Metabolic control of cell adhesion and migration through beta1A integrin acetylation : be aware of the acetylation switch!

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Thesis

Reference

Metabolic control of cell adhesion and migration through beta1A integrin acetylation : be aware of the acetylation switch!

KASTBERGER, Birgit

Abstract

Alpha5beta1A integrins form two types of cell-matrix adhesions with differential functions:

short, peripherally located focal adhesions (FAs) that provide strong anchorage to the substrate and intracellular signalling; and long, centrally located fibrillar adhesions (FBs) that enable the formation of fibronectin (FN) fibrils in the pericellular space. Despite the critical role of alpha5beta1A integrins for FN deposition during fibrosis, the integrins' relevant signalling is not well understood. Interestingly, beta1A integrin bears a lysine acetylation site located within integrin's kindlin recognition sequence in the cytoplasmic tail. Genetic deletion of kindlin has been demonstrated to handicap cell spreading and the formation of thin, centrally located FBs. These facts suggest a functional link between integrin acetylation, kindlin binding affinities, FB formation and FN fibrillogenesis capacities. Therefore, we hypothesized that acetylation of beta1A integrin might function as regulatory switch which defines for each beta1A integrin its FA or FB character.

KASTBERGER, Birgit. Metabolic control of cell adhesion and migration through beta1A integrin acetylation : be aware of the acetylation switch!. Thèse de doctorat : Univ.

Genève, 2015, no. Sc. 4875

URN : urn:nbn:ch:unige-801528

DOI : 10.13097/archive-ouverte/unige:80152

Available at:

http://archive-ouverte.unige.ch/unige:80152

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITÉ DE GENÈVE

Département de médecine FACULTÉ DES SCIENCES

Département de physiologie cellulaire et métabolisme Professeur B. Wehrle-Haller

Section de chimie et biochimie FACULTÉ DES SCIENCES

Département de biochimie Professeur J. Gruenberg

Metabolic Control Of Cell Adhesion And Migration Through β

1A

Integrin Acetylation

Be aware of the acetylation switch!

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie

par

Birgit KASTBERGER

de Autriche

Thèse N^o 4875

GENÈVE Atelier ReproMail

2015

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Be aware of the acetylation switch!

by B

^IRGIT

K

^ASTBERGER

Metabolic control of cell Adhesion and Migration

through β 1A integrin Acetylation

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i

4.3.1 Deacetyl-β1A and β1D integrins show slow integrin dynamics and form stable cell-matrix adhesions . 68 4.3.2 Adhesions composed of deacetyl-β1A integrins “slide” faster as adhesions composed of acetylated β1A integrin 70 4.4 THE ROLE OF ACETYLATED INTEGRINS IN FIBRONECTIN POLYMERISATION ... 73

4.4.1 Acetyl-β1A integrins show enhanced FN fibrillogenesis capacities ... 73

4.5 WHICH HISTONE-DEACETYLASES CAN DEACETYLATE ACETYL-β1A? ... 75

4.5.1 Fibronectin binding assay in an KDAC overexpressing environment ... 75

5 DISCUSSION ... 77

5.1 AIM OF THE STUDY, SUMMARY AND PERSPECTIVE ... 77

5.2 β1A ACETYLATION WORKS AS SWITCH BETWEEN FAS AND FBS ... 78

5.3 β1A ACETYLATION HAS AN INFLUENCE ON FN AFFINITY AND EXTRACELLULAR FIBRILLOGENESIS ... 81

5.4 β1A ACETYLATION REGULATES ADHESION STABILITY, TURNOVER AND MOTILITY ... 82

5.5 β1A ACETYLATION INFLUENCES CELL MORPHOLOGY AND CELL EDGE MOTILITY ... 82

5.6 KDAC6 AND KDAC7 MODULATE β1A INTEGRIN’S AFFINITY FOR FN... 83

5.7 THE (β1A INTEGRIN) ACETYLATION SWITCH AS A THERAPEUTIC TARGET ... 84

5.7.1 A perspective to treat cancer via the (β1A integrin) acetylation switch ... 84

5.7.2 A perspective to treat fibrotic disease/muscle disease via the (β1A integrin) acetylation switch ... 86

5.7.3 A perspective to treat neurodegenerative diseases via the (β1A integrin) acetylation switch ... 87

5.7.4 KDAC inhibitors in disease ... 89

6 REFERENCES ... 91

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iii

I Acknowledgements

This research project was a wonderful and overwhelming journey of research, culture and the exploration of impressive, talented individuals. I am very grateful to so many people for making this project possible. I would first like to thank my thesis supervisor Prof. Bernhard Wehrle-Haller whose selfless time and care were sometimes all that kept me going. His unbreakable believe in me and our research question and very importantly his sense of observation were guiding me to the answers I never imagined to be able to find. He took care about my constant growth and teached me experimental set-ups, scientific presentations and people management. Thanks to him, I learnt that ideas do not have any hierarchy and that problem solving is my passion. Thanks for the marvellous research opportunity that I was offered in your lab.

Thanks to Prof. Daniel Bouvard and Prof. Marc Block who greatly supported me with cells and fibronectin fragments and willingly answered all my open questions about their material during this thesis project. I thank you also for your enthusiasm and your contribution to our idea by applying your knowledge of kindlin pull downs to analyse acetylation dependent β1A integrin binding specificities to kindlin. Also many thanks to Prof. Daniel Bouvard for accepting to be part of my thesis committee and agreeing on reading and evaluating my thesis.

Thank you to my repondant Prof. Jean Gruenberg who advised me well during the first difficult starting period for my research project and who impressed me with his immense knowledge, kindness and flexibility.

Thanks are also due to Prof. Thierry Soldati and Prof. Didier Perret who helped me through bureaucratic formalities of the PhD program. I am very grateful for their support and their personal care that I pass all required steps. Furthermore, I really appreciated the atmosphere and discussion of the yearly mini-symposia animated by Prof. Thierry Soldati, many thanks to all participants of these reunions. Your ideas and critical questions animated me to dig deeper into my research topic.

