FIELD OF THE INVENTION

The invention relates to modulators of IREl activity and to uses thereof.

BACKGROUND OF THE INVENTION

Signalling of the Unfolded Protein Response (UPR) is mediated by three endoplasmic reticulum (ER) resident transmembrane proteins - PERK, ATF6 and IREl. IREl (or ERN1, endoplasmic reticulum to nucleus signalling 1) is a transmembrane sensor of ER stress with a luminal domain containing binding sites for the chaperone BiP and a cytosolic region which shows two principal catalytic elements: a serine/threonine kinase and an endoribonuclease domain. Under basal conditions, IREl is thought to exist as a monomer. However recent structural studies in S. cerevisiae showed that the luminal domain of IREl exists as a dimer and suggested that it could bind directly to non-folded peptides, similar to MHC class I molecules. The dimeric status of IREl was confirmed by structural studies carried out on the cytosolic domain of IREl in S. cerevisiae which revealed a dimer conformation subjected to conformational changes upon ER stress that leads to activation of its kinase and RNAse activities. This suggested that in response to the accumulation of misfolded proteins in the ER, IREl could dimerize and transautophosphorylate thus triggering its endoribonuclease activity. In a more recent study, the cytosolic domain of S. cerevisiae IREl was crystallized in an oligomeric form in the presence of kinase inhibitors such as Sunitinib. This highly ordered molecular structure was found to be physiologically relevant in S. cerevisiae. A revised model for the IREl activation process was proposed in which different IREl dimers promote the trans-autophosphorylation which is observed in vivo in oligomeric structures. This re-orders the RNAse domain and creates a binding surface for the mRNA substrates. However, this model does not take into account the capacity of IREl to induce JNK and ERK signalling cascades and does not provide any insights into the mechanisms by which these kinases are activated downstream of IREl . Regardless, IREl endoribonuclease activity initiates the unconventional splicing of unspliced XBP1 (uXBPl) mRNA that introduces a translational frame-shift leading to the synthesis of a potent transcription factor (sXBPl) (Yoshida, H. et al., Cell 107, 881-91 (2001)). It has also been shown that IREl activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins, through the so-called Regulated IREl -Dependent Decay (RIDD) (Hollien et al., J. Cell. Biol. Vol.186 No.3 323-331). Finally, IREl endoribonuclease activity was shown to be partly independent of IREl phosphorylation (Sha, H. et al., Cell Metab 9, 556-64 (2009)), suggesting the existence of alternative modes of regulation. Although the precise mechanisms of IREl activation and signalling specificity are yet to be fully understood, this protein has been shown to play a key role in many diseases associated with the accumulation of misfolded proteins in the ER, which suggests its potential relevance as a therapeutic target (Ron, D. et al., Nat Rev Mol Cell Biol 8, 519-29 (2007); Moenner, M. et al., Cancer Res 67, 10631-4 (2007)). Moreover, it has been demonstrated that artificially sustaining IRE1 XBP1 signalling using mutant IREl proteins gave a survival advantage to cells subjected to ER stress (Lin, J.H. et al., Science 318, 944-9 (2007); Han, D. et al., Cell 138, 562-75 (2009)).

There is thus a need to provide compounds able to modulate IREl activity in order to enhance or decrease ER stress resistance, e.g. able to treat diseases associated with the protein secretory pathway or stress conditions.

SUMMARY OF THE INVENTION

To date both activators (quercetin, flavonols) and inhibitors of IREl RNAse activity (IREstatins) have been characterized. The inventors have now identified modulators of IREl activity susceptible to favour specific IREl dependent signalling cascades. More particularly, they have found modulators of the first steps of the IREl activation process.

The invention pertains to fragments of 16 to 55 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of:

- SEQ ID NO: l, and

- amino acid sequences having at least 80% identity with SEQ ID NO: 1 , or derivatives thereof.

The invention also pertains to nucleic acid sequences comprising a sequence capable of encoding a fragment or a derivative thereof according to the invention.

The invention also relates to plasmids comprising a nucleic acid sequence according to the invention. The invention still relates to expression vectors comprising a nucleic acid sequence according to the invention or capable of expressing a fragment or a derivative thereof according to the invention.

The invention also relates to host cells comprising a plasmid or an expression vector according to the invention.

The invention still relates to cells which express a fragment according to the invention. The invention also relates to conjugates comprising a fragment or a derivative thereof according to the invention, linked to a Cell Penetrating Peptide.

The invention also relates to fragments or derivatives thereof, or the nucleic acid, or the plasmid, or the expression vector, or the host cell or the cell, or the conjugate according to the invention, for use in a method of treatment of the human or animal body.

The invention also relates to fragments or derivatives thereof, or the nucleic acid, or the plasmid, or the expression vector, or the host cell or the cell, or the conjugate according to the invention, for use in a method for modulating the activity of IREl protein in the human or animal body.

DETAILED DESCRIPTION OF THE INVENTION

Fragments of the cytosolic domain of IREl

The invention first relates to a fragment of 16 to 55 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of:

- SEQ ID NO: 1 , and

- amino acid sequences having at least 80% identity with SEQ ID NO: 1.

Indeed, the inventors have found that it is possible to modulate the activity of the protein IREl with fragments of the cytosolic domain of the IREl protein. The amino acid sequence of IREl is shown in SEQ ID NO: 14 and the cytosolic domain of the protein IREl is shown in SEQ ID NO:l. The fragments of the cytosolic domain of IREl according to the invention are able to interfere with the enzymatic activities of IREl, and thereby to modulate the activity of IREl , i.e. to inhibit or improve the activity of the IREl.

In particular, the fragments according to the invention are able to:

1. inhibit or activate the RJDD (Regulated IREl -Dependent Decay) of mRNAs, and/or 2. enhance or prevent the splicing of unspliced XBP1 (uXBPl) mRNA, and/or

3. inhibit or activate the JNK signalling cascade.

The fragments according to the invention are fragments of SEQ ID NO: 1 or of amino acid sequences having at least 80% identity with SEQ ID NO: 1. Indeed, the amino acid sequence of the fragments according to the invention may be modified, e.g. by substitution, deletion or addition of at least one amino acid as compared to the native sequence SEQ ID NO: l . These modifications of the amino acid sequence of the fragments can be useful to enhance stability, affinity, activity, etc., of the fragments, as illustrated in the experimental section. The fragments according to the invention are thus fragments of SEQ ID NO: l or of amino acid sequences having at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO: 1.

As used herein, the percentage of sequence identity refers to comparisons among amino acid sequences, and is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical amino acid residue occurs in both sequences or an amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981 , Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1 70, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. ScL USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided. In a particular embodiment, the invention relates to a fragment of 16 to 55 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of: - SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, and

- amino acid sequences having at least 80% identity with at least one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9.

In another particular embodiment, the invention relates to a fragment of 16 to 55 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of SEQ ID NO: 5 and amino acid sequences having at least 80% identity with SEQ ID NO:5. Particular fragments of SEQ ID NO:5 are SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13.

In particular embodiment, the fragment or a derivative of the invention comprises at least the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 13. In a particular embodiment, the fragments according to the invention consist of 16 to 50 amino acids. In another embodiment, the fragments according to the invention consist of 16 to 45, particularly of 16 to 40, 16 to 35, 16 to 30, 16 to 25, or 16 to 20 amino acids.

In one embodiment, the fragment or a derivative of the invention is a fragment of 16 to 55 amino acids of an amino acid sequence, wherein said amino acid sequence is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13.

In one embodiment, the fragments according to the invention are selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13.

In a preferred embodiment, the fragment or a derivative of the invention is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 13. Typically, the fragments or derivatives according to the invention alter IRE1 oligomerization.

Typically, the alteration of IREl oligomerization is measured by AlphaScreen® assay. To alter IRE1 oligomerization may be to promote ΙΚΕ * ¹⁰ oligomeric structures formation or to compete with dimer formation.

Typically, the fragments according to the invention alter the AlphaScreen® signal in IRE1 oligomerization assay (by altering it is meant decreasing in a statistically significant manner at a p value < 0.05). The measurement of AlphaScreen® signal alteration in IRE1 oligomerization assay is performed with an AlphaScreen® GST Detection Kit (PerkinElmer®), following a protocol adapted from the protocol as described by Bouchecareilh M. et al., Journal in Biomolecular Screening, 15(4), 2010. The measurement of the alteration of the AlphaScreen® signal in IRE1 oligomerization assay according to the invention is performed according to a test A, which comprises the following steps:

1 ) mix in Reaction Buffer to a final volume of 10 μί:

- 2 μL· of GST-IRE 1 (0.05 k xL final concentration in 20 iL comprising Reaction buffer and beads),

- 7 yiL of serial dilutions of the fragment to be tested (0 to 10 ^"6 M),

- 1 μΐ, ATP (20 mM),

2) incubate for 45 min at 23°C,

3) add

- 5 μΐ- GSH Acceptor beads (20 g/mL final in 20 xL reaction)

- 5 μί, GSH Donor Beads (20pg mL final in 20 \xL reaction)

4) incubate lh at 23 °C in the dark,

5) read on Envision® plate reader.

All the reagents are diluted in 20 mM Tris-HCl, pH 7.4, 50 mM NaCl, ImM MgC12, I mM MnCl ₂ and the beads in 20mM Tris-HCl, pH 7.4, 50mM NaCl, I mM MgC12, 1 mM MnCl ₂₎ 0.5 mM DTT.

