Influence of caspase-binding ability of the X-linked Inhibitor of Apoptosis Protein (XIAP) on serine protease inhibitor-sensitive apoptosis

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Influence of caspase-binding ability of the X-linked Inhibitor of Apoptosis Protein (XIAP) on serine protease inhibitor-sensitive apoptosis

DISSERTATION

zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.)

des Fachbereichs für Biologie an der Universität Konstanz

vorgelegt von

Michael Hans-Martin Walliser

Tag der mündlichen Prüfung: 20.07.2005 1. Referent: Prof. Dr. Albrecht Wendel

2. Referent: PD Dr. Christian Schudt

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Meiner Frau.

Meinen Kindern.

«Niemals gegen den Zweifel leben!

Sondern immer und in jeder Lage mit dem Zweifel!»

Joachim Fest

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Danksagung

Die vorliegende Arbeit wurde im Zeitraum von Dezember 2001 bis Mai 2005 unter der Leitung von Prof. Dr. Albrecht Wendel am Lehrstuhl für Biochemische Pharmakologie im Fachbereich Biologie der Universität Konstanz angefertigt.

Mein größter Dank gilt denn auch Prof. Dr. Wendel für die freundliche Aufnahme an seinem Lehrstuhl, seine Unterstützung meiner Arbeit sowie für die hervorragenden Arbeitsbedingungen.

Für die Betreuung möchte ich mich bei Dr. Gerald Künstle und Dr. Thomas Meergans bedanken.

Letzterem vor allem für seine stete Bereitschaft zur umfassenden und kritischen Diskussion sowie für die daraus resultierenden Ideen. Außerdem für die Bereitstellung einiger entscheidender experimenteller Modelle.

Weiter war meine Aufnahme in das von Prof. Dr. Albrecht Wendel zusammen mit Prof. Dr. Klaus P. Schäfer (ALTANA Pharma AG, Konstanz) gegründete und geleitete Graduiertenkolleg „Biomedizinische Wirkstoff-Forschung“ eine große Ehre. Als dessen Mitglied wurden mir exzellente Möglichkeiten geboten meine Promotion in einem größeren Rahmen als Ausbildung zu verstehen. Darunter zählten nicht nur die teils im Ausland besuchten hervorragenden Fortbildungskurse und Seminare sondern auch die freundschaftlichen Kontakte innerhalb des Kollegs.

Darum gilt ein besonderer Dank ALTANA Pharma, vor allem Prof. Dr. Schäfer, für das großartige Engagement, PD Dr. Jutta Schlepper-Schäfer für die hervorragende Koordination innerhalb des Graduiertenkollegs und PD Dr. Christian Schudt für die Begutachtung der vorliegenden Arbeit.

Prof. Dr. Werner Hofer möchte ich für seine freundliche Verbundenheit und seine Bereitschaft mir als Prüfer zur Verfügung zu stehen herzlich danken. Letzteres gilt auch für Prof. Dr. Alexander Bürkle. Für die Durchsicht des Manuskripts danke ich Georg Dünstl, Thomas Meergans und Rupert Barensteiner.

Frau Gudrun Kugler möchte ich ganz herzlich für ihre zahllosen Hilfeleistungen danken.

Astrid Leja, Verena Tautorat, Georg Dünstl und Markus Müller sowie all meinen Laborkollegen besonders Petra Gais, Carolin Rauter, Petra Richl, Christof Schläger, Timo Weiland und Markus Weiller danke ich für eine wunderbare Zeit.

Den meinen Weg begleitenden Freunden Jürgen Härtenstein, Oliver Bauerle und Steffen Sterkel danke ich eben dafür.

Mein größter Dank jedoch gilt meiner großen und – besonders – meiner kleinen Familie, allen voran meiner Frau Mirjam Christina und meinen Söhnen Ruben Johan-Ernst und Jonah Johan-Richard für ihre übergroße Geduld mit mir und meiner Arbeit – und für ihre Liebe.

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TABLE OF CONTENTS

4.2.2.3.1 Inhibition of cell death by XIAP ∆RING 39 4.2.2.3.2 Lack of the RING domain results in absence of auto-ubiquitination 40 4.2.2.3.3 Ubiquitin ligase activity contributes to inhibition 40 4.2.2.4 A cellular assay for direct inhibition of caspase-3 by XIAP mutants 41 4.2.2.5 Caspase-binding ability is essential for inhibition by XIAP 43 4.2.2.6 Co-expression of the reciprocal mutants XIAP DWE and XIAP ∆RING 44 4.3 A death receptor-independent model of HeLa cell apoptosis 45

4.3.1 Auto-activation and inhibition of over-expressed caspase-8 45 4.3.2 XIAP inhibits cell death caused by caspase-8 over-expression 46 4.3.3 TLCK inhibits cell death caused by caspase-8 expression after caspase arrest 47 4.4 Breakdown of mitochondrial membrane potential and apoptosis 48

5 DISCUSSION 50

5.1 Execution of death receptor agonist-induced apoptosis 50 5.2 Protection of cells by XIAP and XIAP variants 53

5.2.1 Mutant forms of XIAP 54

5.2.2 Direct inhibition of caspase-3 by XIAP mutants 57 5.2.3 Caspase binding ability is essential for protection 58 5.2.4 Complete protection needs the XIAP subunits on one molecule 58 5.3 A death receptor-independent model of HeLa cell apoptosis 60 5.4 Breakdown of mitochondrial membrane potential and apoptosis 62

6 SUMMARY 64

7 ZUSAMMENFASSUNG 65

8 REFERENCES 66

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1 INTRODUCTION

1.1 Programmed cell death

At present two main forms of cell death - necrosis and programmed cell death (PCD) – have been identified. Necrosis is a violent and quick form of cell death affecting large areas within the tissue. Necrotic cells show characteristics like cytoplasm swelling, destruction of organelles, and disruption of the plasma membrane. The poured out cytoplasm leads to necrotic death of neighboured cells and in consequence inflammation of large areas. It is obvious that this form of cell death is limited to pathological events ¹ like physical or chemical injury, acute anoxia, or massive shortage of nutrients ². A remarkable type of cell death distinct from necrosis is programmed cell death. It has been defined for developmental processes underlying a genetic “program”, classically. Nevertheless, the term “programmed cell death” nowadays is used for all sequentially executed cell deaths, including apoptosis. Therefore, Assuncao et al. proposed to characterize programmed cell death as “a sequence of events based on cellular metabolism that lead to cell destruction” ¹. This type of cell death can be observed both in pathological and physiological processes ³. Since this work is subjected to death receptor-mediated apoptosis, only programmed cell death accompanied with apoptotic morphology will be looked at in the following.

