identification of a novel binding partners for tumor suppressor pten

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Cowden disease and Bannayan-Zonana syndrome [1]. Pten encodes a tumor suppressor protein PTEN, which shares a high degree of homology with the family.
Experimental Oncology 26, 15-19, 2004 (March) Exp Oncol 2004 ORIGINAL CONTRIBUTIONS 26, 1, 15-19

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IDENTIFICATION OF A NOVEL BINDING PARTNERS FOR TUMOR SUPPRESSOR PTEN BY A YEAST TWO-HYBRID APPROACH Olena Gorbenko 1, Vitaliy Kuznetsov 1, Olexandr Kukharenko 1, Alexandr Zhyvoloup 1, Ganna Panasyuk 1, Ivan Nemazanyy 1, Valeriy Filonenko 1, Ivan Gout1,2* 1 Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine 2 University College London, Department of Biochemistry and Molecular Biology, London WC1E 6BT, United Kingdom Aim: To identify novel PTEN-binding partners. Methods: The technique of yeast two-hybrid screening was used in this study. A panel of bait constructs was created, containing the C-terminal domain of PTEN, full length PTEN, activated and phosphatase-dead mutants. The expression of LexA-fused baits, their nuclear localization and autoactivation potential were tested according to the standard protocol of Duplex A system. CDNA libraries from Colon Cancer, HeLa and Mouse Embryo were screened with two selected bait constructs. Isolated positive clones were further analysed by mating assay and identified by automated DNA sequencing and database searching. Results: Extensive screening of cDNA libraries with the full length and the C-terminal domain of PTEN led to the identification of 43 positive clones, which were confirmed in mating assay. Sequence analysis indicated that two clones encode AEBP1 (Adipocyte Enhancer Binding Protein 1). Conclusion: Our data indicate that the interaction between PTEN and AEBP1 is mediated by their C-terminal and N-terminal domains, respectively. The functional importance of PTEN-AEBP1 interaction is currently under investigation.

Pten (phosphatase and tensin homologue deleted on chromosome ten) also referred to as MMAC (mutated in multiple advanced cancers) is a tumor suppressor gene, which is frequently mutated in a wide range of human cancers, including glioblastoma, melanoma, prostate, breast and endometrial cancers. Germline PTEN mutations are present in patients with Cowden disease and Bannayan-Zonana syndrome [1]. Pten encodes a tumor suppressor protein PTEN, which shares a high degree of homology with the family of protein-tyrosine phosphatases and cytoskeletal protein tensin. PTEN structural domains include an N-terminal phosphatase domain within which the consensus of phosphatase signature motif lies, a lipid-binding C2 domain, and a 50 amino acid C-terminal tail that contains a PDZ-binding sequence (usually found in proteins that serve as scaffolds for the assembly of multiprotein complexes) [2, 3]. Tumor suppressor function of PTEN is directly associated with its lipid phosphatase activity which dephosphorylates the lipid second messenger phosphatidylinositol 3, 4, 5-triphosphate and by doing so antagonizes the action of phosphoinositide 3-kinase (PI3-kinase) [4]. Akt/PKB kinase is a key indirect target of PTEN. It is well established that the products of PI3-kinase activity, PtdIns-3,4-P2 and PtdIns-3,4,5P3, activate Akt/PKB by binding to its pleckstrin homology domain. Activated Akt/PKB modulates a number of downstream targets that affect cell growth, proliferation and the cell cycle. In addition, Akt/PKB promotes cell survival by inhibiting the function of Received: February 12, 2004. *Correspondence: E-mail: [email protected] Abbreviations used: Pten — phosphatase and tensin homologue deleted on chromosome ten.

