Patent Application
20100184709
The present invention relates to assays and screening methods, a method for detecting Malt1-specific cleavage, isolated polypeptides, antibodies and inhibitors. The present invention also relates to the use of Malt1 as a proteolytic enzyme and the use of compounds comprising a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47 as substrates susceptible for specific proteolytic cleavage.
Triggering of the antigen receptor of B or T cells leads to the initiation of multiple signalling pathways that regulate cellular proliferation and survival of immature and naive lymphocytes, and the effector functions of mature B and T cells. The signalling pathway that leads from antigen-receptor triggering to the activation of transcription factors of the nuclear factor-κB (NF-κB) family has a crucial role in these processes, and is controlled by highly similar molecular events in B and T cells. Genetic deficiencies in NF-κB-family members or signalling components that act upstream of NF-κB have been linked to immune deficiencies, whereas aberrant constitutive NF-κB activation has been associated with the development of autoimmune and neoplastic disorders. The understanding of the molecular mechanisms that control NF-κB activation in lymphocytes is therefore the focus of intense investigation. Recent studies have identified Carma1 (caspase recruitment domain, CARD, membrane-associated guanylate kinase, MAGUK, protein 1), Bcl-10 (B-cell lymphoma 10) and Malt1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) as signalling compounds that have crucial and specific roles in T-cell receptor (TCR)- and B-cell receptor (BCR)-induced NF-κB activation.
Malt1-deficient lymphocytes show impaired antigen receptor-induced activation of the transcription factor nuclear factor kappa B (NF-κB) and, as a consequence, impaired cytokine production and proliferation. Moreover, chromosomal translocation and abnormal expression or activity of Malt1 has been associated with formation of B-cell lymphomas of the mucosa-associated tissue.
More recently, the role of Bcl-10 and Malt1 has been extended to a role in lymphocyte adhesion, to activating signalling functions in other leukocytes such as phagocytes, mast cells and natural killer cells and to a signalling function downstream of G-protein coupled receptors, such as the receptors for lysophosphatidic acid or angiotensin II in non immune cells. This suggests that these proteins may have relevant immunomodulatory functions, but also be relevant for example in inflammation, tumour promotion and blood pressure regulation.
Dhaval N. Ghosalia at al., “Functional phenotyping of human plasma using a 361-fluorogenic substrate biosensing microarray”, Biotechnology and Bioengineering, 94, 6, p. 1099-10 (2006) report enzymatic activity of 10 different plasma proteases on as many as 361 different fluorogenic substrates. The goal behind this study is to provide a microarray that gives quick information on enzymatic activity of plasma for diagnostic purposes, for example. In Table 1 a peptide substrate Ac-ARSR-ACC is disclosed. This reference does not concern enzymatic activity of hMalt1, the latter occurring inside cells in nature.
WO2002/33058 reports the nucleotide sequences encoding hMalt1, which is indicated as BAA83099. This reference mentions cysteine proteases, including hMalt1, and a ligand binding to the cysteine protease and inhibiting its protease activity. However, this reference does not disclose the nature of the protease activity of hMalt1 and, therefore, does not disclose a real inhibitor either.
In needs to be noted that Malt1 is known to comprise a putative active-site cysteine residue in the caspase-like domain of Malt1, as established by sequence alignments with other caspases. However, attempts to demonstrate caspase-like proteolytic activity led to the conclusion that Malt1 does not have aspartate-specific proteolytic activity (Snipas, S. J. et al., “Characteristics of the caspase-like catalytic domain of human paracaspase”, Biol. Chem. 385, 1093-1098 (2004)).
In other words, while an enzymatic activity for Malt1 was hypothesised on the basis of sequence analysis, extensive experimental efforts failed to demonstrate such a cleaving activity, which is why presently the role of hMalt1 in signalling pathways is obscure.
In view of the above, it is an objective of the present invention to elucidate the role of Carma1, Bcl-10, and/or Malt1 signalling pathways, for example in B or T cells.
It is a further objective to determine the mechanism by way of which these cell components function in the signalling pathways.
It is a still further objective to provide a possibility of interfering in this pathway so as to be able to remedy the consequences and symptoms of abnormal expression or activity of Malt1, and/or other components of this or other signalling pathways.
It is, more particularly, an objective of the present invention to provide means, such as enzyme assays or screening methods, that permit the testing or screening for bioactive principles putatively interfering in a signalling pathway of cells, such as leukocytes, and in particular lymphocytes such as B and T cells.
It is also an objective to provide research tools for assisting the search and development of bioactive principles that remedy disorders and/or diseases, such as immune deficiencies, autoimmune or neoplastic disorders. Preferably, such principles are capable of affecting the cellular proliferation and survival of lymphocytes.
It is also an objective of the present invention to provide new peptide sequences, preferably useful in research and/or medicine.
Background knowledge on the field of the present invention is found in the review publications of Thome “Carma1, Bcl-10 and Malt1 in Lymphocyte Development and Activation”, Nat. Rev. Immunol., 4, 348-359 (2004); and Rawlings et al., “The CARMA1 signalosome links the signalling machinery of adaptive and innate immunity in lymphocytes”, Nat. Rev. Immunol., 6, 799-812 (2006).
Remarkably, the present inventors report a proteolytic activity for human Malt1. Interestingly, this activity is induced by T cell receptor (TCR) stimulation. It is also remarkable that the cleaving sequence recognized by Malt1 represents a new peptide cleaving sequence. There is currently no protease known having the same cleaving sequence specificity. These findings are useful in new enzyme assays and screening methods. They are also useful for in silico screening of potential Malt1-substrates.
Accordingly, the present invention provides, in a first embodiment, a method for detecting the occurrence of Malt1-specific cleavage, the method comprising the steps of assessing the consumption of a substrate cleaved by Malt1 enzymatic cleaving activity and/or the presence of a product obtained by Malt1 enzymatic cleaving activity. The invention also provides an assay for conducting this method.
The present invention further provides, in a second embodiment, an enzyme assay suitable to assess the consumption of a substrate and/or the production of a product, said assay comprising a substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, wherein said substrate is intended to be enzymatically cleaved after the C-terminal Arginine of said amino acid sequence.
In a third embodiment, the present invention provides a screening assay comprising a proteolytic enzyme and a substrate, said substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, said substrate being susceptible of being cleaved by said enzyme.
In a fourth embodiment, the present invention provides a method of assessing cleaving activity on a substrate having a bond to be cleaved following an amino acid sequence according to SEQ ID NO: 1-47, preferably 4-47, said method comprising the steps of contacting the substrate with a sample for which cleaving activity is to be assessed.
In a third embodiment, the present invention provides a screening assay comprising a proteolytic enzyme and a substrate, said substrate comprising a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47, said substrate being susceptible of being cleaved by said enzyme.
In a fourth embodiment, the present invention provides a method of screening for a bioactive principle, the method comprising the steps of:
In a fifth embodiment, the present invention provides a compound capable of inhibiting the proteolytic cleavage catalysed by Malt1. The invention also provides a compound that is capable of inhibiting the proteolytic cleavage of human Bcl10 or a homologue thereof at a position corresponding to Arg228 of SEQ ID NO: 50.
In an embodiment, the present invention provides a compound compound comprising a peptide sequence according to any one of SEQ ID NO. 1-47, preferably 4-47, linked at the C-terminal end of the respective sequence to a group selected from chloromethylketone (cmk), fluoromethylketone (fmk), aldehyde (—CHO), and, said compound being optionally further substituted at the N-terminal end of said peptide.
