Patent Application
20080044815
a first fluorescent reporter gene cassette comprising the gfp gene encoding green fluorescent protein placed operably under control of a chimeric yeast operable LexA/GAL1 promoter having 8 LexA operator sites, and upstream of the yeast ADH1 terminator;
a second fluorescent reporter gene cassette comprising the cobA gene encoding a fluorescent protein placed operably under control of a chimeric cI/GAL1 promoter having 3 cI operator sites;
a wild-type yeast operable selectable marker gene (ADE2) for conferring adenine auxotrophy oh cells expressing said gene;
a selectable marker gene for conferring resistance to the antibiotic kanamycin in bacteria;
a bacterial origin of replication (colE1); and
a eukaryotic origin of replication (2 Ori).
a is a graphical representation showing mutation data from reverse two-hybrid screening, indicating the mutations identified in the 16 mutant JNK sequences. Mutations were calculated per region of JNK secondary structure and then normalized for the length of the secondary structure. Two regions were identified with 50% hits/length (#1 and #2), and point mutations were designed to address the importance of these regions (Leu-110-His and Val-219-Asp, respectively).
b is a diagrammatic representation showing four views of the JNK protein (i-iv) to illustrate all faces of the three-dimensional structure, with the positions of mutated amino acids shown in black JNK mutants containing 5 or less mutations per JNK sequence. Limitation of mutations to this level per molecule reduces background interference. This resulted in 27 identified amino acid mutations (Lys-Glu, Gln-102-Arg, Leu-110-His, Leu-110-Pro, Met-121-Lys, Asp-124-Tyr, Leu-131-Arg, Leu-131-Phe, Net-135-Lys, Lys-140-Glu, Lys-166-Glu, Tyr-190-His, Asn-205-Asp, Cys-213-Ser, Val-219-Asp, Glu-261-Lys, Asn-262-Ser, Leu-279-Pro, Asn-287-Tyr, Ser-292-Cys, Arg-309-Trp, Asp-313-Gly, Tyr-320-His, Asp-339-Tyr, Trp-352-Arg, Met-361-Val, Glu-365-Val). Note that Leu-110 and Leu-131 were mutated on two separate occasions. The positions of these mutated amino acids in JNK1 were mapped onto the crystal structure of the JNK3 protein.
a is a diagrammatic representation showing four views of the JNK protein (i-iv) to illustrate all faces of the three-dimensional structure, with the positions of single point mutations indicated and positions of mutated amino acids shown in black. Single point mutants define important residues on JNK for its interaction with TI-JIP. Point mutants of JNK were constructed by site-directed mutagenesis to assess the relative contribution of different hot-spots to the JNK-TI-JIP interaction. Amino acids located in putative mutational hot-spots were targeted for further investigation.
b is a representation of β-galactosidase overlay assay results (left) showing the ability of JNK mutants to interact with TI-JIP and Western blot assay data to detect the HA-tagged full length JNK1 mutant proteins (right). Of the nine point mutations tested, three point mutations (Leu-131-Arg, Arg-309-Trp, Tyr-320-His) rendered JNK incapable of interaction with TI-JIP. Western blotting was performed to ensure that the lack of interaction did not arise from problems associated with protein expression. Two independent colonies were tested for each mutation to confirm the results of the overlay assay and Western blotting.
a is a photographic representation showing expression of wild-type (WT) JNK and mutants (n=2) in transfected COS cells. The wild type JNK construct was pCMV-FLAG-JNK1. Equivalent constructs with point mutations corresponding to JNK1(Leu-131-Arg), JNK1(Arg-309-Trp) and JNK1(Tyr-320-His) were also used.
b is a representation showing a typical autoradiograph (upper panel) illustrating phosphorylation of GST-c-Jun(1-135) by wild-type (WT) JNK and mutants (n=2) for COS cells transfected as described in the legend to
c is a photographic representation showing a typical autoradiograph (upper panel) illustrating phosphorylation of GST-c-Jun(1-135) by wild-type (WT) JNK and mutants (n=2) for COS cells transfected as described in the legend to
a is a representation showing that JNK mutants were not activated by constitutively-active MKK4 (MKK4(ED)) or MKK7. COS cells were transfected with pCMV-FLAG-JNK1, or equivalent constructs with point mutations corresponding to JNK1(Leu-131-Arg), JNK1(Arg-309-Trp) and JNK1(Tyr-320-His). JNK proteins were immunoprecipitated from transfected cell lysates, and immunoprecipitates were used as the substrates in in vitro kinase assays with GST-MKK4(ED). Following separation by SDS-PAGE, activation of JNK and mutant proteins was assessed by autoradiography (upper panel) (n=2). Coomassie Blue staining (lower panel) confirmed substrate loading.
b is a representation showing that JNK mutants were not activated by constitutively-active MKK4 (MKK4(ED)) or MKK7. COS cells were transfected with either, JNK or mutant constructs alone, or co-transfected with pEBG-MKK7β1. Lysates were separated by SDS-PAGE and then transferred to nitrocellulose. Immunoblotting was performed using an antibody directed towards the dual-phosphorylated activated form of JNK to detect the amount of JNK activation stimulated by co-expressed MKK7 (upper panel) (n=2). Total JNK protein expression was assessed using antibodies directed against JNK1 and the FLAG epitope tag. The Tyr-320-His mutant consistently had a reduced SDS-PAGE mobility relative to wild-type JNK1, despite sequencing the construct to confirm its identity.
One aspect of the present invention provides a method for identifying the interaction interface between two protein binding partners. In one embodiment there is provided a method for identifying a region in a protein of interest that mediates the ability of the protein to bind to a binding partner protein in a protein complex that comprises more than two proteins, said method comprising expressing a mutated form of the protein of interest and the “native form of the binding partner protein and native forms of one or more other proteins that bind to the protein of interest such that the binding of the mutated form of the protein of interest to the native form of the binding partner protein and each other protein operably controls the expression of a different reporter gene, and selecting for modified expression of the reporter gene that is operably under the control of a binding between the protein of interest and the binding partner protein and unmodified expression of each other has reporter gene, wherein said modified expression indicates that the mutation is within a region in the protein of interest that mediates the ability of the protein to bind to the binding partner protein.
By “interaction interface” is meant the portion or region of one protein that is in close physical proximity or relation with another in a protein complex, such as, for example, a protein complex having a function in vivo. As will be known to those skilled in the art, an interaction interface will comprise one or more amino acid residues in one of the protein binding partners that are essential for such binding or interaction to occur and/or that mediate binding of one protein to another protein. The amino acid residues in the interaction interface may be contiguous or non-contiguous with respect to the primary structure (i.e., the amino acid sequence) of the protein.
Those skilled in the art will be aware that an interaction interface is useful in its isolated form as a dominant negative mutant to inhibit a protein-protein interaction. Accordingly, notwithstanding that an interaction interface may consist of a single amino acid residue, the term “interaction interface” shall be taken for practical purposes to encompass any peptides consisting of at least 5 contiguous amino acid residues in length derived from the amino acid sequence of a protein wherein said contiguous amino acid residues comprise one or more amino acid residues in the protein that are essential for binding of that protein to another protein, or mediate an interaction between that protein and another protein. Thus, an interaction interface includes amino acid residues flanking an amino acid residue that is required for binding in the primary structure of a protein.
It is to be understood that the “interaction interface” of a protein will not extend to any peptides consisting of or comprising an amino acid sequence of a full-length protein. In fact, an interaction interface will generally have an upper length of about 50 amino acid residues that are contiguous with the primary sequence of a protein. In a preferred embodiment, the interaction interface of a protein will comprise an amino acid sequence consisting of about 5-10 amino acid residues that are contiguous with the primary sequence of a protein, or about 15-20 contiguous amino acid residues in length or about 20-25 contiguous amino acid residues in length or about 25-30 contiguous amino acid residues in length.
Those skilled in the art will also understand that the term “protein binding partner” means a protein that is involved in a close physical relation or association with another protein in a protein complex. As used throughout this specification and in the claims unless the context requires otherwise, the term “protein binding partner” shall be taken to mean a specific proteinaceous species, including peptides and polypeptides that is involved in a close physical relation or association with a specified protein of interest.
The term “protein of interest” as used herein shall be taken to mean a protein species in which one or more amino acid residues that are essential for binding to the “protein binding partner” are being determined, or are the subject of a claim.
Preferably, a direct interaction between the protein of interest and the protein binding partner, or a direct interaction between a fusion protein comprising the protein of interest and a fusion protein comprising the protein binding partner, is sufficient to bind to the upstream region (5′-UTR) of a reporter gene and activate its expression. Alternatively, there may also be one or more additional proteins in the assay that bind, to the protein binding partner or to the protein of interest, to produce a functional protein complex that is capable of binding to and activating expression of a reporter gene.
As used herein, the “other protein” shall be taken to mean a protein that binds to a protein of interest and optionally to a protein binding partner of the protein of interest, the only requirement being that the other protein does not inhibit the interaction between the protein of interest and the protein binding partner such that said interaction is abrogated. In one embodiment, the other protein(s) will bind to a different site in the protein of interest to the interaction site between the protein of interest and the protein binding partner.
The interaction between the other protein and the protein of interest may be direct or indirect. In one embodiment, an “adaptor” protein or peptide can be included in the assay to mediate or enhance the interaction. For example, the protein of interest may comprise a DNA binding domain fusion between the GAL4 DNA or LexA operator binding domain of a transcription factor and an amino acid sequence that dimerizes with the adaptor polypeptide, whilst the other protein comprises an activation domain fusion between a transcriptional activator domain, such as the GAL4 activator domain, and an amino acid sequence that dimerizes with the adaptor protein. Alternatively, there may be direct interaction between the protein of interest and the other protein, without a requirement for an adaptor protein to facilitate their dimerization.
Moreover, because the “other protein” is included as an internal control for the correct conformation of the protein of interest, it is not necessary for the “other protein” to be a protein that forms part of a naturally occurring protein complex with both the protein of interest and the protein binding partner. For example, the protein of interest may interact with the protein binding partner under a specified environmental condition or at a particular stage of development that is different to the environmental/developmental milieu in which the protein of interest binds to the other protein(s). In this case, the method of the present invention will require an artificial combination in vitro of distinct protein complexes that occur in vivo. In an alternative embodiment, the protein of interest may interact with the protein binding partner in vivo under a specified environmental condition or at a particular stage of development that is the same as the environmental/developmental milieu in which the protein of interest binds to the other protein(s). In this case, the method of the present invention may require an artificial combination in vitro of distinct protein complexes that occur in vivo, or alternatively, rely upon the reconstitution in vitro of a protein complex that is known to occur in vivo.
In another preferred alternative embodiment, the ‘protein partner’ and the ‘other protein’ may represent two allelic or mutant forms of the same protein or even two orthologues of the protein encoded by the genomes of distinct species.
Fragments of a protein of interest, fragments of a protein binding partner, and fragments of the other protein(s) that retain the ability of the full-length protein to bind to another protein in the method of the present invention can also be used. Accordingly, the terms “protein of interest”, “protein binding partner” and “other protein” clearly encompass such functionally equivalent fragments. In fact, in many instances it is preferred to express such fragments, because gene, constructs for their expression are easier to produce than gene constructs expressing full-length proteins.
As used herein, the term “native form” with reference to a protein binding partner or other protein shall be taken to mean a full-length protein that has an amino, acid sequence corresponding to the sequence of a naturally-occurring isoform of the protein, or a fragment of the full-length protein.
It will be understood from the preceding description that the selection of a particular species of protein of interest, protein binding partner, and other protein, for use in the inventive method will vary according to the interaction interface being determined. In view of the general applicability of the present invention to determining any interaction interface, the only requirement being that the protein of interest is capable of binding to more than one protein or peptide, the present invention is not to be limited to particular species of proteins or peptides or a particular species of interaction.
Notwithstanding the preceding paragraph, several protein-protein interactions are described below for the purposes of exemplification of the invention. In one embodiment, the protein of interest is a MAP kinase protein, such as, for example, a stress-activated MAP kinase protein selected from the group consisting of a p38 protein, an SAPK protein, a JNK protein and an ERK protein.