proapoptotic proteins, such as BAD and caspase 9 [5]. Recently, signaling via PI3-K/Akt pathway has been implicated in the regulation of glucose and lipid metabolism [6]. PTEN exhibits also protein phosphatase activity. It has been demonstrated that PTEN dephosphorylates FAK, a key mediator of integrin-mediated signaling pathway. The interaction between PTEN and FAK induces dephosphorylation of tyrosine-phosphorylated FAK, resulting in inhibition of cell migration, spreading and focal adhesion. Studies from several laboratories have established that the expression of FAK is often elevated in proliferating cells of advanced cancers [7, 8]. In addition to its role in regulating the PI3K/Akt cell survival pathway, PTEN also inhibits growth factor-induced phosphorylation of an adapter protein Shc and suppresses the mitogen-activated proteins kinase (MAPK) signaling pathway [9]. The data presented above demonstrate that PTEN is involved in the regulation of diverse cellular processes by modulating the function of key players in different signaling pathways. The majority of PTEN missense mutations are located in the phosphatase domain causing the loss of PTEN phosphatase activity. It is also important to note that a large number of PTEN nonsense or frame-shift mutations are targeted to the C-terminal domain, suggesting an important role of this domain in the regulation of the PTEN tumor suppressor activity [10]. To date, a significant progress has been made in structure-function analysis of PTEN and deciphering its involvement in malignant transformation and the progression of the disease [11, 12]. However, very little is known about the mechanisms controlling the expression and regulation of PTEN. So far, only few PTENbinding partners have been identified, mainly through

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Experimental Oncology 26, 15-19, 2004 (March)

the use of yeast two-hybrid system. In 2000, a group from the Department of Molecular Oncology at GENETECH (San Francisco) identified the first partner of PTEN in an extensive yeast two-hybrid screening using as bait the N-terminal phosphatase domain fused to LexA protein. It was found to be a novel protein, termed MAGI-3, which belongs to the MAGUK-family (membrane-associated guanilate kinases) and is highly homologous to rat MAGI-1 and human AIP-1/S-SCAM (Atrophin-Interacting Protein/Synaptic-Scaffolding molecule) [13]. The authors found that the interaction of SCAM and MAGI-1 with PTEN is mediated via their PDZ domains and the C-terminal PDZ-binding motif in PTEN. Later on, MAGI2 (a truncated form of MAGI3) was also found to interact with PTEN [14]. To identify novel PTEN partners we have created several PTEN bait constructs for the use in yeast twohybrid system [15]. Analysis of generated constructs indicated that only two of them were suitable for screening libraries. Extensive screening of three cDNA libraries from Mouse Embryo, HeLa and Colon Cancer led us to the isolation of 43 cDNA clones which were further confirmed in mating assay. The identity of isolated cloned was found by DNA sequencing and database searching. Two clones encoded AEBP1 (adipocyteenhancer binding protein). AEBP1 is a transcription factor with carboxypeptidase activity, whose function is linked to signal transduction and lipid metabolism [16]. We have suggested the model of interaction and regulation of AEBP1 transcription activity by tumor suppressor PTEN.

MATERIALS AND METHODS DupLexATM yeast two-hybrid system, developed by OriGene Technologies Inc. (USA) has been employed in the study. The following components of the system have been used: yeast strains EGY48 and RFY206; bacterial strain E.coli KC8; reporter plasmids pSH18-34, PJK101; control plasmids pRHFM1, pSH17-4, pEG202-pLexA-Max, pBait, pTarget; vectors for bait and prey constructs pEG202, pEG202-NLS, pYESTrp and pJG4-5. Creation of bait-constructs. Four forms of PTEN were used for making bait constructs: wild type, phosphatase-dead (Cys124 substituted for Ser, primers 5'GTCGAATTCATGACAGCCATCATCAAAGAG-3', 5'-CGAGGATCCTCAGACTTTTGTAATTTGTGTATGC-3'), C-terminal region (primers 5'-CGTCGAATTCGAAACTATTCCAATGTTCAGTGG-3', 5'-CGAGGATCCTCAGACTTTTGTAATTTGTGTATGC-3') and PTEN activated mutant generated in our laboratory by PCR-based site-directed mutagenesis (Ser370 substituted for Asp using following primers 5' ACACCAGATGTTGATGACAATGAACCTGATCA-3', 5'-GTTCATTGTCATCAACATCTGGTGTTACAGAAG-3'). DNA fragments were amplified by Vent polymerase (New England BioLabs, UK). PCR-amplified DNA fragments were cloned into ðÅG202 and pEG202-NLS vectors using ligation kit from “Takara” (Japan).

