Protein-Protein Interaction Between Wilms

1
Protein-Protein Interaction Between Wilms Tumor
1(WT1) and Nuclear Receptors

• Sandra Franco1, Kwang-Hoon Song Ph.D1

2
Background Information

• What are nuclear receptors (NR)?1

• Homeostasis
• Differentiation
• Embryonic Development
• Organ physiology

http//fig.cox.miami.edu/cmallery/150/memb/c11x10
hormone-receptors2.jpg
3

• Nuclear receptor structure 2

A/B Modulator
C DBD
D Hinge
E LBD
F
a
Ligand and coactivator Binding pockets
CoR-box
Zn CTE
AF-2
AF-1
DNA binding
AF-1 and 2 Transcriptional activation functions.
DBD and LBD Recognize specific DNA sequences
and ligands, respectively
4

• Orphan Nuclear Receptors1

No defined ligand (hormone)
 
 
 
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• Wilms Tumor 1 (WT1) Gene2
• WT1 gene is expressed during mammalian embryonic
  development in many
• tissues, and is fundamental during the
  formation of the gonads.
• Research has demonstrated that the interaction
  between SF-1 orphan nuclear receptor and WT1
  (specifically KTS isoforms) regulate and promote
  the expression of the hormone Mullerian
  Inhibiting Substance (MIS) in males.

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• Structure of WT13

KTS Lys-Thr-Ser
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Mullerian Inhibiting Substance (MIS)
Mullerian Inhibiting Substance is secreted by the
testes and the expression of MIS causes
regression of the Mullerian Duct in the
developing male embryo.
http//www.ansi.okstate.edu/course/3443/study/Note
s/sexdeter/
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Diseases
Mutations in WT1 are associated with diseases
such as
Denys-Drash syndrome (DDS), which is a type of
male pseudohermaphroditism
Kidney tumors
Fraser syndrome
WAGR (Wilms tumor, Aniridia, Genitourinary
abnormalities, and mental Retardation)
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Purpose

• The purpose of this research was to observe if
  any protein interaction occurred between the wild
  type WT1 and various orphan nuclear receptors
  (NUR77, SHP, Dax-1, ERR?, CAR, Beta 2, CRIF, and
  HNF, SF-1 ).
• In addition, we investigated the protein-protein
  interaction between a mutant WT1 isoform and
  various orphan nuclear receptors (SF-1, Nur77,
  and Beta-2).

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Methodology Yeast Two-Hybrid Technique
X protein of interest (nuclear receptor)
Y potential binding protein (Wilms Tumor 1)
X (nuclear receptor) Y (WT1) Transcriptional
activator
http//www.bioteach.ubc.ca/molecularbiology/ayeast
twohybridassay

• BD DNA-binding domain Binds to DNA
• AD Activation domain Activates transcription
  of DNA
• Transcriptional Activator Transcribes a
  reporter gene which is a gene whose
• protein product can be measured.

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How to make the construct?
Or yeast
BD DNA binding domain
Transcribed DNA
Or hunter

• Construct the bait and hunter
• bait nuclear receptor
• hunter Wilms Tumor 1

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• Two plasmid constructs
• The bait (X), which is the protein of interest
  (nuclear receptor) fused to a GAL4 binding domain
  (BD)
• The hunter (Y), which is the potential binding
  partner (Wilms Tumor 1) fused to a GAL4
  activation domain (AD)

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• Constructs are introduced
• into a host yeast cell by
• transfection.

During transfection, the outer- membrane of a
yeast cell is treated. This produces holes so
that the plasmid can cross the membrane and
enter the cell.

• After several days, they are plated on a media.
  Only cells with both
• plasmids will survive.

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Blue colonies indicates a positive
protein-protein interaction
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Results
B42
Result (24h/48h)
-/- -/- -/- -/- -/- -/- -/- -/- -/-
Only Nur77 SF-1 mCAR ERRr SHP DAX-1 TRR dHAND
Lex A-WT1KTS
(Lys-Thr-Ser)
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B42
Result (24h/48h)
-/- / / -/ -/- -/ -/ -/ -/ /
Only Nur77 SF-1 mCAR ERRr SHP DAX-1 CRIF HNF4 BETA
2
Lex A-WT1-KTS
(Lys-Thr-Ser)
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B42
Result (24h/48h)
Only Nur77 SF-1 BETA2
-/- -/- / -/-
Lex A-WT1-LLAA
(Leu-Leu-Ala-Ala)
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Discussion

• The result of the yeast two-hybrid system
  demonstrated a positive interaction between the
  wild type WT1-KTS and orphan nuclear receptors
  SF-1, Nur77, and Beta-2.
• Furthermore, when the protocol was replicated
  using the mutant WT1 isoform and different
  nuclear receptors our results indicated a
  positive interaction between LLAA and orphan
  nuclear receptor SF-1.

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Conclusion

• Our results are very primitive and are believed
  to be the foundation of the basic understanding
  of the protein-protein interaction between WT1
  and orphan nuclear receptors and the role they
  play in embryonic development and disease.
  Further research
• should include and not be limited to the
  following
• Find the physiological function between orphan
  nuclear receptor SF-1 and the mutant form of WT1
  LLAA as well as wild type WT1 and orphan
• nuclear receptors Beta-2 and Nur77.
• Determine the structural location of the
  protein-protein interaction of the
  aforementioned.
• Determine the physiological location of the
  protein-protein interaction in vivo of the
  aforesaid (testis, ovary, and/or kidney) by using
  mammalian Two-Hybrid Technique.

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References

• Giguere V. 1999. Orphan Nuclear Receptors
  From Gene to Function. Endocrine Reviews
  20(5) 689-725.
• Nachitigal W. M, Hirokawa, Y, VanHouten E. L. D,
  Flanagan N. J, Hammer D. G, Ingraham, A. H 1998.
  Wilms Tumor 1 and Dax-1 Modulate the Orphan
  Nuclear Receptor SF-1 in Sex-Specific Gene
  Expression. Cell 93 445-454.
• Tajida K, Carroll J, Roberts T.C. 1999.
  Regulation of Insulin-Like Growth Factor I
  Receptor Promoter Activity by Wild-Type and
  Mutant Versions of the WT1 Tumor Suppressor.
  Endocrinology 140 (10) 4713-4724.
• http//bioteach.ubc.ca/MolecularBiology/AYeastTwoH
  ybridAssay/
• http//fig.cox.miami.edu/cmallery/150/memb/c11x10
  hormone-receptors2.jpg
• http//www.ansi.okstate.edu/course/3443/study/Note
  s/sexdeter/
• http//www.gfmer.ch/Genetic_diseases/Developmental
  _genetic_diseases/Branchiootorenal_dysplasia.htm
• http//www.icomm.ca/geneinfo/ambgen.htm
• http//www.nlm.nih.gov/medlineplus/ency/imagepages
  /9056.htm