I would like to express my gratitude to Prof. Joszef Kiss, Dr. Kristof Egervari, Dr. Volodymyr Petrenko, Prof. Vesa Hytönen, Prof. Gregory Giannone, Prof. Gerald Kastberger, Dr. Stephanie Frank and Dr. Michael Bachmann representing our precious collaborators in Switzerland, France, Finland, Austria and Germany. They helped us to get more insights into the function of β1A

integrin acetylation. Thank you for the enriching discussions and your encouragement to go further with our research. Dr. Egervari, thank you for introducing me into the world of FRAP experiments, for your constant good mood and your kindness. Prof. Kiss and Dr. Petrenko thank you for your support by in vivo experiments that underlined the importance of β1A integrin acetylation for whole organisms. Prof. Hytönen, thank you for your interest in our idea and your willingness to add your expertise and tool set to our project. Prof. Giannone thanks for hosting me two weeks in Bordeaux and giving me the opportunity to learn more on FRET and trafficking experiments. Prof. Kastberger, thank you for your contribution of your skills in programming and image analysis and taking over the adhesion motility analysis part. Dr. Frank, Dr. Michael Bachmann many thanks for incorporating β1A acetylation into your research, which gave us a

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iv unique view of the behaviour of acetylated β1A integrin on fibronectin dot-gradients as well as its substrate preferences.

Thanks to Prof. Nicolas Mermod for providing the MAR element, Prof. Roger Thierry and Eleonora Ciarlo for sharing with us their library of different KDAC constructs.

Tamara Bollmann, thank you for making yourself available for my questions. You always lent a sympathetic ear to all my professional and personal little problems.

I am so grateful for the financial support from the FSRMM (Foundation Suisse de Recherche sur les Maladies Musculaires) and the DIP UNI. I appreciated the little congresses of FSRMM at Macolin each two years, which permitted me to get to know other young researchers with common interests in muscle disease research.

A special thanks to all former and current members of the lab including our lab technicians Monique Wehrle-Haller, Marie-Claude Jacquier, Nicole Aebischer; our PostDoc Dr. Severine Tabone-Eglinger; my PhD student colleagues Dr. Perrine Pinon, Patricia Vazquez, Zuleika Calderin Sollet, Dr. Martinho Soto Ribeiro; as well as to my interns Rebecca Brogli, Linnea Lagerstedt, Ilena Vincenti, Tatiana Fomekong and Dr. Magali Schnell. I very much appreciated our daily coffee breaks, our laughing, our team spirit, the kindness, the sincerity, the shoulder to lean on and the always open door to each other. Thank you girls for these magnificent four years.

Thank you to my beloved husband Nabil Horr, you are my rock in breaking waves, my motivator, my listener, my everything. I am indebted to your understanding that research also means endless working hours and even weekly shuttling between two homes in our case. Thank you for your patience.

I would like to express my sincerest thanks to my dad and his wife Prof. Gerald Kastberger and Prof. Ilse Kranner who have always helped me with words and deeds during my thesis period.

You comforted me when I lived setbacks, encouraged me in my ideas and had very treasurable feedback to share. Many thanks for your optimism and support.

Last, I would like to thank all my colleagues from the CMU (Centre Médicale Universitaire) and the cell physiology department at the University of Geneva. I acknowledge your advice, ideas, openness, your “Bonjour” in the corridors and the smiles I collected every day, we have spent a great time together.

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II Abbreviations

1G or 4.5G 1g/l or 4.5g/l glucose AcCoA acetyl-coenzyme A AD Alzheimer’s disease

ALS amyotrophic lateral sclerosis CI confidence interval

DMEM dulbecco’s modified eagle medium

DMSO dimethyl sulfoxide DOC deoxycholate detergent ECM extracellular matrix EGF epidermal growth factor EtOH ethanol

FA focal adhesion

FAK focal adhesion kinase FB fibrillar adhesion

FBS fetal bovine serum

FERM 4.1, ezrin, radixin, moesin domain

FN fibronectin

FX focal complexes

h hours

HD Huntington’s disease

ILK integrin linked kinase KMT lysine methyl transferase

LAP long actin promoter

LIM Lin11, Isl-1, Mec-3 mAb monoclonal antibody MAR matrix attachment region MIDAS metal-ion-dependent

adhesive site

min minutes

MP membrane-proximal

nt not transfected

o/n over night

pAb polyclonal antibody PD Parkinson’s disease

PH pleckstrin homology

PTB phosphotyrosine binding PTM posttranslational modification pTyr phospho-tyrosine

RGD Arg-Gly-Asp ROI region of interest

SAHA suberoylanilide hydroxamic acid

SAP short human actin promoter SBHA suberoyl bishydroxamic acid

sec seconds

SEM standard error of mean SH2 Src homology

SH3 Src homology 3

Smb SAP MAR best

sns statistically not significant TIRF total internal reflection

fluorescence

TM transmembrane

TSA trichostatin A

VN vitronectin

WB western blot

wt wild type

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III Résumé

Les intégrines sont des récepteurs hétérodimeriques transmembranaires qui contiennent une chaine α et une chaine β. Ils relient mécaniquement la matrice extracellulaire et le cytosquelette d’actine formant ainsi des adhésions entre la cellule et la matrice. Ces adhésions, dépendantes des intégrines, représentent des sites de signalisation intracellulaire et contrôlent le remodelage de la matrice extracellulaire. L’activation d’intégrine est contrôlée allostériquement par le recrutement des adaptateurs talin et kindlin sur le domaine cytoplasmique de la chaine β de l’intégrine. L’intégrine activée peut se lier avec un ligand extracellulaire. Les intégrines α5β1A

