Patrick Mpho Rakgotho Title of M Sc dissertation: Identification of proteins that interact with DWNNdomain of SNAMA a member of a novel protein superfamily. A dissertation submitted in fulfillment of the requirements for the Degree Master of Science in the School of Molecular and Cell Biology at the University of the Witwatersrand Supervisor: Dr M. Ntwasa University of the Witwatersrand Room 514 Gate House School of Molecular and Cell Biology P/Bag X3 Wits 2050, South Africa Tel: +27 11 7176335/54 Fax: +27 11 7176351 TABLE OF CONTENTS Page DECLARATION i ACKNOWLEDGEMENTS ii ABSTRACT iii LIST OF ABBREVIATIONS iv SECTION 1 1 1. INTRODUCTION 1 1.1 DWNN has a ubiquiti n lik e fold 3 1.2 DWNN associat e d domains 7 1.2.1 RING finger 7 1.2.2 Cystein e rich domains 9 1.2.3 PACT interacts with p53 12 1.3 SNAMA fa mily me mbers 16 1.4 Apoptosis 18 1.5 Apopto s i s in Drosophila melanogaster 19 1.6 Ai m of this study 23 SECTION 2 24 2. MATERIALS AND METHODS 24 2 . 1 Yeast strain s 24 2.2 Bacteria l strains 24 2.3 Cell cultur e s 25 2.4 Plasmi d s 25 2.5 Bacteri a l growth condit i o n s 26 2.5.1 Bacteria 26 2.5.2 Select i o n of transfo r me d cells and recombi n a n t clones 26 2.6 Yeast 27 2.6.1 Selectio n of transfor me d cells a nd screening for interactors 27 2.6.2 Intera c t i o n mating 27 2.7 Polyme r a s e chain reacti o n 28 2.8 Extracti o n of DNA from agarose gels 28 2.9 Restric t i o n endonuc l e a s e diges tion of plasmi d DNA 29 2.10 Re moval of 5 ? end phospha t e overhan g s 29 2.11 Recover y of DNA from liquid mixture 29 2.12 Ligation of DNA molecules 30 2.13 Prepar a t i o n of comp et e n t 30 2.13.1 Bacter i a l compet e n t cells 30 2.13.2 Yeast compet e n t cells 31 2.14 Transf o r ma t i o n s 31 2.14.1 Transfor mation of bacterial cells 31 2.14.2 Transfor ma t i o n of yeast cells 32 2.15 Isolation of plasmi d DNA fro m Escherichia coli 32 2.15.1 Small scale plasmi d prepar a t i o n 33 2.15.2 Large scale plasmid preparation 33 2.16 Protein extraction and pur ification 34 2 . 1 6 . 1 Extract i o n of soluble protein 34 2.16.2 Extract i o n from embryos 34 2.16.3 Extrac t i o n from adult flies 35 2.17 Polyac r y l a mi d e gel electro p h o r e s i s 35 2.18 Staining SDS PAGE gels 36 2.19 Immunopr e c i p i t a t i o n s 36 2.20 Western blottin g 37 SECTION 3 38 RESULTS 38 3 . 1 SNAMA interact s with othe r protein s 38 3.1.1 Yeast two hybrid assay 40 3.1.1a Cloning into pGEM-T Easy vector 40 3.1.1b Constr u c t i o n of the bait plasmi d 41 3.1.1c Preparation of the bait 44 3.1.1d Transfor ma tion of bait strain with cDNA library clones 44 3.1.1e Interac t i o n hunt 44 3.1.2 Immunopr e c i p i t a t i o n assays 45 3.1.2a Heterologous expression of DWNN in E. coli cells 45 3.1.2b Immuno p r e c i p i t a t i o n s with the GST-DWNN fusion protein 46 3.1.2c Interact i o n s with Dmp53 49 3.1.2d Western analysis of immunop recipitated proteins 52 SECTION 4 55 DISCUSSION 55 4.1 Protein-protein interactions using the yeast two-hy b r i d system 57 4.2 Protein-protein interactions using immunoprecipitation 58 SECTION 5 61 CONCLUSION AND FUTURE PROSPECTS 61 SECTION 6 63 APPENDIX 1 63 M e d i a and Suppl e me n t s 63 General stock buffer s and kits compon e n t s 64 APPENDIX 2 67 APPENDIX 3 70 REFERENCES 75 Declaration I decla r e that I dentification of proteins that in teract with DWNN domain of SNAMA a member of a novel protein superfamily i s m y own work, that h a s not been subm it t e d f o r any degree in any other instit u t i o n , and that a ll th e sourc e s I h a ve used or quote d have been indica t e d and acknow l e d g e d by com p le t e refere n c e s . Na m e of candida t e : Patrick Mpho Rakgoth o Signed on this day:????????????????????? Signature of candidate:???????????????????. i Acknowledgements I would like to thank Dr M. Ntwasa for givi ng m e an opportun i t y to work in his lab. His patience , support and guidance have given m e enough streng t h to com p let e this course . I would also like to thank m y colleag u e s (Rodne y , Zam eer , Chris, Arshad and Shune) in the fly lab f o r being there during good and ba d tim e s in the la b. I would also like to exten d m y since r e g r ati t u d e to all tho s e pe ople in the school of m o lecu l a r and cell biology (Technic a l staff, fellow students and staff m e m b ers) for their help with som e aspect s of the projec t . I would also like to thank Zukile, Tshepo and Arshad for those long and fruitfu l discuss i o n s . Finally I woul d like to thank the NRF and my fa m ily for their financ i a l suppor t . ii ABSTRACT SNAMA is a 142 kDa Drosophila melanogaster protein, which consists of the uncharacterized conserved domain with no name (DWNN), zinc and RING finger-like motifs. The primary structure of SNAMA suggests that it might play an important role in cell cycle regulation and apoptosis. Previous studies revealed that homozygous SNAMA mutants underwent ectopic apoptosis which resulted in recessive lethality. SNAMA orthologues such P2P-R, PACT and RBBP6 are involved in cell cycle regulation, whereas Mpe1 is involved in mRNA processing. The aim of this study was to map out the role of SNAMA by isolating proteins which interact with it. DWNN was inserted into pGEX6P-2, phylexzeo plasmid (bait) and the Drosophila 0-12 hours cDNA library inserted in pJG4-5 (prey). The bait and the prey plasmid were used to transform appropriate yeast cells to probe for interacting proteins in yeast two hybrid assays, whereas the pGEX6P-2 was used for heterologous overexpression of DWNN in E. coli. Immunoprecipitation assays were also carried out with the crude protein extract from embryos, adult wild type, SNAMA mutant flies and the overexpressed protein using antibodies against SNAMA, Drosophila p53 Human DWNN and GST. The hybrid assay did not produce any interactors. Some of the proteins obtained from the immunoprecipitations were isolated and sequenced. The proteins identified were hsp82, Hsp70 and CG2985-PA. Data obtained from the immunoprecipitations suggest that SNAMA like Dmp53 might be involved in cell cycle regulation. iii LIST OF ABBREVIATIONS BDGP Berkeley Drosophila Genome Project CIDE Cell death inducing DFF-like effector CPF Cleavage and Polyadenylation Factor DFF DNA fragmentation factor DMF Dimethyl formamide DMSO Dimethyl Sulfoxide DTT Dithiothreitol DWNN Domain with no name FOG Friends of GATA GST Glutathione-S-transferase GATA Cluster of tetranucleotide G A T A HUB-1 Homologous to ubiquitin IPTG Isopropyl ?-D-1-thiogalactopyranoside IAP Inhibitor of apoptosis LB Luria-bertani LiPEG Lithium acetate Tris EDTA Poly ethylene glycol 330 LiTE Lithium acetate Tris EDTA solution MDM2 Murine double minute-2 NCBI National Center for Biotechnology Information P2P-R Proliferation Potential Protein-Related PACT P53 Associated Cellular-protein-Testis derived PBS Phosphate buffered saline PI-3 Phosphatidylinositol PMSF Phenylmethylsulphonylfluoride PVDF Polyvinylidene Fluoride RBQ1/RBBP6 Retinoblastoma Binding Protein 6 RING Really Interesting New Gene SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SR Serine rich SUMO-1 Small ubiquitin related modifier-1 TAE Tris-acetate EDTA buffer TE Tris-EDTA TEMED N,N,N,N ? -Tetramethylethylenediamine TFB Transformation Buffer Ubl Ubiquitin like protein Ubp Ubiquitin based protein YNB Yeast Nitrogen Base YPD Yeast Peptone Dextrose X-gal 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside iv 1 Identification of proteins that interact with DWNN domain of SNAMA a member of a novel protein superfamily SECTION 1 1. INTRODUCTION Domain with no name (DWNN) is a c onse r v e d domai n that chara c t e r i s e s a superf a mi l y of protei n s found in most eukar y o t e s but not in proka r y o t e s . Genomi c searc h e s using the conse r v e d domai n revea l e d that the SNAMA has one gene in lower eukar y o t e s such as mosqu i t o e s ( Anopheles gambiae ) , frui t flie s ( Drosophila melanogaster ) and nemat o d e s ( Caenorhabditis elegans ) . In humans , the gene has two trans c r i p t s , which encode the 13 kDa DWNN domain only and a 200 kDa multi d o ma i n compl e x prote i n (Mbit a , 2004) . The Drosophila homolo g u e is calle d SNAMA (Math e r et al . , 2005). SNAMA is a Xhosa word, which means some t h i n g that stick s . It has a predi c t e d molec u l a r weight of 142 kDa and cons ists of zinc and RING ( Re a l l y I n t e r e s t i n g New G e n e ) finge r - l i k e motif s close l y assoc i a t e d with the N-termi n a l DWNN (figur e 1). Even though the exact role of SNAMA is unknow n , its primar y struc t u r e sugge s t s that the prote i n might play a role in trans c r i p t i o n and cell cycle regul a t i o n . Zinc finge r prote i n s tend to promo t e prote i n - p r o t e i n and prote i n - D N A inter a c t i o n s (Matt h e w s et al . , 2000; Hart et al . , 1996). Adams et al. (2000) repor t e d that about half of the trans c r i p t i o n facto r s in Drosophila are zinc finge r prote i n s . 