The term “p38 protein” shall be taken to refer to a stress-activated serine/threonine protein kinase of mammals, such as, for example, a human, rat or mouse protein, belonging to the MAP kinase superfamily and having an estimated molecular mass of about 38 kDa. The term “p38” further encompasses proteins designated “CSBP” or “RK” or “p38 MAPK” or “SAPK-2” or an isoform of p38 selected from the group consisting of “p38-alpha”, “p38-beta”, “p38-gamma” and “p38-delta”. Those skilled in the art will readily be able to obtain and identify a p38 protein from the literature (see, eg., Cano and Mahadevan, Trends Biochem. Sci. 20, 117-122, 1995; Davis, Trends Biochem. Sci. 19, 470-473, 1994; Eyers et al., Chem and Biol 5, 321-328, 1995; Jiang et al, J Biol Chem 271, 17920-17926, 1996; Kumar et al, Biochem Biophys Res Comm 235, 533-538, 1997; Stein et al., J Biol Chem 272, 19509-19517, 1997; Li et al., Biochem Biophys Res Comm 228, 334-340, 1996; Wang et al., J Biol Chem 272, 23668-23674, 1997; Wang et al., J Biol Chem 273, 2161-2168, 1998; and the references cited therein). An exemplary human p38 amino acid sequence is provided by Han et al., Science 265, 808-811, 1994 or Lee et al., Nature 372, 739-746, 1994, and Bernd et al. U.S. al. U.S. Ser. No. 10/197,315 (Publication No. 20030059881) which are incorporated herein by reference. The term “p38” shall also be understood to encompass any variants of the sequences disclosed by Han et al., Science 265, 808-811, 1994 or Lee et al., Nature 372, 739-746, 1994, and Bernd et al. U.S. Ser. No. 10/197,315 (Publication No. 20030059881) which are functionally equivalent to a p38 protein as defined herein.
Diverse extracellular stimuli, including ultraviolet light, irradiation, heat shock, high osmotic stress, pro-inflammatory cytokines and certain mitogens, trigger a stress-regulated protein kinase cascade culminating in activation of p38 through phosphorylation on a TGY motif within the kinase activation loop (ie., residues Thr180 to Tyr182). The p38 protein appears to play a major role in apoptosis, cytokine production, transcriptional regulation, and cytoskeletal reorganization, and has been causally implicated in sepsis, ischemic heart disease, arthritis, human immunodeficiency virus infection, and Alzheimer's disease. The availability of specific inhibitors helps to clarify the role that p38 plays in these processes, and may ultimately offer therapeutic benefit for certain critically ill patients.
The terms “SAPK protein” or “JNK protein” shall be taken to refer to a stress-activated protein kinase of mammals, including but not limited to JNK1, JNK2, JNK3, an isoform of JNK1, JNK2 or JNK3 (Gutta et al., EMBO J., 1996, 15, 2760), or another member of the JNK family of proteins whether they function as Jun N-terminal kinases per se (that is, phosphorylate Jun at a specific amino terminally located position) or not. Preferred JNK proteins are capable of reversibly binding and phosphorylating the transcription factor cJun and/or the activator protein 1 (AP-1) transcription factor complex comprising c-Jun and/or c-Fos. SAPK/JNK effectively acts as a universal pivot point, with targets to both a ternary complex transcription factor (ELK-1) and activating transcription factor 2 (ATF-2). The ternary complex factor ELK-1, once activated by SAPK/JNK, leads to positive regulation of the c-Fos promoter resulting in increased expression of the c-Fos protein with concomitant increases in AP-1 levels. Targeting of ATF-2, which can form heterodimers with c-Jun, is another suitable route to initiate increases in AP-1 expression. Given the myriad of possibilities for activating AP-1, it is quite apparent that the SAPK/JNK is a model transduction junction for amplifying a given extracellular, signal. The SAPK/JNK proteins are encoded by at least three genes, and as with all MAPKs, each SAPK/JNK protein isoform contains a characteristic Thr-X-Tyr phospho-acceptor loop domain, where X indicates any amino acid structurally suitable for a loop domain.
An exemplary SAPK/JNK protein is described by Derijard et al Cell 76 (6), 1025-1037, 1994 which is incorporated herein by way of reference. For the purposes of nomenclature, the amino acid sequence of this JNK protein is set forth herein as SEQ ID NO: 1. Preferred JNK proteins will comprise an amino acid sequence that is at least about 70% identical to the sequence set forth in SEQ ID NO: 1.
The term “extracellular regulated protein kinase” or “MAP2 kinase” or “ERK” shall be taken to refer to a stress-activated protein kinase of mammals, including but not limited to a protein selected from the group consisting of ERK1, ERK2, ERK3, ERK4, an isoform of ERK1, ERK2, ERK3 or ERK4, or another member of the ERK/MAP-2 kinase family of proteins whether they function as MAP-2 kinases per se (that is, phosphorylate MAP-2) or not MAP-2 kinases or ERKs are generally expressed in the central nervous system, and comprise a phospho-acceptor sequence of Thr-Glu-Tyr, an amino-terminal kinase domain followed by an extensive carboxy-terminal tail of unknown function that comprises several proline-rich motifs indicative of binding sites with SH3 domains. The SH3 adaptor proteins are instrumental in linking the initial activation of the kinase to the downstream components of any signal transduction pathway. Although the stimuli that recruit ERK have not been well identified, environmental stresses such as osmotic shock and oxidant stress have been shown to substantially activate ERK and similar substrates.
The amino acid sequences of several ERK proteins are described by Boulton et al U.S. Ser. No. 6,297,035 and U.S. Ser. No. 6,303,358, which are incorporated herein by reference:
In accordance with this embodiment the protein binding partner and other protein(s) are proteins that bind to the MAP kinase protein, such as for example, a protein substrate of the MAP kinase. Such proteins will be known to those skilled in the art. Preferred protein binding partners and other proteins are selected from the group consisting of: These transcription factors include c-Jun (SEQ ID NO: 2), JIP2 (SEQ ID NO: 3), JunD (SEQ ID NO: 5), JunB (SEQ ID NO: 6), ATF-2 (SEQ ID NO: 7), CREB2 (SEQ ID NO: 8), Elk1 (SEQ ID NO: 9), NF-kappaB (SEQ ID NO: 10), human WOX3 (SEQ ID NO: 17), human WOX1 (SEQ ID NO: 18) and murine WOX1 (SEQ ID NO: 19). Other AP1 family proteins, such as, for example, v-Jun or Fas can also be used. MKK3 (Davis et al., U.S. Ser. No. 6,541,605), MKK4/SEK1 (Davis et al., U.S. Ser. No. 6,541,605), MKK7, a Bcl-2 family protein (eg., BIM), cdc47, and S6 kinase protein are also useful.
In a preferred embodiment, the method of the present invention is applied to the identification of an interaction interface in a JNK protein. In accordance with this embodiment, the protein of interest is a JNK protein, and the protein binding partner is a protein selected from the group consisting of an AP-1 family protein (eg p53, JunD, JunB, c-Jun, v-Jun, or Fas), and a fragment of an AP-1 family protein that interacts with JNK, and the other protein is a protein selected from the group consisting of ATF-2, Elk1, CREB, NF-kappaB and a WOX protein, a fragment of ATF-2 that interacts with JNK, a fragment of Elk1 that interacts with JNK, a fragment of CREB that interacts with JNK, a fragment of NF-kappaB that interacts with JNK and a fragment of a WOX protein that interacts with JNK.
Alternatively, wherein the protein of interest is a JNK protein, and the protein binding partner is a protein selected from the group consisting of ATF-2, Elk1, CREB; NF-kappaB, a fragment of ATF-2 that interacts with JNK, a fragment of Elk1 that interacts with JNK, a fragment of CREB that interacts with JNK, a fragment of NF-kappaB that interacts with JNK, a fragment of WOX1 that interacts with JNK and a fragment of WOX3 that interacts with JNK, and the other protein is a protein selected from the group consisting of an AP-1 family protein (eg p53, JunD, JunB, c-Jun, v-Jun, or Fas), and a fragment of an AP-1 family protein that interacts with JNK.
Other combinations of proteins for identifying the interaction site(s) of JNK are not to be excluded.
In an alternative embodiment, the protein of interest is the oncoprotein SCL or a dimerization region of SCL, and, the protein binding partner and other protein are selected from the group consisting of: LMO1, LMO2, DRG, mSin3A, E47, a dimerization region of LMO1, a dimerization region of LMO2, a dimerization region of DRG, a dimerization region of mSin3A, and a dimerization region of E47.
Preferably, the protein of interest, protein binding partner and other protein are presented in the inventive method as a fusion protein with the DNA binding domain (DBD) of a transcription factor or a transcription activator domain (AD). In accordance with this embodiment, those skilled in the art of hybrid screening approaches will be aware that two proteins that interact with each other are generally expressed separately as a fusion with a DBD and an AD. Similarly, in the present context, it is preferred that the protein of interest is expressed as a fusion protein with an AD and the protein binding partner and other protein are each expressed as fusion proteins with a different DBD to avoid inappropriate docking on the wrong reporter gene.
When the appropriate association between proteins occurs, a functional transcription factor is reconstituted, and expression of a reporter gene placed under the control of the reconstituted transcription factor occurs.
Preferred DNA binding domains include, for example, the GAL4 DNA binding domain or LexA DNA binding protein which binds to the lexA operator.
Preferred activation domains include, for example, the GAL4 activation domain, the VP16 activation domain, the mouse NF κB activation domain and fortuitous activation domains such as the B42 activation domain encoded by the E. coli genome.
Preferably, but not necessarily, each interaction will utilize a different DNA binding domain.
For example, fusion proteins may be constructed between an oncoprotein and a DNA binding domain and/or a DNA activation domain. For example, a sequence of nucleotides encoding or complementary to a sequence of nucleotides encoding $CL may be fused to a transcriptional activation domain and a nucleotide sequence encoding LMO1 may be fused to the LexA DNA binding domain while the E47 protein may be fused to the the CI DNA binding domain.
Alternatively, wherein the protein of interest is a transcription factor with an endogenous transcriptional activation domain, such as, for example, the Fos transcription factor that binds to JUN, expression of that protein as a fusion protein with a DNA binding domain or an activation domain may not be required, provided that the protein fused to an appropriate domain to enable it to bind to the upstream region of a promoter to which a reporter gene is linked and provided that the protein is able to activate expression of the reporter gene in the host organism of the screen such as yeast.
Mutated Form of a Protein of Interest
In a preferred embodiment, the present invention further comprises the step, of producing a mutated from of the protein of interest.
As used herein, the term “mutated form” with reference to a protein species shall be taken to mean a variant of the protein that comprises one or more amino acid substitutions, deletions or additions relative to the amino acid sequence of the native polypeptide. By “native polypeptide” is meant a form of a polypeptide that is functional in binding to a native form of a protein binding partner.
Those skilled in the art will be aware of several methods for producing a mutated form of a protein.
In one embodiment, the nucleotide sequence encoding the protein of interest is mutated by a process such that the encoded peptide, varies by one or more amino acids compared to the “template”-nucleic acid fragment. The “template” may have the same nucleotide sequence as the original nucleic acid fragment in its native context (ie. in the gene from which it was derived). Alternatively, the template may itself be an intermediate variant that differs from the original nucleic acid fragment as a consequence of mutagenesis. Mutations include at least one nucleotide difference compared to the sequence of the original fragment. This nucleic acid change may result in for example, a different amino acid in the encoded peptide, or the introduction or deletion of a stop codon. Mutations that introduce amino acid substitutions are preferred, however not essential to the present invention, because the screening process selects against or nonsense mutations.
In one embodiment, nucleic acid encoding the protein of interest or a fragment thereof is modified by a process of mutagenesis selected from the group consisting of, mutagenic PCR, replicating the nucleic acid in a bacterial cell that induces an accumulation of a random mutations through defects in DNA repair, by site directed mutagenesis, of by replicating the nucleic acid in a host cell exposed to a mutagenic agent such as for example radiation, bromo-deoxy-uridine (BrdU), ethylnitrosurea (ENU), ethylmethanesulfonate (EMS) hydroxylamine, or trimethyl phosphate. Alternatively, the nucleic acid can be exposed to the the mutagenic agent in vitro, prior to transformation.
In a preferred embodiment, the nucleic acid is modified by amplifying a nucleic acid fragment using mutagenic PCR. Such methods is include a process selected from the group consisting of: (i) performing the PCR reaction in the presence of manganese; and (ii) performing the PCR in the presence of a concentration of dNTPs sufficient to result in misincorporation of nucleotides.
Methods of inducing random mutations using PCR are well known in the art and are described, for example, in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995). Furthermore, commercially available kits for use in mutagenic PCR are obtainable, such as, for example, the Diversify PCR Random Mutagenesis Kit (Clontech) or the GeneMorph Random Mutagenesis Kit (Stratagene).
In one embodiment, PCR reactions are performed in the presence of at least about 200 μM manganese or a salt thereof, more preferably at least about 300 μM manganese or a salt thereof, or even more preferably at least about 500 μM or at least about 600 μM manganese or a salt thereof. Such concentrations manganese ion or a manganese salt induce from about 2 mutations per 1000 base pairs (bp) to about 10 mutations every 1000 bp of amplified nucleic acid (Leung et al Technique 1, 11-15, 1989).
