Nuclear Transport And Its Effect On Breast Cancer Tumor Cells
pax8b
1. Homology Modeling of theHomology Modeling of the
human PAX8 Protein andhuman PAX8 Protein and
mechanisms for sequencemechanisms for sequence
specific DNA recognitionspecific DNA recognition
Abhishek DabralAbhishek Dabral
School of Biology,School of Biology,
Georgia Institute of TechnologyGeorgia Institute of Technology
2. What is PAX?What is PAX?
The PAX gene family encodes a group ofThe PAX gene family encodes a group of
transcription factors that have been conservedtranscription factors that have been conserved
through millions of years of evolution and playthrough millions of years of evolution and play
roles in early development.roles in early development.
Pax proteins are transcriptional regulators thatPax proteins are transcriptional regulators that
have critical roles in mammalian development, thehave critical roles in mammalian development, the
mutations of PAX genes cause profoundmutations of PAX genes cause profound
developmental defects.developmental defects.
3.
4. PAX OrganizationPAX Organization
► All PAX proteins have aAll PAX proteins have a paired domain (PD)paired domain (PD),,
which spans 128 amino acids near the N-terminuswhich spans 128 amino acids near the N-terminus
and consists of two helix-turn-helix (HTH) motifs.and consists of two helix-turn-helix (HTH) motifs.
► Sequence conservation among PAX proteins isSequence conservation among PAX proteins is
highest in the paired domainhighest in the paired domain but can also bebut can also be
extended to a paired-typeextended to a paired-type homeodomain (HD)homeodomain (HD) andand
to a stretch of residues between paired domainto a stretch of residues between paired domain
and homeodomain calledand homeodomain called octapeptide (OP)octapeptide (OP)..
5. PAX StructurePAX Structure
► PD is composed of amino andPD is composed of amino and
carboxy terminal subdomainscarboxy terminal subdomains
each of which are made up of 3each of which are made up of 3
alpha helices resembling thealpha helices resembling the
HTH (helix-turn-helix) motifHTH (helix-turn-helix) motif
found in all HD.found in all HD.
► Third helix of PD and HDThird helix of PD and HD
proteins interacts with the majorproteins interacts with the major
groove of the DNA.groove of the DNA.
► PDs have the ability to not adoptPDs have the ability to not adopt
a fixed structure unless it isa fixed structure unless it is
bound to DNA, this lends it abound to DNA, this lends it a
great diversity as a protein.great diversity as a protein.
6. In mammals,In mammals, 99 PAXPAX genes have been identified.genes have been identified.
PAX genes divided intoPAX genes divided into 4 subgroups4 subgroups based on:based on:
► Genomic StructureGenomic Structure
► Sequence SimilaritySequence Similarity
► Conserved FunctionConserved Function
PAX SubgroupsPAX Subgroups
7. PAX 8 is the only member of the family expressed in the
thyroid tissue.
PAX 8 cooperates with TTF1 (Thyroid Transcription Factor 1) to
influence thyroid specific gene regulation.
Pax8 is extremely important for the correct development of the thyroid gland
because inactivation of the Pax8 gene causes absence of follicular cells,
and
therefore absence of thyroid hormone .
PAX 8 co-expresses with Wilms’ tumor gene (WT1) during
kidney development suggesting a possible interaction.
The PAX FamiliesThe PAX Families
8. Splice Variants in PAX 8Splice Variants in PAX 8
Alternative splicing in PAX gene by inclusionAlternative splicing in PAX gene by inclusion
or exclusion of exons 7 and/or 8 hasor exclusion of exons 7 and/or 8 has
produced several known products but theproduced several known products but the
biological significance of the variants isbiological significance of the variants is
unknown.unknown.
The human PAX8 gene generates at leastThe human PAX8 gene generates at least
five different alternatively spliced transcriptsfive different alternatively spliced transcripts
encoding different PAX8 isoforms.encoding different PAX8 isoforms.
10. What questions could a PAX 8What questions could a PAX 8
model answer?model answer?
Better understanding of :Better understanding of :
Paired Domain-DNA interactionPaired Domain-DNA interaction––
► Biological function of PDBiological function of PD
Function of N and C subdomainsFunction of N and C subdomains ––
► Specific DNA contacts made by themSpecific DNA contacts made by them
► Do they cooperate with each other, does one affect theDo they cooperate with each other, does one affect the
function of the other and how?function of the other and how?
Effects of mutationsEffects of mutations
► Relation to the abnormal phenotypeRelation to the abnormal phenotype
11. Why Homology modeling?Why Homology modeling?
►No solved X- Ray structureNo solved X- Ray structure for ourfor our
Target protein ie. PAX 8Target protein ie. PAX 8
Moreover:Moreover:
►X-Ray structure is both time consumingX-Ray structure is both time consuming
and expensiveand expensive
►Only a small number of proteins can beOnly a small number of proteins can be
made to form crystals and crystal ismade to form crystals and crystal is
not the protein’s native state.not the protein’s native state.
12. Why Homology modeling?Why Homology modeling?
►No solved NMR structureNo solved NMR structure for ourfor our
Target protein ie. PAX 8Target protein ie. PAX 8
Moreover:Moreover:
►NMR does not work too well for proteinNMR does not work too well for protein
complexes.complexes.