2 DW NN Zinc fing e r CCHC RING fing e r - lik e motif Worms Flies Human s Fun g i Ascidi a n Plants Human s Othologues of SNAMA such as P53 Associ a t e d Cellu l a r - p r o t e i n Testi s deriv e d (PACT) (Simon s et al . , 1997), Retino b l a s t o ma Bindin g Protei n 6 (RBBP6 ) (Sakai et al . , 1995) and Proli f e r a t i o n Poten t i a l Prot e i n - R e l a t e d P2P-R (Witt e and Scott , 1997) , were repor t e d to inter a c t with p53 and possi b l y regul a t e its activ i t y (Scot t et al . , 2003) , thus sugges t i n g that SNAMA might play a simila r role in flies. P53 is a tumor suppr e s s o r , which preve n t s growt h and survi v a l of stres s e d cells . It is a sequen c e - s p e c i f i c trans c r i p t i o n facto r , wh ich media t e s activ a t i o n and repre s s i o n of cell cycle genes (Ryan et al . , 2001). Figure 1: DWNN superfamily domain organization. Homozygous SNAMA knocko u t mutan t s under w e n t ectop i c apopt o s i s durin g embryo n i c stage and never devel o p e d furth e r into adult h o o d (Math e r et al . , 2005). The human homol o g u e of this protei n was is ola t e d in cells , which were resis t a n t to cytot o x i c T cell killi n g due to the di srup t i o n of the DWNN gene, thus implyi n g 3 a certai n role of the DWNN protei n in apopto s i s (Pugh et. al . , 2006). Taken toget h e r these facts stren g t h e n the hypot h e s i s that SNAMA plays an import a n t role in pathw a y s invol v e d in cell cycle regul a t i o n and apopt o s i s . 1.1 DWNN has a ubiquitin like fold Struct u r a l predi c t i o n sugge s t s that the domai n will have an ubiqu i t i n like fold. The alpha Helix and the four beta sh eets of DWNN were found to correspo n d to the arran g e me n t obser v e d in ubiqu i t i n (f igu r e 2). Even thoug h seque n c e ident i t y between the DWNN and Drosophila ubiqui t i n is only 22% (figu r e 3), the struc t u r a l predi c t i o n sugge s t s that the domai n might under g o ubiqu i t i n like inte r a c t i o n s (Mat h e r et al . , 2005). 4 Figure 2: A. Computer generated model of DWNN based on data of (B) ubiquitin structure . DWNN image was creat e d using Insig h t II model l e r (Acce l r y s Inc ) , where a s ubiqu i t i n struc t u r e was created with PDB viewer (Guex and Peitsch , 1997). Both DWNN and ubiquit i n have the ? - ? - ?- ? - ?- ? struc t u r e (Vija y - K u ma r et al . , 1987) Figure 3: Sequence alignment of DWNN orthologues and ubiquitin. DWNN1 (AAI100 4 2 ) is a human 13kDa transc r i p t , DWNN2 (XP219 2 9 6 ) is a 200kDa trans c r i p t and Mpe1 (NP01 2 8 6 4 ) is the yeast ortho l o g u e (Vo et al . , 2001). The sequenc e s were aligned using DNAMAN progra mme versio n 4.03 (Lynnon Biosoft ) . Protei n such as Small Ubiqu i t i n relat e d Modifi e r - 1 (SUMO - 1 ) ; an ubiqu i t i n - l i k e (ubl) prote i n has low homol o g y to ubiqu i t i n and modif i e s prote i n s by a proce s s calle d sumoy l a t i o n . It is vital in prote i n traff i c k i n g and stabi l i s a t i o n A B 5 (Yeh, et. al . , 2000) . SUMO-1 regul a t e s p53 stabil i t y by covale n t ligat i o n , and induct i o n of confor ma t i o n a l chang e (Rodr i g u e s et al., 1999). The DWNN in SNAMA lacks the C-ter mi n a l di-gl y c i n e resid u e s which chara c t e r i s e s ubiqu i t i n and ubl prote i n s , thus sugge s t i n g that it might belon g to anoth e r set of ubiqi u t i n - l i k e prote i n s such as Homolo g o u s to Ub iqui t i n - 1 (HUB1 ) (Figu r e 4). Luders et al . (2003 ) repor t e d that HUB 1 intera c t s and coval e n t l y attac h e s to other prote i n s even thoug h it lacks the C-ter mi n a l di-gl y c i n e resid u e s . HUB1 thoug h has conse r v e d c-ter mi n a l di-ty r o s i n e and also inter a c t s with other prote i n s via its N- ter mi n a l regio n . Figure 4: Alignment of ubiquitin and ubiquitin like domains . Ubiqui t i n [AAH14880; Vijay-Kumar et al . , 1987] and most of its relate d protei n s (SUMO1 [NP001005781; Saitoh et al., 1997], Rub1p [NP010423; Hochstrasser, 1996], NEDD8 [AAH04625; Kuma r et al . , 1993] are chara c t e r i s e d by the N-ter mi n a l di- glyc i n e residue s . HUB1 [Q6Q546 ; Luders et al . , 2003] underg o ubiqui t i n like inter a c t i o n s even thoug h it lacks the di -gly c i n e resid u e s ) . HUB1 homolo g u e s are ubiqui t i n like protei n 5 (ubl-5 ) [AAP3 6 0 1 9 ; McNal l y et al ., 2003] and CG3450 [AAF573 9 8 ] found in humans and flies respect i v e l y . Ubiqui t i n is a 76 amino acid prote i n , which tags prote i n s desti n e d for degra d a t i o n by the prote a s o me pathw a y . It exist s as a monome r , or usual l y as 6 fusio n molec u l e taggi n g prote i n s for de gra d a t i o n (Vars h a v s k y , 1997) . Protei n s targe t e d for degra d a t i o n are coval e n t l y bound via the lysin e resid u e s to the glyci n e at the C- termi n a l end of ubiqu i t i n and event u a l l y degra d e d by the 26S prote a s o me (Ciec h a n o v e r , 1994; Vars ha v s k y , 1997; Hershk o and Ciecha n o v e r , 1998) . The prote a s o me pathw a y is a majo r intra c e l l u l a r prote o l y t i c pathw a y for maint a i n i n g prote i n turno v e r in eukar y o t e s (Figu r e 5). Ubiqui t i n a t i o n invol v e s three enzyme s ; ubiqu i t i n - a c t i v a t i n g enzyme (E1), ubiqu i t i n - c o n j u g a t i n g enzyme (E2) and ubiqu i t i n prote i n ligas e (E3) (Obin et al . , 1996; Shang et al . , 1997). The initi a l step in the pathw a y is ATP depen d e n t and resul t s in the activ a t i o n of ubiqu i t i n . The activ a t e d ubiqu i t i n is then transfe r r e d to E2. The E2 enzyme eithe r catal y s e s the attac h me n t of the ubiqu i t i n to the subst r a t e direc t l y , or requi r e the third enzyme E3 (Gilc h r i s t et al . , 1997; Jahnge n - H o d g e et al . , 1997; Shang et al . , 1997) . The ubiqu i t i n a t e d prote i n is event u a l l y degra d e d by the 26S prote a s o me with the liber a t i o n of the ubiqu i t i n moeit y . 7 Figure 5: The Ubiquitin-Proteosome Pathway. Illust r a t i o n adapt e d from Hershk o and Ciechn o v e r , (1998) . 1.2 DWNN associated domains 1.2.1 RING finger T h e RING finger is a modifie d zinc finge r motif (Free mo n t et al . , 1991) . It is highl y conse r v e d and most commo n motif s are chara c t e r i s e d by a core C3HC4 sequenc e . Known RING finger contain i n g prote i n s have ubiqu i t i n ligas e activ i t y and thus might play a role in prote i n tr aff i c k i n g and turno v e r via the prote a s o me pathw a y . The pathw a y is vital in regul a t i n g cellu l a r prote i n s , like degra d a t i o n of 2 6 s p r o t e a s o m e E 3 P r o t e i n P r o t e i n u b E 2 u b E 1 u b E 2 E 1 E 2 u b u b Targe t p rot e i n Pepti d e s 8 short - l i v e d regul a t o r y prote i n s such as transc r i p t i o n and DNA repair facto r s , kinas e s , phosp h a t a s e s and tumor suppr e s s o r s . It also plays an impor t a n t role durin g morph o g e n e s i s and organ e l l e bioge n e s i s (Hata k e y a ma et al . , 2001). The RING finge r motifs catal y s e the ligat i o n of activ a t e d ubiqu i t i n with the targe t prote i n . Mutati o n s in the RING finge r of some prote i n s are linke d to devel o p me n t a l abnor ma l i t i e s (Joaz e i r o and Weiss ma n , 2000) and disea s e s such as Parkin s o n s (Sati j n and Otte, 1999). Figure 6: The cross-brace mo del of RING finger motifs. Conser v e d cyste in e resid u e s high lig h te d . The lin e s repr e s e n t othe r amin o acid s . Adapted from Free mo n t et al. (19 9 1 ) . Homolog y search e s reveal e d SNAMA and all other ortho l o g u e s have an unusu a l RING finge r - l i k e motif becau s e the histi d i n e in posit i o n 4 is subst i t u t e d by a serin e (figu r e 6). This arran g e me n t of amino acids sugge s t s that the motif might belong to a new set of unchar a c t e r i s e d RING finger s (Mathe r et al . , 2005). C C H C C C C C Zn 2 + Z n 2 + 9 1.2.2 Cysteine rich domains Zinc finge r s are cyste i n e / h i s t i d i n e rich motifs widely found within many prote i n s in eukar y o t e s and are chara c t e r i s e d by their abili t y to bind zinc ions. They are unusu a l l y small self- f o l d i n g domai n s of which a zinc atom is cruci a l comp o n e n t of its terti a r y stru c t u r e ; th ey are prese n t in struc t u r e s of most regul a t o r y prote i n s where they act as nucle i c acid bindi n g domai n s (Kawa g a s h i r a et al . , 2001). In Drosophila most zinc finge r s have been impli c a t e d in array s of prote i n - p r o t e i n inter a c t i o n s and many