In another embodiment, PCR reactions are performed in the presence of an elevated or increased or high concentration of dGTP. It is preferred that the concentration of dGTP is at least about 25 μM, or more preferably between about 50 μM and about 100 μM. Even more preferably the concentration of dGTP is between about 100 μM and about 150 μM, and still more preferably between about 150 μM and about 200 μM. Such high concentrations of dGTP result in the misincorporation of nucleotides into PCR products at a rate of between about 1 nucleotide and about 3 nucleotides every 1000 bp of amplified nucleic acid (Shafkhani et al BioTechniques 23, 304-306, 1997).
PCR-based mutagenesis is preferred for the mutation of the nucleic acid fragments of the present invention, as increased mutation rates is achieved by performing additional rounds of PCR.
In another preferred embodiment, the nucleic acid encoding the protein of interest is mutated by inserting said nucleic acid into a host cell that is capable of mutating nucleic acid. Such host cells are deficient in one or more enzymes, such as, for example, one or more recombination or DNA repair enzymes, thereby enhancing the rate of mutation to a rate that is rate approximately 5,000 to 10,000 times higher than for non-mutant cells. Strains particularly useful for the mutation of nucleic acids carry alleles that modify or inactivate components of the mismatch repair pathway. Examples of such alleles include-. alleles selected from the group consisting of mutY, mutM, mutD, mutt, mutA, mutC and mutS. Bacterial cells that carry alleles that modify or inactivate components of the mismatch repair pathway are well known in the art, such as, for example the XL-1Red, XL-mutS and XL-mutS-Kanr bacterial cells (Stratagene).
Alternatively the nucleic acid is cloned into a nucleic acid vector that is preferentially replicated in a bacterial cell by the repair polymerase, Pol I. By way of exemplification, a Pol I variant strain will induce a high level of mutations in the introduced nucleic acid vector. Such a method is described by Fabret et al (In: Nucl. Acid Res, 28, 1-5 2000), which is incorporated herein by reference.
In a further preferred embodiment, alanine scanning mutagenesis is carried out. Those skilled in the art will be aware that alanine scanning mutagenesis introduces substitutions of alanine residues in a protein for other amino acid residues. Commercially available methods and reagents are available for performing alanine scanning mutagenesis of nucleic acid encoding the protein of interest, such as, for example, by cloning said nucleic acid into a suitable expression vector e.g., pcDNA3.1 (Stratagene) and using the resulting recombinant vector with the Quickchange Mutagenesis kit supplied by Stratagene.
Preferably, mutagenesis is performed under conditions such that the coding region of the nucleic acid encoding the protein of interest is saturated with mutations across the mutant library, however each molecule that is mutated comprises only a single or a few mutations. Preferably, the, mutated nucleic acid should encode a variant or mutated form of the protein of interest that differs from the native form by less than about 5 amino acid substitutions and more preferably only 1 or 2 amino acid substitutions. Accordingly, a library of mutants is produced wherein the aligned sequences of the encoded proteins have mutations spanning the entire protein sequence.
Each mutant form of the protein of interest is then separately expressed with the native form of the protein binding partner and other protein. This is achieved, for example, by transformation of suitable host cells expressing the protein binding partner and other protein and containing nucleic” acid comprising each reporter gene with the library of mutants under conditions such that a single mutant sequence is introduced to each transformant.
Reporter Genes
As used herein, the term “reporter gene” shall be taken to mean a genomic gene, cDNA or other nucleic acid encoding a protein that is physically measurable or detectable, wherein the level of expression of the protein can be measured and/or correlated with a change in the binding activity between the protein of interest and the protein binding partner or between the protein of interest and the other protein(s). Reporter genes are well known in the art, and include, but are not limited to, nucleic acids encoding proteins that fluoresce, for example the red fluorescent protein (i.e, cobA gene product) or green fluorescence protein (i.e., the gfp gene product), nucleic acids encoding proteins that induce a colour change in the presence of a substrate, for example E coli β-galactosidase or LacZ or GusA, and nucleic acids encoding proteins that confer growth characteristics on a cell by (for example) complementing auxotrophic mutations (such as for example the HIS3 gene). Genes that confer resistance to an antibiotic (eg., ampicillin, kanamycin, G418, tetracycline, neomycin, etc), or other toxic chemical compound are also useful in this context.
Counter selectable reporter genes encode a lethal product when expressed in a cell, or alternatively, encode a protein or enzyme that converts a non-toxic substrate to a toxic product. Counter selectable reporter genes suitable for this purposes include, for example, the yeast URA3, structural gene which is lethal to yeast cells when expressed in the presence of 5-fluororotic acid. (5-FOA); the yeast CYH2 gene which is lethal when expressed in the presence of the drug cycloheximide; and the yeast LYS2 gene which is lethal in the presence of the drug αα-aminoadipate (α-AA). Those skilled in the art will be aware that reverse n-hybrid screens routinely employ such counter selectable reporter genes. (e.g, WO 99/35282).
The only requirement for a suitable reporter gene is the capability of being expressed in a manner that is readily detected, such as by the phenotype said expression confers on the cell (for, example, restoration of prototrophy for a particular nutrient by complementation, or conditional lethality in the presence of a particular substrate), or alternatively, by expressing an enzyme activity, or a protein detectable by immunoassay or colorimetric detection, or fluorescence.
Suitable reporter genes include those encoding Escherichia coli β-galactosidase enzyme, the firefly luciferase protein (Ow et al, Science 234:856-859, 1986; Thompson et al, Gene 103:171-177, 1991) the green fluorescent protein (Prasher et al, Gene 111:229-233, 1992; Chalfie et al, Science 263:802-805, 1994; Inouye and Tsuji, FEBS Letts 341:277-280, 1994; Cormack et al, Gene, 1996; Haas et al, Curr. Biol. 6:315-324, 1996; see also GenBank Accession No. U55762); and the red fluorescent proteins of Discosoma (Matz et al, Nature Biotechnology 17: 969-973, 1999) or Propionibacterium freudenreichii, (Wildt and Deuschle, Nature Biotechnology 17: 1175-1178, 1999). Additionally, the HIS3 gene (Larson et al. EMBO J. 15 (5):1021, 1996; Condorelli et al., Cancer Research 56:5113, 1996; Hsu et al., Mol. Cell. Biol. 11:3037, 1991; Osada et al., Proc. Natl. Acad. Sci. USA 92:9585, 1995) and LEU2 gene (Mahajan et al., Oncogene 12:2343, 1996), the GUSA and LYS2 genes (are also useful.
It will be apparent from the preceding description that each interaction in the inventive method (i.e., the interaction between the protein of interest and the protein binding partner, and each additional interaction between the protein of interest and each other protein), operably regulates the expression of a different reporter gene. The selection of suitable reporter genes will largely influence the manner in which the selection of modified expression of the reporter gene that is operably under the control of a binding between the protein of interest and the binding partner protein and modified or unmodified expression of each other reporter gene is performed.
In a preferred embodiment, the reporter gene that is operably under the control of the interaction between the protein of interest and the protein binding partner is a counter selectable reporter gene, preferably a counter selectable reporter gene selected from the group consisting of URA3, CYH2 and LYS2. In accordance with this embodiment, modified expression of the reporter gene is carried out under conditions such that cells expressing the reporter gene do not survive selection on 5-FOA (in the case of URA3), or cycloheximide (in the case of CYH2) or α-AA (in the case of LYS2). Also in accordance with this embodiment, the reporter gene(s) placed operably the control of the interaction(s) between the protein of interest and the other protein(s) will be a reporter gene other, than the aforementioned counter selectable reporter gene, since those interactions are to be maintained.
It will be apparent to those skilled in the art that a reporter gene other than a counter selectable reporter gene can also be used for detecting the interaction between the protein of interest and the protein binding partner, since reduced expression of a reporter gene when the interaction is abrogated is generally detectable using such systems.
In a particularly preferred embodiment, the reporter gene/s operably under the control of the interaction between the protein of interest and the protein binding partner is at least one a counter selectable reporter gene selected from the group consisting of URA3, CYH2 and LYS2, or a gene encoding a fluorescent protein such as GFP, and the reporter gene(s) placed operably the control of the interaction(s) between the protein of interest and the other protein(s) is selected from the group consisting of LYS2 and cobA. In accordance with this embodiment, modified expression of the reporter gene is carried out under conditions such that cells expressing the reporter gene do not survive selection on 5-FOA (in the case of URA3), or cycloheximide (in the case of CYH2) or α-AA (in the case of LYS2), however cells in which the interaction between the protein of interest and the other protein(s) is maintained are selected by their ability to fluoresce at an appropriate wavelength (in the case, of fluorescent reporters) or grow in media lacking a certain nutrient such as lysine or leucine.
Combinations of a counter selectable reporter gene with one or more genes that encode fluorescent proteins are particularly preferred for high throughput applications, where large numbers of samples are screened in batches. By virtue of the phenotype that counter selectable reporter genes produce on a cell, they are particularly preferred for rapidly eliminating background in which the interaction between the protein of interest and the protein binding partner is not abrogated. Additionally, fluorescence generated from fluorescent proteins is readily assayed by fluorometry or fluorescence activated cell sorting (FACS), a technique known to those skilled in the art.
The expression of multiple reporter genes can also be placed operably under the control of the interaction between the protein of, interest and the protein binding partner, to reduce background effects and the selection of “false positives” in the screening process. Preferably, such multiple reporter genes will include at least one counter selectable reporter gene and at least one gene encoding a fluorescent protein.
Persons skilled in the art will be aware of how to utilize reporter genes in performing the invention described herein, without undue experimentation. For example, the coding sequence of the gene encoding such a reporter molecule may be modified for use in the cell line of interest (e.g. human cells, yeast cells) in accordance with known codon usage preferences. Additionally the translational efficiency of mRNA derived from non-eukaryotic sources may be improved by mutating the corresponding gene sequence or otherwise introducing to said gene sequence a Kozak consensus translation initiation site (Kozak, Nucleic Acids Res. 15: 8125-8148, 1987). Likewise the promoter sequences controlling expression from the reporter genes may be modified to minimise background expression and to put them more tightly under the control of factors binding to introduced exogenous elements such as lexA operators.
Expression of Proteins and Reporter Genes
Expression of the protein of interest, protein binding partner, other protein(s) and reporter genes, requires nucleic acid encoding each protein and nucleic acid comprising each reporter gene to be placed operably in connection with a promoter sequence.
Reference herein to a “promoter”. is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical genomic gene, including the TATA box which is required for accurate transcription initiation in eukaryotic cells, with or without a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers). Promoters may also be lacking a TATA box motif, however comprise one or more “initiator elements” or, as in the case of yeast-derived promoter sequences, comprise one or more “upstream activator sequences” or “UAS” elements. For expression in prokaryotic cells such as, for example, bacteria, the promoter should at least contain the −35 box and −10 box sequences.
A promoter is usually, positioned upstream or 5′ of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within about 2 kb of the start site of transcription of the gene.
In the present context, the term “promoter” is also used to describe a synthetic or fusion molecule, or derivative that confers, activates or enhances expression of the subject reporter molecule in a cell.
Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the gene and/or to alter the spatial expression and/or temporal expression. For example, regulatory elements which facilitate the enhanced expression of a gene by galactose or glucose or copper may be placed adjacent to a heterologous promoter sequence driving expression of the gene. Promoters comprising regulatory elements of the GALL or CUP1 promoters are particularly preferred for titration of the, expression of one or more proteins in response to galactose or copper, respectively, in the culture medium in which the host cell is grown.
Suitable promoters also include those from genes that are induced by the absence of a nutrient, for example the PHO5 gene is induced by a reduction in the amount of phosphate in the media in which a cell is cultured.
Placing a gene operably under the control of a promoter sequence means positioning the said gene such that its expression is controlled by the promoter sequence. Promoters are generally positioned 5′ (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
Examples of promoters suitable for use in regulating expression of the protein of interest or the protein binding partner or the other protein include viral, fungal, yeast, insect, animal and plant promoters, especially those that can confer expression in a eukaryotic cell, such as, for example, a yeast cell or a mammalian cell.
Those skilled in the art will recognise that the choice of promoter will depend upon the nature of the cell being transformed and the molecule to be expressed. Such persons will be readily capable of determining functional combinations of minimum promoter sequences and operators for cell types in which the inventive method is performed.
Whilst the invention can be performed in yeast cells, the inventors clearly contemplate modifications wherein the invention is performed entirely in bacterial or mammalian cells or in non-cellular systems (e.g., ribosome display, mRNA display or covalent display), utilizing appropriate promoters that are operable therein to drive express ion of the various assay components under such conditions. Such embodiments are within the ken of those skilled in the art.
