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«Developing quantitative GTPase affinity purification (qGAP) to identify interaction partners of Rho GTPases Dissertation zur Erlangung des ...»

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Developing quantitative GTPase affinity purification

(qGAP) to identify interaction partners of Rho GTPases

Dissertation

zur Erlangung des akademischen Grades

doctor rerum naturalium

(Dr. rer. nat.)

eingereicht im Fachbereich Biologie, Chemie, Pharmazie

der Freien Universität Berlin

vorgelegt von

FLORIAN ERNST RUDOLF BENJAMIN PAUL (DIPL.-BIOCHEM.)

Januar 2014

ii

Die vorliegende Arbeit wurde von Oktober 2008 bis Januar 2014 am

Max-Delbrück-Centrum für Molekulare Medizin unter der Anleitung von Prof. Dr. MATTHIAS SELBACH angefertigt.

1. Gutachter: Prof. Dr. MATTHIAS SELBACH Cell Signalling and Mass Spectrometry Max-Delbrück-Centrum, Berlin

2. Gutachter: Prof. Dr. UDO HEINEMANN Institut für Chemie / Biochemie Freie Universität Berlin / Max-Delbrück-Centrum, Berlin Disputation am 8. Mai 2014 iii iv “Study hard what interests you the most in the most undisciplined, irreverent and original manner possible.” ― Richard P. Feynman v vi Contents 1 Introduction

1.1 Rho GTPases

1.1.1 The GDP/GTP cycle

1.1.2 Rho GTPases as part of the Ras superfamily

1.1.3 The family of Rho GTPases

1.2 Regulation of Rho GTPases

1.2.1 Guanine nucleotide exchange factors

1.2.2 GTPase activating proteins

1.2.3 Guanine dissociation inhibitors

1.2.4 Subcellular localization

1.3 General concepts of Rho GTPase signaling

1.3.1 Cell polarity

1.3.2 Actin cytoskeleton

1.3.3 Gene expression

1.3.4 Enzymatic activity and cell cycle

1.4 Disease relevance of Rho GTPases

1.5 Mass spectrometry for Protein identification

1.5.1 Quantitative mass spectrometry

1.5.2 Protein Identification and Quantification

1.6 Protein Interactions and q-AP/MS

1.6.1 Identification of Rho GTPase interaction partners

1.7 Objectives

2 Materials & Methods

2.1 Materials

2.1.1 Chemicals

2.1.2 Media and buffers

2.1.3 Enzymes/Proteins

2.1.4 Antibodies

2.1.5 Kits

2.1.6 Bacteria strains

2.1.7 Plasmids

2.1.8 Cell lines

2.1.9 cDNA clones

2.2 Molecular biology methods

2.2.1 Primer design and Oligonucleotides

2.2.2 Polymerase Chain Reaction

vii 2.2.3 DNA purification

2.2.4 Restriction digest

2.2.5 Agarose gel electrophoresis

2.2.6 DNA extraction

2.2.7 Ligation

2.2.8 Transformation of chemically competent E. coli

2.2.9 Long term storage of E. coli cultures

2.2.10 Plasmid preparation

2.3 Biochemical Methods

2.3.1 Antibiotics

2.3.2 Protein expression in E. coli

2.3.3 E. coli cell lysis and preparation of the cytosolic fraction

2.3.4 Glutathione affinity chromatography

2.3.5 Size exclusion chromatography

2.3.6 Nucleotide loading

2.3.7 Determination of protein-bound nucleotide

2.3.8 Protein concentration determination

2.3.9 Protein concentration and storage

2.3.10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

2.3.11 Western blotting

2.4 Cell cultivation, fractionation and lysis

2.4.1 Cell media/SILAC

2.4.2 Cell cultivation

2.4.3 Preparation of cytosolic extracts

2.4.4 Transient transfection of mammalian cells

2.4.5 Lysis of adherent mammalian cells

2.4.6 Preparation of mice brain

2.4.7 Immunoprecipitation (GFP-fusion proteins)

2.4.8 Pull down assay

2.4.9 Protein ethanol precipitation

2.4.10 Proximity ligation assay

2.4.11 Microscopy and processing of images

2.5 Mass spectrometry

2.5.1 In solution digestion

2.5.2 In gel digestion

2.5.3 Stage-tip purification

2.5.4 Liquid-chromatography mass spectrometry (LC-MS)

2.6 Data processing and analysis

viii 2.6.

