Alexander Almér

Dag Westerståhl

Review of Relativism and Monadic Truth

Introduction

Truth relativism is once again hotly debated in philosophy. Not the old and much attacked claim that all truths are relative, but the claim that certain important kinds of truths, in certain important kinds of discourse, are relative. Typical kinds are discourse about matters of taste, about humor, about morals, about probabilities, about what might have been, about knowledge, and about the future. Herman Cappelen and John Hawthorne's book Relativism and Monadic Truth is thus a timely anti-relativist contribution this debate. However, their target is wider than these modern forms of relativism. The main culprits, in their opinion, are the fathers of what has today become a widely accepted way to deal with at least modal and temporal linguistic phenomena, i.e. David Lewis and David Kaplan. Although these authors didn't think of themselves as relativists, the central role they attached to relative propositions -entities that in addition to a world need a time, or a location, or some other index, in order to have a truth value -is what C&H are criticizing. According to them, this very idea is a false start, almost a category mistake: propositions are true or false simpliciter, not relative to something.

Whether truth is relative or not depends on what one takes as truth bearers.

If they are sentences, or occurrences of sentences, or utterances, one might consider having a language as a parameter; cf. Tarski's notion 'true in L' in [START_REF] Tarski | The semantic conception of truth[END_REF].

But this relativization is fairly uninteresting; we may as well fix the language. 1 1 It might be interesting if it were frequently the case that the same sentence belonged to 1 When the truth bearers are sentences in a formal language, we are also familiar with the notion 'true in a model M', which may seem relative. But it isn't.

The model just specifies the meanings of the (non-logical) atomic expressions, again something which belongs the given interpreted language. These issues, fortunately, are irrelevant to C&H, who focus on what most people take to be the archetypal truth bearers, i.e. propositions. One thing that most people agree on concerning propositions is that although linguistic means can be used to express them, propositions are not language-specific. 2 The same proposition can be expressed by different sentences or utterances in the same language, but also in different languages. What theorists disagree about, however, is whether propositions are relative to worlds, times, and other indices.

How does one establish if a certain notion is relative or not? Put slightly differently, how does find out whether a property or relation has more arguments than it seems to have? There appears to be no well-established method.

One may appeal to a distinction between linguistic and metaphysical form, or between surface form and LF, or between arguments and adjuncts, or between 'being about' versus 'concern'. But although some cases are fairly clear, many are still debated, and truth is one of these.

C&H's book consists mainly of a wealth of counter-arguments against familiar arguments, mostly from the recent literature, that truth has some extra parameter. They spend less space on positive support for their own claim, which is that truth is monadic, and that this is not a technical issue, it is what emerges "when one carves linguistic and psychological reality at its joints." (p.

3) In our opinion, the collection and discussion of arguments is the main asset of the book. The lack of positive support could be seen as one of its problems.

We'll come back to this, after overview of its contents. different languages. Although this happens, it is not very frequent for natural languages (in contrast with the logicians' use of (partially) uninterpreted languages).

2 Unless you are a Whorfian linguistic relativist, but this version of relativism is not discussed by C&H.

The book

So many arguments are discussed in this book that it would be impossible to even mention them all here. We focus on the ones we think raise the most interesting issues.

Chapter 1

Chapter 1 presents the view to be defended, smartly called Simplicity (Who could be against it?) and the kinds of arguments against Simplicity the book aims to rebut. Simplicity is characterized by T1-T5, which say that propositions are: true or false simpliciter (T1), the semantic values of sentences in contexts (T2), the objects of propositional attitudes (T3) and of illocutionary acts (T4), as well as of agreement and disagreement (T5). Although one may introduce relative notions of truth, e.g. in possible worlds semantics, these are less fundamental than absolute truth. Two further important properties of propositions are mentioned: Contingency is the claim that a proposition which is (absolutely) true could have been (absolutely) false; Temporality says that some true propositions will be false (or have been false). Both are said to be perfectly compatible with Simplicity, although the authors realize that, especially in the case of Temporality, this is not quite trivial; see below.

The rest of the chapter is about relativism and its motivations. (a) Proliferation is the idea that if we already accept with Lewis and Kaplan that truth is relative to at least worlds and times, truth simpliciter is already abandoned. Therefore, adding a few further parameters, such as assessors or standards, is not a radical step.

(b) Disquotation is about how relativists can deal disquotationally with truth in the object language. The fact that speakers pass effortlessly from It is cold to It is true that it is cold doesn't make object language truth monadic for the relativist. Relativization to, say, an assessor enters on both sides of the disquotational scheme: The proposition that it is true that it is cold is true for Aristotle iff the proposition that it is cold is true for Aristotle, and similarly for other kinds of relative propositions.

(c) Non-relativity of semantic value and belief reports is, first, the position that although certain (utterances of) sentences express contents that are relative to, say, a standard of taste, the semantic claim that such a sentence expresses such a content is not itself relative to standards of taste. This is clearly the position taken by modern relativists. Second, C&H extend this non-relativity to belief reports. And it does seem reasonable that the claim that Tim believes that apples are delicious is not itself relative to Tim's or anyone else's standard of taste. Prima facie, however, the relativist could take such non-relativity in two ways. She may construe Tim's belief object as thick : that apples are delicious according to Tim's standard. Or it may be thin: the proposition that apples are delicious according to x. C&H argue that relativists must choose the thin alternative: otherwise they cannot say that when two people disagree about taste, they have contrary beliefs.

It is not quite clear if features (a) -(c) define relativism as C&H are using the term. Also, their account is "in part normative" (p. 9); a reconstruction of (what they think is) the best case for relativism, and so neither is it always clear who (if anyone) holds this view. These two circumstances sometimes render their discussion less transparent than it could have been, in particular the discussion of assessor sensitivity and of non-indexical contextualism.

First, the main contrast to relativism, concerning statements about taste, humor, morals, epistemic modals, etc. is contextualism, (unobjectionably) described as the view that seemingly contrary claims about whether something is funny, for example, express fully compatible propositions (of the simple kind), i.e. that it is funny according to A, and that it is not funny according to B, and so disagreement is only apparent.

In order to salvage disagreement, relativists posit assessor sensitivity. Now there are two kinds of assessor sensitivity: with respect to semantic values (propositions) and with respect to assertions or utterances. C&H start with the former: the same proposition may be true for one assessor and false for another.

But then they claim that sensitivity of assertions follows from this, once one accepts "the eminently natural principle that an assertion is true iff the semantic value of that assertion is true" (p. 18). So then the same assertion/utterance can be true for one onlooker and false for another.

But it is not clear that relativists of any kind should believe in this transfer principle, however natural. Rather, their version of it is: an assertion/utterance is true iff its semantic value is true relative to the operative assessor. Moreover, they differ as to where this assessor comes from. According to non-indexical contextualists, the operative assessor is determined by the utterance context, along with the operative time, world, etc. This is precisely what characterizes that position: it treats parameters like assessors or standards just as Kaplan treats time. Other relativists on the other hand, notably Lasersohn and MacFarlane (e.g. [START_REF] Lasersohn | Context dependence, disagreement, and predicates of personal taste[END_REF] and [START_REF] Macfarlane | Making sense of relative truth[END_REF]), think of assessment as a separate act, independent of the utterance. This is why an utterance can differ in truth value between assessors.

Both kinds of relativist accept (their respective version of) the revised transfer principle, but that principle cannot be used to get from assessor sensitivity of content to assessor sensitivity of assertions.

An analogous unclarity pertains to C&H's presentation of non-indexical contextualism (pp. 20-24). They follow the account in MacFarlane [START_REF] Macfarlane | Non-indexical contextualism[END_REF], arguing at the same time that the position is patently absurd and therefore can be left aside. As they and MacFarlane point out, a non-indexical contextualist, call her B, in a situation where A utters S in context c (thereby expressing proposition P) may have to accept both of the following:

(1) a. A's utterance of S at c is true.

b. What A says in uttering S at c (i.e. P) is false. This is why C&H think non-indexical contextualism is absurd. But again, if we bear in mind that while utterance truth is absolute for B, truth of propositions is relative to standards (so (1-b) implicitly uses B's standard), the air of absurdity disappears. In other words, absurdity only follows under the above-mentioned transfer principle. 3The remainder of Chapter 1 is mainly a warm-up for Chapter 2.

Chapter 2

Chapter 1 identified the recent relativist challenge as one of the two main attacks on Simplicity. For what it's worth, we don't find this so obvious. But it must be admitted that intuitions in cases like this are rather weak, and it is hard not to let those intuitions be biased by whether you already accept Simplicity or not. Perhaps intuitions can be sharpened, for example by making more fine-graded distinctions as to exactly how possible circumstances are allowed to differ. Lacking this, the application of Agree in purely modal cases still seems problematic. C&H regiment operator arguments into several subclaims, such as Sententiality (that, linguistically, the operators take sentences as arguments), Parameter Dependence (the just mentioned dependence of Kaplanian propositions on circumstantial parameters), and Uniformity (the idea that a sentence makes the same semantic contribution when it stands alone as when it is a subexpression of some larger expression). They argue that each operator argument, for each kind of circumstantial parameter, has serious flaws, but not that there is one single fault common to all of them.