forment deux types d’adhésion avec la matrice extracellulaire ayant des fonctions différentes : des adhésions focales courtes et localisées en périphérique cellulaire qui établissent un ancrage fort avec le substrat et permettent la signalisation intracellulaire; et les adhésions fibrillaires longues et localisées au centre de la cellule qui ont un rôle dans la production des fibrilles de fibronectine dans l’espace pericellulaire. Malgré le rôle critique des intégrines α5β1A dans la déposition de fibronectine lors de la fibrose, les mécanismes impliqués dans la signalisation des intégrines ne sont guère compris dans cette pathologie. Curieusement, β1A porte un site d’acétylation sur la lysine K794 qui se trouve sur la queue cytoplasmique de l’intégrine dans la région du site de liaison avec la kindlin. Par ailleurs, il a été démontré que le knock-out de kindlin mène à un défaut de l’étalement cellulaire et au développement d’adhésions fibrillaires fines et centrales. Ces données indiquent un lien fonctionnel entre l’acétylation de l’intégrine β1A, son affinité pour la kindlin, la création des adhésions fibrillaires et la capacité de fibrillogenèse de la fibronectine. C’est pourquoi nous avons émis l’hypothèse que l’acétylation de la lysine 794 de l’intégrine β1A pourrait fonctionner comme un régulateur qui définit pour chaque β1A intégrine son caractère d’adhésion focale ou fibrillaire. Pour répondre à cette question, nous avons fait des analyses quantitatives d’image, nous avons évalué la conglomération des intégrines et nous avons mesuré la capacité de fibrillogenèse des cellules β1 -/- GD25 ré-exprimant des mutants d’intégrine qui imitent la déacetylation (K794R /ou A) ou l’acétylation (K794Q). En parallèle, et dans le but d’évaluer la régulation des intégrines via l’acétylation, nous avons aussi traité les intégrines endogènes d’une cellule avec des inhibiteurs de déacétylase et des activateurs d’acétyltransferase. Ces populations d’intégrine β1A acétylées, obtenues par traitement avec les drogues ou avec la mutation K794Q, s’organisent dans des adhésions fibrillaires longues et dynamiques qui se dégradent moins, peut-être à cause du blocage de l’ubiquitination des lysines via l’acétylation présente. De plus, ces intégrines, mimant l’acétylation de la lysine, augmentent la capacité de fibrillogénèse de la fibronectine des cellules mais réduisent l’affinité des cellules pour la fibronectine soluble. Les intégrines β1A K794Q stimulent le développement des filopodes des cellules ce qui rend les cellules plus exploratoires avec des bords qui migrent plus vite. À l’opposé, les populations d’intégrine β1A deacétylées, obtenues par des drogues ou avec la mutation K794R β1A, forment des adhésions plus courtes, similaires aux adhésions focales et aux adhésions des intégrines β1D spécifiques au muscle. Ces adhésions des intégrines β1A deacétylées ont plus de stabilité et montrent une affinité élevée pour la fibronectine. En outre, la dégradation des intégrines β1A déacétylées est probablement stimulée par l’ubiquitination des lysines, facilitée par l’absence du groupe acétyl. Les adhésions des intégrines β1A K794R affichent aussi des motilités élevées témoignant d’une plus grande mobilité de l’actine dans ces zones

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viii d’adhésion focale comparée aux zones centrales des adhésions fibrillaires. En résumé, nos résultats confirment qu’une acétylation sur le résidu K794 de l’intégrine β1A active les propriétés d’adhésion fibrillaire du récepteur, induit des changements de morphologie cellulaire et régule sa capacité de modélisation de la matrice interstitielle. Etonnamment, des maladies comme le cancer, les maladies musculaires et neurodégénératives ne sont pas seulement connues pour une déposition de matrice extracellulaire défectueuse, une morphologie cellulaire altérée et des adhésions troublées mais aussi pour un spectre d’acétylation des protéines dérégulé. Ces observations laissent spéculer que la dérégulation de l’acétylation des intégrines β1A peut sous- tendre ces pathologies, ce qui rendrait le site d’acétylation K794 sur les intégrines β1A une cible médicamenteuse attractive.

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IV Abstract

Integrins are heterodimeric transmembrane (TM) receptors consisting of one α and one β- subunit. They provide the mechanical link between the extracellular matrix (ECM) and the actin cytoskeleton and form cell-matrix adhesions. These integrin-dependent cell adhesions are the site of intracellular signalling and are used to remodel the ECM. Integrin function is allosterically controlled by ligand binding to the extracellular domain and recruitment of the cytoskeletal adapter proteins talin and kindlin to the cytoplasmic domain of the β-integrin chain. The α5β1A integrins form two types of cell-matrix adhesions with differential functions:

short, peripherally located focal adhesions (FAs) that provide strong anchorage to the substrate and intracellular signalling; and long, centrally located fibrillar adhesions (FBs) that enable the formation of fibronectin (FN) fibrils in the pericellular space. Despite the critical role of α5β1A

integrins for FN deposition during fibrosis, the integrins’ relevant signalling is not well understood. Interestingly, β1A integrin bears a lysine acetylation site (K794) located within integrin’s kindlin recognition sequence in the cytoplasmic tail. Genetic deletion of kindlin has been demonstrated to handicap cell spreading and the formation of thin, centrally located FBs.

These facts suggest a functional link between integrin acetylation, kindlin binding affinities, FB formation and FN fibrillogenesis capacities. Therefore, we hypothesized that lysine 794 acetylation of β1A integrin might function as regulatory switch which defines for each β1A

integrin its FA or FB character. To address this issue, we performed quantitative image analysis, evaluated integrin clustering and FN fibrillogenesis of deacetylation (K794R/or A) or acetylation (K794Q) mimetic β1A integrin mutants that were expressed in β1-/- GD25 cells. Alternatively, cells expressing endogenous β1A-integrin were treated with deacetylase inhibitors or acetyltransferase activators, in order to evaluate acetylation dependent integrin regulation.

Acetyl-β1A integrin populations obtained via drug treatment or K794Q mutation organized into long, dynamic FBs with reduced degradation, probably due to blocked lysine ubiquitination.

Furthermore, acetyl-mimetic integrins raised the cell’s FN fibrillogenesis capacity but lowered cell affinities for soluble FN. K794Q β1A integrins stimulated cells to form filopodia-like structures, which rendered the cells more explorative with quicker moving cell edges. In contrast, deacetylation mimetic K794R β1A integrins as well as drug-induced β1A integrin deacetylation provided shorter FA-like integrin clusters that equalled in several properties the ones of muscle specific β1D integrins. These deacetyl-β1A integrins displayed increased stability and stronger FN binding affinities. Moreover, deacetylated β1A integrins stimulated integrin degradation probably due to disburdened lysine ubiquitination. K794R β1A integrins also displayed higher adhesion motilities reflecting the raised F-actin speeds in FA areas compared to central FB zones. Briefly, our results confirm that the K794 β1A acetylation switches the integrin’s adhesion properties towards a FB character, provoking changes in cell morphology and the remodelling of the interstitial ECM. Strikingly, diseases like cancer, muscle or neurodegenerative disorders show not only altered ECM deposition, cell morphology, and pathological adhesion characteristics but are also associated with exaggerated acetylation signalling. These observations let us speculate that a deregulated β1A integrin acetylation might underlie such pathologies rendering the K794 site acetylation of β1A integrins into an attractive drug target.

