Structural and Functional Studies of Wilms Tumour Protein - WT1; Novel Insights into Functionality of Zinc Finger Proteins

Elmar Nurmemmedov

Research output: ThesisDoctoral Thesis (compilation)

Abstract

The molecular process under which message in DNA is relayed onto RNA is called transcription, and it accomplished through involvement of so-called transcription factors. Zinc finger proteins are one of the most abundant classes of regulatory proteins in eukaryotic cells where they play central roles in a variety of cellular activities. A classical zinc finger is a small protein motif with a simple beta-beta-alpha secondary structure stabilized by a zinc ion that is coordinated by two conserved cysteines and two histidines.

Wilms tumour protein (WT1) is a transcription factor, which in normal cells regulates a vast network of genes during development of the kidney and the genitourinary system; its malfunction may lead to serious abnormalities such as WAGR syndrome, Denys Drash syndrome and Frasier syndrome. WT1 has been interesting for researchers as a zinc finger protein, and as a transcription factor having both oncogenic and tumor suppressor functions.

WT1 contains four C2H2-type zinc fingers and it specifically binds to GC-rich sequences in the promoter regions of its target genes, which are either up- or down-regulated. It has a dual functionality: it can bind specifically to DNA and RNA. Alternative splicing of the immature WT1 mRNA at exon 5 and exon 9 produces 4 distinct isoforms with 17 and 3 amino acids (KTS) insertions, respectively. The KTS insertion into the linker between zinc fingers 3 and 4 abrogates binding to the DNA. On the other hand, it differentially affects affinity of WT1 for its single-stranded RNA targets ? this is not yet understood. Thus, two unique features of WT1 differentiate it from other zinc finger proteins of the same class: the KTS insertion and an unconventional amino acid composition of its zinc finger 1.

We have undertaken an interdisciplinary approach in order to better understand the nucleic acid-binding features of WT1. In the Paper I we describe a study where we express, purify the WT1 protein and make its biochemical characterization. In Paper II a surface plasmon resonance-based study addresses the DNA-binding properties of WT1. Paper III describes an analogous study where RNA-binding properties of WT1 are investigated. Finally, Paper IV is a Bacterial 1-Hybrid System-based study that is directed towards elucidation of the DNA-binding preferences of zinc finger 1. Interesting conclusions about the functionality of the KTS insertion and the zinc finger 1 are reached.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • Thunnissen, Marjolein, Supervisor
Award date2007 Oct 26
Publisher
ISBN (Print)978-91-7422-176-3
Publication statusPublished - 2007

Bibliographical note

Defence details

Date: 2007-10-26
Time: 13:15
Place: Kemicentrum, Lecture Hall B

External reviewer(s)

Name: Ward, Andrew
Title: Professor
Affiliation: University of Bath, UK

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Subject classification (UKÄ)

  • Biological Sciences

Free keywords

  • Naturvetenskap
  • Natural science
  • function
  • structure
  • transcription factor
  • cancer
  • affinity
  • kinetics
  • Biacore
  • RNA binding
  • DNA binding
  • Wilms tumour (WT1)
  • zinc finger

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