devel o p me n t a l proce s s e s (Hart et al ., 1996) . These prote i n s const i t u t e a major group of trans c r i p t i o n facto r s and play impor t a n t roles in gene expre s s i o n and cellu l a r signa l trans d u c t i o n (Otsu k a et al ., 1996) . Zinc finge r s are extre me l y commo n and easil y disti n g u i s h a b l e by their natur e and spaci n g of the zinc coord i n a t i n g resid u e s . These motif s tend to mediat e intera c t i o n s with DNA, RNA and protei n s (Lai et al . , 2000). Most common and abunda n t is the Cys 2 His 2 (CCHH ) zinc finger which is charac t e r i s e d by the X- Cys - X 2 ,4 - Cys - X 3 - P h e X 5 - L e u - X 2 - His - X 3 -5 - His sequence. The CCHH zinc finge r motif was first disco v e r e d in studi e s of the Xenopus laevis transc r i p t i o n facto r IIIA (TFII I A ) and the protei n was shown to contai n bound zinc ions (Mil l e r et al., 1985) . Zinc finger s are most common motif s in eukar y o t i c prote i n s (Lait y et al., 2001; Jantz et al., 2004). The ??? fold chara c t e r i s e s these motif s . This proto t y p e fold was obser v e d in the trans c r i p t i o n facto r Zif26 8 , which has three zinc finger s (Lait y et al . , 2001). Other trans c r i p t i o n facto r s such as GATA (Clus t e r of tetra n u c l e o t i d e G u a n i n e Ad e n i n e Th y mi d i n e Ad e n i n e ) famil y conta i n sever a l zinc finge r s locat e d at both 10 ends of the protei n . The Cys 4 (CCCC ) finge r s locat e d at the N-ter mi n a l of these prote i n s tends to be speci f i c a l l y for in ter a c t i o n with co-fa c t o r s , where a s the classi c a l zinc finger s (CCHH ) inter a c t s with DNA (Macka y et al., 1998). GATA- 1 has 3 zinc finger s , where half were s hown to promo t e inter a c t i o n s with other trans c r i p t i o n a l facto r s such as FOG ( Fr i e n d s O f G A T A ) (Macka y et al., 1998; Fox et al. , 1999). SNAMA contain s one of the unusual zinc fingers , the CCHC. These are the retro v i r a l type zinc finge r s (Tzaf a t i et al., 1995) . In eukar y o t e s these motif s were repor t e d to promo t e prote i n - p r o t e i n inter a c t i o n s (Matt h e w s et al., 2000). The motif s inter a c t with zinc finge r s of othe r prote i n s as obser v e d in prote i n s that inter a c t with the trans c r i p t i o n factor GATA, such as FOG (Fox et al., 1999). FOG is a trans c r i p t i o n a l co-fa c t o r , whic h inter a c t s with GATA result i n g in the activa t i o n and gene expres s i o n . GATA fact o r s and its co-ac t i v a t o r s panni e r and serpe n s contr o l ultima t e fate of cells durin g devel o p me n t in Drosophila (Fox et al., 1998). Several other CCHC contai n i n g prote i n s such as LIM domai n s (Spec i a l i s e d doubl e zinc- f i n g e r motif s ) (Kuro d a et al., 1996), U-shap e d (Matt h e w s et al . , 2000) and CBP (cAMP- r e s p o n s e eleme n t - b i n d i n g prote i n - b i n d i n g prote i n ) (Newt o n et al., 2000) were also repor t e d to inter a c t with other protei n s . Unlike the DNA bindin g zinc finge r prote i n s with sever a l motif s close to each other, the CCHC finger s exist in most prote i n s as singl e motif s . Althou g h these zinc finge r s are known to pre do mi n a n t l y promo t e prote i n - p r o t e i n inter a c t i o n , some yeast zinc finge r pr otei n s such as MPE1 (YKL05 9 c ) , are invol v e d in singl e stran d e d nucle i c acid proce s s i n g . Even thoug h the exact role in 11 the proce s s i n g is still uncle a r it is one of the prote i n s in the yeast ? s cleav a g e and polyad e n y l a t i o n facto r compl e x (Vo et al . , 2001). Figure 7: The prototype fold of the retroviral type zinc finger motifs CCHC. A d a p t e d from Willi a ms et al . (2002) . Other zinc finger s such as the CCCH have only been found in a handful of prote i n s such as trist e t r a p r o l i n (TTP) (DuBo i s et al . , 1995). The exact role of these unus u a l zinc finge r s in cell activ i t i e s is not clear l y known . Genera l l y most zinc finge r prote i n s tend to have a regul a t o r y role. TTP was repor t e d to be the physi o l o g i c a l regul a t o r of tumor necro s i s facto r - ? and granu l o c y t e - ma c r o p h a g e colo n y - s t i mu l a t i n g facto r (Carb a l l o et al . , 2000) ; it also regul a t e s their mRNA stabi l i t y in norma l cell s (Lai et al. , 2000). Divers i t y of the zinc finge r conta i n i n g prote i n s makes them inter e s t i n g . Despi t e their diver s i t y , resea r c h has shown that they can be divid e d into subcl a s s e s based on their mode of funct i o n . Their speci f i c i t y rende r s them effec t i v e tools that may be impor t a n t in futur e appli c a t i o n s . Recen t l y , sever a l arti fi c i a l zinc fing e r moti fs have been synth e s i s e d and were obser v e d to mimi c C C H C Z n 2 + 12 act i v i t i e s of their natu r a l coun t e r p a r t s (Hur t et al., 2003; Libri et al., 2004; Wolfe et al., 2003; Nguyen - H a c k e l e y et al., 2004). This opens a new field of resear c h where DNA bindin g and protei n bindin g zinc finge r s might be used in targe t e d drug deliv e r y and gene thera p y . 1.2.3 PACT interacts with p53 The region downst r e a m of the DW NN domain is highly homolo g o u s to PACT (p53 assoc i a t e d cellu l a r prote i n , te sti s deriv e d ) . PACT is a murine polyp e p t i d e , which was initi a l l y ident i f i e d as a p53 assoc i a t e d prote i n that has the abili t y to bind wild type p53 and Retin o b l a s t o ma (Rb) and inter f e r e s with their DNA binding sides (Simon s et al . , 1997). PACT has an N-termi n a l RING finger and highl y basic C-ter mi n u s . The prote i n has an alter n a t i v e l y splic e d regio n adjac e n t to the serin e rich (SR) regio n . The alter n a t i v e l y splic e d varia n t of PACT was later repor t e d to be proli fe r a t i o n poten t i a l prote i n - r e l a t e d (P2P- R ) (Scot t et al . , 2003). P53 is a 393 amino acid nucle a r phosph o p r o t e i n , sequen c e specif i c DNA- b i n d i n g trans c r i p t i o n facto r which acts as a tetra me r (Lan e and Hall , 1997 ) . It is a trans c r i p t i o n fact o r with the prima r y stru c t u r e divid e d into four disti n c t domai n s ; Trans a c t i v a t i o n (N-te r mi n a l ) , DNA bindi n g (Core ) , the oligo me r i z a t i o n and regul a t i o n domai n s (C-te r mi n a l ) (Hupp et al . , 2000; Prive s , 1994) . It is a tumor suppr e s s o r prote i n , which regul a t e s trans c r i p t i o n of genes requi r e d for cell- c y c l e arres t and apopt o s i s (Cadw e l l and Zambe t t i , 2001; Jin et al . , 2000). The protei n also plays an impor t a n t role in prote c t i n g the integ r i t y of the genome follo w i n g 13 DNA damage and other physi o l o g i c a l stre s s (Priv e s , 1994) . The C-ter mi n a l regul a t o r y domai n of p53 conta i n s the phosp h o r y l a t i o n and acety l a t i o n sites , which modul a t e s prote i n - p r o t e i n inter a c t i o n s with SUMO- 1 (Hupp et al . , 2000), where a s the N-ter mi n a l trans a c t i v a t i o n domai n inter a c t s with Murin e doubl e minute - 2 (MDM2) and p300 (Hupp et al . , 2000) . The tumor suppr e s s i o n abili t y of p53 is also cell type depen d e n t , as it is known to induc e senes c e n c e in G1 and cell cycle arres t in G2 (Greg o r c et al . , 2003; Lohrum and Vousde n , 2000; Moll et al . , 2001) . P53 preve n t s cell progr e s s i o n in late G1 phase by modul a t i n g the functi o n of p21/WAF - 1 , which inhibi t s G1 cycli n depen d e n t kinas e s thus preve n t i n g the cell from progr e s s i n g to the next stage of cell divis i o n (Kim et al ., 2002) . Variou s forms of cellu l a r stres s such as ioniza t i o n radiat i o n and DNA damag e leads to activ a t i o n and stabi l i z a t i o n of the prote i n (Pete r s et al . , 2002). DNA damagi n g agents induce p53 stabil i z a t i o n , by phosp h o r y l a t i o n of certa i n serin e resid u e s withi n the N-ter mi n a l a nd C-ter mi n a l domai n s (Mich a e l and Oren, 2003) . Phosph o r y l a t i o n is carri e d out by a speci f i c set of enzyme s calle d Phosp h a t i d y l i n o s i t o l - 3 (PI-3 ) kinas e s (Lato n e n et al . , 2002). The protei n has been impli c a t e d to play a role in tumou r i g e n e s i s due to its high level s of expre s s i o n in tumor cells, where the DNA bindin g dom ai n regio n of this prote i n is predo mi n a n t l y mutat e d (Hala z o n e t i s et al . , 1993; Sling e r l a n d et al . , 1993). P53 is maint a i n e d at low level s in norma l ce lls , by rapid prote i n turno v e r (Rodr i g u e z et al . , 1999). Regula t i o n of this is a highl y comple x proces s , which involv