Chapter 3

For example, as to Sententiality, it is fairly clear that expressions like in Boston or on Tuesday are not sentential operators in English. Although In Boston it is raining and On Tuesday he mowed the lawn are well-formed sentences, at no reasonable syntactic level do these expressions appear as sentence operators. 4 In so far as an operator argument relies on such sentences, it appears to be weak. Things get more interesting for expressions like possibly, somewhere, or sometimes, which at least seem to operate on sentences. As to somewhere, there is a well-known argument in Lewis [START_REF] Lewis | Index, context, and content[END_REF], cited by C&H, to the effect that propositions cannot be functions from just worlds to truth values: If they were, the proposition expressed by an utterance of (3) It is raining.

in a context c would be something like that it is raining-at-l c , where l c is the location of c (disregarding tense here), but this proposition is useless for calculating the proposition expressed by uttering (4) Somewhere it is raining.

in c, since the location is shifted. In other words, says Lewis, taking these propositions as semantic values would make the semantics non-compositional.

But if we take location too to be an argument, compositionality is restored.

However, C&H observe, this reasoning presupposes Sententiality. Clearly, an adequate semantic analysis of (4) has to quantify over locations at some point, but if we, for example, take the logical form of (3) to contain a variable for location, normal existential quantification of that variable can occur directly in the sentence, and there is no need for location-dependent propositions. But then, at the level of logical form, (4) doesn't have (3) as a subsentence, instead it has the corresponding subformula with a free variable. Similarly, Uniformity fails: the semantic contribution of the stand-alone (3) (where the variable is fixed to l c ) isn't the same as the contribution of the same phrase in (4).

In the case of tense, it is even easier to argue that Sententiality and Uniformity fail. It requires quite significant 'rewriting' to get The girls danced contain a tenseless The girls dance. Moreover, the use of the past tense in the former sentence is referential, and thus not obtainable with a (standard) tense operator. And for cases when quantification over times does occur, C&H mention influential arguments from King [START_REF] King | Tense, modality, and semantic values[END_REF] and others that this is done by explicit quantification over times and not by tense operators (see also below).

Now consider the case where the Kaplan style analysis seems to stand at its strongest: modals using necessarily and possibly. Here, no arguments similar to those around (4) work, for C&H are emphatic that simple non-modal sentences have no hidden world parameter or variable, and no reference to the actual world. Also, this is where the strongest case can be made for Sententiality. But if one accepts all of this, what to say about simple sentences like the following?

(5) Possibly, the girls are dancing.

Here C&H are a bit vague. Sometimes they take refuge in the position that their job is not to propose theories of modality or tense, but to criticize arguments against the simple view. Still, for sentences as central as [START_REF] Lasersohn | Context dependence, disagreement, and predicates of personal taste[END_REF] it would be strange to have nothing to say. And in fact they do say two things. On the one hand, they say that the usual possible worlds account, in terms of a relation true at, is perfectly fine, but that this is compatible with the view that the monadic true is "more fundamental". On the other hand, when pressed to explain how the more fundamental account would go, their story is simply disquotational. To take one of their examples, It could be the case that there are talking donkeys is true simpliciter iff it could be the case that there are talking donkeys. And to someone who objects that this is no analysis at all, they reply that to require more would be "to endorse a wildly implausible form of semantic reductionism" (p. 80, fn. 21). For now, we leave to the reader to ponder this, but come back to similar questions in Section 3.

Chapter 3 contains much more. There is a brief but, we think, important discussion of how Contingency and Temporality go together with Simplicity, that we come back to below. There is an even more briefly argued claim that the Agree test gives strong evidence against the Kaplanian approach. The argumentation in Chapter 3 is intentionally swift, and doesn't go into the intricacies of current linguistic debate on these matters, although numerous of the debated topics are mentioned. The authors' contention is that even a quick look at this debate shows that the Lewis/Kaplan approach to at least tense and location is in serious trouble.

Chapter 4

Predicates of personal taste, epistemic uses of might, and probability operators, to mention a few of the relativist's favorite cases, have some features in common. They seem to pass agreement-based tests, and trigger a sense of faultless disagreement, at least with some observers. Moreover, they prompt seemingly appropriate assessments of truth and correctness that vary with context. In Chapter 4, C&H critically re-examine these data, aiming to show that with a proper analysis, contextualism has the resources to account for them. This occupies the main part of the chapter, which is is rounded off with an exposition of some weaknesses pertaining to relativism: it over-generates predictions of contradictory beliefs, it has trouble making sense of factive verbs applied to sentences containing relative predicates, and it loses contact with our common (monadic) notion of truth. We restrict attention here to some central themes developed in the first part of the chapter.

The strategy deployed is to provide contextualist semantics for predicates of personal taste, like fun, tasty, and disgusting, with the understanding that the analysis could be generalized to other relativism-friendly predicates. They begin by providing a contextualist analysis of the word filling that can handle cases of agreement and disagreement. This model is then generalized to other, more relativism-prone, predicates of taste. 5 The upshot is that there is no such phenomenon as faultless disagreement in matters of taste. Taste sentences do not really pass agreement-based tests, impressions notwithstanding. Much of the discussion centers around uncovering sources of these mistaken impressions.

The first misunderstanding concerns the alleged contradiction afflicting utterances of a taste sentence and its negation when both are true. For example, in many cases we are not prone to say that an utterance of That is fun in a conversational context contradicts an utterance of its negation (with the same target), just as traditional contextualism predicts. C&H recognize, however, that if the response is No. That is not fun, we do perceive a contradiction. In their opinion, such use of No indicates a corrective intention of the hearer. But this contradiction can be given a contextualist account, if due attention is paid to the fact that interlocutors may use the predicate term auto-or exocentrically (Lasersohn's terminology). They list three possible interpretations, noting that there are others. First, the speaker might use fun autocentrically whereas the hearer exocentrically (referring to speaker standard) points out that the speaker is wrong about his own standard. Second, the speaker might use fun referring to a group standard; while the hearer corrects the speaker since not all members of the group find the target fun. And finally, the speaker utters That is fun autocentrically, whereas the hearer responds to an exocentric interpretation of that utterance. The first case involves an ordinary disagreement, and the second case exhibits a similar cognitive mistake. The third scenario involves a hearer response based on a misinterpretation.

All this is good news for contextualism, but the relativist would insist that there are disputes in matters of taste that do not fall neatly into any of these categories. Arguments for relativism are supposed to gain support from cases where no interlocutor has made any epistemic or cognitive mistake. But C&H's claim is that such cases of faultless disagreement hardly exist, or at least are too rare to carry any weight. They consider the cases analyzed as more or less prototypical: what looks like relativism-friendly data evaporates on a closer look.

They do mention, however, cases where these prototypes do not fit the felt disagreement. In particular, predicates codifying sensations of (physical) disgust seem to provoke persistent disputes, where interlocutors as well as eavesdroppers report a sense of disagreement and intuitions of contradiction. A set of examples involves Vinnie, a speaking vulture who loves rotten flesh, and Jones, his human friend who hates it. Vinnie says (roughly) Rotten flesh is not disgusting, Jones says It is disgusting. We can imagine both resisting claims that they don't disagree. C&H recognize that quite a few informants report intuitions of contradiction, both in a conversational setting and in a situation where Vinnie and Jones are not participants in the same conversation.

This may seem embarrassing to contextualism, but C&H suggest a way out.

First, they predict that a number of sufficiently talented speakers would cease using corrective phrases in situations like Vulture vs. Jones, if properly taught that such standards are subjective. This form of linguistic therapy C&H call Phyrronian reflection. The fact that linguistic therapy is sometimes required to convert believers in faultless disagreement into enlightened contextualists can be explained by appeal to a "dose of semantic blindness" (p. 118). This is an error-theory, building on the assumption that some sensibilities are connected to physical or emotional responses, for example disgust, such that humans tend to behave as if there are superior standards of sensitivity. It is also assumed that most people would respond positively to Phyrronian therapy.

C&H make several points in Chapter 4 that are well worth considering, and the discussion in this chapter is more detailed than in the others. But taking onboard an error-theory of this type is a rather heavy commitment. Talk of folk perspectives grounded in psycho-biology (sensibilities, and emotive or physical responses) that guide erroneous linguistic behavior is not self-evidently feasible.

What establishes the correct use of terms like agree, and why would semantic blindness have survival value?

3 How simple is Simplicity?

In a book whose main thesis is that truth is a monadic property of propositions, one might expect a crucial issue to be what propositions are. Yet C&H take this rather lightly. Their approach is to criticize arguments against Simplicity, not to offer independent reasons for it.

There is no denying that each of T1-T5 has intuitive support. But the book's dialectic leaves open the possibility that there is no viable notion of proposition satisfying all of them. It wouldn't be the first time a set of intuitions is incoherent. In any case, remaining neutral on what propositions really are, and refuting arguments against T1-T5, isn't enough to show that the simple view itself is coherent.

Presumably, C&H just take for granted that it is. But it seems to us that this is at least a problem for discussion, and moreover that the authors are sometimes driven, in spite of their professed neutrality, to views about propositions that are neither simple nor unproblematic. We end by discussing some putative problems with the book's account of simple propositions.

Compositionality

We saw above how C&H want to defuse the argument in Lewis [START_REF] Lewis | Index, context, and content[END_REF] against propositions that depend only on worlds and not on locations: it presupposes Sententiality and Uniformity. In fact, their critique summarizes the more detailed reasoning around Lewis's argument in King [START_REF] King | Tense, modality, and semantic values[END_REF]. Now recall that Lewis's point was that if merely world-dependent propositions are used as semantic values, the semantics would not be compositional. So suppose we accept, with King and C&H, that (again ignoring tense) the logical form of (4) uses an explicit location variable l; we can take this form to be ∃l rain(l). Is it then obvious that we

Contingency

The modern idea of propositions starts with Frege and Russell. Despite the familiar differences between these two about the constituents of propositions (i.e. whether they are real objects and relations, or senses), their propositions are absolutely true or false, and they serve as objects of propositional attitudes.

