Structure-Based, Rational Design of T Cell Receptors.

Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D = ∼1 - 5 μM. Beyond the affinity threshold at K D < 1 μM we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.

Unraveling the complex network of cuticular structure and function.

A hydrophobic cuticle is deposited at the outermost extracellular matrix of the epidermis in primary tissues of terrestrial plants. Besides forming a protective shield against the environment, the cuticle is potentially involved in several developmental processes during plant growth. A high degree of variation in cuticle composition and structure exists between different plant species and tissues. Lots of progress has been made recently in understanding the different steps of biosynthesis, transport, and deposition of cuticular components. However, the molecular mechanisms that underlie cuticular function remain largely elusive.

Molecular design of the Calphabeta interface favors specific pairing of introduced TCRalphabeta in human T cells.

A promising approach to adoptive transfer therapy of tumors is to reprogram autologous T lymphocytes by TCR gene transfer of defined Ag specificity. An obstacle, however, is the undesired pairing of introduced TCRalpha- and TCRbeta-chains with the endogenous TCR chains. These events vary depending on the individual endogenous TCR and they not only may reduce the levels of cell surface-introduced TCR but also may generate hybrid TCR with unknown Ag specificities. We show that such hybrid heterodimers can be generated even by the pairing of human and mouse TCRalpha- and TCRbeta-chains. To overcome this hurdle, we have identified a pair of amino acid residues in the crystal structure of a TCR that lie at the interface of associated TCR Calpha and Cbeta domains and are related to each other by both a complementary steric interaction analogous to a "knob-into-hole" configuration and the electrostatic environment. We mutated the two residues so as to invert the sense of this interaction analogous to a charged "hole-into-knob" configuration. We show that this inversion in the CalphaCbeta interface promotes selective assembly of the introduced TCR while preserving its specificity and avidity for Ag ligand. Noteworthily, this TCR modification was equally efficient on both a Mu and a Hu TCR. Our data suggest that this approach is generally applicable to TCR independently of their Ag specificity and affinity, subset distribution, and species of origin. Thus, this strategy may optimize TCR gene transfer to efficiently and safely reprogram random T cells into tumor-reactive T cells.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Design of potent inhibitors of human RAD51 recombinase based on BRC motifs of BRCA2 protein: modeling and experimental validation of a chimera peptide.

We have previously shown that a 28-amino acid peptide derived from the BRC4 motif of BRCA2 tumor suppressor inhibits selectively human RAD51 recombinase (HsRad51). With the aim of designing better inhibitors for cancer treatment, we combined an in silico docking approach with in vitro biochemical testing to construct a highly efficient chimera peptide from eight existing human BRC motifs. We built a molecular model of all BRC motifs complexed with HsRad51 based on the crystal structure of the BRC4 motif-HsRad51 complex, computed the interaction energy of each residue in each BRC motif, and selected the best amino acid residue at each binding position. This analysis enabled us to propose four amino acid substitutions in the BRC4 motif. Three of these increased the inhibitory effect in vitro, and this effect was found to be additive. We thus obtained a peptide that is about 10 times more efficient in inhibiting HsRad51-ssDNA complex formation than the original peptide.

Role of CD44H carbohydrate structure in neuroblastoma adhesive properties.

BACKGROUND: CD44 represents a heterogeneous group of surface glycoproteins involved in cell-cell and cell-matrix interactions. CD44H is the major receptor for hyaluronate, and most if not all CD44H known functions are attributed to its ability to recognize hyaluronate. We have previously demonstrated a lack of CD44 expression in high stages and NMYC-amplified tumors and further have shown that NMYC-amplified cell lines either did not express CD44 at all or expressed a nonfunctional receptor. On the other hand, nonamplified cells constitutively expressed an active receptor, suggesting that absence of CD44-mediated hy aluronate binding could be related to increased malignancy in human neuroblastoma. PROCEDURE: In the present study we have compared the glycosylated structure of CD44 expressed by NMYC amplified vs. nonamplified cell lines in relation to their adhesive properties for hyaluronate. These adhesive properties were measured after modifications of the carbohydrate structure with enzymes and inhibitors of N- or O-linked glycosylation. RESULTS AND CONCLUSIONS: Our results indicate that increased sialylation, defective N-linked glycosylation, and substitution of the CD44 glycoprotein with keratan sulfate glycosaminoglycan might include modifications observed on neuroblastoma cells that could account for the inability of the receptor to bind hyaluronate.

