Peptide name : Interferon gamma (IFN-gamma)

Source/Organism : Chimpanzee

Linear/Cyclic : Not found

Chirality : Not found

Peptide length: 166

C-terminal modification: Not found

N-terminal modification : Not found

Non-natural peptide information: None

Assay type : Antibody-based assay

Assay time : 48h

Activity : Not found

Cell line : U937

Cancer type : Not specified

Other activity : Not found

Amino acid composition bar chart :

Molecular mass : 19348.0813 Dalton

Aliphatic index : 0.751

Instability index : 30.2946

Hydrophobicity (GRAVY) : -0.577

Isoelectric point : 9.4979

Charge (pH 7) : 9.6534

Aromaticity : 0.114

Molar extinction coefficient (cysteine, cystine): (15930, 16055)

Hydrophobic/hydrophilic ratio : 0.72916666

hydrophobic moment : -0.304

Missing amino acid : None

Most occurring amino acid : K

Most occurring amino acid frequency : 21

Least occurring amino acid : W

Least occurring amino acid frequency : 1

Secondary structure fraction (Helix, Turn, Sheet): (0.3, 0.2, 0.3)

SMILES Notation: CC[C@H](C)[C@H](NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CS)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCSC)[C@@H](C)O)[C@@H](C)CC)[C@@H](C)CC)C(C)C)C(C)C)C(C)C)[C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H](Cc1c[nH]cn1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCC(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)O)[C@@H](C)O)C(C)C)[C@@H](C)CC)[C@@H](C)CC)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)O)C(C)C)[C@@H](C)CC)[C@@H](C)O)C(C)C)[C@@H](C)CC)C(C)C)[C@@H](C)CC)[C@@H](C)CC

1 : Fabunmi RP, et al. Interferon gamma regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies. J Cell Sci. 2001; 114:29-36. doi: 10.1242/jcs.114.1.29

2 : Landar A, et al. Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma. J Mol Biol. 2000; 299:169-79. doi: 10.1006/jmbi.2000.3734

3 : Thiel DJ, et al. Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex. Structure. 2000; 8:927-36. doi: 10.1016/s0969-2126(00)00184-2

4 : Gerhard DS, et al. The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome Res. 2004; 14:2121-7. doi: 10.1101/gr.2596504

5 : Goshima N, et al. Human protein factory for converting the transcriptome into an in vitro-expressed proteome,. Nat Methods. 2008; 5:1011-7. doi: 10.1038/nmeth.1273

6 : Gray PW and Goeddel DV. Structure of the human immune interferon gene. Nature. 1982; 298:859-63. doi: 10.1038/298859a0

7 : Gray PW, et al. Expression of human immune interferon cDNA in E. coli and monkey cells. Nature. 1982; 295:503-8. doi: 10.1038/295503a0

8 : Ooi J, et al. Unrelated cord blood transplantation after myeloablative conditioning in patients over the age of 45 years. Br J Haematol. 2004; 126:711-4. doi: 10.1111/j.1365-2141.2004.05130.x

9 : Lee SM, et al. The regulation and biological activity of interleukin 12. Leuk Lymphoma. 1998; 29:427-38. doi: 10.3109/10428199809050903

10 : Taya Y, et al. Cloning and structure of the human immune interferon-gamma chromosomal gene. EMBO J. 1982; 1:953-8. doi: 10.1002/j.1460-2075.1982.tb01277.x

11 : Rinderknecht E, et al. Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation. J Biol Chem. 1984; 259:6790-7.

12 : Lah TT, et al. Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages. FEBS Lett. 1995; 363:85-9. doi: 10.1016/0014-5793(95)00287-j

13 : Kerner G, et al. Inherited human IFN-γ deficiency underlies mycobacterial disease. J Clin Invest. 2020; 130:3158-3171. doi: 10.1172/JCI135460

14 : Nishi T, et al. Cloning and expression of a novel variant of human interferon-gamma cDNA. J Biochem. 1985; 97:153-9. doi: 10.1093/oxfordjournals.jbchem.a135039

15 : El Bougrini J, et al. Arsenic enhances the apoptosis induced by interferon gamma: key role of IRF-1. Cell Mol Biol (Noisy-le-grand). 2006; 52:9-15.