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1

1 Introduction

1.1 Posttranslational modifications

The human genome with 20.000 to 25.000 coding regions (International Human Genome Sequencing, 2004) is much less complex than the human proteome of more than one million proteins (Figure 1.1). Thus, single genes must be the precursor of multiple proteins. So, what mechanism triggers this 50fold diversity increase from the gene recipe to the amino acid chain product? The solution to the puzzle is both, gene transcription variability as well as protein modification after accomplished translation. Hence, each gene can be transcribed into up to six diverse mRNA variants, a number caused by genomic recombination as well as alternative transcription initiation, termination and splicing. Each of these mRNA species gives rise to around 10 differently co- and post-translationally chemically processed proteins in the next regulation level. Overall, this increase in complexity from the gene template to its translated protein product pushes predictions of the human proteome size to a number of 1.5 million (25.000 x 6 x 10) different protein species (Duan and Walther, 2015; Jensen, 2004). This demonstrates that posttranslational modifications (PTMs) of proteins take strongly part in the proteome

diversification. PTMs affect the protein’s conformation, activity, stability, subcellular localization, and its interaction profile with other biomolecules for signal transduction like DNA, cofactors, lipids and partner proteins (Beltrao et al., 2013; Cain et al., 2014; Choudhary et al., 2014; Close et al., 2010; Lu et al., 2013; Venne et al., 2014; Woodsmith et al., 2013). By controlling biological protein function, PTMs are engaged in the regulation of the whole cell physiology including gene

Figure 1.1 - PTMs diversify the proteome: The genome encodes for about 25.000 genes. On average, one gene is transcribed into six distinct mRNAs. After translation of the mRNA into the protein molecule, the protein will be further modified by PTMs. One protein sequence can undergo the covalent addition of chemical groups on one or several amino acids with equal or different modifications. One specific pattern of modifications on all modifiable amino acid residues of a protein is called “mod-form”. Due to the huge number of combinatorial possibilities, each protein can give rise to many different mod-forms at the same time and also over time. Meaning, one protein population may be composed of molecules of different mod- forms at one moment that will further change individually over time (Prabakaran et al., 2012).

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2 expression, cell-to-cell communication along with cell-to-pericellular space signalling (Deribe et al., 2010; Wang et al., 2014). Over the last decade, a growing number of distinct PTM types were revealed to be abundantly present on nuclear and non-nuclear proteins in both, eukaryotes and prokaryotes (Cain et al., 2014; Close et al., 2010; Eichler and Adams, 2005; Walsh et al., 2005;

Wellen and Thompson, 2012). PTMs have been divided into two groups:

Figure 1.2 - PTM frequency on proteins: Statistics of experimentally observed PTM distribution in the Swiss- Prot Knowledgebase on all analysed eukaryotic and prokaryotic proteins show that phosphorylation is currently the most abundant PTM listed followed by acetylation. Last revised: 06/17/2014 (Khoury et al., 2011).

The first category incorporates all covalent additions of a chemical moiety to an amino acid’s side chain of a protein. Over 200 different residue additions are known, each recognized by specific PTM recognition domains (Deribe et al., 2010). Hinging on the type of residue, these covalent reactions vary in formation speed and reversibility (Venne et al., 2014). Consequently, some PTMs are more stable than others, the same or different PTM types often turn up at the same moment on one single protein. In contrast, different PTMs can also occur over time on one single amino acid. The mass of combinatorial possibilities makes the number of potential molecular states extremely elevated (Prabakaran et al., 2012). The chemical residue additions are mainly catalysed by PTM enzymes however several studies point to a second non-enzymatic amino acid modification route via an equilibrium reaction caused by locally high metabolite concentrations (Choudhary et al., 2014; Walsh et al., 2005). The second class of PTM imbeds cleavage of protein backbones (Walsh et al., 2005).

More than a half of all PTM sites annotated in Swiss-Prot are currently amino acid phosphorylations, acetylation is the second most frequent PTM observed followed by glycosylation (Figure 1.2). Methylation represents only a portion of 2% of all measured PTM sites on proteins these days (Khoury et al., 2011). Different PTMs on a single protein of eukaryotes and prokaryotes are assumed to cross-talk with each other to allow coordination between several different protein states (Beltrao et al., 2013; Lu et al., 2011; Venne et al., 2014).

Interestingly, when we compare the number of experimentally validated PTM substrate sites per amino acid, Lysine turns out to be a signalling hub for PTMs. Thus, it is the second heavily modified amino acid evoking affinities for 27 distinct forms of PTMs (Figure 1.3).

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3

Figure 1.3 – Several PTMs compete for one amino acid: Several modifications target the same amino acid residue. Cysteine and lysine show the highest variability and can bear 29 and 27 different PTMs respectively.

Last revised: 02/10/2014 (Lu et al., 2013).

1.1.1 Acetylation

N-terminal and internal protein acetylation reactions can be distinguished: The acetylation of the N-terminus is irreversible and belongs to the class of cotranslational modifications. This chemical reaction takes place in the cytosol during protein translation mostly on N-terminal alanine, serine or methionine residues (Lu et al., 2013; Park et al., 2015; UniProt, 2015). Internal lysine acetylation is the most frequent amino acid acetylation modification (Prabakaran et al., 2012). It is occurring posttranslationally by the covalent addition of an acetyl group to lysine’s epsilon- amino side chain. In contrast to the N-terminal acetylation, this covalent reaction is reversible.

The acetylation process neutralizes the lysine’s positive charge, this hampers electrostatic interaction and renders lysine’s normally hydrophilic character more hydrophobic. The sites where hydrogen bonds can form are also altered by internal acetylation. This provokes a change in protein stability and docking sites. Thus, although the acetyl-residue promotes new hydrogen bonds through its carbonyl group, it also impedes former established hydrogen bonds on lysine’s epsilon-amine (Friis et al., 2009; Soppa, 2010).

The acetyl reaction is balanced through the two complementary enzymes lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) (Figure 1.4) that catalyse the addition or erasure of acetyl groups at internal lysines (Choudhary et al., 2014). Acetylation and deacetylation speeds were identified to achieve half of maximal signal changes within one to ninety minutes (min) (Evertts et al., 2013; Waterborg, 2002). Acetyl groups are originating from

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4 acetyl Coenzyme A (AcCoA) donor molecules. Bromodomains, which are also present in some KATs, are known to recognize and bind such acetyl-lysine groups (Mujtaba et al., 2007). Over the last decade, it has become clear that the acetylation signal machinery is not restricted to the nucleus. Generally speaking, more than 50% of all acetylated proteins in humans are located in the cytoplasm (Lu et al., 2011). Nevertheless, the stoichiometry of the different protein mod- forms varies from one organ to another (Lundby et al., 2012a). Of special interest for this project is the abundancy of acetylation signals identified on cytoplasmic proteins, which are implicated

Figure 1.4 - Lysine acetylation mechanism: The donor AcCoA transfers the acetyl group to the epsilon-amine of lysine’s side chain. The catalysing enzymes in this chemical moiety addition or subtraction process are KAT and KDAC. Several drugs such as resveratrol or SBHA are known to either enhance or block the enzymes’

activity leading to an increase or decrease of an organism’s overall protein acetylation level. glucose and EtOH substrates can be salvaged to become AcCoA suppliers. The increased cytoplasmic AcCoA concentrations may then stimulate enzymatic and non-enzymatic protein acetylation. Adapted from (Choi et al., 2013; Evertts et al., 2013; Friis et al., 2009; Lundby et al., 2012a; Okanishi et al., 2013; Prabakaran et al., 2012; Walsh et al., 2005; Waterborg, 2002; Zhang and Chen, 2012).

