Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-␣ (IFN-␣) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-␣. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-␣ receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficient in vitro IFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-␣. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.The intracellular protein-tyrosine kinases of the Janus kinase (JAK) family play an essential role in cytokine signaling: they interact with receptor components and undergo tyrosine phosphorylation and enzymatic activation upon ligand binding. Their activation is the first step of a cascade of intracellular phosphorylation events ultimately leading to the transcriptional activation of target genes (1, 2). Signaling through the receptor for type I interferons (IFN-␣ and -␤) requires two members of the JAK family, Tyk2 and JAK1 (3, 4). Both enzymes are associated with the receptor, which is composed of IFN-␣ receptor (IFNAR) 1 (5) and IFNAR2-2, the longer splice variant of the IFNAR2 gene (6, 7). Tyk2 was shown to interact with the membrane-proximal region of IFNAR1 (8, 9), and JAK1 with IFNAR2-2 (10, 11). The stoichiometry of the activated receptor͞JAK complex is not known and might differ for the different type I IFNs. Ligand-induced dimerization or oligomerization of the receptor components leads to asymmetric trans-phosphorylation and consequent catalytic activation of Tyk2 and JAK1 (4,12,13).Tyk2 shares with the other members of the JAK family a unique structural framework (see Fig. 1A) comprising seven conserved JAK homology (JH) regions (14). The most Cterminal one (JH1) is a tyrosine kinase (TK) domain, which is flanked by the JH2 or kinase-like (KL) domain of unknown function. The remaining five blocks of homology (JH3 to JH7) extend toward the N terminus of the protein and exhibit variable degrees of conservation among the family members, the most conserved being JH4 with a central core of 18 identical...

Bombesin/gastrin-releasing peptides (BN/GRP) were shown to bind selectively to cell surface receptors, stimulating the growth of various types of malignancies in murine and human models. The novel BN/GRP synthetic receptor antagonist, RC-3095, was able to produce long-lasting tumor regressions in murine and human tumor models in vitro and in vivo. Animal toxicology studies showed no detectable organ toxicity apart from local irritation at the injection site. The purpose of this study was to determine the safety and feasibility of the administration of RC-3095 by daily subcutaneous injections in patients with advanced and refractory solid malignancies. Twenty-five patients received RC-3095 once or twice-daily at doses ranging from 8 to 96 ug/kg. Dose was escalated in groups of 3-5 patients per dose level. The only toxicity observed was local discomfort in the injection site at the highest doses. A single dose administration of RC-3095 at the highest dose level (96 ug/kg) was tested in a clearly hypergastrinemic individual with the Zollingen-Ellison syndrome and produced a decrease in plasma gastrin down to 50% of basal levels in 6 h. There was no objective tumor responses in patients included in the study. A short-lasting minor tumor response was observed in a patient with a GRP-expressing progressive medullary carcinoma of the thyroid. Due to problems with the analytical method, plasma pharmacokinetic data was obtained only from two patients included at the highest dose level. In these patients, RC-3095 reached plasma concentrations >100 ng/mL for about 8 h, which were within therapeutic levels on the basis of prior data obtained in mice and rats. The plasma elimination half-life was between 8.6-10.9 h. Due to the occurrence of local toxicity at the injection site, the dose escalation procedure could not be fully evaluated up to a maximum tolerated dose. Thus, a recommended dose of RC-3095 for Phase II trials could not be clearly established. Considering the novelty of its mechanism of action and impressive preclinical anti-tumor activity, further studies exploiting new formulations of RC-3095 for human use, such as slow-release preparations, and analogues with a more favorable pharmacokinetics are warranted.