But as far as we know, neither Frege nor Russell entertained the idea that some true propositions might have been false, or that they will be false. But suppose we ignore these worries and accept that some propositions that are true simpliciter will be false. Exactly what kinds of things are these propositions? In the modal case we might perhaps be able to find a way (though it seems unlikely) to explain how a true proposition could be false without invoking something like worlds. But there is absolutely no way to explain how a true proposition will be false without invoking times. Thus, these propositions must be things that are true at some times and false at others. But then we don't really see how to tell them apart from temporal propositions: entities that C&H insist we must avoid! 9 So Temporality seems to work against Simplicity:

8 In footnote 22, C&H refer to Ch. 5 in Williamson [START_REF] Williamson | The Philosophy of Philosophy[END_REF] for "more on semantics with a modal metalanguage that proceeds in this vein [i.e. without possible worlds]" (p. 80). Here

Williamson analyzes necessity and possibility in terms of counterfactuals, arguing that speakers often know fairly well how to assess counterfactual claims. But he is very clear that he is not doing semantics, "which concern[s] the truth conditions of counterfactuals, not how subjects attempt to find out whether those truth conditions obtain" ( [START_REF] Williamson | The Philosophy of Philosophy[END_REF], p. 142). The latter is his main occupation in that chapter, and he maintains that a preliminary investigation into the epistemology of counterfactuals can be undertaken even before a precise semantics has been given. There is no claim, as far as we can see, that such a semantics could be done without something like possible worlds. 9 It doesn't seem to help to add the word simpliciter here. What could the difference be between saying that P is true at t, and saying that P is true simpliciter at t? it posits propositions that do require a time argument. 10

A technical issue?

Here is a final more general worry, also related to time. In contrast to worlds, most sentences make reference to time in some form, and the best way to deal with temporal phenomena in language is a crucial issue in semantics. Kaplan chose to proceed as in intensional logic, letting time be a parameter or circumstance of evaluation. C&H incline towards the first-order approach recommended in King [START_REF] King | Tense, modality, and semantic values[END_REF] with explicit quantification over times, which in particular requires practically all properties and relations to have an extra time argument.

Indeed, they present several arguments for that position, and note that it is favored by many contemporary linguists. But there are dissenting voices, e.g.

Recanati [START_REF] Recanati | Perspectival Thought: A Plea for Moderate Relativism[END_REF]. One argument is precisely that it is just unnatural to treat simple properties like cold, red, or sitting as binary relations with a time argument.

Without going into this debate, we note here the striking similarity with the issue about the property truth. If the choice is between adding a time argument to cold, indeed to practically all ordinary properties and relations, and adding a time argument to true, aren't both forms of relativism? Also, the choice bears on technical issues; intuitions about simplicity will not be enough.

In conclusion

Relativism and Monadic Truth is an eminently readable book. The pace is fast, the style is witty, a wealth of interesting issues are raised in only 148 pages.

Some of these issues are cursorily treated, but this is intentional. The idea is to create the impression that there are overwhelmingly many pieces of evidence, some strong, others more speculative, but all pointing in the same direction:

Truth is monadic, propositions are true or false simpliciter. The evidence is mostly indirect: purported arguments against Simplicity are faulty.

One problem with this approach is that critics will not find the discussion detailed enough. Unless impressed with the sheer number of (counter-)arguments, they will hardly be convinced that they are wrong. So in a sense the book is not written for specialists in the philosophy of language, but for a more general audience. Which is fine, of course. Moreover, both specialists and a philosophically interested general audience may be inspired by it, or provoked by it, to undertake a deeper scrutiny of the attractions of Simplicity.

  Lewis and Kaplan style relativity to worlds, times, etc. is only sketched; it is the focus of Chapter 3. The bulk of Chapter 1 discusses recent more far-reaching relativisms due to Kölbel, Lasersohn, MacFarlane, Egan, and others. Three characteristic traits of such relativism are identified: Proliferation, Disquotation, and Nonrelativity of semantic value and belief reports.

Chapter 3

 3 is perhaps the most radical one in the book, taking on the whole tradition from Lewis and Kaplan. The most detailed discussion centers around what C&H call Operator arguments, to the effect that certain linguistic phenomena show that operators on worlds, times, locations, or standards of precision are present in language, and that these parameters belong to what Kaplan calls the circumstances of evaluation, not the context of utterance. The latter fixes the reference of indexicals and determines which proposition is expressed, the former are needed to determine whether that proposition is true or false.

  First, C&H observe that although apparently context-sensitive expressions like smart, ready, enough can be used in constructions that pass say tests, this no longer holds for agree. A and B can use Nicola is ready to mean very different things (e.g. that she is ready to go out in the rain, and that she is ready for an exam, respectively), but even though there may be a sense in which A and B both said that Nicola was ready, they hardly agreed about it. This of course undermines the extensive use of say tests in Cappelen and Lepore[START_REF] Cappelen | Insensitive Semantics: A Defense of Minimalism and Speech Act Pluralism[END_REF] to argue for a semantic minimalism assigning a context-independent content to Nicola is ready, and indeed C&H do not hold such a view. The chapter ends with an application to an example used by MacFarlane[START_REF] Macfarlane | Relativism and disagreement[END_REF] to show that besides the proposition under discussion, the possible world concerned by an attitude is needed to decide whether it is in (dis)agreement with another attitude. Jane asserts correctly that Mars has two moons (P), whereas her counterpart June in a merely possible world where Mars has three moons asserts that Mars doesn't have two moons (not-P). Still they do not disagree, MacFarlane says, since the circumstances concerned are different. This would go against both Agree and Simplicity. C&H respond by first emphasizing that talk about disagreement between actual individuals and their counterparts may lead intuitions in the wrong direction. Instead, we should consider what existing persons would have said by uttering S or not-S, had things been different from what they are. As they note, MacFarlane essentially agrees that this way of putting it is preferable. But then they find it "entirely obvious" to say that if June under the different circumstances would have asserted not-P, what she would have said is in disagreement with what Jane actually asserted.

The relativist arguments are based on certain data about expressions that seem context-insensitive with respect to content, yet prompt truth ascriptions that vary with context. Chapter 2 examines two types of tests for identity (non-relativity) of semantic value across contexts: speech reports involving says, believes, and cognates, and reports of agreement or disagreement.

C&H first consider a simple test, called Says-That (ST), which roughly amounts to the following: If A utters sentence S in a context c, and if it is easy to claim of that utterance, though in another context c , that A said that S is true, this is evidence that there is a level of content in S not sensitive to the difference between c and c . To cast doubt on the reliability of ST, C&H present obviously context-sensitive expressions that yet pass the test, such as locationsensitive expressions like left, nearby, and local. For example, if A says Naomi went to a nearby beach referring to a location close to A, B can report that utterance from any location by: A said that Naomi went to a nearby beach. It would be absurd to conclude that nearby makes no context-bound contribution to sentences containing it, hence ST seems flawed.

Having proved ST ineffective, C&H suggest a diagnosis: due to flexible mechanisms of saturation, an utterance can be parasitic on contexts other than the utterance context. Consequently, a test based on collective reports, Collective-Says-That (CST), blocking the possibility for a reporter to parasite upon one single context, might be more promising. However, they argue that CST fails too, in the following kind of situation. C reports: A and B said that Naomi went to a nearby beach. Even assuming that A and B where significantly differently located when uttering Naomi went to a nearby beach, we may find C's report felicitous and reasonable. This is so, it seems, since we are able to understand that Naomi went to a beach nearby A and B, respectively. C&H note that it is easy to explain why CST fails to produce the expected result in these cases, if the logical form is understood in terms of lambda abstraction:

A and B λx(x said that Naomi went to a beach nearby (to x))

That is, both A and B are individuals x such that x said that Naomi went to a beach nearby to x (p. 47). Similar lambda abstraction is used anyway to account for other linguistic phenomena, such as so-called sloppy reading of elliptic statements like John likes his mother and so does Bill.

In Part 2 of the chapter, C&H examine data about constructions containing the verbs agree and disagree, by designing a test, called Agree, for agreement reports. The test comes in three versions, but essentially it goes as follows: If it is easy to find pairs of contexts c and c such that utterances of S in c and c , respectively, cannot be reported as agreement, this is an indication that S has context-sensitive content. Conversely, if it is hard to find such context pairs for a sentence type, content identity is indicated. Corresponding principles hold for disagreement reports. The reliability of Agree can be checked in the same way as for ST and CST, i.e. whether there are sentences containing obviously context-sensitive expressions which pass the test.

Similarly, whereas ST and CST failed for nearby, agreement tests give intuitively correct results. This is because the verb agree doesn't distribute over the individuals or opinions disquoted, in contrast with say. Thus, in the situation just discussed when A and B both utter Naomi went to a nearby beach, it is not appropriate to report this by saying that A and B agree that Naomi went to a nearby beach. The lambda-abstracted interpretation is no longer available. C&H find additional evidence for the superiority of Agree with mixed quotation reports. Briefly, say lends itself to mixed quote readings in ways which agree doesn't. (2-a) is possible as a mixed quote report -indicating that both used the word gay, though perhaps A meant 'happy' and B meant 'homosexual' -but (2-b) is just infelicitous.

(2) a. A and B both said that they are gay, but they meant very different things by that.

b. A and B agree that they are gay, but they mean very different things by that.

In sum, C&H find (dis)agreement tests quite reliable for tracking sameness and difference of content.