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5 in cellular adhesion, communication and cytoskeleton modulation (Choudhary et al., 2009; Lu et al., 2011).

Unexpectedly, KAT mediated acetylation can be circumvented due to locally high acetyl-CoA concentrations, which enable kinetically slow but biologically relevant non-enzymatic protein acetylation reactions (Verdin and Ott, 2015). Moreover, several compounds were demonstrated to either inhibit or increase KAT or KDAC activities. Suberoyl bishydroxamic acid (SBHA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) are claimed KDAC inhibitors (Kim and Bae, 2011). Anacardic acid is an example of a KAT inhibitor (Ghizzoni et al., 2012; Ning et al., 2008; Ravindra et al., 2009) and resveratrol and SRT-501 are declared deacetylase activators (Gertz et al., 2012; Shepard et al., 2010). The overall lysine acetylation can be enhanced when the AcCoA donor concentration rises (Figure 1.5). AcCoA originates from several pathways such as caloric intake, autophagy and bacterial fermentation processes (Choudhary et al., 2014; Zhao et al., 2010). Regularly, acetylation is found surrounded by neighbouring acetylation or phosphorylation signals. This signal accumulation in close vicinity would suggest PTM crosstalk

Figure 1.5 – Normal physiological sources of AcCoA: AcCoA can be delivered in the cell cytoplasm (c), in mitochondria (m) or the nucleus (n) through the fat, sugar and EtOH metabolism, by autophagy in times of nutrient restriction as well as via breakdown of acetate originating from colon’s bacteria fermentation metabolism. The increase of available AcCoA leads to an increase of overall protein acetylation in the concerned cell’s compartment. Acetate is degraded with the help of AcCoA synthetases (AceCS). EtOH is converted through a three step process to AcCoA; first alcohol dehydrogenase (ADH) forms acetaldehyde, then aldehyde dehydrogenase (ALDH) catalyses the conversion to acetate which is again transformed to AcCoA by AceCS. Interestingly the enzymes ADH, ALDH and AceCS are not only restricted to the liver but expressed, albeit at lower levels, in many tissues including cardiac and skeletal muscle (Crabb et al., 2004;

Fujino et al., 2001; Hurley and Edenberg, 2012; Loikkanen et al., 2002). Adapted from (Choudhary et al., 2014; Marino et al., 2014).

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6 to be a common protein signalling mechanism applied for the regulation of cellular function. In fact, in silico modelling confirms the impact of acetylation signals on nearby, maximal five residues distant phosphorylation sites by influencing their kinase binding capacity and their proper phosphorylation status (Lu et al., 2011). Acetylation appears to be often deregulated in disease. For instance, Huntington, Alzheimer (AD), Parkinson’s disease (PD) patients as well as bacterial infections come with significantly enriched protein acetylation. Moreover, KDAC expression and consequently protein acetylation are disequilibrated in oncogenic disease. Lung and pancreatic cancer were identified to have reduced KDAC levels, which leads to an enriched acetylated lysine profile. On the other hand, gastric cancer is linked to lower numbers of acetylated proteins provoked by increased KDAC levels (Lu et al., 2011; Singh et al., 2010a).

The KDACs are classified into four groups (Figure 1.6) based on their sequence similarity to yeast KDACs. Moreover, they can be divided into two families according to their different cofactors:

Zn²⁺ or NAD⁺. In humans out of 18 KDACs 10 can shuttle to the cytoplasm. TSA can compete with Zn²⁺ binding and therefore succeed in inhibiting KDAC classes I/II and IV. KDAC class III is on the contrary due to its different cofactor TSA insensitive (Kim and Bae, 2011; Rajendran et al., 2011;

Singh et al., 2010a). The KATs are grouped into five families in relation to related KAT domains found in yeast, metazoan or fungi: Gcn5/PCAF, HAT1, MYST, p300/CBP and Rtt109. The catalytic mechanism of each KAT family differs remarkably, some KATs also require cofactors. In the Gcn5/PCAF KAT family the bromodomains were revealed to recognize and bind the acetyl-lysine residue (Yuan and Marmorstein, 2013).

1.1.2 Phosphorylation

The chemical addition of a phosphate group (PO4-3 ) from an ATP donor molecule to the hydroxyl group of a target amino acid like tyrosine, threonine or serine is called protein phosphorylation

Figure 1.6 - KDAC classes, cofactors, cytoplasmic and tissue locations: The KDAC classes have different tissue expression patterns, while class I and III are rather ubiquitously expressed, class II and IV are more specific for muscle. Moreover, KDAC 3/4/5/6/7/9/10 and SIRT 2 were demonstrated to shuttle from the nucleus to the cytoplasm allowing cytoplasmic protein deacetylation.

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7 (Figure 1.7). Thereof, serine is the most frequently modified. The transfer loads negative charge onto the concerned amino acid modulating the protein’s structure, activity and available docking sites (Lanouette et al., 2014). Enzymes charged of phosphorylation are called kinases; opposed

Figure 1.7 - Amino acid phosphorylation mechanism: Protein phosphorylation is rapid and reversible.

Kinases phosphorylate within 20min half of all phospho-sites whereas phosphatases take up to 4 h to process dephosphorylation of half of all the concerned sites. To increase phosphorylation, one can either administer okadaic acid or hydrogen peroxide to block phosphatase activity, or add growth factors like EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) to increase kinase activity. In contrast, drugs like imatinib and trastuzumab block kinase activity, and in consequence reduce protein phosphorylation levels.

Adapted from (Arena et al., 2005; Baumann et al., 2010; Bennetzen et al., 2010; Prabakaran et al., 2012;

Weiser and Shenolikar, 2003).

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8 to them are phosphatases, which manage the complementary hydrolytic process of dephosphorylation. Tyrosine-phosphorylation signals are recognized by binding domains such as Src homology 2 (SH2) and phospho-tyrosine binding (PTB) domains, and serine/threonine phosphorylation sites by binding proteins 14-3-3 and domains such as WD40 (Tzivion et al., 2001).

