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WO2001085933A2 - Proteines interagissant avec la plakoglobine - Google Patents

Proteines interagissant avec la plakoglobine Download PDF

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WO2001085933A2
WO2001085933A2 PCT/EP2001/004872 EP0104872W WO0185933A2 WO 2001085933 A2 WO2001085933 A2 WO 2001085933A2 EP 0104872 W EP0104872 W EP 0104872W WO 0185933 A2 WO0185933 A2 WO 0185933A2
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plakoglobin
pla
interacting
polypeptide according
isolated
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WO2001085933A3 (fr
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Frans Van Roy
Stefan Bonne
Ann Vanlandschoot
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Vlaams Instituut voor Biotechnologie VIB
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Vlaams Instituut voor Biotechnologie VIB
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel proteins interacting with human plakoglobin. More particularly, the present invention relates to novel proteins that are involved in transduction of a plakoglobin related-signal to the nucleus.
  • the cadherin superfamily represents several cadherins with function in cell-cell adhesion, morphogenesis and tissue homeostasis (Takeichi, 1991 ; Kemler, 1992; Suzuki, 1996; Nollet et al., 2000).
  • the transmembrane glycoprotein E-cadherin is the best-studied prototype of this family and has been identified as a potent suppressor of invasion (Behrens et al., 1989; Frixen et al., 1991 ; Vleminckx et al., 1991).
  • catenins catena means chain
  • Armadillo proteins e.g. ⁇ -catenin, plakoglobin and p120 ct ⁇
  • vinculin-like ⁇ -catenins were also found to be associated with the cytoplasmic tumor suppressor gene product APC (adenomatous polyposis coli) (Peifer, 1993; Su et al., 1993).
  • APC cytoplasmic tumor suppressor gene product
  • the armadillo protein plakoglobin is a structural component of the intercellular cadherin/catenin adhesion complex.
  • Desmosomal cadherins can also bind to plakoglobin, localizing the plakoglobin in the desmosomes. (Mathur et al., 1994; Troyanovsky et al., 1994a; Troyanovsky et al., 1994b; Chitaev et al., 1996).
  • plakoglobin mediates a link between desmosomal cadherins and the cytokeratin cytoskeleton via desmoplakin (Korman et al., 1989; Kowalczyk et al., 1997).
  • plakoglobin like the homologous armadillo protein ⁇ -catenin, is found in a macromolecular complex with the tumor suppressor protein APC (Rubinfeld et al., 1993; Su et al., 1993; Shibata et al., 1994; Rubinfeld et al., 1995).
  • the ⁇ -catenin protein shows a high homology with plakoglobin, especially in the Arm domain, while the amino- and carboxyterminal sequences are less conserved (McCrea et al., 1991 , Butz et al., 1992).
  • Knock-out mice for the plakoglobin gene are lethal, revealing a specialized and indispensable function for plakoglobin in the desmosomes, particularly in heart (Ruiz et al., 1996; Bierkamp et al., 1996).
  • Knock-out mice for ⁇ - catenin are as well lethal, but differ significantly from the plakoglobin knock-out phenotype (Haegel et al., 1995). Truncation of the plakoglobin protein can result in Naxos disease (McKoy et al., 2000). For the ⁇ -catenin knock-out mice, it was shown that the development of the embryonic ectoderm was affected (Haegel et al., 1995).
  • LEF-1 lymphocyte enhancer-binding factor-1
  • ⁇ -catenin an architectural transcription factor
  • ⁇ -catenin/LEF-1 complex target genes induced by the ⁇ -catenin/LEF-1 complex are the c-myc proto- oncogene (He et al., 1998) and the cyclin-D1 gene (Shtutman et al., 1999; Tetsu and McCormick, 1999).
  • said proteins are involved in transduction of a plakoglobin mediated and/or a plakoglobin related signal to the nucleus.
  • One embodiment of the invention is a plakoglobin interacting polypeptide comprising the novel amino acid sequence depicted in SEQ ID N° 6 or an amino acid sequence at least 60%, preferably at least 70%, more preferably at least 80% identity to SEQ ID N° 6, as calculated on the whole sequence using an advanced Blast search (Altschul et al., 1997), or a functional fragment thereof.
  • Examples of functional fragments are plakoglobin interacting polypeptides, comprising SEQ ID N°2 and/or SEQ ID N°4. Preferably, said functional fragments are essentially consisting of SEQ ID N°2 or SEQ ID N° 4.
  • Another embodiment of the invention is a plakoglobin interacting polypeptide comprising a novel amino acid sequence as depicted in SEQ ID N° 8. It is another aspect of the invention to provide a nucleic acid sequence encoding a plakoglobin interacting protein according to the invention.
  • One preferred embodiment is a nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID N° 5.
  • nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID N° 1 and/or comprising a nucleic acid sequence as depicted in SEQ ID N° 3.
  • nucleic acid sequence comprising a nucleic acid sequence as depicted in SEQ ID N° 7.
  • Another aspect of the invention is an expression vector, comprising a nucleic acid sequence encoding a plakoglobin interacting polypeptide according to the invention.
  • Still another aspect of the invention is a host cell, transformed or transfected with said expression vector; said host cell can be a prokaryotic host cell, such as Escherichia coli or Bacillus subtilis, or it can be an eukaryotic host cell.
  • a eukaryotic host cell may be any eukaryotic host cells including but not limited to yeast cells, insect cells and mammalian cells.
  • Another aspect of the invention is a method to produce a plakoglobin interacting polypeptide, according to the invention. Preferentially, this method is comprising the use of a host cell according to the invention. Methods to produce said polypeptide using said host cell are known to the person skilled in the art.