Transformation and selection of recombinant clones. Yeast strains were transformed with generated constructs and DupLexA system plasmids by PEG-lithium method as recommended by OriGene Technologies. Transformation of E.coli KC8 (Trp-) with DNA plasmids was performed by electroporation using Electroporator 2510 (Eppendorf, Germany) device under 14 kV/cm2 voltage and 4-5 msec conditions. Selection of Trp+ -clones was performed as recommended by OriGene protocol. SDS-PAGE electrophoresis and immunoblotting. Yeast protein extracts were prepared in accordance with CLONTECH Yeast Protocols Handbook. Cells were lysed on ice in equal volume of buffer A (50 mM ammonium acetate, 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1 mM PMSF, 0.03 mM leupeptin, 145 mM benzamidine, 0.37 mg/ml aprotinin, 0.1 mg/ml of pepstatin A), glass beads and 20% trichloracetic acid. The mixture was vortexed for 2 min before centrifugation. Proteins from prepared lysates were resolved on a 4–12% SDS-PAGE gradient gel and transferred to PVDF-membrane using semidry Trans-Blot SD device (BioRad, USA). The expression of PTEN LexA-fused proteins was analyzed with polyclonal anti-LexA antibodies, kindly provided by Dr. Erica Golemis (USA) [17]. The blot was developed using the Western Lightning chemoluminiscence reagent kit (ECL “Amersham”, UK) Transactivation and nuclear localization tests of PTEN-baits. The ability of PTEN LexA-fused proteins to transactivate the system was examined according to the manufacturer’s recommendations. Transactivation activity has been estimated by the level of colony coloring in X-Gal containing medium and by growth in leucine-depleted medium. The ability of PTEN LexA-fused proteins to translocate to the nucleus and to bind to the LexA-specific DNA operator sequence was detected by β-galactosidase activity of LacZ reporter gene. Yeast two-hybrid screen. The two-hybrid screening of three cDNA libraries from Mouse Embryo (OriGeneTM Technologies), HeLa cells and Colon Cancer (Invitrogen) was carried out using the C-terminal domain and activated mutant of PTEN. Bait constructs, reporter plasmids and cDNA libraries were sequentially transformed into EGY48 yeast strain using the method of yeast transformation in semi-fluid agarose [18]. The primary selection of positive yeast clones by Leu+ phenotype was carried out on agarized galactose-containing Leu-minimal medium which was selective for reporter, bait and prey plasmids (–Ura, –His, –Trp, respectively) as recommended by the protocol. To fulfill a color LacZ selection, the colonies were overlaid with X-Gal containing solution of agarose. The blue color selection was used to identify transformants in which the interaction between PTEN bait-proteins and library prey-proteins took place. The secondary screening of transformants was carried out by comparative analysis on 4 types of media with or without galactose inductor: –HUTdex (–Ura,

Experimental Oncology 26, 15-19, 2004 (March) –His, –Trp, +glucose), -HULTdex (–Ura, –His, –Trp, –Leu, +glucose), –HUTgal (–Ura, –His, –Trp, +galactose), –HULTgal (–Ura, –His, –Trp, –Leu, +galactose). Positives were selected by their ability to grow on –HULTgal/not to grow on –HULTdex, and to become blue on –HUTgal/not to become blue on –HUTdex. The quantitative analysis of LacZ and Leu+ phenotypes after galactose induction was performed in 96-well plates and calculated by formulae: NlacZ = (Gal405/Glu405) x (Glu620/Gal620), where Galxxx, Gluxxx-values of E405 and E620 wavelengths for galactose- and glucose-containing media, respectively. Nleu = (LEUGal620/LEUGlu620) x (Glu620/Gal620), where LEUGal620, LEUGlu620 are values of E620 of cell cultures in galactose-, glucose-containing media from Leu+ phenotype analysis, Glu620, Gal620 are values of Å620 of cell cultures in galactose-, glucose-containing media from LacZ phenotype analysis [19]. Analysis of plasmid DNAs. The analysis of plasmid DNAs of selected positive (LacZ Leu+) yeast clones was performed according to yeast two-hybrid system protocol. E.coli bacteria were transformed with isolated prey-DNA plasmids from libraries and grown on LB selective medium with 50 µg/ml of ampicillin. HindIII, XhoI and EcoRI, XhoI restrictases were used to digest HeLa, Colon Cancer and Mouse Embryo DNA plasmids, respectively. Comparative analysis of DNA restricted products was carried out by electrophoresis in 1.2% agarose gel. Mating assay. RFY206 (MATa) yeast strain was transformed with isolated prey-plasmids and systemic pTarget plasmid. Mating between RFY206 (MATa) and EGY48 (MATα strains containing two variants of bait vectors and systemic pBait plasmid together with PSH18-34 reporter plasmid) was performed by the method adapted for 96-well plates. MATa and MATα transformed strains were grown on selective medium (–Trp for MATa and –Ura, –His for MATα) in plate wells to OD600 = 1. Later on, 25 µl of yeast suspension was mixed with enriched YPD medium in various combinations “MATa x MATα” and incubated for 36 h at 30 oC. Cell suspensions were washed in water and used for quantitative analysis of LacZ and Leu+ phenotypes of the mated clones, as described earlier, and on plates with agar medium according to OriGENE protocol.