  Theseadditional features, i.e. Contingency and Temporality, are important parts of C&H's view of propositions, however.The idea of Contingency is most naturally explained in terms of some notion of possible world. Indeed, C&H freely avail themselves of possible worlds throughout the book. But they think of the relation of truth at a world as The one argument one finds in the book is, as we already noted, that speakers have a primitive understanding of what it means that P could be true, and no further elucidation is necessary or even desirable. But recall that the whole point of introducing propositions in the first place is to do semantics. And, it seems to us, theorists usually think of the possible worlds idea as something that clarifies the semantics of 'could be true'. But as soon as you accept this (whether propositions are sets of possible worlds or structured entities with 'holes' that can be filled by worlds), truth becomes true at this world, and 'could be true' becomes 'true at some world'. The relation true at cannot be eliminated.8 3.3 TemporalityIt is even harder to make Temporality fit Simplicity. The plausibility of the claim that truth simpliciter is the same as truth in the actual world rests on the idea that there is only one real world. As the authors realize, the corresponding claim for time leads to Presentism: there is only one real time, the present. But although Presentism has its champions, it is hardly a majority view. It is not a simple view. A reader initially attracted to Simplicity might be surprised to find herself saddled with such a heavy ontological commitment.

a technical one, "less fundamental" than monadic truth. Although this is a repeated claim, it is hardly elaborated or explained. The following quote is characteristic: "[There is] . . . no deep threat to Simplicity posed by the hypothesis that the semantic value of some speech is true at some worlds and false at others. For this hypothesis is quite compatible with the hypothesis that this semantic value is true or false simpliciter -one needs merely to be careful not to jumble alethic properties." (p. 94) This is not very enlightening. To be sure, we can always replace 'true at the actual world' by 'true simpliciter ', but the issue is what to do with 'true at w' when w is not the actual world. No hint has been given of how to eliminate this from the semantics, and the reader remains in the dark as to exactly how Contingency is supposed to be compatible with Simplicity.

For similar reasons, it is not clear if non-indexical contextualism is a version of relativism or not, according to C&W. They are content to say that it is "less relativist-sounding" and that it posits Proliferation and Disquotation (p. 20). No reason is given why it should not also embrace Non-relativity of semantic value and belief reports. Although the terminological issue is unimportant, it bears emphasizing that non-indexical contextualism is a position taken by several theorists, some of whom think of themselves as relativists (e.g. Kölbel[START_REF] Kölbel | Faultless disagreement[END_REF]), whereas others (e.g. Brogaard[START_REF] Brogaard | Moral contextualism and moral relativism[END_REF]) reserve that label for the position of Lasersohn and MacFarlane.

Rather, they are modifiers, placed at the front by a rule of fronting.

There are also interesting discussions about the importance of distinguishing between dispositional and non-dispositional uses of taste predicates, and between generic and particular applications of them; we forego that here.

Does Presentism make this problem go away? We admit that we do not see how.

can take the semantic values of sentences in context to be location-independent propositions 6 without running into trouble with compositionality?

First, what is the logical form of (3)? One suggestion is that it is rain(l), and that the function assigning semantic values in context, let us call it cont for 'content', is such that cont(rain(l), c) = rain(l c ) , the proposition that it rains at l c . This seems in fact to be what C&H suggest. 7 But that doesn't work, because then we cannot calculate cont(∃l rain(l), c) from cont(rain(l), c) (and the meaning of ∃), so cont is not compositional, just as Lewis said. We seem to be forced to say that the logical form of ( 3) is something like rain(l c ) instead.

But this too is a bit hard to swallow, at least if one assumes that logical form pertains to expressions, independently of extra-linguistic context.

Second, suppose this could somehow be solved. We still need to associate a content with rain(l), i.e. we need to say what cont(rain(l), c) is, in order to compositionally derive the proposition expressed in (4). In the present set-up, this value must be something like a set of locations (the set of locations where it rains). Indeed, one could argue that sentences and formulas are expressions of the same category, and therefore should have the same kind of semantic values. Only, when there are no free variables, as in (4), this will be either the empty set or the set of all locations. In that case we may be able to associate a proposition of the kind C&H or King wants. Still, having the right content for formulas is crucial to compositionality. And these contents are very much like location-dependent propositions. Thus, location-dependent propositions do not really go away, even if they may be avoided, with some effort, as values of closed sentences. This is not surprising, since whether one uses location operators or King's alternative account, quantification over locations occurs in sentences like [START_REF] Kölbel | Faultless disagreement[END_REF]. Also, as we saw, the details of a compositional account may be more tricky than they seem. 6 It doesn't matter here whether these are absolute as in the book or functions from worlds and standards of precision to truth values as in King's paper.

7 "[O]ne might hypothesize that the deep structure of 'It is raining' is 'It is raining at x ', and then allow that in some contexts the hidden pronoun refers to some salient location while in others it is bound by a quantifier." (p. 83)


Geert Van Tetering

Niels Bovenschen

Jan Meeldijk

Paul J Van Diest

Marc Vooijs
email: marc.vooijs@maastrichtuniversity.nl

Cleavage of Notch1 by Granzyme B Disables Its Transcriptional Activity

Keywords: Notch signaling, Granzyme B, cleavage, J-secretase, proteolysis, serine protease

Granzyme-mediated cell death is the main pathway for cytotoxic lymphocytes to kill virus-infected and tumor cells. A major player in this process is granzyme B (GrB), which triggers apoptosis in both caspase-dependent and caspase-independent manners. A caspase-independent substrate of GrB is the highly conserved transmembrane receptor Notch1. The GrB cleavage sites in Notch1 and functional consequences of Notch1 cleavage by GrB were unknown. We confirmed that Notch1 is a direct and caspase-independent substrate of GrB. We demonstrate that GrB cleaved the intracellular Notch1 domain at least at two distinct aspartic acids D1860 and D1961. Granzyme B cleavage of Notch1 can occur in all subcellular compartments, during maturation of the receptor, at the membrane, and in the nucleus. GrB also displayed perforin-independent functions by cleaving the extracellular domain of Notch1. Overall, cleavage of Notch1 by GrB resulted in a loss of transcriptional activity, independent of Notch1 activation. We conclude that GrB disables Notch1 function, likely resulting in anti-cellular proliferation and cell death signals.

Introduction

The immune system uses two types of cells as its primary defense against tumor cells and viral pathogens, i.e. natural killer (NK) cells and cytotoxic T-lymphocytes (CTL). Both types of cytotoxic cells harbor cytotoxic granules that are released upon target cell recognition. These granules contain serine proteases, also called granzymes, and the pore-forming protein perforin. While perforin pores facilitate the entry of granzymes into the target cell, the latter trigger apoptosis by cleaving critical death substrates [START_REF] Chowdhury | Death by a thousand cuts: granzyme pathways of programmed cell death[END_REF].

Of the five human granzymes, granzyme B (GrB) is the most potent granzyme in inducing apoptosis [START_REF] Afonina | Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B[END_REF]. GrB initiates target cell apoptosis in two ways. First, it cleaves the BH3 domain protein Bid thereby activating the Bax pathway that leads to mitochondrial cytochrome C release. This leads to the formation of the Apaf1 apoptosome, resulting in the activation of pro-caspase 9/3 and subsequent DNA fragmentation and cell death. Second, GrB triggers apoptosis by directly cleaving and activating pro-caspase 3 [START_REF] Afonina | Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B[END_REF]. Caspase activity can be efficiently and irreversibly inhibited by specific synthetic peptides, like Benzyloxycarbonyl-Val-Ala-Asp (OMe) Fluoromethylketone (z-VADfmk) [START_REF] Slee | Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32[END_REF].

Besides activating caspases, GrB also has substrates that contribute to a caspase-independent cell death. One of these substrates of GrB is the intracellular domain of Notch1, a type I transmembrane receptor [START_REF] Loeb | Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis[END_REF]. The Notch1 receptor is part of a highly conserved signaling pathway essential in controlling spatial patterning, morphogenesis and cellular homeostasis in embryos and adults. Notch receptors (N1 to N4) are transmembrane glycoproteins that transduce signals by binding to membrane bound ligands (i.e., Delta, Jagged) on adjacent cells. During maturation and transport Notch1 is cleaved in the Golgi-system by a furin-like convertase (S1) resulting in a heterodimeric receptor. Upon ligand binding at the cell surface, Notch receptors undergo two successive proteolytic cleavages: an ectodomain cleavage (S2) followed by intramembrane proteolysis by J-secretase (S3; Figure 1A) [START_REF] De Strooper | A presenilin-1-dependent gamma-secretaselike protease mediates release of Notch intracellular domain[END_REF]. This process releases the Notch intracellular domain (NICD), which translocates to the nucleus and binds the transcription factor CBF1/ Suppressor of Hairless/Lag-1 or CSL (RBP-JN in mice) participating in a complex activating its target genes [START_REF] Christensen | lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis elegans, is homologous to human CBF1 and Drosophila Su(H)[END_REF][START_REF] Fortini | The suppressor of hairless protein participates in notch receptor signaling[END_REF][START_REF] Fryer | Mastermind mediates chromatin-specific transcription and turnover of the Notch enhancer complex[END_REF][START_REF] Schroeter | Notch-1 signalling requires ligandinduced proteolytic release of intracellular domain[END_REF][START_REF] Stifani | Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins[END_REF]. Notch proteolysis is controlled by conformational changes in the extracellular domain induced by ligand binding or cancer-prone oncogenic mutations [11]. Increasing evidence suggests that additional proteases are capable of activating or inactivating Notch receptor signalling, however the significance of this remains unclear.