  • Another aspect of the invention is a novel plakoglobin interacting protein, according to the invention, for the use as a medicament. Still another aspect of the invention is the use of a plakoglobin interacting protein, according to the invention, for the manufacture of a medicament to treat plakoglobin related diseases.
  • Such proteins may be novel proteins as described above, or known proteins for which the plakoglobin interaction has been demonstrated for the first time, such as NRF1 or NDP52.
  • Plakoglobin related diseases include, but are not limited to, cancers, especially skin carcinomas such as basal cell carcinoma, squamous cell carcinoma or extramammary Paget's disease, Naxos disease, heart diseases, skin blistering and acantholytic diseases such as subcorneal acantholysis, Grover's disease, Hailey-Hailey's disease or Darier's disease.
  • Still another aspect of the invention is a plakoglobin interacting protein, that is also interacting with plakophilin.
  • plakoglobin interacting protein to treat plakophilin related diseases, such as ectodermal dysplasia/skin fragility syndrome (McGrath et al., 1997; Whittock et al., 2000).
  • plakophilin related diseases such as ectodermal dysplasia/skin fragility syndrome (McGrath et al., 1997; Whittock et al., 2000).
  • said plakoglobin interacting protein comprises SEQ ID N°6, SEQ ID N° 2 or SEQ ID N° 4, or a functional fragment thereof.
  • the terms 'medicament' or 'use for the manufacture of a medicament to treat' or 'pharmaceutical composition' relate to a composition comprising plakoglobin interacting proteins according to the invention, or homologues, derivatives or functional fragments as described above and a pharmaceutically acceptable carrier or excipient (both terms can be used interchangeably) to treat diseases as indicated above.
  • Suitable carriers or excipients known to the skilled man are saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers and preservatives.
  • suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers.
  • the 'medicament' may be administered by any suitable method within the knowledge of the skilled man. The dosage and the method of administration will depend upon the individual. Generally, the medicament is administered so that the compound of the present invention is given at a dose between 1 ⁇ g/kg and 10 mg/kg, more preferably between 10 ⁇ g/kg and 5 mg/kg, most preferably between 0.1 mg/kg and 2 mg/kg.
  • Still another aspect of the invention is the use of a plakoglobin interacting polypeptide according to the invention and/or a functional fragment thereof to screen compounds that interfere with the interaction of said plakoglobin interacting polypeptide with plakoglobin.
  • polypeptides as depicted in SEQ ID 2, 4, 6, or 8 can be used, as well as the polypeptides NRF1 or NDP52.
  • Still another aspect of the invention is the use of a plakoglobin interacting polypeptide according to the invention and/or a functional fragment thereof to screen compounds that interfere with the interaction of said plakoglobin interacting polypeptide with plakophilin.
  • polypeptides as depicted in SEQ ID 2, 4, or 6 can be used Screening methods for such compounds are known to the person skilled in the art and have been described, as a non limiting example, in WO9813502 and in WO9923116.
  • Another aspect of the invention is said screening method, comprising a plakoglobin interacting polypeptide according to the invention.
  • Still another aspect of the invention is a compound, isolated with said screening method.
  • Another aspect of the invention is a pharmaceutical composition comprising one or more plakoglobin interacting polypeptides according to the invention, or one or more said compounds, isolated with the screening method according to the invention.
  • Such pharmaceutical compositions may be used to treat cancers, especially skin carcinomas, or acantholytic diseases such as subcorneal acantholysis, Graver's disease, Hailey-Hailey's disease or Darier's disease, or ectodermal dysplasia/skin fragility syndrome.
  • Plakoglobin interacting polypeptide means that said protein may interact with plakoglobin, preferentially human plakoglobin, or a functional fragment of said plakoglobin as can be measured as a non limiting example by a yeast two hybrid test.
  • a functional fragment of human plakoglobin is a fragment that comprises at least amino acid residues 37-404 and/or amino acid residues 405-570 of the human plakoglobin sequence.
  • Plakoglobin interacting protein is a fragment that still can interact with plakoglobin, or with a functional fragment of plakoglobin as defined above.
  • Polypeptide as used here means any proteineous structure, independent of the length and includes molecules such as peptides, phosphorylated proteins and glycosylated proteins.
  • Interacting or interaction means any interaction, be it direct or indirect.
  • a direct interaction implies a contact between the binding partners.
  • An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two compounds. This interaction can be completely indirect, with the help of one or more bridging compounds, or partly indirect, where there is still a direct contact that is stabilized by the interaction of one or more compounds.
  • Interfere with the interaction can be any way of interference, both positive or negative. It can mean an enhancement of the interaction, a weakening of the interaction or a complete inhibition of the interaction.
  • Compound means any chemical of biological compound, including simple or complex inorganic molecules, peptides, peptido-mimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.
  • Figure 1 Retransformation of yeast cells with plakoglobin fragments (bait plasmids) and PLA_2H12 (prey plasmid). Protein-protein interactions, indicated by + or ++, were demonstrated by expression of both the HIS3 and the LacZ reporter genes. Minus sign, no interaction is detected. Lines indicate the plakoglobin fragments (initiating and terminating amino acid residues are indicated by the appropriate codon numbers), as described at the right and encoded by the plasmids listed at the left. Boxes covering the lines represent armadillo repeats.
  • Figure 2 Northern blot analysis of the PLA_2H12 mRNA in several human tumor cell lines and human tissues. 28S and 18S are ribosomal size markers.
  • Figure 3 Sequence of human PLA_2H12 cDNA. Spaces separate blocks of 10 nucleotides (nt). The predicted amino acid sequence of the ORF is indicated in bold (one-letter code). The start codon and the stop codon are boxed in black. Also indicated is the sequence of the predicted bipartite nuclear localisation signal (shaded box). The predicted SH3 domain is indicated with open arrows. Two leucine zipper motifs are underlined.