In a sixth embodiment, the present invention provides a compound capable of inhibiting the proteolytic cleavage of a substrate peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, for example 4-47, said inhibited proteolytic cleavage being at the carboxyl end of the carboxy terminal Arginine amino acid residue of said amino acid sequence. Similarly, the present invention provides a compound capable of inhibiting an enzyme selected from human Malt1 (SEQ ID NO: 48), an animal homologue of human Malt1 or a functional variant of any of the two aforementioned.
In a seventh embodiment, the present invention provides an isolated polypeptide selected from:
(a) a polypeptide having an amino acid sequence SEQ ID. NO 50 (Bcl10 large cleaved fragment);
(b) a homologue of SEQ ID. NO 50, or,
(c) a variant of SEQ ID NO: SEQ ID. NO 50.
Preferably, the polypeptide of (a), (b) and/or (c) has a C-terminal —S—R amino acid sequence at the end. More preferably, the C-terminal ending of the polypeptide corresponds to the amino acid sequence according to any one selected from SEQ ID NO: 1-47, preferably 4-47. More particularly, the C-terminal sequence is selected from the groups of preferred Malt1 target sequences mentioned herein. The present invention also provides the use of such polypeptides for detecting the occurrence of enzymatic activity of Malt1.
In a further embodiment, the present invention provides a peptide having an amino acid sequence according to SEQ ID NO: 49.
In still a further embodiment, the present invention provides an isolated polypeptide selected from (a) a polypeptide having amino acid sequence SEQ ID. NO 48 including isoforms; (b) a functional homologue of SEQ ID. NO 48; and, a variant of any of the polypeptides defined under (a) or (b), wherein said polypeptide has proteolytic activity, in particular human Malt1-specific proteolytic activity.
In further embodiments, the present invention provides the use of human Malt1, functional homologues or variants thereof as a specific protease, and a method of cleaving a substrate, the method comprising the step of contacting the substrate with human Malt1, a functional homologue and/or a variant thereof. The present invention also encompasses the use of human Malt1 in enzyme assays.
In still further embodiments, the present invention provides the use of substances comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, for example 4-47 as cleavable substrates, for example in enzyme assays and/or screening methods.
In yet another embodiment the present invention provides an antibody specifically binding to a product of Malt-1 specific cleavage. For example, the antibody binds to Bcl10, a homologue or variant thereof as cleaved by Malt1. Preferably, the antibody does not bind to Bcl10 not cleaved by Malt1, the antibody thus specifically detecting only the cleaved specimen.
In another embodiment, the present invention provides a method of preparing and/or designing an inhibitor of Malt1 proteolytic cleaving activity, the method comprising the steps of: (a) providing a peptide comprising an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4-47 or any of the preferred sequences selected from SEQ ID NO: 1-47 as defined in the present specification; (b) modifying said peptide, preferably by using peptidomimetics approaches, thereby obtaining a modified peptide; (c) exposing said modified peptide to enzymatically active Malt1, a homologue and or functional variant thereof; (d) determining the presence or absence of inhibition of Malt1 proteolytic cleaving activity; and, if no inhibition is found, (e) repeating the previous steps, whereby in step (b) modification of the peptide is such that a modified peptide is obtained that is structurally different from any modified peptide obtained previously.
In yet another embodiment the present invention provides a method for in silico screening for putative substrates of Malt1, a homologue or variant thereof, the method comprising the steps of systematically looking for any of SEQ ID NO: 1-47, for example 4-47 or for the nucleotide sequences encoding any of SEQ ID NO: 1-47, for example 4-47 in a database comprising amino acid or nucleotide sequences of an organism, for example the genome of an organism. For example, the open reading frames of the genome of the organism may be screened.
Further aspects and embodiments of the present invention are provided in the appended claims.
a-c show western blots of lysates of a T cell-line (Jurkat), human cytotoxic T cell clones and a B-cell line (Raji) as indicated following stimulation of the cells with various TCR stimulants or reagents mimicking T- or B-cell activation. The stained proteins are involved in signalling and are used here to assess efficient lymphocyte activation. In all Figures, black arrowheads indicate the position of unmodified Bcl10, while white arrowheads indicate a faster migrating form of Bcl10.
a-e show western blots of lysates of different T cell-lines as indicated following stimulation of the cells with various TCR stimulants. In
a shows a western blot of Bcl10 in a 2 dimensional SDS PAGE gel, revealing the presence of a faster migrating Bcl10 species at a pI more acidic than the wildtype form of Bcl10.
b shows an alignment of the C-terminal amino acid sequence of human and murine Bcl10 with previously identified metacaspase cleavage sites identified in Serpin1 and metacaspase9 of A. thaliana (AtSerpin1 and Atmc9) and in metacaspase type II of P. abies (mcIIa-Pa).
c and d show western blots of lysates of transduced and transfected cells. In
a-c show lack of an effect of various protease inhibitors targeting lysosomal proteases (NH4/leupeptin), proteasomal activities (MG132) or caspases (Z-VAD-fmk) on the appearance of a faster migrating Bcl10 species.
a and b show Malt1-dependent cleavage activity of lysates of T cells following T-cell activation and unstimulated control cells. For assessing cleavage activity, LRSR-peptide bound fluorogenic compound, Ac-LRSR-amc, was used. In
a and b show sequence alignments of amino acid sequences originating from different species and also from a virus. The sequences are aligned on a target cleaving sequence that can be cleaved by hMalt1.
a-f illustrate the inhibition of hMalt1 cleaving activity by Malt1 inhibitors of the present invention. In
The attached sequence listing is integral part of the present specification. SEQ. ID. NOs: 1-47 show target sequences that are specifically recognized and cleaved at the C-terminal Arginine residue by Malt1. SEQ ID NO: 48 is the amino acid sequence encoding hMalt1, which was described previously. SEQ ID NO: 49 is the shorter of two proteolytic cleavage products of Malt1 on hBcl10, while SEQ ID NO: 50 shows the larger of the two peptide products. SEQ ID NO: 51 is the amino acid sequence encoding hCARMA1 as previously described. SEQ ID NOs: 52-61 are extracts of sequences of various organisms, aligned for comparison and for the determination of a consensus sequence in
The embodiments of the present invention are based on the finding of a proteolytic activity of human Malt1 (hMalt1) and on the finding of so far unknown peptide target cleavage sequences for which hMalt1 is specific. The present inventors further found that the proteolytic activity of hMalt1 plays an important role in the adaptive immune response through its contribution in signalling pathways of the B-cell and T-cell receptors. The proteolytic activity of Malt1 described herein is equally important for other receptors that signal via Malt1. For example, these include receptors that signal via ITAM (immunoreceptor tyrosine-based activation motifs), such as for example receptors of the FcR family and pathogen receptors similar to Dectin-1, and G-protein coupled receptors such as for example the receptors for lysophosphatidic acid or for angiotensin II, which has pro-inflammatory effects and effects on blood pressure. The involvement of Malt1 in these pathways is reviewed in an article by Wegener and Krappmann: “CARD-Bcl10-Malt1 signalosomes: Missing link to NF-kB”, SciSTKE2007, pe21 (2007).
For the purpose of the present invention, the expression “Malt1-specific cleavage” refers to cleavage resulting from enzymatic cleaving activity of the enzyme hMalt1, a homologous protein of hMalt1 of another species and from a functional variant of any of the foregoing. The expression “hMalt1” encompasses different isoforms of the protein according to SEQ ID NO: 48 found in humans.
