US 200300.40089A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0040089 A1 Legrain et al. (43) Pub. Date: Feb. 27, 2003 (54) PROTEIN-PROTEIN INTERACTIONS IN Publication Classification ADIPOCYTE CELLS (76) Inventors: Pierre Legrain, Paris (FR); Stefano (51) Int. Cl." .......................... G01N 33/53; G06F 19/00; Marullo, Paris (FR); Jockers Ralf, G01N 33/48; G01N 33/50; Bures Sur Yvette (FR) C07H 21/04; C12N 9/00 (52) U.S. Cl. ......................... 435/183; 435/69.1; 435/7.1; Correspondence Address: 435/325; 435/320.1; 536/23.2; LERNER, DAVID, LITTENBERG, 702/19 KRUMHOLZ & MENTLIK 600 SOUTHAVENUE WEST (57) ABSTRACT WESTFIELD, NJ 07090 (US) Disclosed are protein-protein interactions in adipocytes. Also disclosed are complexes of polypeptides or polynucle (21) Appl. No.: 10/038,010 otides encoding the polypeptides, fragments of the polypep (22) Filed: Jan. 2, 2002 tides, antibodies to the complexes, Selected Interacting Domains (SID(R) which are identified due to the protein Related U.S. Application Data protein interactions, methods for Screening drugs for agents which modulate the interaction of proteins and pharmaceu (60) Provisional application No. 60/259,377, filed on Jan. tical compositions that are capable of modulating the pro 2, 2001. tein-protein interactions. Patent Application Publication Feb. 27, 2003 Sheet 1 of 19 US 2003/0040089 A1 pBl Alias: pAS2DD Application: 2HY (bait) --- Backbone: Specificity: 287 NSBfrB Selection:rare a amplicillin0 - Scal 6720 287 Ppu 1 Of COIStructedConstructed by:by Bpm 6327 1285 x 1288 B.Se R AWN 5760 pBl -- 1748 Bsp El 841 Pn Sap 5226 7159 base pairs 1956 Apa Bf Unique Sites 2. 2174 BSX Pst 4776 Sa 4770 Bar H 4764 Xnal 4759 Sgt E. 2880 Dra Nicolty 4749 2986 Naee Bst DS 4749 3289 Npe Ngo MIV Sf 4747 32.93 EC 136 MSc 4746 3293 Sac Hpal 4570 3384 Mill C BSf B 426 3465 Ppu M BSa B 3797 3515 ReA Oligo 160 gagagtagtaacaaaggtel AAAGACAGTTGACTGTATCGCCG GAA TTT AT Sf S" BamHI sali Pst G GCC ATG GAG GCC CCG GGG ATC CGT CGA CCT GCA GCC Nico I Oligo 161 AAG CTA ATT ccggg.cgaatttctatg Oligo 1605 GAGAGTAGTAACAAAGGTC3 Oligo 161 5’ CATAAGAAATTCGCCCGG 3 FIGURE I Patent Application Publication Feb. 27, 2003 Sheet 2 of 19 US 2003/0040089 A1 Alias: paS2DDNSI Application: 2HY (bait) 287 Bfirst Backbone: pAS2DD 287 NS Specificity. Sfinon-oriented Sca. 6761 287 Ppu 1 Ol Selection: ampicillin V Constructed by SW Bpm. 6368 1285 Xern 1288 Bse R Awn 5801 pB5 -- 18411748 BspPrin El Sap 5267 7200 base pairs s: 2E S Unique Sites 2124 Bst 36 2174 BSf X Pst 4817 a Bsp. 120 4805 Apa 4805 oSf 4797 Pac 4788 Bri 4782 2880 Dra Spe. 4774 V 2986 Nae Eagt 4767 2986 Ngo MV Not 4766 3289 N. Sac 4764 3293 Ech 136 MI 113 4764 3293 Sac a S. 4753 3384 Mul Styl 4749 3465 Ppu Mi Nico 4749 3515 ReA Msc 4746 Hpal 4570 BSfB 4026 Bsa B 3797 Oligo 160 gagagtagtaacaaaggtelAAAGACAGTTGACTGTATCGCCG GAA TTT ATG Sf Sac II Spe I Ban HI GCC ATG GCC GCA GGG GCC GCG GCC GCA CTA GTG GGG ATC C Nico I