Polypeptides
OwnerSimon Dowler, David Campbell +3inventors
InventorsSimon Dowler, David Campbell, Alexander Gray, Peter Downes, Dario Alessi
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Abstract
The use of a polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, in a screening method for identifying a compound suitable for modulating signalling by PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2. The polypeptide preferably comprises a PH domain which binds specifically to one of PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2. The PH−domain preferably has at least five of the six residues of a PutativePtdIns(3,4,5)P3 Binding Motif (PPBM).
Claims58
The use of a polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, in a screening method for identifying a compound suitable for modulating signalling by PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2.
The use of claim 3 wherein the polypeptide binds specifically to PtdIns(3,4)P2 and is a polypeptide as defined in claim 27 (TAPP) or a fragment, variant, derivative or fusion thereof, or a fusion of a said fragment, variant or derivative.
The use of claim 3 wherein the polypeptide binds specifically to PtdIns4P and is a polypeptide as defined in claim 28 (FAPP) or a fragment, variant, derivative or fusion thereof, or a fusion of a said fragment, variant or derivative.
The use of claim 3 wherein the polypeptide binds specifically to PtdIns3P and is a polypeptide as defined in claim 29 (PEPP) or AtPH1 or a fragment, variant, derivative or fusion either thereof, or a fusion of a said fragment, variant or derivative.
The use according to any of the previous claims wherein the method comprises the steps of (1) exposing the said polypeptide to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2, in the presence of a test compound;
- (2) determining whether the test compound modulates binding of the said phosphoinositide to the said polypeptide;
- and (3) selecting a compound which modulates binding of the said phosphoinositide to the said polypeptide.
A method of identifying a compound that modulates the phospholipid binding activity of a polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, the method comprising contacting a compound with the said polypeptide or a suitable variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof and determining whether the phospholipid binding activity of the said polypeptide or said variant, fragment, derivative or fusion thereof or a fusion of a variant, fragment or derivative thereof is changed in the presence of the compound from that in the absence of said compound.
A method of identifying a compound capable of disrupting or preventing the interaction between a polypeptide that is capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, and a polypeptide that is capable of binding to the said phosphoinositde-binding polypeptide (interacting polypeptide) wherein the said phosphoinositide-binding polypeptide or a suitable variant, fragment, derivative or fusion or a fusion of a variant, fragment or derivative thereof, and/or the interacting polypeptide are exposed to the said compound and the interaction between the phosphoinositide-binding polypeptide or variant, fragment, derivative or fusion and the interacting polypeptide in the presence and absence of the compound is measured.
A method of identifying a compound that is capable of binding to a polypeptide that is capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3 (interacting polypeptide), wherein the said polypeptide or suitable fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative is exposed to the compound and any binding of the compound to the said polypeptide or fragment, variant, derivative or fusion thereof, or fusion of a variant, fragment or derivative is detected and/or measured.
A method of identifying a polypeptide (interacting polypeptide) that interacts with a polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, the method comprising 1) contacting a) the said phosphoinositide-binding polypeptide or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative with b) a composition that may contain such an interacting polypeptide, 2) detecting the presence of a complex containing the said phosphoinositide-binding polypeptide or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative and an interacting polypeptide, and optionally 3) identifying any interacting polypeptide bound to the said phosphoinositide-binding polypeptide or a suitable fragment, variant, derivative or fusion thereof or a suitable fusion of a fragment, variant or derivative.
The method according to 9 or 10 in which the said binding activity or interaction is decreased.
The method according to 9 or 10 in which the said binding activity or interaction is increased.
A method of detecting and/or quantifying PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 in a sample wherein the sample is exposed to a polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3 and the binding of the said polypeptide to any said phosphoinositide present is detected.
A substantially pure polypeptide capable of binding to PtdIns(3,4)P2, PtdIns3P, PtdIns4P or PtdIns(3,5)P2 but not capable of binding to PtdIns(3,4,5)P3, wherein the polypeptide is not full length centaurin-β2 or full length AtPH1 [19].