PACIC Instrument: disentangling dimensions using published validation models.

OBJECTIVE: To better understand the structure of the Patient Assessment of Chronic Illness Care (PACIC) instrument. More specifically to test all published validation models, using one single data set and appropriate statistical tools. DESIGN: Validation study using data from cross-sectional survey. PARTICIPANTS: A population-based sample of non-institutionalized adults with diabetes residing in Switzerland (canton of Vaud). MAIN OUTCOME MEASURE: French version of the 20-items PACIC instrument (5-point response scale). We conducted validation analyses using confirmatory factor analysis (CFA). The original five-dimension model and other published models were tested with three types of CFA: based on (i) a Pearson estimator of variance-covariance matrix, (ii) a polychoric correlation matrix and (iii) a likelihood estimation with a multinomial distribution for the manifest variables. All models were assessed using loadings and goodness-of-fit measures. RESULTS: The analytical sample included 406 patients. Mean age was 64.4 years and 59% were men. Median of item responses varied between 1 and 4 (range 1-5), and range of missing values was between 5.7 and 12.3%. Strong floor and ceiling effects were present. Even though loadings of the tested models were relatively high, the only model showing acceptable fit was the 11-item single-dimension model. PACIC was associated with the expected variables of the field. CONCLUSIONS: Our results showed that the model considering 11 items in a single dimension exhibited the best fit for our data. A single score, in complement to the consideration of single-item results, might be used instead of the five dimensions usually described.

Expanding molecular modeling and design tools to non-natural sidechains.

Protein-protein interactions encode the wiring diagram of cellular signaling pathways and their deregulations underlie a variety of diseases, such as cancer. Inhibiting protein-protein interactions with peptide derivatives is a promising way to develop new biological and therapeutic tools. Here, we develop a general framework to computationally handle hundreds of non-natural amino acid sidechains and predict the effect of inserting them into peptides or proteins. We first generate all structural files (pdb and mol2), as well as parameters and topologies for standard molecular mechanics software (CHARMM and Gromacs). Accurate predictions of rotamer probabilities are provided using a novel combined knowledge and physics based strategy. Non-natural sidechains are useful to increase peptide ligand binding affinity. Our results obtained on non-natural mutants of a BCL9 peptide targeting beta-catenin show very good correlation between predicted and experimental binding free-energies, indicating that such predictions can be used to design new inhibitors. Data generated in this work, as well as PyMOL and UCSF Chimera plug-ins for user-friendly visualization of non-natural sidechains, are all available at http://www.swisssidechain.ch. Our results enable researchers to rapidly and efficiently work with hundreds of non-natural sidechains.

The alternatively spliced domain TnFnIII A1A2 of the extracellular matrix protein tenascin-C suppresses activation-induced T lymphocyte proliferation and cytokine production.

Several lines of evidences have suggested that T cell activation could be impaired in the tumor environment, a condition referred to as tumor-induced immunosuppression. We have previously shown that tenascin-C, an extracellular matrix protein highly expressed in the tumor stroma, inhibits T lymphocyte activation in vitro, raising the possibility that this molecule might contribute to tumor-induced immunosuppression in vivo. However, the region of the protein mediating this effect has remained elusive. Here we report the identification of the minimal region of tenascin-C that can inhibit T cell activation. Recombinant fragments corresponding to defined regions of the molecule were tested for their ability to inhibit in vitro activation of human peripheral blood T cells induced by anti-CD3 mAbs in combination with fibronectin or IL-2. A recombinant protein encompassing the alternatively spliced fibronectin type III domains of tenascin-C (TnFnIII A-D) vigorously inhibited both early and late lymphocyte activation events including activation-induced TCR/CD8 down-modulation, cytokine production, and DNA synthesis. In agreement with this, full length recombinant tenascin-C containing the alternatively spliced region suppressed T cell activation, whereas tenascin-C lacking this region did not. Using a series of smaller fragments and deletion mutants issued from this region, we have identified the TnFnIII A1A2 domain as the minimal region suppressing T cell activation. Single TnFnIII A1 or A2 domains were no longer inhibitory, while maximal inhibition required the presence of the TnFnIII A3 domain. Altogether, these data demonstrate that the TnFnIII A1A2 domain mediate the ability of tenascin-C to inhibit in vitro T cell activation and provide insights into the immunosuppressive activity of tenascin-C in vivo.

