Analysis of Interleukin 12 Bioinformaticians Wednesday Sok Joshua

Analysis of Interleukin 12 Bioinformaticians: Wednesday Sok Joshua Corey Harbindar Singh Mandeep Singh Protein Project February 1, 2002

Interleukin 12 w Interleukin 12, aka IL 12 is only one of many Interleukins present in the body. It is a more recent IL to be studied, therefore there is less information known on the protein. The newer information however could help us better understand ourselves and to find treatment for some genetic diseases.

Background of IL 12 w w w It is a cytokine, a hormone-like substance that regulates the activity of cells involved in an immune response. IL 12 is one of many naturally produced biological response modifiers (BRMs). It was first discovered in the late 1980 s. By 1991, two genes necessary for IL 12 production were identified and cloned. IL 12 is made by a select group of immune cells that normally are the first to encounter diseasecausing organisms in the body and react to them.

Known activities Activates other immune cells called T cells, B cells, dendritic cells and natural killer cells w promotes antibiody production by B cells and stimulates dendritic cells to multiply w shown to inhibit angiogenesis, the development of new blood vessles within tumors w immune cells seek and destroy antigens (cancer, tumor cells), IL 12 increases the production by these cells of interferon gamma, another BRM which augments the killing ability of immune cells w

IL 12 instead of IL 2 By activating specific immune cells early in development of an anti-cancer immune response, IL 12 is associated with less toxicity than IL 2, which is a BRM used in clinical trials for advanced cancer in the last 10 years. w IL 2 is normally released by T cells late in development of an immune response. IL 2 stimulates a strong and immediate immune response which is associated with side effects when administered to patients. w

Recent Research in Laboratory Suggests IL 12 can effectively treat a wide variety of cancers with minimal toxicity. w Investigations at Univ. of Pittsburg Cancer Institute (UPCI) and several other sites around the country now are exploring the use of IL 12 in clinical trials for patients with advanced cancer. w IL 12 effectively eliminates cancer in lab animals that are given small doses of the substance. w

Laboratory Results w Mice having advanced cancer eradicated with IL 12 don’t develop the same cancer again if they are artificially re-introduced into the body, which suggests their bodies have developed an immune memory of the cancers.

UPCI Developed 2 protocols w Protocol 1 w w IL 12 protein is injected into patients with advanced cancer; study accrued patients with different cancers, administered IL 12 in various doses to assess safety and max. tolerated dose of drug, in order to explore the IL 12 ability to fight cancer w Protocol 2 Another clinical trial began Aug 1995 involving the IL 12 gene transfer to patients with advanced cancer, to allow continuous production of cytokines inside the body. The IL 12 gene delivery system developed by investigators at the UPGenetics Inst is being produced at the UP Biotechoogy Center.

Structure Similarity of IL 12 to IL 6 Protein made up of 3 alpha chains, beta sheets, and a side chain, the functional unit w IL 12 contains an immunoglobulin C-2 type domain and fibronectin type III domain( on beta) w IL 12 is produced and secreted hormone- like to activate other cells, inferring that it is involved in intracellular signaling. w It is similar to other cytokines, but most similar to IL 6 and ciliary neuroptic factor receptor (CNER). w

IL 12 Information IL 12 is a complex protein to separate for the genes that codes for its two subunits w For Mice: w • alpha subunit found on chromosome 3 • beta subunit found on chromosome 11 w For Humans: • alpha subunit found on chromosome 3 • beta subunit found on chromosome 5

3 d Structure http: //www. rcsb. org/pdb/cgi/explore. cgi? job=graphics& pdb. Id=1 F 45&page=0&pid=241861012580580

Mutant Versions of IL 12 Alpha subunit p 35 w 1026 bp in length w 8 exons, 7 introns w 3 SNPs w • @ 634 bp A/G • @ 799 bp T/C • @ 807 bp T/C w w w Beta subunit p 40 2318 bp in length 8 exons, 7 introns 1 SNP @ 1188 bp variation between A &

IL 12 Studied in Many Organisms Humans, mouse, horse, dog, sheep, cows, pigs, cat, woodchuck, red deer, and sooty mangabey and rhesus macaque(primates) w There is a substantial interest that exists for the study of IL 12, but it will take several years for any therapy to become standard. w

Comparison of Mouse and Human protein sequences MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITW 60 MC Q+L ISWF++V L SPL+A+WEL+KDVYVVE+DW PDAPGE V LTCDTPEED ITW MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITW 60 TSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNF 120 T DQ V+GSGKTLTI VKEF DAGQYTCHKGGE LSHS LLLHKKE+GIWST+ILK+ TLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQ 120 ---KNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKV 177 KNKTFL+CEA NYSGRFTC WL + DL F++KSS S D + VTCG A+LSAE+V KEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERV 180 TLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPK 237 D ++YE YSV CQED CP AEE+LPIE+ ++A + KYENY++SFFIRDIIKPDPPK RGDNKEYE-YSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPK 239 NLQMKPLKNS-QVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLV 296 NLQ+KPLKNS QVEVSWEYPD+WSTPHSYFSL F V++Q K ++ K K NLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREK----KDRVFT 291 EKTSTEVQC-KGGNVCVQAQDRYYNSSCSKWACVPC 331 +KTS V C K ++ V+AQDRYY+SS S+WA VPC DKTSATVICRKNASISVRAQDRYYSSSWSEWASVPC 327

Homology Between Mouse and Human There is a 70% homology between the human and mouse p 40 (beta chain) and 60% homology between the p 35 chains of IL 12. w Human IL 12 is inactive on mouse cells, mouse IL 12 is active on human cells. w

The conserved regions on the alignment are color coded in light blue, the regions of non-conserved regions are coded in white, and the dark blue regions are all the regions where there is an exact match.

Phylogenetic Trees Shows all species being in common, all are vertebrates and mammals. w Rooted tree shows humans being closely related to macmu and certo, and that the human and mouse diverged from the same species. w Unrooted tree illustrates same information. w

Grease Analysis w IL 12 appears to be mostly hydrophilic, with a small hydrophobic region

Acknowledgments w Tools used in biology workbench • • • Ndjinn-for database searches Clustal. W-alignment of the protein sequences Drawtree-unrooted phylogenetic tree Drawgram-rooted phylogenetic tree Blast. N-compare nucleotide sequence to databases DDB(protein databank)-viewing of structures Non-Redundant Protein Databaes Blast. P-compare protein sequence to database Protdist-comparing similarities of proteins Texshade-graphical comparison of alignment Boxshade

Acknowledgments w w w Journal of Medical Virology 60: 264 -268 (2000) Clinical Immunology. Volume 98, Issue 1. January 2001, pages 119 -124. Journal of Clinical Investigation. Volume 108, Issue 12. December 2001, pages 1749 -1758. American Journal of Human Genetics. Volume 67, Issue 1. July 2000, pages 67 -81. International Immunology. Volume 13, Issue 5. May 2001, pages 685 -694. Journal of Immunology. Volume 166, Issue 6. March 2001, pages 3749 -3756.

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