Post-translational modifications of Hepatoma-derived growth factor (HDGF) [Elektronische Ressource] / submitted by Ketan Thakar

Ketan

Sujets

• Biologie

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2010
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Post-translational modifications of
Hepatoma-derived growth factor (HDGF)

+NH
3
-
OO
OH SH
N NN N N
H H H H H
O O O O O
Post-translational
modifications

O
NHSUMO

OH CO SR3
P
O SH
OH
N N N N N
H HH H H
O O O O O


Ketan Thakar



Universität Bremen
2010
O
-
O O-


Post-translational modifications of
Hepatoma-derived growth factor (HDGF)







Dissertation submitted as a partial fulfillment for procuring the degree
Doctor of Natural Science (Dr. rer. nat.)



Submitted to

Fachbereich 2 Biologie/Chemie
Universität Bremen




Submitted by

Ketan Thakar
M.Sc. Biochemistry and Molecular Biology



Bremen 2010
Examination Committee


Reviewers:

1. Prof. Dr. Sørge Kelm
Universität Bremen, Fachbereich 2, Biochemie
Postfach 330440, 28334 Bremen

2. Dr. Kathrin Mädler
Universität Bremen, Fachbereich 2, Biochemie
Leobener Straße NW2, B2060, 28359 Bremen


Examiners:

1. Dr. Frank Dietz
Universität Bremen, Fachbereich 2, Biochemie
Postfach 330440, 28334 Bremen

2. Prof. Dr. Reimer Stick
Universität Bremen, Fachbereich 2, Institut für Zellbiologie
Leobener Straße NW2 A3290, 28359 Bremen


Date of Public defense:

28 May, 2010TABLE OF CONTENTS
Table of Contents

I. Summary………………………………………………………………………………………………….. vii
II. Zusammenfassung…………………………………………………………………………………… viii

1. Introduction…………………………………………………............................................................. 1
1.1 The HDGF related protein (HRP) family………………………………………………………… 1
1.1.1 Hepatoma-derived growth factor……………………………………………………………. 2
1.1.2 Hepatoma-deactor related protein-1 (HRP-1)…………………… 2
1.1.3 Hepatoma-derived growth factor relate-2 (HRP-2) …………………………….. 2
1.1.4 Hepatoma-dead protein-3 (HRP-3) ………………….. 3
1.1.5 Hepatoma-derived growth factor relate-4 (HRP-4)……………………………… 3
1.1.6 Lens epithelium derived growth factor (LEDGF)………………………………... 3
1.2 HDGF structure-function relationship………………………………………………………….. 4
1.2.1 HDGF is a modular protein with two structurally independent domains………………… 4
1.2.2 HDGF PWWP domain………………………………………………………………………... 4
1.2.2a HDGF binds to DNA through the N-terminal PWWP domain……………………………. 7
1.2.2b HDGF PWWP domain as a potential protein-protein interaction domain………………. 7
1.2.3 HDGF and heparin binding specificity………………………………………………………. 7
1.3 Biological roles of HDGF………………………………………………………………………….. 9
1.3.1 HDGF in cancer development, prognosis and diagnosis…………………………………. 9
1.3.2 HDGF and its role in developmental regulation………………………………… 10
1.3.3 HDGF in organ remodeling after injury………………………………………….. 10
1.3.4 HDGF involvement in apoptosis……………………………………….. 11
1.4 Concept of the thesis ……………………………………………………………………………… 12

2. Materials and Methods…………………………………………………………………………….. 13
2.1 Cell lines……………………………………………………………………………………………… 14
2.2 Plasmid vectors and Primers…………………………………….. 14
2.3 Kits………………………………………………………………………………………… 14
2.4 Antibodies………………………………………. 15
2.5 Growth media……………………………………………………………………………………….. 16
2.6 Solutions and buffers…………………………………… 16
2.7 Molecular cloning and plasmid isolation……………………………………………. 18
2.7.1 Bacterial Transformation……………………………………………………………………... 18
2.7.2 Colony PCR……………………………………………………………………………………. 18
2.7.3 Plasmid Purification………………………………… 18
iv TABLE OF CONTENTS
2.7.3a Plasmid purification using the NucleoBond Xtra Midi kit (Macherey-Nagel) …………… 19
2.7.3b urifising the GeneJET Plasmid Miniprep kit (Fermentas) …………… 19
2.7.4 Measurement of plasmid concentration and level of purity………………………………. 19
2.7.5 Agarose gel electrophoresis………………………………………………………. 19
2.8 Plasmid construction………………………………………………………………………………. 20
2.8.1 Production of untagged mHDGF D205G and hHDGF G205D mutants………………… 20
2.8.2 Site-directed mutagenesis …………………………………………………………………... 20
2.8.3 Preliminary assessment of the site-directed mutagenesis…………………….. 21
2.8.4 DNA Sequencing PCR reaction……………………………………………………………... 21
2.9 Recombinant protein expression in eukaryotic cell lines…………………………………... 21
2.9.1 Culturing eukaryotic cell lines………………………………………………………………... 21
2.9.2 In vitro transfection……………………………………………. 22
2.9.3 Cell Harvesting……………………………………………………………………… 22
2.10 Protein Analysis…………………………………………………………………………………….. 22
2.10.1 Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) …………. 22
2.10.2 Western blotting……………………………………………………………………………….. 23
2.10.2a Transferring resolved proteins on PVDF membrane………………… 23
2.10.2b Immunodetection……………………………………………………………………………… 23

