Antigen-specific CD8+ T-cell tolerance, induced by myeloid-derived suppressor cells (MDSCs), is one of the main mechanisms of tumor escape. Using in vivo models, we show here that MDSCs directly disrupt the binding of specific peptide-major histocompatibility complex (pMHC) dimers to CD8-expressing T cells through nitration of tyrosines in a T-cell receptor (TCR)-CD8 complex. This process makes CD8-expressing T cells unable to bind pMHC and to respond to the specific peptide, although they retain their ability to respond to nonspecific stimulation. Nitration of TCR-CD8 is induced by MDSCs through hyperproduction of reactive oxygen species and peroxynitrite during direct cell-cell contact. Molecular modeling suggests specific sites of nitration that might affect the conformational flexibility of TCR-CD8 and its interaction with pMHC. These data identify a previously unknown mechanism of T-cell tolerance in cancer that is also pertinent to many pathological conditions associated with accumulation of MDSCs.

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.

The membrane‐tethered mucins are cell surface‐associated dimeric or multimeric molecules with extracellular, transmembrane and cytoplasmic portions, that arise from cleavage of the primary polypeptide chain. Following the first cleavage, which may be cotranslational, the subunits remain closely associated through undefined noncovalent interactions. These mucins all share a common structural motif, the SEA module that is found in many other membrane‐associated proteins that are released from the cell surface and has been implicated in both the cleavage events and association of the subunits. Here we examine the SEA modules of three membrane‐tethered mucins, MUC1, MUC3 and MUC12, which have significant sequence homology within the SEA domain. We previously identified the primary cleavage site within the MUC1 SEA domain as FRPG/SVVV a sequence that is highly conserved in MUC3 and MUC12. We now show by site‐directed mutagenesis that the F, G and S residues are important for the efficiency of the cleavage reaction but not indispensable and that amino acids outside this motif are probably important. These data are consistent with a new model of the MUC1 SEA domain that is based on the solution structure of the MUC16 SEA module, derived by NMR spectroscopy. Further, we demonstrate that cleavage of human MUC3 and MUC12 occurs within the SEA domain. However, the SEA domains of MUC1, MUC3 and MUC12 are not interchangeable, suggesting that either these modules alone are insufficient to mediate efficient cleavage or that the 3D structure of the hybrid molecules does not adequately recreate an accessible cleavage site.

Organ-specific homing of malignant cells involves interactions mediated through cell adhesion molecules and their receptors on the cell surface. Identification of peptides that mimic these receptor-ligand interactions is critical for analyzing the functional role of these proteins and is therapeutically significant to target or block organ-specific homing of tumor cells. Following three cycles of in vivo biopanning using a phage display peptide library injected into mice, we identified 11 unique peptides that were specific for homing to lung, liver, bone marrow, or brain. We developed a bioinformatics strategy to identify putative cell adhesion molecules (CAM) involved in tumor cell migration, invasion, and metastasis based on identified organ-specific peptides. Structural information, including surface exposure and the binding preference of any of these residues in the identified proteins, was examined. These studies resulted in identification of Semaphorin 5A (mouse, Sema5A; human, SEMA5A) and its receptor Plexin B3. The gene expression profile of these proteins in tumors and tumor cell lines was assessed using virtual microarray and serial analysis of gene expression (SAGE) databases and was further confirmed using reverse transcriptase polymerase chain reaction (RT-PCR). Our data demonstrate an association between the expression of SEMA5A and Plexin B3 and the aggressiveness of pancreatic and prostate cancer cells. In summary, using a combined experimental and bioinformatics approach, we have identified functional tumor-specific CAMs, which may be critical for organ-specific metastasis.

The resolved X-ray crystal structures of the glutamate-binding domain (S1/S2 domains) of the GluR2 and NR1 glutamate receptor subunits were used to model the homologous regions of the N-methyl-D-aspartate (NMDA) receptor's NR2 subunits. To test the predictive value of these models, all four stereoisomers of the antagonist 1-(phenanthren-2-carbonyl) piperazine-2,3-dicarboxylic acid (PPDA) were docked into the NR2B glutamate-binding site model. This analysis suggested an affinity order for the PPDA isomers of D-cis Ͼ L-cis Ͼ L-trans ϭ D-trans and predicted that the 2-position carboxylate group of the cis-PPDA isomers, but not of the trans-PPDA isomers, may be interacting with histidine 486 in NR2B. Consistent with these predictions, cis-PPDA displays a 35-fold higher affinity for NR2B-containing NMDA receptors than trans-PPDA. In addition, mutating NR2B's H486 to phenylalanine decreased cis-PPDA affinity 8-fold but had no effect on trans-PPDA affinity. In contrast, the NR2B H486F mutation increased the affinity of the typical antagonists CGS-19755 [(2R*,4S*)-4-phosphonomethyl-2-piperidine carboxylic acid] and 4-(3-phosphonopropyl) piperidine-2-carboxylic acid. In the NR1-based NR2 models, there were only four subunit-specific amino acid residues exposed to the ligand-binding pocket (and six in the GluR2-based models). These residues are located at the edge of the binding pocket, suggesting that large antagonists may be necessary for subtype specificity. Of these residues, mutational analysis and modeling suggest that A414, R712, and G713 (NR2B numbering) may be especially useful for developing NR2C-and NR2D-selective NMDA receptor antagonists and that residues A414 and T428 may determine subunit variations in agonist affinity.