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The beta-carboline alkaloids found in medical plants and in a variety of foods, beverages and cigarette smoke have a range of action in various biological systems. In vitro studies have demonstrated that these alkaloids can act as scavengers of reactive oxygen species. In this paper, we report the in vivo antioxidative properties of the aromatic (harmane, harmine, harmol) and dihydro-beta-carbolines (harmaline and harmalol) studied by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their antimutagenic activity was also assayed in S. cerevisiae and the antigenotoxicity was tested by the comet assay in V79 cell line, when both eukaryotic systems were exposed to H(2)O(2). We show that the alkaloids have a significant protective effect against H(2)O(2) and paraquat oxidative agents in yeast cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon ␣/␤ receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein.Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon ␣ signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon ␣/␤ receptor signaling.The Janus kinase (JAK) 1 family of non-receptor tyrosine kinases consists of four mammalian proteins (Tyk2, JAK1, JAK2, and JAK3) that play a critical role in initiating signaling cascades of a large number of cytokine receptors (1, 2). All JAK proteins possess a carboxyl-terminal tyrosine kinase (TK) catalytic domain, a central kinase-like (KL) domain, and a large amino-terminal (N) region, which has been subdivided into five JAK homology regions (JH7 to JH3) based on sequence conservation (3). The specific and noncovalent association of these kinases to the intracellular region of cytokine receptors governs their activation upon ligand binding (2). We are interested in understanding the mode of action and specific roles of Tyk2, which is activated, together with another JAK family member, by the type I interferons (IFN) (several ␣ and one ␤ subtypes), by interleukin (IL) 6, IL-10, and IL-12 (4 -9).The IFN-␣/␤ receptor is present at low numbers on the surface of all cell types and consists of two transmembrane proteins called IFNAR1 and IFNAR2 (10, 11). The IFNAR2 gene generates several alternatively spliced forms, but only the product harboring a long intracytoplasmic domain (IFNAR2c) is part of a functional IFN-␣/␤ receptor (12). Whereas the stoichiometry and spatial organization of these components within the receptor complex are unknown, the epitopes on the IFN molecule contacting IFNAR1 and IFNAR2 are being identified (13). High affinity binding of IFN-␣/␤ to the receptor results in tyrosine phosphorylation and enzymatic activation of the associated JAK1 and Tyk2 in a defined temporal order, which is thought to result from the topology of each kinase within the complex (14, 15). Studies of kinase-deficient mutant cell lines showed that in the absence of either kinase, high affinity IFN-␣ binding is impaired, demonstrating a structural role...

No abstract

MxA is a GTPase encoded by an interferon-inducible human gene. Its constitutive expression renders transfected mammalian cells resistant to infections with several different RNA viruses, including vesicular stomatitis virus (VSV). Differences in viral RNA levels of VSV-infected cells either expressing or lacking MxA indicated that VSV mRNA synthesis is the principal target of MxA action. We now used purified histidine-tagged MxA (His-MxA) that we produced in Escherichia coli to successfully inhibit VSV in vitro transcription, a reaction catalyzed by VSV ribonucleoprotein complexes isolated from virus-infected cells or from purified virions. MxA was inactive when added to preformed VSV mRNAs, arguing against the possibility that it has a negative effect on viral RNA stability. MxA inhibited both leader RNA and mRNA synthesis of VSV, suggesting that it interfered with transcription initiation. The degree of VSV inhibition correlated directly with the specific GTPase activities of the various wild-type MxA preparations. No inhibition of viral mRNA synthesis was observed when a C-terminally truncated, GTPase-inactive variant of His-MxA was added to the transcription reactions. Purified His-MxA-E645R, a mutant of MxA with normal GTPase activity whose range of antiviral activity in vivo is altered so that it no longer inhibits VSV, showed no inhibitory effect on VSV in vitro transcription. Since MxA inhibited VSV RNA synthesis in the presence of GMP-PNP or GTP gamma S, GTP analogs that are readily accepted by the viral polymerase but cannot be hydrolyzed by MxA, the possibility was excluded that MxA acts by depleting the viral polymerase for its nucleotide substrates. Thus, binding of GTP rather than its hydrolysis seems of importance for the anti-VSV activity of MxA.

MxA is a GTPase encoded by an interferon-activated human gene which inhibits the multiplication of several RNA viruses. Recombinant histidine-tagged MxA protein (His-MxA) was expressed in Escherichia coli and purified to near homogeneity. Gel filtration showed that it formed high molecular weight oligomers. Purified His-MxA exhibited specific GTP hydrolysis rates of up to 350 nmol of GTP/min/mg of protein, corresponding to a turnover number of 27 min-1. The Km for this reaction was 260 microM. Guanine nucleotides did not copurify with His-MxA. Binding experiments in solution with fluorescent-labeled nucleotides confirmed that His-MxA binds guanine nucleotides rather weakly and further showed that the fluorescent GDP analog N-methylanthraniloyl (mant)-GDP had a much lower affinity for His-MxA (Kd 20 microM, koff 8.5 s-1) than the nonhydrolyzable GTP analog mant-5'-guanylyl-beta,gamma-imidotriphosphate (mant-GMP-PNP) (Kd 0.75 microM, koff 0.012 s-1). Competitive binding studies with nonlabeled nucleotides revealed a similar binding preference of His-MxA for GTP over GDP: the Kd for GTP was 20 microM, whereas the Kd for GDP was 100 microM. Thus, a high percentage of MxA molecules may be complexed with GTP in vivo.