16 : Grzesiek S, et al. 1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma. Biochemistry. 1992; 31:8180-90. doi: 10.1021/bi00150a009

17 : Yamamoto S, et al. Studies on the sugar chains of interferon-gamma from human peripheral-blood lymphocytes. J Biochem. 1989; 105:1034-9. doi: 10.1093/oxfordjournals.jbchem.a122762

18 : Walter MR, et al. Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor. Nature. 1995; 376:230-5. doi: 10.1038/376230a0

19 : Akiyama K, et al. Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing. FEBS Lett. 1994; 343:85-8. doi: 10.1016/0014-5793(94)80612-8

20 : Devos R, et al. Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells. Nucleic Acids Res. 1982; 10:2487-501. doi: 10.1093/nar/10.8.2487

21 : Hisamatsu H, et al. Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma. J Exp Med. 1996; 183:1807-16. doi: 10.1084/jem.183.4.1807

22 : Greenlund AC, et al. Interferon-gamma induces receptor dimerization in solution and on cells. J Biol Chem. 1993; 268:18103-10.

23 : Pan YC, et al. Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus. Eur J Biochem. 1987; 166:145-9. doi: 10.1111/j.1432-1033.1987.tb13494.x

24 : Ealick SE, et al. Three-dimensional structure of recombinant human interferon-gamma. Science. 1991; 252:698-702. doi: 10.1126/science.1902591

Paper title : Interferon gamma regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies.

Doi : https://doi.org/10.1242/jcs.114.1.29

Abstract : PA28 is an interferon (gamma) (IFN(gamma)) inducible proteasome activator required for presentation of certain major histocompatibility (MHC) class I antigens. Under basal conditions in HeLa and Hep2 cells, a portion of nuclear PA28 is concentrated at promyelocytic leukemia oncoprotein (PML)-containing bodies also commonly known as PODs or ND10. IFN(gamma) treatment greatly increased the number and size of the PA28- and PML-containing bodies, and the effect was further enhanced in serum-deprived cells. PML bodies are disrupted in response to certain viral infections and in diseases such as acute promyelocytic leukemia (APL). Like PML, PA28 was delocalized from PML bodies by expression of the cytomegalovirus protein, IE1, and in NB4 cells, an APL model line. Moreover, retinoic acid treatment, which causes remission of APL in patients and reformation of PML-containing bodies in NB4 cells, relocalized PA28 to this site. In contrast, the proteasome, the functional target of PA28, was not detected at PML bodies under basal conditions in HeLa and Hep2 cells, but IFN(gamma) promoted accumulation of 'immunoproteasomes' at this site. These results establish PA28 as a novel component of nuclear PML bodies, and suggest that PA28 may assemble or activate immunoproteasomes at this site as part of its role in proteasome-dependent MHC class I antigen presentation.

Paper title : Design, characterization, and structure of a biologically active single-chain mutant of human IFN-gamma.

Doi : https://doi.org/10.1006/jmbi.2000.3734

Abstract : A mutant form of human interferon-gamma (IFN-gamma SC1) that binds one IFN-gamma receptor alpha chain (IFN-gamma R alpha) has been designed and characterized. IFN-gamma SC1 was derived by linking the two peptide chains of the IFN-gamma dimer by a seven-residue linker and changing His111 in the first chain to an aspartic acid residue. Isothermal titration calorimetry shows that IFN-gamma SC1 forms a 1:1 complex with its high-affinity receptor (IFN-gamma R alpha) with an affinity of 27(+/- 9) nM. The crystal structure of IFN-gamma SC1 has been determined at 2.9 A resolution from crystals grown in 1.4 M citrate solutions at pH 7.6. Comparison of the wild-type receptor-binding domain and the Asp111-containing domain of IFN-gamma SC1 show that they are structurally equivalent but have very different electrostatic surface potentials. As a result, surface charge rather than structural changes is likely responsible for the inability of the His111-->Asp domain of to bind IFN-gamma R alpha. The AB loops of IFN-gamma SC1 adopt conformations similar to the ordered loops of IFN-gamma observed in the crystal structure of the IFN-gamma/IFN-gamma R alpha complex. Thus, IFN-gamma R alpha binding does not result in a large conformational change in the AB loop as previously suggested. The structure also reveals the final six C-terminal amino acid residues of IFN-gamma SC1 (residues 253-258) that have not been observed in any other reported IFN-gamma structures. Despite binding to only one IFN-gamma R alpha, IFN-gamma SC1 is biologically active in cell proliferation, MHC class I induction, and anti-viral assays. This suggests that one domain of IFN-gamma is sufficient to recruit IFN-gamma R alpha and IFN-gamma R beta into a complex competent for eliciting biological activity. The current data are consistent with the main role of the IFN-gamma dimer being to decrease the dissociation constant of IFN-gamma for its cellular receptors.

