Andrea S. Llera

Three-Dimensional Structure of the Complex between a T Cell Receptor β Chain and the Superantigen Staphylococcal Enterotoxin B

Abstract Superantigens (SAGs) are a class of immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the Vβ domain of the T cell antigen receptor (TCR). The three-dimensional structure of the complex between a TCR β chain (mouse Vβ8.2) and the SAG staphylococcal enterotoxin B (SEB) at 2.4 A resolution reveals why SEB recognizes only certain Vβ families, as well as why only certain SAGs bind mouse Vβ8.2. Models of the TCR-SEB-peptide/MHC class II complex indicate that Vα interacts with the MHC β chain in the TCR-SAG-MHC complex. The extent of the interaction is variable and is largely determined by the geometry of Vα/Vβ domain association. This variability can account for the preferential expression of certain Vα regions among T cells reactive with SEB.

The role of the matricellular protein SPARC in the dynamic interaction between the tumor and the host

Tumor growth is essentially the result of an evolving cross-talk between malignant and surrounding stromal cells (fibroblasts, endothelial cells and inflammatory cells). This heterogeneous mass of extracellular matrix and intermingled cells interact through cell–cell and cell–matrix contacts. Malignant cells also secrete soluble proteins that reach neighbor stromal cells, forcing them to provide the soil on which they will grow and metastasize. Different studies including expression array analysis identified the matricellular protein SPARC as a marker of poor prognosis in different cancer types. Further evidence demonstrated that high SPARC levels are often associated with the most aggressive and highly metastatic tumors. Here we describe the most recent evidence that links SPARC with human cancer progression, the controversy regarding its role in certain human cancers and the physiological processes in which SPARC is involved: epithelial–mesenchymal transition, immune surveillance and angiogenesis. Its relevance as a potential target in cancer therapy is also discussed.

Structures of two streptococcal superantigens bound to TCR beta chains reveal diversity in the architecture of T cell signaling complexes

Superantigens (SAGs) crosslink MHC class II and TCR molecules, resulting in an overstimulation of T cells associated with human disease. SAGs interact with several different surfaces on MHC molecules, necessitating the formation of multiple distinct MHC-SAG-TCR ternary signaling complexes. Variability in SAG-TCR binding modes could also contribute to the structural heterogeneity of SAG-dependent signaling complexes. We report crystal structures of the streptococcal SAGs SpeA and SpeC in complex with their corresponding TCR beta chain ligands that reveal distinct TCR binding modes. The SpeC-TCR beta chain complex structure, coupled with the recently determined SpeC-HLA-DR2a complex structure, provides a model for a novel T cell signaling complex that precludes direct TCR-MHC interactions. Thus, highly efficient T cell activation may be achieved through structurally diverse strategies of TCR ligation.

Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor.

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of α and β, or γ and δ, polypeptide chains with variable (V) and constant (C) regions. In contrast to αβ TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), γδ TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certainγδ T cells,. These studies indicate that antigen recognition by γδ TCRs may be fundamentally different from that by αβ TCRs. The three-dimensional structures of several αβ TCRs and TCR fragments, and their complexes with peptide–MHC or superantigens, have been determined. Here we report the crystal structure of the Vδ domain of a human γδ TCR at 1.9 Å resolution. A comparison with antibody and αβ TCR V domains reveals that the framework structure of Vδ more closely resembles that of VHthan of Vα, Vβ or VL(where H and L refer to heavy and light chains), whereas therelative positions and conformations of its complementarity-determining regions (CDRs) share features of both Vα and VH. These results provide the first direct evidence that γδ TCRs are structurally distinct from αβ TCRs and, together with the observation that the CDR3 length distribution of TCR δ chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by γδ TCRs may resemble antigen recognition by antibodies.