Derivatives of the fragments according to the invention

The invention also relates to the derivatives of the fragments according to the invention, wherein said derivatives alter the AlphaScreen® signal of IRE- 1.

The derivatives typically consist of fragments according to the invention which are chemically or biologically modified.

Examples of derivatives are fragments according to the invention wherein: - at least one amino acid of the fragment has been substituted, inserted or deleted, and/or

- at least one amino acid of the fragment is chemically altered or derivatized. Such "chemically altered or derivatized" amino acids include, for example, naturally occurring amino acid derivatives, for example 4-hydroxyproline for proline, 5- hydroxyl sine for lysine, homoserine for serine, ornithine for lysine, and the like. Other "chemically altered or derivatized" amino acids include, e.g., a label, such as fluorescein, tetramethylrhodamine or cyanine dye Cy5.5; or one or more post- translational modifications such as acetylation, amidation, formylation, hydroxylation, mefhylation, phosphorylation, sulfatation, glycosylation or lipidation. Indeed, certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of peptides in human serum (Powell et al, Pharma Res 1993: 10: 1268-1273). "Chemically altered or derivatized" amino acids also include those with increased membrane permeability obtained by N-myristoylation (Brand, et al, Am J Physiol Cell Physiol 1996; 270:C1362-C1369). It is understood that a derivative of a fragment according to the invention may contain more than one of the "chemically altered or derivatized" amino acids.

Other derivatives of the fragments according to the invention are peptidomimetics of said peptides. Peptidomimetics refer to a synthetic chemical compound, which has substantially the same structural and/or functional characteristics of the fragments according to the invention. The mimetic can be entirely composed of synthetic, non- natural amino acid analogs, or can be a chimeric molecule including one or more natural amino acids and one or more non-natural amino acid analogs. The mimetic can also incorporate any number of natural amino acid conservative substitutions that do not destroy the mimetic's activity. Routine testing can be used to determine whether a mimetic has the requisite activity, using Test A according to the invention. The phrase "substantially the same", when used in reference to a mimetic or peptidomimetic, means that the mimetic or the peptidomimetic has one or more activities or functions of the referenced molecule, e.g. alteration of the AlphaScreen® signal of IRE-1. The techniques for developing peptidomimetics are conventional. For example, peptide bonds can be replaced by non-peptide bonds or non-natural amino acids that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure. The development of peptidomimetics can be aided by determining the tertiary structure of the original fragment/peptide, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling. Once a potential peptidomimetic compound is identified, it may be synthesized and its capability of altering the AlphaScreen® signal of IRE-1 can be assayed.

Peptidomimetics can contain any combination of non-natural structural components, which are typically from three structural groups: residue linkage groups other than the natural amine bond ("peptide bond") linkages; non-natural residues in place of naturally occurring amino acid residues; residues which induce secondary structural mimicry (e.g. beta turn, gamma turn, beta sheet, alpha helix conformation); or other changes which confer resistance to proteolysis.

For example, lysine mimetics can be generated by reacting lysinyl with succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transamidase- catalyzed reactions with glyoxylate.

One or more residues can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L- configuration (which can also be referred to as R or S, depending upon the structure of the chemical entity) can be replaced with the same amino acid or a mimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.

As will be appreciated by one skilled in the art, the peptidomimetics of the present invention can also include one or more of the modifications described herein for the "chemically altered or derivatized" amino acids, e.g., a label, or one or more postradiational modifications. The fragments, derivatives and peptidomimetics can be produced and isolated using any method known in the art. The fragments according to the invention can be synthesized, whole or in part, using usual chemical methods. Techniques for generating peptide and peptidomimetic libraries are well known, and include, for example, multipin, tea bag, split-couple-mix techniques and SPOT synthesis.

Conjugates

The invention also relates to conjugates comprising a fragment or a derivative thereof according to the invention, linked to a Cell Penetrating Peptide or CPP.

Indeed, to facilitate the uptake of the fragments or derivatives thereof according to the invention across cell membranes, such as the plasma membrane and/or the nuclear membrane of a cell, it is very useful to conjugate those fragments with a "cell penetrating peptide" (CPP). CPPs are well known peptides which can be conjugated to cargos to facilitate transport of the cargos across the membranes. CPP are for instance well described by Lebleu B. et at, Advanced Drug Delivery Reviews 60 (2008) 517- 529 and by Said Hassane F. et at, Cell. Mol. Life Sci. (2010) 67:715-726. Any CPP can be used to improve the cytoplasmic delivery of fragments or derivatives thereof according to the invention.

Examples of CPP which can be conjugated with the fragments or derivatives thereof according to the invention are the following:

Name Sequence

Tat GR KRRQRRRPPQ

RXR RXRRXRRXRRXR

Bpep RXRRBRRXRRBRXB

Pip2b RXRRXRRXRIHILFQNrRMKWHK

wherein:

- X = aminohexyl, β-alanyl, p-aminobenzoyl, isonipecotyl, or 4-aminobutyryl - B = betaAlanine

- small letter = D-amino acid (D-amino acids may be replaced by L-amino acids).

The CPP is typically linked to the N-terminal or C-terminal end of the fragment or derivative thereof according to the invention, preferably to the C-terminal. Chemical linkage may be performed via a disulphide bond, thioether or thiol-maleimide linkage. In a particular embodiment, the fragment or derivative thereof according to the invention is linked to the CPP through a linker. Any type of linker can be used by the skilled person, provided that said linker allows chemical linkage of the fragment to the CPP. A wide range of linkers are possible, including amino acid sequences having a C- terminal Cysteine residue that permits formation of a disulphide, thioether or thiol- maleimide linkage. Other ways of linking the fragment according to the invention to the CPP include use of a C-terminal aldehyde to form an oxime. Still another type of linkers use click chemistry.

Examples of linkers are amino acid or amino acid sequences chosen from the group comprising: C, BC, XC, GC, BBCC, BXCC, XBC, X, XX, B, BB, BX or XB, wherein:

- X = aminohexyl, β-alanyl, p-aminobenzoyl, isonipecotyl, or 4-aminobutyryl - B = betaAlanine.

In one preferred embodiment, the conjugate of the invention has the amino acid sequence set forth as SEQ ID NO :93. This conjugate, called below TAT-P4, is a conjugate wherein the fragment having the amino acid sequence set forth as SEQ ID NO : 13, called below P4, is linked to TAT peptide.

TAT-P4 : GRKKRRQRRRPQAHGKIKAMIEDFGLCKKL (SEQ ID NO:93)

In another preferred embodiment, the conjugate is a peptidomimetic, all-D retroinverso, of TAT-P4, called below FIRE.

Nucleic acid sequences

The invention also relates to a nucleic acid sequence comprising a sequence capable of encoding a fragment or a derivative thereof according to the invention.

The term "nucleic acid sequence" as used in this document includes genomic DNA, cDNA, synthetic DNA, and RNA. Preferably it means DNA, more preferably cDNA sequence coding for a fragment according to the invention. The nucleotide sequence may be of genomic or synthetic or recombinant origin, which may be double-stranded or single-stranded whether representing the sense or anti-sense strand. Plasmids

The invention still relates to plasmids comprising a nucleic acid sequence according to the invention. As used herein, "plasmids" refer to double-stranded generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmids are typically used as vectors, i.e. as vehicles to transfer a nucleic acid sequence ("insert") into a host cell. Plasmids minimalistically consist of an origin of replication that allows for semi- independent replication of the plasmid in the host and also the nucleic acid sequence insertion. Plasmids may also comprise many more features, notably a "multiple cloning site" which includes nucleotide overhangs for insertion of an insert, and multiple restriction enzyme consensus sites to either side of the insert. Expression vectors

The invention also relates to expression vectors comprising a nucleic acid sequence according to the invention or capable of expressing a fragment according to the invention.

An expression vector according to the invention can be any vector which is capable of expressing a fragment according to the invention in a selected host organism, and the choice of vector will depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, i.e. a vector that exists as an episomal entity, the replication of which is independent of chromosomal replication, such as, for example, a plasmid, a bacteriophage or an episomal element, a minichromosome or 1640 an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome. The expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes. The expression vector normally comprises control nucleotide sequences encoding a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes. For expression under the direction of control sequences, the nucleic acid sequence is operably linked to the control sequences in proper manner with respect to expression. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.

Cells

The invention also relates to host cells comprising a plasmid according to the invention or an expression vector according to the invention.

The invention still relates to cells which express a fragment according to the invention. A cell or a host cell according to the invention can be prokaryotic or eukaryotic. Typically, prokaryote cells are bacterial cells. Eukaryotes cells are for instance selected from plant cells, animal cells, or fungal cells. Preferably, the cells are animal cells, and more preferably human cells.

Applications of the fragments according to the invention

The invention also relates to the use of the fragments or derivatives thereof according to the invention, or of the conjugates according to the invention, or of the nucleic acid sequences according to the invention, or of the plasmids according to the invention, or of the expression vectors according to the invention, or of the host cells or of the cells according to the invention, for modulating the activity of IRE1 protein.

The invention still relates to a method for modulating the activity of IRE1 protein, said method comprising a step consisting of contacting said protein with a fragment or a derivative thereof according to the invention.