Paper title : Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex.

Doi : https://doi.org/10.1016/s0969-2126(00)00184-2

Abstract : BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.

Paper title : The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

Doi : https://doi.org/10.1101/gr.2596504

Abstract : The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Paper title : Human protein factory for converting the transcriptome into an in vitro-expressed proteome,

Doi : https://doi.org/10.1038/nmeth.1273

Abstract : Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

Paper title : Structure of the human immune interferon gene.

Doi : https://doi.org/10.1038/298859a0

Abstract : Sequence determination of cloned cDNAs and genes of the three classes of interferon (IFN-alpha, -beta and -gamma) has revealed more than a dozen members of the human IFN-alpha gene family and a single gene for IFN-beta. These genes are found on chromosome 9 and contain no introns. We recently reported that the 146-amino acid sequence of mature IFN-gamma deduced from the nucleotide sequence of a cloned cDNA was quite unrelated to those of the other IFNs, and that the gene for IFN-gamma contains at least one intron. We now describe the isolation, characterization and DNA sequence of the human IFN-gamma gene. It contains three introns, a repetitive DNA element, and is not highly polymorphic. All our evidence to date and the present data suggest that this is the only gene for IFN-gamma and that the resolution of IFN-gamma into two components is probably the result of post-translational processing of the protein.

Paper title : Expression of human immune interferon cDNA in E. coli and monkey cells.

Doi : https://doi.org/10.1038/295503a0

Abstract : Not available

Paper title : Unrelated cord blood transplantation after myeloablative conditioning in patients over the age of 45 years.

Doi : https://doi.org/10.1111/j.1365-2141.2004.05130.x

Abstract : We report the results of unrelated cord blood transplantation (CBT) after myeloablative conditioning in 21 patients over the age of 45 years. Among the patients the median age was 48 years (range, 45-53 years), the median weight was 58.6 kg (range, 43.6-76.2 kg) and the median number of cryopreserved nucleated cells was 2.45 x 10(7)/kg (range, 1.63-3.71 x 10(7)/kg). Nineteen patients had myeloid reconstitution and the median time to more than 0.5 x 10(9)/l absolute neutrophil count was 22 d. A self-sustained platelet count more than 50 x 10(9)/l was achieved in 17 patients at a median time of 49 d. Acute graft-versus-host disease (GVHD) above grade II occurred in 7 of 19 evaluable patients and chronic GVHD occurred in 14 of 16 evaluable patients. Among 14 chronic GVHD patients, in seven patients the disease was extensive. Fifteen patients were alive and free of disease at between 217 and 1798 d after transplantation. With a median follow-up of 847 d, the probability of disease-free survival at 2 years was 71.4%. These results suggest that patients over 45 years of age without suitable related or unrelated bone marrow donors should be considered as candidates for CBT.

Paper title : The regulation and biological activity of interleukin 12.