Cell death with apoptotic morphology can be triggered by different stimuli, including death receptor-mediated signalling and intracellular stress. All these stimuli feed into a conserved sequence of execution of cell death. The dominant enzyme family herein is that of so-called caspases. These cysteine-proteases are held responsible for most of the visible changes within the cell undergoing apoptosis ⁴. The changes are characterized by cell shrinkage, blebbing of the plasma membrane and condensation of DNA, which occurs at the molecular level as internucleosomal cleavage of the DNA. Although this is seen late and not very distinct in necrotic cells, it is an early event in apoptosis ^2,5. During apoptotic cell death, the organelle integrity is maintained whereas the asymmetric distribution of membrane phospholipids like phosphatidylserine (PS) is destroyed. Phosphatidylserine exposure on the surface of the cell serves as stimulus for macrophages to phagocyte the apoptotic cell or parts of it ². This ensures removal before any cytosolic content of the cell can damage

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1 INTRODUCTION

neighbour cells ¹. Additionally, to avoid an inflammatory reaction against apoptotic cells, enzymes responsible for inflammatory induction are inactivated ⁶.

1.1.1 Caspases 1.1.1.1 Classification

The name “caspase” was composed of the “c” derived from their cysteine protease mechanism and the term “asp-ase”, referring to their cleavage ability after an aspartic acid ⁷. Caspase analogues are evolutionary wide spread and found in yeast ⁸, worms up to humans. So-called meta- and paracaspases are found even in some bacteria, plants, fungi, protists, and animals ⁹. Caspases are expressed as latent zymogens, the pro-caspases. In humans, three caspases (caspases 1, 4, and 5) are related to the activation of pro-inflammatory Cytokines whereas caspase-14 is discussed to play a role in keratinocyte differentiation. The other seven (caspases 2, 3, and 6-10) are employed by the apoptosis machinery. For the last, a classification has established due to their mode of action: initiator and executioner caspases.

Caspase-2, and 8-10 belong to the group of initiator caspases ^1,7,10. These caspases convert apoptotic signal into proteolytic activity ⁷. Adapter molecules that bridge to apoptotic signal transduction achieve recruitment and activation of caspases. This interface is a critical point in living and dying of the cell. At this point, the integration of death signals, their amplification, and their regulation take place ⁷. All other apoptosis-related caspases are called executioner caspases, particularly caspase-3, 6, and 7 ^1,7,10. They take part in proteolytic inactivation and execution of the apoptotic signals. These proteases specifically cleave up to 100 proteins during apoptosis ⁷. For a better understanding, the caspase substrates can be classified within three groups ⁴.

Group I: Other caspase proteases.

Examples are pro-caspase-1, pro-caspase-3, pro-caspase-7 ², pro-caspase-8, pro- caspase-9 ¹¹, and pro-caspase-10.

Group II: Cellular proteins that need to be inactivated for cell death to occur.

For instant, PARP (DNA repair), ICAD (inhibitor of caspase-activated DNAse), and GRASP65 (Golgi reassembling and stacking protein) are identified as loss of function proteins after cleavage. Caspase-3 and 7 cleave all this proteins. Nevertheless, other caspases are cleaving group II substrates, as well. For instant, caspase-8 cleaves the

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1 INTRODUCTION

pro-apoptotic Bcl-2 member Bid ⁷. Finally, lamins, fodrin, and actin are important structural proteins necessary for maintenance of cell shape.

Group III: Cellular proteins whose activation is required for execution of cell death.

Protein Kinase Cδ and ROCK I ⁷ become activated after cleavage and are implicated in membrane blebbing and apoptotic body formation. Therefore, they are classified as gain of function proteins after cleavage ^4,7.

1.1.1.2 Structure

The two groups – initiators and executioners – of caspases share domains that are very similar among the family such as the large and small catalytic domain separated by a linker region. Proteolytic cleavage of this linker liberates the two catalytic subunits during activation. Additional cleavage removes the N-terminal prodomain and the liberated large and small catalytic subunit join to form the active site within this heterodimer. Next, two of these heterodimers join to form the entire active tetramer ².

Aside from this similarity, they differ at their N-termini. Initiator or apical caspases (from the point of activation sequence) tend to have long N-terminal pro-domains containing protein-protein interaction motifs that are also present in adapter proteins.

With the domains CARD (CAspase Recruitment Domain) and DED (Death Effector Domain) the adapter molecules are able to recruit the caspases ^7,12.

In contrast the executioner caspases posses only a small N-terminal pro-domain that is removed during apoptosis. This N-terminal pro-domain is called N-peptide ⁷ sometimes.

1.1.1.3 Caspase activation

1.1.1.3.1 Induced-proximity model

The member of the TNF-receptor family ¹³ Fas (CD95/Apo-1) has been chosen to investigate the mechanisms of activating caspase cascade. Since an activating antibody against Fas is sufficient to induce apoptosis, it was possible to co-precipitate components of the DISC. Surprisingly, it revealed that only Fas, the adapter molecule FADD, and caspase-8 are forming an active DISC. These results showed that the action of the death receptor is limited to recruitment. Moreover, this recruitment brings the caspase molecules into close proximity, that enables dimerization of the

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1 INTRODUCTION

caspase-8 monomers, and it seems to be sufficient to form the active site. Cleavage appears not to be required but seems to provide stability to the dimer generation on the DISC. Following dimerization, the N-terminal DED domains are cleaved off, presumably allowing the activated caspase to be released into the cytosol ^14-18. To understand the activation by dimerization, it is required to take notice of the unusual properties of caspase zymogens that set them apart from most known proteases.

Unlike other proteases, simple expression of caspase zymogens (e.g. in E. coli) usually results in their activation. The zymogenicity, defined as the ratio of the activity of a processed protease to its zymogen, is known for some caspases (see Table 1.1).

In stark contrast to caspase-3, caspase-8 and 9 show only little differences between their activities as zymogen and the entirely activated protease, 100 and 10times, respectively. Consistent with this, a caspase-8 mutant that cannot be cleaved anymore exhibited 1% of the cleavable wild type caspase activity ¹⁴. Confirming that, a non-cleavable caspase-9 mutant displayed 10% of complete activity under conditions similar to cytochrome c release from mitochondria ¹⁵. These results arouse suspicion that cleavage alone is not sufficient to generate active caspase-9. Instead, the activity is achieved by small-scale rearrangements of the surface of the molecule, which can be attained by dimerization of the Caspase-9 monomers at the apoptosome. Interestingly, these dimers have only one active site because of steric obstruction whereas the non-active domain is almost identical with the zymogen form of caspase-7 ¹⁸. Though this circumstance facilitates the activation by local proximity, it makes a tight system of repressing unwanted proteolytic activity necessary.

Possibilities are either permanent inhibition by cellular IAPs ¹⁶ or specific subcellular localization ¹⁹.

Protease Zymogenicity

Caspase-3 >10,000

Caspase-8 100

Caspase-9 10

Table 1.1 | Zymogenicities of some caspases

The zymogenicity is defined as the ratio of the activities of the processed protease to its zymogen.