The high dissociation constant of ATP makes kinases unlike KATs operate close to independently from the ATP donor concentration. Phosphorylation adds a relatively big mass of 80-95 Da to the appropriate amino side chain. Phosphate group addition is a rapid process with not more than 20 min to reach half of maximal phospho-modification; dephosphorylation is possible within less than 4 hours (h) (Bennetzen et al., 2010).

Several human cancers, like the HER-2 (kinase) amplification in breast cancer, the KIT (kinase) receptor point mutations in gastrointestinal cancer as well as SHP2 (phosphatase) mutations in solid tumours were linked to gain or loss of function mutations on these kinase or phosphatase genes. That is why a therapeutic strategy consists of blocking or interfering with the enzyme’s activity. So, imatinib inhibits KIT kinases and trastuzumab blocks HER-2 receptor tyrosine kinases.

On the other hand okadaic acid is a serine/threonine phosphatase blocker and H2O2 is known to inhibit tyrosine phosphatases like SHP2 is one (Arena et al., 2005; Baumann et al., 2010;

Bennetzen et al., 2010; Prabakaran et al., 2012; Weiser and Shenolikar, 2003).

1.1.3 Methylation

DNA, nuclear and cytoplasmic proteins can be methylated by the transfer of a methyl group from the S-adenosylmethionine donor molecule (UniProt, 2015). Among proteins, methylations occur on N- and C-termini of proteins (Bedford and Clarke, 2009) as well as on a variety of carbon-, oxygen-, and sulphur-groups of amino acid side chains (Walsh et al., 2005). Nevertheless, lysine represents the most prevalently methylated amino acid (Lu et al., 2013; Yang and Seto, 2008) . Lysine methyl transferases (KMTs) can catalyse the binding of one, two or three methyl groups (Figure 1.8) on lysine’s epsilon-amino group (Prabakaran et al., 2012) which concurrently blocks acetylation access and by this means increases the protein’s stability (Egorova et al., 2010).

Lysine-Demethylases (KDM) allow methylation reversibility, it could be demonstrated that a trimethylated status of lysine shows the lowest turnover with a stability of one day (1440min) before the amine gets demethylated. Methylation provokes only a small mass change with 14, 28 or 42 Da, compared to phosphorylation, and does not alter the positive charge of lysine. Each methyl group addition removes a potential hydrogen bond donor and increases hydrophobicity of the former hydrophilic lysine. Due to lysine methylation, protein shape and docking sites are modulated leading to altered protein/DNA and protein/protein interaction (Lanouette et al., 2014; Walsh et al., 2005). Methylation can interact with other PTMs. One reader domain for methylated lysine is known to be the chromodomain. Strikingly, the chromodomain is present in an acetyltransferase serving as activation switch to trigger lysine acetylation (Egorova et al., 2010). Accordingly, also other examples of crosstalk mechanisms between lysine methylation and neighbouring phosphorylation, acetylation or ubiquitination signals have been detected (Beltrao et al., 2013; Venne et al., 2014; Woodsmith et al., 2013; Yang and Seto, 2008).

We are now aware of the implication of lysine methylation in oncogenic signalling pathways responsible for cell growth, proliferation and differentiation; even many kinases are confirmed

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9 substrates for KMTs. Therefore, learning about deregulated KMTs and an aberrant methylation profile in several types of cancer may come as no surprise (Biggar and Li, 2015; Lanouette et al., 2014). Furthermore, methylation has been linked to bacterial pathogenicity, helping bacteria to adhere, invade or to protect themselves against proteolytic cleavage. KMT and KDM modulating drugs, like trifluoroacetate salt or GSK J4 HCl, may be a means to treat the revealed methyl signal dysregulation for above-mentioned human pathologies.

1.1.4 Glycosylation

Glycosylation, one of the three most prevalent protein modifications (Figure 1.3), is referred to as the enzymatic covalent binding of mono- or oligosaccharides (glycan) to asparagine via an N-protein sugar linkage, serine and threonine via an O-linkage and tryptophan through a C-link (UniProt, 2015). Glycoproteins bear one or more of these N-, O- or C linked glycans, carbohydrates that typically have a bigger size than the naked protein substrate itself and consequently, constitute up to 80% of the glycoprotein’s mass (Rhodes, 1997). In mammals, not more than ten distinct monosaccharides, comprising amongst others glucose, galactose, N-acetylglucosamine or N-acetylgalactosamine, form the basic building block for a myriad of more or less complex glycostructures that all differ in sugar numbers, sequence and branching. N- and C-glycosylations are irreversible, occurring co- (endoplasmatic reticulum) and posttranslationally (Golgi apparatus) on membrane-, extracellular- and secreted-proteins. Despite the claimed irreversibility of an established asparagine-sugar link, the attached glycan moiety may experience dynamic alterations in sugar composition and branching during a protein’s life.

Figure 1.8- Lysine methylation mechanism: Trimethylated lysine is a rather stable PTM with measured half lives on histones of about 1 day (1440 min). Lysine gets methylated by KMTs, the more methyl groups are added the longer it takes for the methylation process. The drugs trifluoroacetate salt or UNC0631 inhibit KMT’s activity. Demethylation by KDMs takes principally less time than methylation and displays the same trend like KMTs with the longest processing times for tri-methylated lysines. GSK J4 HCl is a drug that is capable of inhibiting KDM activity (Fodor et al., 2006; Zee et al., 2010).

+

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10 In contrast, O-linked glycosylation is reversible and may take place post translation in the Golgi apparatus, cytosol or nucleus on many intracellular and extracellular proteins. Serine or threonine O-linked glycosylation may compete with phosphorylation signals on the same substrates. By this means, O-linked glycomodification may take part in the regulation of signalling pathways (Stowell et al., 2015; Theillet et al., 2012; UniProt, 2015; Walsh et al., 2005).

Interestingly, glycosylation status depends on the nutritional cell environment. High blood glucose concentrations (hyperglycemia), observed in diabetes or obesity, are known to foster abnormally elevated O-glycoprotein levels (Baudoin and Issad, 2014). Enzymes that transfer the sugar molecules from UDP-, GDP- or CMP-“activated” sugar donors to concerned protein substrates are named glycosyltransferases (GT). Their opponents catalysing the cleavage of sugar blocks are known as glycosidases. Glycosylation is known not only in eukaryotes but also prokaryotes, archea and viruses. Protein glycosylation patterns are like acetylation, tissue- and cell specific (Stowell et al., 2015; Theillet et al., 2012; UniProt, 2015).