  • leucine residues that are part of the consensus sequence of a leucine zipper (L-X 6 -L-X 6 -L-X 6 -L) are marked in gray.
  • the poly-A signal is indicated in bold.
  • Exon-exon junctions are marked. Between black arrows is indicated the original cDNA fragment that was found by us to interact with plakoglobin in a yeast 2- hybrid screen.
  • FIG. 4 Alignment of the SH3 domains of PLA_2H12 (human), ZO-1 (human and mouse), ZO-2 (human and dog), ZO-3 (dog). The conserved amino acids are in bold (one letter code).
  • the SH3 domain of human PLA_2H12 was detected by BLAST using the ProDom database (Altschul et al., 1997).
  • Figure 5 Genomic structure of the human PLA2H12 gene
  • Figure 6 Rapid-scan RT-PCR expression analysis of the PLA_2H12 mRNA.
  • Figure 7 Sequence of the human PLA_2H34 cDNA fragment, found to encode a polypeptide interacting with plakoglobin in a in a yeast 2-hybrid screen. Spaces separate blocks of 10 nucleotides (nt). The predicted amino acid sequence of the ORF is indicated in bold (one-letter code). A stop codon is boxed.
  • Figure 8 Retransformation of yeast cells with bait plasmids, encoding plakoglobin fragments (listed at the right; see also Fig. 1) and prey plasmids, encoding various protein fragments (listed on top; see also Table 6). Protein-protein interactions, indicated by + to +++, were demonstrated by expression of both the HIS3 and the LacZ reporter genes. Minus signs, no interaction is detected. Lines indicate the plakoglobin fragments (initiating and terminating amino acid residues are indicated by the appropriate codon numbers). Boxes covering the lines represent armadillo repeats.
  • Figure 9 Retransformation of yeast AH109 with plakoglobin encoding bait plasmids, listed on the left, and prey plasmids listed on the right, encoding various parts of the human PLA_2H12 protein. Growth and blue-staining on SD-LWHAde+alphaX-gal plates indicate interacting protein (fragments) (a, a'). Panel b and b' represent control plates indicative for the ability of the transformed yeasts to grow on medium independent of any interaction between bait and prey. For the numbering see Figure 1.
  • Eschehchia coli DH5 ⁇ (supE44, hsdRM, deoR, rec ⁇ , endM , /acZDM15) and E. coli HB101 (st/pE44, mcrB, mrr, hsdS20, recAI) were used for transformations, plasmid propagation and isolation.
  • the bacteria were grown in LB medium supplemented with 100 ⁇ g/ml ampicillin.
  • transformed HB101 bacteria were grown on minimal M9 medium, supplemented with 50 ⁇ g/ml ampicillin, 40 ⁇ g/ml proline, 1 mM thiamine-HCI and 1 % of the appropriate amino acid drop-out solution. After selection the bacteria were maintained in LB medium supplemented with 50 ⁇ g/ml ampicillin.
  • Colon adenocarcinoma cell lines SW620 (CCL-227) and SW1116 (CCL-233) were obtained from the American Tissue Culture Collection (ATCC, Rockville, MD).
  • DLD1/R2/7 was a round cell variant subcloned from DLD1 (CCL-221). (Vermeulen et al., 1995; Watabe-Uchida et al., 1998).
  • Cell lines LICR-HN2, LICR-HN3, LICR-HN5 and LICR-HN6 are derived from head and neck squamous cell carcinoma (Easty et al., 1981).
  • MKN45 is a gastric carcinoma cell line (Motoyama and Watanabe, 1983).
  • Restriction enzymes were purchased from Gibco BRL Life Technologies (Paisley, UK) or from New England Biolabs (Beverly, MA, USA). Restriction enzymes were used according to manufacturers' recommendations. All PCR reactions were performed using VentTM (Biolabs) DNA polymerase. The primers for PCR amplification were either home made (University of Ghent) or obtained from Gibco BRL. The standard PCR mixture, in a reaction volume of 100 ⁇ l, contained template cDNA (plasmid), 25 pmol of both specific primers, 200 ⁇ M dXTPs and the PCR buffer supplied with VentTM DNA polymerase. Unless otherwise stated, no additional MgSO 4 was added. VentTM DNA polymerase was used at 1 U/reaction.
  • the DNA amplification was performed in the PTC-200 Peltier Thermal Cycler PCR System (MJ Research, Watertown, MA).
  • the PCR program started with a DNA denaturating step at 94°C for 3 min, followed by 80°C for 1 min. Cycling conditions were 94°C for 1 min, 50-60°C for 30 sec and 72°C for 2 min. This was repeated for a total of 35 cycles and was followed by a final extension step at 72°C for 10 min.
  • the plasmid pHPG Ca2.1 containing full-length human plakoglobin cDNA was obtained from Dr. Goldschmidt M. and Dr. W. Franke (German Cancer Research Center, Heidelberg) (Genbank Ace. No. M23410) (Franke et al., 1989).
  • the PCR product was digested with the EcoRI and Sac ⁇ restriction enzymes.
  • Another fragment of human plakoglobin cDNA was generated by digestion of the plasmid pHPG Ca2.1 with Sad and BglW restriction enzymes. Digestion of the plasmid with the restriction enzymes BglU and Pst ⁇ resulted in the isolation of a third fragment. Ligation of these three fragments into the EcoRI-Psfl digested pGBT9 vector (MatchmakerTM, Clontech, Palo Alto, CA) resulted in the construction of the pGBT9Plako (227-2340) plasmid.
  • the fragment was ligated into the EcoRI-SacI digested pBluescriptllKS- vector (Stratagene, La Jolla, CA) to construct the pBSKSIIPIako (227-560).
