Distinct conformational stability and functional activity of four highly homologous endonuclease colicins

The family of conserved colicin DNases E2, E7, E8, and E9 are microbial toxins that kill bacteria through random degradation of the chromosomal DNA. In the present work, we compare side by side the conformational stabilities of these four highly homologous colicin DNases. Our results indicate that the apo-forms of these colicins are at room temperature and neutral pH in a dynamic conformational equilibrium between at least two quite distinct conformers. We show that the thermal stabilities of the apo-proteins differ by up to 20 degrees C. The observed differences correlate with the observed conformational behavior, that is, the tendency of the protein to form either an open, less stable or closed, more stable conformation in solution, as deduced by both tryptophan accessibility studies and electrospray ionization mass spectrometry. Given these surprising structural differences, we next probed the catalytic activity of the four DNases and also observed a significant variation in relative activities. However, no unequivocal link between the activity of the protein and its thermal and structural stability could easily be made. The observed differences in conformational and functional properties of the four colicin DNases are surprising given that they are a closely related (> or =65% identity) family of enzymes containing a highly conserved (betabetaalpha-Me) active site motif. The different behavior of the apo-enzymes must therefore most likely depend on more subtle changes in amino acid sequences, most likely in the exosite region (residues 72-98) that is required for specific high-affinity binding of the cognate immunity protein.     

Regulation of encephalitogenic T cells with recombinant TCR ligands

We have previously described recombinant MHC class II beta1 and alpha1 domains loaded with free antigenic peptides with potent inhibitory activity on encephalitogenic T cells. We have now produced single-chain constructs in which the peptide Ag is genetically encoded within the same exon as the linked beta1 and alpha1 domains, overcoming the problem of displacement of peptide Ag from the peptide binding cleft. We here describe clinical effects of recombinant TCR ligands (RTLs) comprised of the rat RT1.B beta1alpha1 domains covalently linked to the 72-89 peptide of guinea pig myelin basic protein (RTL-201), to the corresponding 72-89 peptide from rat myelin basic protein (RTL-200), or to cardiac myosin peptide CM-2 (RTL-203). Only RTL-201 possessed the ability to prevent and treat active or passive experimental autoimmune encephalomyelitis. Amelioration of experimental autoimmune encephalomyelitis was associated with a selective inhibition of proliferation response and cytokine production by Ag-stimulated lymph node T cells and a drastic reduction in the number of encephalitogenic and recruited inflammatory cells infiltrating the CNS. The exquisitely selective inhibition could be observed between molecules that differ by a single methyl group (the single amino acid residue difference between RTL-200 (threonine) and RTL-201 (serine) at position 80 of the myelin basic protein peptide). These novel RTLs provide a platform for developing potent and selective human diagnostic and therapeutic agents for treatment of autoimmune disease.     

Anergy induction by dimeric TCR ligands

T cells that recognize particular self Ags are thought to be important in the pathogenesis of autoimmune diseases. In multiple sclerosis, susceptibility is associated with HLA-DR2, which can present myelin-derived peptides to CD4(+) T cells. To generate molecules that target such T cells based on the specificity of their TCR, we expressed a soluble dimeric DR2-IgG fusion protein with a bound peptide from myelin basic protein (MBP). Soluble, dimeric DR2/MBP peptide complexes activated MBP-specific T cells in the absence of signals from costimulatory or adhesion molecules. This initial signaling through the TCR rendered the T cells unresponsive (anergic) to subsequent activation by peptide-pulsed APCs. Fluorescent labeling demonstrated that anergic T cells were initially viable, but became susceptible to late apoptosis due to insufficient production of cytokines. Dimerization of the TCR with bivalent MHC class II/peptide complexes therefore allows the induction of anergy in human CD4(+) T cells with a defined MHC/peptide specificity.     

Ligand-independent down-regulation of IFN-gamma receptor 1 following TCR engagement

Activated T lymphocytes modulate the level of many molecules on their cell surface, including cytokine receptors. This regulation of cytokine receptor expression affects the ability of T cells to respond to cytokines and thus influences the outcome of an immune response. The receptor for IFN-gamma, a proinflammatory cytokine, consists of two copies of a ligand binding chain (IFN-gammaR1) as well as two copies of a second chain (IFN-gammaR2) required for signal transduction. The expression of IFN-gammaR2 is down-regulated at the mRNA level on CD4+ T cells when they differentiate into the Th1, but not the Th2, phenotype. This down-regulation has been demonstrated to depend on the ligand, IFN-gamma, which is produced by Th1 but not Th2 T cells. The regulation of the cell-surface expression of IFN-gamma receptors during primary T cell activation has not been reported. Naive and differentiated T lymphocytes express IFN-gammaR1 at the mRNA level and as a cell-surface protein. In this study, we present evidence that cell-surface expression of IFN-gammaR1 is transiently down-regulated on the surface of naive CD4+ T cells shortly after TCR engagement. Furthermore, this down-regulation is not mediated by the ligand, IFN-gamma, but results from TCR engagement and can be inhibited by cyclosporin A.     