In vivo, “Malt1-specific cleavage” refers to intracellular enzymatic cleavage activity. Of course, enzyme assays using isolated and/or recombinant hMalt1, a functional homologue or variant thereof may be provided, so that the hMalt1 specific cleavage is conducted in a system, which is free of cells and/or in the extracellular matrix.
“Malt1-specific cleavage” is preferably characterised by the specificity of suitable peptide target sites where cleavage can occur. Accordingly, “Malt1-specific cleavage” refers to the cleavage of molecules comprising the specific target cleaving sites mentioned herein. In particular, it refers to cleavage of molecules comprising any one of SEQ ID NO: 1-47, in particular 4-47, and/or molecules comprising any one of SEQ ID NO 52-61 (Sequence alignment of
Accordingly, the present invention provides a method, an assay and a screening method in which the Malt1-specific proteolytic activity is assessed.
The term “assess” or “assessment” used with respect to the enzyme or screening assay of the invention and also with respect to the methods of the invention, refers to one or more activities related to the qualitative finding of presence or absence of an enzymatic activity as specified, the quantitative approximation of such activity, for example in terms of rate of product consumption, relative comparison of activities, and/or also the quantitative measurement of such activity, for example.
Proteolytic activity or proteolytic cleavage, in the context of the present invention, preferably refers to the cleavage of a covalent bond of one or more compounds of formula (I) to (IV) below,
wherein the covalent bond being cleaved is indicated as a dashed line and wherein R1 and R2 refer to organic residues including organometallic compounds and complexes, and wherein R2 can also be H. Preferably, the compounds of formula (I)-(IV) comprise an amino acid sequence selected from any one of SEQ ID NO: 1-47, in particular 4-47, and/or any one of the amino acid sequences which are underlined in
For example, R2 is -Leu-R3, with R3 being an organic residue including organometallic compounds or —H. Preferably, R1 is —NH—R3. An organic residue is a hydrocarbon optionally substituted and optionally comprising one or more heteroatoms. For example, R1 and R2 comprise and/or consist of peptides such as mono-, di-, tripeptides, oligopeptides (3-10 amino acids) and/or polypeptides (>10 amino acids). Preferably, these peptides are peptides comprising and/or consisting of L-amino acids, more preferably amino acids selected from the 20 standard proteinogenic amino acids. R1 and R2 may be the same or different. R1 and/or R2 may be proteins having a specific enzymatic activity. Furthermore, R1 and R2 may be compounds having any specific biologic activity or fluorescence or phosphorescence, for example.
The covalent bond to be cleaved preferably is a C—N bond, for example a peptide bond. It may also be a C—C bond, or a C—O bond, for example an ester or ether bond.
In compounds (I)-(IV), the amino acid moieties Arg, Ser and/or Thr may be chemically modified, for example in order to improve cell permeability of the substrate. For example, any one or both of the free amino group of the Arg-residues may be C1-C4-alkylated, preferably methylated.
Herein below, compounds (I)-(IV), the principle substrates of the enzyme assay and screening method of the present invention, will also be referred to as R1-R-S-R-R2 and R1-R-T-R-R2, with R, S and T standing for Arginine, Serine, and Threonine. For the purpose of the present specification, if peptide sequences are indicated with amino acids abbreviated by the one- or three letter code from left to right, the N-terminal end of the sequence is indicated left and the C-terminal end of the sequence is indicated right, as is conventional.
Accordingly, the term “substrate” for the present invention encompasses dipeptides, tripeptides, oligopeptides and polypeptides as well as derivatives thereof. In this sense, the term substrate includes compounds comprising a peptide comprising an amino acid sequence as defined in any one selected from SEQ ID NO: 1-47, in particular 4-47. Preferably, the substrate comprises and/or consists of a tri- or an oligopeptide having 3 to 10 amino acids.
The sequences of SEQU ID NO: 1-47, preferably 4-47, are also referred herein as the target cleaving sequence.
According to a preferred embodiment, the substrate comprises a peptide comprising an amino acid sequence of Xaa-R- (SEQ ID NO: 1), wherein Xaa stands for the any one amino acid selected from the groups of S, T, V, and G. Preferably, the substrate comprises a peptide comprising an amino acid sequence of —S/T-R— (selected from the group of SEQ ID NO: 2 and 3). More preferably, the substrate comprises a peptide comprising an amino acid sequence of —S—R— (SEQ ID NO: 2), wherein said substrates are cleaved by Malt1 following the carboxyl terminus of the R (Arg) residue. For example, according to an embodiment, the substrate comprises a peptide comprising an amino acid sequence of —R—S/T-R— (selected from the group of SEQ ID NOs.: 4 and 5). For example, said amino acid sequences of said substrates are provided in a larger peptide comprising further amino acids or amino acid sequences at the C-terminal of the indicated target sequence.
Preferably, the substrate comprises a peptide that comprises an amino acid sequence selected from any one of SEQ ID NO: 4, 5, 8-47, in particular any one of 5, 4, 11, 13, 23, 31, 33 and/or 43.
Preferably, the substrate comprises an amino acid sequence as defined by the general consensus sequences according to SEQ ID NO: 6 and/or 7, with Xaa standing for any amino acid but preferably L, P or H. According to a preferred embodiment, the substrate comprises an amino acid sequence selected from LRSR and LRTR.
According to an embodiment, the substrate comprises an amino acid sequence of at least 5 amino acids, said five amino acids being an N-terminal Proline followed by an amino acid sequence comprising 4 amino acids according to any one of SEQ. ID. NO 8-47 (P-G-R-S-R, P-A-R-S-R, etc.), preferably, PLRSR, PHRSR, PPRSR, PLRTR, PHRTR, and/or PPRTR.
The substrate comprising an amino acid sequence according to any one of SEQ ID NO: 1-47, preferably 4-47, may be provided in the form of a modified peptide. Modifications contemplated by the invention include, but are not limited to, pegylation (PEG linkage), glycosylation, amidation, carboxylation, phosphorylation, or addition of an acetyl, myristic, palmitic, stearic, or acidic group, creation of acid addition salts, amides, esters, in particular C-terminal esters, and N-acyl derivatives of the peptides of the invention. The peptides also can be modified to create peptide derivatives by forming covalent or noncovalent complexes with other moieties. Covalently-bound complexes can be prepared by linking the chemical moieties to functional groups on the side chains of amino acids comprising the peptides, or at the N- or C-terminus. These modifications may also occur at amino acids that form part of the target cleaving sequence according to any one of SEQ ID NO: 1-47, preferably 4-47.
Accordingly, substrates according to the invention are Bcl10 of Homo sapiens (NCBI accession number CAH71557), Mus musculus (NP 033870), Rattus norvegicus (NP 112618) or Bos Taurus (AAI18327), these proteins all being characterised by the target cleaving sequence LRSR. Another substrate is vE10 of Equine herpesvirus 2, which contains a LRTR target sequence. Furthermore, Carma1 of Homo sapiens (NP 115791), Mus musculus (NP 780571), Bos taurus (XP 593388) or Canis familiaris (XP 547005) are also potential substrates.
According to an embodiment, the substrate is (a) human Bcl10 (hBcl10), (b) a non-human homologue thereof, or (c) a variant of any of (a) or (b). HBcl10 corresponds to the peptide according to SEQ ID NO: 50 with the short peptide SEQ ID NO: 49 being added at the C-terminal end of SEQ ID NO: 50.