CRYSTAL STRUCTURE OF IMAGINAL DISC GROWTH FACTOR-2: A MEMBER OF A NEW FAMILY OF GROWTH-PROMOTING GLYCOPROTEINS FROM DROSOPHILA MELANOGASTER

Imaginal disc growth factor-2 (IDGF-2) is a member of a recently described family of Drosophila melanogaster-soluble polypeptide growth factors that promote cell proliferation in imaginal discs. Although their precise mode of action has not been established, IDGFs cooperate with insulin in stimulating the growth of imaginal disc cells. We report the crystal structure of IDGF-2 at 1.3-Å resolution. The structure shows the classical (βα)8 barrel-fold of family 18 glycosyl hydrolases, with an insertion of an α + β domain similar to that ofSerratia marcescens chitinases A and B. However, amino acid substitutions in the consensus catalytic sequence of chitinases give IDGF-2 a less negatively charged environment in its putative ligand-binding site and preclude the nucleophilic attack mechanism of chitin hydrolysis. Particularly important is the replacement of Glu by Gln at position 132, which has been shown to abolish enzymatic activity in chitinases. Nevertheless, a modest conservation of residues that participate in oligosaccharide recognition suggests that IDGF-2 could bind carbohydrates, assuming several conformational changes to open the partially occluded binding site. Thus, IDGFs may have evolved from chitinases to acquire new functions as growth factors, interacting with cell surface glycoproteins implicated in growth-promoting processes, such as the Drosophila insulin receptor.

Structure of the Vδ domain of a human γδ T-cell antigen receptor

Antigen recognition by T lymphocytes is mediated by cell-surface glycoproteins known as T-cell antigen receptors (TCRs). These are composed of α and β, or γ and δ, polypeptide chains with variable (V) and constant (C) regions. In contrast to αβ TCRs, which recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC), γδ TCRs appear to recognize proteins directly, without antigen processing, and to recognize MHC molecules independently of the bound peptide. Moreover, small phosphate-containing non-peptide compounds have also been identified as ligands for certainγδ T cells,. These studies indicate that antigen recognition by γδ TCRs may be fundamentally different from that by αβ TCRs. The three-dimensional structures of several αβ TCRs and TCR fragments, and their complexes with peptide–MHC or superantigens, have been determined. Here we report the crystal structure of the Vδ domain of a human γδ TCR at 1.9 Å resolution. A comparison with antibody and αβ TCR V domains reveals that the framework structure of Vδ more closely resembles that of VHthan of Vα, Vβ or VL(where H and L refer to heavy and light chains), whereas therelative positions and conformations of its complementarity-determining regions (CDRs) share features of both Vα and VH. These results provide the first direct evidence that γδ TCRs are structurally distinct from αβ TCRs and, together with the observation that the CDR3 length distribution of TCR δ chains is similar to that of immunoglobulin heavy chains, are consistent with functional studies suggesting that recognition of certain antigens by γδ TCRs may resemble antigen recognition by antibodies.

Functional Analysis of the TCR Binding Domain of Toxic Shock Syndrome Toxin-1 Predicts Further Diversity in MHC Class II/Superantigen/TCR Ternary Complexes

Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR β-chain (Vβ). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central α helix adjacent to the N-terminal α helix and the β7-β9 loop as well as with two universally conserved SAG residues (Leu137 and Tyr144 in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly16, Trp116, Glu132, His135, Gln136, and Gln139), each individually critical for functional activity as well as direct interaction with the human TCR Vβ2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vβ2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vβ specificity of TSST-1.