Focal localization of placental protein 14 toward sites of TCR engagement

TCR signal transduction is amplified by the dynamic accumulation of accessory molecules at APC-T cell contact sites, along with the simultaneous exclusion from these sites of negative regulators, such as certain tyrosine phosphatases and large glycosylated proteins. However, given the general nature of the cytoskeleton-driven clustering mechanism underlying molecular segregation events at the APC-T cell interaction site, the possibility exists that negative regulators might similarly be segregated at these sites. Using fluorescence microscopy, we have demonstrated that placental protein 14 (PP14), a direct T cell inhibitor, focuses toward APC-T cell contact sites in conjunction with conjugate formation. We have further established that the function of PP14 is dependent upon its localization to the sites of TCR triggering, where it negatively regulates T cell activation. Thus, PP14 provides an example of a soluble negative T cell regulator whose inhibitory activity is linked to modulation of the APC-T cell contact site, thereby hindering early events triggered by the TCR.     

Recruitment of transferrin receptor to immunological synapse in response to TCR engagement

T cell receptor engagement by an APC induces the formation of a highly organized complex of surface receptors and intracellular signaling molecules, known as the immunological synapse, at the site of cell-cell contact. The transferrin receptor (TfR, CD71) is normally present in the plasma membrane and recycling endosomes. In this study, we show that, although the TfR is typically absent from lipid rafts at steady state, stimulation with a mitogenic mixture of anti-CD3 Abs of human Jurkat T cells leads to a rapid compartmentalization of the TfR into lipid rafts accompanying that of CD3epsilon and activated Lck. This change occurs very rapidly and is accompanied by an increase in the surface expression of the TfR, probably by translocation from an internal endosomal pool. TfR recruitment to lipid rafts was also observed in primary T cells treated with mitogenic anti-CD3 Abs and in Jurkat T cell-APC conjugates. The use of beads coated with Abs indicates that the surface and endosomal TfR pools redistribute to the contact site region in response to engagement of CD28 and CD3. In T cell-APC conjugates, the T cell TfR endosomal pool relocates beneath the contact site, whereas surface TfR localizes to the peripheral ring of the immunological synapse. In the presence of specific anti-TfR Abs, the total number of T cell-APC contacts and the percentage of conjugates with CD3 and Lck translocated to the contact site were reduced. Our results therefore suggest the involvement of the TfR in the formation of the immunological synapse.     

Rapid production of TNF-alpha following TCR engagement of naive CD8 T cells

The acquisition of effector functions by naive CD8 T cells following TCR engagement is thought to occur sequentially with full functionality being gained only after the initiation of division. We show that naive CD8 T cells are capable of immediate effector function following TCR engagement, which stimulates the rapid production of TNF-alpha. Stimulation of splenocytes from naive mice of differing genetic backgrounds with anti-CD3epsilon mAb resulted in significant production of TNF-alpha by naive CD8 T cells within 5 h. Moreover, naive lymphocytic choriomeningitis virus-specific TCR-transgenic CD8 T cells stimulated with either their cognate peptide ligand or virus-infected cells produced TNF-alpha as early as 2 h poststimulation, with production peaking by 4 h. Naive CD8 T cells produced both membrane-bound and soluble TNF-alpha. Interfering with TNF-alpha activity during the initial encounter between naive CD8 T cells and Ag loaded dendritic cells altered the maturation profile of the APC and diminished the overall viability of the APC population. These findings suggest that production of TNF-alpha by naive CD8 T cells immediately after TCR engagement may have an unappreciated impact within the local environment where Ag presentation is occurring and potentially influence the development of immune responses.     

Distinct chaperone affinity to folding variants of homologous recombinant proteins

Two -galactosidase fusion proteins, VP1LAC and LACVP1, contain the same viral capsid protein fused to either the amino or carboxy termini of the enzyme, respectively. Once produced in Escherichia coli, these fusions undergo a rapid, site-limited proteolysis releasing active -galactosidase fragments indistinguishable from the native enzyme. In vivo binding preferences of DnaK and GroEL chaperones for these homologous protein fragments have been observed, indicating that accessibility of chaperone target sites in degradation products could be determined by the folding pathway undergone by the larger polypeptide before the proteolytic attack.     

Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.     

Fyn regulates the duration of TCR engagement needed for commitment to effector function

In naive T cells, engagement of the TCR with agonist peptide:MHC molecules leads to phosphorylation of key intracellular signaling intermediates within seconds and this peaks within minutes. However, the cell does not commit to proliferation and IL-2 cytokine production unless receptor contact is sustained for several hours. The biochemical basis for this transition to full activation may underlie how T cells receive survival signals while maintaining tolerance, and is currently not well understood. We show here that for CD8 T cells commitment to proliferation and cytokine production requires sustained activation of the Src family kinase Lck and is opposed by the action of Fyn. Thus, in the absence of Fyn, commitment to activation occurs more rapidly, the cells produce more IL-2, and undergo more rounds of division. Our data demonstrate a role for Fyn in modulating the response to Ag in primary T cells.     

