The interferon gamma (IFN-gamma) receptor: a paradigm for the multichain cytokine receptor.

With the purification and cloning of the interferon gamma (IFN-gamma) receptor chains the mechanism of IFN-gamma action and the resultant signal transduction events were delineated in remarkable detail. The interferon gamma (IFN-gamma) receptor complex consists of two chains: IFN-gammaR1, the ligand-binding chain, and IFN-gammaR2, the accessory chain. Binding of IFN-gamma causes oligomerization of the two IFN-gamma receptor subunits, IFN-gammaR1 and IFN-gammaR2, which initiates the signal transduction events: activation of Jak1 and Jak2 receptor associated protein tyrosine kinases, phosphorylation of the IFN-gammaR1 intracellular domain on Tyr440 followed by phosphorylation and activation of Stat1alpha, the latent transcriptional factor. With all these steps established, the IFN-gamma receptor complex has provided the basic model for understanding the receptors for other members of the family of class II cytokine receptors.

Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-1 beta synthesis in Escherichia coli.

A new dual-replicon recombinant plasmid, pPR53-tsr, has been constructed; it is a derivative of the expression vector pPR-TGATG-1 [Mashko et al., Gene 88 (1990) 121-126]. In contrast to its progenitor, pPR53-tsr is a low-copy-number (low-Cop) plasmid amplifiable in temperature-dependent fashion. In addition to both the replicon and the par locus from plasmid pSC101, providing segregational stability and a low Cop at 28 degrees C, the new plasmid contains a mutant ColE1 replicon whose RNAII is synthesized under the control of the pL promoter. The presence of a thermolabile repressor, cIts857, allows the thermo-inducible amplification of pPR53-tsr; the increased plasmid Cop is estimated at approx. 200 per genome 6 h after thermal induction at 42 degrees C. Thus, pPR53-tsr can be used as a donor of the thermo-inducible dual-replicon fragment for recombinant plasmids. Here, we employ such an approach for optimization of production of human interleukin-1 beta (hIL-1 beta) in Escherichia coli at a high level. The thermo-induced level of recombinant hIL-1 beta (re-hIL-1 beta) biosynthesis was around 9% of total cellular protein when the dual-replicon high-Cop vector was used. A method based on acidification of the water-soluble protein fraction to pH 4.0 has been developed that allows for the isolation of 80%-pure re-hIL-1 beta. The homogeneous material was obtained by two subsequent hydrophobic sorbent chromatographies. The protein yield ranged between 3-5 mg of re-hIL-1 beta/g of wet cells. The re-hIL-1 beta specific activity was about 2 x 10(8) units/mg, coinciding with that of the authentic hIL-1 beta.

[Effect of Bacillus subtilis A-50 "streptomycin" mutations on the formation of molecular forms of subtilisin, an extracellular alkaline proteinase]. / Vliianie "streptomitsinovykh" mutatsii Bacillus subtilis A-50 na obrazovanie molekuliarnykh form subtilizina--vnekletochnoi shchelochnoi proteinazy.

The patterns of subtilisin molecular forms of streptomycin-resistant (Strr) and streptomycin-dependent (Strd) mutants of Bacillus subtilis A-50, as well as the revertants of Strd to streptomycin-independence (Str1) were studied. Strr mutants had different quantitative pattern of the same subtilisin molecular forms as compared with the initial strain A-50 (the forms with Rf 0.08, 0.16 and 0.3). In comparison with the initial strain A-50, Strd mutants and Str1 revertants revealed three additional forms of the active enzyme with Rf 0.02, 0.5 and 0.7 and the molecular weights less than 35,000, 28,000 and 20,000 respectively. It was suggested that the rate and character of the enzyme secretion of the degree of its post-translational modifications might result in the different pattern of subtilisin molecular forms produced by these streptomycin mutants.

[Obtaining human recombinant (serine-17) beta-interferon by the method of oligonucleotide-directed mutagenesis and its expression in Escherichia coli]. / Poluchenie rekombinantnogo (serin-17) beta-interferona cheloveka metodom oligonukleotidnapravlennogo mutageneza i ego ékspresiia v Escherichia coli.

The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis. On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed. It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.

