage from phage-peptide libraries is described. The methodology employs a chemically modified form of streptavidin, termed nitrostreptavidin, which exhibits a reversible attraction for biotin. The new approach emulates conventional procedures in that a biotinylated probe, in this case biotinylated alpha-bungarotoxin, is attached to an immobilized streptavidin matrix. The phage library is introduced, and interacting phage particles are released under conventional acidic conditions (pH 2.2). At this stage, the primary peptide sequences characterizing the released phage are found to be identical with those previously known to interact with the toxin. However, other phage particles, which presumably interact more strongly than those released by acid, remain attached to the immobilized toxin. These can be released by virtue of the reversible biotin-binding properties of nitrostreptavidin. For this purpose, alkaline solutions (pH 10) or free biotin can be used. Using this approach, phage particles that recognize alpha-bungarotoxin were isolated; their peptide sequences were found to be similar to, but clearly distinct from, those collected by conventional acid elution. The affinity of the isolated phage was dramatically higher than that of phage obtained by the conventional methodology. In contrast, their synthetically prepared 15-mer peptides actually exhibited a lower affinity for the toxin than that shown by peptides prepared on the basis of the sequence obtained from conventional acid-eluted phage. This apparent discrepancy can be explained by an altered conformational state of the peptides in solution, compared to the epitopes expressed in situ on the phage surface.

Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR.

Identification of a hexapeptide that mimics a conformation-dependent binding site of acetylcholine receptor by use of a phage-epitope library.

Monoclonal antibody (mAb) 5.5 is directed against the ligand-binding site of the nicotinic acetylcholine receptor. The epitope for this antibody is conformation-dependent, and the antibody does not react with synthetic peptides derived from the receptor sequence. We have identified a ligand peptide that mimics this conformation-dependent epitope from a phage-epitope library composed of filamentous phage displaying random hexapeptides. Among 38 positive phage clones, individually selected from the library, 34 positive clones carried the sequence Asp-Leu-Val-Trp-Leu-Leu (DLVWLL), 1 positive clone had the sequence Asp-Ile-Val-Trp-Leu-Leu (DIVWLL), and 3 positive clones expressed the sequence Leu-Ile-Glu-Trp-Leu-Leu (LIEWLL), none of which are significantly homologous with the nicotinic acetylcholine receptor alpha subunit sequence. All of these phages bind specifically to mAb 5.5. The synthetic peptide DLVWLL inhibits binding of mAb 5.5 to the related peptide-presenting phage and to the nicotinic acetylcholine receptor in a concentration-dependent manner; the IC50 value is of the order of 10(-4) M. Bioactivity of the peptide "mimotope" DLVWLL was demonstrated in vivo in hatched chickens by inhibition of the mAb 5.5 effect by the peptide. The neuromuscular block and myasthenia gravis-like symptoms that are induced in chicken by passive transfer of mAb 5.5 were specifically abolished by DLVWLL. This study shows the potential of a random peptide phage-epitope library for selecting a mimotope for an antibody that recognizes a folded form of the protein, where peptides from the linear amino acid sequence of the protein are not applicable.