RESULTS AND DISCUSSION To identify binding partners, which can specifically recognize various conformational forms of PTEN, we have created a panel of baits containing wild type, activated and phosphatase-dead forms of PTEN. Structural studies of PTEN indicated that the phosphatase and C2 domains associate across an extensive surface [20]. To remove the constrains of the phosphatase domain on the C2 domain and PDZ domain recognition motif, we have also created the bait with the C-terminal domain of PTEN. The schematic presentation of LexA/PTEN fusion constructs is shown in Fig. 1. The decision to make a whole panel of bait constructs could be also justified by the fact that many types of

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Fig. 1. Schematic representation of LexA/PTEN fusion constructs in pEG202-NLS

bait do not fulfill the criteria of suitability for yeast twohybrid screening. We have used a standard pEG202 plasmid and pEG202-NLS plasmid, which contains nuclear localization signal at the N-terminus. The expression of recombinant LexA/baits was examined by SDS-PAGE electrophoresis under denaturing conditions and immunoblotting with anti-LexA antibodies. As shown in Fig. 2, total lysates of transformed yeasts contain LexA fused proteins of predicted sizes and a control protein LexA-Max. Then, we tested all bait constructs for nuclear localization and in an autoactivation assay. The analysis indicated that all bait constructs do not autoactivate reporter gene transcription, however only two of them, LexA/C-term PTEN and LexA/ActPTEN are capable to translocate to the nucleus (data not shown). Based on these criteria, we have selected LexA/C-term PTEN and LexA/ActPTEN baits for screening of cDNA libraries from Mouse Embryo, HeLa cells and Colon Cancer.

Fig. 2. Expression analysis of various LexA/PTEN bait constructs in yeast. 1: LexA/PTEN-dead form in pEG202-NLS; 2: LexA/ PTEN C-terminal region in pEG202-NLS; 3: LexA/PTEN-activated form in pEG202-NLS; 4: LexA-Max protein as an expression control. Western blot with anti-LexA antibodies

After transformation with cDNA libraries, colonies were grown on selective galactose-containing medium without leucine. The selection of positive clones was carried out by blue color (in the presence of X-Gal) and by their ability to grow on leucine-depleted medium. Nearly 600 primary transformants were identified and stroked simultaneously on four types of media (see Materials and Methods). Secondary selection was performed by the ability of yeast colonies to grow on leu-