  • This plasmid was digested with EcoRI-Psfl and the fragment was ligated into the EcoRI-Psfl digested pGBT9 vector to yield the pGBT ⁇ PIako (227-461) plasmid.
  • the pBSKSIIPIako (227-1335) plasmid was constructed by ligation of the EcoRI-H/ncll fragment of the pSE280Plako (227-2340) plasmid into the EcoRI-H/ticll digested pBluescriptllKS- vector (Stratagene). This plasmid was used to isolate an EcoRI-Sa/l fragment. This fragment of human plakoglobin cDNA was finally ligated into the EcoRI- Sa/I digested pGBT9 to construct the ⁇ GBT9Plako (227-1335).
  • the pBSKSIIPIako (1335-2340) plasmid was constructed by ligation of the Hinc ⁇ -Xho ⁇ fragment of pSE280Plako(227-2340) into the Hinc ⁇ -Xho ⁇ digested pBluescriptllKS- vector (Stratagene).
  • the pBSKSIIPIako (1335-2340) plasmid was digested with the EcoRI-Xmal restriction enzymes and the resulting fragment was ligated into the EcoRI- Xma ⁇ digested pGBT9 vector.
  • the final plasmid was called pGBT9Plako (1335-1853).
  • a fragment of the plakoglobin cDNA was isolated by digestion of the pSE280Plako (227-2340) plasmid with Xmal and Pst ⁇ restriction enzymes. This fragment was ligated into the Xma ⁇ -Pst ⁇ digested pBluescriptllKS- vector to obtain the pBSKSIIPIako (1832- 2340) plasmid. This plasmid was subsequently digested with BamH ⁇ and Psti restriction enzymes and ligation of this fragment into the Bamb ⁇ -Pst ⁇ digested pGBT9 vector resulted in the construction of the pGBT9Plako (1853-2340) plasmid.
  • the pBSKSIIPIako (227-1335) plasmid was digested with Ec/136ll and Xhol restriction enzymes. This fragment was ligated into the Sma ⁇ -Xho ⁇ digested pBluescriptllKS- plasmid to yield the pBSKSIIPIako (560-1332) plasmid. A ⁇ amHI-Psfl restriction fragment of this plasmid was isolated and ligated into the pGBT9 vector digested with the appropriate restriction enzymes. The constructed plasmid was called pGBT ⁇ PIako (558-998) plasmid.
  • the pGBT9-Plako (1335-2120) plasmid was constructed by ligation of the EcoRI-Sacl fragment of the pGBT9Plako (1335-2340) plasmid into the pGBT9 vector.
  • Saccharomyces cerevisiae strain HF7c (Mata, ura3-52, his3- ⁇ 200, ade2-101 , Iys2-801 , trp1-901 , leu2-3, 112, gal4-542, gal80-538, lys2::GAL1-H!S3, URA3::GAL4 17-mers)-CYC1-LacZ) (MatchmakerTM, Clontech, Palo Alto, CA) was used for most of the assays.
  • the HF7c yeast strain carries two reporter genes, HIS3 and LacZ, both integrated into the yeast genome and under the control of GAL4 responsive elements, the GAL1 UAS and the UAS G -i 7m er- It has also two auxotrophic markers, trpl and Ieu2, which are used for plasmid selection upon transformation.
  • Yeast cultures were grown at 30°C in either complete YPD medium (1 % yeast extract, 2% peptone and 2% glucose) or SD minimal medium (0.5% yeast nitrogen base without amino acids, 2% glucose, and 1% of the appropriate amino acid drop-out solution).
  • yeast strain AH109 (MATa, trp1-901 , leu2-3, 112, ura3-52, his3-200, gal4 ⁇ , gal ⁇ O ⁇ , LYS2::GAL1 UAS -GAL1 T ATA-HIS3, GAL2UAS-GAL2 T ATA-ADE2, URA3::MEL1u A s-MEL1 TA T A -lacZ, Clontech) was used.
  • the AH109 strain carries three reporter genes (ADE2, HIS3, and lacZ) under the control of distinct GAL4 upstream activating sequences (UASs) and TATA boxes. These promoters yield strong and specific responses to GAL4.
  • the strain has two auxotrophic markers, trpl and Ieu2, used for plasmid selection upon transformation.
  • Yeast cultures were grown in either complete YPD medium (see above) supplemented with adenine to a final concentration of 0.003% adenine (YPDA), or SD minimal medium (see above).
  • Plasmids encoding the GAL4 hybrid proteins were introduced into the HF7c yeast reporter strain by the lithium acetate (LiAc) transformation procedure (Gietz et al., 1992). Transformants were selected for the presence of the plasmids by growing on appropriate media at 30°C. They were allowed to grow until the colonies were large enough to perform a ⁇ -galactosidase filter assay, usually for 3-4 days. The transformed cells were then transferred onto an 82-mm nitrocellulose membrane (Sartorius, Goettingen, Germany), permeabilized by freezing the membranes in liquid nitrogen for one minute, followed by thawing at room temperature.
  • LiAc lithium acetate
  • the membranes are soaked with 1.5 ml of Z-buffer containing 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactosidase (X-gal) and incubated at 30°C until the appearance of blue colonies. This takes usually 30 min to 12 h. The membranes are then dried, analyzed and stored.
  • Z-buffer containing 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactosidase (X-gal)
  • the plasmid pGBT9Plako (227-1853) was used to screen a human fetal kidney cDNA library, cloned in the GAL4 activation domain vector pGAD10 (Clontech, Palo Alto, CA).
  • the plasmids were introduced into the HF7c yeast strain by using sequential transformation by the lithium acetate (LiAc) method described by Gietz et al. (Gietz et al., 1992).