Notch1 was identified as a GrB substrate in an in vitro proteomics screen [START_REF] Loeb | Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis[END_REF]. In vitro validation studies demonstrated Notch1 is cleaved by GrB primarily at a single-unidentified-site, yet more fragments are observed at higher GrB concentrations indicative of multiple cleavages. Experiments exposing cell lysates to purified GrB showed this cleavage occurs in cells in a caspase independent manner. Moreover, NK cell mediated killing of intact K562 cells resulted in similar cleavage of Notch1 as seen in vitro and in cell lysates. This indicated physiological granzyme levels are sufficient to cleave Notch1 in a caspase-independent manner. Although Loeb et al. identified Notch1 as a physiological GrB substrate several questions remain regarding the mechanism and the consequence of GrB cleavage of Notch1. In the present study, we addressed where in the cell Notch1 is cleaved by GrB and at which cleavage sites, and its effect on Notch1 signaling activity. We show that Notch1 is cleaved by GrB at multiple sites in vitro and in living cells independent of its activation. Importantly, GrB cleavage occurs in all subcellular compartments and results in a loss of Notch1 transcriptional activity.

Materials and methods

Cell lines -HeLa FRT cells were generated by transfecting the FRT-LacZeo plasmid according to the manufacturer's instructions (Invitrogen). Single integration was determined using Southern Blotting (not shown). pcDNA5-FRT-Notch1 constructs were stably integrated into the FRT site after selection of HeLa cells with 200 μg/ml Hygromycin B. The selected single integrant cells were maintained at 200 μg/ml Hygromycin B.

GrB production and purification -Active recombinant human GrB and inactive control GrB-SA were expressed in Pichia pastoris and purified by cation-exchange chromatography as previously described [START_REF] Bovenschen | NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network[END_REF][START_REF] Bovenschen | Granzyme K displays highly restricted substrate specificity that only partially overlaps with granzyme A[END_REF]. GrB preparations were dialyzed against 50 mM Tris (pH 7.4), 150 mM NaCl (TBS) and stored at -80°C. GrB, but not GrB-SA, was active as determined by the small synthetic chromogenic substrate IETD-pNA (Bachem) (data not shown).

Plasmids and vectors -All mNotch1 plasmids were initially cloned into pCS2+ 6Myc as described [START_REF] Kopan | Signal transduction by activated mNotch: importance of proteolytic processing and its regulation by the extracellular domain[END_REF]. Notch1 full length, LNR, NEXT, NICD were constructed as described previously, HA-N1FL-6MT was a gift from R. Kopan (Washington University, St. Louis, USA). Mutations in Notch molecules were introduced by PCR-directed cloning. For in vitro transcription all mNotch1 variants were cloned in the pSensor backbone (Promega), replacing the luciferase. For stable expression in FRT cell lines Notch was subcloned into pcDNA5 vector (Invitrogen). A promoter fragment containing 12 x CSL synthetic binding sites in tandem (kindly provided by S. Boyle (Washington University, St. Louis, USA)) was subcloned into pGL4.24 (Promega) and was used for Notch transcription assays. For normalization of transcription assays a CMV-driven Gaussia luciferase (Gluc, NEB) was used. N-terminally tagged Gluc-N1FL was created using PCR-directed cloning from the pGluc-Basic (NEB).

In vitro translation -Notch1 was produced in vitro using the TNT ® SP6 Coupled Wheat Germ Extract System (Promega) in combination with FluoroTect™ GreenLys in vitro Translation Labeling System (Promega). Gels were analyzed on a Typhoon scanner (GE Healthcare).

GrB assay -Cells were pre-treated with 100 μM z-VAD-fmk in serum-free medium for 1 hour. Cells were washed twice with PBS before incubation with the GrB mix, which consists of the perforin analog streptolysin O (SLO) (5μg/ml), zVAD-fmk (100 μM), GrB or GrB-SA (diluted in TBS). The mix was incubated on the cells for 30 minutes at 37qC, after which the cells were washed with serum free medium and lysed by the addition of 17 μl 4x Laemmli buffer to 50 μl serum-free medium. In case of ɶ-secretase inhibition cells were treated with dibenzazepine ((DBZ) 200 nM) for 16 hours before GrB incubation. For inhibition of ER-Golgi transport Brefeldin A (1 mg/ml stock in methanol) was used at a concentration of 2 μg/ml for 4 hours at 37qC. GrB activity was blocked using up to 300 nM of the inhibitor Ac-IETD-CHO (Merck). The caspase-3/7 GLO assay (Promega) was used according to the manufacturer's instructions.

Western Blotting -Samples were boiled for 5 minutes at 95 °C and run on an SDS-PAGE gel, blotted onto a PVDF membrane, and incubated with antibodies overnight at 4°C. Antibodies used were Anti-Myc (9E10) 1:5000, Anti-cleaved Notch (Val1744, Cell Signaling) 1:1000, Anti-cleaved caspase 3 (Asp175, Cell Signaling) 1:1000, Anti-HA (12CA5), Anti-Actin (MP Biomedicals) 1:10000.

Transcription assays -HeLa cells were transfected with a pGL4.24 12 x CSL Firefly luciferase reporter, pBasic-CMV Gaussia luciferase was used as a transfection control. Cell samples were lysed using Passive Lysis Buffer and media was collected, spun down and diluted in Passive Lysis Buffer before analysis with the Dual luciferase kit (Promega) on a luminometer. Firefly values were corrected for the secreted Gaussia luciferase values, resulting in relative light units (RLU). Membrane fractions -HeLa FRT N1 wt cells were grown in a T165 until confluency. Cells were washed 3x 20 ml PBS, incubated with 14 ml hypotonic PBS (5% PBS in ddH 2 O) for 15 min at room temperature. Cells were washed with vesiculation buffer (2ml 5M NaCl, 50μl 1M MgCl 2 , 500μl 1M KCl, 709 mg Na 2 HPO 4 , pH 8.8) and incubated with 10 ml vesiculation buffer for 90 minutes at 37qC. The buffer was collected and spun at 250 g for 5 minutes at 4qC to get rid of cell debris. The supernatant was spun at 100,000 g at 4qC for 1 hour. The pellet was resuspended in serum-free medium and divided over 3 wells of a 96-well plate for GrB incubation.

Apoptosis assay -Cells were grown in 96 wells. 8 hours after GrB incubation cells were trypsinized and spun down at 1500rpm for 5 minutes. Cells were resuspended in 50μl Annexin V binding buffer (140 mM NaCl, 4 mM KCl, 0.75 mM MgCl 2 , 10 mM HEPES, 1.5 mM CaCl 2 , pH 7.4) containing Annexin-V-Fluos (Roche) and 1μg/ml Propidium Iodide (PI) and incubated for 15 minutes at room temperature. Before FACS analysis samples were diluted with 150 μl Annexin V binding buffer.

Results

Notch1 is directly cleaved in vitro by granzyme B

To investigate cleavage of Notch1 by GrB in more detail we produced a Notch1 protein mimicking the membrane bound form that lacks most of the extracellular domain (termed NEXT), containing a Cterminal Myc epitope tag (Figure 1). NEXT proteins are constitutively active and a substrate for Jsecretase [START_REF] Mumm | A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1[END_REF][START_REF] Van Tetering | Metalloprotease ADAM10 is required for Notch1 site 2 cleavage[END_REF]. NEXT6Myc recombinant protein was produced in vitro and visualized by SDS-PAGE after incorporation using fluorescently labeled Lysines. In vitro translated NEXT proteins were incubated with increasing concentrations of a catalytically active GrB and a control mutant GrB-SA, which has attenuated cleavage activity. GrB cleavage of Notch1 was completely inhibited using a GrB specific inhibitor, ruling out possible contaminants present in the P.pastoris culture (Supplementary Figure 1). We observed the appearance of several cleavage products with increasing concentrations of GrB within 60 minutes of incubation (Figure 2A). At the lowest concentration tested (5 nM of GrB) already a cleavage product with an estimated size of 55 kDa was observed. With higher concentrations of GrB an additional cleavage product of around 45 kDa was detected. At the highest concentrations, the GrB-SA also generated the 55 kDa cleavage product, however to a much lesser extent than the wild type GrB. Under these conditions no 45 kDa product was observed with the GrB-SA whereas full conversion from the 55 kDa precursor had occurred with wild type GrB. Others have previously reported that the single SA mutation attenuated but does not completely abrogate granzyme activity in vitro [START_REF] Giesubel | Cell binding, internalization and cytotoxic activity of human granzyme B expressed in the yeast Pichia pastoris[END_REF][START_REF] Wu | Structural basis for proteolytic specificity of the human apoptosis-inducing granzyme M[END_REF]. To further investigate GrB cleavage products we made use of the Carboxy-terminal Myc epitope. Myc immunoblotting revealed that both the 55 and the 45 kDa could be readily detected, supporting the notion that the 45 kDa product was produced from the 55 kDa precursor (Figure 2B). To see whether sequences within the transmembrane or extracellular domains were required for GrB cleavage we also investigated whether the non-membrane bound form, called NICD (mimicking the J-secretase cleaved form of Notch1), was also a substrate for GrB. In vitro produced NICD1 proteins were also efficiently cleaved by GrB producing similar cleavage products 55 and 45 kDa, respectively (Figure 2C,D). These results indicate that the intracellular domain of Notch1 is a direct substrate of GrB in vitro.