A homologue of a specific peptide of reference (the “original” peptide) mentioned herein is another peptide occurring in another species than the original peptide Homo sapiens but sharing the ancestry with the original peptide. Preferably, a homologue according to the present invention may or may not perform the same function in its natural environment as the original peptide reported herein. Rodent homologues of human Bcl10, for example, are indicated above.
A variant peptide is a peptide differing from an original peptide in that the original peptide has been chemically modified. For example, amino acid residues of the original peptide have been deleted, added or changed, but which still is capable of fulfilling substantially the same function as the original peptide. Variant polypeptides encompass peptides in which specific amino acid residues have been replaced by similar amino acids, such as amino acids having similar properties. For examples, a variant peptide would be a peptide in which an amino acid having a hydrophobic residue has been replaced by an amino acid having another hydrophobic residue, or in which an acidic amino acid by another, similarly acidic amino acid, and so on. Variant peptides also include compounds resulting from chemical modification of an original peptide, in particular original peptides comprising markers suitable to detect and/or quantify the peptide or a fragment thereof. A variant also encompasses a peptide obtained by gene shuffling using a nucleotide sequence encoding the original peptide. Gene shuffling is often used to improve the desired activity of an enzyme by creating mutated genes encoding a variant of the original enzyme and selecting those variants having improved characteristics.
Preferably, in a variant peptide, the domain or amino acid sequence corresponding to that of the original peptide (excluding added sequences e.g. with fusion peptide variants) has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 97% amino acid sequence identity with the original peptide as referred to herein.
The reference to a protein and/or peptide, for the purpose of the present invention, also includes a reference to naturally occurring isotypes. Preferably, these isotypes are able to fulfil the same function as required for the purpose of the present invention, or the isotype is provided in a variant form capable of fulfilling the function.
According to a preferred embodiment, the substrate comprises a marker molecule, for example marker peptides, suitable for assessing, preferably quantifying, the consumption of the substrate and/or the production of the product.
For example, suitable marking systems could be substrates in which cleavage induces the loss of fluorescence activated energy transfer (FRET) between suitable donor/acceptor pairs that would be added on suitable sites within the substrate which are preceding and following the cleavage site (for example the N- and C-terminus of a substrate peptide). This would allow to assess a loss of FRET upon cleavage because of the resulting separation of acceptor and donor moieties. A similar principle could be used to change (for example, destroy or obtain) any physical, chemical, or biological (e.g. enzymatical) property depending on proximity of two subunits/protein parts for functionality. For example, it is possible for a marker of hMalt1-specific proteolytic activity to obtain measurable physical, chemical or biological property, such as fluorescence, enzymatical or other biological activity through cleavage of the substrate.
It would also be possible to immobilise the substrate via one particular part of it (for example the free amino terminus of the peptide) and to assess proteolytic release of a measurable enzymatic or radioactive or fluorescent activity that has been covalently or not covalently linked to the substrate (for example to its C-terminus as part of R1) and that would be released into the supernatant upon incubation with samples containing proteolytically active Malt1.
For example, the substrate comprises a fluorescent compound, for example 7-amino-4-methylcoumarin (AMC) or a fluorescent derivative thereof, linked to the C-terminal Arginine of the target cleaving sequence, and wherein substrate consumption or product production is assessed by measuring, approximating and/or comparing fluorescence intensity of cleaved AMC or the fluorescent derivative thereof.
An antibody specifically binding to a product of Malt1-specific cleavage is encompassed by the invention and may also be used as a marker for detecting Malt-1 cleaving activity, in particular to a product obtained from Malt1 specific cleavage. Preferably, the antibody does not bind to a substrate from which said product is obtained by Malt1-specific cleavage. Preferably, the antibody of the invention specifically binds to cleaved Bcl10, such as the polypeptide of SEQ ID NO: 49, but does not bind to a Bcl10 not having the C-terminal amino acid sequence of SEQ ID NO:49. This C-terminal amino acid sequence preferably involves a sequence of the at least 4, more preferably the at least 6 and most preferably the at least 8 amino acids of the C-terminus of SEQ ID NO: 49.
In the assay and the method of screening of the present invention, a substrate consumption and/or product production is preferably catalysed by human Malt1, a homologue thereof and/or a variant thereof. For example, a variant hMalt1 comprising and/or consisting essentially of the caspase-like domain of hMalt1 or of a homologue thereof may be suitable. The caspase-like domain of hMalt1 is the sequence stretching from aa 336-572 of SEQ ID NO: 48. Depending the authors and sequence analysis program used, the caspase-like domain of hMalt1 may comprise a few amino acids more or less on either side. These caspase-like domains provide the cleaving activity of hMalt1, homologues and/or functional variants thereof. Variants comprising all or part of the caspase-like domain are therefore examples of functional variants of hMalt1. Of course, in variants comprising all or part of aa 336-572 of SEQ ID NO: 48, individual amino acids may be exchanged and/or omitted, while the cleaving activity is retained. For example, amino acids may be classified as polar and apolar, as acidic, neutral and (weakly) basic. It is common to provide peptide variants in which amino acids having similar properties while retaining the activity. Such variants are also included herein.
Other enzymes, so far not described, may have the same cleaving activity as defined above and may be used instead. With the teaching of the present invention it is now possible to more specifically search and identify other enzymes having the same activity.
Accordingly, the enzymatic activity reported herein may be accomplished by hMalt1 according to SEQ ID NO: 48. Various isoforms of hMalt1 have been reported so far, for example under accession number BAA83099 in the NCBI protein database. Isoforms of proteins mentioned herein are also encompassed by the present invention.
The assay of the present invention may be provided in the form of a kit comprising various components, one of which being, for example, a substrate as defined above susceptible of hMalt1-specific cleavage. The term assay as used in this description also encompasses a kit.
The enzyme having hMalt1-specific cleaving properties may be a component of the assay, the assay being provided in the form of a kit comprising various components. Preferably, however, the enzyme may be included in a sample to be tested and is, in this case, provided by the user of the assay.
The assay may be used to assess the activity of an enzyme by exposing cells to a substrate as defined herein. Cells may be any cells of interest, including human and/or non-human cells. In particular, the cells may be human, animal, plant and/or bacterial cells. The cells may be provided in the form of a sample of human or animal, for example, rodent, tissue. The sample to be tested may be provided in the form of a slice of tissue of a human or an animal. The sample may contain blood cells, in particular lymphocytes, for example B- or T-lymphocytes. In particular, the sample may be a tumor tissue sample, for example a lymphoma sample.
Cells to be tested may or may not be pre-treated (“activated”) before testing enzymatic hMalt1-specific cleaving activity. For example, cells may be exposed to specific substances for testing the effect of these compounds on the hMalt1-specific cleaving. Such substances may be substances activating TCR- or BCR-mediated cell signalling cascades, such as those mentioned in the examples below. Such substances may be part of the assay or may be separately obtained and applied by a user of the assay. Such activating substances may form part of the assay.
Preferably, cells are preferably destroyed, for example lysed, to provide a sample for which the occurrence of Malt1-specific cleavage can be detected. Since Malt1-specific cleavage occurs in the intracellular space, destruction of cells naturally expressing Malt1 is preferred in order to assess the consumption of a substrate and/or the presence of a product of Malt1-specific cleavage. Lysis facilitates the contact of the substrate with hMalt1-specific cleaving enzymes present in the cells.
