An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

Peptide array X-linking (PAX): a new peptide-protein identification approach

Many protein interaction domains bind short peptides based on canonical sequence consensus motifs. Here we report the development of a peptide array-based proteomics tool to identify proteins directly interacting with ligand peptides from cell lysates. Array-formatted bait peptides containing an amino acid-derived cross-linker are photo-induced to crosslink with interacting proteins from lysates of interest. Indirect associations are removed by high stringency washes under denaturing conditions. Covalently trapped proteins are subsequently identified by LC-MS/MS and screened by cluster analysis and domain scanning. We apply this methodology to peptides with different proline-containing consensus sequences and show successful identifications from brain lysates of known and novel proteins containing polyproline motif-binding domains such as EH, EVH1, SH3, WW domains. These results suggest the capacity of arrayed peptide ligands to capture and subsequently identify proteins by mass spectrometry is relatively broad and robust. Additionally, the approach is rapid and applicable to cell or tissue fractions from any source, making the approach a flexible tool for initial protein-protein interaction discovery.     

Interaction of Peptides with Sequences from the Newcastle Disease Virus Fusion Protein Heptad Repeat Regions

Typical of many viral fusion proteins, the sequence of the Newcastle disease virus (NDV) fusion protein has several heptad repeat regions. One, HR1, is located just carboxyl terminal to the fusion peptide, while the other, HR2, is located adjacent to the transmembrane domain. The structure and function of a synthetic peptide with a sequence from the region of the NDV HR1 region (amino acids 150 to 173) were characterized. The peptide inhibited fusion with a half-maximal concentration of approximately 2 microM; however, inhibition was observed only if the peptide was added prior to protease activation of the fusion protein. This inhibition was virus specific since the peptide had minimal effect on fusion directed by the Sendai virus glycoproteins. To explore the mechanism of action, the potential HR1 peptide interaction with a previously characterized fusion inhibitory peptide with a sequence from the HR2 domain (J. K. Young, R. P. Hicks, G. E. Wright, and T. G. Morrison, Virology 238:291-304, 1997) was characterized. The results demonstrated an interaction between the two peptides both functionally and directly. First, while the individual peptides each inhibit fusion, equimolar mixtures of the two peptides had minimal effect on fusion, suggesting that the two peptides form a complex preventing their interaction with a target protein. Second, an HR2 peptide covalently linked with biotin was found to bind specifically to HR1 peptide in a Western blot. The structure of the HR1 peptide was analyzed by nuclear magnetic resonance spectroscopy and found to be an alpha helix.     

Molecular parameters that control the association of low density lipoprotein apo B-100 with chondroitin sulphate

The association of low density lipoprotein (LDL) with proteoglycans of the intima, in particular chondroitin 6-sulphate proteoglycans, may contribute to LDL accumulation during atherogenesis. We studied the interactions of apolipoprotein B-100 (apo B-100) peptide segments and model peptides with chondroitin 6-sulphate. The ability of these peptides to inhibit complex formation between LDL and chondroitin 6-sulphate was used as a measurement of the interaction. Results from earlier studies suggest that surface located segments of apo B-100 are responsible for the interaction of LDL with heparin and chondroitin sulphate-rich arterial proteoglycans. Therefore 16 hydrophilic apo B-100 peptides were selected for studies and synthesized with a peptide synthesizer. These synthetic peptides were 7 to 26 amino acids long. Four of the peptides inhibited the association of LDL with chondroitin 6-sulphate, namely apo B segments 4230–4254, 3359–3377, 3145–3157 and 2106–2121. The 3359–3377 segment was the most efficient. A common feature betweeb the interacting peptides was an excess of positively charged side chains and based on these results we synthesized nine model peptides that shared sequence characateristics with the interacting apo B-100 peptides. Five of these: RSGRKRSGK, RSSRKRSGK, RGGRKRGGK, RSRSRSRSR AND RGRGRGRGR were shown to block the LDL-chrondroitin-6-sulphate association, RSRSRSRSR being the most effective. The results suggest that the optimal association of the peptides with chrondroitin 6-sulphate is obtained with a minimal chain length of nine amino acids and a minimum of five positive charges and that flexibility in the binding region is important.     

Analysis of HLA-E peptide-binding specificity and contact residues in bound peptide required for recognition by CD94/NKG2

The MHC class Ib molecule HLA-E is the primary ligand for CD94/NKG2A-inhibitory receptors expressed on NK cells, and there is also evidence for TCR-mediated recognition of this molecule. HLA-E preferentially assembles with a homologous set of peptides derived from the leader sequence of class Ia molecules, but its capacity to bind and present other peptides remains to be fully explored. The peptide-binding motif of HLA-E was investigated by folding HLA-E in vitro in the presence of peptide libraries derived from a nonameric leader peptide sequence randomized at individual anchor positions. A high degree of selectivity was observed at four of five total anchor positions, with preference for amino acids present in HLA-E-binding peptides from class Ia leader sequences. Selectivity was also observed at the nonanchor P5 position, with preference for positively charged amino acids, suggesting that electrostatic interactions involving the P5 side chain may facilitate assembly of HLA-E peptide complexes. The observed HLA-E peptide-binding motif was strikingly similar to that previously identified for the murine class Ib molecule, Qa-1. Experiments with HLA-E tetramers bearing peptides substituted at nonanchor positions demonstrated that P5 and P8 are primary contact residues for interaction with CD94/NKG2 receptors. A conservative replacement of Arg for Lys at P5 completely abrogated binding to CD94/NKG2. Despite conservation of peptide-binding specificity in HLA-E and Qa-1, cross-species tetramer-staining experiments demonstrated that the interaction surfaces on CD94/NKG2 and the class Ib ligands have diverged between primates and rodents.     