A substantially pure polypeptide comprising the amino acid sequence
- 30 MPYVDRQNRICGFLDIEENENSGKFLRRYFILDTREDSFVWYMDNPQNLP SGSSRVGAIKLTYISKVSDATKLRPKAEFCFVMNAGMRKYFLQANDQQDL VEWVNVLNKAIKITVPKQSDSQPNSDNLSRHGECGKKQVSYRTDIVGGVP IITPTQKEEVNECGESIDRNNLKRSQSHLPYFTPKPPQDSAVIKAGYCVK QGAVMKNWKRRYFQLDENTIGYFKSELEKEPLRVIPLKEVHKVQECKQSD IMMRDNLFEIVTTSRTFYVQADSPEEMHSWIKAVSGAIVAQRGPGRSASS EHPPGPSESKHAFRPTNAAAATSHSTASRSNSLVSTFTMEKRGFYESLAK VKPGNFKVQTVSPREPASKVTEQALLRPQSKNGPQEKDCDLVDLDDASLP VSDV
- (human TAPP1 amino acid sequence) or
- 31 RGEREARRVWQADPEIPGARRTRRPEGRPRPM*RAPPEPRPLHGGG*CEQ SPGMPYVDRQNRICGFLDIEEHENSGKFLRRYFILDTQANCLLWYMDNPQ NLAMGAGAVGALQLTYISKVSIATPKQKPKTPFCFVINALSQRYFLQAND QKDMKDWVEALNQASKITVPKGGGLPMTTEVLKSLAAPPALEKKPQVAYK TEIIGGVVVHTPISQNGGDGQEGSEPGSHTILRRSQSYIPTSGCRASTGP PLIKSGYCVKQGNVRKSWKRRFFALDDFTICYFKCEQDREPLRTIFFKDV LKTHECLVKSGDLLMRDNLFEIITSSRTFYVQADSPEDMHSWIKEIGAAV QALKCHP
- (partial human TAPP2 amino acid sequence) or
- 32 MPYVDRQNRICGFLDIEENENSGKFLRRYFILDTREDSFVWYMDNPQnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nMNAGMRKYFLQANDQQDLVEWVNVLNKAIKITVPKQSDSQPASDSLSRQ GDCGKKQVSYRTDIVGGVPIITPTQKEEVNECGESLDRNNLKRSQSHLPY FAPKPPSDSAVIKAGYCVKQGAVMKNWKRRYFQLDENTIGYFKSELEKEP LRVIPLKEVHKVQECKQSDIMMRDNLFEIVTTSRTFYVQADSPEEMHSWI KAVSGAIVAQRGPGRSSSSnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
- (partial mouse TAPP1 amino acid sequence;
- the run of n's indicates a gap of unknown length) or
- 33 MPYVDRQNRICGFLDIEDNENSGKFLRRYFILDTQANCLLWYNDNPQNLA VGAGAVGSLQLTYISKVSIATPKQKPKTPFCFVINALSQRYFLQANDQKD LKDWVEALNQASKITVPKAGTVPLATEVLKNLTAPPTLEKKPQVAYKTEI IGGVVVQTPISQNGGDGQEGCEPGTHAFLRRSQSYIPTSGCRPSTGPPLI KSGYCVKQGNVRKSWKRRFFALDDFTICYFKCEQDREPLRTIPLKDVLKT HECLVKSGDLLMRDNLFEIITTSRTFYVQADSPEDMHSWIEGIGAAVQAL KCHPREPSFSRSISLTRPGSSTLTSAPNSILSRRRPPAEEKRGLCKAPSV ASSWQPWTPVPQAEEKPLSVEHAPEDSLFMPNPGESTATGVLASSRVRHR SEPQHPKEKPFVFNLDDENIRTSDV
- (mouse TAPP2 amino acid sequence) or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant, fragment, fusion or derivative thereof.