Glycosylation influences protein localization, stability and activity. Protein glycosylation prevents protein aggregation during the folding process by rendering proteins more soluble. Glycosylation influences the protein’s conformation, stability, it allows the correct targeting to intra- and extracellular destinations, inhibits random proteolysis and provides docking and recognition sites for carbohydrate recognizing proteins such as lectines. Glycosylation is a common modification of cell membrane proteins that participate in cell-environment and cell-cell interactions. Immune responses and hormone action depend also on the glycosylation signals of proteins (Wang et al., 2014). However, glycosylation should not be confused with glycation, a non-enzymatic, over time irreversible, N-glycosylation reaction of α-amino group of the N-terminal amino acid of proteins, or on the amino group of lysine, hydroxylysine, arginine, histidine as well as with cysteine side chains. Glycation is induced by exposure to glucose and provokes changes in protein conformation and function. The early sugar-peptide adducts are still reversible but can transform into irreversible so called advanced glycation end products (AGEs) implicated in various diseases that come with abnormal protein glycation like diabetes and a range of age-related neurodegenerative disorders including AD, PD, and Huntington disease (Li et al., 2012; Neelofar and Ahmad, 2015). Defects in the glycosylation machinery have a strong impact on the human development and lead to organ dysfunctions like in the rare congenital disorder of glycosylation (CDGs). In many diseases, like rheumatoid arthritis, cystic fibrosis, AIDS and a range of cancers, the glycosylation pattern is altered due to a disease related glycosyltransferase/glycosidase activity or expression changes (Anugraham et al., 2014; Lisowska and Jaskiewicz, 2001).

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11

1.2 Integrins

Integrins are heterodimeric TM receptors consisting of one α and one β-subunit, they provide the mechanical link between the ECM and the actin cytoskeleton and form cell-matrix adhesions (Figure 1.9). These integrin cell adhesions remodel the ECM, a process, which is mediated by intracellular adaptor signalling. Not all integrin heterodimers are in the same state. Integrins that a capable of binding to extracellular ligands are termed “active”. The inactive integrin population shows low ligand affinity and does not signal. Integrin activity is allosterically switched-on by the recruitment of the cytoskeletal adaptor proteins - including talin and kindlin - to the cytoplasmic domain of the β-integrin chain.

Integrins mediate cell adhesion, cell migration and signal transduction processes that are involved in tissue organization, cell development like cell growth, differentiation, apoptosis and gene regulation, host defence and haemostasis. Consequently, integrins play a crucial role during embryogenesis by guiding cells to their final destination for building up organs. Integrins help maintain organ and tissue structures by exhibiting firm cell-matrix adhesions, and allow cell dynamics for tissue repair mechanisms. However, bacterial and viral pathogens exploit integrins for cell attachment or internalisation (Kerr, 1999; Stewart and Nemerow, 2007). Integrin mutation or abnormal integrin function was demonstrated to lead to aberrant development and diseases such as cancer metastasis, bleeding disorders, leukocyte-adhesion deficiencies, skin blistering or muscular dystrophies (Campbell and Humphries, 2011; Harburger and Calderwood,

Figure 1.9 -Integrin structure and functions: Integrins are TM proteins that link the ECM with the actin cytoskeleton. The intracellular integrin tail is a signalling hub where different adaptor proteins bind in order to exchange information between the cytoplasm and the pericellular space. Several integrin receptor molecules cluster together into adhesions on the cell membrane, which enables the cell to attach, polymerize ECM proteins into fibres and migrate.

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12 2009; Hynes, 2002; Mayer, 2003; Moser et al., 2009; Pinon and Wehrle-Haller, 2011; Yamada et al., 2003).

In humans, 18 α-subunits (α1-11, αD, αE, αL, αM, αV, αX, αIIB) can associate with eight different β- chains (β1-8) to form 24 distinct integrin heterodimers in a restricted manner and each exhibiting a specific ligand binding affinity. One gene encodes for one α or β-subunit (Takada et al., 2007).

The membrane-proximal (MP) sequence at the α-chain associates non-covalently by electrostatic interactions with the β-chain (Banno and Ginsberg, 2008; Shattil et al., 2010). The α-chain superfamily has a length between 1000 and 1150 amino acids, with a sequence similarity of 18- 63% and a mature size of 120-210 kDa. β-subunits exhibit 28-55% sequence similarities, they are composed of 730-800 amino acids and their protein weight ranges between 90 and 130 kDa (Krissansen and Danen, 2001; Takada et al., 2007).

Integrins are composed of one long and two short domains. Thus, both integrin subunits can be divided into three regions (Figure 1.11), an N-terminal large extracellular ecto-domain comprising more than 700 amino acids, one TM domain of about 25-29 amino acid residues and a C-terminal short cytoplasmic domain with 13-70 amino acids length, except for the β4 subunit which has ten times longer cytoplasmic domain of about 1000 amino acids (Banno and Ginsberg, 2008; Zhang and Chen, 2012). All integrins are glycoproteins carrying over 20 potential N-glycosylation sites on their extracellular domain (Janik et al., 2010). The integrin head provides the ligand binding structure and the following separate stalks link the globular head domains to the plasma membrane (Krissansen and Danen, 2001). The α and β integrin subunits form together a globular head in the extracellular integrin domain, which is capable of binding to ECM ligands like fibronectin (FN) or vitronectin (VN). As discussed above, integrins that possess a high ligand binding affinity are called activated. Different extracellular divalent cations are known to trigger

β₁

α₁ α₂ α₃ α₄ α₅ α₆ α₇ α₈ α₉ α₁₀ α₁₁ α_V

β₃ β₅ β₆ β₈

α_IIB β₄

β₇ α_E β₂

α_D α_L α_M α_X

Figure 1.10 – Integrin heterodimers in humans: In humans 24 different integrin heterodimers can form. β1

integrin shows the biggest combination capacity, contributing by twelve different integrin receptors. The α chains in violet contain an additional I-domain subunit. The αvβ3 integrin is a multiligand receptor capable of binding several ECM proteins including FN, VN, tenascin or thrombospondin. α5β1 integrin is more specific for FN. Adapted from (Hynes, 2002; Krissansen and Danen, 2001).