Not STOP Sf Pst TT AAT TAAGGG CCA CTG GGG CCC CTC GAC CTG CAGCCA Pac Oligo 161 AGC TAA TT|ccgggcgaatttctatg Oligo 160 5 GAGAGTAGTAACAAAGGTC3 Oligo 161 5’ CATAAGAAATTCGCCCGG3 FIGURE 2 Patent Application Publication Feb. 27, 2003 Sheet 3 of 19 US 2003/0040089 A1 pB6 Application: 2HY (bait) 287 Bir B Backbone: pAS2DD 287 NS Specificity: Sfi oriented Scat 6761 287 Ppu 10) Selection: ampicillin Constructed by: SW Bpm 6368 1285 Xcin 288 Bse R Afwn 5801 pB6 -- 1748 Bsp El 1841 Pri Sap 15267 7200 base pairs 1956 Apa B Unique Sites 1956 BSAP 2124 Bsu 36 2174 BSX Pst 4817 Bsp120 4805 Apa 4805 oS 4797 Pact 4788 Ban H 4782 2880 Dral Spel 4774 2986 Nae Eag 4767 2986 Ngo MV No. 4766 3289 Niru Saci. 4764 3293 EC 136 M113 4764 3293 Sac Sf 4753 3384 Mill Styl 4749 3465 Ppu Mi Nico 4749 3515 ReA Aisc 4746 HpaI 4570 Est B 4026 Bsa B 3797 Oligo 160 gagagtagtaacaaaggtoAAAGACAGTTGACTGTATCGCCG GAATTT ATG Sf Sac II Spe I Ban HI GCC ATG GCC GGA CGG GCC GCG GCC GCA CTA GTG GGG ATC C Nico I Not STOP sf, I Apal Pst TT AAT TAAGGG CCA CTGGGG CCC CTC GAC CTG CAGCCA Pac Oligo 161 AGC TAA TTccgggegaatcitatg Oligo 160 5 GAGAGTAGTAACAAAGGTC3 Oligo 161 5’ CATAAGAAATTCGCCCGG3? FIGURE 3 Patent Application Publication Feb. 27, 2003 Sheet 4 of 19 US 2003/0040089 A1 pB13 Alias: pGBT9NSl Application: 2HY (bait) Backbone: pCBT9 165 Bsts 651 Scil Specificity. Sfinon-oriented 651 Xho Selection: amplicillin 709 Hpa Constructed by: CR 724 BSrG 885 MSc 888 Nicol 888 Sty Ppu 10 5290 903 Milu 113 Wsi 5290 903 Sac Birs 5290 905. Not w 906 Eag 913 Spel Sna B 4936 921 Barr H 927 Pac 944. Apa 944 Bsp 12O 2 g56 Psi 1168 Nae pB13 1168 Ngo MW Aat 4160 -- 1440 BSF 5580 base pairs Unique Sites 1590 estx 1604 corv 1645 Bsu 36 1747 Mife Scal 3720 1803 Xibal 1809 Apa Bl Pwu 3609 809 BSAP 1929 Piml 217 Pvu Bpn 3327 V 2226 Sap Ahd 3237 2349 Af 2349 Bsp U11 2760 Alwin Oligo 160 AAAGACAGTTGACTGTATCGCCG GAA TTT ATG Sfi Sac II Spe I Bam H. GCC ATG GCC GCA GGG GCC GCG GCC GCA CTA GTG GGG ATC C Nico Not STOP Sf Pst TT AAT GGG CCA CTG GGG CCC CTC GAC CTG CAG CCA Pac Oligo 161 AGC TAA TT ccgggcgaatttctatg Oligo 1605 GAGAGTAGTAACAAAGGTC3 Oligo 161 5’ CATAAGAAATTCGCCCGG 3 FIGURE A Patent Application Publication Feb. 27, 2003 Sheet 5 of 19 US 2003/0040089 A1 pB14 Alias: pgET9NS2 Application: 2HY (bait) 165 Bst B Backbone: pCBT9 651 Sci Specificity. Sfi oriented 651 Xho Selection: ampicillin 709 Hipal 724 BSFG Constructed by: CR 885 Msc 888 Nool B88 Sty Ppu 10 529D 903 Milu 113 Nisi 5290 903 Sac. It 905 Nof Birb 5290, 906 Eagi 13 Spel Sna B 4936 21 Ban.H. g27 Pac 944 Apa 944 Bsp 12Ol 2. 