Doi : https://doi.org/10.3109/10428199809050903

Abstract : Interleukin 12 (IL-12) is a pleiotropic cytokine and mediates several biological activities on human T and natural killer (NK) cells, including induction of IFN-gamma production, enhancement of cell-mediated cytotoxicity and comitogenic effects on resting T-cells. The major cellular sources producing IL-12 are antigen-stimulated monocytes, macrophages, and B-cells isolated from peripheral blood mononuclear cells (PBMC). Our laboratory has investigated the regulation of IL-12 gene expression in both cord blood and adult PBMC, and the effects of IL-12 on induction of IFN-gamma production, NK, and lymphokine-activated killer (LAK) cytotoxicity. IL-12 mRNA expression and protein production in LPS-stimulated cord blood MNC were 3-4 fold decreased when compared with adult PBMC. There were no differences between cord blood and adult PBMC in both basal levels of transcription or the degree of transcriptional activation of the IL-12 gene. Additionally, the half-life of IL-12 p40 mRNA was 3-fold lower in activated cord blood compared to adult PBMC. Exogenous IL-12 induced a significant increase of IFN-gamma from both cord and adult PBMC. Cord MNC has significantly reduced levels of NK activity, and IL-12 significantly enhanced cord blood NK cytotoxicity up to similar levels in adult PBMC. IL-12 also significantly enhanced cord blood NK and LAK activities against a broad range of neuroblastoma, leukemia, and lymphoma cell lines. Lower doses of IL-12 and IL-15 concomitantly generated either synergistic or additive effects on cord blood NK and LAK cytotoxicities. In light of the important biological functions of IL-12, reduced expression and production of IL-12 from activated cord blood may contribute to the immaturity of cord blood cellular immunity and contribute, in part, to decreased severe graft vs. host disease following unrelated cord blood stem cell transplantation. IL-12 enhancement of IFN-gamma, NK, and LAK activity in activated cord blood MNC up to comparable levels in adult PBMC suggests that exogenous IL-12 stimulation can compensate for the immaturity in cord blood cellular immunity. These characteristics of IL-12 biological activity strongly suggest its potential usefulness in future cancer immunotherapy.

Paper title : Cloning and structure of the human immune interferon-gamma chromosomal gene.

Doi : https://doi.org/10.1002/j.1460-2075.1982.tb01277.x

Abstract : Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.

Paper title : Natural human interferon-gamma. Complete amino acid sequence and determination of sites of glycosylation.

Doi : https://doi.org/Not available

Abstract : Fresh human peripheral blood lymphocytes were induced with desacetylthymosin -alpha 1 and staphylococcal enterotoxin B. The induced gamma interferon (or IFN-gamma, immune interferon, type II interferon) was purified to homogeneity utilizing controlled-pore glass, concanavalin A-Sepharose, Bio-Gel P100, or Sephacryl S-200, and reversed phase high performance liquid chromatography. This procedure resulted in two active species with apparent Mr = 20,000 and 25,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both species were found to have identical amino acid sequences with a pyroglutamate residue as NH2-terminus. In both cases six different COOH termini were found. They are, at least qualitatively, identical in both species. There are two possible Asn-X-Ser/Thr glycosylation sites. Both carry carbohydrates in the Mr = 25,000 species whereas in the Mr = 20,000 species only one site is glycosylated. This likely explains the difference in apparent molecular weight between the two species and the expected molecular weight based upon the amino acid sequence.

Paper title : Gamma-interferon causes a selective induction of the lysosomal proteases, cathepsins B and L, in macrophages.

Doi : https://doi.org/10.1016/0014-5793(95)00287-j

Abstract : Previous studies have indicated that acid-optimal cysteine proteinase(s) in the endosomal-lysosomal compartments, cathepsins, play a critical role in the proteolytic processing of endocytosed proteins to generate the antigenic peptides presented to the immune system on major histocompatibility complex (MHC) class II molecules. The presentation of these peptides and the expression of MHC class II molecules by macrophages and lymphocytes are stimulated by gamma-interferon (gamma-IFN). We found that treatment of human U-937 monocytes with gamma-IFN increased the activities and the content of the two major lysosomal cysteine proteinases, cathepsins B and L. Assays of protease activity, enzyme-linked immunosorbant assays (ELISA) and immunoblotting showed that this cytokine increased the amount of cathepsin B 5-fold and cathepsin L 3-fold in the lysosomal fraction. By contrast, the aspartic proteinase, cathepsin D, in this fraction was not significantly altered by gamma-IFN treatment. An induction of cathepsins B and L was also observed in mouse macrophages, but not in HeLa cells. These results suggest coordinate regulation in monocytes of the expression of cathepsins B and L and MHC class II molecules. Presumably, this induction of cysteine proteases contributes to the enhancement of antigen presentation by gamma-IFN.