The role of matrix metalloproteinase mmp-9 in peripheral neural system regeneration

Multiple lines of evidence show that matrix metalloproteinases (MMPs) are involved in the peripheral neural system degenerative and regenerative processes. MMP-9 was suggested in particular to play a role in the peripheral nerve after injury or during Wallerian degeneration. Interestingly, our previous analysis of Lpin1 mutant mice (which present morphological signs of active demyelination and acute inflammatory cell migration, similar to processes present in the PNS undergoing Wallerian degeneration) revealed an accumulation of MMP-9 in the endoneurium of affected animals. We therefore generated a mouse line lacking both the Lpin1 and the MMP-9 genes in order to determine if MMP-9 plays a role in either inhibition or potentiation of the demyelinating phenotype present in Lpin1 knockout mice. The inactivation of MMP-9 alone did not lead to defects in PNS structure or function. Interestingly we observed that the double mutant animals showed reduced nerve conduction velocity, lower myelin protein mRNA expressions, and had more histological abnormalities as compared to the Lpin1 single mutants. In addition, based on immunohistochemical analysis and macrophage markers mRNA expression, we found a lower macrophage content in the sciatic nerve of the double mutant animals. Together our data indicate that MMP-9 plays a role in macrophage recruitment during postinjury PNS regeneration processes and suggest that slower macrophage infiltration delays regenerative processes in PNS.

Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

Autosomal-dominant distal myopathy associated with a recurrent missense mutation in the gene encoding the nuclear matrix protein, matrin 3.

Distal myopathies represent a heterogeneous group of inherited skeletal muscle disorders. One type of adult-onset, progressive autosomal-dominant distal myopathy, frequently associated with dysphagia and dysphonia (vocal cord and pharyngeal weakness with distal myopathy [VCPDM]), has been mapped to chromosome 5q31 in a North American pedigree. Here, we report the identification of a second large VCPDM family of Bulgarian descent and fine mapping of the critical interval. Sequencing of positional candidate genes revealed precisely the same nonconservative S85C missense mutation affecting an interspecies conserved residue in the MATR3 gene in both families. MATR3 is expressed in skeletal muscle and encodes matrin 3, a component of the nuclear matrix, which is a proteinaceous network that extends throughout the nucleus. Different disease related haplotype signatures in the two families provided evidence that two independent mutational events at the same position in MATR3 cause VCPDM. Our data establish proof of principle that the nuclear matrix is crucial for normal skeletal muscle structure and function and put VCPDM on the growing list of monogenic disorders associated with the nuclear proteome.

Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

The effects of physiologically plausible connectivity structure on local and global dynamics in large scale brain models.

Functionally relevant large scale brain dynamics operates within the framework imposed by anatomical connectivity and time delays due to finite transmission speeds. To gain insight on the reliability and comparability of large scale brain network simulations, we investigate the effects of variations in the anatomical connectivity. Two different sets of detailed global connectivity structures are explored, the first extracted from the CoCoMac database and rescaled to the spatial extent of the human brain, the second derived from white-matter tractography applied to diffusion spectrum imaging (DSI) for a human subject. We use the combination of graph theoretical measures of the connection matrices and numerical simulations to explicate the importance of both connectivity strength and delays in shaping dynamic behaviour. Our results demonstrate that the brain dynamics derived from the CoCoMac database are more complex and biologically more realistic than the one based on the DSI database. We propose that the reason for this difference is the absence of directed weights in the DSI connectivity matrix.

Rational design of indoleamine 2,3-dioxygenase inhibitors.

Indoleamine 2,3-dioxygenase (IDO) is an important therapeutic target for the treatment of diseases such as cancer that involve pathological immune escape. We have used the evolutionary docking algorithm EADock to design new inhibitors of this enzyme. First, we investigated the modes of binding of all known IDO inhibitors. On the basis of the observed docked conformations, we developed a pharmacophore model, which was then used to devise new compounds to be tested for IDO inhibition. We also used a fragment-based approach to design and to optimize small organic molecule inhibitors. Both approaches yielded several new low-molecular weight inhibitor scaffolds, the most active being of nanomolar potency in an enzymatic assay. Cellular assays confirmed the potential biological relevance of four different scaffolds.