Preferably, the enzymatic activity is assessed in presence of a specific buffer enabling hMalt-specific enzymatic activity. Buffers suitable for assessing hMalt1-specific enzymatic activity are preferably buffers with physiological pH and ionic strength, for example 20 mM Tris/HCl and 150 mM NaCl, and preferably comprising a reducing agent, for example dithiothreitol or mercaptoethanol. Preferably, the buffer contains Ca2+ ions. Furthermore, buffers may contain inhibitors of proteases and/or phosphatases to avoid degradation of the substrate by other, non-specific proteases.
Suitable protease and/or phosphatase inhibitors not inhibiting hMalt1-specific cleavage and/or suitable buffers may be part of the assay.
The present invention also provides an assay, in particular a screening assay and/or a screening method. One goal of these aspects of the present invention is to provide a possibility of assessing the effect of a principle to be screened on hMalt1-specific cleaving.
In a step of the screening method, a system comprising an enzyme capable of hMalt1-specific cleaving is provided. For example, the system may comprise hMalt1, a homologue and/or derivative there from. The enzyme may, for example, be provided in the form of cells as defined above, said cells comprising the enzyme. For example, cells transfected or transduced to express increased amounts of hMalt1 may be used. Instead of using cells, it is preferable to use purified and/or isolated enzyme, for example enzyme produced in a suitable expression system. Preferably, the enzyme is recombinant.
The system comprising the enzyme preferably comprises co-factors assisting the proteolytic cleavage. In particular, the system preferably comprises a peptide selected from human Carma1 (SEQ ID NO: 51), a non-human homologue thereof and/or a functional variant thereof. It was shown in the present invention that Carma1, in particular its binding to Bcl10 and its oligomerization, may play a role in optimal hMalt1-specific cleavage. As with the enzyme providing hMalt1-specific cleaving, Carma1 may be provided in the form of cells expressing Carma1, or in a recombinant, preferably purified form.
The system also preferably comprises a peptide selected from Bcl10, a non-human homologue thereof and/or a functional variant thereof, since the capacity of Carma1 to bind Bcl10 (which mediates formation of a Carma1-Bcl10-Malt1 complex) seems to be required for activation-induced Bcl10 cleavage by Malt1.
Of course, isolated Carma1, hMalt1 and further selected components may be provided together in a purified preparation. These components may be provided in any method and/or assay of the present invention. For example, Carma1 and/or Bcl10 may be provided in a purified preparation, or even all three together.
Preferably, if recombinant Malt1 is used for the purpose of the present invention, for example in the screening method, a recombinant Malt1 form is selected that contain oligomerization domains or that contain protein domains that can be inducibly oligomerized by addition of suitable bi-valent or multivalent compounds. The recombinant Malt1 may also be chemically modified to allow inducible oligomerization. The necessity of oligomerization is explained by the fact that a role of Carma1 seems to be the induction of an oligomerization of Malt1.
The components used for the screening method, such as the enzyme as well as the substrate and optional cofactors, buffers, and the like may be used as indicated with respect to the assay described above. For example, the system comprising the enzyme is an aqueous solution or suspension to which a purified form of the enzyme and, optionally cofactors have been added. Accordingly, the present invention also relates to a screening assay suitable for conducting the screening method. The screening assay may also be provided in the form of a kit.
Preferably, the screening method of the present invention comprises the step of comparing the measured proteolytic activity of the enzyme to a control proteolytic activity measured in absence of the principle to be screened.
Preferably, the screening method of the present invention comprises the step of comparing the measured proteolytic activity of the enzyme to a control proteolytic activity measured in presence of a control principle of which the effect (e.g. activation, inhibition or stabilisation) on the enzyme is known.
Generally, the screening method comprises the step of assessing if the principle to be screened exerts an effect on said proteolytic activity by comparing the measured proteolytic activity with the control proteolytic activity and by finding an effect if said activity and said control activity differ from each other.
The effect that the principle to be screened exerts may be selected from an inhibitory effect or an activating effect, wherein an inhibitory effect is found if said proteolytic activity is lower than said control proteolytic activity and/or wherein an activating effect is found if said proteolytic activity is higher than said control proteolytic activity.
The present invention also provides an inhibitor, in particular (1) a compound capable of inhibiting the proteolytic cleavage of a substrate polypeptide at the carboxyl end of the Arginine amino acid residue at a cleavage site having an amino acid sequence selected from any one of SEQ ID NO: 1-11, or from any one of 4, 5, and 8-11; (2) a compound capable of inhibiting the proteolytic cleavage of human Bcl10 or a homologue thereof at a position corresponding to Arg228 of SEQ ID NO: 49 (hBcl10, cleaved).
Such an inhibitor can be an antibody and/or a peptide comprising an amino acid sequence selected from any one of SEQ ID NO: 1-47, preferably 4-47.
The inhibitor may be a monoclonal or polyclonal antibody.
Preferably, the inhibitor is a compound capable to bind to the proteolytically active site of human Malt1, including compounds binding to or covering sites Cys464 and/or His415 of SEQ ID NO: 48. Alternatively, the inhibitor binds to or associates with other portions of Malt1, for example portions outside the caspase-like domain, said compound inhibiting proteolytic activity of Malt1 by said binding or association to said other portions of Malt1, for example by inducing conformational changes affecting the proteolytically active site.
The present invention particularly relates to inhibitors comprising the target peptide sequence. These are substrate-mimetic inhibitors that comprise the Malt1 recognition sequence (any one of SEQ ID NO: 1-47, in particular 4-47, such as SR and preferably RSR, RTR, in particular RPR) linked to reactive groups such as chloromethylketone (cmk) or fluoromethylketone (fmk), for example, that irreversibly inhibit; or aldehyde (CHO) reactive groups that reversibly inhibit the active site of Malt1. To increase cell permeability, the peptides can be N-terminally modified with Ac (acetyl) or Z (benzyloxycarbonyl) groups and the amino groups of the arginine residues can be alkylated, for example with a C1-C10, preferably C1-C5 alkyl, preferably methylated, for example. Other ways of increasing cell permeability are also encompassed within the scope of the present invention. As another example, the peptides may be biotinylated, for example N-terminally A biotinylated inhibitor is useful as a research tool to inhibit and subsequently isolate caspases, for example by pull-down assays using avidin- or streptavidin-coupled supports. According to yet another embodiment, the inhibitor comprises a fluorescent grouping. For example, fluorescent FITC (Fluorescein isothiocyanate) may be linked N-terminally to the peptide group of the inhibitor to yield FITC-RSR-fmk, or FITC-VRPR-fmk, for example.
According to an embodiment, at least one amino group of an arginine residue of said peptide is alklyated, for example with an C1-C10, preferably a C1-C5 alkyl.
According to an embodiment, the inhibitor is the compound X-VRPR-Y, wherein X is selected from H, Ac, Z, biotyl, a fluorescent substituent and from other substituents providing a desired functionality to the compound, and Y is selected, independently of X, from fmk, cmk and —CHO.
In general, inhibitors of the present invention may be functionalised as desired, preferably at the N-terminal end of the peptide fragment, in order to adapt it to a specific purpose, which increases its utility as a research tool.
