Transcript Determination

Determination of Sites of CYP1B1
Mutations in Aligned Sequences of
Cytochrome P450 Family Members
and 3D-Structural Model
by: Betsabeh Khoramian Tusi
EYE
• The human eye is filled with a liquid known
as the aqueous humor. The aqueous humor
is produced by the ciliary body, a stucture which
lies behind the iris.
• This liquid circulates in the chambers of the eye, then gets drained away via
a pathway which lies at the angle between the cornea and the iris (the
drainage angle).
• Anything that block this exit path will result in liquid accumulation and
produce high intraocular pressure (IOP), which, if left untreated, leads to
optic-nerve damage and ultimately blindness. This condition is called
Glaucoma.
Primary Congenital Glaucoma
(PCG)
• PCG is a form of glaucoma, manifested during the
neonatal or infantile period (prior to the age of 3).
• PCG is likely to be due to maldevelopment of the
anterior chamber angle of the eye, thus interfering
with the aqueous humor outflow.
• The incidence of this disease in the Middle East is
approximately four times greater than in Western
countries.
CYP1B1
• Three loci for Primary Congenital Glaucoma have been
identified through linkage analysis in affected families:
GLC3A, GLC3B, GLC3C.
(Locus is really ,an approximate address of a gene but we don’t know the exact gene)
• Only the gene associated with GLC3A has been found:
CYP1B1. It codes for the protein cytochrome P4501B1.
• Mutations in CYP1B1 account for approximately 1/3 of PCG
cases in populations studied (very variable!!).
Why do mutations in this gene cause
maldevelopment of the eye?
Really, we don’t know !!!
But we do know:
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CYP1B1 is expressed in many tissues.
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CYP1B1 is a member of a superfamily of genes (many related genes)
whose protein products are all oxygenases. Oxygenases have roles in
detoxification reactions.
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Mutations in CYP1B1occur in many cancers.
WE DO NOT KNOW WHY CYP1B1 DAMAGE SHOULD HAVE
SPECIFIC EFFECTS IN THE EYE!!!
CYP1B1 gene structure
• CYP1B1 has three exons,that only two of them are
translated in to protein.(exon no.2& the first part of
the exon no.3)
OUR RESEARCH
1.
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We have collected blood samples and isolated DNA from
100 Iranian PCG patients.
We sequenced the three exons and neighboring intronic
sequences of the gene in 50 (exon1) to 70 (exon 3) of the
patients.
We have identified 15 CYP1B1 mutations, five of which have
not been previously reported:
E173K, D291G, G329V, R368C, I399V.
We used bioinformatics tools and did protein homology
modeling of the CYP1B1 protein using information at
databases and located our mutations and other known
CYP1B1 mutations in derived 3D-structure.
• The cytochrome P450 superfamily in the
human genome has 22 members.
• The amino acid sequences of these 22
cytochromes were obtained from SwissProt and
Genebank.
• We did multiple sequence alignment using the
ClustalW program.
We determined the positions of
putative disease mutations
(including new mutations found in
the Iranian population) in the
multiple sequence alignment.
Mutation spectrum:
Only nucleotide substitution mutations were
analyzed.
i.e. deletions and insertions which cause
frameshifts and are likely to be damaging
wherever they occur, are not considered.
NEXT:
Construction of a three dimensional model
of CYP1B1 gene product by homology
modeling.
(homology modeling using the Swiss-Model Program)
Protocol for Modeling
1. The complete protein sequence of CYP1B1 was screened
against the PDB structure database in order to identify the
template structures appropriate for modeling.
2. From a series of templates, we selected those of four proteins
with the highest homology. The four proteins were:
1po5,1suo,1nr6 and 1n6b .(all of these proteins are kinds of
cytochromeP450)
3. We entered our cytochrome P450 amino acid sequence and
browsed the known 3D structures of the selected homologous
proteins, and then asked SWISS-Model to construct a 3Dmodel for cytochrome P450.
Results
Mutation Sites
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Cytochrome P450 proteins all have approximately 500
aa’s; CYP1B1 has 543 aa’s.
As the 22 cytochrome P450 proteins aligned are not very
closely related, only 25 sites were well conserved and
only 9 of these were strictly (100%) conserved.
The 9 strictly conserved residues all lie within a 122 aa
region.
12 of the 28 mutations analyzed lie within the highly
conserved 122 aa region; five of these mutations lie at or
adjacent to the strictly conserved positions (including one
of the new Iranian mutations).
Multiple sequence alignment for 22 different
members of the cytochrome P450 superfamily
LOGO
NB: Although the amino acids at many sites are not highly
conserved, but the chemical properties of amino acids at most sites
are well conserved e.g. hydrophobicity, hydrophilicity as shown here.
Three-dimensional model of the
CYP1B1 protein
See rotating model of 3-D structure
Site of mutations in 3D-model of CYP1B1 protein
NOTE:
1. Sites of 12 mutations in conserved sequence region
are within heme binding site.
2. Many of the remaining mutations are on part of
surface region of the protein, suggesting this region
has an important role----- perhaps interaction with
other proteins.
3. Many of our novel mutations lie within the surface
region.
• Secondary structure derived from 3-D model of
cytochrome p450 1B1 protein using DSSP
(Definition of secondary structure of proteins)
• Structural alignment
NB: Structural alignment shows that structural
elements such as alpha helices and beta strands are
more conserved than residue types and residue
properties.
The End
Thanks a lot for your attention