►Very time consumingVery time consuming
13. Obtain Target Sequence
Get Information about
Target Protein
Template Selection
(Crystal Structures)
Initial Model
Validate
Model
Sequence Database (Genbank)
WHAT IF,
PROCHECK,
3D JIGSAW,
Esypred,
SWISS Model,
FUGUE
RAMPAGE
MODELER TOOLBOX
Blastp, CDD
BLAST PDB database
Clean PDB files
Create alignment of target
with template sequences
(Convert aln to ali)
MODELER
CLUSTALW
Steps for HomologySteps for Homology
ModelingModeling
14. The Template StructureThe Template Structure
PAX6PAX6
5822580|pdb|6PAX|A5822580|pdb|6PAX|A Chain A,Chain A, Crystal Structure Of The HumanCrystal Structure Of The Human
Pax-6 Paired Domain-Dna Complex Reveals A GeneralPax-6 Paired Domain-Dna Complex Reveals A General
Model For Pax Protein-Dna InteractionsModel For Pax Protein-Dna Interactions
Length = 133Length = 133
Score = 198 bits (503),Score = 198 bits (503),
Expect = 3e-52Expect = 3e-52
Identities = 92/123 (74%),Identities = 92/123 (74%),
Positives = 107/123 (86%)Positives = 107/123 (86%)
Query: 10Query: 10
HGGLNQLGGAFVNGRPLPEVVRQRIVDLAHQGVRPCDISRQLHGGLNQLGGAFVNGRPLPEVVRQRIVDLAHQGVRPCDISRQL
RVSHGCVSKILGRYYETG 69 H G+NQLGG FVNGRPLP+RVSHGCVSKILGRYYETG 69 H G+NQLGG FVNGRPLP+
RQRIV+LAH G RPCDISR L+VS+GCVSKILGRYY TG Sbjct: 2RQRIV+LAH G RPCDISR L+VS+GCVSKILGRYY TG Sbjct: 2
HSGVNQLGGVFVNGRPLPDSTRQRIVELAHSGARPCDISRILQHSGVNQLGGVFVNGRPLPDSTRQRIVELAHSGARPCDISRILQ
VSNGCVSKILGRYYATG 61 Query: 70VSNGCVSKILGRYYATG 61 Query: 70
SIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRDRLLASIRPGVIGGSKPKVATPKVVEKIGDYKRQNPTMFAWEIRDRLLA
EGVCDNDTVPSVSSIN 129 SIRP IGGSKP+VATP+VV KI YK++EGVCDNDTVPSVSSIN 129 SIRP IGGSKP+VATP+VV KI YK++
P++FAWEIRDRLL+EGVC ND +PSVSSIN Sbjct: 62P++FAWEIRDRLL+EGVC ND +PSVSSIN Sbjct: 62
SIRPRAIGGSKPRVATPEVVSKIAQYKQECPSIFAWEIRDRLLSESIRPRAIGGSKPRVATPEVVSKIAQYKQECPSIFAWEIRDRLLSE
GVCTNDNIPSVSSIN 121 Query: 130 RII 132 R++ Sbjct: 122GVCTNDNIPSVSSIN 121 Query: 130 RII 132 R++ Sbjct: 122
RVL 124RVL 124
19. ValidationValidation
1. Swiss Model(http:swissmodel.expasy.org)1. Swiss Model(http:swissmodel.expasy.org)
WhatCheck Report generated for your SWISS MODEL request :WhatCheck Report generated for your SWISS MODEL request :
► No errors in amino acid nomenclatureNo errors in amino acid nomenclature
► Improper Dihedral angle distribution OK —Improper Dihedral angle distribution OK —
The RMS Z-score for all improper dihedrals in the structure is within normal range.The RMS Z-score for all improper dihedrals in the structure is within normal range.
► Normal bond angle variability.Normal bond angle variability.
► A few residues had abnormal backbone torsion angles.A few residues had abnormal backbone torsion angles.
► A few pair of atoms had abnormally short interatomic distances.A few pair of atoms had abnormally short interatomic distances.
Overall the model conforms to the common refinement constraintsOverall the model conforms to the common refinement constraints
21. Main Chain-Side Chain ContactsMain Chain-Side Chain Contacts
Source:Source: MolProbity, an interactive macromolecular structure validation tool provided by the RichardsonMolProbity, an interactive macromolecular structure validation tool provided by the Richardson
laboratory, Duke University.laboratory, Duke University.
22. LimitationsLimitations
► Could not model the entire protein due to lack ofCould not model the entire protein due to lack of
homologous structures and extensive loop regionhomologous structures and extensive loop region
which is tough to model.which is tough to model.
► The paired box region may undergo someThe paired box region may undergo some
structural changes in the presence of the partialstructural changes in the presence of the partial
homeodomain (cooperativity in DNA binding).homeodomain (cooperativity in DNA binding).
► The DNA contacts made by the model may differThe DNA contacts made by the model may differ
from the template due to presence of other non-from the template due to presence of other non-
identical residues.identical residues.
28. 6.6.
Simon C. Lovell, Ian W. Davis, W.Simon C. Lovell, Ian W. Davis, W.
Bryan Arendall III, Paul I. W. deBryan Arendall III, Paul I. W. de
Bakker, J. Michael Word, Michael G.Bakker, J. Michael Word, Michael G.
Prisant, Jane S. Richardson, David C.Prisant, Jane S. Richardson, David C.
Richardson (2003)Richardson (2003)
Structure validation by C-alpha geometry: phi,Structure validation by C-alpha geometry: phi,
psipsi, and C-beta deviation., and C-beta deviation. Proteins:Proteins:
Structure, Function, and Genetics.Structure, Function, and Genetics. 5050::
437-450.437-450.
7.