e s p53 being the induc e r of its maste r regul a t o r MDM2. MDM2, an ubiqui t i n ligas e , tags p53 which leads to its degrad a t i o n (Fuch s et al . , 1998). Wild type p53 in some tumor s is respo n s i b l e for activ a t i o n and overe x p r e s s i o n of MDM2 (Perr y et al . , 2000) . The oncoge n i c poten t i a l of the MD M2 gene produ c t is attri b u t e d to 14 the fact that it binds to the trans a c t i v a t i o n domai n and thus inhib i t s p53-me d i a t e d tran s a c t i v a t i o n of antip r o l i fe r a t i v e targ e t gene s (Bot t g e r et al . , 1999). Genome wide searc h e s revea l e d that Drosophila melanogaster lacks MDM2 homolo g u e . Express i o n of MDM2 did induce a popt o s i s in flies (Sutc l i f f e et al . , 2003; Jin et al . , 2000) and SNAMA may be a candida t e pr ote i n which might fulfi l l the role of MDM2 in flies. Drosophila melanogaster p53 D mp 5 3 was discove r e d throug h homol o g y searc h e s using the human homolo g u e . Althou g h the overal l homolo g y w ith the human p53 is low, there was signi f i c a n t simil a r i t y in the trans a c t i v a t i o n and DNA bindin g domain s locate d in the N-ter mi n a l regio n and the C-ter mi n a l locat e d oligo me r i z a t i o n domai n (figu r e 8) (Ollma n n , 2000) . Bourdo n et al (2007 ) have recen t l y shown that the human and the Drosop h i l a p53 have severa l isofo r ms and they have differ e n t expre s s i o n patte r n s . Unlike the human p53 Dmp53 lacks MDM2 bindin g sites thus imply i n g evolu t i o n a r y diver g e n c e (Sutc l i f f e et al . , 2003). Experi me n t a l evide n c e showe d that overex p r e s s i o n of Dmp53 result s in apopt o s i s but not cell cycle arres t . Th eref o r e , the evolu t i o n a r y role of p53 appea r s to be cell death induc t i o n , where a s in postmi t o t i c reti n a l cell s the prot e i n prote c t e d cells again s t UV irrad i a t i o n (Jass i m et al . , 2003) . It only induce s apopt o s i s in proli f e r a t i n g cells as is the case in retin a l cells , where a s in wing disk cells it plays a promi n e n t role in promo t i n g apopt o s i s (Lee et al . , 2003). 15 Loss of functi o n mutan t s flies also re vea l e d that Dmp53 inter a c t s with the RHG ( Reaper, Head-involution defective[hid] , Grim and Sickle ) locus prote i n s and induces apopto s i s . Known Drosophila apopto s i s induc e r s reape r and sickl e are downst r e a m effect o r s of Dmp53- me d i a t e d apopt o s i s in flies . Death induc i n g signa l s like ionis i n g radia t i o n and DNA damag e tends to media t e reape r depen d e n t cell death . Reaper has a Dm p53 - b i n d i n g regio n withi n the ionis a t i o n radia t i o n - i n d u c i n g domai n (Stel l e r , 2000) . Dmp53 appear s to preser v e genomi c stabi l i t y by regul a t i n g cell death in devel o p i n g cells (Soga mme et al . , 2003), where a s it also prote c t s postmi t o t i c cells again s t hid media t e d apopt o s i s (Jass i m, 2003). The regula t i o n mechan i s m of Dmp53 is unknow n , but Jin et al . (2000) sugge s t e d that the prote i n might be regula t e d by a mechan i s m used by PEST contai n i n g prote i n s . Expres s i o n of this pr ote i n in norma l cells is very low like in human s , thus imply i n g that it has a strong regul a t o r y mecha n i s m. Figure 8 : Domains arrangement of human p53 [hp53] (upper panel) and Drosophila melanogaster p53 [Dmp53] (lower panel). T h e N-ter min a l regio n of hp53 conta in s bind in g sites for MDM2 (bla c k box) . Gree n box is the tran s a c tiv a tio n , red box the DNA bind in g and the blue box the regu la tio n doma in . Adap ted from Hupp et al. (200 0 ) and Jin et al. (20 0 0 ) . Transa c t i v a t i o n DNA Bindi n g (Core Domai n ) Tetramerisation Regul a t i o n T r a n s a c t i v a t i o n DNA Bind i n g Domai n Tetramerisation 16 1.3 SNAMA family members Blast search e s with SNAMA revea l e d that the protei n is conser v e d and found in most eukar y o t e s . The prote i n seque n c e s were align e d and used to const r u c t the pylog e n e t i c tree (figu r e 9). Even thoug h most of the prote i n s in the phylo g e n e t i c tree are uncha r a c t e r i s e d , the score on the tree sugge s t s that the prote i n s evolv e d from a common ances t o r . Databa s e s earc h e s also reveal e d that SNAMA is simil a r to previ o u s l y chara c t e r i s e d prote i n s RBBP6 (Saka i et al ., 1995), PACT (Simo n s et al . , 1997), P2P-R (Witte and Scott, 1997) and Mpe1 (Vo et al . , 2001). P2P-R is a nuclea r protei n , which is highl y expre s s e d in proli f e r a t i n g cells (Gao et al . , 2002) . The prote i n also has the abili t y to restr i c t mito t i c prog r e s s i o n at prome t a p h a s e and induc e mitot i c apopt o s i s when overe x p r e s s e d (Gao and Scott , 2002) . Mpe1 was repor t e d to play a role in RNA process i n g . This functio n was also observe d with P2P-R. 17 S. po mb e/C A A21 8 0 0 0.350 A. nid ula ns/E A A 6 20 1 1 0.367 S. cer e visia e /N P _ 01 28 6 4 0.368 0.024 0.030 U . ma yd i s/X P _ 7 56 4 68 0.417 E . c u nic ul i / X P _9 55 5 860.435 0.017 0.016 A. melli fe r a /XP _ 0 0 11 2 05 6 1 0.089 D . rer io /XP _ 70 7 46 3 0.130 R. no rve gic us/ X P _ 219 2 96 M. mula tta / X P _0 01 0 968 870.098 0.107 0.070 T . nigr o vir id is/C AF9 3 9 600.032 X. tro p ic a lis/NP _ 98 8 98 10.103 0.132 M . musc ul us/X P _6 2 0 500 0.271 0.005 H . sap ien/ AA H 6 3 5 2 4 0.186 G . gall us/X P _ 4 1 487 0 0.099 E . cab a llus/ A A O3 7 7 6 50.139 0.085 0.113 0.012 O. sativ a/ AAP 5 39 0 0 0.221 A. th a lia na/ N P _ 1 99 5 54 0.215 0.243 0.015 D . mela no g ast e r / A AF4 7 1 620.300 A. ga mb i a e / X P _ 30 9 42 8 0.274 0.149 C. ele ga n s/NP _ 4 9 242 4 0.448 0.004 0.05 S. p o mb e/C A A21 8 0 0 A. nid ula ns/E A A 6 20 1 1 S. c er e visia e /N P _ 01 28 6 4 U . ma yd i s/X P _ 7 56 4 68 E . c u nic ul i / X P _9 55 5 86 A. me lli fe r a /XP _ 0 0 11 2 05 6 1 D . re r io /XP _ 70 7 46 3 R. no rve gic us/ X P _ 219 2 96 M. mula tta / X P _0 01 0 968 87 T . nigr o vir id is/C AF9 3 9 60 X. tr o p ic a lis/NP _ 98 8 98 1 M . musc ul us/X P _6 2 0 500 H . s a p ie n/ AA H 6 3 5 2 4 G . gall us/X P _ 4 1 487 0 E . c a b a llus/ A A O3 7 7 6 5 O. sativ a/ AAP 5 39 0 0 A. th a lia na/ N P _ 1 99 5 54 D . mela no g ast e r / A AF4 7 1 62 A. ga mb i a e / X P _ 30 9 42 8 C. e le ga n s/NP _ 4 9 242 4 Figure 9 : Phylogenetic analysis of SNAMA and related proteins. The tree was gener a t e d using DNAMA N (Ly nn o n Bioso f t ) multi p l e seque n c e align me n t progr a m. The whole prote in sequ e n c e s were used in the alig n me n t. The numb e r s on the tree sign if y bran c h dista n c e s betw e e n diff e r e n t orga n is ms . 18 1.4 Apoptosis Apopto s i s or progr a mme d cell death is a major form of cell suici d e , by which anima l s elimi n a t e unwan t e d , dama g e d or harmf u l cells (Garc i a - D o mi n g o et al ., 1999; Song et al . , 2000) . The proce s s is highl y conse r v e d and is trigg e r e d in a well defin e d patte r n of cellu l a r and bioch e mi c a l event s indep e n d e n t of the origi n of the death stimu l u s (Clav e r i a et al . , 2002) . It commo n l y occur s durin g anima l devel o p me n t and metamor p h o s i s (Colu s s i et al . , 2000; Dorsty n et al . , 2002) . It is mostl y a gene- d i r e c t e d proce s s , orch e s t r a t e d by a handfu l of genes in C. elegans and D. melanogaster (Green, 2000; Melino , 2001). Apopto s i s is chara c t e r i s e d by a serie s of bioch e mi c a l event s , which are divid e d into three funct i o n a l compo n e n t s : caspa s e s , caspa s e activ a t o r s and a famil y of apopt o t i c regul a t o r s and inhib i t o r s (Fras e r et al., 1999; Whi t e et al ., 1994) . Sever a l input s invol v i n g signa l trans d u c t i o n pathw a y s are integ r a t e d into the apopt o t i c pathw a y , which contr o l s the respo n s e of cells to exter n a l stimu l i (Widma n n et al . , 1998). The proces s is define d by morph o l o g i c a l chara c t e r i s t i c s such as cell shrink a g e , membr a n e blebb i n g , chroma t i n conde n s a t i o n and DNA fragme n t a t i o n (Yue et al . , 1999) . In metaz o a n s it usual l y occur s durin g devel o p me n t , immun e selec t i o n , maint e n a n c e of cellu l a r integ r i t y and tissu e homeo s t a s i s (Sah et al . , 1999). Apoptos i s can be trigge r e d by many differ e n t stimu l i , like death facto r s , stero i d hormon e s , DNA damage , cytoto x i c cells, remo v a l of extra c e l l u l a r surv i v a l sign a l s , antic a n c e r drugs and viral infec t i o n (Song et al . , 2000; Yokoyama et al . , 2000) . Althou g h most proapo p t o t i c signa l s are inter n a l , some exter n a l facto r s , such as ultra v i o l e t (UV) irrad i a t i o n , may also 19 trig g e r this proce s s . Regar d l e s s of the stimu l i , most of the downs t r e a m cell elimi n a t i o n is carri e d out by the activ a t i o n of conse r v e d seque n c e speci f i c prote a s e s known as caspa s e s (cyst e i n e aspar t a s e s ) . 