GrB cleaves Notch1 in vivo independent of caspases

To address if GrB also cleaves Notch1 under physiological conditions, we produced HeLa cells stably expressing a full-length constitutively active Notch1 receptor carrying a mutation (L1594P) similar to those found in human acute T-cell leukemias [START_REF] Weng | Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia[END_REF]. N1L1594P HeLa cells show ligand-independent Notch1 cleavage and activity as shown by immunoblotting and transcriptional reporter assays which is fully blocked by incubation of J-secretase inhibitor dibenzazepine (DBZ) (Supplementary figure 2A,B). Next, we incubated GrB with N1L1594P cells, in combination with Streptolysin-O (SLO) acting as a pore-forming protein allowing GrB to enter the cell (Figure 3A). Immunoblotting revealed a dose dependent appearance of a 55 kDa fragment reaching a maximum with 450 nM of GrB. This 55 kDa fragment appeared similar to the 55 kDa fragment observed by in vitro cleavage of Notch1 by GrB. Notably at lower concentrations of GrB, little if no cleavage occurred compared to 600 nM of the catalytic site mutant GrB-SA. Interestingly, whereas in vitro cleavage by GrB led to the consecutive cleavage of the 55 kDa precursor to produce a smaller 45 kDa cleavage product this was not readily observed in the Notch1 expressing stable cell line exposed to GrB; only upon enrichment the fragment could be detected (Figure 5B). Importantly, Notch1 cleavage could only be detected in the presence of both GrB and SLO indicating that cellular uptake of GrB was required for cleavage (Supplementary figure 3A,B). To address whether the cleavage of Notch1 in vivo is caused by GrB through activation of a caspasedependent pathway, we incubated cells with GrB and the cell-permeable pan-caspase inhibitor z-VAD-fmk, which irreversibly binds to the catalytic site of caspases and inactivates its proteolytic activity including autoproteolysis. Incubation of GrB with HeLa N1L1594P cells caused a dosedependent increase in the appearance of the 55 kDa cleavage product largely independent of 3B, upper panel). Under these conditions, z-VAD-fmk efficiently blocked caspase autoproteolysis as shown by immunoblotting for activated caspase-3 (Figure 3B, lower panel) and by a caspase-3/7 pro-fluorescence substrate assay (Supplementary Figure 4). Finally, zVAD blocked GrB-induced apoptosis as measured by AnnexinV-Propidium-iodide labeling and Flow cytometric analysis. Thus, GrB cleavage of Notch1 in vivo does not depend on caspase-dependent cleavage.

To address whether prolonged GrB activity led to a full conversion of the 55 kDa fragment and the formation of additional cleavage products, we incubated HeLa N1L1594P with 300 nM GrB for 30 minutes after which cells were washed and incubated again for varying time-points. We observed robust cleavage and production of the 55 kDa fragment after 30 minutes of GrB incubation. Continued incubation for up to 120 minutes reduced the presence of the 55 kDa cleavage product. Furthermore this reduction was accompanied by a concomitant reduction of the Notch1 full-length unprocessed and S1 cleaved Notch1 TMIC isoforms (Figure 3C). By Myc immunoblotting we could not detect the formation of the 45 kDa product nor of any other additional cleavage products generated from the precursor fragment within 2 hours after GrB addition (Figure 3C). Taken together, these results indicate that GrB cleaves Notch1 in vivo independent of caspases.

GrB cleavage is J J-secretase independent Notch activity requires proteolytic processing by a metalloprotease and J-secretase [START_REF] Mumm | A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1[END_REF]. To address whether GrB induced cleavage is dependent on J-secretase activity, we pre-incubated HeLa N1L1594P cells with the J-secretase inhibitor (GSI) dibenzazepine after which GrB (with z-VAD-fmk) was added for 30 minutes and cell extracts were analyzed by immunoblotting. Whereas the GSI efficiently blocked cleavage of Notch1 at Val1744, it did not influence GrB cleavage. Both in the presence and absence of GSI the 55 kDa cleavage product was readily observed (Figure 4). These data point to a role for GrB in cleavage of the membrane bound Notch1 isoforms as well as the S3/Jsecretase cleaved NICD. This is supported by the observation that the full-length uncleaved Notch1 isoform also appears to be a GrB substrate (Figure 3C,4).

GrB cleaves Notch in all subcellular compartments.

The previous experiments support a role for GrB cleavage of Notch1 independent of J-secretase. Our results indicate that both the truncated constitutively active and full-length unprocessed forms of Notch1 are GrB substrates. To investigate in which subcellular compartments Notch is cleaved by GrB we determined cleavage patterns in a step-by-step manner. First to address whether full-length unprocessed Notch1 is a GrB substrate we treated HeLa N1 L1594P cells with Brefeldin A; an inhibitor of anterograde ER-Golgi transport. Brefeldin A treatment blocked Notch S1 processing as expected [START_REF] Blaumueller | Intracellular cleavage of Notch leads to a heterodimeric receptor on the plasma membrane[END_REF]21] leading to an accumulation of full-length unprocessed form of Notch at the expense of TMIC (Figure 5A). Upon incubation of these cells with GrB, cleavage of the unprocessed full-length precursor into the 55 kDa fragment was observed. Next, we determined if the cell surface bound signaling competent Notch receptor is also a substrate for GrB. To address this we enriched for plasma membrane bound Notch1 receptors from cells using an osmotic based vesiculation process [START_REF] Cohen | Osmotic swelling of phospholipid vesicles causes them to fuse with a planar phospholipid bilayer membrane[END_REF], whereby vesicles bud off the plasma membrane containing surface molecules. To circumvent the presence of S2 and S3 Notch processing fragments in these vesicles we used HeLa cells expressing the wild type Notch1 receptor (HeLa N1wt) and all preparations contained J-secretase inhibitor. We incubated isolated vesicles directly with GrB and addressed whether membrane-bound Notch is a substrate. A clear enrichment of membrane bound Notch1 (TMIC) was observed and very little unprocessed Notch1 on the membrane as expected [START_REF] Bush | Ligand-induced signaling in the absence of furin processing of Notch1[END_REF]. Notch cleavage products of 55 kDa and 45 kDa were readily detected in vesicles exposed to GrB but not to GrB-SA. Further a loss of the unprocessed full-length isoform was observed as well (Figure 5B). These results indicate that the membrane S1-cleaved signaling competent Notch 1 receptor is also a GrB substrate. Both our in vitro and in vivo data suggest that NICD1 may be a direct GrB substrate (Figure 2). To address if NICD1 is a direct substrate in living cells, HeLa cells transfected with NICD1 were treated with GrB and lysates were analyzed on immunoblots. In the absence of active GrB, NICD1 was expressed at the expected molecular weight as determined by C-and N-terminal antibodies (Figure 5C). Upon incubation of GrB the expected 55 kDa processing fragment was observed by Myc immunoblotting. This indicates that the membrane cleaved activated Notch1 isoform NICD is also a GrB substrate. So far our data have shown that all intracellular forms of Notch are subject to GrB cleavage, but recent observations point to a role of extracellular activity of GrB which is perforin-independent [24][START_REF] Chamberlain | Perforin-independent extracellular granzyme B activity contributes to abdominal aortic aneurysm[END_REF][START_REF] Prakash | Active and zymogen forms of granzyme B are constitutively released from cytotoxic lymphocytes in the absence of target cell engagement[END_REF]. Therefore, we investigated whether GrB can induce cleavage in the mature cell surface exposed Notch extracellular domain (NECD) as well. We expressed a full-length Notch1 protein fused to Gaussia Luciferase at the extracellular amino-terminus and with a 6 Myc epitope tags at the intracellular carboxy-terminus (Gluc-N1 wt). Release of Gluc in the medium can be measured by a sensitive enzymatic assay directly on cell culture medium. HeLa cells were transfected with Gluc-N1 wt together with the multimerized 12 x CSL binding site containing Notch reporter plasmid driving Firefly luciferase expression. The fusion of Gluc to the Notch receptor did not induce activation of the receptor (not shown). These cells were incubated with GrB in the absence of SLO and Gluc was measured in the medium 8 hours later. A three-fold significant increase in Gluc in the medium was detected when cells were incubated with 300 nM GrB but not with GrB-SA (Figure 5D). Although GrB induced release of Gluc into the medium, this did not lead to an increase in Notch transcriptional activity as measured by the 12 x CSL Notch reporter assay. To determine if GrB is capable of cleaving Notch in the non-matured intracellularly located NECD, we expressed an N-terminally HA-tagged and C-terminally Myc-tagged Notch molecule (HA-N1-6MT) in HeLa cells. After treatment of these cells with GrB in the presence of SLO we analyzed the Nterminus on HA immunoblots. As expected Myc immunoblots showed the GrB induced expected 55 kDa fragment, in addition HA blots showed a concomitant reduction in size from the full-length unprocessed Notch1 isoform of approximately 55 kDa (Figure 5E). Interestingly, we did not observe additional cleavage products suggesting that NECD is not a direct substrate for GrB.