Table adapted from Salvesen and Dixit, 1999 ¹⁶

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1 INTRODUCTION 1.1.1.3.2 Proteolytic activation

In contrast to caspases-8 and 9, the executioner pro-caspases-3 and 7 exist at the cytosolic concentrations as inactive dimers already ¹⁷. These zymogen dimers require cleavage of the linker for their activation by another caspase ^2,17,18. The difference between caspases 8, 9 and 3, 7 concerning the dimerization is the fact that the dimerization interface of the last group is much more hydrophobic. Specifically, kD for dimerization of caspase-3 is more than three orders of magnitude tighter than of caspase-8 ¹⁸.

1.1.2 Initiation of apoptosis

The currently known pathways prompting apoptosis can be divided into three main groups ¹. The extrinsic pathway triggered by death receptors, the intrinsic pathway triggered by events concerning cell organelle integrity, and strategies of direct target cell killing by immune cells. Cells treated with death receptor agonists can execute apoptosis by activation of caspase-3 either by caspase-8 directly ²⁰ or by mitochondrial reaction to caspase-8 activity. These different types of cells are called

“type I” and “type II”, respectively ¹. In some cases, caspase activity could be restored to basal level and the cell survived after the apoptotic stimulus was removed. This reversible caspase activation could be a regulatory process in the initiation of apoptosis somehow ²¹.

1.1.2.1 Death receptors

First evidence for triggering of apoptosis by receptors as well as caspase aggregation came from the death receptor Fas. The death receptors TRAIL-R1 (TNF-Related Apoptosis Inducing Ligand Receptor), TRAIL-R2, TNFR1 (TNF Receptor), and Fas/CD95 are a subgroup of the tumour necrosis factor (TNF) superfamily that possess a death domain (DD) ¹³. This domain is responsible for recruitment of the adapter molecules like FADD and TRADD which, in turn, bring caspases into close proximity that activates them by dimerization ^12,16,18. Several publications discuss the differences initiating apoptosis by Fas and TNFR1 ^22,23. Recently some groups published the presumption that, in contrast to Fas-induced FADD recruitment, signal transduction from TNF receptor to caspase-8 is organized in a separate complex within the cytosol. The hypothesis arose since it was not possible to detect DISC- bound FADD in Jurkat cells ²². However, this extrinsic apoptosis initiation will be

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1 INTRODUCTION

completed by successive activation of the executioner caspases. Another important activity of the TNF receptor signalling is the induction of transcription by NFκB ^24,25, which suppresses apoptosis ²⁶. The apoptosis-restraining TNF receptor-associated factors (TRAFs) as well as some IAPs (c-IAP1/2) are target genes of NFκB activation ²⁶. In addition, the transcription factor AP-1 is activated through the activation of JNK ²⁷ by TNF ²⁶, too.

1.1.2.2 Cytochrome c and the apoptosome

The intrinsic pathway of initiation of apoptosis ⁷ uses a different mechanism to activate caspase-9. A large number of chemicals are known to induce this pathway, especially DNA-damaging agents. Finally, all of their effects seem to act on the level of the Bcl-2 family members, like Bax, Bak, and Bid. For instant, the cytosolic monomers Bax and Bak undergo conformational changes upon apoptotic signals, which include oligomerization and insertion into the mitochondrial outer membrane.

The subsequent release of inter-membrane space proteins like cytochrome c ²⁸ can be completely prevented by members of the Bcl-2 family ^7,28-30.

The released cytochrome c binds to the cytosolic molecule Apoptotic Protease Activating Factor 1, Apaf-1, that initiates recruitment of caspase-9 together with subsequent binding of dATP/ATP. Caspase-9 binds to the hub of the now heptameric wheel-like apoptosome (see Figure 1.1) ^28,31,32. Since caspase-9 needs no proteolytical activation to obtain its complete activity ¹⁵, the induced-proximity model fits in this case, too ¹⁴ (see section 1.1.1.3). However, cleavage by caspase-3 seems to stabilize the assembly of the apoptosome ¹⁴ since caspase-3 is needed for caspase-9 procession in MCF-7 cells essentially ³³.

Cytochrome c deficient cells ³⁴ as well as cells with scavenged cytochrome c (peptide inhibitor) ³⁵ are resistant to intrinsic apoptosis, underlining the importance of the Apaf- 1-mediated apoptosis. However, the extrinsic stimuli are not affected ³⁴ or reduced ³⁵, respectively.

These events lead to the apoptotic cascade and death of the cell, finally. Caspase-8 and 10 in conjunction with caspase-3 could potentially feedback onto mitochondria, thereby establishing a feed-forward mechanism to accelerate cell death. This idea is supported by the finding that in some cellular systems inhibition of caspase-3 is sufficient to lower the apoptosis rate dramatically although caspase-9 is not inhibited at all ³⁶.

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1 INTRODUCTION

TNFα

Cell membrane

TRADD TRADD

Mitochondrium D

D D DD D

Figure 1.1 | Caspase activation pathways

Two major pathways to caspase activation are illustrated. Binding of TNFα to its receptor induces oligomerization and thereby activation of caspase-8 through the adapter molecule FADD (extrinsic pathway). Activated caspase-8 processes and activates caspase-3 leading to further caspase-activation and execution of apoptosis. Caspase-8 also activates the intrinsic pathway by cleaving Bid, leading to the release of Cytochrome c and formation of the apoptosome. The hollow circles indicate the active sites.^2,11,32,37

1.1.2.3 Other caspase-activating mechanisms

Whilst Fas-mediated killing is largely reserved for the removal of thymocytes during their maturation and after activation, the primary mechanism employed for the killing of target cells is granule-mediated apoptosis. Briefly, the lytic granules of cytotoxic

largesmallpro

DED DED D DD

D DD

DD FADD

RIP Bid

Cytochrome c

TRAF2 Apaf-1

pro

dATP

Pro-

or caspase-9 large

small

Caspase-8 Pro-caspase-8

NFκB

^cFLIP

p large small

Pro-caspase-3 dimer

Apoptosome Caspase-3

Apoptosis Survival

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1 INTRODUCTION

T lymphocytes contain perforin and the serine proteases granzyme A and B. After granule exocytosis, the granzymes gain access through the perforin channels that have formed in the target cell membrane. Caspase-3 is a substrate of granzyme and the cleavage leads to its activation ². Therefore, the target cell dies through its own caspase machinery that is switched on by another cell.

1.1.3 Activation of the most important caspases 1.1.3.1 Caspase-3

Caspase-3 is the most important executioner caspase and its lack has fatal consequences in cancer development ³⁸. Its activity is very high and it needs proteolytic activation. The preferred cleavage motif is DExD ². Caspase-3 knock out mice survive birth but suffer post partum from high mortality caused by developmental defects correlated to decreased apoptosis. Nevertheless, many caspase-3-deficient cell types die apoptotically after stimulation with Fas. However, this cell death is characterized by absence of DNA fragmentation ². It is reported for different experimental systems ² like the caspase-3 deficient breast carcinoma cell line MCF-7 ³⁹. Caspase-3 can activate caspase-2, 6 ⁴⁰, and 9 and it can be activated by caspases 6 ⁴⁰ to 10 ². Nowadays it is evident that caspases-3 and 7 are almost synonymous concerning their substrate and inhibitor specificity ⁷.