1.5 Apoptosis in Drosophila melanogaster The apopt o t i c machi n e r y is highl y conse r v e d in metazo a n s , where b y compo n e n t s that media t e apopt o s i s in highe r eukar y o t e s , have homol o g o u e s in simpl e eukar y o t e s like Drosophila melanogaster and Caenorhbditis elegans . The death recep t o r media t e d pathw a y in flies consi s t s of trans me mb r a n e prote i n s simil a r to the Fas/C D 9 5 Tumor Necro s i s Recep t o r Famil y . For insta n c e Eiger ( Drosophila tumor necro s i s facto r ) is a type II trans me mb r a n e prote i n (Igak i , 2002) , where a s Wegen (memb e r of the Drosophila tumor necro s i s facto r recep t o r (TNFR ) super f a mi l y ) is a type III tran sme mb r a n e protei n with a TNFR homolo g y domain (Kanda , 2002). Transme mb r a n e prote i n s like Fas/CD 9 5 and Tumor necro s i s facto r recep t o r famil y are some of the best- c h a r a c t e r i z e d pathw a y s invol v e d in the initi a t i o n of apopt o s i s (Yue et al . , 1999) . Fas is a type I membr a n e recep t o r , where a s its ligan d is synth e s i z e d as a type II membr a n e prote i n . They are member s of the TNF recept o r and TNF famil y of prote i n s respe c t i v e l y and they have their conse r v e d C-ter mi n a l regio n in the extra c e l l u l a r regio n and the varia b l e N-te r mi n a l in the cytop l a s mi c regio n (Naga t a , 1997) . The intra c e l l u l a r regio n s of these death recep t o r s are com pos e d of compon e n t s like death effect o r domain (DED) or death domain (DD). Th e recep t o r s inter a c t via the DD and DED to bind and cleav e initi a t o r caspa s e s (casp a s e 8 and 10). The activ a t e d initi a t o r caspa s e s then cleav e and activ a t e downs t r e a m effec t o r caspa s e s ; caspa s e 20 3, 6 or 7, which in turn cleave a nd activa t e endonu c l e a s e s such as DNA fragme n t a t i o n facto r (DFF) and cell deat h- i n d u c i n g DFF45- l i k e effect o r (CIDE) . This mecha n i s m of cell death is widel y known as an extri n s i c pathw a y . This mecha n i s m of cell death begin s in the envir o n me n t outsi d e the cell, the stimu l i trigg e r s the casca d e of event s that l eads to cell death , where a s the intri n s i c pathw a y is trigg e r e d by an inter n a l stimu l i . Throug h seque n c e homol o g y searc h e s proap o p t o t i c genes in the nemat o d e Caenorhbditis elegans were observ e d to be highly conse r v e d i n which some genes ( ced- 9 and ced-3 ) were found to have a high sequen c e homol o g y with the mamma l i a n ( bcl-2 ) fami l y of genes (Frase r and Evan, 1997). In Drosophila much of the progr a mme d cell death occur s durin g devel o p me n t media t e d mostl y by activ a t i o n of the kille r genes . Studie s have uncov e r e d these cell death genes in the fruit fly Drosophila melanogaster (Garcia - D o mi n g o et al ., 1999) . The four closel y linke d genes Reaper, Hid, Grim a n d Sickle are locat e d at region H99 of the third chromos o me (Haini n g et al . , 1999; Igaki et al . , 2000; Clave r i a et al., 2002) . They act as death switc h e s regul a t e d at the level of trans c r i p t i o n . Ectopi c expre s s i o n of these genes leads to apopt o s i s in viabl e cells and their inact i v a t i o n preve n t s apopt o s i s in the cells that were fated to die (Bergma n n et al . , 1998) . The N-ter mi n a l regio n of these prote i n s is simil a r to a proap o p t o t i c mitoc h o n d r i a l prote i n homol o g o u s to the proap o t o t i c prote i n secon d mitoc h o n d r i a - d e r i v e d activ a t o r of caspa s e s / D i r e c t IAP Bindin g protei n with Low pI (Smac/D I A B L O ) (Verhag e n and Va ux, 2002). Smac/DI A B L O is release d durin g stres s relat e d apopt o s i s and inact i v e inhib i t o r of apopto s i s (IAP) (Hu and Yang, 2003). 21 Even thoug h these genes are close l y linke d in the chromos o me , their regul a t i o n and funct i o n appea r to be diffe r e n t . Reape r and Grim are only expre s s e d in the cells that are going to die, where a s Hid has been detec t e d even in the cells that were going to live (Berg ma n n et al . , 1998) . Clave r i a et al . (1998) also repor t e d that Grim a n d Reape r trigg e r e d the apopt o s i s progr a mme via the Drosophila caspase 1 (DCP1) and Hid a c t i v a t e d apopt o s i s via a caspa s e that is still to be ident i f i e d , thus sugge s t i n g that Hid might be playi n g a disti n c t role in devel o p me n t a l l y regul a t e d apopt o s i s (Kura d a and White , 1998) . Hid, grim a n d reape r were found to have diffe r e n t expre s s i o n patte r n s , their N-ter mi n a l seque n c e s bind to inhib i t o r s of a popt o s i s and are conse r v e d (Clav e r i a et al ., 2002) . Althou g h apopt o s i s is a highly re gul a t e d and speci f i c progr a mme , some defec t s in the machi n e r y are linke d to a numbe r of patho l o g i c a l condi t i o n s . Variou s disor d e r s such as cance r and auto i mmu n e disea s e s have been attri b u t e d to the failu r e of progr a mme d cell death (Sesh a g i r i et al . , 1999), wherea s , diseas e s like AIDS, neuro d e g e n e r a t i v e disor d e r s and cell loss are assoc i a t e d with incre a s e d apopt o s i s (Gil- G o me z et al . , 1998). As many proap o p t o t i c enzyme s like caspa s e s are almos t alway s prese n t in the cell, the cell must have the means of pr eve n t i n g the trigg e r i n g of that pathw a y (Jone s et al. , 2000) . As menti o n e d earli e r , the a popt o t i c machi n e r y consi s t s of the proap o p t o t i c and the anti- a p o p t o t i c eleme n t s , which the latte r eleme n t s are also impor t a n t in the cell cycle . The anti- a p o p t o t i c activ i t y is provi d e d by a batte r y of cell death inhib i t o r s , which are prese n t in vario u s steps of the apopt o t i c machi n e r y . The inhib i t o r s of apopt o s i s (IAP) were first disco v e r e d in the 22 bacu l o v i r u s e s , which are arthr o p o d - s p e c i f i c double strand e d DNA viruse s (Sesh a g i r i et al . , 1999). These IAP antago n i z e d the host defens e mechan i s ms by atten u a t i n g the apopt o t i c pathw a y and thus assis t i n g in the propa g a t i o n and the spread i n g of the pathog e n (List o n et al . , 1996). In Drosophila IAP ortholo g s name ly DIAP 1 and DIAP2, have simila r struc t u r a l domai n s like RING finge r motif s and bacul o v i r u s inhib i t o r of apopt o s i s type repeat s BIR (Salve s e n and Ducket t , 2002). Jones et al . (2000 ) repor t e d a new type of IAP in the fly, namely de ter i n , which has some deviat i o n s from tradit i o n a l struc t u r e s of DIAP. The pr otei n has only one BIR and no ring finger moti fs . 23 1.6 Aim of this study The aim of this study was to charac t e r i s e SNAMA by identi f y i n g the protei n s which inter a c t with it using th e yeast two hybri d syste m and immun o p r e c i p i t a t i o n s assay s and also by deter mi n i n g its role in cell cycle regul a t i o n . The prima r y aim of this proje c t was to ident i f y genes codin g for protei n s , which intera c t with SNAMA. The yeast two-h y b r i d syste m was chose n , as it would enabl e the isola t i o n of the ge nes codin g for the inter a c t i n g prote i n s . The coding region of the DWNN domain of SNAMA was inser t e d into phybl e x / z e o plasmi d and the prote i n produ c e d was used as bait to trap any prot e i n that might inter a c t with the domai n in y east two-h y b r i d assay s . The prey vecto r used was pJG4-5 , and contai n e d the Drosophila 0-12 hours embryo cDNA libra r y . The yeast two hybri d syste m is an in-vi v o expre s s i o n syste m which can mimi c inter a c t i o n likel y to happe n in th e organi s m from which the genes codin g for the prote i n s being inves t i g a t e d were clone d from. Since sever a l prote i n s requi r e postt r a n s l a t i o n a l modif i c a t i o n the some inter a c t i o n s might not be detec t e d using the yeast two hybri d syste m. The immun o p r e c i p i t a t i o n s were carri e d out to probe for inte r a c t i o n s like l y to occur in organi s m of intere s t . The region encod i n g DWNN was also insert e d into a pr okar y o t e expres s i o n vector pGEX6p . Antibo d i e s again s t the fusio n prote i n were immob i l i z e d on a prote i n A matri x and were used