A substantially pure polypeptide comprising the amino acid sequence
- 34 MEGSRPRSSLSLASSASTISSLSSLSPKKPTRAVNKIHAFGKRGNALRRD PNLPVHIRGWLHKQDSSGLRLWKRRWFVLSGHCLFYYKDSREESVLGSVL LPSYNIRPDGPGAPRGRRFTFTAEHPGMRTYVLAADTLEDLRGWLRALGR ASRAEGDDYGQPRSPARPQPGEGPGGPGGPPEVSRGEEGRISESPEVTRL SRGRGRPRLLTPSPTTDLHSGLQMRRARSPDLFTPLSRPPSPLSLPRPRS APARRPPAPSGDT
- (partial human PEPP1 amino acid sequence) or
- 35 MEGSRPRSSLSLASSASTISSLSSLSPKKPTRAVNKIHAFGKRGNALRRD PNLPVHIRGWLHKQDSSGLRLWKRRWFVLSGHCLFYYKDSREESVLGSVL LPSYNIRPDGPGAPRGRRFTFTAEHPGMRTYVLAADTLEDLRGWLRALGR ASRAEGDDYGQPRSPARPQPGEGPGGPGGPPEVSRGEEGRISESPEVTRL SRGRGRPRLLTPSPTTDLHSGLQMRRARSPDLFTPLSRPPSPLSLPRPRS APARRPPAPSGDTAPPARPHTPLSRIDVRPPLDWGPQRQTLSRPPTPRRG PPSEAGGGKPPRSPQHWSQEPRTQAHSGSPTYLQLPPRPPGTRASMVLLP GPPLESTFHQSLETDTLLTKLCGQDRLLRRLQEEIDQKQEEKEQLEAALE LTRQQLGQATREAGAPGRAWGRQRLLQDRLVSVRATLCHLTQERERVWDT YSGLEQELGTLRETLEYLLHLGSPQDRVSAQQQLWMVEDTLAGLGGPQKP PPHTEPDSPSPVLQGEESSERESLPESLELSSPRSPETDWGRPPGGDKDL ASPHLGLGSPRVSRASSPEGRHLPSPQLGTKAPVARPRMNAQEQLERMRR NQECGRPFPRPTSPRLLTLGRTLSPARRQPDVEQRPVVGHSGAQKWLRSS GSWSSPRNTTPYLPTSEGHRERVLSLSQALATEASQWHRMMTGGNLDSQG DPLPGVPLPPSDPTRQETPPPRSPPVANSGSTGFSRRGSGRGGGPTPWGP AWDAGIAPPVLPQDEGAWPLRVTLLQSSL
- (human PEPP1 amino acid sequence) or
- 36 CKHPVTGQPSQDNCIFVVNEQTVATMTSEEKKERPISMINEASNYNVTSD YAVHPMSPVGRTSRASKKVHNFGKRSNSIKRNPNAPVVRRGWLYKQDSTG MKLWKKRWFVLSDLCLFYYRDEKEEGILGSILLPSFQIALLTSEDHINRK YAFKAAHPNMRTYYFCTDTGKEMELWMKAMLDAALVQTEPVKRVDKITSE NAPTKETNNIPNHRVLIKPEIQNNQKNKEMSKIEEKKALEAEKYGFQKDG QDRPLTKINSVKLNSLPSEYESGSACPAQTVHYRPINLSSSENKIVNVSL ADLRGGNRPNTGPLYTEADRVIQRTNSMQQLEQWIKIQKGRGHEEETRGV ISYQTLPRNMPSHRAQIMARYPEGYRTLPRNSKTRPESICSVTPSTHDKT LGPGAEEKRRSMRDDTMWQLYEWQQRQFYNKQSTLPRHSTLSSPKTMVNI SDQTMHSIPTSPSHGSIAAYQGYSPQRTYRSEVSSPIQRGDVTIDRRHRA HHPKVK
- (partial human PEPP2 amino acid sequence) or