Identification of GrB cleavage sites in Notch

There are five predicted GrB cleavage sites with an aspartic acid in the P1 position within the Notch1 intracellular domain [START_REF] Loeb | Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis[END_REF][START_REF] Thornberry | A combinatorial approach defines specificities of members of the caspase family and granzyme B[END_REF](Figure 6A). To identify the main cleavage site of GrB in Notch, we performed mutation analysis on the three conserved aspartic acid residues most proximal to the membrane (VLPD1823, VDAD1860, VISD1902). Either or both of the mutations D1823Q and D1902E did not result in an abrogation of Notch cleavage since both 55 and 45 kDa fragments could be observed in vitro (Supplementary Figure 5). The aspartic acid mutant D1860A however revealed a blockade in Notch processing demonstrated by the accumulation of the full-length unprocessed NEXT molecule as well as a loss of the 55 kDa fragment (Figure 6B). The 45 kDa fragment, however, could be detected. This indicates that the 55 kDa fragment results from a cleavage at VDAD1860. Interestingly, a shift in cleavage was observed resulting in a 65 kDa fragment reflecting cleavage at the more proximal aspartic acid VLPD1823. By combining the D1860A and D1823Q mutations we observed an accumulation of full-length NEXT and a loss of the 65 and 55 kDa fragments, respectively. Yet, the 45 kDa fragment could still be observed indicating GrB is capable of cleaving this site directly, independent of cleavage at D1860, albeit less efficient. Only after mutating D1961A production of the 45 kDa fragment was blocked and only full-length NEXT protein could be observed (Figure 6B). Myc immunoblotting revealed that in addition to the 55 and 45 kDa fragments also the larger 65 kDa fragment was observed (not shown). In order to determine whether the cleavage sites identified in vitro correspond to the in vivo situation we transfected HeLa cells with a truncated Notch1 LNR constructs containing the same cleavage site mutations. As observed in vitro, mutation of D1823Q and D1902E did not abrogate Notch cleavage in cells (not shown), however the single D1860A mutant shows an accumulation of the full-length Notch precursor and TMIC, and the appearance of the 65 kDa fragment similar to in 6C). Mutating both D1823Q and D1860A completely blocked cleavage in cells as well. In this cell based assay we could detect the 45 kDa fragment in cells in the double/triple mutant, indicating cleavage can take place at this site in vitro and in vivo independent of a proximal cleavage. The additional mutation at D1961A resulted in a complete loss of cleavage as seen in the in vitro results (Figure 6C). These results indicate that the main GrB cleavage sites in Notch1 are the aspartic acids D1860 and D1961.

GrB cleavage disables Notch activity

To address whether GrB cleavage affects Notch activation, we performed gene reporter assays to measure transcriptional response upon Notch1 activation in the presence or absence of GrB. HeLa N1 L1594P cells were transfected with a multimerized 12 x CSL binding site reporter driving Firefly luciferase expression pre-incubated with GSI and z-VAD-fmk after which they were exposed to GrB for 30 minutes, washed and allowed to recover for an additional 4 hrs after which extracts were analyzed for reporter gene luciferase activity. As expected GSI reduced reporter gene activity up to 95% in HeLa N1 L1594P cells that normally have high constitutive Notch activation (Figure 7). Exposure of cells to 300 nM of GrB for 30 minutes significantly reduced transcriptional output after 4 hours suggesting that GrB cleavage of Notch1 reduces Notch1 signaling activity. Post incubation of GrB-treated cells for longer time-points resulted in a non-specific inhibition of reporter activity due to SLO alone (not shown). These results demonstrate that GrB cleavage of Notch leads to the inactivation of Notch1 by interfering with the ability of NICD to activate gene transcription. Notch receptors are GrB substrates irrespective of their activation status. Our results indicate that GrB can cleave Notch at multiple levels, during maturation, at the cell surface and after release from the membrane in the nucleus. Previous results indicate nuclear access of GrB to be primarily caspasedependent [START_REF] Pinkoski | Nuclear translocation of granzyme B in target cell apoptosis[END_REF]. In our study, making use of caspase inhibitors during the course of GrB treatment, we observe GrB cleavage of NICD suggesting that GrB may also enter the nucleus and cleave Notch independent of caspase activity. Cleavage of the Notch effector NICD by GrB leads to direct inactivation of transcriptional activity. Yet, cleavage of the precursor and membrane-bound forms of the receptor results in a lower number of intact Notch molecules at the cell surface able to receive a signal, thereby decreasing signaling capacity indirectly. Thus GrB can inactivate Notch signalling at all levels, either directly or indirectly.

The optimal substrate cleavage site of GrB is P4 I/VxxD-P1 with critical I/V and D residues at P4 and P1 respectively. Considering the Notch1 proteins used here this leaves five possible GrB scissile bonds, at VLPD1823, VDAD1860, VISD1902, VSAD1961 and VLLD2071, all of which are conserved between mouse and human Notch1. We systematically mutated the P1 aspartic acids and found that mutation of both D1823 and D1902 in Notch still resulted in cleavage by GrB similar to wild type controls in vitro and in vivo (not shown). Only upon mutation of D1860 a cleavage defect was observed both in vitro and in vivo. Strikingly, this induced a shift in cleavage in vitro to VLPD1823, resulting in a 65 kDa fragment which disappeared upon additional mutation of D1823. This could indicate that cleavage takes place at two residues simultaneously. However, experiments using the single D1823 mutant ruled out the possibility of subsequent cleavage starting at D1823 (not shown). The D1823Q/D1860A double mutant still showed the 45 kDa cleavage fragment to be present, demonstrating GrB is able to cleave this site directly although less efficient. Mutation of D1961 showed a loss of the smaller 45 kDa fragment, revealing the second cleavage site of GrB. Strikingly, in vivo the 45 kDa fragment is also produced but detection was more difficult for reasons unknown. Only upon high Notch expression or enrichment of Notch molecules the 45 kDa fragment could be detected in living cells. Detection of the 45 kDa may also be hampered because of processing by other proteases or degradation by the proteasome.

GrB cleavages at D1860 and D1961 both resulted in a loss of the RAM domain, yet maintaining part of the ankyrin repeats. The RAM domain is required for NICD binding to CSL as well as inducing derepression of CSL target genes [29], thus loss results in a block of transcription. The CDC10/ankyrin repeats, responsible for CSL binding [START_REF] Wilson | Crystal structure of the CSL-Notch-Mastermind ternary complex bound to DNA[END_REF], can activate Notch signalling in the absence of a RAM domain [START_REF] Aster | Essential roles for ankyrin repeat and transactivation domains in induction of T-cell leukemia by notch1[END_REF][START_REF] Kato | Involvement of RBP-J in biological functions of mouse Notch1 and its derivatives[END_REF][START_REF] Kurooka | Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region[END_REF]. However, this is several-fold lower than wild type NICD1. Since part of the ankyrin repeats is removed by the second cleavage at D1961 this may further attenuate Notch1 transcriptional activity. Thus, both cleavage scenarios lead to a loss of transcriptional activity, thereby effectively blocking transcriptional activation. [START_REF] Kim | Notch interferes with the scaffold function of JNK-interacting protein 1 to inhibit the JNK signaling pathway[END_REF], by inhibiting p53 [START_REF] Beverly | Suppression of p53 by Notch in lymphomagenesis: implications for initiation and regression[END_REF][START_REF] Mungamuri | Survival signaling by Notch1: mammalian target of rapamycin (mTOR)-dependent inhibition of p53[END_REF][START_REF] Nair | Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway[END_REF], or the PKB/Akt pathway [START_REF] Nair | Activated Notch1 inhibits p53-induced apoptosis and sustains transformation by human papillomavirus type 16 E6 and E7 oncogenes through a PI3K-PKB/Akt-dependent pathway[END_REF][START_REF] Sade | The anti-apoptotic effect of Notch-1 requires p56lckdependent, Akt/PKB-mediated signaling in T cells[END_REF]. Breast epithelial cells are protected against apoptosis due to the induction of PKB/Akt signaling in response to Notch1 activation [START_REF] Meurette | Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells[END_REF]. In addition, downregulation of Notch1 in pancreatic cells leads to increased apoptosis [START_REF] Wang | Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells[END_REF]. In tumor cells NICD1 can prevent the degradation of the X-linked inhibitor of apoptosis protein (XIAP) thereby increasing cell survival [START_REF] Liu | Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation[END_REF]. Loss of Notch1 signaling in either of these cases would lead to a loss of anti-apoptotic signals, which may eventually lead to cell death. This supports the hypothesis that GrB cleavage may attenuate Notch1 mediated cell survival and thereby promote apoptotic potential in tumor cells.

T-cell proliferation and maturation is a highly Notch1 dependent process, resulting in various types of T-cells. In a subset of T-cells, mature Th2 cells, cell death induced by GrB is caspase independent [START_REF] Devadas | Granzyme B is critical for T cell receptor-induced cell death of type 2 helper T cells[END_REF]. Consistently, genetic loss or inhibition of GrB leads to Th2 proliferation. Since Th2 cells depend on Notch1 activation for their proliferation [START_REF] Amsen | The different faces of Notch in T-helper-cell differentiation[END_REF], this suggests that GrB could trigger cell death by downregulating Notch1 signalling by directly cleaving the receptor. This would ensure control over Th2 lymphocyte numbers and activity after executing their function in the immune response, thereby tightly controlling cell numbers to avoid an excessive number of activated T-cells. However, in CTLs Notch1 is thought to positively regulate the expression of GrB and perforin by directly binding to their promoters [START_REF] Cho | Notch regulates cytolytic effector function in CD8+ T cells[END_REF]. One could see the GrB cleavage of Notch1 as an activating cleavage thereby inducing an auto-regulatory loop by upregulating GrB and perforin. However, we have clearly shown that cleavage of Notch1 by GrB leads to inactivation of signaling making it very unlikely that within the CTL there is such a positive auto-regulatory loop. The function of GrB cleaving Notch1 within Tcells therefore remains obscure.

