Structural and functional characterization of the interactions of platelet-derived chemokines CCL5, CXCL4 and CXCL4L1 [Elektronische Ressource] / Alisina Sarabi

Alisina Sarabi

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2011
• Nombre de lectures: 30
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Structural and Functional Characterization
of the Interactions of Platelet-Derived Chemokines
CCL5, CXCL4 and CXCL4L1



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation
vorgelegt von

Diplom-Biologe
Alisina Sarabi
aus
Kabul, Afghanistan



Berichter:
Universitätsprofessor Christian Weber
Universitätsprofessor Rainer Fischer

Tag der mündlichen Prüfung: 31.01.2011




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. The results of this work were in part published in:


Rory R Koenen, Philipp von Hundelshausen, Irina V Nesmelova, Alma Zernecke, Elisa A
Liehn, Alisina Sarabi, Birgit K Kramp, Anna M Piccinini, Søren R Paludan, M Anna
Kowalska, Andreas J Kungl, Tilman M Hackeng, Kevin H Mayo & Christian Weber
(2009). Disrupting functional interactions between platelet chemokines inhibits
atherosclerosis in hyperlipidemic mice. Nat Med 15 (1): 97-103.


Alisina Sarabi, Birgit K Kramp, Maik Drechsler, Tilman M Hackeng, Oliver Soehnlein,
Christian Weber, Rory R Koenen and Philipp von Hundelshausen (2010). CXCL4L1
inhibits angiogenesis and induces undirected endothelial cell migration without affecting
endothelial cell proliferation and monocyte recruitment. J Thromb Haemost 9 (1): 209-19
[Epub ahead of print DOI:10.1111/j.1538-7836.2010.04119.x.] PMID: 20961394.















II
Table of Contents

Table of Contents
Table of contents…………………………………………………………...…………….i
Abbreviations…………………………………………………………………….............vi

I Introduction...........................................................................................................1
I.1 Atherosclerosis......................................................................................................1
I.2 Chemokines2
I.3 Interaction of chemokines ..................................................................................... 4
I.4 Chemokine receptors.............................................................................................6
I.4.1 Alternative signaling mechanism...............................................................8
I.5 Platelets.................................................................................................................. 8
I.6 Platelet-derived chemokines CXCL4, CXCL4L1 and CCL5 ............................. 10
I.6.1 CXCL4.....................................................................................................10
I.6.2 CXCL4L1.................................................................................................14
I.6.3 CCL5........................................................................................................15
I.7 Aims of the study 17
II Material and methods..........................................................................................18
II.1 General equipment...............................................................................................18
II.2 Molecular biology19
II.2.1 General work with E. coli......................................................................... 19
Bacteria growth media.............................................................................. 20
Preparation of heat-shock competent E. coli............................................ 20
i Table of Contents

Heat-shock transformation of competent E. coli...................................... 21
II.2.2 Bacterial strains........................................................................................21
II.2.3 Plasmids....................................................................................................21
II.2.4 Polymerase chain reaction........................................................................ 21
II.2.5 Cloning of CXCL4 and CXCL4L1 .......................................................... 22
II.2.6 Miniprep: Small-scale purification of plasmid DNA ............................... 23
II.2.7 Midiprep and maxiprep: Large-scale purification of plasmid DNA ........ 24
II.2.8 Restriction endonuclease digestion of DNA ............................................ 24
II.2.9 Agarose gel electrophoresis......................................................................24
II.2.10 Quantification of DNA ............................................................................. 25
II.2.11 Sequencing of DNA.................................................................................25
II.3 Protein analysis....................................................................................................25
II.3.1 Protein concentration assay25
II.3.2 SDS-polyacrylamide gel electrophoresis (PAGE) ................................... 26
II.3.3 Tricine gel.................................................................................................27
II.3.4 Coomassie blue staining ........................................................................... 28
II.3.5 Silver staining...........................................................................................28
II.3.6 Western blot analysis................................................................................ 28
II.3.7 Dot blot.....................................................................................................29
II.3.8 Primary antibody......................................................................................29
II.3.9 Secondary antibody..................................................................................30
II.4 Protein expression and purification ..................................................................... 30
II.4.1 CCL5 expression and purification............................................................ 30
II.4.2 CCL5 FPLC (fast protein liquid chromatography) .................................. 31
Sephacryl S-100HR 31
HiTrap chelating HP................................................................................. 31
™MonoS 5/50 GL ..................................................................................... 32
™Resource Reverse Phase Chromatography (RPC) ................................. 32
ii Table of Contents

II.4.3 CCL5 HPLC (high performance liquid chromatography)........................ 33
II.4.4 CXCL4 and CXCL4L1.............................................................................34
II.4.5 CXCL4 and CXCL4L1 FPLC .................................................................. 34
™HiLoad 16/10 SP-Sepharose 34
™ ™ MonoS 5/50 GL or Capto S................................................................. 35
™Resource RPC ........................................................................................ 35
II.4.6 CXCL4 and CXCL4L1 HPLC 35
II.4.7 Chemical synthesis of CXCL4L1............................................................. 36
Peptide synthesis and native chemical ligation ........................................ 36
II.5 Biochemical analysis of the CCL5-CXCL4 heterodimer.................................... 39
II.5.1 Molecular dynamics simulations.............................................................. 40
II.5.2 Differential scanning calorimetry.............................................................40
II.5.3 Interaction of CXCL4 or CXCL4L1 with CCL5 ..................................... 41
Ligand blot................................................................................................ 41
Isothermal fluorescence titration .............................................................. 41
II.6 Cell culture..........................................................................................................42
II.6.1 General.....................................................................................................42
II.6.2 Cell lines...................................................................................................42
II.6.3 Culturing of adherent cell monolayers ..................................................... 42
II.6.4 cells in suspension ............................................................... 43
II.6.5 Freezing and thawing of cells................................................................... 43
II.7 Functional assays.................................................................................................43
II.7.1 Angiogenesis in vitro................................................................................ 43
II.7.2 Endothelial migration...............................................................................43
Transwell assay ........................................................................................ 43
iii Table of Contents

The µ-Slide Chemotaxis chamber ............................................................ 44
II.7.3 Endothelial cell proliferation....................................................................44
II.7.4 Monocyte recruitment...............................................................................45
In vitro ..