An inhibitor that showed inhibition of Malt1-specific cleaving is z-VRPR-fmk. This inhibitor may be used as such, for example in research and/or in vitro testing, such as in vitro enzymatic assays of this invention. This inhibitor may also be useful for in vivo applications, for example as a medicament and in the treatment and/or prophylaxis of the conditions mentioned below. According to preferred embodiment, an inhibitor having improved in vivo characteristics, such as fewer side effects and comparatively low toxicity and overall improved pharmacological properties is designed on the basis of the indicated z-VRPR-fmk by peptidomimetics. Such an inhibitor may be synthesized according to the methodology disclosed in the following publications: Bioorganic & Medicinal Chemistry, Volume 5, Issue 5, May 1997, Pages 797-807. E. Ponnusamy, U. Fotadar, A. Spisni and D. Fiat, A novel method for the rapid, nonaqueous tert-butoxycarbonylation of some O-17-labeled amino-acids and O-17-NMR parameters of the products, Synthesis (Stuttgart) 1 (1986), pp. 4849.
Accordingly, the present invention encompasses peptidomimetics of the target sequence peptides of SEQ ID NO 1-47, in particular 4-47, in particular RSR, RTR and RPR, optionally comprising further amino acids. The peptidomimetics may comprise the same chemical modifications as indicated for the peptide substrate sequences SEQ ID NO 1-47 above (pegylation, glycosylation, etc.)
The present invention also provides a method for obtaining an inhibitor capable of inhibiting Malt1-specific cleaving and/or a compound for preventing and/or treating the conditions indicated below. The method comprises the steps of designing and testing peptidomimetic compounds for their Malt1-specific cleaving inhibitory activity. Drug design according to peptidomimetics has been discussed in the art. For example, Walensky et al., Science 2004; Sep. 3; 305(5689): 1411-3, describe hydrocarbon stapling for obtaining peptidomimetics having improved pharmacological properties. Li L. et al, Science 2004 Sep. 3. describe a small molecule mimic of Smac, a pro-apoptotic protein. Rubin-Carrez C. published on the internet a report entitled “des peptidomimetiques peptidiques aux mimétiques non peptidiques: sur la voie du medicament.”, which sets out some of the methodology currently use for developing peptidomimetics: http://ist.inserm.fr/BASIS/elgis/fqmat/atelier/DDD/1235.doc
For example, the substitution of classic peptide bonds by isosteric amides resistant to peptidases is mentioned in this report, amongst many other ways. The above references are entirely incorporated herein by reference. The present invention thus encompasses peptidomimetic compounds of the peptide-based inhibitors reported herein.
The method of screening of the present invention may be a method for screening for principles having at least one of the effects selected from: (1) immunomodulation, (2) modulation of inflammatory processes, (3) suppressing an immune reaction, (4) reducing or avoiding an immune reaction against a transplant, (5) reducing and/or increasing blood pressure, (6) reducing cellular proliferation. Medicaments with these effects may be prepared from active principles selected by the screening method of the present invention.
The screening method of the present invention is susceptible of identifying inhibitors and/or principles that are useful as a medicament. For example, the principle may be used for the prevention or treatment of (1) lymphomas and/or leukaemia, (2) an immunological disorder, (3) multiple sclerosis, (4) an autoimmune disease, (5) diabetes, (6) allergies, (7) hemic disease, in particular hemic tumours, (8) tumours in general, (9) inflammatory disorders, for example colitis, arthritis and/or artheriosclerosis, or combinations of two or more of the aforementioned.
The present invention also provides inhibitors identified by the screening method, the inhibitors potentially having the characteristics indicated above. Accordingly, inhibitors may be used as medicaments.
The present invention will now be illustrated by way of examples. These examples do not limit the scope of this invention which is defined by the appended claims.
Blood samples were obtained from two HLA-A*0201 positive healthy volunteers containing readily detectable frequencies of EBV- and CMV-specific CD8+ T-cells in ex-vivo analyses. From these blood samples, peripheral blood mononuclear cells (PBMCs) were obtained by density centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) and cryopreserved in RPMI 1640 supplemented with 40% FCS and 10% DMSO (1×107-2×107 cells per vial) until further use. Phycoerythrin-labeled HLA-A*0201/peptide multimers were prepared with NLVPMVATV peptide (CMV/pp65495-503) and were kindly provided by P. Guillaume and I. Luescher (Ludwig Institute for Cancer Research, Epalinges, Switzerland). Multimer-positive CD8+ T-cells were sorted by a FACSVantage® SE using CellQuest software (Becton Dickinson), cloned by limiting dilution, and expanded in RPMI 1640 medium supplemented with 8% human serum (HS), 150 U/ml of recombinant human IL-2 (rIL-2; a gift from GlaxoSmithKline), 1 μg/ml phytohemagglutinin (PHA; Sodiag, Losone, Switzerland) and 1×106/ml irradiated allogeneic PBMC (3000 rad) as feeder cells. Positive EBV- and CMV-specific T-cell clones (cytotoxic T lymphocytes, CTL) were periodically (every 15 days) re-stimulated in 24-well plates with PHA, irradiated feeder cells, and rIL-2.
The human cell lines Jurkat (J77 clone 20), Hut78 and Raji cells (all are gifts of Oreste Acuto, Pasteur Institute, Paris) were grown at 37° C. in RPMI 1640 supplemented with 10% FCS and antibiotics.
For short term stimulations, cells were re-suspended at a density of 5×107 cells/ml in RPMI with 0.5% serum and pre-warmed for 10 min at 37° C. before addition of the stimulating agent.
T-cell stimulation was initiated by addition of PMA (10 ng/ml, Alexis) and ionomycin (1 μM, Calbiochem) (P/I) or a combination of anti-human CD3ε (10 μg/ml of OKT3, Apotech) and anti-CD28 (10 μg/ml of CD28.2, Immunotech) antibodies (CD3/CD28), immediately followed by addition of 5 μg/ml of cross-linking goat anti-mouse antibody (Southern Biotech).
Pooled human CMV-specific CTL clones were stimulated by 10 μg/ml MHC-pep tetramers (HLA-A*0201/peptide multimers with NLVPMVATV peptide corresponding to CMV/pp 65495-503, kindly provided by P. Guillaume and I. Luescher, Ludwig Institute, Epalinges, Switzerland) together with 10 μg/ml of anti-CD28 (CD28.2, Immunotech).
Raji B cells were stimulated using P/I as described above. Stimulations were conducted for various time intervals, depending on the cell type, as indicated in the figures.
Stimulations were stopped by addition of ice-cold Tris-NaCl buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl), and pelleted cells were lysed in Tris-NaCl lysis buffer containing 1% NP40, proteinase inhibitors (Complete, Roche) and phosphatase inhibitors (cocktails I and II, Sigma).
Postnuclear cell lysates were boiled with reducing SDS-sample buffer and analyzed on 16×20 cm gels by 15% SDS-PAGE according to Anderson, N. L. & Anderson, N. G. Analytical techniques for cell fractions. XXII. Two-dimensional analysis of serum and tissue proteins: multiple gradient-slab gel electrophoresis. Anal Biochem 85, 341-54 (1978).
Primary antibodies used are monoclonal mouse anti-phospho ERK (Sigma), rabbit anti-phospho-IκBα (5A5, Cell Signalling), rabbit anti-IκBα (Sigma), and rabbit anti-Bcl10 (H-197, Santa Cruz). Western blots were revealed using HRP-coupled goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch).
The results are shown in
In all Figures, black arrowheads indicate the position of unmodified Bcl10, while white arrowheads indicate a faster migrating form of Bcl10.
