Crystal structures of interleukin-2 tyrosine kinase and their implications for the design of selective inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.     

Ligand-induced, p38-dependent apoptosis in cells expressing high levels of epidermal growth factor receptor and ErbB-2

Increased expression of the epidermal growth factor (EGF) receptor (EGFR) and ErbB-2 is implicated into the development and progression of breast cancer. Constant ligand-induced activation of EGFR and ErbB-2 receptor-tyrosine kinases is thought to be involved in the transformation of fibroblasts and mammary epithelial cells. Data herein show that ligand stimulation of cells that express both the EGFR and the ErbB-2 may result either in cell proliferation or apoptosis depending on the expression levels of EGFR and ErbB-2. Mammary tumor cells that express low levels of both receptors or high levels of ErbB-2 and low levels of EGFR survive and proliferate in the presence of EGF. In contrast, fibroblastic cells or mammary tumor cells, which co-express high levels of EGFR and ErbB-2 invariably undergo apoptosis in response to EGF. In these cells persistent activation of p38 MAPK is an essential element of the apoptotic mechanism. Also, the data implicate a p38-dependent change in mitochondrial membrane permeability as a downstream effector of apoptosis. Ligand-dependent apoptosis in cells co-expressing high levels of EGFR and ErbB-2 could be a natural mechanism that protects tissues from unrestricted proliferation in response to the sustained activation of receptor-tyrosine kinases.     

The amitochondriate eukaryote Trichomonas vaginalis contains a divergent thioredoxin-linked peroxiredoxin antioxidant system

Trichomonas is an amitochondriate parasitic protozoon specialized for an anaerobic lifestyle. Nevertheless, it is exposed to oxygen and is able to cope with the resultant oxidative stress. In the absence of glutathione, cysteine has been thought to be the major antioxidant. We now report that the parasite contains thioredoxin reductase, which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants. Thioredoxin reductase and thioredoxin also reduce cystine and so may play a role in maintaining the cellular cysteine levels. The importance of the thioredoxin system as one of the major antioxidant defense mechanisms in Trichomonas was confirmed by showing that the parasite responds to environmental changes resulting in increased oxidative stress by up-regulating thioredoxin and thioredoxin peroxidases levels. Sequence data indicate that the thioredoxin reductase of Trichomonas differs fundamentally in structure from that of its human host and thus may represent a useful drug target. The protein is generally similar to thioredoxin reductases present in other lower eukaryotes, all of which probably originated through horizontal gene transfer from a prokaryote. The phylogenetic signal in thioredoxin peroxidase is weak, but evidence from trees suggests that this gene has been subject to repeated horizontal gene transfers from different prokaryotes to different eukaryotes. The data are thus consistent with the complexity hypothesis that predicts that the evolution of simple pathways such as the thioredoxin cascade are likely to be affected by horizontal gene transfer between species.     

Phylogenetic reconstruction of the evolution of stylar polymorphisms in Narcissus (Amaryllidaceae)

We investigated the origin of stylar polymorphisms in Narcissus, which possesses a remarkable range of stylar conditions and diverse types of floral morphology and pollination biology. Reconstruction of evolutionary change was complicated by incomplete resolution of trees inferred from two rapidly evolving chloroplast regions, but we bracketed reconstructions expected on the fully resolved plastid-based tree by considering all possible resolutions of polytomies on the shortest trees. Stigma-height dimorphism likely arose on several occasions in Narcissus and persisted across multiple speciation events. As proposed in published models, this rare type of stylar polymorphism is ancestral to distyly. While there is no evidence in Narcissus that dimorphism preceded tristyly, a rapid transition between them may explain the lack of a phylogenetic footprint for this evolutionary sequence. The single instances of distyly and tristyly in Narcissus albimarginatus and N. triandrus, respectively, are clearly not homologous, an evolutionary convergence unique to Amaryllidaceae. Floral morphology was likely an important trigger for the evolution of stylar polymorphisms: Concentrated-changes tests indicate that a long, narrow floral tube may have been associated with the emergence of stigma-height dimorphism and that this type of tube, in combination with a deep corona, likely promoted, or at least was associated with, the parallel origins of heterostyly.     