Mapping of an epitope of human leukocyte alpha interferon A which is recognized by the murine monoclonal antibody NK2.

An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope.

Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10).

We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system.

[Isolation of proteinase I from E. coli cells using affinty chromatography on bacitracin-Sepharose]. / Izuchenie proteinaz E. coli. Vydelenie proteinazy I s pomoshch'iu affinnoi khromatografii na batsitratsin-sefaroze.

A serine proteinase (proteinase I) was isolated in a homogeneous state from E. coli K12 cells, using bacitracin-Sepharose 4B affinity chromatography. The enzyme effectively cleaved N alpha-acetyl-L-phenylalanine beta-naphthyl ester. The proteinase was inhibited by diisopropylphosphofluoridate and phenylmethanesulphonyl fluoride, but was resistant to EDTA and natural trypsin or subtilisin protein inhibitors. The enzyme did not cleave trypsin and subtilisin synthetic substrates, possessing a narrow substrate specificity. The amino acid composition of the enzyme was determined. The enzyme molecular weight was found to be about 20 000.

The intracellular domain of interferon-alpha receptor 2c (IFN-alphaR2c) chain is responsible for Stat activation.

Type I IFNs activate the Jak-Stat signal transduction pathway. The IFN-alpha receptor 1 (IFN-alphaR1) subunit and two splice variants of the IFN-alphaR2 subunit, IFN-alphaR2c and IFN-alphaR2b, are involved in ligand binding. All these receptors have been implicated in cytokine signaling and, specifically, in Stat recruitment. To evaluate the specific contribution of each receptor subunit to Stat recruitment we employed chimeric receptors with the extracellular domain of either IFN-gammaR2 or IFN-gammaR1 fused to the intracellular domains of IFN-alphaR1, IFN-alphaR2b, and IFN-alphaR2c. These chimeric receptors were expressed in hamster cells. Because human IFN-gamma exhibits no activity on hamster cells, the use of the human IFN-gamma receptor extracellular domains allowed us to avoid the variable cross-species activity of the type I IFNs and eliminate the possibility of contributions of endogenous type I IFN receptors into the Stat recruitment process. We demonstrate that Stat recruitment is solely a function of the IFN-alphaR2c intracellular domain. When chimeric receptors with the human IFN-gammaR1 extracellular domain and various human IFN-alpha receptor intracellular domains were expressed in hamster cells carrying the human IFN-gammaR2 subunit, only the IFN-alphaR2c subunit was capable of supporting IFN-gamma signaling as measured by MHC class I induction, antiviral protection, and Stat activation. Neither the IFN-alphaR2b nor the IFN-alphaR1 intracellular domain was able to recruit Stats or support IFN-gamma-induced biological activities. Thus, the IFN-alphaR2c intracellular domain is necessary and sufficient to activate Stat1, Stat2, and Stat3 proteins.

Identification of the functional interleukin-22 (IL-22) receptor complex: the IL-10R2 chain (IL-10Rbeta ) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes.

Interleukin-10 (IL-10)-related T cell-derived inducible factor (IL-TIF; provisionally designated IL-22) is a cytokine with limited homology to IL-10. We report here the identification of a functional IL-TIF receptor complex that consists of two receptor chains, the orphan CRF2-9 and IL-10R2, the second chain of the IL-10 receptor complex. Expression of the CRF2-9 chain in monkey COS cells renders them sensitive to IL-TIF. However, in hamster cells both chains, CRF2-9 and IL-10R2, must be expressed to assemble the functional IL-TIF receptor complex. The CRF2-9 chain (or the IL-TIF-R1 chain) is responsible for Stat recruitment. Substitution of the CRF2-9 intracellular domain with the IFN-gammaR1 intracellular domain changes the pattern of IL-TIF-induced Stat activation. The CRF2-9 gene is expressed in normal liver and kidney, suggesting a possible role for IL-TIF in regulating gene expression in these tissues. Each chain, CRF2-9 and IL-10R2, is capable of binding IL-TIF independently and can be cross-linked to the radiolabeled IL-TIF. However, binding of IL-TIF to the receptor complex is greater than binding to either receptor chain alone. Sharing of the common IL-10R2 chain between the IL-10 and IL-TIF receptor complexes is the first such case for receptor complexes with chains belonging to the class II cytokine receptor family, establishing a novel paradigm for IL-10-related ligands similar to the shared use of the gamma common chain (gamma(c)) by several cytokines, including IL-2, IL-4, IL-7, IL-9, and IL-15.

