Visualization and Targeted Disruption of Protein Interactions in Living Cells

172 Pages·2013·18.61 MB·English

VISUALIZATION AND TARGETED
DISRUPTION OF PROTEIN INTERACTIONS
IN LIVING CELLS

WEN DENG

MÜNCHEN 2013
Visualization and Targeted Disruption of
Protein Interactions in Living Cells

WEN DENG

Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München

vorgelegt von
Wen Deng
aus Hanzhong, China
München, den 25. 11. 2013
Erstgutachter: Prof. Dr. Heinrich Leonhardt
Zweitgutachter: Prof. Dr. Marc Bramkamp

Tag der mündlichen Prüfung: 17.01.2014
CONTENTS
CONTENTS
SUMMARY ......................................................................................................................................................... 1
1. Introduction ...................................................................................................................... 3
1.1 Methods to study protein interactions ............................................................................... 3
1.1.1 Genetic assay - Y2H .......................................................................................................... 3
1.1.2 Affinity purification based methods................................................................................ 4
1.1.3 Molecule proximity based biophysical/biochemical assays ......................................... 6
1.1.4 Fluorescence based dynamic assays ............................................................................. 11
1.2 The p53-Mdm2 interaction ................................................................................................ 15
1.2.1 The cellular role of p53 .................................................................................................. 15
1.2.2 Mdm2 and its relationship with p53 ............................................................................. 16
1.2.3 Inhibitors of the p53-Mdm2 interaction ....................................................................... 18
1.3 Centromere characterization ............................................................................................. 20
1.3.1 Centromeric DNA ............................................................................................................ 20
1.3.2 Specific histones at centromeres ................................................................................... 21
1.3.3 Epigenetic features of the centromere .......................................................................... 23
1.3.4 CENP-A deposition at centromeres ............................................................................... 24
1.4 The kinetochore structure and CCAN (Constant Centromere Associated Network) .... 27
1.4.1 The kinetochore structure ............................................................................................. 27
1.4.2 Constant centromere associated network .................................................................... 28
1.5 Aims of this work ................................................................................................................ 33
2. Results ............................................................................................................................. 35
2.1 Visualization and Targeted Disruption of Protein Interactions in Living Cells ............. 35
2.2 CENP-C Facilitates the Recruitment of M18BP1 to Centromeric Chromatin................. 57
2.3 Step-Wise Assembly, Maturation and Dynamic Behavior of the Human CENP-
P/O/R/Q/U Kinetochore Sub-Complex ............................................................................. 81
2.4 Binding of the Heterogeneous Ribonucleoprotein K (hnRNP K) to the Epstein-Barr
Virus Nuclear Antigen 2 (EBNA2) Enhances Viral LMP2A Expression ........................... 99
3. Discussion ..................................................................................................................... 121
3.1 F3H – a versatile tool for protein manipulation and interaction studies ..................... 121
3.1.1 F3H as a method to study protein-protein interactions ............................................ 121
3.1.2 Application of the F3H assay for high-throughput screens ....................................... 127
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CONTENTS
3.1.3 Developing a method for protein interaction inhibitor studies ............................... 130
3.1.4 Application of the GFP binding protein and nanobodies .......................................... 132
3.2 Cell cycle coupled control of CENP-A incorporation ..................................................... 133
3.2.1 Recruitment of Mis18bp1 to the centromere ............................................................ 133
3.2.2 Mis18 complex regulates the epigenetic state of centromeric chromatin .............. 134
3.2.3 Cell cycle-dependent regulation of the Mis18 complex and CENP-A incorporation
....................................................................................................................................... 135
3.3 Assembly of CENP-O class protein ................................................................................. 138
3.3.1 Dependency of CCAN protein assembly ..................................................................... 138
3.3.2 The CENP-P/O/R/Q/U is not a pre-assembled complex .......................................... 138
3.3.3 Self-assembly of the CENP-P/O/R/Q/U at kinetochores .......................................... 139
4. Annex ............................................................................................................................. 141
4.1 References ........................................................................................................................ 141
4.2 Abbreviations ................................................................................................................... 157
4.3 Contributions ................................................................................................................... 160
4.4 Declaration ....................................................................................................................... 161
4.5 Acknowledgements.......................................................................................................... 162
Curriculum Vitae ..................................................................................................................... 163

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SUMMARY
Summary
Protein-protein interactions are directly or indirectly the basis of all biological processes.
Due to their importance, many methods have been developed to detect them. Each
method has advantages and disadvantages, and is suitable for different applications. Some
methods test the protein in an artificial environment or require expensive equipments,
high expertise and complicated experimental processes. Therefore, we developed a cell
based protein interaction detection system – F3H assay, which can be widely used to
study protein interactions in living cells. This F3H method takes the advantage of the GFP
binding protein, a GFP recognizing single VHH domain antibody, to target GFP fusion
protein to an artificial chromosomal locus. By analyzing the colocalization of the
coexpressed RFP fusion proteins at the locus protein interactions could be identified.
Due to their fundamental role in biological activities, protein-protein interactions are also
important targets for drug design. Since the F3H assay could visualize protein interaction
in living cells in real-time, I further developed the assay to monitor the disruption of
protein interactions after interaction inhibitor treatment. Using the p53-Mdm2 interaction
as a test system, I successfully observed disruption of the interaction between p53-Mdm2
by several inhibitors, including both small molecular compounds and peptide inhibitors.
To make this protein-protein interaction inhibitor study system more efficient, I expanded
this assay to 96-well plates and established an automated analysis protocol, which allows
screening for protein-protein inhibitors in a high throughput way. Our data demonstrated
the versatility of the F3H method in protein interaction studies and its strong potential in
protein inhibitor screening.
I applied this F3H assay to study mammalian kinetochore assembly in vivo. The
kinetochore is a multi-protein complex formed by different classes of proteins. The
CCAN (constitutive centromere-associated network) proteins are important components
of functional kinetochores. Using the F3H assay, I systematically studied the interactions
among the CCAN members and found some new interactions which may contribute to the
assembly of the CCAN. A group of the CENP-P/O/R/Q/U proteins took our special
interest, and based on interaction data, we proposed a stepwise self-assembly model for
the kinetochore assembly of the CENP- P/O/R/Q/U proteins in mammalian cells.
Besides, the CENP-A incorporation mechanism was also studied in this work using our
F3H assay. M18bp1 is one component of the Mis18 complex which is necessary for
CENP-A incorporation after cell division. But how Mis18 complex is recruited to
centromeres was unclear. I screened the interactions between M18bp1 and the CCAN
proteins and found that CENP-C interacts with M18bp1. This interaction facilitates the
recruitment of M18bp1 to centromere and is important for proper CENP-A incorporation.
Furthermore, we studied the interactions between EB virus protein EBNA2 and its host
cell protein huRNP K, revealing a role of hnRNP K in up-regulation of viral gene
expression. In conclusion, the F3H assay is a powerful method to study protein
interactions and also to screen protein interaction inhibitors.
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