For activation, the zymogen has to be cleaved at two sites to become the mature caspase-3. First, the small catalytic subunit is set free then the large subunit is liberated from the prodomain. It has been revealed that lack of the prodomain increases autocatalytic activation ⁴¹.

1.1.3.2 Caspase-8

Caspase-8 is the important player for death receptor-mediated apoptosis. Its zymogenicity is low and therefore its not-cleaved form exhibits a rather high activity.

This fits very well with the activation by dimerization. For caspase-8 it has been reported that it can activate caspase-3, 10, and – through the mitochondrial pathway – caspase-9. Caspase-6, in turn, is able to activate caspase-8 ². However, in embryonic development caspase-8 serves as an essential protein with absolute necessary non-apoptotic activities ⁴².

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1 INTRODUCTION 1.1.3.3 Caspase-9

Caspase-9 is the key of the intrinsic pathway. Its activation takes place by dimerization on the apoptosome. Caspase-9 can activate caspase-3 and 7 and becomes cleaved by caspase-3 and activated by the mitochondrial pathway through caspase-8. Caspase-9, Apaf-1, and cytochrome c knockouts have shown the absolute importance of these molecules ².

1.1.4 Other proteins involved in apoptosis 1.1.4.1 Apoptotic factors and DNAses

Mitochondria release several pro-apoptotic proteins during apoptosis. For example, proteases, DNAses, and promoting factors are known. From the last class cytochrome c is one of the best understood, as discussed before. Another is the Apoptosis Inducing Factor AIF. This molecule has two functions already identified.

One activity is a NADH oxidase and has a function presumably in the mitochondria.

The other is an allosterically activating activity of DNAses ^43-45. It has been shown for several models of apoptosis that AIF can be released in a caspase-dependent manner ⁴⁶ downstream of cytochrome c release ⁴⁷. However, most reports postulate a caspase-independent release. For this reason and the little understanding in this field it is reasonable to suggest two mechanisms of apoptosis execution by AIF ⁴³. This hypothesis is supported by the finding of selective release of proteins as cytochrome c and AIF. In this case, the release of AIF takes part without the caspase-activating release of Cytochrome c and leads, however, to execution of apoptosis. In samples of acute myeloid leukaemia (AML) patients, inhibition of caspases totally failed to protect cells from undergoing apoptosis induced by chemotherapeutics. However, AIF was released from mitochondria in the presence or absence of caspase inhibitor ⁴⁸. From an evolutionary view, caspases seem much more recent than AIF and AIF probably represents an ancient death style cells died from before evolution invented caspases ⁴³.

Another protein released by mitochondria is the sequence-unspecific RNA / DNA endonuclease EndoG. Several functions in maintaining cell viability have been discovered, e.g. generation of primers for mitochondrial DNA amplification ⁴⁴. Upon apoptotic stimuli including Bcl-2 family members, EndoG translocates to the nucleus, where it extensively degrades nuclear DNA into oligonucleosomal fragments, similar

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1 INTRODUCTION

to those generated by caspase-activated DNAse (CAD) ⁴⁴. Moreover, orthologs of AIF and EndoG in C. elegans have revealed that they could cooperate in the nucleus in caspase-independent cell death (CICD).

Some other pro-apoptotic mitochondrial proteins were recently discovered. One of them is WOX1. This molecule has been shown to enhance TNFα-mediated apoptosis by down-regulation of anti-apoptotic Bcl-2 proteins, upregulation and direct binding of p53 ^44,49. In addition, WOX1 can interact with molecules associated to the TNF receptor, such as TRADD and TRAF2 ⁴⁴. After apoptotic stimulation (TNFα, staurosporine) WOX1 translocates to the cytosol and the nucleus where interaction with p53 takes place ⁴⁹. For the protein p53, a broad range of effects has been described. However, p53 is able to initiate apoptosis even without showing the largely known mechanisms ⁴⁹. Furthermore, WOX1 over-expression leads to apoptosis showing condensation of cytoplasm and nuclei in TNF-insensitive cells but neither caspase nor serine protease inhibitors could inhibit this apoptosis ⁵⁰.

1.1.4.2 Non-caspase proteases

Proteolytic damage by any of several proteases, not uniquely caspases, can trigger cell death even with apoptotic morphology ^4,51. In literature, there are many examples of death associated with protease activity from lysosomal, proteasomal ⁵¹, and mitochondrial proteases ⁴⁴ as well as with granzyme B and matrix metalloproteinases ⁵¹. Some of the best evidence for caspase-independent cell death originates from studies of calpains, cathepsins, and granzymes (cysteine or aspartate, cysteine, and serine proteases, respectively). Though these proteases cooperate in caspase-dependent apoptosis, they are able to induce caspase-independent cell death with most morphological changes seen in classical apoptosis ⁵².

Serine proteases are an important family of proteases with respect to several physiological conditions of the cell. Sharing a highly reactive cysteine residue in the reactive centre named this family. The proteases have been roughly classified into three subgroups according to their substrate specificity, chymotrypsine, trypsine, and elastase ⁵³.

Several investigations have not only linked serine protease activity to cell death with apoptotic morphology ^54,55 but also demonstrated the importance in chromatin degradation ⁵⁵. Furthermore, they are able to execute the entire cell death pathway by

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1 INTRODUCTION

death receptors ^1,56, long term-culture, chemotherapeutics ⁵⁷ or ionizing radiation ⁵⁸. Moreover, even phagocytosis can take place under caspase-inhibiting conditions ⁵⁹. The over-expression of a serpin (SERine Protease INhibitor) PAI-2 (plasminogen activator inhibitor type 2) can inhibit TNFα-mediated apoptosis without inhibition of caspases. Unfortunately, the inhibited serine protease remained unidentified since it was not uPA (urokinase-type Plasmin Activator), the actual target of PAI-2 ⁵⁶. Another example of a serpin is the Leucocyte Elastase Inhibitor LEI. The endonuclease L- DNAse II derives from LEI and cannot be inhibited by caspase inhibitors ^1,57. It has been shown that TNFα induces an increase in L-DNAse II activity, depending on the serine protease activity of AP24 ^4,60. This serine protease was able to interact with LEI and transform it into L-DNAse II, a serine protease inhibitor can inhibit L-DNAse II activation, in turn. Furthermore, the couple AP24/LEI-L-DNAse may be analogous to caspase-mediated DNAse (CAD) activation ⁶¹, which occurs after cleavage of its inhibitor ICAD by caspase-3. Instead of caspase activity, a serine protease activity seems to be important ^57,60. In 2000, the serine protease Omi/HtrA2 has been identified. Omi/HtrA2 is a mitochondrial protein, which upon apoptotic stimuli is released to the cytosol to bind IAPs (Inhibitor of Apoptosis Proteins) ⁶². This binding disrupts the inhibitory interaction of IAPs with caspases, inactivates XIAP by cleavage ^53,62, and results in caspase activation. However, Omi/HtrA2 is able to induce apoptosis in a caspase-independent manner, too. These effects seem to be linked to its serine protease activity ^44,63.