Plasma of healthy individuals contains low levels of GrB, whereas patients suffering from viral infections, inflammation, or auto-immune diseases show a several-fold increase in GrB levels [24, [START_REF] Spaeny-Dekking | Extracellular granzymes A and B in humans: detection of native species during CTL responses in vitro and in vivo[END_REF][START_REF] Tak | The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis (RA)[END_REF]. This could be due to leakage out of the immunological synapse, explaining the higher GrB levels in patients since the number of immune reactions is increased. However, recently it was found that GrB is actively secreted into the extracellular milieu [START_REF] Prakash | Active and zymogen forms of granzyme B are constitutively released from cytotoxic lymphocytes in the absence of target cell engagement[END_REF]. Moreover, perforin-independent functions of GrB have been reported [24][START_REF] Chamberlain | Perforin-independent extracellular granzyme B activity contributes to abdominal aortic aneurysm[END_REF][START_REF] Prakash | Active and zymogen forms of granzyme B are constitutively released from cytotoxic lymphocytes in the absence of target cell engagement[END_REF]. Here we show that the extracellular domain of Notch1 receptor is a substrate to cleavage induced by GrB in the absence of perforin. We observed a significant threefold increase in GrB induced Notch1 shedding, however, without the induction of transcriptional activation. This suggests that the cleavage must take place at the N-terminal end upstream of the NRR domain, since removal of this domain leads to activation of Notch. Ligand binding to Notch is thought to induce a substantial conformational change of the NRR leading to consecutive S2 and S3 cleavage, producing NICD [START_REF] Van Tetering | Metalloprotease ADAM10 is required for Notch1 site 2 cleavage[END_REF][START_REF] Gordon | Structural basis for autoinhibition of Notch[END_REF]48]. The GrB induced cleavage of NECD however does not lead to such a conformational change sufficient for receptor activation. Yet, this cleavage could shed off the ligand-binding domain, leaving the receptor inactive at the cell surface unable to signal. In this way GrB can inactivate the receptor indirectly, resulting in an increased pro-apoptotic environment. Interestingly, there is good correspondence between the cleavage fragments observed using N-and C-terminal epitope tags after GrB cleavage. Whereas Myc immunoblotting detects the appearance of the 55 kDa cleavage product the N-terminal HA tag detects a similar reduction in the size of the fulllength precursor. Importantly, no other cleavage events were observed suggesting that the Gluc release in the medium seen after GrB exposure of cells is an indirect effect of GrB. Thus the NECD is cleaved by a protease which is activated by GrB. It will be interesting to identify the proteolytic activity induced by GrB responsible for this NECD cleavage.

Collectively, our results demonstrate that Notch1 is an efficient direct substrate for GrB. Cleavage of Notch1 leads to inactivation of signaling both by the action of intra-as well as extracellular GrB. Direct cleavage of active NICD, but also downregulating total numbers of Notch1 precursors and receptors at the membrane decreases signaling capacity. This downregulates anti-apoptotic and proproliferative signals achieving a faster and more efficient apoptotic response. This example clearly shows an important caspase-independent function of granzymes. Since tumor cells recognized by CTLs will not only encounter secreted GrB, but a mixture of granzymes [START_REF] Bovenschen | Orphan granzymes find a home[END_REF] it will be of interest to determine the caspase-independent spectrum of substrates of the granzymes altogether to gain more insight into the cytotoxic killing mechanisms involved in cancer. In HeLa cells expressing activated Notch molecules GrB induces efficient cleavage. D) HeLa cells expressing N-terminally tagged Gluc-Notch1 was incubated with GrB in the absence of perforin. Gluc activity was measured in the medium, and form lysates the 12xCSL driven Notch activity was determined. A significant (* p=0.0495) increase was observed upon 300 nM GrB which did not lead to Notch activation. Graph is representative of at least two independent experiments in triplo, the pvalue is calculated using a Kruskall-Wallis non-parametric test. E) Expression of N-terminally tagged HA-Notch-FL-6Myc indicated the NECD was not a direct substrate of GrB. Arrow indicates HA-tagged NECD.

Biochemical

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Biochemical

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  Biochemical Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110226 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110226 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited caspase activity (Figure

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110226 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110226 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited vitro cleavage of recombinant substrates (Figure

Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110226 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110226 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Discussion Granzyme B is essential for NK cells and CTLs induced cell death in pathogen-infected and tumor cells. This cell death is mainly mediated by the action of granzymes packaged in cytotoxic granules that will be released into the target cell upon recognition. Previously, by NK-mediated cytotoxicity Loeb et al. showed physiological levels of GrB are able to cleave Notch1 in a caspase-independent manner. It cleaves Notch1 into at least a single fragment at an unidentified site [4]. In the present study, we confirmed that GrB cleaves the Notch1 receptor irrespective of caspase activation. Proteolyis of Notch by GrB results in at least two fragments in vitro, cleaving after D1860 and D1961. Cell based assays show GrB cleaves Notch at the same sites only in the presence of the pore forming protein SLO demonstrating GrB cellular entry is required to cleave Notch. Furthermore, we show that GrB can cleave Notch receptors irrespective of its activity and location; it cleaves the immature Notch precursor, the cell surface bound receptor as well as the membrane cleaved activated NICD. Notch1 cleavage by GrB inactivates the signaling cascade directly by blocking transcriptional activity.

Biochemical

  Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110226 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110226 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2011 The Authors Journal compilation © 2011 Portland Press Limited Loeb et al. suggest the cleavage of Notch1 will lead to an increase in apoptosis in tumor cells. This coincides with our findings that cleavage of Notch1 results in a block of transcriptional activity. Inhibition of apoptosis by Notch1 appears to occur via several mechanisms. Notch1 has been shown to protect cells from apoptosis acting via JNK activation

Biochemical

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Biochemical

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Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. In vitro cleavage of Notch1 by GrB in a dose-dependent manner. A) SDS-PAGE analysis showing in vitro produced NEXT-6Myc proteins with incorporated fluorescently labeled Lysine residues are cleaved upon GrB incubation into 55 kDa and 45 kDa fragments as indicated by arrows. * indicates an alternative translation product. B) Immunoblotting shows both the 55 kDa and 45 kDa fragments are C-terminally 6Myc tagged. C) Similar to NEXT, also the shorter NICD construct is a substrate of GrB resulting in two fragments of 55 and 45 kDa fragments. D) Similar to NEXT, also these fragments are C-terminally tagged with 6Myc.

Figure 3 .

 3 Figure 3. In vivo cleavage of Notch1 by GrB. A) N1 L1594P-HeLa cells incubated with various concentrations of GrB show a 55 kDa cleavage fragment derived from Notch1 as determined by Myc immunoblots. Ponceau S staining of the same immunoblot serves as a loading control. B) Upper panel: N1 L1594P-HeLa cells incubated with the caspase inhibitor z-VAD-fmk show cleavage of Notch1 upon GrB treatment. Lower panel: z-VAD-fmk inhibits the autoproteolysis of caspase 3 (p17) as determined with activated caspase 3 immunoblot.Actin staining of the same immunoblots serves as a loading control. C) After GrB incubation N1 L1594P HeLa cells were additionally incubated for 2 hours. This prolonged incubation demonstrated a decrease in the 55 kDa fragment yet no 45 kDa fragments are observed. In addition, a loss of the full-length and TMIC isoforms of Notch is observed (arrows). * indicates an aspecific product.

Figure 4 .

 4 Figure 4. GrB cleaves Notch independent of activation. N1 L1594P HeLa cells were treated with the J-secretase inhibitor dibenzazepine (DBZ), inhibiting the activating cleavage of Notch1. Upon GrB incubation still 55 kDa fragments could be observed on the Myc immunoblot. The Val1744 immunoblot stains Notch S3 cleavage products.

Figure 5 .

 5 Figure 5. Intra-and extracellular GrB cleavage of Notch. Analysis of GrB action in different subcellular locations: A) Brefeldin A treated cells, blocking ER to Golgi transport, show cleavage of Notch upon GrB incubation. B) Fractions enriched for plasma membrane bound Notch1 show cleavage upon GrB treatment. Arrow indicates 45 kDa fragment. C)In HeLa cells expressing activated Notch molecules GrB induces efficient cleavage. D) HeLa cells expressing N-terminally tagged Gluc-Notch1 was incubated with GrB in the absence of perforin. Gluc activity was measured in the medium, and form lysates the 12xCSL driven Notch activity was determined. A significant (* p=0.0495) increase was observed upon 300 nM GrB which did not lead to Notch activation. Graph is representative of at least two independent experiments in triplo, the pvalue is calculated using a Kruskall-Wallis non-parametric test. E) Expression of N-terminally tagged HA-Notch-FL-6Myc indicated the NECD was not a direct substrate of GrB. Arrow indicates HA-tagged NECD.

Figure 6 .

 6 Figure 6. Cleavage occurs at D1860 and D1961. A) The diagram shows the five predicted cleavage sites in the intracellular part of the Notch1 receptor. B) In vitro mutation analysis reveals that wildtype Notch1 NEXT 6Myc shows the two cleavage fragments, whereas mutation of VDAD1860 shows a loss of the 55 kDa fragment, yet a new 65 kDa fragment appears, which upon additional mutation of VLPD1823 is lost maintaining the 45 kDa fragment. Only upon an additional third mutation of VSAD1961 a complete loss is seen in Notch cleavage upon GrB treatment. * indicates an alternative translation product. C) This could be confirmed in vivo by GrB incubation of HeLa cells expressing Notch1 LNR 6Myc carrying the various

Figure 7 .

 7 Figure 7. GrB cleavage results in a block of transcription. Transcriptional Notch1 activation assay shows a significant (* p=0.0495) loss of activity upon GrB treatment. Graph is representative of at least two independent experiments in triplo, the p-value is calculated using a Kruskall-Wallis non-parametric test.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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Acknowledgements:

We thank S. Boyle and R. Kopan at Washington University Saint Louis for reagents.

Funding: This study was supported by the Dutch Cancer Society (NKB-KWF) [grant UU-2009-4302 to NB and UU2006-3623 to MV] the Netherlands Organization for Scientific Research (NWO) [grant 916.66.044 to NB] and by the European Research Council under the European Community Seventh Framework Programme [FP7/2007-2013/ERC Grant 208259 to MV]. Biochemical Journal Immediate Publication. Published on 06 May 2011 as manuscript BJ20110226 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20110226

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.