18 cine-depleted medium with galactose induction/not to grow on leucine-depleted medium with glucose and become blue on galactose medium/not to become blue on glucose medium. The quantitative assay of Leu+/ LacZ phenotypes allowed us to select 50 positive clones (46 of which interacted with the C-terminal PTEN bait and 4 with the activated form of PTEN. Further analysis of isolated clones in mating assay confirmed the specificity of interaction for 43 clones (among those, 7 clones exhibited a very weak interaction). The identity of isolated clones was found by automated DNA sequencing and database searching. Two clones (N76 and N91), isolated by screening of Colon Cancer library with the C-terminal PTEN showed 96 and 100 % homology to human AEBP1 (adipocyte enhancer binding protein 1). AEBP1 is a transcriptional repressor with carboxypeptidase (CP) activity. There are three splicing forms of AEBP1, which have different N-terminal sequences, encoding for proteins of 82, 95 and 175 kDa. Restriction and sequencing analysis indicated that both clones have the inserts of approximately 600 bp, initiating at amino residue 35 of the shortest splicing form of AEBP1. Taking together, these results indicate that the N-terminal region of AEBP1 mediates the interaction with the C-terminal region of PTEN. The N-terminal region of AEBP1 possesses DLD domain (Discoiding-like domain), which is implicated in lipid binding. Interestingly, the C-terminal region of PTEN encodes another type of lipid-binding module, C2 domain. The mechanisms, which regulate the association between AEBP1 and PTEN, and its functional consequences remains to be elucidated. AEBP1 represent a new type of transcription factor that might regulate gene expression by cleavage of proteins involved in transcription. AEBP1 belongs to B-like carboxypeptidase family involved in different metabolic and signal transduction processes [21]. Biochemical and genetic studies of different splicing forms of AEBP1 indicate their involvement in cell differentiation [22]. The model of adipocyte differentiation has been used extensively to study the expression and the role of AEBP1 in this process. It was found that AEBP1 binds to the regulatory sequence AE (adipocyte enhancer) in the proximal part of aP2-gene promoter encoding for FABP4 (fatty acid-binding protein) and negatively regulates its transcription [16]. On the other side, a tumor suppressor PPARγ is known to activate the expression of aP2 gene, which correlates with adipogenesis and preadipocyte differentiation [23]. The mechanism of PPARγ action is still unclear, but it was shown that the activation of PPARγ increases PTEN expression in pancreatic cancer cells [24]. Furthermore, overexpression of fatty acid synthase (FAS) in PTEN-null prostate cancer cell line PC3 has been observed [25]. This phenomenon is attributed to the increase of Akt/PKB activity, since the expression of FAS is known to be regulated via PI3-K signaling pathway. It has been also demonstrated that PI3-kinase inhibitor LY 294002 as well as reintroduction of PTEN considerably decreases FAS expression in PC3 cells [25].

Experimental Oncology 26, 15-19, 2004 (March) The data presented in this study establish a potential link between PTEN and AEBP1, a negative regulator of adipogenesis. This finding may give us the insight into the role of PTEN in non-cancer related diseases, such as diabetes. It is tempting to propose a working model, linking PI3-K pathway to the regulation of adipogenesis via AEBP1 (Fig. 3). We suggest that PTEN can regulate the expression of adipogenic genes at two levels: a) by dephosphorylating the products of PI3-K activity, which activate Akt/PKB pathway, b) by direct modulation of AEBP1 transcriptional activity. We are currently investigating the specificity of PTEN/AEBP1 interaction in mammalian cells and its functional importance.

Fig. 3. Regulation of adipogenesis via PI3-K/PTEN signalling pathway and a transcriptional repressor AEBP1

ACKNOWLEDGEMENTS This work was supported in part by FEBS Short Term Fellowship. Also we want to thank our collaborators in Laboratory of Cell Growth Regulation and scientists from Ludwig Institute for Cancer Research for their time and help.

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19 17. Toby GG, Golemis EA. Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions. Methods 2001; 24: 201–17. 18. Panasyuk G, Nemazanyy I, Filonenko V, Zhyvoloup A. Large-scale yeast transformation in low-percentage agarose medium. Biotechniques 2004; 36: 40–2. 19. Zhyvoloup AM, Nemazanyy IO, Pobigaylo NV, Panasyuk GG, Palchevskyy SS, Kukharenko OP, Savinska LO, Ovcharenko GV, Vudmaska MI, Gout IT, Matsuka GKh, Filonenko VV. The use of yeast two-hybrid screen in search of S6K1 and S6K2 binding proteins Biopolymers Cell 2002; 18: 102–9 (in Ukrainian). 20. Georgescu MM, Kirsch KH, Akagi T, Shishido T, Hanafusa H. The tumor-suppressor activity of PTEN is regulated by its carboxyl-terminal region. Proc Natl Acad Sci USA 1999; 96: 10182–7. 21. Muise AM, Ro HS. Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA. Biochem J 1999; 343: 341–5. 22. Ro HS, Kim SW, Wu D, Webber C, Nicholson TE. Gene structure and expression of the mouse adipocyte enhancer-binding protein. Gene 2001; 280: 123–33. 23. Pelton PD, Zhou L, Demarest KT, Burris TP. PPARgamma activation induces the expression of the adipocyte fatty acid binding protein gene in human monocytes. Biochem Biophys Res Commun 1999; 261: 456–8. 24. Farrow B, Evers BM. Activation of PPARγ increases PTEN expression in pancreatic cancer cells. Biochem Biophys Res Com 2003; 22: 50–3. 25. Van De Sande T, De Schrijver E, Heyns W, Verhoeven G, Swinnen JV. Role of the phosphatidylinositol 3'-kinase/PTEN/Akt kinase pathway in the overexpression of fatty acid synthase in LNCaP prostate cancer cells. Cancer Res 2002; 62: 642–9.