  • the interaction screen was carried out in the yeast strain HF7c on media lacking leucine, tryptophan, histidine, and containing 5 mM 3-aminotriazole (3-AT).
  • the ⁇ -galactosidase activity in yeast was measured using a ⁇ -galactosidase filter assay. Yeasts harboring interacting proteins were used for plasmid isolation. The obtained plasmid mixture was transformed into Eschehchia coli HB101 electrocompetent cells. HB101 has a defect in the leuB gene, which can be complemented by LEU2 from yeast. So, this strain can be used for selection of the library plasmid, which carries the yeast LEU2 transformation marker. From these transformants, the library plasmids were isolated and introduced into Eschehchia coli DH5 ⁇ . Plasmids isolated from this strain were further characterized by DNA sequence.
  • the cDNA insert of clone 11 b10 comprises nt 324-3948 of the full length PLA__2H12 (Fig. 3). Both fragments were simultaneously ligated into the pGBKT7 vector, which was SamHI cut, blunted and Sail cut, yielding a full length PLA_2H12 cDNA in frame with the GAL4-DNA binding domain (pGBKT7hPLA_2H12FL). Both the PCR fragment and the cloning sites of this construct were sequenced to ensure no mutations had occurred.
  • the pGBKT7hPLA_2H12FL plasmid was subsequently cut with EcoRI and Sa/I, the insert of interest was purified and cloned in the EcoRI and Xhol sites of pGADT7, yielding pGADT7hPLA_2H12FL. Again, the cloning sites were sequenced to ensure no mutations had occurred and the insert was in frame with the GAL4-DNA activation domain.
  • the full length human plakophilin-1 (PKP1) ORF cloned in the pEGFP-C2 vector (Clontech) was a gift from dr. M. Hatzfeld (Halle, Germany).
  • This construct was cut with EcoRI and Sa/I, the insert of interest was purified and cloned in the EcoRI and Sa/I sites of the pGBKT7 vector yielding pGBKT7hPKP1. Sequencing of the cloning sites was performed.
  • the full-length human plakophilin-3 (PKP3) ORF was amplified using primers FVR1846F and FVR1847R (Table 2b). These primers contain an artificial EcoRI and Sa/I site, respectively.
  • the amplified product was purified, cut with EcoRI and Sa/I and cloned in the EcoRI and Sa/I sites of the pGBKT7 vector yielding pGBKT7hPKP3. This construct was fully sequenced to ensure no mutations had occurred. All constructs were checked for auto-activation in the two-hybrid system.
  • PLA_2H12 was estimated by Northern blot analysis. Total RNA (30 ⁇ g) was glyoxylated, size fractioned on a 1% agarose gel and transferred to a Hybond-N+ membrane (Amersham). The probe was radioactively labeled using random priming (RadPrime DNA labeling system; Gibco BRL Life Technologies, Gent, Belgium). The probe used was the EcoRI-generated insert of the originally isolated two-hybrid prey clone PLA_2H12. Hybridizations were performed as described elsewhere (Bussemakers et al., 1991). The blot was exposed for 10 d to a P-imager screen (Molecular Dynamics).
  • FVR1039R GSP1 ; 5'-AGCTGCCTCCCCAGC-TGCTTCTG-3'; Table 1
  • FVR1040R GSP2; 5'-GGCCTGGTCTCGCTCCTTCTCAA-3'
  • the 3' RACE primers used were FVR1041 F (GSP1 ; 5'-GGCCCAACTGGAGGAGATTGAGA-3', Table 1) and FVR1042F (GSP2; 5'-GCAAC-TGGGGAGGCAGCTGTCAT-3').
  • the fragments were further characterized by cloning into pGEM ® -T cloning system (Promega, Madison, Wl) and by subsequent DNA sequence analysis by using the M13 forward (5'- CGCCAGGGTTTTCCCAGTCACGAC-3'; FVR283; Table 4) and M13 reverse primer (5'-TCACACAGGAAACAGCTATGAC-3'; FVR284).
  • the specificity of the fragments was determined using the DNAstar software packages (DNASTAR Inc, Madison, USA), and by the Staden gap4 software (Bonfield et al., 1995).
  • a Rapid-Screen cDNA library panel of human lung was used (OriGene Technologies, Inc., Rockville, MD) to elongate further the PLA_2H12 cDNA.
  • the screening was performed according to the manufacturers' protocol with the PLA_2H12-specific primers FVR731F (5'-CGCAGGACAGCAGGCAGGAG-3'; Table 1) and FVR733R (5'- CAGGATGGAAAGCCGATTGA-3').
  • the PCR in a reaction volume of 25 ⁇ l, contained 5 ⁇ l of template cDNA, 50 pmoles of the PLA_2H12-specific primers FVR731 F and FVR733R (Table 1), 50 ⁇ M dXTPs, 5 U of Taq DNA polymerase (Boehringer) and the buffer supplied with the DNA polymerase.
  • a programmable thermal cycler GeneAmp PCR system 9700, Perkin Elmer Cetus was used.
  • the PCR program started with a DNA denaturating step at 94°C for 3 min. Cycling conditions were 94°C for 1 min, 60 for 1 min and 72°C for 1 min 30 sec.
  • the fragments were further characterized by cloning into pGEM ® -T cloning system (Promega, Madison, Wl) and by subsequent DNA sequence analysis by using the M13 forward (5'-CGCCAGGG- TTTTCCCAGTCACGAC-3'; FVR283; Table 4) and M13 reverse primer (5'- TCACACAGGAAACAGCTATGAC-3'; FVR284). Again, the specificity of the fragments was determined using the software packages DNAstar and Staden gap4. Since 5' RACE experiments did not enable us to complete the 5' end of the human PLA_2H12 cDNA sequence, another strategy was followed.