The appearance of this Bcl10 species was delayed with respect to IκB phosphorylation (
To test the relevance of signalling components acting upstream and downstream of Bcl10 in the NF-κB pathway, and in particular the association of Bcl10 with its upstream binding partner Carma1 through a caspase recruitment domain (CARD), a number of further experiments were conducted.
In a first approach, Jurkat cells were pre-incubated with either the pan-PKC inhibitor bisindolyl-maleimide VIII (BIM) or with Gö6976, an inhibitor of classical (Ca2+- and DAG-dependent) PKC isoforms, before stimulation with CD3/CD28 as indicated in Example 1. For pre-incubation, the cells were incubated with 5 mM Gö6976 (Calbiochem) or 500 nM Bisindoleylmaleimide VIII acetate (Alexis), or solvent control for 30 min at 37° in RPMI with 0.5% serum directly prior to stimulation.
In a further approach, Jurkat cells were lentivirally transduced with Carma1-specific or control shRNA constructs as described in Rueda, D. et al. “Bcl10 controls TCR- and FcγR-induced actin polymerisation”. J. Immunol. 178, 4373-4384 (2007). These cells were stimulated with CD3/CD28 or with P/I as described in Example 1.
Moreover, two types of Malt1-silenced Jurkat cells were exposed to a CD3/CD28 stimulation as described in Example 1 above. The first type (#1) was silenced by lentiviral silencing vector for Malt1 as described by Rueda, D. et al (see above), and the second, independent Malt1 silencing vector (#2) was obtained from OpenBiosystems (Clone TRCN0000073826).
Furthermore, Jurkat cells expressing constructs for VSV-tagged wt or CARD-mutated, dominant negative Carma1 (Carma1-DN) were prepared by retroviral transduction of Jurkat cells, as disclosed in Gaide, O. et al. “CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation” Nat. Immunol. 3, 836-843 (2002)). This construct has a non-functional CARD motif that does not allow binding to Bcl10 which is required for the formation of a Carma1-Bcl10-Malt1 complex (reviewed in Thome 2004, cited above). These cells were stimulated with CD3/CD28 as described in Example 1.
In another experiment, Jurkat cells were lentivirally transduced with a VSV-tagged expression construct for the Carma1 coiled coil domain (or empty vector), see also A Tanner, M. J., Hanel, W., Gaffen, S. L. & Lin, X. CARMA1 Coiled-coil domain is involved in the oligomerization and subcellular localization of CARMA1, and is required for T cell receptor-induced NF-kappa B activation. J Biol Chem (2007). This construct lacks the N-terminal CARD domain of Carma 1 as well as part of the C-terminus and prevents oligomerization of endogenous Carma1 in the cell by interfering with the oligomerization process.
Cell lysis and western blots was done as in Example 1. The primary antibody for the experiment with VSV-tagged wt or VSV-tagged Carma1-DN and Carma1-coiled-coil construct is monoclonal mouse anti-VSV (P5D4, Sigma). For Carma1, a rabbit anti-Carma1 (AL220, Alexis) antibody was used.
The outcome of these studies can be seen in
Consistent with a described role for PKCtheta (a Ca2+-independent, so-called novel PKC isoform) upstream of the Carma1-Bcl10-Malt1 complex in T cells, BIM but not Gö6976 prevented the generation of the faster migrating Bcl10 species (
In order to determine if proteolytic cleavage was at the origin of the faster migrating Bcl10 species, Jurkat cells were stimulated for 30 min by PMA and ionomycin and cell extracts were prepared and analysed by two dimensional gel electrophoresis and anti-Bcl10 Western blot as described by Rueda et al. 2007, see above.
The Western blot is shown in
The faster migrating species thus most likely resulted from proteolytic cleavage at the C-terminus of Bcl10, which contains a positively charged Arg residue (Arg 232) whose proteolytic removal could explain the more acidic pI value (
To determine whether cleavage occurred after the preceding Arg residue 228, we stably expressed a Bcl10 wt form or an Arg 228/Gly mutant in Jurkat cells and tested the effect of PMA/ionomycin stimulation on these constructs.
The Bcl10 point mutant was generated by a standard double PCR approach, subcloned into expression vectors derived from pCR3 (Invitrogen) and verified by sequencing in both directions before further subcloning into the lentiviral vector pRDI—292 (a gift of R. Iggo, ISREC, Epalinges, Switzerland) that allows expression of constructs under the EF1 promotor. The equivalent lentiviral expression vector for Bcl10 wt has been described (Rueda et al. 2007). Transduction of Jurkat T cells was performed as described before (Rueda et al. 2007).
Stimulation and Western blots were done as described in Example 1 above, using monoclonal mouse anti-FLAG (M2, Sigma).
The result is shown in
Human embryonic kidney cells (293T cells) were co-transfected with combinations of VSV-tagged Malt1 and FLAG-tagged Bcl10 mutant constructs that were prepared with the same method (double PCR approach, etc) as detailed above. Transfection of 293T cells was done as in Rueda et al. 2007. Transfected samples included: cells transfected with mock plasmid (devoid of functional Malt1), with VSV-Malt 1 (functional wt Malt1), with VSV-Malt1 point mutated at Cys 464 (putative active site) of Malt1 (=“C464/A”), and cells with VSV-Malt1 with a deletion of the caspase-like domain (aa 336 to aa 566 of Malt1) (=“Δcasp”) all with FLAG-tagged wt Bcl10. Further cells were prepared by transfection with mock plasmid and VSV-wt Malt1, both with FLAG-tagged, point mutated Bcl10 (R228/G).
The outcome of these experiments is shown in
In order to analyse the C-terminal nature of the fast and slow migrating forms of Bcl10, FLAG-tagged Bcl10 was immunoprecipitated from 293T cells co-transfected with FLAG-Bcl10 and Malt1.
In particular, pooled lysates from ten 10 cm dishes of FLAG-Bcl10- or FLAG-Bcl10- and VSV-Malt1-transfected 293T cells were used for anti-FLAG immunoprecipitation on 120 μl of anti-FLAG-agarose (Sigma). Samples were processed on 15% Anderson SDS-PAGE (Anderson et al. 1978, see above), and Coomassie-stained bands corresponding to uncleaned and cleaved Bcl10 were cut out and analyzed by in-gel Glu-C digest and mass spectrometric (MALDI-MS) analysis (TOPLAB, Munich, Germany).
The results are shown in
In conclusion of the experiments conducted under Example 3 Malt1 is identified as an Arg-specific protease cleaving Bcl10 after Arg 228.
For testing if the observed proteolytic activity is an indirect effect of Malt1 on other proteases, Jurkat cells were pretreated with the lysosomal or proteasomal inhibitors (NH4+/leupeptin or MG132) or solvent control before stimulation with PMA and ionomycin, and postnuclear lysates were analyzed by Western blot as indicated. As is shown in
In an analogue experiment, Jurkat cells were pretreated with the pan-caspase inhibitor zVAD-fmk or solvent control, and stimulated with anti-CD3 and anti-CD28 antibodies or hexameric recombinant FasL for the indicated times. Postnuclear lysates were analyzed by Western blot with the indicated primary antibodies (
In conclusion, it is unlikely that Bcl10 cleavage is mediated by an indirect effect of Malt1 on other proteases, since inhibition of lysosomal, proteasomal or caspase-type proteolytic activities did not affect Bcl10 cleavage.
In this example, the capacity of Malt1 to cleave a Bcl10-derived fluorogenic substrate upon T-cell activation was assessed.