Research on apoptotic cell death independent of caspases is an emerging field. After all, in most cases the two well-differentiated pathways leading to apoptosis, caspase- dependent and -independent, may operate together in eukaryotic cells ⁴⁴, sequentially ^64-67 or probably in parallel 44,64,68,69. Finally, the classically defined morphology can be achieved either by caspase activation or through other families of proteases. Hence, it is no surprise that the cellular mechanisms and the morphology can vary among these various types of apoptosis. For instant, MCF7 cells lacking caspase-3 have the same apoptosis rate like caspase-3 substituted MCF7 cells. Only the latter showed nuclear condensation ⁷⁰.

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1 INTRODUCTION 1.2 Inhibition of apoptosis

1.2.1 Inhibition of apoptosis at the level of caspases

Inhibition of initiator and executioner enzymes of apoptosis is a useful and common way for the repression of apoptosis.

However, some other mechanisms are known. FLICE-inhibitory proteins (FLIPs) prevent recruitment of caspase-8 to the Death Inducing Signalling Complex (DISC) and its subsequent activation ⁷¹. In addition, on the level of mitochondria (Bcl-2 members) as well on the level of the plasma membrane (volume regulatory responses and ionic repression of apoptosis) apoptosis-inhibitory mechanisms are described (see Bortner and Cidlowski, 2002 ⁷¹).

1.2.1.1 Endogenous caspase inhibitors

Endogenous inhibitors of apoptosis at the level of caspases are known as members of the IAP (Inhibitor of Apoptosis Protein) family. Several members have been identified in a number of species ^72,73. Characteristics of IAPs are at least one BIR domain along with the ability to inhibit apoptosis ^71,74. Since the first IAP was identified in baculovirus, the domain got the name Baculoviral IAP Repeat (BIR) ²⁶. The BIR domain consists of about 70 amino acids with a conserved spacing of cysteine and histidine residues, which suggests that this structure represents a novel zinc-binding fold ^26,74. Homology to this domain has been used to identify members of this family, like the mammal forms XIAP, c-IAP1, c-IAP2, NAIP, and survivin. XIAP and survivin consist of three and one BIR domains, respectively and both can associate via this conserved baculovirus IAP repeats ⁷⁵. The human c-IAP1 and c-IAP2 are unique in that they contain a CARD (CAspase Recruitment Domain) domain. Several IAPs also contain ubiquitin-conjugating domains like UBC in the human IAP Apollon ⁷¹ or RING in the XIAP protein.

1.2.1.1.1 XIAP

In respect of using only XIAP in this work, this section will be confined to XIAP, the X- linked Inhibitor of Apoptosis Protein which is named “X-linked” for its unique gene location on the X chromosome (Xq25) ⁷⁴. XIAP (or hILP ⁷⁶) was observed in all adult and foetal tissues tested except leucocytes, indicating that it is a ubiquitous protein ⁷⁴. The generation of XIAP-deficient mice revealed no changes in development,

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1 INTRODUCTION

histology, and not even in the induction of caspase-dependent as well -independent apoptosis. That is probably due to the compensation of the loss by upregulation of c- IAP1 und c-IAP2 ⁷⁷. XIAP consists of four domains, BIR1, BIR2, BIR3, and RING ^26,78. XIAP is able to prevent apoptosis by direct inhibition of distinct caspases ⁷⁹. During apoptosis, XIAP can be specifically cleaved in to the fragments BIR1-2 and BIR3- RING, thus resulting in a decreased efficacy of inhibition ⁸⁰. Over-expressing XIAP has been shown to prevent apoptosis induced by several stimuli, including TNF, Fas, staurosporine, etoposide ^71,74, cytochrome c release ^4,74,81 and growth factor withdrawal ^71,74. XIAP can prevent or delay ⁷¹ apoptosis by inhibition of caspase-3, 7 ⁷⁶ and 9 ^28,71,74 whereas XIAP did not at all inhibit caspase-1, 6, 8 ⁷⁶, and 10 ⁷⁴. Additional functions are described as well. XIAP over-expression enhances the phosphorylation of AKT ⁸² and also induce signalling pathways like NFκB, Smad, and JNK ⁸³. Investigations on the translation of XIAP has revealed a rare and (for IAPs) unique mechanism. Since the mRNA possesses an Internal Ribosome Entry Site (IRES) ^84-86, it can be translated by a rare cap-independent translation mechanism ⁸⁶. Therefore, translation of XIAP can be dramatically increased in response to conditions that lead to a general inhibition of normal protein biosynthesis.

Structural and functional studies have revealed that XIAP inhibits caspase-3 and 7 by its DxxD sequence motif ²⁶ within the flexible linker between the BIR1 and BIR2 domain ^28,36,87. However, BIR1 is essential for inhibition and BIR2 can interact with caspase-3 ²⁶. In the presence of XIAP, caspase-3 becomes cleaved at the linker between the large and small catalytic sub-domains (e.g. by caspase-8) and this activated caspase is then inhibited by XIAP ^4,76. The second cleavage to remove the prodomain cannot take place anymore ^4,76. This means that for inhibition of caspase-3 and 7 by XIAP the caspases have to be in an activated form ^71,74. A very low inhibition constant could be determined for this inhibition, 0.2 and 0.7nM ^26,74, respectively.

Therefore, XIAP is one of the most effective caspase-3 inhibitor identified so far ^74,88. Intriguingly, XIAP inhibits caspase-3 by steric inhibition of the active site ⁷¹ whereas the synthetic inhibitors occupy the substrate-binding site ^28,71. In contrast, caspase-7 is inhibited by XIAP and by synthetic inhibitors at the substrate-binding site ^28,71. Surprisingly, only caspase-3 binding to XIAP is abolished after the inhibitors have bound ⁸⁷. These findings indicate that caspase-3 and 7 are inhibited by use of two different binding sites ⁸⁹. There are also reports discussing transcriptional regulation

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1 INTRODUCTION

of XIAP. It has been shown that NFκB is able to up-regulate XIAP transcription in response to TNFα exposure ^90,91.

While for inhibition by XIAP caspases-3 and 7 have to be proteolytically active this is in stark contrast to caspase-9 ⁷¹. This initiator caspase can be inhibited by XIAP in its zymogen as well as in its activated form ^71,74. That can be explained by the differences in their activation mechanism ⁷⁴ (see section 1.1.1.3.1). It has been shown that the BIR3 domain of XIAP binds directly to the small catalytic domain of caspase-9 and to its small subunit in the cleaved protein, respectively ^28,92. Thus, XIAP sequesters caspase-9 in a monomeric state, which serves to prevent catalytic activity ⁹³.