The role of TCR engagement and activation-induced cell death in sepsis-induced T cell apoptosis

Sepsis induces extensive apoptosis in T and B cells suggesting that the loss of immune effector cells could be one explanation for the profound immunosuppression observed in this disorder. Unfortunately, the mechanisms responsible for lymphocyte apoptosis in sepsis remain unknown. In T cells, apoptosis can occur through activation-induced cell death (AICD) in which engagement of the Ag receptors by cognate Ag or polyclonal activators such as bacteria-derived superantigens induces activation, proliferation, and apoptosis. We examined whether proliferation and AICD are necessary for apoptotic cell death in sepsis using normal and TCR transgenic mice. Results show that although sepsis resulted in activation of a small percentage of T cells, no proliferation was detected during the first 48 h following onset, a time when extensive apoptosis is observed. We also observed that T cells do not enter the cell cycle, and stimulation via the TCR in TCR transgenic animals does not enhance or decrease cell death in sepsis. Interestingly, T cells recovered from septic mice retained their ability to proliferate and synthesize cytokines albeit at reduced levels. With the exception of IL-10, which was increased in lymphocytes from mice with sepsis, sepsis caused a decrease in the production of both proinflammatory and anti-inflammatory cytokines. We conclude that lymphocyte apoptosis in sepsis does not require proliferation, TCR engagement, or AICD. Thus the immunosuppression observed in sepsis cannot be the result of T cell deletion via the TCR.     

Temperature dependent dynamics in highly homologous adenylate kinases

To correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.     

The magnitude of TCR engagement is a critical predictor of T cell anergy or activation

Fast dissociation rate of peptide-MHC complexes from TCR has commonly been accepted to cause T cell anergy. In this study, we present evidence that peptides that form transient complexes with HLA-DR1 induce anergy in T cell clones in vitro and specific memory T cells in vivo. We demonstrate that similar to the low densities of long-lived agonist peptide-MHC, short-lived peptide-MHC ligands induce anergy by engagement of approximately 1000 TCR and activation of a similar pattern of intracellular signaling events. These data strongly suggest that short-lived peptides induce anergy by presentation of low densities of peptide-MHC complexes. Moreover, they suggest that the traditional antagonist peptides might also trigger anergy by a similar molecular mechanism. The use of short-lived peptides to induce T cells anergy is a potential strategy for the prevention or treatment of autoimmune diseases.     

A1/Bfl-1 expression is restricted to TCR engagement in T lymphocytes

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.     

Distinct functional roles of homologous Cu+ efflux ATPases in Pseudomonas aeruginosa

In bacteria, most Cu(+) -ATPases confer tolerance to Cu by driving cytoplasmic metal efflux. However, many bacterial genomes contain several genes coding for these enzymes suggesting alternative roles. Pseudomonas aeruginosa has two structurally similar Cu(+) -ATPases, CopA1 and CopA2. Both proteins are essential for virulence. Expressed in response to high Cu, CopA1 maintains the cellular Cu quota and provides tolerance to this metal. CopA2 belongs to a subgroup of ATPases that are expressed in association with cytochrome oxidase subunits. Mutation of copA2 has no effect on Cu toxicity nor intracellular Cu levels; but it leads to higher H(2) O(2) sensitivity and reduced cytochrome oxidase activity. Mutation of both genes does not exacerbate the phenotypes produced by single-gene mutations. CopA1 does not complement the copA2 mutant strain and vice versa, even when promoter regions are exchanged. CopA1 but not CopA2 complements an Escherichia coli strain lacking the endogenous CopA. Nevertheless, transport assays show that both enzymes catalyse cytoplasmic Cu(+) efflux into the periplasm, albeit CopA2 at a significantly lower rate. We hypothesize that their distinct cellular functions could be based on the intrinsic differences in transport kinetic or the likely requirement of periplasmic partner Cu-chaperone proteins specific for each Cu(+) -ATPase.     

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.     

Different catalytic properties of two highly homologous triosephosphate isomerase monomers

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.     

Antigen-driven selection of TCR In vivo: related TCR alpha-chains pair with diverse TCR beta-chains

Ag-driven selection mediates effective T cell help and the development of Th cell memory in vivo. To analyze the dynamics of interclonal competition during the selection process in vivo, we use the I-Ek-restricted murine response to pigeon cytochrome c (PCC). The dominant PCC-specific clonotype expresses Valpha11Vbeta3 V regions with preferred sequence features in the third hypervariable regions (CDR3). In the current study we define and quantitatively monitor four subdominant PCC-specific clonotypes that express Valpha11 paired with non-Vbeta3 TCR beta-chains (Vbeta6, Vbeta8.1/8. 2, Vbeta8.3, and Vbeta14). The subdominant clonotypes emerge with similar dynamics to the dominant clonotype and together amount to similar numbers as the dominant clonotype in vivo. These subdominant clonotypes do not efficiently enter germinal centers, although they enter the memory compartment and rapidly re-emerge upon secondary challenge. Analysis of CDR3 diversity in the TCR alpha-chains identifies many preferred sequence features expressed by the dominant clonotype. These studies quantitatively demonstrate selection for diverse Th cells in vivo and highlight TCR alpha-chain dominance in Ag-driven selection for best fit.