Paper title : Inherited human IFN-γ deficiency underlies mycobacterial disease.

Doi : https://doi.org/10.1172/JCI135460

Abstract : Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients' herpesvirus Saimiri-immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

Paper title : Cloning and expression of a novel variant of human interferon-gamma cDNA.

Doi : https://doi.org/10.1093/oxfordjournals.jbchem.a135039

Abstract : A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.

Paper title : Arsenic enhances the apoptosis induced by interferon gamma: key role of IRF-1.

Doi : https://doi.org/Not available

Abstract : Interferons (IFNs) and arsenic trioxide (As2O3) are known inhibitors of cell proliferation and have been used in the treatment of certain forms of malignancy. IFNgamma treatment of cells leads to tyrosine phosphorylation of STAT1 followed by dimerization that accumulates in the nucleus. This is followed by DNA binding, activation of target gene transcription, dephosphorylation, and return to the cytoplasm. We have shown earlier that IFNgamma and As2O3 act synergistically in acute promyelocytic leukemia cells to upregulate IRF-1 expression and to induce apoptosis. Here, we show that in the human fibrosarcoma cell line 2fTGH, As2O3 prolongs IFNgamma-induced STAT1 phosphorylation resulting in persistent binding of STAT1 to GAS motif leading to an increase in IRF-1 expression which correlated with both higher anti-proliferative effect and increased apoptosis. These biological responses induced by IFNgamma alone or in combination with As2O3 were abolished when IRF-1 expression was down-regulated by RNA interference, thus demonstrating the key role of IRF-1.

Paper title : 1H, 13C, and 15N NMR backbone assignments and secondary structure of human interferon-gamma.

Doi : https://doi.org/10.1021/bi00150a009

Abstract : 1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.

Paper title : Studies on the sugar chains of interferon-gamma from human peripheral-blood lymphocytes.

Doi : https://doi.org/10.1093/oxfordjournals.jbchem.a122762

Abstract : Sugar chains of interferon-gamma (IFN-gamma) from human peripheral-blood lymphocytes (PBL) were liberated by hydrazinolysis. After N-acetylation, the reducing end residues of the sugar chains were tagged with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and reversed-phase HPLC. Five major PA-sugar chains were obtained. The structure of each PA-sugar chain was estimated by comparing its elution times on anion exchange, reversed-phase, and size-fractionation HPLC with those of PA-sugar chains of IFN-gamma from the human myelomonocyte cell line HBL-38 (Yamamoto, S. et al. (1989) J. Biochem. 105, 547-555) as standards, and also by comparison of their elution times after partial desialylation. The results showed that IFN-gamma (PBL) contained mono- and disialo-biantennary structures with 0 or 1 mol of fucose residue, as found for IFN-gamma (HBL-38), but the N-acetylneuraminyl alpha 2-6 linkage was dominant in IFN-gamma (PBL), unlike IFN-gamma (HBL-38), which contains both N-acetylneuraminyl alpha 2-3 and alpha 2-6 linkages.

Paper title : Crystal structure of a complex between interferon-gamma and its soluble high-affinity receptor.

Doi : https://doi.org/10.1038/376230a0

Abstract : The crystal structure of interferon-gamma bound to the extracellular fragment of its high-affinity cell-surface receptor reveals the first view of a class-2 cytokine receptor-ligand complex. In the complex, one interferon-gamma homodimer binds two receptor molecules. Unlike the class-1 growth hormone receptor complex, the two interferon-gamma receptors do not interact with one another and are separated by 27 A. Upon receptor binding, the flexible AB loop of interferon-gamma undergoes a conformational change that includes the formation of a 3(10) helix.

