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URN: urn:nbn:de:bsz:25-opus-68257
URL: http://www.freidok.uni-freiburg.de/volltexte/6825/


Mattay, Dinah

Targeting the coiled-coil domain of the leukemia-relevant protein AF10

Angriff auf die Coiled-Coil Domäne des Leukämie-relevanten Proteins AF10

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Kurzfassung in Englisch

Every cellular process depends on a functioning network of specific protein-protein interactions, and deregulation is associated with numerous diseases. One of the major causes of human cancers is chromosomal translocation. Through non-random chromosomal translocation, the C-terminal part of the transcription factor AF10 becomes fused to the Nterminal part of MLL or CALM and subsequently causes acute myeloid and lymphoblastic leukemia. The MLL-AF10 fusion, which results from the t(10;11)(p12;q23) translocation, is found mainly in acute myeloid leukemia. MLL has been described as the fusion partner of more than 40 other genes in various types of leukemia. CALM-AF10 is found in several subtypes of leukemia and in lymphoma, resulting from the t(10;11)(p13;q14-21) translocation. Interestingly, in all cases the coiled-coil domain of AF10 was found to play a major role.
The coiled-coil motif is one of the most abundant protein interaction domains. It consists of two or more alpha-helices, built from repeating heptads, wrapped around each other in a lefthanded super coil. Coiled coils occur in virtually every physiological system because of their ability to function specifically in a fine-tuned network of homo- and heterotypic interactions.
The coiled-coil domain of AF10 is also responsible for binding to the gene regulator proteins GAS41 and hDOT1L, and little is known about these interactions to date. For hDOT1L a model exists, in which it was shown that a defective hDOT1L mutant could prevent recruitment of the wild-type hDOT1L and so leading to inhibition of specific gene overexpression and leukemogenesis
In this work, the wild-type interaction of AF10 and GAS41 should be investigated further, and an inhibitor for this interaction should be found. Theoretical considerations and circular dichroism measurements were used to characterize the interaction of AF10 and GAS41 and map the interacting region to the C-terminal part of the GAS41 helix. The Tm values of AF10 were highest in complex with the last register of GAS41, GAS41reg4, which is located at the
C-terminus of the GAS41 helix.
In addition, targeting the AF10 coiled-coil domain should inhibit inappropriate or adventitious protein interactions. In a semi-rational approach based on maximizing charge attractions and minimizing repulsions as well as steric hindrance, a genetic library was generated, using the coiled-coil domain of GAS41 as a scaffold. Through phage display, a peptide, named “anti- AF10”, was selected, that binds AF10 with a nine-fold higher affinity than the natural interaction partner GAS41, which was shown in phage ELISA experiments. The interaction and the inhibition potential was characterized qualitatively and quantitatively by phage ELISA, circular dichroism, surface plasmon resonance and FRET measurements, as well as PCA growth assays in bacteria and cross-linking experiments. In addition, it was found by an electrophoretic mobility shift assay that the helices are able to interact both in a parallel and in
an anti-parallel manner.
Furthermore, the stability of the anti-AF10/AF10 complex was improved by mutating the outer positions of the coiled coil using phage display selections of three different libraries. The results were analyzed statistically by phage ELISA and by surface plasmon resonance measurements. The outcome of these experiments enhanced our understanding of proteinprotein interaction principles.
In a further approach, two cell lines which both express the fusion protein CALM-AF10, DG-75 and U-937, were transfected with anti-AF10. After characterizing the cell lines by microscopy, Western blot and real-time PCR, different transfection methods were tested and the reaction conditions were optimized. First investigations concerning the effect of anti- AF10 on gene expression in the cells were performed by real-time PCR. Until now, no effect of anti-AF10 on gene expression was shown by investigating the RNA of the two cell lines by real-time PCR.
Natural proteins represent only a small fraction of possible protein sequences available and are not always perfectly optimized. Protein design strategies by introducing mutations into a natural protein were used to improve its stability and binding affinity to a target protein. In future, interfering peptides recognizing and blocking specific targets might become important in diagnostics and therapeutics. On the one hand, this work represents a first step in argeting the leukemia relevant protein AF10, while on the other hand, it inhances our understanding of protein-protein interaction principles.