- 37 MAADLNLEWISLPRSWTYGITRGGRVFFINEEAKSTTWLHPVTGEAVVTG HRRQSTDLPTGWEEAYTFKGARYYINHNERKVTCKHPVTGQPSQDNCIFV VNEQTVATMTSEEKKERPISMINEASNYNVTSDYAVHPMSPVGRTSRASK KVHNFGKRSNSIKRNPNAPVVRRGWLYKQDSTGMKLWKKRWFVLSDLCLF YYRDEKEEGILGSILLPSFQIALLTSEDHINRKYAFKAAHPNMRTYYFCT DTGKEMELWMKAMLDAALVQTEPVKRVDKITSENAPTKETNNIPNHRVLI KPEIQNNQKNKEMSKIEEKKALEAEKYGFQKDGQDRPLTKINSVKLNSLP SEYESGSACPAQTVHYRPINLSSSENKIVNVSLADLRGGNRPNTGPLYTE ADRVIQRTNSMQQLEQWIKIQKGRGHEEETRGVISYQTLPRNMPSHRAQI MARYPEGYRTLPRNSKTRPESICSVTPSTHDKTLGPGAEEKRRSMRDDTM WQLYEWQQRQFYNKQSTLPRHSTLSSPKTMVNISDQTMHSIPTSPSHGSI AAYQGYSPQRTYRSEVSSPIQRGDVTIDRRHRAHHPKHVYVPDRRSVPAG LTLQSVSPQSLQGKTLSQDEGRGTLYKYRPEEVDIDAKLSRLCEQDKVVH ALEEKLQQLHKEKYTLEQALLSASQEIEMHADNPAAIQTVVLQRDDLQNG LLSTCRELSRATAELERAWREYDKLEYDVTVTRNQMQEQLDHLGEVQTES AGIQRAQIQKELWRIQDVMEGLSKHKQQRGTTEIGMIGSKPFSTVKYKNE GPDYRLYKSEPELTTVAEVDESNGEEKSEPVSEIETSVVKGSHFPVGVVP PRAKSPTPESSTIASYVTLRKTKKMMDLRTERPRSAVEQLCLAESTRPRM TVEEQMERIRRHQQACLREKKKGLNVIGASDQSPLQSPSNLRDNPFRTTQ TRRRDDKELDTAIRENDVKPDHETPATEIVQLKETEPQNVDFSKELKKTE NISYEMLFEPEPNGVNSVEMMDKERNKDKMPEDVTFSPQDETQTANHKPE EHPEENTKNSVDEQEETVISYESTPEVSRGNQTMAVKSLSPSPESSASPV PSTQPQLTEGSHFMCV
- (human PEPP2) or
- 38 MSNKTGGKRPATTNSDIPNHNMVSEVPPERPSVRATRTARKAIAFGKRSH SMKRNPNAPVTKAGWLFKQASSGVKQWNKRWFVLVDRCLFYYKDEKEESI LGSIPLLSFRVAAVQPSDNISRKHTFKAEHAGVRTYFFSAESPEEQEAWI QAMGEAARVQIPPAQKSVPQAVRHSHEKPDSENVPPSKHHQQPPHNSLPK PEPEAKTRGEGDGRGCEKAERRPERPEVKKEPPVKANGLPAGPEPASEPG SPYPEGPRVPGGGEQPAQPNGWQYHSPSRPGSTAFPSQDGETGGHRRSFP PRTNPDKIAQRKSSMNQLQQWVNLRRGVPPPEDLRSPSRFYPVSRRVPEY YGPYSSQYPDDYQYYPPGVRPESICSMPAYDRISPPWALEDKRHAFRNGG GPAYQLREWKEPASYGRQDATVWIPSPSRQPVYYDELDAASSSLRRLSLQ PRSHSVPRSPSQGSYSRARIYSPVRSPSARFERLPPRSEDIYADPAAYVM RRSISSPKVPPYPEVFRDSLHTYKLNEQDTDKLLGKLCEQNKVVREQDRL VQQLRAEKESLESALMGTHQELEMFGSQPAYPEKLRHKKDSLQNQLINIR VELSQATTALTNSTIEYEHLESEVSALHDDLWEQLNLDTQNEVLNRQIQK EIWRIQDVMEGLRKNNPSRGTDTAKHRGGLGPSATYSSNSPASPLSSASL TSPLSPFSLVSGSQGSPTKPGSNEPKANYEQSKKDPHQTLPLDTPRDISL VPTRQEVEAEKQAALNKVGVVPPRTKSPTDDEVTPSAVVRRNASGLTNGL SSQERPKSAVFPGEGKVKMSVEEQIDRMRRHQSGSMKEKRRSLQLPASPA PDPSPRPAYKVVRRHRSIHEVDISNLEAALRAEEPGGHAYETPREEIARL RKMELEPQHYDVDINKELSTPDKVLIPERYIDLEPDTPLSPEELKEKQKK VERIKTLIAKSSMQNVVPIGEGDSVDVPQDSESQLQEQEKRIEISCALAT EASRRGRMLSVQCATPSPPTSPASPAPPANPLSSESPRGADSSYTMRV
- (human PEPP3 amino acid sequence) or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant, fragment, fusion or derivative thereof.
