Cutting edge: identification of a pre-ligand assembly domain (PLAD) and ligand binding site in the IL-17 receptor

IL-17 is the hallmark cytokine of the newly described "Th17" lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a "pre-ligand assembly domain" (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.     

Mapping the structure of folding cores in TIM barrel proteins by hydrogen exchange mass spectrometry: the roles of motif and sequence for the indole-3-glycerol phosphate synthase from Sulfolobus solfataricus

To test the roles of motif and amino acid sequence in the folding mechanisms of TIM barrel proteins, hydrogen-deuterium exchange was used to explore the structure of the stable folding intermediates for the of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus (sIGPS). Previous studies of the urea denaturation of sIGPS revealed the presence of an intermediate that is highly populated at approximately 4.5 M urea and contains approximately 50% of the secondary structure of the native (N) state. Kinetic studies showed that this apparent equilibrium intermediate is actually comprised of two thermodynamically distinct species, I(a) and I(b). To probe the location of the secondary structure in this pair of stable on-pathway intermediates, the equilibrium unfolding process of sIGPS was monitored by hydrogen-deuterium exchange mass spectrometry. The intact protein and pepsin-digested fragments were studied at various concentrations of urea by electrospray and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, respectively. Intact sIGPS strongly protects at least 54 amide protons from hydrogen-deuterium exchange in the intermediate states, demonstrating the presence of stable folded cores. When the protection patterns and the exchange mechanisms for the peptides are considered with the proposed folding mechanism, the results can be interpreted to define the structural boundaries of I(a) and I(b). Comparison of these results with previous hydrogen-deuterium exchange studies on another TIM barrel protein of low sequence identify, alpha-tryptophan synthase (alphaTS), indicates that the thermodynamic states corresponding to the folding intermediates are better conserved than their structures. Although the TIM barrel motif appears to define the basic features of the folding free energy surface, the structures of the partially folded states that appear during the folding reaction depend on the amino acid sequence. Markedly, the good correlation between the hydrogen-deuterium exchange patterns of sIGPS and alphaTS with the locations of hydrophobic clusters defined by isoleucine, leucine, and valine residues suggests that branch aliphatic side-chains play a critical role in defining the structures of the equilibrium intermediates.     

Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis

The proteins encoded by gene 7 of the severe acute respiratory syndrome coronavirus (SARS-CoV) have been demonstrated to have proapoptotic activity when expressed from cDNA but appear to be dispensable for virus replication. Recombinant SARS-CoVs bearing deletions in gene 7 were used to assess the contribution of gene 7 to virus replication and apoptosis in several transformed cell lines, as well as to replication and pathogenesis in golden Syrian hamsters. Deletion of gene 7 had no effect on SARS-CoV replication in transformed cell lines, nor did it alter the induction of early apoptosis markers such as annexin V binding and activation of caspase 3. However, viruses with gene 7 disruptions were not as efficient as wild-type virus in inducing DNA fragmentation, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, indicating that the gene 7 products do contribute to virus-induced apoptosis. Disruption of gene 7 did not affect virus replication or morbidity in golden Syrian hamsters, suggesting that the gene 7 products are not required for acute infection in vivo. The data indicate that open reading frames 7a and 7b contribute to but are not solely responsible for the apoptosis seen in SARS-CoV-infected cells.     

The inhibition of TNF-alpha anti-tumoral properties by blocking antibodies promotes tumor growth in a rat model

Tumor necrosis factor (TNF) antagonists represent a milestone in the therapy of autoimmune conditions. Anti-TNF antibodies have been approved for clinical use and during the last eight years thousands of patients have been treated. However, the long-term sequelae of anti-TNF agents in promoting carcinogenesis remain unclear. This study sought to define the role of intra-tumor TNF-alpha production on cancer cell progression and to determine whether TNF-alpha antibodies can suppress anti-tumoral immunity. Using an experimental animal tumor model we demonstrate that anti-TNF-alpha antibodies hinder anti-tumor immune responses and promote growth of immunogenic rat colon tumors (REG) that are always rejected by immunocompetent untreated rats. The major role of TNF-alpha in the anti-tumoral immune response was confirmed by transfecting progressive and tolerogenic rat colon tumor cells (PRO) with the TNF-alpha gene. PRO tumor cells secreting TNF-alpha induce tumor-infiltrating dendritic cell (DC) activation. This triggers a potent immune response leading to tumor rejection and long-lasting immunity. Therefore, the prominent role of TNF-alpha in anti-tumoral immune responses underscores the need for caution and close surveillance following the administration of TNF inhibitors.     