3. Publications……………………………………………………………………………………………. 25
3.1 Publication 1………………………………………………………………………………………… 27
3.2 Publication 2…………………………………… 44
3.3 3………………………………………………………………………………………… 66

4. Additional results…………………………………………………………………………………….. 85
4.1 SUMOylation of HDGF by SUMO isoforms in mammalian cells…………………………… 86
4.2 HDGF is processed C-terminally at a potential caspase cleavage site………… 87

5. Discussion………………………………………………………………………………………………. 89
5.1 SUMOylation of HDGF…………………………………………………………………………….. 89
5.2 Phosphorylation dependent regulation of HDGF secretion and processing……………. 91
5.3 HDGF dimerisation…………………………………………………………………………………. 92
5.4 HDGFHRP-2 interaction……………………………….. 93
5.4.1 Differential expression of HRP-2 corresponds to alternatively spliced isoforms………. 93
5.4.2 HDGF/HRP-2 interaction studies……………………………………………………………. 94
5.5 Caspase dependent cleavage of HDGF………………………………………………………… 95
5.6 Conclusion and perspectives…………………...................................................................... 96
v TABLE OF CONTENTS
6. References………………………………………………………………………………………………. 99

A. Appendix…………………………………………………………………………………………………. 107
A.1 Abbreviations…………………………………………………………………………………………108

A.2 List of Tables and Figures………………………………………………………………………… 109
A.2.1 List of Tables……………………………………………………………………………...…… 109
A.2.2 List of Figures…………………………………………………………………………...…….. 109
A.3 Vector maps…………………………………………………………………………...…………….. 110
A.4 List of Manufacturers…………………………………………………………………………........ 112
A.4.1 Chemicals and consumables……………………………………………………….............. 112
A.4.2 Devices……………………………………………............................................... 113

Acknowledgements……………………………………………………………………........................... 114

Erklarüng…………………………................................................ 115

vi SUMMARY
I. Summary

Post-translational modifications (PTM’s) are modifications that occur during or after protein translation.
Nascent or folded protein can be subjected to an array of specific enzyme-catalyzed modifications on the
amino acid side chains or the peptide backbone. Two broad categories of protein PTM’s occur; the first
includes all enzyme-catalyzed covalent additions of different lower molecular chemical groups up to
complex proteins to amino acid side chains in the target protein, whereas the second category comprises
structural changes and the cleavage of peptide backbones in proteins either by action of proteases or,
less commonly, by autocatalytic cleavage. PTM’s can modulate the function of proteins by altering their
activity state, localization, turnover, and interactions with other proteins.

Hepatoma-derived growth factor (HDGF) is the prototype of a family of six proteins comprising HDGF, the
four HDGF-related proteins (HRP-1–4), and the lens epithelium-derived growth factor (LEDGF). HDGF
exhibits growth factor properties and has been implicated in organ development and tissue differentiation
of the intestine, kidney, liver, and cardiovascular system. Recently, the role of HDGF in cancer biology has
become a main focus of its research. HDGF was found to be over-expressed in a large number of
different tumor types. Although a direct influence of HDGF on tumor biology is still unclear, its expression
is correlated with metastasis and tumor recurrence in multiple studies. HDGF appears to be a novel
prognostic marker for different types of cancer. Growth promoting as well as other activities of HDGF, like
the suppression of differentiation; possible role in apoptotic processes; or its angiogenic properties have
been suggested to play a role in tumor induction and/or cancer progression.

Interestingly, until now, very little is known about how PTM’s are involved in modulating HDGF function.
Therefore, the main aim of the thesis was the identification of HDGF PTM’s and their consequence on its
function. At first, we have identified that HDGF is post-translationally modified by SUMO-1 at a
non-consensus site and SUMOylated HDGF does not associate with chromatin i