  • a typical PCR reaction contained 25 pmol of forward and reverse primers, 1 ⁇ l 10mM dXTP, 5 ⁇ l 10 x reaction buffer, 5 ⁇ l SW620 cDNA, 2.5 U pfu DNA polymerase and deionized water to a final volume of 50 ⁇ l.
  • PCR reactions were run on a PTC-200 thermal cycler (MJ Research) and PCR programs were as follows: an initial denaturation step at 95°C for 3 min, followed by 38 cycles of 45 sec at 94°C, 40 sec at 65°C and 90 sec at 75°C. Final extension was 10 min at 75°C.
  • fragments were incubated with TAQ DNA polymerase and dXTP for 25 min at 72°C and this allowed, after purification of the fragments, to clone these fragments using the pGEM-T vector system (Promega). Cloned fragments were checked by subsequent sequencing. Alternatively, PCR products were directly sequenced using internal primers. DNA sequence analysis was performed as described above.
  • the PCR in a reaction volume of 25 ⁇ l, contained 5 ⁇ l of a 100-times diluted template cDNA, 25 pmoles of the PLA_2H12- specific primers FVR731 F (Table 1 ; 5'-CGCAGGACAGCAGGCAGGAG-3') and FVR733R (5'-CAGGATGGAAAGCCGATTGA-3'), 200 ⁇ M dXTPs, 1.25 U of AmpliTaq DNA polymerase (Perkin Elmer) and the buffer supplied with the DNA polymerase.
  • a programmable thermal cycler GeneAmp PCR system 9700, Perkin Elmer Cetus was used.
  • the PCR program started with a DNA denaturating step at 94°C for 10 min.
  • the nested PCR was done on 2.5 ⁇ l (1/10 of the end volume) of the first PCR reaction. PCR reactions using ⁇ -actin-specific primers, provided in the kit, were performed as a positive control on each cDNA, derived from the different tissues, examined.
  • Example 1 Two-hybrid cDNA library screening
  • the pGBT9Plako (227-1853) plasmid was used as bait.
  • This comprises a large cDNA fragment, encoding amino acid residues 37-570 of the human plakoglobin, fused to the GAL4 DNA binding domain in the pGBT9 vector (Clontech).
  • This bait plasmid was assayed for interaction with proteins encoded by a GAL4 activation domain cDNA library from human fetal kidney (Clontech).
  • three H/S3-positive, acZ-positive clones were isolated.
  • the library plasmids could be isolated and the sequences of the inserts were determined.
  • the first plasmid designated by us PLA_2H01 (PLAkoglobin 2Hvbrid clone 1), encoded almost the entire cytoplasmic domain of human N-cadherin.
  • PLA_2H01 PLAkoglobin 2Hvbrid clone 1
  • PLA_2H03 contained a fragment of human P-cadherin.
  • Several library plasmids contained fragments of cytokeratin 8 cDNA.
  • the extracellular domain of human Notch 2 was also isolated as a putative interaction partner of plakoglobin.
  • Two cDNA clones encoded nuclear proteins such as NRF1 (nuclear respiratory factor 1; Chan et al., 1993) and NDP52 (nuclear dot protein 52), also known as nuclear domain 10 protein (Korioth et al., 1995).
  • These prey plasmids comprised, respectively, nt 1431-1984/aa 168-322 of human NRF-1 cDNA/protein (Genbank Acc. No. HUMNRF1A; genpept Ace. No.
  • NRF1 belongs to the CNC-bZIP family of transcription factors forming a complex with transcription factors of the Fos/Jun family (Novotny et al., 1998; Venugopal and Jaiswal, 1998), whereas NDP52 has been reported to localize mainly in the cytoplasm besides the nucleus (Sternsdorf et al., 1997).
  • PLA_2H12 contained unknown cDNAs.
  • Two of these (PLA_2H12 and PLA_2H15) turned out to be identical; their sequence (indicated between black arrows in Fig.3) is disclosed in SEQ ID N° 5. These two identical clones were chosen for further examination.
  • the sequence of PLA_2H34 ( Figure 7) is disclosed as SEQ ID N° 7.
  • the primary purpose of this test was to check the specificity of the interaction between plakoglobin and the polypeptides encoded by the abovementioned clones, more particularly NRF-1 , NDP52, PLA_2H12 and PLA_2H34.
  • the isolated two-hybrid plasmids were retransformed into the HF7c strain and were combined with various fragments of plakoglobin fused to the GAL4-DBD ( Figures 1 and 8). Assuming that only few of the fragments of plakoglobin will interact, we considered the non-interacting fragments as negative and the interacting fragments as positive controls. In this way, we could also narrow down the PLA_2H12 binding region of plakoglobin.
  • the isolated two-hybrid clone PLA_2H12 was retransformed into the HF7c strain.
  • This PLA_2H12 prey clone was then combined with either one of the following bait plasmids (Fig. 1): the original bait plasmid pGBT9Plako (227-1853), the pGBT9-Plako (227-461), the pGBT ⁇ PIako (227-1028), the pGBT9Plako (227-1335), the pGBT9-Plako (1335- 1853), the pGBT9Plako (1335-2120), the pGBT9Plako (1853-2340), the pGBT9Plako (558-998) and the pGBT9-Plako (558-2120).
  • the PLA_2H12 plasmid transformants exhibited the desired H/S3-positive, ⁇ -galactosidase-positive phenotype upon combination with pGBT9Plako (227-1853) as expected, but also upon combination with the pGBT9Plako (227-1335), the pGBT9Plako (1335-1853) or the pGBT9Plako (558-2120) bait plasmids (Fig. 1).