Identification of an interleukin 17F/17A heterodimer in activated human CD4+ T cells

IL-17F and IL-17A are members of the IL-17 pro-inflammatory cytokine family. IL-17A has been implicated in the pathogenesis of autoimmune diseases. IL-17F is a disulfide-linked dimer that contains a cysteine-knot motif. We hypothesized that IL-17F and IL-17A could form a heterodimer due to their sequence homology and overlapping pattern of expression. We evaluated the structure of recombinant IL-17F and IL-17A proteins, as well as that of natural IL-17F and IL-17A derived from activated human CD4+ T cells, by enzyme-linked immunosorbent assay, immunoprecipitation followed by Western blotting, and mass spectrometry. We find that both IL-17F and IL-17A can form both homodimeric and heterodimeric proteins when expressed in a recombinant system, and that all forms of the recombinant proteins have in vitro functional activity. Furthermore, we find that in addition to the homodimers of IL-17F and IL-17A, activated human CD4+ T cells also produce the IL-17F/IL-17A heterodimer. These data suggest that the IL-17F/IL-17A heterodimer may contribute to the T cell-mediated immune responses.     

Cellular characterization of a novel focal adhesion kinase inhibitor

Focal adhesion kinase (FAK) is a member of a family of non-receptor protein-tyrosine kinases that regulates integrin and growth factor signaling pathways involved in cell migration, proliferation, and survival. FAK expression is increased in many cancers, including breast and prostate cancer. Here we describe perturbation of adhesion-mediated signaling with a FAK inhibitor, PF-573,228. In vitro, this compound inhibited purified recombinant catalytic fragment of FAK with an IC(50) of 4 nM. In cultured cells, PF-573,228 inhibited FAK phosphorylation on Tyr(397) with an IC(50) of 30-100 nM. Treatment of cells with concentrations of PF-573,228 that significantly decreased FAK Tyr(397) phosphorylation failed to inhibit cell growth or induce apoptosis. In contrast, treatment with PF-573,228 inhibited both chemotactic and haptotactic migration concomitant with the inhibition of focal adhesion turnover. These studies show that PF-573,228 serves as a useful tool to dissect the functions of FAK in integrin-dependent signaling pathways in normal and cancer cells and forms the basis for the generation of compounds amenable for preclinical and patient trials.     

Using the Ralstonia solanacearum Tat secretome to identify bacterial wilt virulence factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

Bacterial diversity associated with Blood Falls, a subglacial outflow from the Taylor Glacier, Antarctica

Blood Falls is the surface manifestation of brine released from below the Taylor Glacier, McMurdo Dry Valleys, Antarctica. Geochemical analyses of Blood Falls show that this brine is of a marine origin. The discovery that 74% of clones and isolates from Blood Falls share high 16S rRNA gene sequence homology with phylotypes from marine systems supports this contention. The bacterial 16S rRNA gene clone library was dominated by a phylotype that had 99% sequence identity with Thiomicrospira arctica (46% of the library), a psychrophilic marine autotrophic sulfur oxidizer. The remainder of the library contained phylotypes related to the classes Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria and the division Bacteroidetes and included clones whose closest cultured relatives metabolize iron and sulfur compounds. These findings are consistent with the high iron and sulfate concentrations detected in Blood Falls, which are likely due to the interactions of the subglacial brine with the underlying iron-rich bedrock. Our results, together with previous reports, suggest that the brine below the Taylor Glacier hosts a viable ecosystem with microorganisms capable of growth, supported by chemical energy present in reduced iron and sulfur compounds. The metabolic and phylogenetic structure of this subglacial microbial assemblage appears to be controlled by glacier hydrology, bedrock lithology, and the preglacial ecosystem.