A substantially pure polypeptide comprising the amino acid sequence
- 39 MEGVLYKWTNYLTGWQPRWFVLDNGILSYYDSQDDVCKGSKGSIKMAVCE IKVHSADNTRMELIIPGEQHFYMKAVNAAERQRWLVALGSSKACLTDTRT KKEKEISETSESLKTKMSELRLYCDLLMQQVHTIQEFVHHDENHSSPSAE NMNEASSLLSATCNTFITTLEECVKIANAKFKPEMFQLHHPDPLVSPVSP SPVQMMKRSVSHPGSCSSERSSHSIKEPVSTLHRLSQRRRRTYSDTDSCS DIPLEDPDRPVHCSKNTLNGDLASATIPEESRLTAKKQSESEDTLPSFSS
- (human FAPP1 amino acid sequence) or
- 40 MEGVLYKWTNYLTGWQPRWFVLDNGILSYYDSQDDVCKGSKGSIKMAVCE IKVHSADNTRMELIIPGEQHFYMKAVNAAERQRWLVALGSSKACLTDTRT KKEKEISETSESLKTKMSELRLYCDLLMQQVHTIQEFVHHDENHSSPSAE NMNEASSLLSATCNTFITTLEECVKIANAKFKPEMFQLHHPDPLVSPVSP SPVQMMKRSVSHPGSCSSERSSHSIKEPVSTLHRLSQRRRRTYSDTDSCS DIPLEDPDRPVHCSKNTLNGDLASATIPEESRLTAKKQSESEDTLPSFSS
- (mouse FAPP1 amino acid sequence) or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant, fragment, fusion or derivative thereof.
A substantially pure polypeptide comprising the amino acid sequence
- 41 DVRAMLRGSRLRKIRSRTWHKERLYRLQED
- or
- 42 FEGTLYKRGALLKGWKPRWFVLNVT (PH30)
- or
- 43 RPGLRALKKMGLTEDEDEDVRAMLRGSRLRKIRSRTWHKERLYRLQEDGL SVWFQRRIPRAPSQHIFFVQHIEAVREGHQSEGLRRFGGAFAPARCLTIA FKGRRKNLDLAAPTAEEAQRWVRGLTKLRARLDAMSQRERLDHWIHSYLH RADSNQDSKMSFKEIKSLLRILV (PH83)
- or
- 44 KEGNLKKKGGGEGGRNWTVRWFKLKND
- (Dictyostelium PH domain polypeptide) or a variant, fragment, fusion or derivative thereof, or a fusion of a said variant, fragment, fusion or derivative thereof.
A polypeptide according to claim 31 wherein the polypeptide comprises a PH domain, preferably a PH domain that has at least five of the six residues of a Putative PtdIns(3,4,5)P3 Binding Motif (PPBM).
A polynucleotide or vector according to any one of claims 32 to 34 or 18 which contains no introns.
A method of determining the susceptibility of a patient to cancer, for example melanoma, comprising the steps of (i) obtaining a sample containing nucleic acid and/or protein from the patient;
- and (ii) determining whether the sample contains a level of PEPP nucleic acid or protein associated with cancer, for example melanoma.
A method of diagnosing cancer, for example melanoma, in a patient comprising the steps of (i) obtaining a sample containing nucleic acid and/or protein from the patient;
- and (ii) determining whether the sample contains a level of PEPP nucleic acid or protein associated with cancer, for example melanoma.
A method of predicting the relative prospects of a particular outcome of a cancer, for example melanoma, in a patient comprising the steps of (i) obtaining a sample containing nucleic acid and/or protein from the patient;
- and (ii) determining whether the sample contains a level of PEPP nucleic acid or protein associated with cancer.
Any novel polypeptide or nucleic acid as herein disclosed.
Dependencies are inferred from the claim text.