In a particularly preferred embodiment, the promoter is a yeast promoter, mammalian promoter, a bacterial or bacteriophage promoter, selected from the group consisting of: MYC, GAL1, CUP1, PGK1, ADH1, ADH2, PHO4, PHO5, HIS4, HIS5, TEF1, PRB1, TDH1, GUT1, SPO13, CMV, SV40, LAC, TEF, EM7, SV40, and T7 promoter sequences. Suitable yeast promoters are known to those skilled in the art and a re listed in standard manuals such as Guthrie and Fink (In: Guide to Yeast Genetics and Molecular and Cell Biology Academic Press, ISBN 01 21822540, 2002).
Typical promoters suitable for expression in viruses of bacterial cells and bacterial cells such as for example a bacterial cell selected from the group comprising E. coli, Staphylococcus sp, Corynebacterium sp., Salmonella sp., Bacillus sp., and Pseudomonas sp., include, but are not limited to, the lacz promoter, the Ipp promoter, temperature-sensitive λL or λR promoters, T7 promoter, T3 promoter, SP6 promoter or semi-artificial promoters such as the IPTG-inducible tac promoter or lacUV5 promoter. A number of other systems for obtaining expression in bacterial cells are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), U.S. Pat. No. 5,763,239 (Diversa Corporation) and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, S. cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
Typical promoters suitable for expression in insect cells, or in insects, include, but are not limited to, the OPEI2 promoter, the insect actin promoter isolated from Bombyx muri, the Drosophila sp. dsh promoter (Marsh et al Hum. Mol. Genet. 9, 13-25, 2000) and the inducible metallothionein promoter. Preferred insect cells for expression of the recombinant polypeptides include an insect cell selected from the group comprising, BT1-TN-5B1-4 cells, and Spodoptera frugiperda cells (eg., sf19 cells, sf21 cells). Suitable insects for the expression of the nucleic acid fragments include but are not limited to Drosophila sp. The use of S. frugiperda is also contemplated.
Promoters for expressing peptides in plant cells are known in the art, and include, but are not limited to, the Hordeum vulgare amylase gene promoter, the cauliflower mosaic virus 35S promoter, the nopaline synthase (NOS) gene promoter, and the auxin inducible plant promoters P1 and P2.
Typical promoters suitable for expression in a virus of a mammalian cell, or in a mammalian cell, mammalian tissue or intact mammal include, for example a promoter selected from the group consisting of, retroviral LTR elements, the SV40 early promoter, the SV40 late promoter, the cytomegalovirus (CMV) promoter, the CMV IE (cytomegalovirus immediate early) promoter, the EF1α promoter (from human elongation factor la), the EM7 promoter, the UbC promoter (from human ubiquitin C).
As will be known to the skilled artisan, the promoter can also be positioned in the expression vector or gene construct into which the prokaryote or eukaryote nucleic acid fragment is inserted.
In one embodiment, the proteins and reporter genes are expressed in vitro. According to this embodiment, a gene construct is produced that comprises a protein-encoding nucleic acid (“open reading frame” or “ORF”) and a promoter sequence and appropriate ribosome binding site which can both be present in the expression vector or added to said nucleic acid before it is inserted into the vector. Typical promoters for the in vitro expression include, but are not limited to the T3 or T7 (Hanes and Pluckthun Proc. Natl. Acad. Sci. USA, 94 4937-4942 1997) bacteriophage promoters.
In another embodiment, the gene construct optionally comprises a transcriptional termination site and/or a translational termination codon. Such sequences are well known in the, art, and is incorporated into oligonucleotides used to amplify the ORF of a reporter gene or an ORF encoding the protein of interest, protein binding partner, or other protein. Alternatively, a transcriptional termination site and/or a translational termination codon can be present in the expression vector or gene construct before the nucleic acid is inserted.
In another embodiment, the ORF is cloned into an expression vector. The term “expression vector” refers to a nucleic acid molecule that has the ability confer expression of nucleic acid to which it is operably connected, in a cell or in a cell free expression system.
Within the context of the present invention, it is to be understood that an expression vector may comprise a promoter as defined herein, a plasmid, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment, or other nucleic acid capable of maintaining and or replicating heterologous DNA in an expressible format. Many expression vectors are commercially available for expression in a variety of cells. Selection of appropriate vectors is within the knowledge of those having skill in the art.
Typical expression vectors for in vitro expression or cell-free expression have been described and include, but are not limited to the TNT T7 and TNT T3 systems (Promega), the pEXP1-DEST and pEXP2-DEST vectors (Invitrogen).
Numerous expression vectors for expression of recombinant polypeptides in bacterial cells and efficient ribosome binding sites have been described, such as for example, PKC30 (Shimatake and Rosenberg, Nature, 292, 128, 1981); pKK173-3 (Amann and Brosius, Gene 40, 183, 1985), pET-3 (Studier and Moffat, J. Mol. Biol. 189, 113, 1986); the pCR vector suite (Invitrogen), pGEM-T Easy vectors (Promega), the pL expression vector suite (Invitrogen) the pBAD/TOPO or pBAD/thio—TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen, Carlsbad, Calif.), the latter of which is designed to also produce fusion proteins with a Trx loop for conformational constraint of the expressed protein; the pFLEX series of expression vectors (Pfizer nc., CT, USA); the pQE series of expression vectors (QIAGEN, CA, USA), or the pL series of expression vectors (Invitrogen), amongst others.
Expression vectors for expression in yeast cells are preferred and include, but are not limited to, the pACT vector (Clontech), the pDBleu-X vector, the pPIC vector suite (Invitrogen), the pGAPZ vector suite (Invitrogen), the pHYB vector (Invitrogen), the pYD1 vector (Invitrogen), and the pNMT1, pNMT41, pNMT81 TOPO vectors (Invitrogen), the pPC86-Y vector (Invitrogen), the pRH series of vectors (Invitrogen), pYESTrp series of vectors (Invitrogen). Particularly preferred vectors are the pACT vector, pDBleu-X vector, the pHYB vector, pJG4-5, pGilda, pEG202, the pPC86 vector, the pRH vector and the pYES vectors, which are all of use in various ‘n’-hybrid assays described herein. Furthermore, the pYD1 vector is particularly useful in yeast display experiments in S. cerevesiae. A number of other gene construct systems for expressing the nucleic acid fragment of the invention in yeast cells are well-known in the art and are described for example, in Giga-Hama and Kumagai (In: Foreign Gene Expression in Fission Yeast: Schizosaccharomyces Pombe, Springer Verlag, ISBN 3540632700, 1997) and Guthrie and Fink (In: Guide to Yeast Genetics and Molecular and Cell Biology Academic Press, ISBN 0121822540, 2002).
A variety of suitable expression vectors, containing suitable promoters and regulatory sequences for expression in insect cells are well known in the art, and include, but are not limited to the pAC5 vector, the pDS47 vector, the pMT vector suite (Invitrogen) and the pIB vector suite (Invitrogen).
Furthermore, expression vector's comprising promoters and regulatory sequences for expression of polypeptides in plant cells are also well known in the art and include, for example, a promoter selected from the group, pSS, pB1121 (Clontech), pZ01502, and pPCV701 (Kuncz et al, Proc. Natl. Acad. Sci. USA, 84 131-135, 1987).
Expression vectors that contain suitable promoter sequences for expression in mammalian cells or mammals include, but are not limited to, the pcDNA vector suite supplied by Invitrogen, the pCI vector suite (Promega), the pCMV vector suite (Clontech), the pM vector (Clontech), the pSI vector (Promega), the VP16 vector (Clontech) and the pDISPLAY vectors (Invitrogen). The pDISPLAY vectors are of particular use in mammalian display studies with the expressed nucleic acid fragment targeted to the cell surface with, the Igκ leader sequence, and bound to the membrane of the cell through fusion to the PDGFR transmembrane domain. The pM and VP16 vectors are of particular use in mammalian two-hybrid studies.
In a particularly preferred embodiment, the expression vector is selected from the group consisting of pDEATH-Trp, (SEQ ID NO: 10), pJFK (SEQ ID NO: 11), pDD (SEQ ID NO: 12), pRT2 (SEQ ID NO: 13), pGMS19 (SEQ ID NO: 15) and pDR10 (SEQ ID NO: 16). These vectors are described in more detail in the figure legends.
Alternatively, or in addition the pGILDA vector described in WO99/35282 can also be used.
Methods of cloning DNA into nucleic acid vectors for expression of encoded polypeptides are well known in the art and are described for example in, Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) or Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
It is preferred that when the gene constructs are to be introduced to and/or maintained and/or propagated and/or expressed in bacterial cells, either during generation of said gene constructs, or screening of said gene constructs, that the gene constructs contain an origin of replication that is operable at least in a bacterial cell. A particularly preferred origin of replication is the ColE1 origin of replication. A number, of gene construct systems containing origins of replication are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), U.S. Pat. No. 5,763,239 (Diversa Corporation) and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
It is also preferred that when the gene constructs are to be introduced to and/or maintained and/or propagated and/or expressed in yeast cells, either during generation of said gene constructs, or screening of said gene constructs, that the gene constructs contain an origin of replication that is operable at least in a yeast cell. One preferred origin of replication is the CEN/ARS4 origin of replication. Another particularly preferred origin of replication is the 2-micron origin of replication. A number of gene construct systems containing origins of replication are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987) and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
Gene constructs will preferably comprise a selectable marker. As used herein the term “selectable marker” shall be taken to mean a protein or peptide that confers a phenotype on a cell expressing said selectable marker that is not shown by those cells that do not carry said selectable marker. Examples of selectable markers include, but are not limited to the dhfr resistance gene, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); the gpt resistance gene, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); the neomycin phosphotransferase gene, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and the hygromycin resistance gene (Santerre, et al., 1984, Gene 30:147). Alternatively, marker genes is catalyse reactions resulting in a visible outcome (for example the production of a blue color when β galactosidase is expressed in the presence of the substrate molecule 5-bromo-4-chloro-3-indoyl-β-D-galactoside) or confer the ability to synthesise particular amino acids (for example the HIS3 gene confers the ability to synthesize histidine).
Recombinant gene constructs capable of expressing the protein of interest, protein binding partner, other protein or reporter gene product are introduced to and preferably expressed within a cellular host or organism. Methods of introducing the gene constructs into a cell or organism for expression are well known to those skilled in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), U.S. Pat. No. 5,763,239 (Diversa Corporation) and (Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001). The method chosen to introduce the gene construct in depends upon the cell type in which the gene construct is to be expressed.
In one embodiment, the cellular host is a bacterial cell. Means for introducing recombinant DNA into bacterial cells include, but are not limited to electroporation or chemical transformation into cells previously treated to allow for said transformation.
In another embodiment, the cellular host is a yeast cell. Means for introducing recombinant DNA into yeast cells include a method chosen from the group consisting of electroporation, and PEG mediated transformation.
In another embodiment, the cellular host is a plant cell. Means for introducing recombinant DNA into plant cells include a method selected from the group consisting of Agrobacterium mediated transformation, electroporation of protoplasts, PEG mediated transformation of protoplasts, particle mediated bombardment of plant tissues, and microinjection of plant cells or protoplasts.
In yet another embodiment, the cellular host is an insect cell. Means for introducing recombinant DNA into plant cells include a method chosen from the group consisting of, infection with baculovirus and transfection mediated with liposomes such as by using cellfectin (Invitrogen).
In yet another embodiment, the cellular host is a mammalian cell. Means for introducing recombinant DNA into mammalian cells include a means selected from the group comprising microinjection, transfection mediated by DEAE-dextran, transfection mediated by calcium phosphate, transfection mediated by liposomes such as by using Lipofectamine (Invitrogen) and/or cellfectin (Invitrogen), PEG mediated DNA uptake, electroporation, transduction by Adenoviuses, Adeno-associated viruses, Papilloma viruses, Lenti-viruses, Herpesviruses, Togaviruses or Retroviruses and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agacetus Inc., WI, USA).
Suitable prokaryotic cells for expression include corynebacterium, salmonella, Eicherichia coli, Bacillus sp. and Pseudomonas sp, amongst others. Bacterial strains which are suitable for the present purpose are known in the art (Ausubel et al, 1987; Sambrook et al, 2001).
Preferred mammalian cells for expression of the nucleic acid fragments include epithelial cells, fibroblasts, kidney cells, T cells, or erythroid cells, including a cell line selected from the group consisting of COS, CHO, murine 10T, MEF, NIH3T3, MDA-MB-231, MDCK, HeLa, K562, HEK 293 and 293T. The use of neoplastic cells, such as, for example, leukemic/leukemia cells, is contemplated herein.
Preferred mammals for expression of the nucleic acid fragments include, but are not limited to mice (ie., Mus sp.) and rats (ie., Rattus sp.).