The fragments according to the invention can be used either for inhibiting the activity of IRE1 or for enhancing/stimulating the activity of IRE1.

The invention also relates to the use of the fragments or derivatives thereof according to the invention, or of the conjugates according to the invention, or of the nucleic acid sequences according to the invention, or of the plasmids according to the invention, or of the expression vectors according to the invention, or of the host cells or of the cells according to the invention, for inhibiting or activating the RIDD (Regulated 1RE1- Dependent Decay) of mRNAs, and/or for inhibiting or activating the splicing of unspliced XBP1 (uXBPl ) mRNA, and/or for inhibiting or activating the INK signalling cascade. In particular embodiment, the invention relates to the use of a fragment of at least 16 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of SEQ ID NO:5 and amino acid sequences having at least 80% identity with SEQ ID NO:5, for enhancing the activity of MEL More particularly, the invention relates to the use of the fragments SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12 and SEQ ID NO: 13 for enhancing the activity of IREl . Still particularly, the invention relates to the use of the fragments SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12 and SEQ ID NO: 13 for inhibiting or activating the RIDD (Regulated IRE1- Dependent Decay) of mRNAs, and/or for inhibiting or activating the splicing of unspliced XBP1 (uXBPl ) mRNA, and/or for inhibiting or activating the JN signalling cascade.

Methods of treatment of the human or animal body

The invention also relates to the fragments or derivatives thereof according to the invention, or to the conjugates according to the invention, or to the nucleic acid sequences according to the invention, or to the plasmids according to the invention, or to the expression vectors according to the invention, or to the host cells or to the cells according to the invention, for use in a method of treatment of the human or animal body.

In particular embodiment, the invention relates to a fragment of at least 16 amino acids of an amino acid sequence, said amino acid sequence being selected from the group consisting of SEQ ID NO:5 and amino acid sequences having at least 80% identity with SEQ ID NO:5, for use in a method of treatment of the human or animal body. More particularly, the invention relates to a fragment selected from SEQ ID NO: 10, SEQ ID NO: l l , SEQ ID NO: 12 and SEQ ID NO: 13 for use in a method of treatment of the human or animal body.

Indeed, modulating of the activity of IREl has numerous applications in therapy: since IREl is involved in the response of ER to stress and more generally in the response of the cell to stress, the fragments, derivatives, conjugates, nucleic acid sequences, plasmids, expression vectors and host cells according to the invention may be used to in methods for treatment of secretory protein misfolding diseases, e.g. cystic fibrosis, alpha 1 antitrypsin deficiency, inflammation (e.g. secretion of the acute phase proteins), in methods for treatment of diseases associated to high stress conditions, e.g. ischemia, stroke, heat shock, hypothermia, or in methods for inhibiting cell apoptosis.

In one embodiment, the fragments or derivatives of the invention are for use in the treatment of secretory protein misfolding diseases such as alpha 1 antitrypsin (alpha 1 AT) deficiency.

In one preferred embodiment, the fragments or derivatives of the invention are for use in a method of treatment of alpha 1 antitrypsin (alpha 1 AT) deficiency.

Indeed, in alpha( 1 )-antitrypsin (alpha 1 AT) deficiency is mainly caused by a polymerogenic mutant form of the secretory glycoprotein alphalAT, alpha lATZ, which is retained in the endoplasmic reticulum (ER) of liver cells. By modulating IRE1 , the accumulation of alpha 1ATZ can be reduced.

Thus, the inventors have shown that by modulating RE homeostasis by fragments or derivatives of the invention, the secretion of alphalAT can be partially restored. In another embodiment, the fragments or derivatives of the invention are for use in the treatment of disorders and conditions associated with transplantation.

Indeed organ transplantation subjects the transplanted organ to major stresses, namely ischemia and reperfusion (IR). ER stress has been shown to be activated in response to both stresses (Emadali J Pathol 2005) and the modulation of ER signalling is therefore a strategy to reduce IR-induced damages to the organs and to favor positive transplantation outcome.

In another preferred embodiment, the fragments or derivatives of the invention are for use in a method of treatment of ischemia and reperfusion. Said methods typically comprise the step of administering a therapeutically effective amount of a fragment, derivative, conjugate, nucleic acid sequence, plasmid, expression vector or host cell according to the invention to a subject in need thereof. In the context of the invention, the term "treating" or "treatment", means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies.

As used herein, "subject" refers to a human or animal that may benefit from the administration of a fragment according to the invention. By a "therapeutically effective amount" of a fragment according to the invention, it is meant a sufficient amount to treat the disease, at a reasonable benefit risk ratio applicable to any medical treatment. Further aspects and advantages of this invention will be disclosed in the following figures and examples, which should be regarded as illustrative and not limiting the scope of this application.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Fragments of the cytosolic domain of IRE1. Eleven overlapping fragments of approximately 6 kDa covering the entire cytosolic domain of IRE1 were expressed as 6xHis fusion proteins and purified. Out of the eleven fragments eight were successfully expressed and purified (Fl, F2, F4, F6, F8, F9, F10, Fl 1).

Figure 2-5: Use of an all-D retroinverso P4-TAT peptide. P4 or P4-TAT all-D retroinverso (FIRE) peptides were labelled with FITC and purified. The peptides (lOOng) were incubated in the culture medium of HuH7 cells for 1 and 4h. The cells were then fixed with 4% PFA for 10 min at room temperature and analyzed by confocal microscopy (data not shown). Figure 2: Comparison of P4 and FIRE peptides on tuni camycin-medi ated toxicity in HuH7 cells. HuH7 cells were treated with tunicamycin ( pg/ml) for 16h in the presence or in the absence of increasing amounts of P4 or FIRE peptides and cell viability was determined using sulforhodamine staining. Figure 3: Cells were treated or not with P4 or FIRE peptides (2μ^π ) in the presence of tunicamycin (5 g/mJ) for 4 hours. Cells were then lyzed and extracts analyzed by immunoblot for XBPls and CHOP expression as well as JNK and eEF2alpha phosphorylation, a representative experiment is shown. Figure 4: Quantification of three independent experiments carried out as in Figure 3. Left: ** p=0.02 compare CTL grey bar to P4 grey bar; * p=0.01 compare white bars FIRE vs. CTL; # p=0.008 compare grey bars FIRE vs. CTL. Right: * p=0.04 compare white bars P4 vs CTL; ** p=0.01 compare grey bars P4 vs CTL; # p=0.009 compare white bars FIRE vs CTL. Figure 5: Impact of FIRE on AIAT secretion. Immunoblot analysis of AIAT secretion by HuH7 cells exposed or not to TG (0.5 uM) and/or FIRE (10 or 50 uM). Top: immunoblot analysis of AIAT secretion. Bottom: quantification of the immunoblot analysis. EXAMPLES

Materials & methods

Antibodies used in this study: anti-IREla (Santa Cruz Biotechnology, Inc. Santa Cruz, CA USA), anti phospho-IREl (Abeam Inc., Cambridge, MA USA), anti phospho-eIF2a(Ser ⁵¹ ) and anti-eIF2a (Cell Signaling Technology, Inc., Dan vers, MA, USA), anti phospho-BAD(Ser" ² ) and anti BAD antibodies were from Cell Signaling Technology, Inc. (Danvers, MA, USA), anti N-His antibodies were from Invitrogen Corp. (Carlsbad, CA USA) and anti HA (Santa Cruz Biotechnology, Inc. Santa Cruz, CA US). F6 peptide derivatives (PI, P2, P3, and P4) were purchased from Genosphere Biotechnologies (Paris, France). Tunicamycin (Tun) was from EMD Biosciences (Merck KGaA, Darmstadt, Germany).

Cloning - IREl ^cyt0 cDNA (AA 470 to 977) and 1 1 IRElcyto fragments were cloned from human liver cD As using either the Gateway® technology (Invitrogen Corp., Carlsbad, CA USA) in pGEX-2TK, pDEST17 or pR 5-myc expression plasmids, or restriction cloning in pTAT-HA (Schwarze SR et al. Science 1999 Sep 3; 285 (5433): 1569-1572) and pCDNA3. Primers used for cloning are listed in Table 1. IREl ⁰⁵ " ⁰ and IRE1 fragment cDNAs devoid of ATG, were amplified by PCR using the Platinium ^® Taq DNA Polymerase High Fidelity (Invitrogen Corp., Carlsbad, CA USA) and the following amplification scheme: denaturation at 94°C for 40 sec, annealing at 60°C for 40 sec, elongation at 68°C for 2 min, 35 cycles. The PCR products were precipitated using PEG8000 and recombined into pDONR201 using the Gateway® BP clonase (Invitrogen Corp., Carlsbad, CA USA). The plasmids were then transformed into competent DH5 cells and positive clones selected and sequenced. The clones were then recombined into destination vectors using LR clonase (Invitrogen Corp., Carlsbad, CA USA). For pTAT-HA cloning, the plasmid was first digested using Kpnl and EcoRI and the linearized vector purified. The PCR products amplified with primers #25 and #26 were digested with the above mentioned enzymes and gel purified prior to ligation into the linearized pTAT-HA vector. Ligation products were transformed into DH5oc cells and amplified. The pTAT-HA vector harbours a N- terminal 6xHis leader followed by the 1 1-amino-acid TAT protein transduction domain flanked by glycine residues (GYGRKKRRQRRRG), a hemagglutinin tag (HA, GYPYDVPDYAG) flanked by glycine residues, and a polylinker in which the F6 fragment was inserted in frame with the above mentioned tags (Schwarze SR et al. Science 1999 Sep 3; 285 (5433): 1569-1572).