Kinetics and substrate specificity of membrane-reconstituted peptide transporter DtpT of Lactococcus lactis

The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[(14)C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, omega-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT(1) and PepT(2) shows that the bacterial transporter is more restrictive in its substrate recognition.     

Adenovirus E1A makes two distinct contacts with the retinoblastoma protein.

Two regions near the amino terminus of the adenovirus E1A protein, which were first identified by sequence conservation among various adenovirus serotypes, have been shown by genetic studies to be essential for E1A-mediated transformation. These same regions are also required for interaction with a number of cellular proteins, including the retinoblastoma protein (pRB). Using synthetic peptides corresponding to portions of these conserved regions, we show that each region can bind independently to pRB. These interactions were observed in both competition and binding assays. In both types of assay, region 2 peptides (E1A amino acids 115 to 132) bound pRB with higher affinity than did region 1 peptides (E1A amino acids 37 to 54), while a peptide combining region 1 and 2 sequences consistently provided the highest-affinity interaction. Cross-blocking experiments using region 1 peptides and region 2 peptides suggested that these two regions of E1A make distinct contacts with pRB. These data support the notion that the pRB-binding domain of E1A contains at least two functional elements.     

Immunochemistry of the dominating antigenic region Ala582 to Cys604 in the transmembranous protein of simian and human immunodeficiency virus

The immunochemistry of two homologous uniquely antigenic peptides representing Ala582 to Cys604 in the transmembrane proteins of simian immunodeficiency virus of rhesus macaque origin, SIVmac (closely related to HIV-2) and HIV-1 (strain HTLV-IIIB) was characterized at the resolution of single amino acids. Five different antigenic sites were identified in the SIVmac peptide by use of 34 mAb against this peptide and two different sites were similarly demonstrated in the HIV-1 peptide by use of 10 peptide-specific mAb. Within some sites the mAb could be subgrouped to show a progressively more narrow epitopic dependence on amino acids in the central part of the site. Three SIVmac peptide mAbs had a remarkably narrow amino acid dependence, Glu584 and Tyr586. Anti-peptide mAbs reacting with the site Trp596 to Gln602 effectively blocked the capacity of the peptide to react with human postinfection HIV-2 antibodies previously demonstrated to have a restricted reactivity involving this site. No similar blocking was seen when mAb specific for Leu587 to Gln590 were used except with a single broadly reacting HIV-2 serum, which depended on an amino acid at a distance of only 6 residues, Trp596. A cross-reacting site involving amino acids Ala582 to Glu588/Lys588 was identified with mAb and rabbit hyperimmune sera against the two peptides. This site was not accessible in the intact transmembrane proteins as determined by ELISA and Western blot tests. Antipeptide mAb against other sites as well as rabbit sera reacted strongly in these tests and can be used as type-specific, component-unique reagents.     

Evidence that HAX-1 is an interleukin-1 alpha N-terminal binding protein

During studies aimed at understanding the function of the N-terminal peptide of interleukin-1 alpha (IL-1 NTP, amino acids 1-112), which is liberated from the remainder of IL-1 alpha during intracellular processing, we identified by yeast two-hybrid analysis a putative interacting protein previously designated as HAX-1. In vitro binding studies and transient transfection experiments confirmed that HAX-1 can associate with the IL-1 NTP. HAX-1 was first identified as a protein that associates with HS1, a target of non-receptor protein tyrosine kinases within haematopoietic cells. Recent data have also revealed interactions between HAX-1 and three disparate proteins, polycystin-2 (derived from the PKD2 gene), a protein linked to polycystic kidney disease, cortactin, and Epstein-Barr virus nuclear antigen leader protein (EBNA-LP). Sequence analysis of different HAX-1 binding domains revealed a putative consensus binding motif that is present in various intracellular proteins. Overlapping peptides comprising the IL-1 NTP were synthesized, and binding experiments revealed that discrete peptides were capable of interacting with HAX-1. HAX-1 may serve to retain the IL-1 NTP in the cytoplasm, and complex formation between the IL-1 NTP and HAX-1 may play a role in motility and/or adhesion of cells.     

Association of Grb-2 and PI3K p85 with phosphotyrosile peptides derived from BTLA

B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory receptor expressed by B and T cells. We previously identified two tyrosine-containing signaling motifs in the cytoplasmic domain of BTLA that interact with the SHP-1 and SHP-2 phosphatases. BTLA has a third conserved tyrosine-containing motif within the cytoplasmic domain, similar in sequence to a Grb-2 recruitment site. To identify specific interacting proteins that would be recruited to this motif, we carried out an unbiased screen by using synthetic peptides in active (e.g., phosphotyrosil-containing) or control (e.g., non-phosphorylated) forms as baits. Using mass spectrometry, we identified two specific interacting proteins, Grb-2 and the p85 subunit of PI3K. Further, we demonstrate that the interaction with Grb-2 is direct, whereas the recruitment of the p85 subunit by BTLA phosphotyrosile-containing peptides may be indirect via its association with Grb-2. These findings may provide biochemical basis for previously unexplained actions of BTLA.     

Role of the Sin3-Histone Deacetylase Complex in Growth Regulation by the Candidate Tumor Suppressor p33ING1

Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.