The nucleic acid encoding the protein of interest, protein, binding partner, other protein or comprising a reporter gene can also be expressed in the cells of other organisms, or entire organisms including, for example, nematodes (eg C. elegans) and fish (eg D. rerio, and T. rubnipes). Promoters for use in nematodes include, but are not limited to osm-10 (Faber et al Proc. Natl. Acad. Sci. USA 96, 179-184, 1999), unc-54 and myo-2 (Satyal et al Proc. Natl. Acad. Sci. USA, 97 5750-5755, 2000). Promoters for use in fish include, but are not limited to the zebrafish OMP promoter, the GAP43 promoter, and serotonin-N-acetyl transferase gene regulatory regions
Placing the expression of a reporter genes operably under the control of an interaction To link reporter gene expression to a protein interaction, the protein of interest, the protein binding partner and any other protein must be expressed at the protein level, as described herein above. Additionally, the reporter gene must be operably linked to a suitable, promoter such that it is capable of being expressed to confer a detectable phenotype. Additionally, the expression of the reporter gene must be capable of being activated, by the binding of one protein to the upstream region of the reporter gene. (5′-UTR) and the interaction of that protein with its cognate binding partner.
Preferred promoters for driving reporter gene expression include those naturally-occurring and synthetic promoters which contain binding sites for transcription factors, more preferably for helix-loop-helix (HLH) transcription factors, zinc finger proteins, leucine zipper proteins and the like. Preferred promoters may also be synthetic sequences comprising one or more upstream operator sequences such as, for example, LexA operator sequences or activating sequences derived from any of the promoters referred to herein such as, for example, GAL4 DNA binding sites. Any of the promoters referred to supra are also suitable for driving reporter gene expression provided that they either naturally contain a suitable cis-acting regulatory sequence to which the protein of interest or the protein binding partner of the other protein can bind, or alternatively, have been engineered to contain such a site.
Preferably, the cis-acting sequence is selected from the group consisting of: LexA operator, GAL4 binding site, and cI operator. In accordance with this embodiment of the invention, it is preferred for the protein of interest or the protein binding partner or the other protein or a fusion protein comprising same to include a DNA binding domain capable of binding to said cis-acting sequence, in which case said DNA binding domain will be selected from the group consisting of: LexA operator binding domain, GAL-4 DNA binding domain; and cI operator binding domain, respectively.
Reporter genes are configured as described supra in a suitable gene construct. Suitably configured reporter genes are then introduced into a cellular host as described.
Host cells capable of expressing the variant protein of interest, and the native forms of the protein binding partner and other protein, and comprising the reporter genes necessary to perform the invention, are grown under conditions sufficient to enable the native form of the protein of interest to associate with the native form of the protein binding partner, and other protein. Conditions will also be selected that facilitate expression of the reporter genes, such as, for example, growth on a suitable media
The association of the variant protein of interest and the protein binding partner will reconstitute an active transcription factor that is capable of activating or enhancing expression of a reporter gene to which either protein docks. Similarly, the association of the variant protein of interest and the other protein will reconstitute an active transcription factor that is capable of activating or enhancing expression of a reporter gene to which either protein docks.
If both reporter genes are activated or enhanced then the mutation in the variant protein of interest is not within the interaction site of the protein of interest with either the protein binding partner or the other protein.
Conversely, if there is no expression of either reporter gene, then the mutation in the variant protein of interest is either a missense mutation encoding an allosteric change in conformation or a nonsense mutation introducing a STOP codon, or within the interaction site of the protein, of interest with both the protein binding partner and the other protein (i.e., the binding sites in the protein of interest for both proteins are either the same, contiguous, or overlap). In either case, such a phenotype is not useful unless the intention is to isolate allosteric mutants defining vulnerable residues to attack in screens for allosteric inhibitors.
In a preferred embodiment, there is expression of only one of the reporter genes, indicating that the mutation in the variant protein of interest is within the interaction site of the protein of interest with either the protein binding partner or the other protein. Accordingly, it is therefore possible to select for expression of a single reporter gene as being indicative that the mutation is within an appropriate binding site. This is made possible by the fact that formation of the different protein-protein interactions are distinguished by virtue of the operable, connection of the target interaction and the non-target interaction to distinct reporter genes, which can be assayed separately or simultaneously, depending upon the reporter genes used.
For example, distinct counter selectable reporter genes can be used, in which case the interactions can be distinguished by survival or growth of cells on particular substrates. In this respect, it is possible to distinguish between an interaction that is operably linked to both URA3 and CYH2 genes, and an interaction linked to the LYS2 gene. Cells in which an interaction is linked to expression of both URA3 and CYH2 genes are detectable, because they are resistant to fluororotic acid (5-FOA) and cycloheximide, and if those cells do not express LYS2, they will not require lysine for growth and/or are sensitive to growth on media containing α-aminoadipate (α-AA).
Similarly, it is possible to distinguish between interactions operably linked to distinct fluorescent protein-encoding reporter genes, by virtue of detecting the different emission wavelengths of the expressed proteins.
Selection of Cells
In accordance with the invention, cells expressing the variant protein of interest, protein binding partner and other protein, and expressing the reporter gene(s) operably connected to the interaction between the protein of interest and the other protein(s), but not expressing the reporter gene operably connected to the interaction between the protein of interest and protein binding partner or having a reduced level of expression thereof, are selected. In such cells, the interaction between the variant protein of interest and the protein binding partner is abrogated, whereas the interaction between the variant protein of interest and the other protein is not. Accordingly, the variant protein of interest will carry an informative mutation in the interaction interface, because it retains the ability of the native protein to interact with the other protein.
Selection of such cells will depend upon the reporter genes used, and can be readily performed using art-recognized procedures. Similarly, culture methods for growing bacterial yeast, or mammalian cells are well-known in the art.
In an alternative preferred embodiment of the invention, where the intention is to discover mutations which cause allosteric changes in folding of the target, cells are, selected and screened for mutations which reduce expression of reporter genes linked to both of the target interactions. Mutant proteins isolated from these yeast will then be expressed and assayed by Western blotting to ensure that the mutations isolated did not unduly effect efficient translation or stability of the protein.
A second aspect of the present invention provides a method for determining an inhibitor of an interaction between a protein of interest and a protein binding partner in a cell, said method comprising:
expressing a mutated form of the protein of interest and the native form of the binding partner protein and native forms of one or more other proteins that bind to the protein of interest such that the binding of the mutated form of the protein of interest to the native form of the binding partner protein and each other protein operably controls the expression of a different reporter gene, and selecting for modified expression of the reporter gene that is operably under the control of a binding between the protein of interest and the binding partner protein and unmodified expression of each other reporter gene, wherein said modified expression indicates that the mutation is within a region in the protein of interest that mediates the ability of the protein to bind to the binding partner protein;
determining a fragment of the mutated form of the protein of interest said fragment comprising the region that mediates the ability of the protein to bind to the binding partner protein; and
determining a fragment in the native form of the protein of interest that is functionally equivalent to (b) wherein said fragment inhibits the interaction between the native form of the protein of interest and the binding partner.
Further steps available to those skilled in the art include the modelling of the position of the critical mutated residues in the tertiary structure of the target protein of interest, if the structure of this protein (or a closely related family member or orthologue) has been solved by standard structural techniques such as X-ray crystallography or Nuclear Magnetic Resonance Spectroscopy.
By “determining a fragment of the mutated form of the protein of interest” is meant that the variant form of the protein is recovered following selection and analysed to determine the nature of the mutation, such as, for example, by determining the nucleotide sequence of the. ORF that encodes it. Naturally, this will involve a comparison with the native nucleotide sequence. In such comparisons or alignments, differences will arise in the positioning of non-identical residues arising from insertion/deletions in the variant, depending upon the algorithm used to perform the alignment. Preferably, such alignments are made using software of the Computer Genetics Group, Inc., University Research Park, Maddison, Wis., United States of America, eg., using the GAP program of Devereaux et al., Nucl. Acids Res. 12, 387-395, 1984, which utilizes the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 443-453, 1970. Alternatively, the CLUSTAL W algorithm of Thompson et al., Nucl. Acids Res. 22, 4673-4680, 1994, is used to obtain an alignment of multiple sequences, wherein it is necessary or desirable to maximize the number of identical/similar residues and to minimize the number and/or length of sequence gaps in the alignment. Alignments can also be performed using a variety of other commercially available sequence analysis programs, such as, for example, the BLAST program available at NCBI.
Preferably, the sequences of several distinct variants of the protein of interest identified in a specific screen are aligned and compared, and more frequently-occurring alleles are determined. Alternatively, or in addition, less frequently-occurring alleles.
Additionally, determination of the length of the encoded variant protein, immunogenic cross-reactivity with the native protein, or a determination of the tertiary or quarternary structure of the variant protein can also be performed to obtain information on the nature and effect of the mutation. Such procedures are well within the ability of the skilled person and can be performed without undue experimentation.
By “determining a fragment in the native form of the protein of interest” is meant that an amino acid sequence in the native protein that encompasses all or part of the mutated site is identified. Such fragments are preferably short, comprising no more than about 50 amino acid residues and preferably no more than about 30 or 20 or 15 or 10 or 5 amino acid residues in length.
As will be apparent to the skilled person, preferred fragments of the native protein will retain the ability to bind to the protein binding partner and thereby have utility as an inhibitor or antagonist of the interaction between the protein of interest and the protein binding partner. Moreover, because such fragments are derived from the interaction site between those two proteins, they are highly specific and preferably do not adversely affect the interaction of the protein of interest with the other protein in vivo or in vitro.
Preferably, based upon the amino acid sequence of the determined fragment of the wild-type or native protein of interest, a peptide consisting of that sequence is synthesized using standard Fmoc/Boc chemistry as described in one or more of the following: J. F. Ramalho Ortigão, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in The Peptides (Gross, E. and Meienhofer, J. eds.), vol. 2, pp. 1-284, Academic Press, New York; Wünsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Müler, E., ed.), vol. 15, 4th edn., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474.
The recovered peptide comprising an interaction interface can be used to validate a therapeutic target (ie. it is used as a target validation reagent). By virtue of its ability to bind to a specific protein, it is well within the ken of a skill artisan to determine the in vivo effect of modulating the activity of the protein by expressing the identified peptide or protein domain in an organism (eg., a bacterium, plant or animal such as, for example, an experimental animal or a human). In accordance with this aspect of the present invention, a phenotype of an organism that expresses the identified peptide or protein domain is compared to a phenotype of an otherwise isogenic organism (ie. an organism of the same species or strain and comprising a substantially identical genotype however does not express the peptide). This is performed under conditions sufficient to induce the phenotype that involves the target protein or target nucleic acid. The ability of the peptide or protein domain to specifically prevent expression of the phenotype, preferably without undesirable or pleiotropic side-effects indicates that the target protein is a suitable target for development of therapeutic/prophylactic reagents.
Accordingly, a third aspect of the present invention provides a method for determining or, validating a protein interaction as a therapeutic drug target or validation reagent comprising:
expressing a mutated form of a protein of interest and the native form of a binding partner protein and native forms of one or more other proteins that bind to the protein of interest such that the binding of the mutated form of the protein of interest to the native form of the binding partner protein and each other protein operably controls the expression of a different reporter gene, and selecting for modified expression, of the reporter gene that is operably under the control of a binding between the protein of interest and the binding partner protein and unmodified expression of each other reporter gene, wherein said modified expression indicates that the mutation is within a region in the protein of interest that mediates the ability of the protein to bind to the binding partner protein;
determining a fragment of the mutated form of the protein of interest said fragment comprising the region that mediates the ability of the protein to bind to the binding partner protein;
determining a fragment in the native form of the protein of interest that is functionally equivalent to (b) wherein said fragment inhibits the interaction between the native form of the protein of interest and the binding partner; and
(d) expressing the fragment at (c) in a cell or organism and determining a phenotype of the cell or organism that is modulated by the target protein or target nucleic acid wherein a modified phenotype of the cell or organism indicates that the protein interaction is a therapeutic target or validation reagent.
Preferably, determining a phenotype of the organism that is modulated comprises comparing the organism to an otherwise isogenic organism that does not express the selected fragment. For example, the phenotype of an organism that expresses a tumor is assayed in the presence and absence of a peptide or protein domain that blocks an interaction between SCL and E47 in a screen of the expression library of the invention. Amelioration of the oncogenic phenotype by the expressed peptide indicates that the SCL/E47 is a suitable target for intervention, wherein the peptide is then suitably formulated for therapeutic intervention directly, or alternatively, small molecules are identified that are mimetics of the identified peptide or protein domain.