1 MaxQuant

2.6.2 Perseus

2.6.3 Cluster analysis of gene ontology (GO) terms

2.6.4 Alignment of protein sequences

3 The quantitative Rho GTPase affinity pull-down (qGAP)

3.1 Development of qGAP

3.1.1 Purification of Rho GTPases

3.1.2 Test of different coupling strategies for qGAP affinity matrix

3.2 Systematic identification of Rho GTPase interaction partners using qGAP

3.2.1 Interaction partners of Rho GTPases in Cell culture

3.2.2 Interaction partners for Rho GTPases in Brain lysates

3.2.3 Enrichment of Rho binding proteins by qGAP

3.2.4 Specificity, sensitivity and reproducibility of qGAP

3.2.5 Validation of identified interaction partners by Proximity Ligation Assay

3.2.6 Effector binding specificity towards GTPases

3.2.7 Cell type specificity of Rho interactions

3.3 Evaluation of alternative strategies for identification of Rho GTPase interactors............ 65 3.3.1 Triplicate label free experiment from cell culture

3.3.2 Immunoprecipitations with tagged CA/DN-variants from SILAC-cell culture............... 66 3.4 A network of Rho GTPase interaction partners

3.4.1 The interactome of Rho GTPases

4 Discussion





4.1 Development of qGAP

4.2 qGAP sensitively and specifically identifies Rho GTPase interaction partners................. 74 4.3 qGAP in comparison to alternative strategies

4.4 A network of Rho GTPase interaction partners

5 Conclusions and Perspectives

6 Supplemental Data

6.1 Additional applications of qGAP

6.1.1 SILAC-qGAP for identification of interaction partners of Arf6

Abbreviations/Units

List of figures

List of tables

Bibliography

Curriculum Vitae

Publications

ix Acknowledgement

Declaration

xSummary

Rho GTPases are central regulators of the actin cytoskeleton. Additionally, they have influence on gene expression, cell division and other biological processes. Classical Rho GTPases function as molecular switches, being active when GTP is bound and inactive in the GDP-bound state. The exchange of nucleotides, the GDP/GTP cycle, is subject to intense regulation. The multiple biological activities of Rho GTPases are mediated by numerous downstream effector proteins. However, a systematic experimental comparison of Rho GTPase interaction partners has not been performed.

This thesis describes development, validation and biological outcome of a new method to identify Rho GTPase effector proteins for both of the nucleotide-loaded forms. We named this assay quantitative GTPase affinity pull-down (qGAP). qGAP combines affinity purification with quantitative mass spectrometry. Recombinant Rho GTPases were purified and loaded with either GDP or GTPγS.

The Rho GTPases were covalently coupled to a sepharose matrix and used for affinity purification.

Quantitative shotgun proteomics was then used to compare the abundance of proteins interacting with the GDP- and GTPγS-loaded forms, to identify loading state-specific binders.

First, qGAP was applied to RhoA, Rac1 and Cdc42 to identify binding partners from cytoplasmic extracts of SILAC-labeled HeLa cells (SILAC-qGAP). Next, lysates from mouse brains were used to identify interaction partners of RhoA, RhoB, RhoC, RhoD, Rac1 and Cdc42 using label free quantification (LF-qGAP). In both variants of qGAP, groups of specific binders could be distinguished from hundreds to thousands of background binders. Interaction partners identified by qGAP where highly enriched with known Rho interaction partners when compared to an unbiased reference database. For LF-qGAP, the sensitivity was good (50%) and the specificity excellent (97%). If further studies show, that the newly identified interaction partners are true, the real sensitivity might be higher. Hierarchical clustering of biological replicate samples showed that qGAP data was highly reproducible. In total, LF-qGAP identified 291 mostly novel interactions. Eleven out of twelve tested novel interactions were confirmed by a newly developed, independent assay.

From the binding data a comprehensive Rho interaction network was constructed. We found the promiscuousness of binding partners to be higher than anticipated. The overlap allowed us to deduce similarities between the network and the phylogeny of Rho GTPases. Altogether, these data show that qGAP is a valuable novel method to study Rho GTPase biology.

xixiiZusammenfassung

Rho GTPasen stellen zentrale Regulatoren des Aktin-Zytoskeletts dar. Zusätzlich können sie die Genexpression, Zellteilung und andere biologische Prozesse beeinflussen. Die klassischen Rho GTPasen sind molekulare Schalter. Wenn sie GTP gebunden haben befinden sie sich im aktiven, bei Bindung von GDP im inaktiven Zustand. Der Austausch dieser beiden Nukleotide, auch GDP/GTPZyklus genannt, wird streng reguliert. Die vielfältigen biologischen Aktivitäten der Rho GTPasen werden durch zahlreiche Effektoren vermittelt. Trotz ihrer Wichtigkeit wurde eine systematische Untersuchung dieser Interaktionspartner noch nicht durchgeführt.

