TITLE PAGE
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATION 8
ABSTRACT 10
Ⅰ. Introduction 11
Ⅱ. Materials and methods 15
Ⅱ. 1. Hyaluronan binding assay 15
Ⅱ. 2. Mammalian transfection of vDUB2 15
Ⅱ. 3. Tet-on system 16
Ⅱ. 4. Real-time PCR analysis 16
Ⅱ. 5. FACS analysis 17
Ⅱ. 6. Localization study and DAPI staining 18
Ⅲ. Results 19
Ⅲ. 1. Hyaluronan-binding properties of vDUB2 19
Ⅲ. 2. Mammalian transfection of vDUB2 24
Ⅲ. 3. Tet-on system and long-term culture observation 26
Ⅲ. 4. Investigation of cell cycle and apoptosis in FACS analysis 28
Ⅲ. 5. Localization study of vDUB2 and observation of the nucleus in HeLa cells 31
Ⅳ. Discussion 34
Ⅴ. Conclusions 38
References 39
Summary in Korean 48
Figure 1. Catalytic domains of vDUB2. (A) Schematic diagram for vDUB2. The domains required for deubiquitinating activity (Cys, Asp1, His, Asp2) are located at the 5' end and in the middle of vDUB2. Two potential hyaluronan binding motifs-R/K(X7)R/K- are located at 401 to 410 and 445 to 453. HABP4_PAI-RBP1 represents a putative domain for hyaluronan/RNA binding domain. The 'RGG' amino acid sequences which are predicted for novel RNA binding motif was found at the C-terminal of vDUB2 20
Figure 2. Hyaluronan/RNA-binding assay. (A) To confirm the interaction between vDUB2 and hyaluronan, CPC precipitation assay was performed. Cell lysate aliquots from HeLa cells transfected with myc-tageed vDUB2 construct were incubated with hyaluronan or RNA and subjected to CPC precipitation. Coprecipitated proteins were subjected to Western blot analysis with an antibody against the myctag. Lane 4 shows that v DUB2 can interact with endogenous hyaluronan. When exogenous hyaluronan was added, the degree 22
Figure 3. The effect of vDUB2 expression in HeLacells. (A) Overexpression of vDUB2 induced apoptosis of HeLa cells. (B) All cells expressing vDUB2 died after 3 weeks during antibiotics selection. However, the mutated form of vDUB2 (C89S) did not induce apoptosis and showed normal morphology similar to control HeLa cells (C) 25
Figure 4. The effect of vDUB2 expression by the Tet-on system. (A) The some of cells expressing vDUB2 by doxycyclin induction showed apoptosis similar to pcDNA3-vDUB2 expressing cells. The irregular morphology considered as apoptosis appenared from the 5th day. (B) Relative amount of initial caspase 3. The amount of caspase 3 cDNA (left) in the control group was calibrated as 100 and right bar indicates relative amounts of caspase 3 cDNA of experimental group which vDUB2 was transfected 27
Figure 5. FACS analysis. Cells expressing vDUB2 or DUB2 (C89S) were harvested I week apart for 3 weeks, fixed with ethanol, and stained with PI (A, B, D). The number of cells shown in sub-G1 population increased in the cased of expressing vDUB2 in HeLa cells (B). The cells expressing vDUB2 (c89S) (C) showed a similar pattern with normal HeLa cells (A). The ratio of each cell cycle phase was compared shown in (G). In apoptosis analysis (D, E, F), the number of cells expressing vDUB2 (E) showed that PI posi 29
Figure 6. The localization of vDUB2 and irregular nuclear morphology in HeLa cells. The GFP-vDUB2 proteins were more concentrated in the nucleus (B). In the nucleus, GFP-vDUB2 proteins are highly condensed at the nucleoli. Some cells exprssing GFP-vDUB2 proteins showed the onset of apoptosis (C) 32