  • the GAL4-DBD fusion proteins encoded by the pGBT9Plako (227-1335) and the pGBT9Plako(1335-2120) plasmids both interact with the polypeptides encoded by the prey plasmids NRF1 and NDP52.
  • This can be explained by a bipartite binding site of plakoglobin for PLA_2H12 (Fig. 1). Stronger binding is achieved when both domains are present. However, one of both domains is sufficient for the binding of PLA_2H12. The same holds true for interaction with the NRF1 and NDP52 polypeptides.
  • Example 3 Northern Blot Analysis
  • RNA of various human cell lines and human tissues was hybridized with a 32 P-labeled EcoRI-fragment of the PLA_2H12 cDNA.
  • a very weak signal could be detected in most cell lines or normal tissues (Fig. 2).
  • a stronger signal was seen in the lanes with SW620, SW1116, DLD1 R2/7 and LICR- HN3 RNA.
  • the positions of 28S and 18S ribosomal RNA on the blot were visualized using methylene blue staining. Using these positions as markers, we could estimate the size of the PLA_2H12 mRNA to be about 3.7 Kb.
  • Example 4 Isolation of the full-length cDNA of PLA 2H12 and in silico analysis of the predicted protein seguence
  • the PLA_2H12 plasmid as isolated from the yeast two-hybrid screen, contained a cDNA insert of about 800 bp (flanked by black arrows in Fig. 3). This cDNA fragment revealed a complete open reading frame, but neither a start, nor a stop codon was present in this sequence.
  • the most interesting feature predicted by the program was a stretch rich in basic amino acids (Arg, Lys) that was annotated as a putative bipartite nuclear localization signal (residues 252-273; Fig. 3). Indeed, the cellular localization as predicted by this program was nuclear, with a reliability of 89%. Moreover, the program also predicts the presence of two leucine zipper domains (Fig. 3).
  • SH3 domains of about 60 aa in length were first identified in the non- catalytic part of several cytoplasmic protein tyrosine kinases, as Src, Abl and Lck. Later on, it has been found in a great variety of other intracellular or membrane- associated proteins (Pawson and Schlessinger, 1993; Pawson, 1995b). SH3 domains may regulate protein localization or enzymatic activity, and often participate in the formation of multiprotein signaling complexes (Morton and Campbell, 1994; Pawson, 1995a). This protein interaction module can be implicated, like other signaling domains such as SH2, PTB, PDZ and WW domains, in a diverse array of signaling events (Pawson, 1995a).
  • plakoglobin can bind to LEF/TCF transcription factors and influence transcription of target genes (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996). In contrast, it was shown that even membrane-anchored plakoglobin can induce a Wnt-like phenotype in Xenopus (Merriam et al, 1997). The latter study shows that nuclear translocation of plakoglobin is not required to induce axis duplication in the early Xenopus embryo.
  • this membrane-anchored plakoglobin did not induce an increase in cytoplasmic, nor in nuclear ⁇ -catenin (Merriam et al., 1997).
  • the plakoglobin-mediated signal should be transduced into the nucleus via other mechanisms, such as the interaction with the PLA_2H12 protein.
  • the original PLA 2H12 clone did not contain the putative nuclear localization signal, nor the SH3 domain.
  • one of both leucine zipper motifs was also excluded from the clone, leaving the second leucine zipper motif as the only domain present.
  • the PLA_2H12 protein might function in this signaling pathway through its SH3 and leucine zipper domains. Another possible mechanism implies the nuclear translocation of very low levels of the plakoglobin protein through its interaction with PLA_2H12, as the latter contains a putative nuclear localization signal.
  • PLA_2H12-specific genomic sequences Two human PLA_2H12-specific genomic sequences (Genbank Ace. No. AL049851 and AL022315) were identified on the basis of a BLASTN search analysis of GenBank (Fig. 5).
  • the PLA_2H12 gene is partly represented in these two BAC clones, which reside within the human chromosomal region 22q13.1.
  • the exon-intron boundaries of the PLA_2H12 gene could be determined by comparison between the cDNA and genomic sequences (Table 8). They all turned out to be consistent with the ag-gt rule (Mount, 1982).
  • the splice donor/acceptor probability scores were determined according to Shapiro and Senapathy (Shapiro and Senapathy, 1987) (Table 8).
  • the gene consists of 21 exons identified so far, ranging in size from 27 nt (exon 11) to 1 ,032 nt (exon 21).
  • the length of intron 11 and intron 12 could not be determined, because the PLA_2H12 gene is only partly represented in these two non- overlapping BACs (Table 8).
  • Example 6 PLA 2H12 expression analysis Using the human Rapid-ScanTM panel, we tried to investigate the expression pattern of the novel plakoglobin-binding protein PLA_2H12 (Fig. 6). No PLA_2H12-specific bands could be detected in the cDNA pool of brain or fetal brain, nor in adrenal gland, muscle, stomach, bone marrow and fetal liver. A weak signal was visualized using heart, placenta, ovary and prostate cDNA as a template. Strong to very strong signals were detectable in cDNA from kidney, spleen, liver, lung, colon, small intestine, testis, salivary gland, thyroid gland, pancreas, uterus, skin and PBL (peripheral blood lymphocytes) (Fig. 6).
  • PBL peripheral blood lymphocytes
  • Example 7 Transformation of the full length human PLA 2H12 and fragments thereof, in the yeast two-hybrid system using yeast strain AH109
  • PLA_2H12 fragments tested in the yeast two-hybrid assay appear to interact with the human Armadillo-like proteins plakophilin-1 (PKP1) and plakophilin-3 (PKP3).
  • PPKP1 and PKP3 proteins belong to the superfamily of Armadillo-related proteins, to which also plakoglobin belongs. This also suggests the Armadillo repeats to be important for interaction with the PLA_2H12 protein, since this is the most conserved domain in these proteins.