The RING domain is defined by seven cysteines and one histidine that can coordinate two zinc atoms ²⁶. XIAP exhibits E3 ubiquitin protein ligase activity which is abolished by the exchange of the zinc-coordinating histidine to alanine (H467A) in the RING domain ⁹⁴. Ubiquitination and the subsequent degradation of proteins have high impact on many cellular functions, including cell cycle control, removal of misfolded or damaged proteins, activation of transcription factors, and generation of antigen peptides for MHC molecules ²⁶. The proteasome multi protein complex mainly accomplishes these tasks. To do so, the proteasome recognizes proteins covalently modified with ubiquitin, an 8kD protein that is ubiquitously expressed ²⁶. The modification of proteins occurs as a multi step process. First, an enzyme called E1 activates ubiquitin at its C-terminal glycine; secondly, with the assistance of an E2 / UBC enzyme an E3 ubiquitin protein ligase transfers the activated ubiquitin to a specific lysine residue on the target protein. Hence, this E3 ligase is responsible for the substrate specificity. Ubiquitin itself undergoes ubiquitination as well, therefore poly-ubiquitin chains can arise ²⁶.

Several targets are identified for ubiquitination by XIAP so far. Ubiquitination has been reported not only for XIAP ^94-96 itself but for caspase-3 ^97,98, caspase-7 ⁹⁹, caspase-9 ¹⁰⁰, and Smac/DIABLO ^100,101. Other proteins than XIAP ¹⁰² can also ubiquitinate proteins of the apoptosis machinery. In addition, the proteasome is a target of caspases, which inhibit proteasome function by cleaving of specific subunits.

Therefore, an additional feed-forward loop is activated ¹⁰³.

Auto-ubiquitination takes place at the residues lysine-322 and lysine-328 within the BIR3 domain ⁹⁵. Ubiquitination of these residues and subsequent degradation may be a mechanism to facilitate apoptosis ^94,95,104. Intriguingly, degradation after

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1 INTRODUCTION

ubiquitination of XIAP can be markedly reduced by phosphorylation (serine-87) of XIAP through the protein kinases AKT1 and AKT2 ^104,105. Since AKT kinases are both strong promoters of cell survival ^106-118 and targets of caspase-3-mediated proteolytic degradation ^82,119 it makes sense that AKT is able to prevent its own degradation in concert with its anti-apoptotic functions ^113,120. Moreover, XIAP over-expression results in an increase of phosphorylated and therefore activated AKT kinase in some cases, as mentioned before ⁸². Additionally, the E3 ligase activity is necessary for the activation of NFκB by XIAP ⁸³.

Loss of E3 activity without a lack of the ability to undergo auto-ubiquitination diminishes inhibition of apoptosis to some extent in apoptotic cells ⁹⁹. Indeed, there is some evidence that ubiquitination of caspases is necessary for efficient inhibition of apoptosis by IAPs ^95,98.

Moreover, c-IAP1 and c-IAP2 are also able to interact with adapter molecules of the TNF receptor complex, namely TRAF1 and TRAF2 ^71,102,121. This is not the case for XIAP ²⁶. Nevertheless, it has been shown that XIAP can interact with the BMP receptor (bone morphogenetic protein receptor) as well as activate the mitogen- activating protein kinase (MAPK) TAK1 (transforming growth factor-beta-activated kinase 1) by TAB1 (TAK1-binding protein 1) which next activates NFκB and another MAP kinase JNK1 ^26,122,123. The activation of the latter by XIAP is selective and very distinct, thus preventing apoptosis induced by caspase-1 ¹²⁴ and TNFα ¹²². This protective activity could be localized between the BIR3 and RING domain ¹²⁴. However, in some other cellular context JNK activity was described as being pro- apoptotic ⁷⁴.

In addition, some functions not directly related to apoptosis of different IAPs have also been described, like cell cycle control (survivin and cIAP1) ^26,125 or the regulation of intracellular copper levels (XIAP) ¹²⁶.

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1 INTRODUCTION

Figure 1.2 | XIAP associates with the apoptosome

After formation of the apoptosome, caspase-3 is also recruited to the apoptosome where it can be activated by either pro-caspase-9 or processed caspase-9. There is some evidence that XIAP is able to bind caspase-9 and caspase-3 at the same time. This might be one reason for the highly efficient and potent inhibition of these caspases by XIAP. Since the precise composition of the apoptosome and the stoichiometry of its components are far from clear, the picture shows only a model of apoptosome assembly.

Apaf-1 Caspase-9

Caspase-3

BIR2

BIR1 BIR3

RING

XIAP

Cytochrome c Apoptosome dATP

adapted from Holcik and Korneluk, 2001 ¹²⁷ and Bratton et al.,2002 ¹¹.

1.2.1.1.2 Inhibition of XIAP

Apart from the indirect inhibition of XIAP through degradation other, direct acting inhibitors are known. Smac/DIABLO, Omi/HtrA2, and XAF1 are well-studied inhibitors of XIAP. However, there are different inhibitors understood to a lesser extend, e.g. the mitochondrial ARTS protein ¹²⁸.

1.2.1.1.2.1 Smac/DIABLO

The mitochondrial protein Smac/DIABLO which is released by a Bcl-2/BclxL sensitive mechanism¹²⁹ during apoptosis antagonizes XIAP-mediated inhibition of caspases by direct binding as a dimer ¹³⁰. Smac/DIABLO contains an N-terminal IAP binding domain that interacts with BIR2 and BIR3 of XIAP. Through binding Smac/DIABLO causes a release of caspase 3, 7 and 9 ^130-132 due to steric hindrance and therefore activates them ¹³⁰. A second effect of Smac/DIABLO binding is the marked reduction of the E3 ubiquitin ligase activity which may interfere with the inhibitory capacities of XIAP ⁹⁹ but does not result in ubiquitination of XIAP ⁹⁶. Stressing that, rapid degradation upon massive ubiquitination mediated by Smac/DIABLO is reported for cIAPs but not for XIAP ¹³³. On the other hand, IAPs are able to regulate the effect of Smac/DIABLO. Thus, rapid degradation of Smac/DIABLO after its release has been reported as well as the ability of XIAP to ubiquitinate Smac/DIABLO in vitro ¹⁰¹.

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1 INTRODUCTION

Furthermore, binding of XIAP to Smac/DIABLO can titrate Smac/DIABLO. This was demonstrated elegantly by heterologous expression of baculoviral IAP (op-IAP) in a mammalian system. While this ancestral IAP failed direct caspase inhibition, it was still able to bind Smac/DIABLO ¹³⁴.

1.2.1.1.2.2 Omi/HtrA2

Omi/HtrA2 contains an N-terminal IAP binding motif like Smac/DIABLO. For further details, see section 1.1.4.2.

1.2.1.1.2.3 XIAP-associated factor 1 (XAF1)

XAF1 antagonizes XIAP activities by direct binding ^63,135. As a nuclear protein, it can affect the redistribution of XIAP from the cytosol to the nucleus ^63,135,136 that can be observed after treatment with some chemotherapeutic drugs ¹³⁷. XAF1 seems to interact with XIAP permanently ⁶³ but on the other hand, XAF1 expression is strongly up-regulated after treatment of cells with interferon ¹³⁸.