[Isolation and properties of monomeric and oligomeric forms of gene-engineered human leukocyte interferon alphaA from Pseudomonas sp]. / Vydelenie i svoistva monomernoi i oligomernykh form genno-inzhenernogo chelovecheskogo leikotsitarnogo interferona alphaA iz Pseudomonas sp.

Using stepwise ion-exchange and gel-permeation high performance liquid chromatography and SDS-PAAG gel electrophoresis, it was demonstrated that the non-reduced gene-engineered interferon alpha A is represented by multiple forms, namely, four monomers, four dimers, two trimers and one tetramer. All the protein forms were obtained in an individual state and characterized in terms of antiviral activity and immunochemical properties. The heterogeneity of the protein is due both to the formation of anomalous intermolecular disulfide bonds and to the existence of reduced S-S bonds. The antiviral activity of the dimers, trimers and tetramers expressed as units per mole of protein is equal to that for the monomeric form, i.e., the interaction of one monomeric subunit of the covalently-linked oligomer is sufficient for the manifestation of the protein antiviral activity. This suggests that the antiviral status of the cell does not depend on the amount internalized interferon molecules of their processing products but is controlled by the cell receptor whose internalization and, possibly, processing stimulate the transcription of genes involved in the triggering of the immune response.

[Isolation and properties of human leukocyte interferon obtained by microbial synthesis]. / Vydelenie i svoistva chelovecheskogo leikotsitarnogo interferona, poluchennogo mikrobnym sintezom.

Highly sensitive, effective and simple methods for determination of leukocytic interferons were developed. The methods are based on formation of immune complexes of interferon with anti-interferon monoclonal and polyclonal antibodies immobilized on an insoluble basis. As a result of interferon alpha 2 purification from the biomass of the interferon-producing bacteria the preparative amounts of the protein were obtained. These amounts were sufficient for the interferon structural and physico-chemical analysis. The isolated interferons are characterized by high stability on storage and may serve a basis for preparation of pharmaceutical agents.

[Analysis of oligomeric forms of recombinant human leukocyte interferons]. / Analiz oligomernykh form genno-inzhenernykh chelovecheskikh leikotsitarnykh interferonov.

Using SDS-PAAG electrophoresis, gel-permeation HPLC and immunoblotting, it was demonstrated that homogeneous preparations of human leukocyte interferons (alpha-INF)-A, -N and -I1 obtained from the biomass of the corresponding producer strains (Pseudomonas sp.) contained several oligomeric forms produced by way of S-S intermolecular cross-linkage and making up to 10-15%, 4-7% and 2-5% of the total monomeric form content in the protein preparations. Immunologic testing with the use of MAB NK-2 and [125I]NK-2 showed that the oligomeric forms of alpha-INF-A, -N and -I1 were present in the protein preparations at all purification stages and seemed to be formed at early steps of interferon synthesis in the cell. The effects of limited proteolysis as well as of acid, alkaline and thermodenaturation on the aggregation and oligomerization of alpha-INF-A were studied. SDS-PAAG electrophoresis performed in the absence of the reducing agents showed that upon denaturation of 10% TCA, the amount of the oligomeric forms in the preparations of homogeneous and especially partly proteolytic INF was significantly increased. The causes and the putative mechanisms of aggregation and oligomerization of INF are discussed.

[Metalloproteinase from Bacillus subtilis: - "intracellular" and extracellular enzymes]. / Metalloproteinaza iz Bacillus subtilis: vnekletochnyi i vnutrikletochnyi fermenty.

"Intracellular" metalloproteinase was purified to homogeneity from Bacillus subtilis 103 crude cell extract, using affinity chromatography on bacitracin-Sepharose 4B. The degree of purification and the yield of the enzyme were about 260-fold and 3%, respectively. In its physico-chemical properties and the amino acid composition the enzyme is very similar, if not identical, to the extracellular metalloproteinase isolated from the culture filtrate of the same strain. Extracellular metalloproteinase-deficient mutant strain Bacillus subtilis SMY-512 does not produce the "intracellular" enzyme either. THe activity of "intracellular" metalloproteinase in the periplasmic space of the cells is about 70% of that in the cytoplasm, thus being indicative of a rather regular distribution of the enzyme throughout the cell compartment.