A fourth aspect of the present invention provides a method for identifying a therapeutic or prophylactic compound comprising:
expressing a mutated form of a protein of interest and the native form of a binding partner protein and native forms of one or more other proteins that bind to the protein of interest such that the binding of the mutated form of the protein of interest to the native form of the binding partner protein and each other protein operably controls the expression of a different reporter gene, and selecting for modified expression of the reporter gene that is operably under the control of a binding between the protein of interest and the binding partner protein and unmodified expression of each other reporter gene, wherein said modified expression indicates that the mutation is within, a region in the protein of interest that mediates the ability of the protein to bind to the binding partner protein;
determining a fragment of the mutated form of the protein of interest said fragment comprising the region that mediates the ability of the protein to bind to the binding partner protein;
determining a fragment in the native form of the protein of interest that is functionally equivalent to (b) wherein said fragment inhibits the interaction between the native form of the protein of interest and the binding partner; and
identifying a mimetic compound of the fragment at (c).
Preferred methods for identifying mimetic compounds are based upon methods described in WO00/68373 and U.S. Ser. No. 10/372,003 for producing expression libraries of mimetic peptides or mimotopes known as “biodiverse gene fragments” (“BGF libraries”), which disclosures are incorporated herein by way of reference in their entirety. In these methods, the BGF libraries are screened to identify those peptides that have the same function as an isolated peptide derived from the protein of interest and comprising the interaction interface of that protein. Accordingly, the BGF libraries are screened to isolate those peptides that inhibit or abrogate the interaction between the protein of interest and the protein binding partner. Preferably, such mimotopes will not adversely affect the interaction of the protein of interest with another protein to which it binds in vivo.
Alternatively, random peptide (synthetic mimetic or mimotope) libraries are produced using short random oligonucleotides produced by synthetic combinatorial chemistry and screened for their ability to inhibit the interaction between the protein of interest and the protein binding partner.
To enhance the probability of obtaining useful bioactive mimetics from random peptide libraries, peptides can be constrained within scaffold structures, eg., thioredoxin (Trx) loop (Blum et al. Proc. Natl. Acad. Sci. USA, 97, 2241-2246, 2000) or catalytically inactive staphylococcal nuclease (Norman et al, Science, 285, 591-595, 1999), to enhance their stability. Constraint of peptides within such structures has been shown, in some cases, to enhance the affinity of the interaction between the expressed peptides and its target, presumably by limiting the degrees of conformational freedom of the peptide, and thereby mining the entropic cost of binding.
Mimotope libraries of up to several thousand polypeptides or peptides can be prepared by gene expression systems and displayed on chemical supports or in biological systems suitable for testing biological activity. For example, genome fragments isolated from Escherichia coli MG1655 can be expressed using phage display technology, and the expressed peptides screened to identify peptides that bind to the protein binding partner and inhibit interaction between the protein of interest and the protein binding partner, essentially as described by Palzkill et al. Gene, 221 79-83, 1998.
Additionally, mimotope libraries can be prepared essentially as described in U.S. Pat. No. 5,763,239 (Diversa Corporation), from uncharacterized environmental samples containing a mixture of uncharacterized genomes. The procedure described by Diversa Corp. comprises melting DNA isolated from an environmental sample, and allowing the DNA to reanneal under stringent conditions. Rare sequences, that are less likely to reanneal to their complementary strand in a short period of time, are isolated as single-stranded nucleic acid and used to generate a gene expression library. Again, the libraries are screened to identify proteins having the ability to bind to the protein binding partner and/or inhibit the interaction of the protein binding partner and the protein of interest eg., using reverse hybrid screens.
Alternatively, knowledge of critical residues required for the dimerisation of the target protein of interest with its partner gained from steps 4a-b above, can be applied to the rational design of peptoid or small molecule inhibitors which interact which such residues and block the interaction and/or folding of the target.
The present invention is further described with reference to the following non-limiting examples.
Introduction
This example describes new approaches to improve our understanding of specific inhibitors of the JNK MAPKs. These protein kinases, first described following their activation in response to stress, have been implicated in the intracellular events culminating in cell death. Because cell death underlies the pathologies of stroke and heart attack that are associated with the ischemia/reperfusion damage, the targeted inhibition of JNK promises an important therapeutic strategy.
Recently, an inventor described a small peptide inhibitor of JNK (MAB3), derived from an organiser/scaffold of the JNK pathway, designated “TI-JIP” (Truncated Inhibitor of JNK based on I). The inventors now have data supporting the efficacy of this inhibitor in protecting neuronal cells following ischemia/reperfusion. Data presented in
The inventors propose that inhibitors of JNK will provide an important strategy following ischemia/ reperfusion damage incurred in diseases such as stroke.
The inventors have continued to refine their understanding of the TI-JIP-JNK interaction, using a reverse two-hybrid screening technology described in WO99/35282, to map 3 critical residues of JNK, each of which prevents JNK interaction with TI-JIP when mutated.
Defining the Interaction Interface on Human JNK1 Using a Reverse Two Hybrid Assay
This example describes the identification and validation of critical residues of JNK that are required for the TI-JP-JNK interaction using a two hybrid assay. This defines amino acids of JNK that must be targeted by an effective and specific JNK inhibitor. This information is critical to the further development and/or discovery of JNK inhibitors targeting this site. The methods described herein have allowed the inventors to rapidly map, in less than 3 months, an interface on JNK that interacts with TI-JIP. This is faster than mapping by conventional co-crystallisation strategies, and reveals the interacting amino acids and the changes that interfere with binding.
Rationale
Following the identification of an effective peptide inhibitor of human JNK1, the inventors are now mapping the regions of JNK involved in this interaction. Improved knowledge of this interaction interface will allow the prediction and/or design of novel JNK inhibitors.
Broad Description of Approach and Results
The direct interaction of TI-JIP and human JNK1 by surface plasmon resonance has been demonstrated. TI-JIP inhibits JNK MAPK but not the closely-related p38 and ERK MAPKs. Four of the 11 amino acids of the TI-JIP, peptide are critical for its efficacy in vitro (MAB3).
The inventors have continued these studies, exploiting the power of yeast screening approaches, to confirm the TI-JIP-JNK interaction and its disruption by single amino acid substitution in TI-JIP. These results highlight the specificity of interactions in the JNK-TI-JIP interface.
To demonstrate that interaction interface can be mapped in a yeast system, the inventors have now exploited reverse two-hybrid screening systems described in WO99/35282.
The inventors constructed a JNK1-mutant library using random PCR mutagenesis. Using TI-JIP in the bait vector, yeast were selected in a single step as described herein for growth on selective media indicating the failure of TI-JIP to interact with mutant JNKs. The significant advance in these protocols has been the introduction of a galactose-titratable expression of the interacting partners thereby allowing greater discrimination of the interactors through continuous adjustment of screening stringency. Full-length JNK mutants were then sequenced.
From a first screen of 0.6×106 diploids, the inventors evaluated six INK mutants. The inventors have subsequently shown that three amino acid residues in JNK, as highlighted in
To further refine the JNK-TI-JIP interface, surface-exposed residues that are within the linear sequence between R309 and Y320 of SEQ ID NO: 1 are evaluated (i.e., the amino acid sequence 309RISVDEALQHPY320). Alternatively, or in addition, sequences flanking this region is evaluated. In particular, multiple JNK mutant libraries are created by site-directed mutagenesis of individual residues to create changes at the following residues:
I311, D313, E314, Q317, P319, K300, W324, E126, and S129.
For each residue, a NN[T/C] codon is introduced to thereby produce a mutated form of a JNK1 protein wherein all amino acids are represented at these positions, with the exception of Q, E and W. Degenerate oligonucleotide pairs are used separately, to create a series of mutant JNK libraries enriched in changes in the region of the proposed interface. This strategy was selected over alternative approaches, such as, for example, the introduction of the degenerate codon NNN, to ensure that a premature translation termination codon is not introduced into the gene, thereby encoding a truncated JNK1 protein. Background is further minimized because there is no carry-through of empty vector. No amino acid residues buried in the kinase domain of JNK1 are mutated.
To confirm that the mutations in JNK1 produce a TI-JIP-resistant JNK sparag, selected mutants are expressed as FLAG-JNK fusion proteins in a mammalian expression vector. Following transient transfection in HEK293 cells, constant expression levels of these mutants is confirmed by immunoblotting with FLAG antibody. Immunoprecipitates of control and mutant forms of FLAG-JNK from stimulated cells are obtained. The activity of control and mutant forms of FLAG-JNK from stimulated cells towards the transcription factor c-Jun is evaluated, along with the activation/phosphorylation of those proteins, such as, for example, by immunoblotting with a phospho-JNK antibody.
Each JNK mutant is tested to ensure that it is not inhibited by TI-JIP. These immunoprecipitation and kinase assays are standard procedures.
Experimental Methods
Plasmid DNA Constructs
Oligonucleotides encoding TI-JIP were annealed to produce a fragment with ends compatible “with EcoRI at the 5′ end and XhoI at the 3′ end. These were ligated into the pGILDA vector (CLONTECH), which had been digested with EcoRI/XhoI, thus generating C-terminal fusion proteins with the LexA DNA-binding domain. The human JNK1 sequence (SEQ ID NO: 1) was PCR-amplified and then digested with MfeI and XhoI. The use of MfeI, which is an isoschizomer of EcoRI, avoided internal digestion within the JNK1 sequence but produced the required sticky ends for subsequent cloning. These fragments were ligated into the pJG4-5 vector (CLONTECH), which had been digested with EcoRI/XhoI, to produce C-terminal fusion proteins with the B42 transcriptional activation domain. DNA sequencing confirmed the identity of these constructs.
Construction of Mutant JNK Library Using Random PCR Mutagenesis
Reactions (50 μL) containing 5U Taq polymerase (ROCHE), 50 pmol forward primer, 50 pmol reverse primer and 10 ng template DNA in Error-prone PCR buffer (final concentrations: 100 mM Tris-HCl pH 8.3, 500 mM KCl, 70 mM MgCl2, 0.1% (w/v) gelatin, 10% (v/v) DMSO, 0.2 mM DATP, 0.2 mM dGTP, 1 mM dCTP, 1 mM dTTP) were performed in 0.25mL PCR tubes. A total of four different mutagenesis reactions were performed, where MnCl2 was added to final concentrations of 0.1 mM, 0.2 mM or 0.3 mM prior to temperature cycling, or MnCl2 was added to a final concentration of 0.3 mM following completion of 10 rounds of temperature cycling. Reactions were subjected to 30 cycles with the following conditions: [94° C. for 1 min; 55° C. for 1 min; 72° C. for 3 min]. Following thermal cycling, reactions were pooled and digested with MfeI/XhoI. The digested products were ligated into EcoRI/XhoI-digested pJG4-5, transformed into ElectroTenBlue™ (Stratagene) electrocompetent E. coli and plated on LB agar containing 100 μg/mL ampicillin. Plates were incubated at 30° C. overnight, then placed at 37° C. for three hours to allow maximum growth of a total of 9×106 single well-isolated colonies. The bacterial library was harvested and DNA was isolated using a QIAGEN Maxiprep Kit. This was introduced into the yeast strain PRT 48, which was derived from the strain SKY 48 (MATα, trp1, ura3, his3, 6lexAop-LEU2, cIop-LYS2) (Serebriiski et al., J. Biol. Chem 274, 17080-17087, 1999) in accordance with the Gietz High Efficiency Transformation Protocol (Agatep, et al., Technical Tips Online 1998), and yeast were grown at 30° C. for 4 days on synthetic complete medium lacking tryptophan and containing 2% Glucose. The resulting 5×105 single well-isolated colonies were harvested and stored at −80° C. in sterile Yeast Freezing Buffer (65% (v/v) glycerol, 0.1 M MgSO4, 25 mM Tris-HCl pH 8.0).
Interaction Mating
The yeast strain PRT 480 (MATa, his3, trp1, ura3, 4 LexA-LEU2, lys2::3 cIop-LYS2, CANR, CYH2R, ade2::2 LexA-CYH2-ZEO, his5::2 LexA-URA3-G418) was constructed from the SKY 473 yeast strain provided by Ilya Serebriiskii, Fox Chase Cancer Center. We introduced into strain PRT 480 the bait plasmid, pGILDA-TI-JIP. We then mated these transformants to PRT 48, which carried either pJG4-5-JNK1, the mutant JNK1 library was constructed in pJG4-5, or the pJG4-5 vector control. In each mating, the total number of cells was 3×108 with a bait:prey ratio of 5:1. Thus, 2.5×108 colony forming units of bait were mated with 5×107 colony forming units of prey. Yeast were resuspended in 200 μL Yeast Extract Peptone Dextrose (YPD) liquid medium (10 g/L Yeast extract, 20 g/L Peptone, 20 g/L Glucose, 20 g/L Bacto-Agar) and then plated on 90 mm YPD agar plates and grown at 30° C. for 12-15 h. Diploids were harvested, washed in sterile H2O and plated on reverse screening plates.