956 Psf 1168 Nae pB14 1168 Ngo MV Aat 4160 -- 1440 BSF 5580 base pairs Unique Sites 1590 BSfx 1604 EcoRV 1645 Bsu 36 1747 Mife Scal 3720 1803 Xiba Pyu 3609 1809. Apa Bl 1809 BsfAP w 1929 Piral 2171 Pvt. Bpm. 3327 V 2226 Sap Ahd 3237 2349A 2349 Bsp U11 2760 Awn Oligo 160 gagagtagtaacaaaggte AAAGACAGTTGACTGTATCGCCG GAA TTT ATG Sf Sac Spe I Bam H. GCC ATG GCC GGA CGG GCC GCG GCC GCA CTA GTG GGG ATC C Nico Not I STOP sfi I Apa I Pst TT AAT GGG CCA CTG GGG CCC CTC GAC CTG CAG CCA Pac Oligo 161 AGC TAA TT ccggg.cgaatttctatg Oligo 160 5 GAGAGTAGTAACAAAGGTC3 Oligo 161 5’ CATAAGAAATTCGCCCGG3 FTCURE 5 Patent Application Publication Feb. 27, 2003 Sheet 6 of 19 US 2003/0040089 A1 pB20 Alias: plex10NS2 Application: 2HY (bait) 161 BSB Backbone: plex 10 (p59) 447 Hic Specificity. Sfi-oriented 447486 Hpal BC Selection:rass a amplicillin- 517 Mul Constructed by: LD 759 BSI 957 Pre 975 Bgll 998 XC Ppu 1 O 5451 04.5 EcoR Nsi 5451 1053 S 8 Bfirst 5451 1064. BSDS Ni387f7 5445 1064 Mu 113 Ava 5445 1064 Sacil O66 No tO67 Eag 1074 Spel 1082 Bar 188 Pac 1097 Sio 1105 Apal 1105 Bsp 120 1117 Ps pB20 1329 Nae - 1329 Ngo MM Aat 4321 -- 5746 base pairs Unique Sites 1601 BSF 1751 BSX Sca. 3881 1806 Est 36 Pyut 3770 1964 xibal 1970 Apa Bl 970 BSAP Bpm 3488 2090 Pin Bsa 3470 2332 Pyt Ahd 3398 2387 Sap AWN 292 2510 Bsp LU11 EcoRI Sf Not Spe I BamHI GAA TTC GGG GCC GGA CGG, GCC GCG GCC GCA CTA GTG GGG ATC C Sac II STOP TT AAT TAAGGG CoA CTG GGG coC CTC GAC CTG CAG Pac Sf Pst FIGURE 6 Patent Application Publication Feb. 27, 2003 Sheet 7 of 19 US 2003/0040089 A1 pP1 Alias: paCTIst Application:2FY (prey) Backbone: paCTI s E; 377 BSa H Specificity: . Xibala 7533 435 Scai Selection: ampicillin 846 BSa Constructed by: SnaB 71.94 913 Ahd 1392 AWNE Hpal 6552 pPl Nr. 646 Bip 6111 1928 Sap Bael 6056 -- 2059 Th111 8134 base pairs 2157 BSnB Sse 8647. 5778 Unique Sites BSEI 5622 Cal 5506 257575 BsgB Age 5407 2764 RSr. 2905 Sex A 2934 M. EcoRV 4909 SES. BSrG 4536 3106 Sma St Oral 4092 I SA 31.17 EcoR 3123 Banil 312.3 Ec136 3123 Sac 3143 Sci 3143Xho ABS GATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATT JC90 Bgl II cgatgatgaagataccCCaCCaaa CCCAAAAAAAGAGATCTGTATGGCTTACCCATACGATGTTCCAG Sf Sna I BamH I ATTACGCTAGCTTGGGTGGTCATATGGCC ATG GAG GCC CCG GGG ATC CGA ATT Nico Sac XhoI Bg II capacoelocaGreeggaeacaceaeCGA GCT CGA CTA GCT AGC TGA CTC GAG AGA TCT ATGAAT 162 ABS2 53 ABS1 5 CGTTTGGAATCACTACAGG 3' JC90. 5’ CGATGATGAAGATACCCCACCAAA 3 162 5 GGGGTTTTTCAGTATCTACG3 ABS2 5’ CACGATGCACAGTTGAAGTG 3' 53 5 GAAATTGAGATGGTGCACGATGCAC3 FIGURE 7 Patent Application Publication Feb. 27, 2003 Sheet 8 of 19 US 2003/0040089 A1 pP2 Application: 2HY (prey) Backbone: paCTIIst Specificity:Sfinon-oriented BC. 7947 95. Selection: amplicillin Xibal 7548 Constructed by:SW 846 Bsal Sna B 7209 913 Ahdi 1392 Alwini Hpa 6567 pP2 Nr. 616 1928 Sap E;2 -- 2O59 Tith 11 8149 base pairs 2157 BSIB Sse 8647. 5793 Unique Sites Bst 5.E. 56375521 2575 Bsg Agel 5422 2764 RSri 2905 Sex A 2934 M. EcoRV 4924 :: Brel 49, i-Si3111 M113 3135 Pac 344 So 3152 Apal 352 Bar 3152 Bsp120) 358 Sci 3158 Xho ABS1 CG cgttggaatcactacagg GATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATT JC90 Bg II cgatgatgaagataccccaccaaa CCCAAAAAAAGAGATCTGTATGGCTTACCCATACGATGTTCCAG Sf Sac ATTACGCTAGCTTGGGTGGTCATATGGCC ATG GCC GCA GGG GCC GCG GCC GCA BamHI -- Pac- Nico CTA GTG.