AlphaScreen® assay - The AlphaScreen® (PerkinElmer, Inc., Waltham, MA USA) assays were performed as previously described (Bouchecareilh M et al. J Biomol Screen 2010 Apr; 15 (4): 406-417). Briefly, AlphaScreen® assays were performed in Costar 384- well microplates in a 25 μ! final reaction volume. GSH Donor beads (PerkinElmer, Inc., Waltham, MA USA) were used at a final concentration of 0.02 mg ml per well. The assays were performed in 20mM Tris-HCl, pH 7.4, 50mM NaCl, ImM MgC12, ImM MnC12, 2mM DTT and 0.1% Tween20. All incubations were performed at 23°C. Laser excitations were carried out at 680 nm, and readings were performed at 520-620 nm using the EnVision ^® (PerkinElmer, Inc., Waltham, MA USA) plate reader. Dimer/oligomer formation assay- Increasing concentrations of GST-IRE l ^cy, °, de-phosphorylated GST-IRE l ^cyw or GST were incubated for 45 min with ImM ATP (Sigma-Aldrich, St. Louis, MO USA) or I mM ATPvS (Sigma- Aldrich, St. Louis, MO USA) or buffer and donor beads. Acceptor beads 0.02 mg mL were then added to the reaction and incubated for an additional hour before reading. INK activation assays were carried out using the phospho-JNK Surefire® kit (TGR Biosciences Pty Ltd, Australia) using the manufacturer's recommendations.

GST-IRE I ^cyto recombinant protein was expressed in DH5 alpha bacteria upon treatment with 1 mM IPTG for 3h at 37°C of 5 liters cultures in Luria Bertani medium. Bacterial culture were centrifu gated and bacterial pellets lysed using 1 mg/ml lysozyme for 30 min on ice then 30 mM Tris-HCl (pH 7.4) and 1 % TXIOO and containing protease inhibitors cocktails (Complete; Roche, Mannheim, Germany) for 30 additional minute on ice. Lysates were then clarified by centrifu gation at 1500 g for 30 min. Clarified lysates were then chromatographied on GSH-Sepharose beads (GE Biosciences) and the purified protein eluated using 10 mM reduced GSH. The purified material was dialyzed and purified again following the same protocol cycles until the protein is purified to homogeneity. For dephosphoryl ati on, 500 xg of purified (3 cycles) GST- IREl ^cyl ° were dephosphorylated using alkaline phosphatase according to the manufacturer's recommendations (Fermantas). Dephosphorylated material was re- purified as described above. The purified proteins were dialyzed and concentrated in the Alphascreen® reaction buffer described above. Nucleotide specificity assay - Optimal concentrations of GST-IRE lcyto or dephosphorylated GST- IRE lcyto (determined experimentally using matrix distribution of the reagents) were incubated for 45 min with varying concentrations of ATP, adenosine diphosphate (ADP), adenosine monophosphate (AMP; 0-10 mM; Sigma- Aldrich), guanosine triphosphate (GTP; 0-10 mM; Sigma-Aldrich), guanosine diphosphate (GDP; 0-10 mM; Sigma-Aldrich), and donor beads. Acceptor beads (0.02 mg/mL) were then added to the reaction and incubated for an additional hour before reading.

Oligomer formation assay - Optimal concentrations of GSTIRE lcyto or dephosphorylated GST-IRE lcyto were incubated for 45 min with increasing concentrations of 6xHis-hIRE lcyto, bovine serum albumin (BSA, Sigma Aldrich) or 6xHis-ScIRE lcyto in the presence of 1 mM ATP and donor beads. Acceptor beads were then added to the reaction and incubated for an additional hour before reading. Phosphorylation assay - Increasing concentrations of GST-IRE lcyto were incubated for 45 min in the presence or absence of ATP. GSH donor beads and Lewis Metal-Fe acceptor beads (PerkinElmer) were added and incubated for an additional hour prior to reading. The assays were performed using thioxene, anthracene, and rubrene containing acceptor beads in all the experiments.

Identification of IRE1 activity modulators using the above-mentioned assays - IRE1 fragments (comprising 50 to 60 amino-acids) derived from IRE1 cytosolic domain were produced as 6xHis tagged recombinant proteins in bacteria and purified on Ni-NTA matrices (Quiagen) following manufacturer's recommendation. Increasing concentrations of the purified fragments (0 to I0 ^"6 M) were added in the oligomer formation assay as described on p7 - 4). Positive hits identified through the afore- mentioned assay were then tested in orthogonal assays as follows.

Unspticed XBP1 (uXBPl) mRNA in vitro transcription - Human uXBPl cDNA was amplified from total RNA extracted from non stressed HuH7 cells using reverse transcription followed by specific PCR amplification with primers 71 and 72 described in Table 1. PCR products were then resolved by agarose gel electrophoresis and purified. The purified PCR products were digested using HindlTI and BamHI restriction enzymes and sub-cloned into pCDNA3. This plasmid was used as a template for T7-mediated in vitro transcription using the Ribomax T7 RNA transcription kit (Promega Corp., Madison, WI, USA) according to the manufacturer's recommendations. RNA was analyzed by ethidium bromide containing agarose gel electrophoresis and visualized using UV trans-illumination.

RNA cleavage assay - Total RNA (10 g) from HepG2 cells or I g of in vitro transcribed uXBPl mRNA was incubated with GST-IREl ^cy,t> (10 μg) at 37°C for 0-10 hrs in 50 mM Tris-HCl pH 7.5, 120 mM NaCl, 1 mM MgCl ₂ , 1 mM MnCl ₂ , 5 mM β- mercaptoethanol, supplemented with 1 mM ATP, or 1 mM ATPyS. Cleaved or uncleaved RNAs were used as a template for reverse transcription using XBP-1 primers, and GAPDH as an internal control.

C. elegems _' b ed analyses - The wild-type strain Bristol N2 was used as the reference strain. C. elegans were maintained at 20°C under standard culture conditions and fed with OP50 (unless otherwise mentioned) as described previously (Jenna S et al. Mol Biol Cell 2005 Apr; 16 (4): 1629-1639). The effect of F6 was performed using two different feeding methods based on our previous study (Delom F et ai. Biochem Biophys Res Commun 2007 Jan 19; 352 (3): 587-591). N2 worms were treated using the alkaline hypochlorite method (0.5M NaOH, 0.8% bleach) to isolate embryos. Eggs were hatched overnight in M9 medium (40 mM Na ₂ HP0 ₄ , 20 mM KH ₂ P0 ₄ , 8 mM NaCl, 20 mM NHjCl) to obtain a LI synchronized population. TAT-F6 or 6xHisF6 expression plasmids were transformed into BL21 bacteria. Isolated transformed bacteria were grown for 12 h at 37"C in Luria-Bertani (LB) medium supplemented with ampicillin (100 g/ml). The culture was diluted 1 :2 in fresh LB medium in the presence of ampicillin and allowed to grow to OD600 = 0.4. IPTG was then added to a final concentration of 1 mM and the culture was incubated with shaking for 3 h at 37 °C. After supplementing with additional ampicillin and IPTG, the bacterial hosts were either directly spotted onto agar plates or were concentrated by centrifugation and then spotted. The agar plates were composed of standard NGM/agar media supplemented with or without 100 μ ηι1 ampicillin and 1 mM IPTG. Synchronized LI worm populations were transferred to these plates. Worms were transferred daily onto new plates until they reached the L3-L4 stage. At this stage the worms were then transferred onto NGM plates containing DMSO as a control or 5 pg ml Tun for 5 h at 20°C, then collected and lysed using Trizol (Invitrogen Corp., Carlsbad, CA USA) for RNA extraction. Worm survival upon tunicamycin was performed as previously described (Caruso ME et al. Mol Cell Biol 2008 Jul; 28 (13): 4261-4274). _IRE1 cyto _{in yitro} cross^ssyug . four μΜ of purified IREl ^cyt0 or de-phosphorylated IREl ^cyt ° were incubated in a final volume of 50 μΐ for 30 min at 4°C. Then either the cross linker DSS (Thermo Fisher Scientific Inc, Waltham, MA), 1 mM final concentration, or DMSO as a control was added and the samples incubated for 2h at 4°C. The reaction was stopped with 20 mM Tris-HCl pH 7.4. The samples were then suspended in 6x Laemmli buffer and the proteins resolved by SDS-PAGE. The gel was then either silver stained or subjected to immunoblot analysis.

Dot blot analyses - Increasing amounts of TAT-fusion fragments were spotted onto PVDF membranes (Millipore Corp., Billed ca, MA USA) and incubated overnight in PBS containing 0.05 % Tween20, 5% BSA. The membrane was then incubated for 1 h with Ni-NTA (QIAGEN Inc., Valencia CA USA) conjugated to horse-radish peroxidase (1:5000 dilution) in PBS containing 0.05 % Tween20 and 1% BSA, then extensively washed with 0.05% Tween20, 1% BSA in PBS and revealed using enhanced chemiluminescence. For IRE1 phosphorylation assays, GST-IREl ^cy, ° protein was incubated in the presence of 1 mM ATP and increasing concentrations of P1-P4 for lh at 30°C and then directly spotted on nitrocellulose membranes using vacuum aspiration. Membranes were then incubated for 45 min with PBS containing 0.05 % Tween20 (PBST) supplemented with 5% BSA and then overnight with and phospho- IREl antibodies. Following extensive washing with PBST, membranes were incubated with anti Rabbit IgG coupled to HRP and revealed using chemiluminescence.