Reverse Two-Hybrid Screening to Isolate JNK1 Mutants That Lost the Ability to Interact with TI-JIP
PRT 480/PRT 48 diploids expressing either pGILDA-TI-JIP/pJG4-5-JNK (positive control), pGILDA-TI-JIP/pJG4-5 (negative control) or pGILDA-TI-JIP/pJG4-5-mutant 10 JNK library (test) were plated at densities of 150,000 diploids per 90 mm plate of synthetic complete medium lacking uracil, histidine and tryptophan (HI) agar plate containing 2% (w/v) Raffinose (Raff), 0.05% (w/v) Glucose (Gluc), 0.08% (w/v) Galactose (Gal) and 0.07% (w/v) 5′fluoroorotic acid (5′FOA). Plates were supplemented with uracil (final concentration of 0.02 mg/mL) to support the growth and 15 survival of yeast prior to any reporter activation. In this novel reverse two hybrid screening system, the screening threshold can be adjusted by modulating the level of sugars in the media. These optimized screening conditions provided maximal death of positive control yeast with minimal death of negative control yeast. Plates were incubated at 30° C. for 72 h, after which time colonies were clearly visible. 20.
Characterisation of Non-Interacting JNK Mutants
Yeast expressing JNK mutants that did not interact with TI-JIP were plated on HW agar containing 2% (w/v) Glucose and grown at 30° C. These yeast were then replica plated onto synthetic complete agar lacking leucine (L agar) containing either 2% (w/v) Gluc, or 0.08% (w/v) Gal and 2% (w/v) Raff, and incubated at 30° C. for 72 h to test for the interaction between JNK and TI-JIP using forward two-hybrid analysis. This control forward analysis was possible due to the 6lexAop-LEU2 reporter carried by the yeast strain PRT 48. Colonies were regarded as false positives if they grew on the L Gal/Raff plates, which indicated an interaction between the mutant JNK protein and TI-JIP. Genuine non-interactors were grown on HW agar containing 0.05% (w/v) Gal and 2% (w/v) Raff for 48 h at 30° C., then vortexed in 20 μL SDS-PAGE Sample
Buffer and snap-frozen in liquid N2. Samples were heated at 100° C. for 5 min prior to separation by SDS-PAGE. Proteins were transferred to nitrocellulose by semi-dry electroblotting and probed for HA-tagged products. Yeast found to express a full-length HA-tagged activation domain-JNK1 fusion protein (58 kDa) were expanded in HW liquid medium containing 2% Gluc, and JNK constructs were rescued by lyticase extraction. These were electroporated into KC8 bacteria, plated on LB agar containing 100 μg/mL ampicillin and grown overnight at 37° C. Colonies were then plated on M9 agar lacking tryptophan (4 g/L Glucose, 1× M9 salts (64 g/L Na2HPO4.7H2O, 15 g/L KH2PO4, 2.5 g/L NaCl, 5 g/L NH4Cl), 2 mM MgSO4, 0.1 mM CaCl2 and 0.75 g/L amino acid dropout mix lacking tryptophan (Ausubel et al ibid.) containing 50 μg/mL kanamycin and grown at 30° C. for 48 h. Mutant pJG4-5-JNK DNA was isolated using a QIAGEN Spin Miniprep Kit prior to sequencing and analysis of mutations. A pool of 16 mutant JNK sequences was identified, each containing from 2 to 11 mutations in the full length JNK sequence. In total, 70 amino acids had been mutated and some mutations were common to more than one mutant JNK sequence.
Identification of Mutational “Hot-Spots” on JNK
From the pool of 16 JNK mutants, the frequency of mutations per region of secondary structure of JNK was calculated and normalized for the length of the structure. This resulted in the identification of secondary “hot-spots”. The mutations were also mapped onto the surface of the JNK3 structure (PDB: 1JNK) using WebLab ViewerLite software. This indicated that some mutations that appeared distant in the protein primary structure were close to each other in the tertiary structure, resulting in tertiary “hot-spots”. To reduce noise, the mutant pool was reduced to those containing five or less point mutations per JNK protein. We then chose nine such regions to target by point mutation, and constructed these point mutants using the Stratagene QuikChange protocol. These were screened for interaction with TI-JIP using forward two-hybrid screening and, β-galactosidase overlay assays (described below). Western immunoblotting for the HA-tagged mutant JNK proteins was performed as described above to confirm that full-length JNK proteins were expressed from the mutant constructs. Point mutants of JNK1 that did not interact with TI-JIP were constructed in the pCMV-FLAG-JNK1 using the Stratagene QuikChange protocol to assess their biochemistry in mammalian cells.
β-Galactosidase Overlay Assays
The RFY 206 strain (MATa, trp1, ura3-52, his3-200, leu2-3, lys2-Δ201, trp1::hisG) carrying the pSH18-34 lacZ reporter plasmid and pGILDA-TI-JIP was mated to the PRT 49 strain derived from the SKY 48 strain (MATα, trp1, ura3, his3, 6-lexAop-LEU2, 3-cIop-LYS2, ade2) carrying JNK mutants in pJG4-5. For qualitative analysis of β-galactosidase activity, these diploids were replica plated onto UHW agar containing either 2% (w/v) Gluc, or 2% (w/v) Raff and 0.05% (w/v) Gal. Following incubation at 30° C. for 48 h, protein-protein interactions were assessed using the chloroform overlay assay technique (adopted from Duttweiler et al., Trends Genet. 12, 340-341, 1996). Yeast grown on agar plates were overlaid with chloroform and incubated at room temperature for 5 min. Plates were then rinsed with chloroform, dried upside down for 5 min, then overlaid with a solution of 1% low-melting agarose in 100 mM potassium phosphate buffer, pH 7.0, containing X-Gal at a concentration of 1 mg/mL. Once the agarose solidified, plates were incubated at 30° C. and monitored for 20 min-3 h for colour changes; Protein-protein interactions were monitored via lacZ reporter, activity converting the colourless X-Gal substrate to a coloured product.
Cell Transfection, Lysis and Immunoblotting
COS cells were transfected with pCMV-FLAG-JNK1 (Derijard et al., Cell 76, 1025-1037, 1994) or equivalent mutant constructs and pEBG-MKK7β1 (provided by A. Whitmarsh, University of Manchester) as specified in the Figures using Lipofectamine and PLUS reagent (Invitrogen) according to the manufacturer's instructions. Following cell, lysis as described in Barr et al., J. Biol. Chem 277, 10987-10997, 2002) and addition of 3× SDS Sample Buffer, proteins were separated using SDS-PAGE. Following protein transfer onto nitrocellulose, immunoblotting was performed using either anti-active JNK (Promega), anti-FLAG M2 (SIGMA) or anti-JNK1 (Santa-Cruz) primary antibodies. Primary antibodies were bound by horseradish peroxidase-conjugated secondary antibodies (PIERCE) and immunocomplexes were visualized using chemiluminescence.
Immunoprecipitation and Protein Kinase Assays
FLAG-JNK1 proteins were immunoprecipitated by addition of anti-FLAG M2 (SIGMA) and incubation for 1 h on ice and then addition of Protein G-Sepharose and incubation at 4° C. for 2 h with rotation. Immunocomplexes were washed three times with lysis buffer, then once with reaction buffer (20 mM HEPES, 20 mM MgCl2, 20 mM β-glycerophosphate, 500 μM DTT, 100 μM Na3VO4; pH 7.6). For assays of JNK activity, the washed complexes were resuspended in 40 μL of reaction buffer containing 10 μg GST-c-Jun (1-135), 20 μM ATP and 1 μCi [λ-32P]ATP and incubated at 30° C. for 30 min. Reactions were stopped by addition of 3× SDS-PAGE Sample Buffer and proteins were separated by SDS-PAGE. JNK activity towards GST-c-Jun (1-135) was visualized by autoradiography and quantitated by Cerenkov counting. For assays of JNK activation, the washed beads were incubated with 30 μL of reaction buffer containing 20 μM ATP, 5 μCi [λ-32P]ATP and 1 μg of GST-MKK4(ED) at 30° C. for 1 h with occasional mixing. After removal of the supernatant, the beads were washed in 200 μl ice-cold lysis buffer and then heated for 5 min at 100° C. in 15 μL of 3× SDS-PAGE sample buffer, prior to separation by SDS-PAGE. Gels were Coomassie-stained, dried and used for autoradiography. Gel bands corresponding to FLAG-JNK were excised from the gels and their radioactivity quantitated by Cerenkov counting. Where immunoprecipitated GST-MKK7β1 was used to activate JNK, reactions were performed as above, but with 1 μCi [λ-32P]ATP and incubation at 30° C. for 30 min.
Results
Random PCR Mutagenesis Created a Library of JNK Mutants
Initially, we constructed a series of directed N- and C-terminal truncations of JNK as fusions with the C-teriminus of the GAL4 transcriptional activation domain, to identify a smaller region of JNK to be subjected to mutagenesis. However, these JNK mutants were poorly expressed in RFY 206/PRT 49 diploids relative to the wild type protein (data not shown), and therefore we proceeded to randomly mutagenise the entire JNK1 sequence. In optimizing the random PCR mutagenesis, we found that reactions containing 0.3 mM MnCl2 resulted in the presence of up to 11 point mutations per full length JNK sequence. Therefore, we used four different mutagenic PCR conditions to generate a library of JNK sequences containing up to 11 point mutations per JNK sequence.
Reverse Two-Hybrid Screening
We employed a reverse two-hybrid method to screen the library of JNK mutants for those that lost the ability to interact with TI-JIP (
Analyzing Colonies That Survived the Reverse Two-Hybrid Screening
Approximately 600 colonies were obtained after plating 600,000 diploids on the. reverse screening plates. Screening by colony PCR using JNK-specific primers indicated that 200 of the 600 colonies contained a prey plasmid with a JNK-insert. A representative selection of this screen is shown in
Summary of Mutation Data
From the reduced pool of 16 mutants, the frequency of mutations per region of secondary structure was calculated and normalised for the length of the structure (
We chose 9 individual JNK residues to target by point mutation. Using site-directed mutagenesis, we altered single residues of JNK to represent the changes that occurred in mutants isolated by reverse two-hybrid screening. Specifically, the point mutations were Leu-110-His, Asp-124-Tyr, Leu-131-Arg, Val-219-Asp, Glu-261-Lys, Arg-309-Trp, Asp-313-Gly, Asp-314-Gly and Tyr-320-His. Locations of the targeted residues are represented on the JNK1 protein structure in
The residues Leu-131 and Tyr-320 were located near each other on a common face of the JNK protein (
Proximity of JNK1 Residues Leu-131, Arg-309 and Tyr-320 to Regions of APKs Previously Reported to Interact with Kinase Interaction Motifs (KIMs)
The acidic “CD” domain of MAPKs is characterized by negatively charged amino acids and is located on the opposite side to the active site in the structure of MAPKs Tanoue et al., EMBO J, 20, 466-479, 2001). In human JNK1, the CD domain residue Asp-326 is conserved, and the acidic Glu-329 might also be considered part of the domain. JNK1 residues Leu-131 and Tyr-320 (
The co-crystallisation of p38 MAPK in complex with KIM-based peptides from substrate MEF2A and activator MKK3b identified a site in the C-terminal domain of the kinase thought to participate in hydrophobic interactions with the KIM peptides (Chang et al., Mol. Cell. 9, 1241-1249, 2002). The equivalent regions of human JNK1 comprise Val-107 to Leu-131 and Val-159 to Leu-165. The JNK1 residue Leu-131 is situated directly within this cluster of residues, and Tyr-320 is situated directly adjacent to these residues (
Whilst our study was nearing completion, it was reported that JNK2 residues Glu-329 and Glu-331 were important for the interaction between JNK2 and JIP-1 (Mooney et. al., J. Biol. Chem. 279, 11843-11852, 2004). In particular, Glu-329 was critical for efficient binding between JNK2 and JIP-1, whereas Glu-331 made a more minor contribution. These residues are conserved in JNK1, and Leu-131 and Tyr-320 are situated a short distance (12-14 Å) from Glu-329 (
In summary, at least Leu-131 and Tyr-320 are situated relatively close to regions of MAPKs thought to mediate interactions with KIMs of interacting partners. Therefore, it seemed possible that in addition to disrupting the JNK-TI-JIP interaction, these mutations would disrupt the interactions between JNK and other KIM-containing partners. To further investigate this hypothesis, we investigated the biochemistry of these JNK1 mutants in mammalian cells.
JNK1 mutants were impaired in their ability to phosphorylate c-Jun following exposure to activating stimuli.
