Transcript Analysis of Interleukin 12 - California State University

Analysis of Interleukin 12
Bioinformaticians:
Wednesday Sok
Joshua Corey
Harbindar Singh
Mandeep Singh
Protein Project
February 1, 2002
Interleukin 12
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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
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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 1980s.
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
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Activates other immune cells called T cells, B
cells, dendritic cells and natural killer cells
 promotes antibiody production by B cells and
stimulates dendritic cells to multiply
 shown to inhibit angiogenesis, the development of
new blood vessles within tumors
 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
IL 12 instead of IL 2
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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.
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.
Recent Research in Laboratory
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Suggests IL 12 can effectively treat a wide
variety of cancers with minimal toxicity.
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.
IL 12 effectively eliminates cancer in lab
animals that are given small doses of the
substance.
Laboratory Results
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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
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Protocol 1
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 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
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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
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Protein made up of 3 alpha chains, beta sheets,
and a side chain, the functional unit
 IL 12 contains an immunoglobulin C-2 type
domain and fibronectin type III domain( on beta)
 IL 12 is produced and secreted hormone- like to
activate other cells, inferring that it is involved in
intracellular signaling.
 It is similar to other cytokines, but most similar to
IL 6 and ciliary neuroptic factor receptor (CNER).
IL 12 Information
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IL 12 is a complex protein to separate for
the genes that codes for its two subunits
For Mice:
• alpha subunit found on chromosome 3
• beta subunit found on chromosome 11
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For Humans:
• alpha subunit found on chromosome 3
• beta subunit found on chromosome 5
3d Structure
http://www.rcsb.org/pdb/cgi/explore.cgi?job=graphics&
pdbId=1F45&page=0&pid=241861012580580
Mutant Versions of IL 12
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Alpha subunit p35
 1026 bp in length
 8 exons, 7 introns
 3 SNPs
• @ 634 bp A/G
• @ 799 bp T/C
• @ 807 bp T/C
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Beta subunit p40
2318 bp in length
8 exons, 7 introns
1 SNP @ 1188 bp
variation between A &
IL 12 Studied in Many Organisms
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Humans, mouse, horse, dog, sheep, cows,
pigs, cat, woodchuck, red deer, and sooty
mangabey and rhesus macaque(primates)
There is a substantial interest that exists for
the study of IL12, but it will take several
years for any therapy to become standard.
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
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There is a 70% homology between the
human and mouse p40 (beta chain) and 60%
homology between the p35 chains of IL 12.
Human IL 12 is inactive on mouse cells,
mouse IL 12 is active on human cells.
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
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Shows all species being in common, all are
vertebrates and mammals.
Rooted tree shows humans being closely
related to macmu and certo, and that the
human and mouse diverged from the same
species.
Unrooted tree illustrates same information.
Grease Analysis
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IL 12 appears to be mostly hydrophilic, with a small
hydrophobic region
Acknowledgments
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Tools used in biology workbench
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Ndjinn-for database searches
ClustalW-alignment of the protein sequences
Drawtree-unrooted phylogenetic tree
Drawgram-rooted phylogenetic tree
BlastN-compare nucleotide sequence to databases
DDB(protein databank)-viewing of structures
Non-Redundant Protein Databaes
BlastP-compare protein sequence to database
Protdist-comparing similarities of proteins
Texshade-graphical comparison of alignment
Boxshade
Acknowledgments
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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.
The End