to immun o p r e c i p i t a t e the fusio n prote i n . The immun o p r e c i p i t a t e d fusio n prote i n was the furth e r expos e d to the crude extra c t from adult flies to probe for any prote i n s which inter a c t with DWNN. Since SNAMA was identified in the scree n for gene invol v e d in cell cycle regul a t i o n the secon d a r y aim was to deter mi n e the exact role of the prote i n in that pathw a y . SECTION 2 2. MATERIALS AND METHOD 2.1 Y east strains Strain Genotype Phenotype Saccharomyces cerevisiae L40 (Invitrogen) MAT? his3?200 trp1-901 leu2- 3112 ade2 LYS2:: (4lexAop-His3) URA3::(8lexAop-lacZ) GAL4 His-, Trp-, Leu-, Ade- S. cerevisiae EGY48 (Gyuris et al., 1993) MAT? ura3 trp1 his3 6lexAop- LEU2 Ura-, Trp-, His-, Leu- S. cerevisiae Rfy206 (Finley and Brent, 1994) MATa trp1?::hisG his3?200 ura3- 52 lys2?201 leu2-3 Ura-, Trp-, His-, Leu- 2.2 Bacterial strains Strain Genotype Escherichia coli XL-1 blue (stratagene) recA1 end A1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F?proAB lacIqZ?M15 Tn 10 (Tetr)] E. coli BL 21 (stratagene) F-ompT [lon] hsdSB(rB-mB-BL 21(DE3) is an E. coli B strain with DE3, a ? prophage carrying the T7 RNA polymerase gene 24 2.3 Cell Cultures Strain Source Drosophila melanogaster Schneider 2 cells Invitrogen 2.4 Plasmids Yeast plasmids Genotype Purpose pHyblex/Zeo (Invitrogen) ZeoR, 2 ? ori, ADH prom. and ter. LexA ORF, TEF1 and EM-7 prom. ColE1 ori, CYC1 ter. Vector ?for bait? protein pJG 4-5 (Gyuris et. al, 1993) TRP1, 2? ori, AmpR, GAL 1 prom. B42-HA tag, NLS, ADH1 ter. Vector for prey protein) pYESTrp2 (Invitrogen) TRP1, 2? ori, AmpR, GAL 1 prom. B42-V5 epitope tag, NLS, ADH1 ter. f1 ori, ColE1 ori, CYC1 ter. Vector for prey protein pSH18-34 (Gyuris et. al, 1993) URA3, 2? ori, AmpR, 8 lexA operators lacZ ORF, ColE1 (pBR322 derived) Reporter 25 Bacterial plasmids Genotype Purpose pGEM-T Easy (Promega) AmpR Cloning vector, for PCR products. pGEX-6P-2 (Pharmacia Biotech) AmpR Overexpression of recombinant proteins 2.5 Bacterial and yeast growth conditions 2.5.1 Bacteria Escherichia coli XL-1 blue and E. coli BL 21(DE3) were used throughout the project. E. coli XL-1 blue cells were used as host cells and for subsequent amplification of recombinant plasmid DNA molecules, whereas E. coli BL 21(DE3) cells were used for heterologous expression of recombinant protein. 2.5.2 Selection of transformed cells and positive clones E. coli XL-1 blue bacterial cells that were transformed with recombinant pGEM-T Easy plasmid were selected on LB agar plates supplemented with 100 ?g/ml ampicillin, 12 ?g/ml tetracycline, 25 ?g/ml X-gal and 100 mM IPTG. Cells containing recombinant clones were white in colour, due to the disruption of the ?-galctosidase coding region. The ?-galactosidase gene produces an enzyme that catalyzes x-gal and cells carrying the non-disrupted gene turn blue when grown on a plate containing x-gal. In instances where other plasmids (pGEX 6P, pHyb lex/zeo and pJG4-5) were used in transformations, cells were selected on 26 plate containing ampicillin (E. coli BL 21(DE3)) or together with tetracycline (E. coli XL-1 Blue), whereas cells transformed with pHyblex/zeo were selected on media containing 100 ?g/ml zeocin. 2.6 Y east Untransformed yeast cells were maintained and grown on Yeast peptone dextrose (YPD) growth media (Appendix 1). Transformed cells were grown under selective pressure on an appropriate yeast nitrogen base (YNB) dropout media (Appendix 1). 2.6.1 Selection of transformed cells and screening for interactors The ?bait? constructs was transformed into an Saccharomyces cerevisiae EGY 48 (Mat? ura 3 his3 leu2::3lexop-LEU2 trp lys2) yeast strain using the Lithium acetate yeast transformation method (Golemis, 1995). This yeast strain contains the pSH18-38 ?-gal reporter plasmid and an internal leucine reporter gene. The transformed organisms were then plated on (YNB) minimal plates lacking uracil supplemented with zeocin and glucose (YNBglu/zeo/ura) to select for pSH18-34 and phyblex/zeo. 2.6.2 Interaction mating The ?prey? plasmid containing the Drosophila library was transformed into the S. cerevisiae RFY 206 (Mata ura3-52 his3 AE200 leu2-3 lys2 AE201 27 trp1::HisG) mating yeast strain using the same method stated above. Transformants were selected by plating the organisms on YPD/glu/ trp- plates. 2.7 Polymerase Chain Reaction (PCR) PCR was performed in the Perkin Elmer Geneamp 2400 thermal cycler. The reaction consisted of 1x PCR buffer (10 mM Tris-HCl, 50 mM KCl; pH 8.3), 1.5 mM MgCl, 3 ?g template DNA, 200 ?M dNTPs, 0.2 ?M of each primer, and 5 units Taq DNA polymerase. The final volume was made up to 50 ?l with sterile distilled water. The run was through 40 cycles: 94oC for 30 seconds (denaturation), 55oC for 1 minute (annealing) and 72oC for 45 sec (extension). The amplification cycles were preceded by 95oC pre PCR denaturation for 5 minutes and followed by 10 minutes final extension step at 72oC. Following the reaction, the PCR product was then qualitatively analysed on a 1% agarose gel. 2.8 Extraction of DNA from agarose gels The gel containing PCR products was subjected to long wavelength ultraviolet light to visualize DNA. The DNA band of interest was excised from the gel and transferred into a clean eppendorf tube. Extraction was performed using the concert matrix gel extraction system from Gibco Life Technologies (11457-017). The system uses silica resin to capture and purify DNA molecules. 28 2.9 Restriction endonuclease digestion of plasmid DNA Restriction analysis reaction was done according to the manufactures instructions. The corresponding 10x buffer constituted 10% of the total reaction with one unit of enzyme for each ?g of DNA. The reaction was carried out at 37oC for 16 hours, whereupon the restricted DNA was analysed on agarose gel. EcoRI and XhoI were used throughout this project and the restrictions were carried out in buffer H (50 mM Tris-HCL, 10 mM MgCl2, 100 mM NaCl, 1mM 1-4 Dithioerythritol [DTE]) from Roche. 2.10 Removal of 5? end phosphate overhangs Calf intestinal phosphatase was used to treat linear plasmid molecules at a ratio of 0.5 units per ?g DNA in a suitable buffer. The reaction was incubated at 37oC for 30 minutes. Heating the tube at 65oC for 15 minutes followed by phenol chloroform isoamyl alcohol (25:24:1) extraction stopped the reaction as outlined in section 2.11. 2.11 Recovery of DNA from liquid mixture The volume of the reaction was adjusted to starting volume of 300 ?l. The sample was extracted twice, first with phenol:chloroform:isoamyl (25:24:1) followed by chloroform. The pure aqueous layer was transferred into a fresh eppendorf tube, followed by DNA precipitation with ethanol acetate for 30 minutes at ?70oC. The DNA was recovered by centrifugation at 12, 000 xg for 10 minutes, washed with 70% ethanol and air dried at room temperature for 10 minutes. DNA was reconstituted with appropriate volume of sterile distilled 29 water. 2.12 Ligation of DNA molecules Ligation into pGEM-T easy vector (Promega) was done according to the manufacturers instructions (A1360). The ligation cocktail consisted of 200 ng of the PCR amplified DNA, 50 ng plasmid and 2.5 units of T4 DNA ligase. Ligations into pHyb/lex zeo and pGEX-6P-2 were conducted in such a way that the ratio of vector to insert was 2:1 where dephosphorylated vector DNA was used. The T4 DNA ligase was at ratio of 1unit/?g of DNA. The reactions were incubated overnight at 4oC. 2.13 Preparation of competent cells and transformation 2.13.1 Bacterial competent cells A single colony of the desired bacterial strain was used to inoculate into 5ml of LB broth (Appendix 1), and the culture was incubated for 16 hours on a shaker platform at 37oC with shaking at 150 xg rpm. The culture was then used to sub- inoculate 100 ml of fresh LB broth medium and incubated further until the cells were at OD550 of between 0.4 and 0.6. The culture was cooled on ice, and was then centrifuged at 1100 xg for 10 minutes at 4oC. The supernatant was removed and the cells resuspended on ice with 35 ml of ice cold transformation buffer (TFB) (Appendix 1). The cell suspension was incubated on ice for 15 minutes after which the cells were harvested by centrifugation (4428 xg) at 4oC. The 30 pellet was gently resuspended in 2 ml of cold TFB. To the cell suspension 150 ?l of 1M dimethyl formamide (DMF) was added followed by 5 minutes incubation after which 150 ?l of 1M Dithiothritol (DTT) was added and the suspension was incubated for further 10 minutes on ice. After incubation 70 ?l of 1 M DMF was added and the cells were kept on ice for 30 minutes followed by centrifugation (13,250 xg) at 4oC. The pellet was resuspended in 0.1 M Calcium chloride/20% glycerol solution and then snap frozen in 0,2ml aliquots using liquid nitrogen. The aliquots were stored at ?70oC. 2.13.2 Yeast competent cells Yeast cells were made competent using modified version of the lithium acetate method by Ito et al., (1983). A single colony of the desired yeast strain was grown in 10 ml YPD broth overnight at 30oC. The overnight culture was used to sub-inoculate 50 ml YPD and grown further for 4 hours after which cells were harvested by centrifugation at 1100 xg. The pellet was resuspended in 40 ml 1x tris-EDTA (TE) buffer, spun again followed by resuspension in 2 ml Lithium acetate-tris-EDTA buffer and the cell suspension was incubated at room temperature for 10 minutes. 