Pull-down assays - Purified 6xHisF6 and F9 peptides were incubated for lh at room temperature with GST-IRE l ^cy1 ° (1 g), after Ni-NTA pre-clearing of the latter, in 20mM Tris-HCl, pH 7.4, 50mM NaCl, ImM MgC12, ImM MnCl ₂₎ 1 mM ATP. Ni- NTA beads were then added for an additional hour. Beads were then spun down and washed three times using 20mM Tris-HCl, pH 7.4, 50mM NaCl. The purified material was resolved by SDS-PAGE and either immunoblotted using anti phospho-IREl and anti-IREl antibodies, or directly in- gel stained using Coomassie Brilliant Blue. The reverse experiment was also carried out using GSH-Sepharose and GST-IRE l ^cyt0 as bait and 6xHis-F6 and -F9 peptides as preys.

Cell Culture and transfection - Human HuH7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum and antibiotics. HuH7 cells were transiently transfected with the pRK5 plasmid or pRK5-F6 plasmid. Plasmid transfections were performed using PromoFectine-Hepatocyte (PromoCell GmbH, Heidelberg, Germany) according to the manufacturer's recommendations. In addition, HuH7 cells (5xl0 ⁵ cells) were transfected with IRE 1 ^-derived peptides (2 μg) using Promofectin-Polypeptide in 24- wells plates (PromoCell GmbH, Heidelberg, Germany) following manufacturer's recommendations. Four hours post-transfection, cells were subjected to tunicamycin treatments as described above.

Determination of toxicity and apoptosis - toxicity and cell survival was determined using a sulforhodamine B staining kit (Sigma, St Louis, MO, USA) and apoptosis was determined using TUNEL staining (TACS-XL kit, R&D systems, Minneapolis, MN USA).

Confocal microscopy - Worm fixation and staining were carried out as previously described by Finney and Ruvkun (Finney M et al. Cell 1990 Nov 30; 63 (5): 895-905) with the exception that mouse anti-HA antibodies were used. Confocal microscopy was performed as previously described (Jenna S et al. Mol Biol Cell 2005 Apr; 16 (4): 1629-1639).

RT-PCR and quantitative RT-PCR (qRT-PCR) analyses - N2 worms were treated with 2 pg/ml tunicamycin for 3 days. They were then collected and lyzed using Trizol (Invitrogen Corp., Carlsbad, CA USA). Total RNA was extracted and reverse- transcribed using the Superscript ΙΪΙ kit (Invitrogen Corp., Carlsbad, CA USA). PGR products were then amplified using the primers described in Table 1 within the linear part of the amplification curve, resolved on agarose gels and visualized following ethidium bromide staining. Transcript expression was normalized to that of AMA1 and/or CRP1 as described previously as the expression of those genes was shown to be unaffected upon Tun treatment (Caruso ME et al. Mol Cell Biol 2008 Jul; 28 (13): 4261-4274). For qRT-PCR analysis, amplification products were generated using primers described in Table 1. Quantitative PGR was performed using an Applied Biosystems StepOne real-Time PGR System. GAPDH mRNA levels (for the human cells) or AMA-1 mRNA levels (for C. elegans), genes whose transcription are not regulated by ER stress, served as internal normalization standards.

Primers - Table 1: Primers used for cDNA cloning and RT-PCR analyses. The Gateway® attBl and attB2 recombination sites (bold italic) were added to the forward and reverse primers respectively. The primers devoid of the recombination sequence were used for semi quantitative RT-PCR analyses. The Kpnl and EcoRI restriction sites for cloning into pTAT were added in primer #2 -#26. Primers #lRv-#26Rv each contain a stop codon upstream of the attB2 recombination sites (#l-#24) or restriction site (#26). Fw: forward; Rv: reverse; FL: full-length.