We constructed the Leu-131-Arg, Arg-309-Trp and Tyr-320-His point mutants of JNK1 in the pCMV-FLAG vector for mammalian expression. COS cells were transfected with these constructs, and Western blotting performed on cell lysates revealed over-expression of FLAG-tagged JNK1 and all three tagged mutants
We then tested the activation of these JNK proteins by two different stimuli. Hyperosmotic shock (0.5 M sorbitol, 30 min) is a well-described activator of mammalian JNK (Bogoyevitch et al., J. Biol. Chem. 270, 297100-29717, 1995). Exposure of COS cells transfected with wildtype JNK1 to 0.5 M sorbitol for 30 min resulted in strong phosphorylation of c-Jun substrate in in vitro kinase assays using FLAG-immunoprecipitation from lysates prepared from these cells, which corresponded to 5.5-fold activation over the corresponding unstimulated cells (
When a constitutively-active form of MEKK1 (CA-MEKK1) was co-transfected into COS cells with wildtype JNK, FLAG immunoprecipitates from these cell lysates displayed a 240-fold increase in c-Jun phosphorylation in in vitro kinase assays relative to the sample prepared from cells transfected with JNK alone (
JNK1 Mutants Were Not Activated by Either MKK4 or MKK7
The impaired c-Jun phosphorylation by the JNK mutants (
Whilst our study was nearing completion, it was reported that a double alanine mutant of JNK2 (Glu-329-Ala, Glu-331-Ala) did not interact with JIP-1, c-Jun or MKK4, but retained the ability to be activated by MKK7 (Mooney et al., J. Biol. Chem. 279, 11843-11852, 2004). Therefore, we tested the ability of the Leu-131-Arg, Arg-309-Trp and Tyr-320-His JNK1 mutants to be activated by MKK7, by phospho-blotting and in vitro kinase assays. Phospho-blotting for dual-phosphorylated JNK indicated that wild-type JNK1 was strongly phosphorylated by MKK7 in co-transfected cells (
Discussion
The JNK MAPK pathway is activated following exposure of cells to a wide range of extracellular stimuli including stress, cytokines and growth factors, but still the role that JNK activation plays remains controversial (reviewed by Bogoyevitch et al., Biochim. Biophys, Acta 1697, 89-101, 2004). Our understanding of this pathway is being enhanced by multiple parallel approaches including gene knockouts and over expression studies, as Well as closer evaluation of the biochemical features of members of this pathway. In addition to studies on the JNKs themselves, or their upstream activators, increasing attention is focused on the regulation of JNK signaling by the JIP family of scaffold proteins. Interestingly, JIPs have been reported to both increase (Whitmarsh et al., Science 281, 1671-1674, 1998) and decrease (Barr et al., J. Biol. Chem 277, 10987-10997, 2002; Bonny et al., Diabetes 50, 77-82, 2001; Dickens et al., Science 277, 693-696, 1997) signaling through the JNK cascade.
We have further investigated the binding interaction between JNK and the TI-JIP peptide, which represents the KIM of the JIP-1 scaffold protein. Using reverse hybrid analysis of a library of mutant JNK1 proteins, we isolated mutant JNKs that lost the ability to interact with TI-JIP. By constructing individual point mutations to assess the relative importance of putative mutational “hot-spots” on the JNK1 protein, we implicated the” residues Leu-131, Arg-309 and Tyr-320 as mediators of the interaction between JNK1 and TI-JIP.
Although site-directed mutagenesis and co-immunoprecipitation analysis are effective for a relatively small number of mutations and for targeting a well-defined region, for many interactions, the potential binding interface is poorly defined. In such cases, mutations targeting many surfaces of the protein can be made and a relatively large number of mutants screened. Random PCR mutagenesis allows the generation of a relatively large pool of mutants; and yeast two-hybrid or N-hybrid assays provide an efficient technique for screening these mutants for non-interactors. The efficiency advantage of reverse two-hybrid and N-hybrid screening over conventional forward two-hybrid screening is that the reverse screening selects against an interaction from up to 10 million mutants, whereas forward two-hybrid screening selects for an interaction. The result of this is that non-interactors are easily obtained with reverse hybrid screening, whereas more extensive forward hybrid screening is required to isolate non-interactors.
It is interesting to note the CD and ED site residues previously reported to mediate the docking interactions between MAPKs and interactors were not involved in the interaction between JNK and JIP-1. This was demonstrated by Mooney et al., J. Biol. Chem. 279, 11843-11852, 2004, who showed that mutation of the CD site residue Glu-326 to asparagines did not disrupt the JNK-JIP-1 interaction, despite its location directly adjacent to Glu-329, which was deemed critical for this interaction. In addition, although the ED site was reported to regulate the specificity of docking interactions for ERK and p38 MAPKs mutation of the region spanning Lys-160 to Asp-162, along with the residue Thr-164 within this site, did not disrupt the JNK—JIP-1 interaction (Mooney et al., J. Biol. Chem. 279, 11843-11852, 2004). It does appear, however, that the residues that mediate JNK binding to JIP-1/TI-JIP are also involved in the interactions of JNK with other activators and substrates, given that the JNK1 mutants in our study were not efficiently activated by MKK4 or MKK7, and that JNK2 mutants that do not bind JIP-1 are not activated by MKK4 and cannot bind c-Jun (Mooney et al., J. Biol. Chem. 279, 11843-11852, 2004). This emphasizes the notion that KIMs bind to similar regions of MAPKs via a combination of both common and distinct binding determinants.
Chang et al., J. Biol. Chem 278, 9195-9202, 2003 showed that murine WOX1 and human WOX3 interact with human JNK1 via the WW domain in the N-terminus of the WOX protein, however human WOX3 protein appears to promote higher endogenous activation of gene expression than murine WOX1. This is presumably due to the presence of an activation domain in WOX3 that is produced as consequence of the deletion in WOX3 that truncates and modifies the C-terminus of the protein relative to WOX1.
The interaction interface between TI-JIP and JNK1 (Example 1) is confirmed using a reverse three hybrid assay PCT/US01/07669). The binding partners assayed are JNK (SEQ ID NO: 1) and TI-JIP (SEQ ID NO: 4) as described in the preceding example, and a WOX protein selected from the group consisting of human WOX3 (SEQ ID NO: 17), human WOX1 (SEQ ID NO: 18) and murine WOX3 (SEQ ID NO:. 19). Alternatively, or in addition, multiple WOX proteins are separately assayed in conjunction with the JNK1/TI-JIP proteins in a reverse three hybrid assay.
In particular, the dual fluorescent reporter construct pRT2 (SEQ ID NO: 14) is transformed into a yeast strain that requires adenine, thereby conferring adenine auxotrophy and enabling selection for maintenance of the vector. Nucleic acid encoding TI-JIP is cloned into the vector pDD (SEQ ID NO: 13) to yield the plasmid pDD-TI-JIP. Nucleic acid encoding a WOX protein is cloned into the plasmid pGMS19 (SEQ ID NO: 15) to yield pGMS19-WOX. Yeast cells carrying the dual reporter gene construct pRT2 are then transformed with pDD-TI-JIP and pGMS19-WOX to thereby express TI-JIP as a fusion with the LexA DNA binding domain, and a WOX protein as a fusion with the DNA binding domain of cI. This yeast is then mated to yeast cells transformed with the mutant JNK library in the pJFK vector (SEQ ID NO: 12). Yeast grown in media lacking adenine, histidine and methionine. Expression of all binding :partners is induced in the presence of Galactose (Gal), the neutral carbon source Raffinose (Raff) and a low concentration of Glucose (Gluc) to reduce background. Yeast cells are assayed by FACS for expression of the GFP and cobA proteins, and yeast cells expressing the red fluorescent protein (cobA) but not the green fluorescent protein (GFP) are selected. The amino acid sequences of the mutant JNK1 proteins in the selected yeasts are determined and compared to the sequences identified in Example 1. The identification of mutations at Leu-131, Arg-309 and Tyr-320 confirms the validity of the assay system. In contrast to the reverse two hybrid assay described in the preceding example, the incidence of uninformative mutations is reduced in a single step.
This example describes the identification of mimetic compounds of TI-JIP that are identified in a screen of a BGF library derived from biodiverse microbial genomes created and validated as described in U.S. Ser. No. 10/372,003. With this BGF library, the inventors will identify new peptides utilizing the JNK-TI-JIP interface. Data already obtained with this library suggests that the encoded peptides yield 10 to 1000-fold better hit rates than the best rates reported from comparable screens of random peptides in aptamer libraries. Using in vitro assays, the inventors will confirm the ability of peptide mimotopes to inhibit JNK and prevent neuronal apoptosis. Non-peptide small inhibitor molecules of JNK are also identified. The technologies used are broadly applicable to emerging approaches to target protein-protein interaction interfaces in general. Novel peptides that also the TI-JIP/JNK interface are identified using the screening approaches described herein to screen BGF libraries.
Screening of 10% of a 2×106 BGF library using Discriminating Blocker Trap reverse two-hybrid technology as described in U.S. Ser. No. 10/372,003 has successfully isolated peptides that block the SCL/E47 interaction but do not bind to either SCL or E47 in the proteins from which they were derived (i.e., in their native context). These peptides also do not block related interactions. (SCL/E2.2 and E47/ID). The peptide fragments range in size from 15 to 29 amino acids, and showed no sequence homology. This suggests conserved structural motifs that are responsible for the inhibition observed.
To select for peptides that block the JNK/TI-JIP interaction, a dual-bait reporter system described herein is used. In this system, the conditional toxicity of the URA3 gene product (in the presence of 5-fluoro-orotic acid) and the CYH2 gene product (in the presence of cycloheximide) allows selection of non-interacting bait and prey. A LacZ reporter is also used to reduce background. Thus, mimetic peptides in the BGF library that block the TI-JIP/JNK interaction permit cell survival in the presence of both 5-fluoro-orotic acid and cycloheximide, and colonies of these cells remain white in medium comprising the chromogenic substrate X-Gal. An added advantage of this approach is the modulation of screening stringency via a galactose-inducible bait/prey expression system. Mimetic peptides having different affinities for the JNK-TI-JIP interface are selected by varying the galactose concentration, with screening under the most stringent conditions identifying the blockers of highest-affinity. About 25 mimetic peptides are identified from a primary screen of about 1×106 clones.
Peptides are synthesized by Auspep Ltd., Australia. For each mimetic peptide, a glycine-spacer and Biotin label are included at the N-terminus, to facilitate subsequent validation testing. For example, this labelling facilitates a determination of the JNK binding cabability of each peptide, using BIAcore surface plasmon resonance.
Each peptide is also tested for its ability to inhibit JNK activity towards c-Jun, and other substrates including Elk1 and ATF-2, using established methods. A range of peptide concentrations (0.001 to 10 μM) is tested.
Using these protocols, the JNK inhibitory properties of 89 peptides based on TI-JIP have been assessed.
In parallel, inhibitory activity of the mimetic peptides toward ERK or p38 MAPKs is determined and those peptides that do modify these pathways are eliminated.
JNK-inhibitory mimetic peptides are delivered to neuronal cells using protein tranduction domain (PTD) technologies. Each peptide is synthesised with the TAT-PTD and a fluorescent FITC label at its N-terminus. Cultured neurons are preincubated with TAT-conjugated peptides (2 μM), exposed to oxygen-glucose deprivation (OGD) to simulate stroke, then maintained in normal medium for 24 h. Cell death is assessed by DAPI staining, with apoptotic cells showing fragmented nuclei, necrotic cells having condensed nuclei, and the nuclei of viable cells being only faintly stained. As shown in
Using data obtained for the mimetic peptides and data on residues important for TI-JIP interactions with JNK, together with the published X-ray crystallographic structure of JNK, the modelling tools Deep View, Wit!P (Novartis) and QXP (as available from Colin McMartin, Thistlesoft Software Co., USA), simulated docking of inhibitory peptides is performed. Deep View defines the binding surface on JNK and ensures consistency between the generated models and experimental results. Refinement utilizes the more powerful programs Wit!P and QXP.
The docked peptides in silico define key binding cavities for inhibitors on the surface of JNK. In the second phase of screening, small non-peptidic, drug-like molecules that have atoms or groups of atoms corresponding to key binding elements of the inhibitory peptides are obtained from database screens and their ability to inhibit JNK activity is determined.
Inhibitors are designed and docked into the JNK model structure. Monte Carlo docking of low molecular weight compounds into the defined binding site is performed with QXP and DOCK, and modeling of the best candidates refined with Wit!P. The leads are refined using the classical optimisation procedures of medicinal chemistry as shown by King In: Medicinal Chemisty-Principles and Practice, 2nd Edition, Royal Soc. Chemistry, 2002.
| Number | Date | Country | Kind |
|---|---|---|---|
| 60474465 | May 2003 | US | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/AU04/00723 | 5/31/2004 | WO | 00 | 4/12/2006 |