Structure-function studies of T-cell receptor-superantigen interactions

Summary: Superantigens (SAGs) are a class of disease‐causing and immunostimulatory proteins of bacterial or viral origin that activate T cells by binding to the Vβ domain of the T‐cell antigen receptor (TCR), The three‐dimensional structure of the complex between a TCR β chain (mouse Vβ8.2‐Jβ2.l‐Cβ1) and the SAG staphylococcal enterotoxin d (SEC3) has been recently determined. The complementarity‐determining region 2 (CDR2) of the β chain and, to lesser extents, CDR1 and hypervariable region 4 (HV4) bind in a cleft between the small and large domains of the SAG, A model of the TCR‐SAG‐peptide/MHC complex constructed from available crystal structures reveals how the SAG acts as a wedge between the TCR and MHC, thereby displacing die antigen in peptide away from the TCR and circumventing the normal mechanism for T‐cell activation by peptide/MHC, To evaluate the actual contribution of individual SAG residues to stabilizing the VβCβ‐SEC3 complex, as well as to investigate the relationship between the affinity of SAGs for TCR and MHC and their ability to activate T cells, we measured the binding of a set of SEC3 mutants to a soluble recombinant TCR β chain and to the human MHC class II molecule HLA‐DR1. We show that there is direct correlation between affinity and ability to stimulate T cells, with SAGs having the highest affinity for the TCR being die most biologically active. We also find that there is an interplay between TCR‐SAG and SAG‐MHC interactions in determining mitogenic potency, such that a small increase in die affinity of a SAG for MHC can overcome a large decrease in the SAGs affinity for die TCR, Finally, we observe that those SEC3 residues that make the greatest energetic contribution to stabilizing the VβCβ‐SEC3 complex are strictly conserved among enterotoxins reactive with mouse Vβ8.2, thereby explaining why SAGs having other residues at diese positions show different Vβ‐binding specificities.

ATP‐dependent modulation and autophosphorylation of rapeseed 2‐Cys peroxiredoxin

2‐Cys peroxiredoxins (2‐Cys Prx) are ubiquitous thiol‐containing peroxidases that have been implicated in antioxidant defense and signal transduction. Although their biochemical features have been extensively studied, little is known about the mechanisms that link the redox activity and non‐redox processes. Here we report that the concerted action of a nucleoside triphosphate and Mg2+ on rapeseed 2‐Cys Prx reversibly impairs the peroxidase activity and promotes the formation of high molecular mass species. Using protein intrinsic fluorescence in the analysis of site‐directed mutants, we demonstrate that ATP quenches the emission intensity of Trp179, a residue close to the conserved Cys175. More importantly, we found that ATP facilitates the autophosphorylation of 2‐Cys Prx when the protein is successively reduced with thiol‐bearing compounds and oxidized with hydroperoxides or quinones. MS analyses reveal that 2‐Cys Prx incorporates the phosphoryl group into the Cys175 residue yielding the sulfinic‐phosphoryl [Prx‐(Cys175)‐SO2PO32−] and the sulfonic‐phosphoryl [Prx‐(Cys175)‐SO3PO32−] anhydrides. Hence, the functional coupling between ATP and 2‐Cys Prx gives novel insights into not only the removal of reactive oxygen species, but also mechanisms that link the energy status of the cell and the oxidation of cysteine residues.

SPARC modulates the proliferation of stromal but not melanoma cells unless endogenous SPARC expression is downregulated

Cell interaction with the extracellular matrix (ECM) has profound influence in cancer progression. The secreted protein, acidic and rich in cysteine (SPARC) a component of the ECM, impairs the proliferation of different cell types and modulates tumor cell aggressive features. This apparent paradox might result either from the biochemical properties of the different SPARC sources or from differential responses of malignant and stromal cells to SPARC. To test these hypotheses, we purified SPARC secreted by melanoma cells (hMel‐SPARC) and compared its activity with different recombinant SPARC preparations, including a new one produced in insect cells. All 5 SPARC species were effective in inhibiting bovine aortic endothelial cell proliferation, adhesion and migration. We then used the melanoma‐derived protein to assess SPARC effect on additional cell types. hMel‐SPARC greatly impaired the proliferation of both normal and transformed human endothelial cells and exerted a moderate biphasic effect on human fetal fibroblasts proliferation, irrespective of their endogenous SPARC levels. However, SPARC had no effect on the proliferation of several human cancer cell lines regardless of their endogenous levels of SPARC expression. Importantly, downregulation of SPARC levels in melanoma cells using either an antisense RNA or a shRNA against SPARC sensitized them to hMel‐SPARC addition in proliferation and migration assays, suggesting that malignant cells developed a SPARC‐resistance mechanism. This was not a general resistance to growth suppressing agents, as melanoma cells with restricted SPARC expression were more resistant to chemotherapeutic agents. Thus, malignant cells expressing or not expressing SPARC developed alternative mechanisms that, in contrary to stromal cells, rendered them SPARC‐insensitive.