NAME SEQUENCE SED Π) NO

1 GGGGA CCACTTTGTA CAAGAAAGCTGGG

15

IRE1 cyto Rv TAtcagagggcgtctggagtc

2 GGGGACAAGTTTGTACAAAAAAGCAGGC

16 IRE1 cyto Fw 7TGctgcccttccacccacct

3 GGGGACAAGTTTGTACAAAAAAGCAGGC

17 Fl Fw 7TGcccctgagcatgcaicag

4 GGGGACCACTTTGTACAAGAAAGCTGGG

18 Fl Rv rAtcactcigagtacgggccag

5 GGGGACAAGTTTGTACAAAAAAGCAGGC

19 F2 Fw 7TGctcctggacacgtctggc

6 GGGGACCA CTTTGTACAAGAAAGCTGGG

20 F2 Rv 7Atcacaccacgctggtttc

7 GGGGACAAG7TTCrACAAAAAAGCAGGC

21 F3 Fw 77Ggacgatggagatgaggaaacc

8 GGGGACCACTTTGTACAAGAAAGCTGGG

22 F3 Rv TAtcatgcgaagctaaaacactc

9 GGGGA CA GTTTGTA CAAAAAAGCA GGC

23 F4 Fw TTGaggatcctccccgagtgt

10 GGGGACCACTTTGTACAAGAAAGCTGGG

24 F4 Rv rAtcactccacatactcttgcagg

1 1 GGGGACAAGTTTGTACAAAAAAGCAGGC

25 F5 Fw 7 Gtgtgcagccaccctgc

12 GGGGACCACTTTGTACAAGAAAGCTGGG

26 F5 Rv Atcagggcatggatatgaggat

13 GGGGACAAGTTTGTACAAAAAAGCAGGC

27 F6 Fw 77XJaagccacacaacatcctcat

14 GGGGACCACTTTGTACAAGAAAGCTGGG

28

F6 Rv rAtcagctcagcatctctggag

15 GGGGA CAAG777X;rACAAAAAAGCAGGC

29 F7 Fw 7TGgaaggctggatcgctcca

16 GGGGACCACTTTGTACAAGAAAGCTGGG

30 F7 Rv 7 ^* Atcaggcacccaggaggat

17 GGGGA CAAG7T7X7rACAAAAAAGCA GGC

31 F8 Fw JTCcagcggcaggccaac

18 F8 Rv GGGGACCACTTTGTACAAGAAAGCTGGG yy TVltcagctccagaagaacgg

GGGGACAAGTTTGTACAAAAAAGCAGGC

33

F9 Fw 77Gcacgtgctcaaacacccg

GGGGACCACTTTGTACAAGAAAGCTGGG

34

F Rv TAtcaagtgatgttctcccgc

G^GACAAG77TGrACAAAAAAG AGGC

35

FlO Fw JTCgtgaagatggactggcgg

GGGGACCACTTTGTACAAGAAAGCTGGG

36

FlO Rv TAtcaggtccccagcgtctc

GGGGACAAGTTTGTACAAAAAAGCAGGC

37

Fl l Fw T Gcctgcagaggtgcggg

GGGGACCACTTTGTACAAGAAAGCTGGG

38

Fl l Rv T!Atcagagggcgtctggagtc

AAAGCCGGT ACC GGTAAGCCA CAC

39

TAT-F6 Fw AACATCTCAT

AAAAAAGAATTCTCA GCT

40

TAT-F6 Rv CAGCATCTCTGGAG

XBP-1 Fw GGAACAGCAAGTGGTAGA 41

XBP- 1 Rv CTGGAGGGGTGACAAC 42

AMA-1 Fw (C.elegans) GCGGTCAGAAAGGCTATCGA 43

AMA-1 Rv {C.elegans) AGCAGTGCCAAATGTCGGTAAT 44

XBP-1 Fw (C.elegans) TTGCACCAGTTGTCGTCGTC 45

XBP-1 Rv (C.elegans) ACCGTCTGCTCCTTCCTCAATG 46

C B-2 Fw (C.elegans) GCTCAAAAACCGTTCACCATCG 47

CKB-2 Rv (C.elegans) GCTTGCACGTCCAAATCAACTC 48

CHT-1 Fw (C.elegans) TGACACCCAACTCAAGACCC 49

CHT-1 Rv (C.elegans) GCAGCCTTCAACTCCTTCAC 50

F22E5.6 Fw TTGATAGCGGAAGCTGGAAAGC 51

F22E5.6 Rv AGAAGCAGTAGGTCCATGACGG 52

HSP-4 Fw (C.elegans) ACGACCACAATCGTCTCAGTCC 53

HSP-4 Rv (C.elegans) CTTCGTCAGTGAGCTTTCCTCC 54

GAPDH Fw ACCACCATGGAGAAGGCTGG 55

GAPDH Rv CTCAGTGTAGCCCAGGATGC 56

BAP31 Fw (C.elegans) TCACCTTCACCCTCCTTCTTCC 57

BAP31 Rv (C.elegans) TGAAGAGCACGAAGCCGAAC 58

T06D8.9 Fw ATGACGACTACTCCAAGCCACC 59

T06D8.9 Rv TCCAGTCCAAAACGATCCACC 60

CREBH Fw TCCACCAAAGAAAACCAGGCAC 61

CREBH Rv (C.elegans) AGACTAGCAGACAGGCAGAGAG 62

COPI Fw (C.elegans) ATTGCCGTGAACCCTGAAGAC 63

COPI Rv (C.elegans) AAACTCCACACAATTCACCCCC 64

EDEM-1 Fw TCACAACATACATGCGTCGAGG 65

EDEM-1 Rv AAGGGCATGATGGCACACAG 66 53 PDI-1 Fw (C.elegans) AGCAATGGCTCCACGCTTAC 67

54 PDI-1 Rv (C.elegans) CACGGAACTTCTTAGCGACCTC 68

55 CDC48 Fw (C.elegans) CGCCGCTACTAATCGTCCTAAC 69

56 CDC48 Rv (C.elegans) G AGC AG AGGG A AGC A AATCAG 70

57 ERpl Fw (C.elegans) AGATCCCirCTCCTCTTGGCAC 71

58 ERpl9 Rv (C.elegans) ACTTGACCCACGCAATATCGTC 72

59 IREl cyto Gateway Fw GGGGACAAGTTTGTACAAAAAAGCAGGC 73

60 IRElcyto Gateway Rv GGGGACCACTTTGTACAAGAAAGCTGGG 74

61 Chop FW (qPCR) CCAAGGGAGAACCAGGAAACG 75

62 Chop Rv (qPCR) TCACCATTCGGTCAATCAGAGC 76

63 Gadd34 Fw (qPCR) CC CTACTTCTGCCTTGTCTCCAG 77

64 Gadd34 Rv (qPCR) TTTTCCTCCTTCTCCTCGGACG 78

65 ERdj4 Fw (qPCR) TGGTGGTTCCAGTAGACAAAGG 79

66 ERdj4 Rv (qPCR) CTTCGTTGAGTGACAGTCCTGC 80

67 He ud Fw (qPCR) TCCTCCTCCTGACGTTGTAAA 81

68 Herpud Rv (qPCR) TGCTCGCCATCTAGTACATCC 82

69 GAPDH Fw (qPCR) AAGGTGAAGGTCGGAGTCAA 83

70 GAPDH Rv (qPCR) CATGGGTGGAATCATATTGG 84

71 XBP-1 Fw (cloning) TGAATTCGATGGTGGTGGTGGCAGCCG 85

72 XBP-1 Rv (cloning) ATTAAGCTTAAGTTAGTTCATTAATGGC 86

73 PDGFRb Fw (qPCR) CTGCGTCTGCAGCACGTGGA 87

74 PDGFRb Rv (qPCR) ACCTGCCCAAAGGCCCCAGA 88

75 Col6 Fw (qPCR) GACCCGGAGGAGAGGCAGGG 89

76 Col6 Rv (qPCR) GCCGTGGTGATGGAGTGGGC 90

77 Sparc Fw (qPCR) GTGCAGAGGAAACCGAA 91

78 Sparc Rv (qPCR AAGTGGCAGGAAGAGTCGAA 92

The FIRE peptide, all-D retro-inverso TAT-P4 was purchased from Selleck Chemicals (Houston, TX, USA) and resuspended in Phosphate Buffered Saline (10 mg/mL) and frozen in 50 g aliquots. Peptide FITC conjugation was carried out using the FTTC- labelling kit from Thermo Scientific (Whaltham, MA, USA). HuH7 cells were cultured as described above and incubated with the indicated amounts of peptides (P4 or FIRE). Cytoprotective effects of the peptides were determined using the sulforhodamine B staining method. Cell extracts were immunoblotted using the indicated antibodies (CHOP, sXBPl, phospho-eDF2alpha, eIF2alpha, phospho-JNK, JNK1).

Results

IREl^-derived peptides alter the formation of lREl^ ⁰ oligomers

This assay is based on the use of GST-IRE l ^cyt0 and AlphaScreen® beads (both donor and acceptor) coated with glutathione (GSH). The GST moieties of GST-IRE 1^° proteins bind to the beads which are then brought into proximity through the association of the IREl ^cyl ° moieties. Using this assay, GST-IRE I' ⁵ " ⁰ concentration which resulted in a maximal AlphaScreen® signal in the presence of 1 mM ATP was determined. We postulated that discrete sequences present in IRE ^cyt0 involved in interfacial contacts between IRE1 monomers might be able to interfere with iRE ^{c t0} oligomerization. To test this hypothesis, eleven overlapping fragments covering the entire cytosolic domain of IRE ^cyt0 were generated as 6xHis fusion proteins. Eight fragments (Fl (SEQ ED NO:2), F2 (SEQ ID NO:3), F4 (SEQ ID NO:4), F6 (SEQ ID NO:5), F8 (SEQ ID NO:6), F9 (SEQ ID NO:7), F10 (SEQ ID NO:8), F) 1 (SEQ ID NO:9)) were expressed as soluble proteins and purified (Figure 1). These fragments were then tested as modulators of IRE1 oligomerization using AlphaScreen®. Increasing concentrations of the peptide fragments were then added as potential reaction modulators. Two of the fragments (F6 and F9) led to significant dose- dependent AlphaScreen® signal decrease, suggesting they may either promote ffiE! ^eyt0 oligomeric structures formation or compete with dimer formation (Bouchecareilh M et al. J Biomol Screen 2010 Apr; 15 (4): 406-417).

To ensure that both F6 and F9 interacted directly with I El ^cy!0 , we monitored the association between GST-IREl ^cyt0 and 6xHis-F6 or 6xHis-F9 using pull -down assays. GST-IREl ^, ° was incubated with identical amounts of 6xHis-F6 or 6xHis-F9 and pulled down using Ni-NTA beads. 6xHis-F6 or 6xHis-F9 peptides were visualized using both immunoblotting with anti His antibodies and Coomassie Brilliant Blue staining. The resulting material was resolved by SDS-PAGE and immunoblotted using either anti phospho-IREl (Ser ⁷²⁴ ) or anti IRE1 antibodies. While both F6 and F9 were able to bind directly to IREl ^cyt0 , F6 displayed a higher affinity for the non phosphorylated B El ^cyt0 . No binding of GST-IRE l ^cyt0 to the Ni-NTA beads alone was detected. The reverse experiment was also carried out by pulling-down GST-IRE 1°^° using GSH Sepharose beads following incubation in the presence of 6xHis-F6 or 6xHis-F9. First the experiment was carried out in the absence of GST-IRE 1^° or in the presence of GST alone, and under these conditions neither F6 nor F9 were found in the pulled-down material. Similarly, when 6xHis-F6 or 6xHis-F9 fragments were incubated with Ni-NTA beads in the absence of GST-IRE l ^cy, °, no anti-GST immunoreactive signal was detected in the pulled-down material. Finally, GST- IRE l ^cyt0 wild-type or kinase defective (K599A) were incubated in the presence of either 6xHis-F6 or 6xHis-F9 and pulled-down using GSH Sepharose beads. Both wild- type and mutant proteins were found to bind efficiently to either 6xHis-F6 and 6xHis- F9 peptides, thus confirming the results observed for the binding of GST-IRE l ^{c i0} to the Ni-NTA beads alone.

Then to test the effect of these fragments on IREl ^cyt0 oligomerization, we used IREl ^cyt0 devoid of GST in cross-linking experiments with disuccinimidyl suberate (DSS) as described previously (Bouchecareilh M et al. J Biomol Screen 2010 Apr; 15 (4): 406- 417). Using increasing concentrations of IREl' ³ " ⁰ , in vitro oligomer formation was saturable and dependent on DSS concentration. The same experiment was then carried out using 0.4 μg of IREl ^{c t0} and increasing concentrations of F6 and F9 (as well as Fl 1 as a control). Quantification of oligomer formation revealed that F6 promoted the formation of higher order oligomers in a dose-dependent manner whereas F9 and Fl 1 did not. The same experiment was carried out using 0.4 μ§ of either IREl ^cyt0 wild type or K599A and 80 nM of F6, F9 or Fl l . This revealed that only F6 promoted IREl ^cytt> oligomerization for both wild-type and K 9A proteins, thus confirming the specific role of F6 in IREl oligomerization.

Because F6 promoted IREl ^cyt0 oligomer formation and was highly conserved across many species, we further tested its effect on IREl -mediated XBP-1 mRNA cleavage. Based on the cross-linking experiments, an optimal concentration of 6xHis-F6 was added to an in vitro RNAse reaction for 90 min in the presence of GST-IRE I ⁰⁵¹⁰ . The relative abundance of unspliced XBP1 mRNA (uXBPl ) was then determined using RT-PCR (Bouchecareilh M et al. J Biomol Screen 2010 Apr; 15 (4): 406-417) and revealed that IREl ^cyt0 RNAse activity was enhanced in the presence of F6. A time- course experiment, using the same assay conditions as for Figure 3D, revealed that in the presence of F6 the n of uXBPl mRNA was 2.5 h compared to 4.7h for control, confirming the results shown in Figure 3D. Moreover, Fl 1 and F9 did not alter IREl activity when compared to control conditions. Thus, increased IREl RNAse activity correlated with enhanced F6 binding to IREl and enhanced oligomer formation. Taken together these results demonstrate that F6 represents a trans-activating entity that enhances IREl endoribonuclease activity through the stimulation of IREl oligomerization.