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13 changes in integrin activation states. Such as Ca²⁺ addition that lowers integrin’s ligand binding affinity, whereas Mn²⁺ strongly and Mg²⁺ to a lesser extent promote integrin activation and adhesiveness (Humphries, 2000; Krissansen and Danen, 2001; Moser et al., 2009; Srichai and Zent, 2010; Takada et al., 2007; Tiwari et al., 2011; Zhang and Chen, 2012). Integrins’ TM domain is composed of hydrophobic residues and has highly conserved features among all 18α and eight β-integrin subunits. Both subunits span the cell membrane by forming an α-helix, however, the integrin α-subunit passes the membrane perpendicularly in contrast to the β-integrin which traverses the cell membrane in a more tilted way (Calderwood et al., 2013; Campbell and Humphries, 2011; Rao et al., 2010; Srichai and Zent, 2010). After the TM domain follows integrin’s short cytoplasmic tail domain starting with a MP region, which has a certain similarity within each group of α and β chains. Generally, cytoplasmic sequences are more conserved in β tails and more heterogeneous in α subunits. At the cytoplasmic tails, adaptor protein binding can establish a dynamic link between the cytoskeleton and the ECM. This cytoplasmic tail signalling of integrins allows allosteric, extracellular regulation of integrin’s ligand binding activity. Almost all β tails possess two highly conserved NXXY motifs. The first membrane proximal NPXY motif is a binding

site for talin and the second membrane distal NXXY region serves as kindlin binding domain (Hynes, 2002; Moser et al., 2009; Srichai and Zent, 2010).

Figure 1.11 - Integrins’ domain architecture: α and β integrins are composed of an ecto-, a transmembrane and a cytoplasmic tail domain. The extracellular portion of the α subunit comprises a ligand binding β- propeller domain, a thigh domain and two calf domains. The β-integrin extracellular domain consists of a ligand binding βI-domain including a metal-ion-dependent adhesive site (MIDAS), a PSI (plexin/semaphorin/integrin) domain, one hybrid domain, four EGF domain repeats and a membrane proximal β-tail domain (Srichai and Zent, 2010). Several α-subunits (α1, α2, α10, α11, αL, αM, αX, αD, αE) have an additional ~200 amino acid long I domain (“I” stands for insertion or interaction) incorporated into the β- propeller sequence and comprises another MIDAS motif. Each MIDAS motif in the β-integrin subunit and in the α-I containing integrin subunit bind one or more of the divalent metal cations Ca²⁺, Mg²⁺, Mn²⁺to regulate protein ligand-binding (Humphries, 2000; Krissansen and Danen, 2001; Moser et al., 2009; Takada et al., 2007). I-domain-less integrin α-subunits include α3, α4, α5, α6, α7, α8, α9, αV, αIIb. Adapted from (Danen, 2001;

Krissansen and Danen, 2001; Moser et al., 2009; Xiao et al., 2004; Zhang and Chen, 2012).

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14 The integrin β-subunits β1, β2, β3, β4 and β5 form several splice variants leading to distinct cytoplasmic domains and alternative localization, function and activity (Danen, 2001). β1 integrin exhibits the biggest combinatorial flexibility with twelve heterodimer types (Figure 1.10) formed due to diverse α-chain combinations, β3 for example can dimerize with only two α-subunit partners (Hynes, 2002). The β1 integrin as well as the β3 integrin subunit are capable of establishing cell-matrix adhesions by clustering together hundreds of α5β1 and/or αVβ3

heterodimers (Solowska et al., 1991; Yamada et al., 2003; Zamir et al., 2000). β1 integrin is expressed in all human cell types except erythrocytes, and β3 integrin is expressed at least at low levels in most normal tissues (Armulik, 2002; Wilder, 2002). The integrin adhesion and signalling machinery is crucial but not specific for metazoa. Thus, the integrin gene family has evolved from unicellular progenitors well before the origin of metazoa and fungi. Interestingly, even one bacterium (T. erythraeum) has been discovered, which expresses one integrin β domain (Sebe- Pedros et al., 2010). In multicellular organisms, the number of integrins expressed is dynamic during development and varies between cell types. That is why, one has to expect two to ten different integrin types present simultaneously in one single cell (Glukhova and Koteliansky, 1995). In higher metazoan, both integrin subunits derive by gene duplication from one common metazoan ancestor - the sponge G. cydonium, which has one α and one β subunit at its disposal.

Adjacently, the integrin superfamily develops and diversifies in multicellular organisms from corals, nematodes and echinoderms to mammals systematically with more complex nervous systems, musculatures, circulatory and immune systems arising. The nematode C. elegans possesses two α and one β subunits allowing the formation of two integrins. The fruit fly D.

melanogaster, can already combine five α subunits with one β integrin. In vertebrates the α- chain’s extra-inserted I domain comes up and the integrin family expands continuously (Hughes, 2001; Hynes, 2002; Krissansen and Danen, 2001; Srichai and Zent, 2010; Takada et al., 2007).

1.2.1 β

1

Integrin family

The β1 integrin subfamily is the building block for twelve heterodimeric adhesion proteins and represents the largest subfamily among the integrin receptors. The human β1 integrin exists as five different splice variants, β1A, β1B, β1C-1, β1C-2, β1D, which differ only in the last 12-29 amino acids of their cytoplasmic domain (Figure 1.12). The β1 integrin splice variants’ transcripts are found in

many human tissues except for β1D, which is only skeletal and cardiac muscle specific. The splice variants do not alter the ligand binding specificity for a given heterodimer but differ in localization and signalling characteristics. β1A, and β1D integrins show significant homology throughout their

Figure 1.12 - β1 integrin cytoplasmic splice variants: Cytoplasmic tail amino acid sequences (from amino acid position 752-798 for β1A integrin) of all five β1 integrin splice variants. The membrane proximal and membrane distal NXXY motifs are marked in violet. Adapted from (Armulik, 2002; Hornbeck et al., 2015; Liu et al., 2014).

Metabolic control of cell adhesion and migration through beta1A integrin acetylation : be aware of the acetylation switch!

Thesis

Reference

Metabolic control of cell adhesion and migration through beta1A integrin acetylation : be aware of the acetylation switch!

Metabolic Control Of Cell Adhesion And Migration Through β

Integrin Acetylation

THÈSE

Birgit KASTBERGER

Be aware of the acetylation switch!

by B

K

Metabolic control of cell Adhesion and Migration

through β 1A integrin Acetylation

Table of Contents

I Acknowledgements

II Abbreviations

III Résumé

IV Abstract

1 Introduction

1.1 Posttranslational modifications

1.1.1 Acetylation

1.1.2 Phosphorylation

1.1.3 Methylation

1.1.4 Glycosylation

1.2 Integrins

1.2.1 β

Integrin family