  • FVR702F SE gcaggacagcaggcaggagc
  • FVR707R SE aggggaactcgctcaggctc
  • FVR729R SE gctgcctccccagttgcttc
  • FVR730R SE ggcctggtctcgctccttct
  • FVR731F SE PCR cgcaggacagcaggcaggag
  • FVR732F SE tggaggcggagcgggatgag
  • FVR733R SE PCR caggatggaaagccgattga
  • FVR1042F SE 3'RA gcaactggggaggcagctgtcat
  • FVR1521R SE gggccatccaggagtctgtt
  • a F (Forward) and R (Reverse) refers to the sense or antisense orientation of the primers.
  • Table 2a Primers used for in-frame cloning of human plakoglobin cDNA into pGBT9 vector
  • Table 2b PCR Primers used for in-frame cloning of the full length human plakophilin-3 open reading frame into the pGBKT7 and pGADT7 vectors
  • FVR1847R atacgtcgacacagccaacccccacctct a R (Reverse) and F (Forward) refers to the sense or antisense orientation of the primers. Restriction sites added are underlined and in bold.
  • FVR217R pGBT9 aaaatcataaatcataagaa
  • FVR283 SE cgccagggttttcccagtcacgac
  • FVR284 SE tcacacaggaaacagctatgac
  • FVR240F ⁇ ' RACE UAP gaattcgtcgactagtac Table 6: PLA 2H clones isolated from a GAL4-DBD yeast two-hybrid fetal kidney cDNA library
  • EST-ID identification number of EST (Expressed Sequence Tag); Clone ID: identification number of the clone; NCBI-ID: identification number according to NCBI database; Genbank-ID: identification number according to the Genbank database; P-score: index which indicates sequence homologies according to the BLAST algoritm (Altschul et al., 1990); Tissue: tissue used to isolate EST
  • Table 8 Overview of the exons and introns of the human PLA 2 H12 gene (part i of 2)
  • Table 8 Overview of the exons and introns of the human PLA 2H12 gene (part 2 of 2)
  • E-cadherin is a tumor/invasion suppressor gene mutated in human lobular breast cancers. EMBO J. 14:6107-6115. Berx, G., Cleton-Jansen, A.-M., Strumane, K., de Leeuw, W.J.F., Nollet, F., van Roy, F.M., and Comelisse, C 1996.
  • E-cadherin is inactivated in a majority of invasive human lobular breast cancers by truncation mutations throughout its extracellular domain.
  • Plakophilin-3 a novel Armadillo-like protein present in nuclei and desmosomes of epithelial cells. J. Cell Sci. 112: 2265-2276.
  • NDP52 a novel protein of the nuclear domain 10, which is redistributed upon virus infection and interferon treatment. J. Cell Biol. 130: 1-13. Korman, N., Eyre, R.W., Klaus-Kovtun, V., and Stanley, J.R. 1989. Demonstration of an adhering junction molecule (plakoglobin) in the autoantigens of pemphigus foliaceous and pemphigus vulgaris. New Engl. J. Med. 321 :631-635.
  • Plakophilins 2a and 2b constitutive proteins of dual location in the karyoplasm and the desmosomal plaque. J. Cell Biol.
  • XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86:391- 399.
  • Nrfl in a complex with fosB, c-jun, junD and ATF2 forms the AP1 component at the TNF alpha promoter in stimulated mast cells.
  • Plakophilin 3 a novel cell-type-specific desmosomal plaque protein
  • the cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway.
  • Cadherin cell adhesion receptors as a morphogenetic regulator.
  • Watabe-Uchida M., Uchida, N., Imamura, Y., Nagafuchi, A., Fujimoto, K., Uemura, T., Vermeulen, S., van Roy, F., Adamson, E.D., and Takeichi, M. 1998.
  • -Catenin-vinculin interaction functions to organize the apical junctional complex in epithelial cells. J. Cell Biol. 142: 847-857.

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Abstract

La présente invention concerne de nouvelles protéines interagissant avec la plakoglobine humaine. Plus particulièrement, l'invention a pour objet de nouvelles protéines qui sont impliquées dans la transduction vers le noyau d'un signal lié à la plakoglobine.
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EP2519633A4 (fr) * 2009-12-29 2014-01-01 Curna Inc Traitement de maladies liées au facteur respiratoire nucléaire 1 (nrf1) par l'inhibition du produit de transcription antisens naturel de nrf1
CN111647063A (zh) * 2020-06-13 2020-09-11 郑州大学 Nfe2l1的截短蛋白及其应用

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WO2001040468A2 (fr) * 1999-12-01 2001-06-07 Millennium Pharmaceuticals, Inc. Nouvelles molecules de la famille de proteines de type card et utilisations de ces dernieres

Cited By (5)

* Cited by examiner, † Cited by third party
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EP2519633A4 (fr) * 2009-12-29 2014-01-01 Curna Inc Traitement de maladies liées au facteur respiratoire nucléaire 1 (nrf1) par l'inhibition du produit de transcription antisens naturel de nrf1
US8921334B2 (en) 2009-12-29 2014-12-30 Curna, Inc. Treatment of nuclear respiratory factor 1 (NRF1) related diseases by inhibition of natural antisense transcript to NRF1
US9663785B2 (en) 2009-12-29 2017-05-30 Curna, Inc. Treatment of nuclear respiratory factor 1 (NRF1) related diseases by inhibition of natural antisense transcript to NRF1
CN111647063A (zh) * 2020-06-13 2020-09-11 郑州大学 Nfe2l1的截短蛋白及其应用
CN111647063B (zh) * 2020-06-13 2021-07-23 郑州大学 Nfe2l1的截短蛋白及其应用

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