2.14 Transformations 2.14.1 Transformation of bacterial cells Transformation was done according to Lederberg and Cohen (1974), where 31 200 ml of competent cells was thawed on ice and 20 ?l of ligation reaction added followed by incubation on ice for 15 minutes. The cells were then heat shocked for 2 minutes at 42oC, after which 900 ?l of cold LB was added and the mixture was incubated at 37oC for 60 minutes. The transformed cells were then plated on LB agar plates containing ampicillin. 2.14.2 Transformation of yeast cells For each transformation 100 ?l yeast suspension aliquot was mixed with 1 ?g plasmid DNA and 100 ?g salmon sperm DNA followed by addition of 700 ?l Lithium acetate polyethyleneglycol 330 buffer. The cells were then incubated at 30oC for 30 minutes followed by addition of 88 ?l dimethylsulfoxide, whereupon they were heat shocked at 42oC for 15 minutes. The cells were centrifuged briefly and the pellet was resuspended in 1x TE and plated on appropriate YNB selective plates. Plates were incubated at 30oC for 3 days. The method was scaled up to 1 liter for transformation with library plasmid and 20 ml aliquots of yeast suspension were mixed with 1 ml (10 mg/ml) of denatured salmon sperm DNA and 500 ?g library DNA. 2.15 Isolation of plasmid DNA from E . coli Plasmid DNA isolation was done according to Birnboim (1983), based on alkaline lysis method. 32 2.15.1 Small-scale plasmid prep aration A colony of transformed E. coli was used to inoculate 2 ml of LB containing 100 ?g/ml of ampicillin, and was grown overnight with shaking at 37oC. A 1.5 ml aliquot of this culture was centrifuged at high speed for 1 minute in a micro- centifuge and the supernatant was carefully removed. The cells were resuspended by vortexing in 100 ?l of cold solution 1 (Appendix 1), followed by 200 ?l of solution 2 (Appendix 1). The cells were mixed gently by inversion, incubated for 3 minutes at room temperature followed by addition of 150 ?l of solution 3 (Appendix 1). The suspension was then mixed gently by inversion and left on ice for 20 minutes. Cell debris was removed by centrifugation at 13250 xg for 10 minutes and the plasmid containing supernatant was transferred to a fresh eppendorf tube. The plasmid was precipitated with 2 volumes of 95% ethanol for 20 minutes at ?20oC prior to centrifugation for 15 minutes. The resulting pellet was washed once with 70% ethanol. The plasmid DNA was recovered by centrifugation for 10 minutes and the DNA was reconstituted with 200 ?l of sterile distilled water. 2.15.2 Lar ge-scale plasmid prepar ation Plasmid DNA was isolated using Qiagen maxiprep kit (12163). A 10 ml overnight culture of transformed E. coli was used to inoculate 500 ml of LB broth containing 100 ?g/ml ampicillin and the mixture was shaken overnight at 37oC. 33 The cells were harvested at 5000 rpm for 10 minutes at 4oC in a Beckman JA 10 rotor. Further on cells were treated according to the manufactures instructions. Isolated DNA was resuspended in 1 ml of sterile distilled water. 2.16 Protein extraction and purification 2.16.1 Extr action of soluble protein A single colony of E. coli BL 21 cells transformed with pGEX-6P-2 recombinant plasmid was inoculated into 10 ml LB broth containing ampicillin and grown overnight with vigorous shaking at 37oC. 5 ml of the overnight culture was used to inoculate 200 ml of fresh LB ampicillin broth and the culture was incubated further until the cells were at OD600 of 0.6. The culture was induced with 0.5 mM IPTG for 4 hours. After induction cells were harvested in pre- weighed centrifuge tubes at 5000 rpm for 10 minutes at 4oC in a Beckman JA 10 rotor. The pellet was resuspended with Bugbuster reagent from Novagen (Cat no:70584) at a ratio of 5 ml per gram of wet cell paste. Sodium benzonase (25 units/ml of bugbuster) and 4 mM Pefabloc were added to the cell suspension followed by incubation on a shaker platform at room temperature for 15 minutes. The insoluble cell debris was removed by centrifugation at 12, 000 xg for 15 minutes and the supernatant was stored for further purification and SDS PAGE analysis. 2.16.2 Extraction from embryos Wild type and l(3)rQ13 mutant fly embryos were collected at different stages of development. Embryos were then washed with distilled water and 34 homogenized by pestle in cell lysis buffer (Appendix1). The homogenate was centrifuged for 10 minutes at 4oC and the supernatant was transferred into a fresh eppendorf tube and stored at -70 oC. 2.16.3 Extraction from adult flies Adult flies were collected into 1.5 ml eppendorf tubes and homogenized with pestle in cell lysis buffer (Appendix1). The supenatant was collected after centrifugation at 12, 000 xg for 5 minutes and stored at -70 oC. 2.17 Polyacylamide gel electrophoresis Denaturing SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) under reducing conditions was used to separate the proteins. Gels were prepared from 30% pre-made stock of 29:1 acrylamide: bisacrylamide (Sigma). The resolving gel consists of 7% acrylamide mix, 0.375 M Tris HCl pH8.8, 0.1% SDS, 0.1% ammonium persulphate and 0.1% Tetramethylethylenediamine (TEMED). The stacking gel added on top of the resolving gel, consisted of 4% acrylamide mix, 0.125 M Tris HCl pH6.8, 0.1% SDS, 0.1% ammonium persulphate and 0.1% TEMED. Samples were prepared for electrophoresis by mixing protein with 2 x SDS PAGE loading buffer (Appendix 1) and then boiling for 5 minutes. The samples loaded onto the gel were electrophoresed in SDS PAGE running buffer (Appendix 1) at 120 V for 2 hours. 35 2.18 S taining SDS PAGE gels Immediately after electrophoresis, the gel was immersed in 5 volumes of the Coomassie blue staining solution (Appendix 1) for 3 hours at room temperature. The gels were then destained until the bands were visible or overnight with gentle agitation. For sensitive applications silver stain plus kit from Bio-Rad (cat no: 161- 0449) and Gelcode blue stain reagent from Pierce biotechnology (cat no: 24592) were used. These reagents were more sensitive and less time consuming as staining was generally completed in less than 2 hours. 2.19 Immunopr ecipitations Protein was extracted from adult flies and embryos in RIPA buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5mM PMSF, 1?g/ml aprotinin, 4.0mM Pefabloc). The lysate was then incubated with 1.0 ?g of antibody (anti-Drosophila p53, anti-Human DWNN and anti-SNAMA) overnight at 4oC. Following incubation 20 ?l of Protein A agarose from Santa Cruz (sc- 2001) was added to the crude protein antibody mixture followed by incubation on a rotating device for 2 hours at 4oC. The protein matrix complex was then washed twice with 1.0 ml RIPA buffer. After final wash the immunoprecipitated proteins were recovered by boiling in SDS sample buffer and analyzed on an SDS PAGE. 36 2.20 Western blotting Denaturing SDS-polyacrylamide gel electrophoresis under reducing conditions was used to separate proteins. Following electrophoresis, the gel was equilibrated in an electroblotting buffer (Appendix 1) for 5 minutes. The gel was then positioned on top of the Whatman filter paper. Wet Hybond PVDF membrane soaked in electroblotting buffer was then placed on top of the gel, taking care to remove air bubbles and another Whatmann paper placed on top of the membrane and the sandwich was placed inside a blotting cassette. Proteins were electroblotted onto the membrane at 300 mA for 3 hours. After blotting, the PVDF membrane was blocked for 1 hour at room temperature in superblock solution, followed by incubation for 1 hour at room temperature or overnight at 4oC with a 1:5000 dilution of rabbit anti SNAMA, anti Human DWNN (provided by Prof J. Rees) or goat anti Drosophila p53 (Santa Cruz: sc-17577) in 1x phosphate buffered saline (PBS). The membrane was washed twice with PBS, 0.1% Tween 20 and then incubated with 1:10 000, dilution of secondary antibody Immunoglobulin G (sc-2768) conjugated to horseradish peroxidase for 1 hour at room temperature. The membrane was finally washed 6 x 10 minutes with PBS, 0.1% Tween 20 and detection of the secondary antibody performed using enhanced chemiluminescence system from Pierce (34080) according to the manufactures instructions. The membrane was then exposed to X-ray film. 37 38 SECTION 3 3. RESULTS