Structural and functional analyses of F6 Because F6 i) is highly conserved throughout evolution, ii) is capable of binding to IREl ^cy, °, iii) enhances El ^cyt0 oligomerization and iv) enhances IREl ^cyt0 endoribonuclease activity, we investigated whether F6 sub-domains were also highly conserved and analyzed the location of these fragments within the structure of IREl ^cyl ° oligomers (Lee KP et al. Cell 2008 Jan 1 1 ; 132 ( 1): 89-100, Korennykh AV et al. Nature 2009 Feb 5; 457 (7230): 687-693). Interestingly a F6 sequence conserved from S. cerevisiae to H. sapiens was located in the kinase active site of IREl and in the vicinity of the nucleotide (or inhibitors, i.e. Sunitinib) present in the nucleotide binding pocket.

To compare the functionality of the entire F6 fragment and that of the conserved region of this fragment, we synthesized a short peptide of 18 amino-acids encompassing the conserved domain from the human sequence (wild-type, PI , SEQ ID NO: 10). The potential structure of this peptide was disrupted by substitution of the alanine residue in position 7 by a proline (destructured, DS, P2, SEQ ID NO: 1 1) and the serine in position 10 by either an alanine residue (non phosphorylable, SA, P3, SEQ ID NO: 12) or a glutamic acid (phosphomimic, SE, P4, SEQ ID NO: 13):

WT - AHGKIKAMISDFGLCKKL (PI)

DS - AHGKIKPMISDFGLCKKL (P2)

SA - AHGKIKAMIADFGLCKKL (P3)

SE - AHGKI AMIKDFGLCRKL (P4)

Using AlphaScreen®, the peptides were first evaluated for their ability to affect

IREl ^cy, ° oligomerization. PI and P4 had the greatest effect on IREl ^cyt0 oligomer formation with an ED50 of 1.4+0.1 μΜ and 3.1+0.5 μΜ, respectively, whereas P2 and P3 did not show any significant effect on the AlphaScreen® signal intensities. These peptides were then tested for their ability to alter IRE^ ⁰ autophosphorylation, oligomerization and IRE ^cyt0 endoribonuclease activity. Interestingly, PI , P3 and P4 promoted rREl ^cyt0 autophosphorylation, oligomerization and endoribonuclease activity whereas P2 did not affect them. In addition although PI and P3 showed similar properties towards IREl activities, P4 was highly effective as an activator for autophosphorylation, oligomerization and endoribonuclease activity. These results confirm that F6 functionally impact on IREl ^cyt0 in vitro activities by enhancing IREl oligomerization, define the conserved domain contained in PI as a critical determinant of these biological effects and identify Ser ⁷¹⁰ as an important residue in the efficiency of the peptide to modulate IREl activity in vitro. Cell-based analysis of F6 functions

To assess the effect of F6 on IRE1 activity in more physiologically relevant systems, F6 cDNA was sub-cloned into the pR 5 vector and transiently transfected into HuH7 cells. Forty-eight hours post-transfection, cells transfected with either an empty vector or the pRK5-F6 vector were subjected to tunicamycin treatment (5 μ /ηι1) for various periods of time. For each time point, RNA was extracted and XBPl mRNA splicing, UPR target gene and RIDD substrate expression were monitored. In a first series of experiments, cells were treated with tunicamycin for 0, 4, 8, 16 and 24h. In agreement with the in vitro observations, expression of F6 in HuH7 cells led to enhanced and prolonged splicing of XBPl mRNA as monitored using qRT-PCR which suggested enhanced activation of IREl endoribonuclease activity. To confirm this observation, the expression of four UPR target genes (CHOP, HERPUD, ERdj4 and GADD34) was also evaluated by qRT-PCR. No major difference between pRK5 and pRK5-F6 transfected cells was observed under basal conditions. When the cells expressing pRK5-F6 were exposed to tunicamycin, the expression of mRNA encoded by genes which are not per se IREl targets such as HERPUD and GADD34 was not significantly increased over that observed for cells expressing pRK5. In contrast the expression of ERdj4 mRNA, which has been described to be a XBPl target gene and to a lesser extent that of CHOP, were subjected to a prolonged induction in F6 expressing cells, thus being consistent with the observed enhanced XBPl splicing and suggesting increased IREl activity toward XBPl mRNA splicing. These data are in agreement with our in vitro observations and indicate that in mammalian cells F6 expression enhanced IREl endoribonuclease signalling upon ER stress. To further characterize the effect of F6 on IREl activity, we monitored the mRNA expression levels of PDGFRb, Collagen6 (Col6) and Sparc, three previously described RIDD substrates. PDGFRb, Collagen6 and Sparc mRNA expression levels were decreased upon ER stress, but mRNA degradation was decreased in cells expressing F6.

Based on this result, we propose that F6 may specifically act on IREl by enhancing its activity toward XBPl mRNA splicing and reducing its RIDD activity. We next monitored JNK1 phosphorylation levels in cells transfected with an empty pRK5 vector, pRK5-F6 or the peptides PI, P2, P3, P4 following tunicamycin treatment. This was carried out using the AlphaS creen® technology and SureFire® kits. In cells expressing the empty vector or transfected with P2, JNK phosphorylation increased after tunicamycin treatment. However, JNK phosphorylation was prevented in cells expressing F6, or transfected with PI , P3 or P4. This indicated that in addition to promoting XBP1 mRNA splicing and to inhibiting RIDD, F6 and its active derivative peptides (PI , P3 and P4) also inhibited IRE 1 -dependent JNK activation. We investigated whether this phenomenon could impact on the activation of other ER stress signalling pathways such as the phosphorylation of the translation initiation factor eiF2 In these experiments we tested the impact of F6, PI (F6 derivative) and P2 (destructured F6 derivative) on tunicamycin-mediated phosphorylation of eiF2a. Expectedly, eiF2a phosphorylation increased upon tunicamycin treatment in control cells, starting after 4h and reaching a peak after 8 hours of treatment, a similar result was observed in P2 transfected cells. Interestingly, eiF2a phosphorylation kinetics was slightly altered in pRK5-F6 or PI transfected cells. Indeed, in both cases a phosphorylation increase was observed as soon as after 2h of tunicamycin exposure and reaching a peak between 4 and 8h of treatment. This indicated that the modulation of IRE1 activity mediated by either F6 or PI impacts on early signalling events triggered in the ER in response to tunicamycin.

We then monitored cell survival using HuH7 cells transfected either with an empty vector or the same vector containing F6 cDNA and then exposed to 5 μg/ml tunicamycin. Increased cell survival was observed for F6 expressing cells following tunicamycin treatment (open circles) when compared to cells expressing the empty vector (open squares). Under basal conditions (no tunicamycin treatment), no difference in cell survival was observed for the F6 expressing cells and the empty vector (control) cells. These results correlated with the induction of apoptosis as monitored using Tunel staining. Indeed, cells expressing F6 displayed reduced Tunel staining upon tunicamycin treatment that cells transfected with an empty vector. The observed decreased in apoptotic cell number (-30%) after 48h of tunicamycin treatment could in part account for the survival increase described in Figure 7C. In an attempt to characterize the mechanisms by which F6 could enhance survival/reduce apoptosis, we monitored the phosphorylation of the pro-apoptotic protein BAD at Serine 1 12. BAD phosphorylation at Serl l 2 increased upon tunicamycin treatment in both pRK5 and pRK5-F6 transfected cells. However, BAD phosphorylation at Serl l 2 appeared more sustained in pRK5-F6 transfected cells than in pRK5 transfected cells. As BAD Serl 12 dephosphorylation is believed to promote BAD apoptotic functions, the prolonged phosphorylation observed in pRK5-F6 transfected cells correlates well with enhanced resistance to ER stress.

Taken together, these results indicate that artificial regulation of IREl by F6 leads to i) increased splicing of XBP1 mRNA and expression of XBP1 target genes, ii) reduced RIDD and JNK activation and iii) enhanced tunicamycin-mediated ER stress resistance through a decrease in apoptosis induction. Effect of F6 on IREl signalling in vivo

To confirm in vivo the observations obtained with the F6 fragment in cultured cells, we used the nematode C. eiegans as a model system. The problem of peptide delivery into the C. eiegans cells was bypassed by using the TAT transduction peptide as a delivery vehicle. A cDNA encoding the F6 peptide was cloned into the pTAT vector downstream of 6xHis, TAT and HA tags and a novel fusion protein was produced in BL21 cells (6xHis-F6 fusion proteins were also produced as a reference). Recombinant F6 expression levels were monitored using dot blot analyses and Ni-HRP and compared to relative quantities of 6xHis-iREl ^cyl0 recombinant proteins. The proteins were then purified and their quality assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining of the gels. To evaluate the efficiency of TAT-F6 delivery into C. eiegans, the worms were grown on BL21 bacteria, or BL21 transformed with pTAT-F6 in the presence or absence of IPTG. Worms were then subjected to i) immunofluorescence analysis using anti-HA antibodies and ii) immunoblot analysis using anti-HA antibodies. TAT-F6 accumulated in intestinal cells upon IP G treatment, whereas only a weak signal was detected in the absence of IPTG which was due to expression leak as indicated by the immunoblot. No signal was observed when non-transfected BL21 or BL21 transformed with an empty pTAT vector were used (data not shown).