Unstimulated or PMA/ionomycin-stimulated Jurkat T cells were mechanically lysed in cleavage assay buffer (50 mM Tris-HCl pH 7.4, 60 mM NaCl, 100 mM CaCl2 and 10 mM DTT) using a dounce homogenizer. Malt1 protease activity was determined upon addition of 50 μM Ac-LRSR-amc (amc=7-amino-4-methylcoumarin) (Peptides International, Inc.) and incubation at 30° C. for 4 h, using a Synergy microplate reader (BioTek). The experiment was repeated for various time-intervals of stimulation (0, 10, 30, 45 and 60 min). In another setting, stimulation was for 20 minutes and the cleavage of Ac-LRSR-amc (Assay time) was followed during indicated times.
The results are shown in
In order to assess if Malt1-mediated cleavage of Bcl10 had an influence on IκB phosphorylation, and, consequently, on NF-kB activation, the following experiment was conducted.
Jurkat cells stably transduced with wildtype Bcl10 or its Arg 228/Gly mutant (see Example 3 and
It was found that intensities and kinetics of IκBα phosphorylation and degradation are similar in Jurkat cells stably expressing the wt or Arg 228/Gly form of Bcl10, indicating that the classical IκBα-dependent pathway of NF-κB activation was unaffected by Bcl10 cleavage.
In another experiment designed to assess the influence of Malt1-mediated cleavage of Bcl10 on NF-κB response, the following experiment was conducted.
For the experiments, cells with inactive Malt 1 as described in Example 3 above were used: VSV-Malt1 (wt with functional Malt1), Δcasp, C464/A, and also another mutant, H415/A, which is mutated by a point mutation at the active site, obtained by the standard double PCR approach as described with respect to C464/A above (Example 3).
These cells were co-transfected with a renilla-luciferase and an NF-κB-firefly luciferase construct and mock plasmid to normalize transfection efficiency as disclosed by Rueda et al. (2007). Relative NF-κB activity was determined 24 h after transfection by dual luciferase assay (Rueda et al. 2007).
The result can be seen in
Central areas of tissue culture dishes (35-mm×10-mm) were coated for 90 min at 25° C. with 50 μl of fibronectin solution (40 μg/ml; Roche). Coated dishes were washed three times with PBS (without Ca2+ and Mg2+) and were blocked for 2 h with 1% (wt/vol) BSA in PBS. Cells were washed with Hank's balanced-salt solution (Gibco) and were left unstimulated or were stimulated at 37° C. for 30 min with MnCl2 (1 mM) or PMA (100 ng/ml), or for 5 min with anti-human CD3 and anti-CD28 (see example 1). Subsequently, cell suspensions were added to fibronectin-coated tissue culture dishes followed by incubation for additional 30 min at 37° C. Non-adherent cells were removed by washing of the dishes three to five times with Hank's balanced-salt solution. Adherent cells were counted in six separate fields with a microscope with an ocular counting reticule.
After activation with either anti-CD3 and anti-CD28 or PMA, cells expressing the R228G mutant had less adhesion to fibronectin than did cells expressing wild-type Bcl-10 (
In order to know whether the cleaved form of Bcl10 is detectable in B-cell lymphomas samples from biopsies were taken and analysed as follows: Lymphoma samples from lymph node, spleen and gastrointestinal biopsies from patients with mucosa-associated lymphoid tissue (MALT) lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL) or follicular cell lymphoma (FCL) were immediately frozen without additives and stored for 3-24 months before lysis. Cells were lysed in 50 mM Hepes pH 7.6, 4 mM EDTA, 150 mM NaCl und 1% Triton X-100 supplemented with the following proteinase and phosphatase inhibitors: 2 mM PMSF, 2 μg/ml aprotinin and 5 μg/ml leupeptin, 2 mM Na3VO4, 100 mM NaF und 50 mM Na4P2O7. Lysates were stored at −80° C. before analysis by SDS-PAGE and Western blot. Controls include lysates of non-stimulated (−) and PMA/ionomycin-stimulated (+) CD8+ T cell clones.
The result can be seen in
To test the efficiency of inhibitors, Jurkat T cells stimulated or not with PMA and ionomycin for 20 min are mechanically lysed in cleavage assay buffer (see example 4) in the absence or presence of increasing concentrations of a irreversible cell permeable inhibitor peptide (z-VRPR-fmk). This inhibitor was designed based on an optimal substrate cleavage sequence reported for the plant metacaspase AtMC9 (see ref. by Vercammen et al. 2006, J. Mol. Biol., 364(4):625-36). Malt1 protease activity is determined as described in example 4. To test the efficiency of the peptide inhibitor on living cells, Jurkat T cells or human primary cytolytic T cells (CTLs) are pre-incubated for 1 h with indicated concentrations of z-VRPR-fmk (see above), before stimulation with PMA and ionomycin for 20 min, lysis of the cells and detection of Bcl10 cleavage by Western blot (see example 1). Moreover, experiments are performed to assess the effect of the inhibitor on anti-CD3/CD28-induced NF-κB activation in Jurkat T cells, on IL-2 production of Jurkat cells stimulated by incubation with antigen-presenting cells (Raji cells in combination with the superantigen SEE) and on stimulation-induced adhesion to fibronectin (see example 7)).
In the presence of the inhibitor peptide, we observe a reduction of the activation-induced LRSR-amc cleavage activity (
Antibodies that exclusively recognize the cleaved form of Bcl10 were produced. To generate these antibodies, a rabbit was immunized with a short peptide comprising the 8 amino acids aminoterminal to the cleavage site (the peptide sequence is MFLPLRSR). The peptide was coupled to the carrier protein KLH and used for repeated immunization of a rabbit. Peptide synthesis, coupling to the carrier and immunization were performed by Eurogentech according to standard procedures. The affinity and/or specificity of the antibodies contained in the serum was further increased by affinity-purification over a CNBr-Sepharose column to which the peptide used for immunization was covalently bound.
In the experiment shown in
Antibodies as produced in this example are useful for the specific detection of Bcl10 cleavage, which serves as a readout for MALT1 activity, for purposes of research, drug development and diagnosis. With such antibodies, cleaved Bcl10 can for example be specifically detected in samples of lysates of lymphocytes that have been stimulated to mimic T-cell activation (see
The examples demonstrate that Malt1 has arginine-directed proteolytic activity and that an increase in this activity is specifically induced by T-cell activation. A C-terminal LRSR228 motif at the C-terminus of Bcl10 was identified as a Malt1 target sequence. Malt1-dependent Bcl10 cleavage is crucial for T-cell activation, since mutation of the Bcl10 cleavage site prevents Malt1-dependent Bcl10 processing and results in impaired TCR-induced T-cell adhesion. Moreover, inhibition of the proteolytic activity of Malt1 using a Malt1 inhibitor impairs various parameters of T-cell activation, including NF-κB activation, IL-2 production and T-cell adhesion. Importantly, processed Bcl10 is also detectable in various forms of B-cell lymphoma. Thus, Malt1 is essential for lymphocyte activation by cleavage of Bcl10 and of additional protein substrates, and therefore represents an interesting target of immuno-modulatory drug development.
| Number | Date | Country | Kind |
|---|---|---|---|
| 60932648 | Jun 2007 | US | national |
| 07112228.7 | Jul 2007 | EP | regional |
| 07123542.8 | Dec 2007 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/IB2008/052122 | 5/30/2008 | WO | 00 | 12/22/2009 |