Paper title : Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing.

Doi : https://doi.org/10.1016/0014-5793(94)80612-8

Abstract : Proteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-gamma) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-gamma appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens.

Paper title : Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Doi : https://doi.org/10.1093/nar/10.8.2487

Abstract : Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.

Paper title : Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma.

Doi : https://doi.org/10.1084/jem.183.4.1807

Abstract : Interferon (IFN) gamma induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair of proteasome subunits expressed reciprocally in response to IFN-gamma. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-gamma, showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-gamma. Thus, IFN-gamma induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, beta-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-gamma may be responsible for acquisition of its functional change.

Paper title : Interferon-gamma induces receptor dimerization in solution and on cells.

Doi : https://doi.org/Not available

Abstract : The extracellular domain (ECD) of the human interferon-gamma (IFN gamma) receptor was stably expressed in Chinese hamster ovary cells and purified to homogeneity. Scatchard analysis of 125I-IFN gamma binding to ECD preparations revealed the formation of a ligand-receptor complex which displayed a Ka of 6.4 +/- 0.9 x 10(8) M-1. Two types of complexes were identified by sucrose density gradient ultracentrifugation. The stoichiometry of the major ECD-ligand complex was determined by high performance liquid chromatography gel filtration. When IFN gamma was incubated with a 2-fold molar excess of ECD, a 190-kDa complex was isolated that contained 2 mol of ECD per 1 mol of IFN gamma. IFN gamma also induced dimerization of IFN gamma receptors expressed at the cell surface as detected by chemically cross-linking receptor bound ligand and analyzing cell lysates by SDS-polyacrylamide gel electrophoresis and immunoblotting. Finally, labeled forms of ECD bound to cells preincubated at 4 degrees C with excess amounts of IFN gamma indicating that the ligand could associate with more than one receptor molecule in the absence of chemical cross-linking agents. These results demonstrate that IFN gamma effects dimerization of its receptor under physiologic conditions and suggest that IFN gamma receptor dimerization may be an important event in inducing IFN gamma-dependent biologic responses.

Paper title : Structural characterization of human interferon gamma. Heterogeneity of the carboxyl terminus.

Doi : https://doi.org/10.1111/j.1432-1033.1987.tb13494.x

Abstract : Natural human interferon gamma(IFN-gamma) was purified from the conditioned medium of peripheral blood leukocytes using selective silica gel adsorption and antibody-affinity chromatography. SDS-PAGE and Western blot analysis demonstrated three major species with molecular masses of 25 kDa, 20 kDa and 17 kDa. Structural analysis of this natural IFN-gamma preparation demonstrated a pyroglutamate residue at the amino terminus and a heterogeneous carboxyl terminus. The longest and most predominant polypeptide was 138 amino acids in length, which is five residues shorter than the sequence predicted from the cDNA. The presence of multiple-carboxyl-terminal forms indicated possible proteolytic processing during induction or protein purification. Limited proteolytic digestion of full-length recombinant IFN-gamma with endoproteinase Lys-C and trypsin revealed that the carboxyl-terminal 15 residues could be released under conditions in which the core portion of the polypeptide chain remained intact. Thus, the heterogeneity of natural IFN-gamma may be explained by partial proteolytic degradation of the molecule and differences in the degree of glycosylation as previously reported [Rinderknecht, E., O'Conner, B. H. & Rodriguez, H. (1984) J. Biol. Chem. 259, 6790-6797].

Paper title : Three-dimensional structure of recombinant human interferon-gamma.

Doi : https://doi.org/10.1126/science.1902591

Abstract : The x-ray crystal structure of recombinant human interferon-gamma has been determined with the use of multiple-isomorphous-replacement techniques. Interferon-gamma, which is dimeric in solution, crystallizes with two dimers related by a noncrystallographic twofold axis in the asymmetric unit. The protein is primarily alpha helical, with six helices in each subunit that comprise approximately 62 percent of the structure; there is no beta sheet. The dimeric structure of human interferon-gamma is stabilized by the intertwining of helices across the subunit interface with multiple intersubunit interactions.