Integrin activation state determines selectivity for novel recognition sites in fibrillar collagens

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs. Recombinant inserted (I) domains of integrin subunits alpha(1), alpha(2) and alpha(11) all bound poorly to all motifs other than GFOGER and GLOGER. Similarly, alpha(2)beta(1) -containing resting platelets adhered well only to GFOGER and GLOGER, while ADP-activated platelets, HT1080 cells and two active alpha(2)I domain mutants (E318W, locked open) bound all motifs well, indicating that affinity modulation determines the sequence selectivity of integrins. GxO/SGER peptides inhibited platelet adhesion to collagen monomers with order of potency F >/= L >/= M > A. These results establish GFOGER as a high affinity sequence, which can interact with the alpha(2)I domain in the absence of activation and suggest that integrin reactivity of collagens may be predicted from their GER content.     

Assessment of screw displacement axis accuracy and repeatability for joint kinematic description using an electromagnetic tracking device

Screw displacement axes (SDAs) have been employed to describe joint kinematics in biomechanical studies. Previous reports have investigated the accuracy of SDAs combining various motion analysis techniques and smoothing procedures. To our knowledge, no study has assessed SDA accuracy describing the relative movement between adjacent bodies with an electromagnetic tracking system. This is important, since in relative motion, neither body is fixed and consequently sensitivity to potential measurement errors from both bodies may be significant. Therefore, this study assessed the accuracy of SDAs for describing relative motion between two moving bodies. We analyzed numerical simulated data, and physical experimental data recorded using a precision jig and electromagnetic tracking device. The numerical simulations demonstrated SDA position accuracy (p=0.04) was superior for single compared to relative body motion, whereas orientation accuracy (p=0.2) was similar. Experimental data showed data-filtering (Butterworth filter) improved SDA position and orientation accuracies for rotation magnitudes smaller or equal to 5.0 degrees, with no effect at larger rotation magnitudes (p<0.05). This suggests that in absence of a filter, SDAs should only be calculated at rotations of greater than 5.0 degrees. For rotation magnitudes of 0.5 degrees (5.0 degrees ) about the SDA, SDA position and orientation error measurements determined from filtered experimental data were 3.75+/-0.30 mm (3.31+/-0.21 mm), and 1.10+/-0.04 degrees (1.04+/-0.03 degrees ), respectively. Experimental accuracy values describing the translation along and rotation about the SDA, were 0.06+/-0.00 mm and 0.09+/-0.01 degrees, respectively. These small errors establish the capability of SDAs to detect small translations, and rotations. In conclusion, application of SDAs should be a useful tool for describing relative motion in joint kinematic studies.     

Golgi duplication in Trypanosoma brucei

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.     

Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also influences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid profiles, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.     

Prolonged production of TNF-alpha exacerbates illness during respiratory syncytial virus infection

CD8(+) CTL are the main effector cells responsible for resolving viral infections. However, the CTL response to respiratory syncytial virus (RSV) infection in mice facilitates viral clearance at the expense of significant immunopathology. Previous reports have shown a strong correlation between the mechanism of CTL activity and the severity of RSV-induced illness. Furthermore, experiments in perforin knockout mice revealed that antiviral cytokine production temporally correlated with RSV-induced illness. In the current study, we show that TNF-alpha is the dominant mediator of RSV-associated illness, and it is also important for clearance of virus-infected cells during the early stages of infection. We also demonstrate that IFN-gamma plays a protective role in conjunction with perforin/granzyme-mediated killing. Preliminary experiments in gld mice that express nonfunctional Fas ligand (FasL) revealed that RSV-induced illness is significantly reduced in the absence of FasL-mediated killing. Antiviral cytokine production was not elevated in the absence of FasL, suggesting a possible link between FasL and antiviral cytokine activity. This work shows that multiple phenotypic subsets of CD8(+) CTLs respond to RSV infection, each with varying capacities for clearance of virus-infected cells and the induction of illness. In addition, the revelation that TNF-alpha is the principal mediator of RSV-induced illness means that administration of TNF receptor antagonists, in combination with antiviral therapy, may be an effective method to treat RSV infections.