Determination of Sites of CYP1B1 Mutations in Aligned Sequences of Cytochrome P450 Family Members and 3D-Structural Model

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Determination of Sites of CYP1B1 Mutations in
Aligned Sequences of Cytochrome P450 Family
Members and 3D-Structural Model

• by Betsabeh Khoramian Tusi

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EYE
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• 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.

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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.

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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 dont 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!!).

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Why do mutations in this gene cause
maldevelopment of the eye?

• Really, we dont know !!!

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• But we do know
• CYP1B1 is expressed in many tissues.
• CYP1B1 is a member of a superfamily of genes
  (many related genes) whose protein products are
  all oxygenases. Oxygenases have roles in
  detoxification reactions.
• Mutations in CYP1B1occur in many cancers.
• WE DO NOT KNOW WHY CYP1B1 DAMAGE SHOULD HAVE
• SPECIFIC EFFECTS IN THE EYE!!!

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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)

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OUR RESEARCH

• 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.

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• 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.

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We determined the positions of putative disease
mutations (including new mutations found in the
Iranian population) in the multiple sequence
alignment.
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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.

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• NEXT
• Construction of a three dimensional model of
  CYP1B1 gene product by homology modeling.
• (homology modeling using the Swiss-Model
  Program)

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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 3D-model for
  cytochrome P450.

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Results
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Mutation Sites

• Cytochrome P450 proteins all have approximately
  500 aas CYP1B1 has 543 aas.
• 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).

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Multiple sequence alignment for 22 different
members of the cytochrome P450 superfamily
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(No Transcript)
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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.
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Three-dimensional model of the CYP1B1 protein
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• See rotating model of 3-D structure

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Site of mutations in 3D-model of CYP1B1 protein
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NOTE

• 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.

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• 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.

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The End
Thanks a lot for your attention