Identification and functional characterization of a second chain of the interleukin-10 receptor complex.

Interleukin-10 (IL-10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL-10Ralpha or IL-10R1), was identified previously. We report here that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co-expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by cross-linking to labeled IL-10. In addition, the IL-10R1 chain was able to be co-immunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrate that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL-10R2 or IL-10Rbeta chain, serves as an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events.

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Two related structural genes coding two homologous serine proteases in the Bacillus subtilis genome.

A Bacillus subtilis intracellular serine protease is homologous in sequence to an extracellular serine protease from the same strain, indicating the presence of two related structural genes in the Bacillus subtilis genome.

[Comparative immunochemical analysis of various human leukocyte interferons]. / Sravnitel'nyi immunokhimicheskii analiz nekotorykh leikotsitarnykh interferonov cheloveka.

Using radioimmunological methods based on the use of mono- and polyclonal antibodies raised against interferon alpha A, it was shown that polyclonal antibodies quantitatively reacted not only with this protein, but also with interferons alpha F and alpha N, whereas all the variants of monoclonal antibodies studied reacted only with interferons alpha A and alpha N. Monoclonal antibodies 5A6, 11E9, 19C10, 258 and 268 are directed against overlapping epitopes of the interferon alpha A molecule, which simultaneously binds not more than two molecules of antibodies with different specificity. The correlation between immunochemical and biological activities of interferon alpha A during temperature denaturation and proteolytic degradation and its ability to form oligomeric complexes were investigated.

The human homologue of the yeast proteins Skb1 and Hsl7p interacts with Jak kinases and contains protein methyltransferase activity.

To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.

[Multiple forms of Bacillus subtilis subtilisin and effects of mutations on the distribution of its molecular forms]. / Mnozhestvennost' subtilizinov Bacillus subtilis i vliianie mutatsii na raspredelenie molekuliarnykh form

Using polyacrylamide-gel electrophoresis, isoelectric focusing and gel-filtration it was demonstrated that the auxotrophic mutant strains of Bac. subtilis A-50 and their prototrophic revertant strains produce multiple molecular forms of subtilisin. Three of them are the same as the corresponding molecular forms of subtilisin from the wild strain A-50. In different mutant strains the relative amounts of the main three forms varies considerably resulting in the absence of certain forms in several strains. There is the additional minor form of subtilisin possessing high electrophoretic mobility in four prototrophic revertant strains and one Arg--auxotrophic strain of Bac. subtilis A-50. It would be reasonable to suppose that different molecular forms of subtilisin derive from the product of its single structural gene as a result of post-translational modifications (limited proteolysis). This enzyme and probably most, if not all secretory proteins may be synthesised as larger precursors and then specifically modified in the bacterial cell membranes. Thus, certain mutations, without affecting the structural gene of this secretory protein -- subtilisin -- have pronounced effects on this structural gene expression, varying the degree of its product modification and the amount of resulting secretory molecular forms of subtilisin.

[Comparative study of the multiplicity of exoenzymes produced by different strains of Bacillus subtilis]. / Sravnitel'noe izuchenie mnozhestvennosti ékzofermentov, produtsiruemykh razlichnymi mutantami Bacillus subtilis.

Molecular forms of two exoenzymes, subtilisin and alpha-amylase, produced by mutants of Bacillus subtilis were studied. The degree of the post-translational modification of subtilisin was less pronounced for P and M mutants than for R mutants. Some of the P and M mutants produced subtilisin having higher molecular weight and hydrophobicity as compared to the R mutants. This form of subtilisin may be the primary product of translation of the structural gene and therefore a precursor of subtilisin. Its appearance outside the cell may be due to the alteration in the cell surface structures in the P and M mutants and abnormal post-translational modification. The P and M mutants produced also differing exocellular proteins as compared to the R mutants, e.g. alpha-amylase forms with the higher isoelectric points.