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

Nurmemmedov, Elmar LU (2007)
Abstract (Swedish)
Popular Abstract in Swedish

Den molekylära processen under vilken DNA-information förs över till RNA-information kallas transkription. Transkription styrs främst i samverkan med många speciella molekyler som kallas transkriptionsfaktorer. Zinkfinger proteiner är en av de vanligast förekommande klasserna av reglerande proteiner i eukaryota celler där de inverkar på en mängd av cellulära aktiviteter. Ett klassiskt zinkfinger är en liten peptid med en enkel beta-beta-alfa sekundärstruktur stabiliserad av en zinkjon, som i sin tur är koordinerad av två bevarade cystein och två histidin aminosyror.



Wilms tumör protein (WT1) är en transkriptionsfaktor, vilken i friska celler reglerar ett vidsträckt nätverk av... (More)
Popular Abstract in Swedish

Den molekylära processen under vilken DNA-information förs över till RNA-information kallas transkription. Transkription styrs främst i samverkan med många speciella molekyler som kallas transkriptionsfaktorer. Zinkfinger proteiner är en av de vanligast förekommande klasserna av reglerande proteiner i eukaryota celler där de inverkar på en mängd av cellulära aktiviteter. Ett klassiskt zinkfinger är en liten peptid med en enkel beta-beta-alfa sekundärstruktur stabiliserad av en zinkjon, som i sin tur är koordinerad av två bevarade cystein och två histidin aminosyror.



Wilms tumör protein (WT1) är en transkriptionsfaktor, vilken i friska celler reglerar ett vidsträckt nätverk av målgener under utvecklingen av njurar och det genitourinära systemet. Dess funktionsfell kan orsaka allvarliga abnormiteter såsom WAGR syndrome, Denys Drash syndrome och Frasier syndrome. WT1 har varit intressant för forskare som ett zinkfinger protein, och en transkriptionsfaktor som har båda onkogeniska och tumörsuppressor funktioner.



WT1 innehåller fyra C2H2-typ zinkfingerar och specifikt binder de till GC-rika sekvenser i målgenernas promotorer so då aktiveras eller de-aktiveras. Den har en dubbel funktion: det kan binda specifikt till DNA och RNA. Alternativ splicing av det omogna WT1 mRNA:t på exon 5 och exon 9 producerar 4 olika isoformer med 17 och 3 (KTS) aminosyrors insertion. KTS insertionen i länken mellan zinkfingrar 3 och 4 stör DNA-bindningen, medan mekanismen fär RNA bindning ännu är okänd. Följakligen, WT1 har två unika egenskaper som skiljar den från andra zinkfinger proteiner: KTS insertionen och en okonventionell aminosyrorasammansättning av dess zinkfinger 1.



Vi har gjort en interdisciplinär studie för att bättre förstå de nukleinsyra-bindande egenskaperna av WT1. I Paper I beskriver vi vår studie om hur vi uttrycker och renar WT1 proteinet, och hur vi karakteriserar dess biokemiska egenskaper. Paper II är en surface plasmon resonance-baserad studie där vi undersöker WT1:s DNA-bindande egenskaper, medan Paper III är en analog studie avseende dess RNA-bindande egenskaper. Till slut, i Paper IV använder vi so kallad Bacterial 1-Hybrid System för att förklara DNA-bindningens preferenserna av zink finger 1. Intressanta slutsatser om betydelsen av KTS insertionen, zinkfinger 1 och WT1:s överallt beteende är gjorda. (Less)
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Ward, Andrew, University of Bath, UK
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Naturvetenskap, Natural science, function, structure, transcription factor, cancer, affinity, kinetics, Biacore, RNA binding, DNA binding, Wilms tumour (WT1), zinc finger
publisher
Molecular Biophysics, Lund University
defense location
Kemicentrum, Lecture Hall B
defense date
2007-10-26 13:15
ISBN
978-91-7422-176-3
language
English
LU publication?
yes
id
a402d473-d315-4c3a-88a3-82af2046e2b0 (old id 599070)
date added to LUP
2007-11-13 08:44:34
date last changed
2016-09-19 08:45:07
@misc{a402d473-d315-4c3a-88a3-82af2046e2b0,
  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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.<br/><br>
<br/><br>
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.},
  author       = {Nurmemmedov, Elmar},
  isbn         = {978-91-7422-176-3},
  keyword      = {Naturvetenskap,Natural science,function,structure,transcription factor,cancer,affinity,kinetics,Biacore,RNA binding,DNA binding,Wilms tumour (WT1),zinc finger},
  language     = {eng},
  publisher    = {ARRAY(0xc4613c8)},
  title        = {Structural and Functional Studies of Wilms Tumour Protein - WT1; Novel Insights into Functionality of Zinc Finger Proteins},
  year         = {2007},
}