In dieser Doktorarbeit sind die Entwicklung, die Validierung und die biologischen Ergebnisse einer neuen Methode zur Identifizierung von Rho GTPase Effektoren beschrieben. Diese Methode wurde von uns „quantitative GTPase affinity pull-down“ (quantitativer GTPasen Affinitäts Pulldown, qGAP) genannt. qGAP vereinigt die Affinitäts-Aufreinigung (Pulldown) mit quantitativer Massenspektrometrie. Dabei werden beide Nukleotid-Ladungszustände berücksichtigt. Zu diesem Zweck wurden rekombinante Rho GTPasen aufgereinigt und entweder mit GDP oder GTPγS geladen.

Die Rho GTPasen wurden kovalent an eine Sepharose Matrix gekoppelt und für Affinitätsaufreinigung eingesetzt. Quantitative shotgun proteomics wurde verwendet, um die Menge von Proteinen zu vergleichen, die mit der GDP- und GTPγS-geladenen Form interagiert haben. Dadurch wurden Ladungs-spezifische Bindungspartner identifiziert.

Zunächst wurde qGAP mit RhoA, Rac1 und Cdc42 auf zytoplasmatische Extrakte von SILACmarkierten HeLa Zellen angewendet (SILAC-qGAP). Im nächsten Schritt wurden Lysate von unmarkierten Maus Gehirnen verwendet um Interaktionspartner von RhoA, RhoB, RhoC, RhoD, Rac1 und Cdc42 zu identifizieren (LF-qGAP). In beiden Varianten von qGAP konnten Gruppe von spezifischen Interaktionspartnern klar von hunderten bis tausenden von Proteinen unterschieden werden. Durch den Vergleich mit einer Interaktionsdatenbank wurde festgestellt, dass diese spezifischen Interaktionspartner klar mit bekannten Rho Effektoren angereichert waren. Für LF-qGAP war die Sensitivität gut (50%) und die Spezifizität exzellent (97%). Falls weitere Studien zeigen, dass die neuen Interaktionspartner wahr sind, könnte die tatsächliche Sensitivität höher liegen. Eine hierarchische Clusteranalyse der biologischen Replikate ergab, dass qGAP sehr reproduzierbar war.

Insgesamt wurden mit qGAP 291, größtenteils neue Interaktionspartner identifiziert. Elf von zwölf getesteten neuen Interaktionen wurden durch eine neu entwickelte, unabhängige Methode validiert.

Die Bindungsdaten wurden verwendet um ein umfassendes Rho Interaktionsnetzwerk zu erstellen. Es wurde eine höhere Promiskuität zwischen Bindungspartnern gefunden als zuvor vermutet. Das Überlappen von Bindungspartnern erlaubte uns auf Ähnlichkeiten zwischen dem Netzwerk und der

–  –  –

Protein-protein interactions (PPIs) are essential for almost every biological process. Specifically, cellular signal transduction depends on the dynamic interaction of proteins with each other. These cascades are highly regulated and influence gene expression, cell death and survival or cell communication among other processes. The activation state of a signaling cascade is key to its control.

Rho (Ras homologues) GTPases are particularly important molecules in cellular signal transduction.

The membrane-anchored proteins are central regulators of the actin cytoskeleton, and determine the morphology and motility of eukaryotic cells1–3. Furthermore Rho proteins have implications for cell polarity, cell division, gene regulation and apoptosis. Their activity is tightly controlled by the GDP/GTP cycle: GTP-bound Rho proteins are active, hydrolysis of the nucleotide to GDP renders them inactive.

PPIs have been systematically studied with the help of a broad range of tools. Most prominently, the yeast two-hybrid approach was used in large scale screening. Good scalability and easy handling made it a very popular method for the discovery of interactions. On the other hand, yeast two-hybrid suffers from a high false positive rate and the cellular background of interactions is not taken into account. In recent years affinity purification followed by quantitative mass spectrometry (q-AP/MS) has become a powerful alternative to identify PPIs.

The objective of this thesis is the systematic analysis of interaction partners of selected Rho GTPases.

To this end, a quantitative proteomic approach is developed and employed. Interaction partners of Rho GTPases are identified in lysates from cell culture and whole organ samples. The quantification is performed with a stable-isotope and a label-free approach. The goal is to provide the scientific community with a comprehensive Rho GTPase interactome for the selected Rho proteins.

In the following introduction, the family of Rho GTPases is described and details about its members and their regulation are presented. Next, the cellular function of Rho GTPases and their relevance for diseases is outlined. Then, the fundamentals of quantitative mass spectrometry-based proteomics are explained with focus on the technologies to study PPIs. Finally the objectives of the thesis are delineated.



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