1.2.1.1.2.4 Small molecular weight inhibitors of XIAP

Despite the lack of a phenotype in XIAP-deficient mice, disruption of the XIAP gene ¹³⁹ or specific down-regulation by siRNA ¹⁴⁰ in several tumour cell lines sensitizes them to apoptotic stimuli ¹³⁹. In these cases, the importance of XIAP to prevent the cells from undergoing apoptosis is quite clear. Hence, development of small molecular inhibitors of XIAP is an emerging field with great hope for cancer treatment ^141,142. Stressing that, no apoptosis inhibitors others than IAPs are able to block the terminal caspases ^86,143. Different inhibitors have been discovered so far.

The most promising are a class of polyphenylureas ¹⁴¹. A structure-based computational screening of a traditional herbal medicine three-dimensional structure discovered another called embelin. Embelin binds XIAP in a similar mode like Smac/DIABLO ^142,144.

Finally, there is some progress in silencing the XIAP gene by siRNA, which is no classical inhibitor but ought to be mentioned for completion 140,145,146.

1.2.1.2 Peptide inhibitors of caspases

For better understanding of cellular apoptotic mechanisms, synthetic peptide inhibitors derived from caspase recognition sites are an established tool. All of them

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1 INTRODUCTION

are very small, containing only three to four amino acids. They are not highly specific for this reason but somehow do appear to affect caspases within particular groups.

For instant the “DEVD” motif (out of the sequence of PARP) blocks caspase-3-like activity, the “VAD” motif is capable of blocking most caspase-mediated apoptosis (caspases 1, 2, 3, 4, 6, and 8 ^4,88]). Addition of a chemical group like ketones (usually flouromethylketone) renders them stable ² and irreversible. For instance, the widely used inhibitor zVAD-fmk inhibits recombinant caspases 1 to 9 with a half-life for irreversible inhibition at 1µM less than 41 minutes, caspases 1, 3, 5, 7, 8, and 9 even within one minute ¹⁴⁷. However, zVAD-fmk has been shown to block cathepsin B activity at a concentration of 1µM in vitro ¹⁴⁸. Furthermore, cells treated with the fluorogenic fam-VAD-fmk inhibitor become more and more labelled because of labelling and simultaneous inhibition of only caspases, exhibiting an active centre ^149,150. Another example is the aldehyde inhibitor AcDEVD-cho that inhibits recombinant caspases 3, 7, and 8 with ki values in the low nanomolar range, 0.23, 1.6, 0.92 ¹⁴⁷, respectively. However, caspase-9 shows only a slight higher ki value of 25 to 60nM ^88,147,151.

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2 AIMS OF THE THESIS

For the hepatoma carcinoma cell line HepG2, it has been shown that these cells could still undergo apoptosis even if caspases are inhibited. These findings together with the ability of XIAP to protect cells by caspase inhibition raised the question if XIAP was able to inhibit caspase-independent cell death, too. Though XIAP is the most investigated anti-apoptotic protein and functions besides caspase inhibition have been discovered, no model of caspase-independent cell death has been employed to test possible effects so far. However, HepG2 cells were no suitable model for investigations that needed transient transfection. The cervix carcinoma derived cell line HeLa is a well-known model. However, caspase-independent cell death has been not described so far.

For this purpose the aims of the present study were:

1) To characterize whether non-caspase proteases are involved in death receptor- mediated cell death after caspase arrest in HeLa cells.

2) To test whether XIAP was able to inhibit caspase-independent cell death induced by TNFα and consequently

3) The cloning of several XIAP mutants and investigations whether they contribute to protection of cell death after caspase arrest.

4) To investigate a serine protease inhibitor-sensitive cell death induced by heterologous expression of caspase-8.

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3 MATERIALS AND METHODS

Abbreviation Full name

AcDEVD-afc N-Acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin AEBSF Pefabloc SC^TM / 4-(2-Aminoethyl)-benzenesulfonyl fluoride

BSA Bovine serum albumin

CHX Cycloheximide

DMSO Dimethyl sulfoxide

DTT Dithiothreitol

ECL Enhanced chemiluminescence EDTA (Ethylenedinitrilo)tetraacetic acid EGFP Enhanced green fluorescence protein

FCS Fetal calf serum

HSA Human serum albumin

MgCl2 Magnesium chloride

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(sulfophenyl)- 2H-tetrazolium, inner salt

NaCl Sodium chloride

NaH2PO4 Sodium dihydrogenphosphate PARP Poly (ADP-ribose) polymerase

PBS Phosphate buffered saline SDS Sodium dodecyl sulfate

TLCK N,p-tosyl-L-lysine chloromethyl ketone

TMRE tetramethylrhodamine, ethyl ester, perchlorate Tris-HCl Tris(hydroxymethyl)aminomethane hydrochloride XIAP X-linked Inhibitor of Apoptosis Protein

zDEVD-fmk N-Benzyloxycarbonyl-Asp-Glu-Val-Asp(O-Me) fluoromethyl zVAD-fmk N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone

HOECHST Hoechst 33342

Ni-NTA agarose Nickel-nitrilotriacetic acid agarose

BCA bichinconinic acid

His-Ubi His-tagged Ubiquitin

ATCC American Type Culture Collection Eagle’s -MEM Eagle’s Minimal Essential Medium

CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate HEPES N-2-Hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid

PES Phenazine ethosulphate

PAGE polyacrylamide gel electrophoresis

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3 MATERIALS AND METHODS 3.1 Materials

3.1.1 Chemicals and reagents

Caspase inhibitors zVAD-fmk and zDEVD-fmk were purchased from Bachem (Weil am Rhein, Germany). Caspase-3 substrate AcDEVD-AFC was delivered from BIOSOURCE (Camarillo, CA, USA). The BCA protein assay reagent was purchased from Uptima (Montlucon, France). CHAPS and HEPES were purchased from ICN Biomedicals (Ohio, USA). Recombinant TNFα: Prof. Dr. D. Männel (University of Regensburg, Germany) generously provided the human form; the mouse form was purchased from Innogenetics (Ghent, Belgium). TRAIL and the activating anti-CD95 antibody (clone CH11) were purchased from Alexis Biochemicals (Gruenberg, Germany) and Biomol (Hamburg, Germany), respectively. Hoechst 33342 and TMRE were delivered from Molecular Probes (Leiden, Netherlands). Fluorescent mounting medium was bought from DakoCytomation (Hamburg, Germany). Pefabloc SC was purchased from ROCHE (Mannheim, Germany), TLCK from Merck Biosciences (Schwalbach/Taunus, Germany). The tetrazolium salt (MTS)-based colorimetric cytotoxicity assay CellTiter96^® AQueous was produced by Promega (Madison, WI, USA). Restriction and DNA modifying enzymes were purchased from New England Biolabs (Frankfurt am Main, Germany) or Fermentas (St. Leon-Rot, Germany).

Clinical grade Saline was obtained from DeltaSelect (Pfullingen, Germany). Most of standard chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) or other established suppliers.