Kurzfassung in Englisch

Every cellular process depends on a functioning network of specific protein- rotein interactions, and deregulation is associated with numerous diseases. One of the major causes of human cancers is chromosomal translocation. Through non-random chromosomal translocation, the C-terminal part of the transcription factor AF10 becomes fused to the Nterminal part of MLL or CALM and subsequently causes acute myeloid and lymphoblastic leukemia. The MLL-AF10 fusion, which results from the t(10;11)(p12;q23) translocation, is found mainly in acute yeloid leukemia. MLL has been described as the fusion partner of more than 40 ther genes in various types of leukemia. CALM-AF10 is found in several subtypes of leukemia and in lymphoma, resulting from the t(10;11)(p13;q14-21) translocation. Interestingly, in all cases the coiled-coil domain of AF10 was found to play a major role.
The coiled-coil motif is one of the most abundant protein interaction domains. It consists of two or more alpha-helices, built from repeating heptads, wrapped around each other in a lefthanded super coil. Coiled coils occur in virtually every physiological system because of their ability to function specifically in a fine-tuned network of homo- and heterotypic interactions.
The coiled-coil domain of AF10 is also responsible for binding to the gene regulator proteins GAS41 and hDOT1L, and little is known about these interactions to date. For hDOT1L a model exists, in which it was shown that a defective hDOT1L mutant could prevent recruitment of the wild-type hDOT1L and so leading to inhibition of specific gene overexpression and leukemogenesis
In this work, the wild-type interaction of AF10 and GAS41 should be investigated further, and an inhibitor for this interaction should be found. Theoretical considerations and circular dichroism measurements were used to characterize the interaction of AF10 and GAS41 and map the interacting region to the C-terminal part of the GAS41 helix. The Tm values of AF10 were highest in complex with the last register of GAS41, GAS41reg4, which is located at the
C-terminus of the GAS41 helix.
In addition, targeting the AF10 coiled-coil domain should inhibit inappropriate or adventitious protein interactions. In a semi-rational approach based on maximizing charge attractions and minimizing repulsions as well as steric hindrance, a genetic library was generated, using the coiled-coil domain of GAS41 as a scaffold. Through phage display, a peptide, named “anti- AF10”, was selected, that binds AF10 with a nine-fold higher affinity than the natural
interaction partner GAS41, which was shown in phage ELISA experiments. The interaction and the inhibition potential was characterized qualitatively and quantitatively by phage ELISA, circular dichroism, surface plasmon resonance and FRET measurements, as well as PCA growth assays in bacteria and cross-linking experiments. In addition, it was found by an electrophoretic mobility shift assay that the helices are able to interact both in a parallel and in
an anti-parallel manner.
Furthermore, the stability of the anti-AF10/AF10 complex was improved by mutating the outer positions of the coiled coil using phage display selections of three different libraries.
The results were analyzed statistically by phage ELISA and by surface plasmon resonance measurements. The outcome of these experiments enhanced our understanding of proteinprotein interaction principles.
In a further approach, two cell lines which both express the fusion protein CALM-AF10, DG-75 and U-937, were transfected with anti-AF10. After characterizing the cell lines by microscopy, Western blot and real-time PCR, different transfection methods were tested and the reaction conditions were optimized. First investigations concerning the effect of anti- AF10 on gene expression in the cells were performed by real-time PCR. Until now, no effect of anti-AF10 on gene expression was shown by investigating the RNA of the two cell lines by real-time PCR.
Natural proteins represent only a small fraction of possible protein sequences available and are not always perfectly optimized. Protein design strategies by introducing mutations into a natural protein were used to improve its stability and binding affinity to a target protein. In future, interfering peptides recognizing and blocking specific targets might become important in diagnostics and therapeutics. On the one hand, this work represents a first step in targeting the leukemia relevant protein AF10, while on the other hand, it enhances our understanding of protein-protein interaction principles.


SWD-Schlagwörter: Coiled coil , Akute myeloische Leukämie , Phagen-Display-Bibliothek , Proteindesign
Freie Schlagwörter (englisch): leukemia , phage display , library , protein design
Institut: Institut für Biologie 3
Fakultät: Fakultät für Biologie
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Dissertation
Erstgutachter: Arndt, Katja M. (Dr.)
Sprache: Englisch
Tag der mündlichen Prüfung: 30.07.2009
Erstellungsjahr: 2009
Publikationsdatum: 04.09.2009
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