ÈÄÅÍÒÈÔÈÊÀÖÈß ÍÎÂÛÕ ÑÂßÇÛÂÀÞÙÈÕ ÏÀÐÒÍÅÐΠÎÏÓÕÎËÅÂÎÃÎ ÑÓÏÐÅÑÑÎÐÀ PTEN ÌÅÒÎÄÎÌ ÄÂÓÃÈÁÐÈÄÍÎÉ ÑÈÑÒÅÌÛ ÄÐÎÆÆÅÉ Öåëü: èäåíòèôèêàöèÿ íîâûõ ïàðòíåðîâ ôîñôàòàçû PTEN. Ìåòîäû: èñïîëüçîâàí ìåòîä äâóãèáðèäíîé ñèñòåìû äðîææåé. Ñêîíñòðóèðîâàííûå áàéò-êîíñòðóêòû, ñîäåðæàùèå Ñ-êîíöåâîé ó÷àñòîê, äèêóþ, ìóòàíòíóþ àêòèâèðîâàííóþ è íåàêòèâíóþ dead-ôîðìû, ïðîâåðåíû â òåñòàõ íà ýêñïðåññèþ, ïðîíèêíîâåíèå â ÿäðî è ñàìîàêòèâàöèþ ñ èñïîëüçîâàíèåì ñòàíäàðòíîãî ïðîòîêîëà Duplex A ñèñòåìû. Âûïîëíåí äâóãèáðèäíûé ñêðèíèíã êÄÍÊ áèáëèîòåê ðàêà êèøå÷íèêà, HeLa è ýìáðèîíà ìûøè ñ ïîñëåäóþùèì îòáîðîì ïîçèòèâíûõ êëîíîâ ìåòîäîì ïîëîâîãî ñëèÿíèÿ. Ïîñëåäîâàòåëüíîñòè èäåíòèôèöèðîâàíû ñ ïîìîùüþ ñèêâåíñ-àíàëèçà è ïîèñêà ãîìîëîãèè â áàçàõ äàííûõ. Ðåçóëüòàòû: ñêðèíèíã êÄÍÊ áèáëèîòåê ñ ïîìîùüþ ïîëíîðàçìåðíîé ôîðìû PTEN è åãî Ñ-êîíöåâîãî äîìåíà ïîçâîëèë îòîáðàòü 43 ïîëîæèòåëüíûõ êëîíà, ïîäòâåðæäåííûõ ìåòîäîì ïîëîâîãî ñëèÿíèÿ. Àíàëèç ïîñëåäîâàòåëüíîñòåé âûÿâèë, ÷òî äâà èç íèõ êîäèðóþò ÀÅÂÐ1 (Adipocyte Enhancer Binding Protein 1). Âûâîäû: ñîãëàñíî íàøèì äàííûì, âçàèìîäåéñòâèå ìåæäó PTEN è AEBP1 îïîñðåäîâàíî èõ Ñ-êîíöåâûì è N-êîíöåâûì ó÷àñòêàìè ñîîòâåòñòâåííî. Ôóíêöèîíàëüíîå çíà÷åíèå PTEN-AEBP1 âçàèìîäåéñòâèÿ èçó÷àåòñÿ.

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