3.1.2 Antibodies

The mouse monoclonal anti-hILP/XIAP was purchased from BD Biosciences Pharmingen (Heidelberg, Germany), mouse monoclonal anti-caspase-8 (Ab-3;

clone 1-3) from Oncogene Research Products (Boston, MA, USA). Rabbit anti- caspase-3 was delivered from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-GFP antibody was obtained from ROCHE Applied Science (Mannheim, Germany).

3.1.3 Cell culture materials

Eagle’s MEM, Trypsin/EDTA, Penicillin/Streptomycin and PBS were purchased from

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3 MATERIALS AND METHODS 3.1.4 Cell lines and bacteria

HeLa cells were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA).

Chemo-competent XL-2-blue MRF’ E. coli bacteria from Stratagene (La Jolla, CA, USA) were used for all cloning.

3.1.5 DNA vectors and constructs

The transfer plasmid pBacMam2 was purchased from Novagene (Houston, TX, USA), the pcDNA5/FRT vector from Invitrogen (Karlsruhe, Germany). The XIAP protein- encoding sequence in the pBacMam2 vector was kindly provided by Astrid Leja / Dr.

Thomas Meergans (University of Konstanz, Germany), the pC3-K (encoding for Caspase-3 lacking the prodomain) ⁴¹ and the pCMV-C8 (encoding for Caspase-8) by Dr. Thomas Meergans (University of Konstanz, Germany). Stratagene (La Jolla, CA, USA) supplied the pEGFP-C1 vector. The pHis-Ubiquitin (His-Ubi) ¹⁵² construct was a kind gift of Prof. M. Scheffner (University of Konstanz, Germany). Interactiva (Ulm, Germany) synthesized all custom-made primers.

3.2 Methods

3.2.1 Construction of plasmids for transfection

Since the multiple cloning site of the pBacMam2 vector provided only two usable restriction sites, we exchanged it for the multiple cloning site of the pcDNA5/FRT vector. To achieve this, a junk sequence was cloned into the BamHI site of the pcDNA5/FRT vector prior the liberation of the PmeI fragment. After ligation into the BglII / MscI cleaved, blunted, and de-phosphorylated pBacMam2, the junk sequence was removed. The improved vector was called pBacMam2A. The XIAP encoding sequence was sub-cloned from the pBacMam2 construct into the pBacMam2A vector by PCR generation using the primer pair 5’-GATCTCGAGCTCGTTTAGTGAACCG and 5’-GATCCCGGGATCCTCTAGAGTC (sense and antisense, respectively). Notice that all constructs have a Flag encoding sequence at the 5’-end.

Site-directed mutagenesis was done by amplifying the 5’-forward fragment up to the site of the exchange with a primer carrying the mutated sequence, the 3’-reverse fragment was done correspondingly.

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For generation of XIAP D148A (hereinafter called XIAP D) primer pairs 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-GTCTGATATAGCTACAACCTGCCCAG for the forward part and 5’-CTGGGCAGGTTGTAGCTATATCAGAC / 5’-GATCCCGGGATCCTCTAGAGTC for the reverse part were used. The two obtained fragments were used as template in a PCR applying the primer pair 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-GATCCCGGGATCCTCTAGAGTC.

For generation of XIAP W310A/E314S (short XIAP WE) primer pairs 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-CAAGGGTCTGAACTGGGCTTCGCATCAG for the forward part and 5’-CTGATGCGAAGCCCAGTTCAGACCCTTG / 5’-GATCCCGGGATCCTCTAGAGTC for the reverse part were used. Again, the primer pair 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-GATCCCGGGATCCTCTAGAGTC was applied to generate the entire sequence.

XIAP H467A (short XIAP E3-) was generated, correspondingly. The primer pairs were 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-CAAGTGACTAGAGCTCCACAAGGAAC and 5’-GTTCCTTGTGGAGCTCTAGTCACTTG / 5’-GATCCCGGGATCCTCTAGAGTC (sense and antisense, respectively). The primer pair 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-GATCCCGGGATCCTCTAGAGTC was used to generate the entire sequence.

XIAP ∆RING was generated with the primer pair 5’-GATCTCGAGCTCGTTTAGTGAACCG / 5’-ATATCCCGGGCTACTCCTCTTGCAGGCGCCTTAG.

The triple mutant XIAP D148A / W310A / E314S (hereinafter called XIAP DWE) was generated utilizing the primer of the XIAP W310A / E314S mutant as described applied to the XIAP D148A template.

All PCR-generated fragments were ligated into the pBacMam2A vector using the restriction sites SacI and SmaI except XIAP H467A. Since mutagenesis of XIAP H467A created a SacI restriction site, the PCR fragment was ligated into a correspondingly MscI and SmaI cleaved XIAP-pBacMam2A construct. Plasmids were prepared using standard kits from Qiagen (Hilden, Germany) or Macherey & Nagel (Düren, Germany).

All computational manipulations of DNA sequences (primer design, restriction site analysis, open reading frame determinations, amino acid translation) were done using the program “GEN22” programmed by Prof. Hofer (University of Konstanz, Germany).

Database search was done using “BLASTN” ¹⁵³ and alignment of two sequences using “lalign” ¹⁵⁴. Both are www-based services provided by Genestream, Institut de Génétique Humaine, Montpellier, France.

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Integrity of protein-encoding sequences was confirmed after ligation by automated sequencing analysis by Sequiserve (Vaterstetten, Germany) and the program

“CHROMAS”.

3.2.2 Cell culture

HeLa cells were grown in Eagle’s MEM containing 10% heat-inactivated FCS, 100µg/ml Penicillin and 100U/ml Streptomycin at 37°C in a humidified incubator with 5% CO2. The cells were passaged twice a week at ratios of 1:6 and 1:12 using Trypsin/EDTA. Cells were seeded for experiments in 48-well plates or 6-well plates at densities of 57,000 and 2.0·10⁵ to 3.0·10⁵ cells per well, respectively. For high quality pictures, cells were grown on sterilized glass cover slips.

3.2.3 Transient transfection

Transient transfection was performed employing Effectene^® from Qiagen (Hilden, Germany) according to the manufacturer’s instructions. Roughly, eight parts of Enhancer were mixed with one part DNA and incubated for four minutes. Then, 10µl Effectene^® reagent was added, mixed and incubated for another 10 minutes. The Complexes were then added to cells provided with fresh medium. Co-transfection was performed with up to three different plasmids. The Ratio of marker plasmid (pEGFP-C1) to other plasmids was about 1:2.5 or lower. In general, cells in 6-well plates were transfected one day after plating and then grown in fresh medium for 16 hours without removing the transfection complexes.

3.2.4 Treatment of cells

HeLa cells were brought into fresh medium containing 100µM CHX and the indicated inhibitors 30 minutes prior to apoptosis induction. As inducers, TNFα, TRAIL or anti- CD95 were employed at final concentrations of 100ng/ml, 10ng/ml, and 50ng/ml, respectively. This cytokines were diluted in saline supplied with 0.1% human serum albumin. All inhibitors were reconstituted with DMSO except TLCK (1mM HCl), CHX and AEBSF (distilled water). Not treated control cells received the vehicle. Final concentration of DMSO never exceeded 1%.