Molecular mechanism of SHP2 activation by PD-1 stimulation.

In cancer, the programmed death-1 (PD-1) pathway suppresses T cell stimulation and mediates immune escape. Upon stimulation, PD-1 becomes phosphorylated at its immune receptor tyrosine-based inhibitory motif (ITIM) and immune receptor tyrosine-based switch motif (ITSM), which then bind the Src homology 2 (SH2) domains of SH2-containing phosphatase 2 (SHP2), initiating T cell inactivation. The SHP2-PD-1 complex structure and the exact functions of the two SH2 domains and phosphorylated motifs remain unknown. Here, we explain the structural basis and provide functional evidence for the mechanism of PD-1-mediated SHP2 activation. We demonstrate that full activation is obtained only upon phosphorylation of both ITIM and ITSM: ITSM binds C-SH2 with strong affinity, recruiting SHP2 to PD-1, while ITIM binds N-SH2, displacing it from the catalytic pocket and activating SHP2. This binding event requires the formation of a new inter-domain interface, offering opportunities for the development of novel immunotherapeutic approaches.

A window of opportunity for cooperativity in the T Cell Receptor.

The T-cell antigen receptor (TCR) is pre-organised in oligomers, known as nanoclusters. Nanoclusters could provide a framework for inter-TCR cooperativity upon peptide antigen-major histocompatibility complex (pMHC) binding. Here we have used soluble pMHC oligomers in search for cooperativity effects along the plasma membrane plane. We find that initial binding events favour subsequent pMHC binding to additional TCRs, during a narrow temporal window. This behaviour can be explained by a 3-state model of TCR transition from Resting to Active, to a final Inhibited state. By disrupting nanoclusters and hampering the Active conformation, we show that TCR cooperativity is consistent with TCR nanoclusters adopting the Active state in a coordinated manner. Preferential binding of pMHC to the Active TCR at the immunological synapse suggests that there is a transient time frame for signal amplification in the TCR, allowing the T cells to keep track of antigen quantity and binding time.

Analysis of interactions between proteins and fatty acids or cholesterol using a fatty acid/cholesterol pull-down assay.

Specific interactions between the polar head groups of membrane lipids and proteins have been described previously. In contrast, the specificity of the interaction between lipid acyl chains with proteins is less understood. By combining a fatty acid or cholesterol pull-down assay with Western blot analysis or mass spectrometry, we identified transmembrane and cytosolic proteins that bound preferentially to short or long acyl chains or to cholesterol. Thus, this approach allows identification of specific fatty acid-protein or cholesterol-protein interactions.

A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells.

BACKGROUND: Although cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer. METHODS: A heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv V(H)CD3-V(L)PSMA and V(H)PSMA-V(L)CD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model. RESULTS: By Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth. CONCLUSIONS: The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease.

Biotinylation of protein complexes may lead to aggregation as well as to loss of subunits as revealed by Blue Native PAGE.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a high-resolution method for studying native protein complexes. Here, the migration behaviour of the B cell antigen receptor (BCR) in BN-PAGE with and without prior biotinylation of the cell surface, from which the complexes were purified, are compared. Non-biotinylated complexes appear as a single band, whereas biotinylated complexes display several bands, indicating that biotinylation leads to aggregation of complexes as well as to loss of subunits. Thus, BN-PAGE has limitations in studying multiprotein complexes. These results are used to gain insight into the stoichiometry of the complex.

An unsolved problem of the clonal selection theory and the model of an oligomeric B-cell antigen receptor.

The B cell antigen receptor (BCR) plays a central role in the development, survival and activation of B lymphocytes. As the pre-BCR, it controls allelic exclusion of heavy chains and the expansion of pre-B cells. As the BCR, it controls the positive and negative selection of immature B cells as well as the survival and activation of mature B cells. Recent studies of receptors have shown that it is the ligand that brings them into the conformation necessary for signaling. How the multiple and structurally diverse antigens could fulfill this task for the BCR is unknown, and we regard this as an unsolved problem of Burnet's clonal selection theory This question and our recent biochemical studies lead us to propose a new model for the BCR, according to which the BCR exists as a precise oligomeric complex on the B cell surface. In this form, it can signal positive selection and survival of B cells. Binding to self- or foreign antigen results in a distortion of the oligomeric complex that gives the signal for negative selection of immature and activation of mature B cells.

Monomeric and oligomeric complexes of the B cell antigen receptor.

The current structural model of the B cell antigen receptor (BCR) describes it as a symmetric protein complex in which one membrane-bound immunoglobulin molecule (mIg) is noncovalently bound on each side by an Ig-alpha/Ig-beta heterodimer. Using peptide-tagged Ig-alpha proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE), and biosynthetical labeling of B cells, we find that the mIg:Ig-alpha/Ig-beta complex has a stoichiometry of 1:1 and not 1:2. An anti-Flag stimulation of B cells coexpressing Flag-tagged and wild-type Ig-alpha proteins results in the phosphorylation of both Ig-alpha proteins, suggesting that on the surface of living B cells, several BCR monomers are in contact with each other. A BN-PAGE analysis after limited detergent lysis provides further evidence for an oligomeric BCR structure.

Stability of the B cell antigen receptor complex.

The B cell antigen receptor (BCR) comprises the membrane-bound immunoglobulin (mIg) molecule and the Ig-alpha/Ig-beta heterodimer. By comparing the stability of the IgD-BCR and IgM-BCR in different detergent lysates, we find that the IgD-BCR is more stable than the IgM-BCR. Analysis of chimeric mIgD molecules suggests that the deltam transmembrane region is responsible for the more stable association of mIgD with the Ig-alpha/Ig-beta heterodimer. Further, the differential glycosylation of Ig-alpha molecules, in the two different BCR complexes, is determined solely by the ectodomains of the mIg molecules. The implications of these findings for the intracellular transport and the signalling capacity of the BCRs are discussed.

Comparison of the tamoxifen regulated chimeric Cre recombinases MerCreMer and CreMer.

The site-directed recombinase Cre can be employed to delete or assemble genes in the genome of living cells. We have constructed expression vectors for chimeric Cre recombinases carrying a mutated hormone binding domain either at the C-terminus only (CreMer) or at both the N and C-termini (MerCreMer). The chimeric Cre proteins can be activated by culturing transfected cells with 4-hydroxytamoxifen. In transfected embryonic stem (ES) cells, we compared the extent of recombination of a floxed gene with the expression levels of the chimeric Cre proteins. Our data demonstrate that the bulky MerCreMer protein is not less active than the CreMer protein. Thus, the tighter control and low background activity of the MerCreMer enzyme is not due to a generally low recombinase efficiency.

Signal transduction: specificity of growth factors explained by parallel distributed processing.

Mutations in proteins of receptor tyrosine kinase signalling pathways might change signalling properties and contribute to the progression towards neoplasia. Ligands for these receptors (growth factors) which elicit the same signal transducing cascades can trigger different developmental pathways in identical cells. For example, PC12 cells differentiate after treatment with NGF, but proliferate after stimulation with EGF. However, their receptors seem to utilize the same signal transducing components. Thus, intracellular signalling specificity remains an enigma. Here, we apply a network model, in which each protein species participating in signal transduction is represented by an element. The elements are connected to each other according to the signalling pathways resembling parallel distributed processes. A general property of these systems is applied to the problem of signal specificity: slight differences in the input may result in completely different outputs, which negates the necessity of specific proteins to each pathway. This line of thinking might explain the specificity of hormones, although they use the same signal transduction network.

The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence.

Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of full-length murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an alpha-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the delta m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.