The effect of TAT-F6 on IREl endoribonuclease activity was then monitored in vivo in C. eiegans fed with bacteria expressing this construct (in the presence or absence of EPTG) and then subjected to tunicamycin treatment to inhibit N-glycosylation and promote ER stress (Caruso ME et al. Mol Cell Biol 2008 Jul; 28 (13): 4261-4274). TAT-F6 significantly increased IREl endoribonuclease activity in vivo as demonstrated by the enhanced splicing of XBP1 mRNA as determined using qRT- PCR. Interestingly, the effect of TAT-F6 on the splicing of XBP1 mRNA in vivo required tuni cam ycin-mediated activation of the UPR since, as noted in mammalian cells, no enhanced splicing of XBP1 mRNA was observed in the absence of tunicamycin treatment. In addition, the observed effect was dependent on the entry of F6 into the cells as the use of 6xHis-F6 instead of TAT-F6 did not lead to any increase in XBP1 mRNA splicing upon tunicamycin treatment. Similarly, the use of BL21 transformed with an empty pTAT vector did not modify XBP1 mRNA splicing under stress conditions (data not shown).

F6-dependent expression of UPR target genes and resistance to stress in C. elegans

To further explore the effects of F6 on the UPR, we selected 13 UPR target genes whose expression was previously reported to be increased in response to ER stress in C. elegans (Shen X et al. PLoS Genet 2005 Sep; 1 (3): e37). HSP4 encodes the ortholog of the mammalian chaperone BiP and is partly under the transcriptional control of XBP1 ; CKB2 encodes a choline kinase whose expression was reported to depend on XBP1 (Caruso ME et al. Mol Cell Biol 2008 Jul; 28 (13): 4261-4274) and CHT1 and F22E5.6 which respectively encode chitinase and polymerase delta- interacting protein PDEP1, were reported to be under the control of ATF6 and PERK as well as IRE1 (Shen X et al. PLoS Genet 2005 Sep; 1 (3): e37). In addition, we evaluated the expression of CDC-48.1 which is the ortholog of Valosin-containing Protein P97 involved in ER associated protein degradation, ERpl9 and PDI-1 which encode ER disulfide isomerases, EDEM-1 (ER degradation enhancing alpha mannosidase -like 1), the transcription factor encoding mRNA CREBh, the caspase 8 substrate encoding mRNA BAP31, SRP-7 (which encodes a protease inhibitor of the serpin family) and finally both Y41C4A.1 1 (which encodes the beta subunit of the coatomer (COPI) complex) and T06D8.9 (which encodes for a protein of unknown function). The expression of these genes was normalized to that of AMAl and/or CRPl, genes which do not undergo UPR-mediated regulation (Shen X et al. PLoS Genet 2005 Sep; 1 (3): e37) and whose expression did not change in the course of our experiments (data not shown). Interestingly, feeding the worms with either BL21 or BL21 expressing F6 under basal conditions in the presence or absence of IPTG treatment did not lead to any significant change in mRNA expression levels of the 13 selected genes and suggested that under basal conditions the expression of F6 did not affect the expression of UPR target genes. In contrast, in the presence of tunicamycin, under feeding conditions using either 6xHis-F6 or the TAT F6 fragments induction of UPR target genes was observed in the absence of IPTG. However when the worms were fed with TAT-F6 expressing bacteria, the expression pattern of most of the genes was altered in an IPTG-dependent manner. Hierarchical clustering of these results showed that selected gene clusters were specifically induced upon F6 expression in the presence of tunicamycin. This was not observed when the worms were fed with 6xHis-F6 transformed bacteria. These data showed that the expression of UPR target genes was significantly modulated upon IPTG induction and exclusively with TAT-F6. This suggested that only TAT-F6 was able to reprogram IRE1 signalling upon tunicamycin treatment. It is interesting to note that the three genes which were subjected to the highest expression induction upon tunicamycin treatment mostly encoded proteins involved in ERAD (EDEM, CDC48.1 and ERpl9). In contrast, the genes whose expression was the highest in the presence of TAT-F6 were mostly related to biological functions linked to protein binding and export from the ER (PDI-1 , BAP31, Y41C4A.1 1, T06D8.9 and F22E5.6). This suggests that enhanced splicing activity of IRE1 upon ER stress may alter cell fate. Finally, we monitored worm resistance to tunicamycin-induced ER stress in the presence or absence of TAT-F6. In the absence of tunicamycin, TAT-F6 did not alter the ability of the worms to develop to the adult stage. In contrast, in the absence of F6, 58.6% of the progeny was arrested in LI after tunicamycin treatment. This phenotype was significantly alleviated when worms were fed TAT-F6 expressing bacteria, with less than 36% of the animals arrested in the LI stage after tunicamycin treatment. Collectively, these results demonstrate that F6 impacts on IREl signalling in vivo in C. elegans subjected to tunicamycin treatment and that this mediates enhanced ER stress resistance and worm survival.

Our results suggest that the in vivo use of ectopic F6 peptide may lead to enhanced IREl activity upon chronic ER stress. This may in turn provide a survival advantage to F6 exposed cells under these stress conditions. This observation may lead to novel strategies for the artificial modulation of the UPR (Unfolded Protein Response).

F6fragment derivative

Preparation of F6 fragment derivatives able to penetrate cells

In this section, we used a modified F6 fragment, which presents a shorter sequence and a modified aminoa-cid Ser to Glu as well as an additional sequence derived from the TAT protein (TAT peptide) to result in the following sequence:

TAT-P4 : GR KRRQRRRPQAHGKIKAMIEDFGLCKKL (SEQ ID NO:93) In addition, to increase the stability of the peptide, it was synthesized using D amino acids as previously demonstrated for other peptides (Borsello et al., Nature Medicine, Vol.9, N°9, 2003). This peptide P4-TAT all-D retroinverso has been called FIRE. As P4 was the most effective peptide modulator of IRE1 activities in vitro, we consequently tested its biological activity in cultured cells. To this end, we modified P4 to make it cell permeable and protease insensitive. To impact on the former, a TAT peptide was attached to the P4 sequence and to modify the latter, TAT-P4 was synthesized as an all-D retroinverso peptide (FIRE). First, to assess FIRE ability to penetrate cells, P4 and FIRE peptides were FTTC labelled and the purified products incubated for lh or 4h with HuH7 cells. Cells were then washed and fixed with 4% PFA and analyzed using confocal microscopy. This showed that FIRE-FTTC was able to penetrate into HuH7 cells whereas P4-FITC was not. After 4h incubation, approximately 70% of the cells add incorporated FIRE-FITC.

F6 fragment derivatives protect cells from ER stress

Based on this result, we next investigated the impact of FIRE on cell resistance to tunicamycin-induced ER stress. A dose response experiment carried out with P4 and FIRE peptides in the absence of tunicamycin treatment revealed that both peptides were not toxic (Fig. 2, open symbols). In contrast, upon tunicamycin treatment, only FIRE showed a dose-dependent cytoprotective effect (with a peak of efficacy at 10μ /ηιΙ). To further describe the signalling events modulated upon ER stress in the presence of FIRE, HuH7 cells were treated for 4h with 2 g/ml P4 or FERE peptides in the presence or not of 5μg m] tunicamycin and the expression of CHOP and sXBPl as well as the phosphorylation of eIF2a and JN 1 were monitored. As observed for F6, FIRE significantly increased the expression of sXBPl and in addition decreased that of CHOP upon tunicamycin treatment (Fig. 3, 4 left). Finally, after 4h of treatment with tunicamycin, FIRE considerably prevented JNK1 phosphorylation and did not impact on eIF2a phosphorylation compared to control cells (Fig. 3, 4 right). These data show that FIRE may act as F6 by modulating IRE1 signalling and displaying cytoprotective effects towards ER stressed cells.

Similar results than ER stress induced by tunicamycin are found with thapsigargin and

DTT. F6 fragment derivatives protect the functionality of the secretory pathway upon

ER stress

The protection of cells from ER stress by induced by tunicamycin, thapsigargin or DTT treatment occurs through the reprogramming of IRE1 signalling towards Xbpl mRNA splicing.

As the translation product of Xbpls mRNA (XBPls) is a transcription factor that promotes the transcriptional activation of genes whose products are involved in the restoration of ER homeostasis (including chaperones), we then tested whether treatment of cells with FIRE could protect the functionality of the secretory pathway upon ER stress.

To test this hypothesis, it has been investigated the secretory capacity of HuH7 cells exposed or not to a thapsigargin treatment and used the alpha 1 antirypsin (AIAT) as a secretion reporter. Briefly, HuH7 cells were treated or not with 0.5 uM thapsigargin in the presence or not of 10 or 50 uM FIRE. The secretion of AIAT was measured over a period of 2h using immunoblot analysis with antibodies against AIAT (Figure 5).

These results indicate that the secretion of AIAT by HuH7 cells is reduced upon TG exposure and that FIRE treatment does not affect its secretion under non-stressed conditions. Interestingly, treatment of HuH7 cells with FIRE restored the secretion of AIAT by HuH7 cells exposed to TG. These results validate our initial hypothesis and confirm that FIRE is able to protect the secretory pathway from ER stress. Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.