Analysis of the interaction of ZAP-70 and syk protein-tyrosine kinases with the T-cell antigen receptor by plasmon resonance.

Tyrosine phosphorylation of a 17-amino acid immunoreceptor tyrosine-based activation motif (ITAM), conserved in each of the signaling subunits of the T-cell antigen receptor (TCR), mediates the recruitment of ZAP-70 and syk protein-tyrosine kinases (PTKs) to the activated receptor. The interaction between the two tandemly arranged Src-homology 2 (SH2) domains of this family of PTKs and each of the phosphotyrosine-containing ITAMs was examined by real-time measurements of kinetic parameters. The association rate and equilibrium binding constants for the ZAP-70 and syk SH2 domains were determined for the CD3 epsilon ITAM. Both PTKs bound with ka and Kd values of 5 x 10(6) M-1.sec-1 and approximately 25 nM, respectively. Bindings to the other TCR ITAMs (zeta 1, zeta 2, gamma, and delta ITAMs) were comparable, although the zeta 3 ITAM bound approximately 2.5-fold less well. Studies of the affinity of a single functional SH2 domain of ZAP-70 provided evidence for the cooperative nature of binding of the dual SH2 domains. Mutation of either single SH2 domain decreased the Kd by > 100-fold. Finally, the critical features of the ITAM for syk binding were found to be similar to those required for ZAP-70 binding. These data provide insight into the mechanism by which the multisubunit TCR interacts with downstream effector molecules.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2.

EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo.

Differential modulation of Th1- and Th2-related cytokine mRNA expression by a synthetic peptide homologous to a conserved domain within retroviral envelope protein.

The influence of a synthetic retroviral peptide, CKS-17, on T helper type 1 (Th1)- or Th2-related cytokines was investigated in human blood mononuclear cells. Cells were stimulated with staphylococcal enterotoxin A, anti-CD3 plus anti-CD28 monoclonal antibodies, or lipopolysaccharide to induce cytokine mRNA. mRNA was detected by a reverse transcription-polymerase chain reaction or Northern blot analysis. CKS-17 down-regulated stimulant-induced mRNA accumulation for interferon gamma (IFN-gamma), interleukin (IL)-2, and p40 heavy and p35 light chains of IL-12, a cytokine that mediates development of Th1 response. CKS-17 up-regulated stimulant-induced mRNA accumulation of IL-10 and did not suppress Th2-related cytokine (IL-4, IL-5, IL-6, or IL-13) mRNA expression. A reverse sequence of CKS-17 peptide, used as a control, showed no such action. Anti-human IL-10 monoclonal antibody blocked ability of CKS-17 to inhibit mRNA accumulation for IFN-gamma but not the CKS-17 suppressive activity of IL-12 p40 heavy chain mRNA. Thus, CKS-17-mediated suppression of IFN-gamma mRNA expression is dependent upon augmentation of IL-10 production by CKS-17. This conserved component of several retroviral envelope proteins, CKS-17, may act as an immunomodulatory epitope responsible for cytokine dysregulation that leads to suppression of cellular immunity.

Quantitative description of the interaction between folate and the folate-binding protein from cow's milk

A detailed study has been carried out on the dependence of folate binding on the concentration of FBP (folate-binding protein) at pH 5.0, conditions selected to prevent complications arising from the pre-existing self-association of the acceptor. In contrast with the mandatory requirement that reversible interaction of ligand with a single acceptor site should exhibit a unique, rectangular hyperbolic binding curve, results obtained by ultrafiltration for the FBP-folate system required description in terms of (i) a sigmoidal relationship between concentrations of bound and free folate and (ii) an inverse dependence of affinity on FBP concentration. These findings have been attributed to the difficulties in determining the free ligand concentration in the FBP-folate mixtures for which reaction is essentially stoichiometric. This explanation also accounts for the similar published behaviour of the FBP-folate system at neutral pH, which had been attributed erroneously to acceptor self-association, a phenomenon incompatible with the experimental findings because of its prediction of a greater affinity for folate with increasing FBP concentration.

Inhibition of protein interactions with the beta(2) sliding clamp of Escherichia coli DNA polymerase III by peptides from beta(2)-binding proteins

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.

Interaction of the Hsp90 cochaperone cyclophilin 40 with Hsc70

The high-affinity ligand-binding form of unactivated steroid receptors exists as a multicomponent complex that includes heat shock protein (Hsp)90; one of the immunophilins cyclophilin 40 (CyP40), FKBP51, or FKBP52; and an additional p23 protein component. Assembly of this heterocomplex is mediated by Hsp70 in association with accessory chaperones Hsp40, Hip, and Hop. A conserved structural element incorporating a tetratricopeptide repeat (TPR) domain mediates the interaction of the immunophilins with Hsp90 by accommodating the C-terminal EEVD peptide of the chaperone through a network of electrostatic and hydrophobic interactions. TPR cochaperones recognize the EEVD structural motif common to both Hsp90 and Hsp70 through a highly conserved clamp domain. In the present study, we investigated in vitro the molecular interactions between CyP40 and FKBP52 and other stress-related components involved in steroid receptor assembly, namely Hsp70 and Hop. Using a binding protein-retention assay with CyP40 fused to glutathione S-transferase immobilized on glutathione-agarose, we have identified the constitutively expressed form of Hsp70, heat shock cognate (Hsc)70, as an additional target for CyP40. Deletion mapping studies showed the binding determinants to be similar to those for CyP40-Hsp90 interaction. Furthermore, a mutational analysis of CyP40 clamp domain residues confirmed the importance of this motif in CyP40-Hsc70 interaction. Additional residues thought to mediate binding specificity through hydrophobic interactions were also important for Hsc70 recognition. CyP40 was shown to have a preference for Hsp90 over Hsc70. Surprisingly, FKBP52 was unable to compete with CyP40 for Hsc70 binding, suggesting that FKBP52 discriminates between the TPR cochaperone-binding sites in Hsp90 and Hsp70. Hop, which contains multiple units of the TPR motif, was shown to be a direct competitor with CyP40 for Hsc70 binding. Similar to Hop, CyP40 was shown not to influence the adenosine triphosphatase activity of Hsc70. Our results suggest that CyP40 may have a modulating role in Hsc70 as well as Hsp90 cellular function.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.