Macromolecular interactions occur in all living organisms thus enabling biological systems to carry out essential functions such as cell differentiation, metabolism and maintenance of cellular homeostasis. These interactions involve essential components such as RNA, DNA and proteins. Execution of processes such as apoptosis relies heavily on these interactions. The main focus of this study was to characterise SNAMA, by isolating proteins which interact with it. The SNAMA homologue was isolated in the process of identifying proteins which play a role in apoptosis. The protein was initially referred to as DWNN (Rees et al., personal communication). Bioinformatics analysis revealed that the human homologue of SNAMA is RBBP6. SNAMA was identified through homology searches using the RBBP6 (Pugh et al., 2006). The hypothesis of this study was that it plays a role in apoptosis. Since apoptosis involves multiple interactions, my hypothesis thus suggests that SNAMA interacts with other proteins such as transcription factors and stress related proteins. SNAMA orthologues such as P2P-R and PACT were previously shown to interact with cell cycle regulators such as p53. In order to test this hypothesis the yeast two-hybrid assay and immunopecipitations were carried out. 3.1 SNAMA interacts with other proteins The yeast two hybrid system is a very powerful tool for identifying protein- protein interactions. The system exploits the fact that eukaryotic transcription factors have multiple domains. A transcription factor can be separated into DNA binding and activation domains, whereby neither of the two domains is capable of activating transcription on its own. The DNA binding (DB) domain is contained in the bait plasmid (phyblex/zeo) whereas the activation domain is in the prey plasmid pJG4-5 (Appendix 3). Interaction of the proteins expressed from both prey and bait plasmids create the complete transcription system. The lexA DB domain in the bait vector and a B42 transcription activation domain in the prey plasmid can only activate reporter genes when brought together by interacting proteins, thus eliminating chances of nonspecific reporter activation. An advantage of this system is immediate isolation of the interacting proteins DNA coding region as compared to the classical biochemical methods, which involve first identification, and later sequencing of the interaction peptides (Van Criekinge and Bayaert, 1999). The Drosophila 0-12 hours embryonic cDNA library in pJG4-5 (prey) plasmid was kindly provided by Professor R. Finley (Finley et al., 1994). Figure 12: Schematic representation of the yeast two hybrid system. Panel A show interaction between the bait and prey and panel B depicts non interacting proteins. Where is the bait protein, stands for the activation domain, DNA binding domain and prey protein. Reporter activation A No expression B 39 3.1.1 Ye ast two hybrid assay 3.1.1a Cloning into pGEM T-easy vector A SNAMA clone in pOT2 was obtained from the Berkely Drosophila Genome Project (AF132177) and used as a PCR template for DWNN amplification. The domain specific primers DWNN1a (5?- GAATTCATGTCGGTACACTAT-3?) and DWNN2a (5?- CTCGAGGGCGATGGGGATGCG-3?) were designed in such a way that they contained EcoRI and XhoI restriction sites respectively (underlined sequences). Following PCR, the amplified DNA product was analysed qualitatively by electrophoresis on 1% agarose gel and the fragm ent size of approximately 200 bp corresponding with the expected size of 213 bp was obtained Figure 13. Figure 13: PCR amplification of DWNN using domain specific primers DWNN1a [forward] a nd DWNN2a [reverse]. Lane1, ? DNA molecular weight marker III sizes of the bands is in kilo base pairs (kb); lane 2 and 3 PCR amplification products. The PCR product was then extracted and ligated into the pGEM-T Easy vector followed by transformation of competent E. coli XL-1 blue cells. White colonies were screened by colony PCR to verify the presence of DWNN in the recombinant plasmids. Furthermore plasmid DNA was isolated the positive (white) colonies and subjected to restriction endonuclease analysis. 40 DWNN (0.2 kb) 21.2kb 0.56kb 1 2 3 41 igure 14 depicts the DNA bands obtained from the screening process. The unr ng colony PCR and restriction endonucleases. The fragments were resolved on 1.1b Construction of the bait plasmid hybrid assays, the DWNN fragment was 3 4 5 F estricted pGEM T-easy recombinant vector in lane 2 and the restricted vector is shown in lane 3 where the DWNN fragment can be seen at the 200 bp region. The colony PCR product in lane 4 was used to further verify that the cloned fragment was indeed DWNN. 1 2 21.2kb 0.56kb DWNN (0.2 kb) Figure 14: Screening of pGE M- T easy- DWNN recombinant plasmid usi a 1% agarose gel with ethidium bromide. Lane 1 contains DNA molecular weight marker III; lane 2 unrestricted pGEM T Easy recombinant plasmid; lane 3 recombinant plasmid restricted with EcoRI and XhoI, lane 4 Colony PCR amplification product; lane 5 Restriction digest product recovered from the gel. 3. To prepare a bait vector for yeast two excised from the pGEM-T easy with EcoRI and XhoI. The resulting fragment was extracted from the gel as outlined in ?Materials and Methods?. The fragment was sub-cloned into dephoshorylated phyblex/zeo plasmid, which was treated with the same restriction endonuclases used to release the DWNN fragment from the primary host vector. The construct was propagated in E. coli XL-1 blue cells and the recombinant plasmid was subjected to restriction analysis to confirm the presence of the insert (figure15). Figure 15: Screening of phyblex/zeo recomb inant plasmids with restriction endonucleases. Gel showing electrophoresis of pHybLex/Zeo DWNN cl ones. Lane 1, DNA molecular weight marker III; lane 2 and 4 Recom binant plasmids restricted with EcoRI and XhoI; lane 3 and 5 Unrestricted recom binant plasmids 1 2 3 4 5 21.2kb 0.56kb DWNN (0.2 kb) GTCACGTTCGCATCTCTAGTAAATTGGAAAAAAATAAAATCGTCGGAGTTTTTATGTGCT || | | || | || | | | | | | GTTTAAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGGGCTGGCGGT TGCAGAGTAGTATTTCTTTCATATGCAACT....ATGTCGGTACACTATAAATTTAAGAG || | | | || |||||||||||||||||||||||||| TGGGGTTATTCGCAACGGCGACTGGCTG GAATTC ATGTCGGTACACTATAAATTTAAGAG TACACTCAACTTTGATACAATTACTTTTGATGGACTTCACATTTCTGTCGGGGACTTAAA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| TACACTCAACTTTGATACAATTACTTTTGATGGACTTCACATTTCTGTCGGGGACTTAAA AAGGGAGATTGTGCAGCAGAAGCGACTGGGCAAAATCATCGACTTTGATCTCCAAATAAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAGGGAGATTGTGCAGCAGAAGCGACTGGGCAAAATCATCGACTTTGATCTCCAAATAAC AAATGCGCAGAGTAAAGAAGAATACAAGGACGATGGGTTCCTTATTCCCAAAAACACAAC |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| AAATGCGCAGAGTAAAGAAGAATACAAGGACGATGGGTTCCTTATTCCCAAAAACACAAC GCTGATCATATCGCGCATCCCCATCGCCCATCCCACAAAAAAGGGCTGGGAGCCACCAGC ||||||||||||||||||||||||||||| | | || GCTGATCATATCGCGCATCCCCATCGCC CTCGAG TCGACCTGCAGCCA Figure 16: Optima l alignment of the cloned DWNN gene fragment (low er sequence) w ith the SNAMA cDNA sequence from NCBI and BDGP (upper sequence) see appendix 2 . Purple coloured nucleotides are restriction sites used for in frame directional ligation of the coding region of the domain domain with lexA DNA binding module. These sequences were aligned using DNAMAN and were found to be 100% com plimentary to the original published sequence. 42 43 Since PCR introduces mutations, plasmid DNA obtained from the positive clones was sequenced and analysed. The cloned DWNN fragment was identical to the one available on the NCBI and BDGP databases and it contained no mutations (figure16). The results also confirmed that the inserted DWNN- encoding fragment was in frame with the coding region for the lexA DB domain (figure17). AGACATTTGAAGGCGTTGGCACGCAAAGGCGTTATGAAATGTTTCCGGCGATCACGNGGG ATTTGTCTGTGCAGAAGAGAAGAAGGGTTGCCGNTGGTAGGTCGTGTGGCTGCCGGTGAA CCACTTCTGGCGCNACAGCATATTGAAGGTCATTATCAGGTCGATCCTTCCTTATTCAAG CCACTTCTGGCGCNACAGCATATTGAAGGTCATTATCAGGTCGATCCTTCCTTATTCAAG CCGAATGCTGATTTCCTGCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATG GATGGTGACTTGCTGGCAGTGCATAAAACTCCAGGATGTACGTAACGGTCAGGTCGTTGT M V T C W Q C I K L Q D V R N G Q V V V CGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAAAAAACAGGGCAATAAAGTCGA A R I D D E V T V K R L K K Q G N K V E ACTGTTGCCAGAAAATAGCGAGTTTAAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTT L L P E N S E F K P I V V D L R Q Q S F CACCATTGAAGGGCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTG GAATTC ATGTC T I E G L A V G V I R N G D W L E F M S GGTACACTATAAATTTAAGAGTACACTCAACTTTGATACAATTACTTTTGATGGACTTCA V H Y K F K S T L N F D T I T F D G L H CATTTCTGTCGGGGACTTAAAAAGGGAGATTGTGCAGCAGAAGCGACTGGGCAAAATCAT I S V G D L K R E I V Q Q K R L G K I I CGACTTTGATCTCCAAATAACAAATGCGCAGAGTAAAGAAGAATACAAGGACGATGGGTT D F D L Q I T N A Q S K E E Y K D D G F CCTTATTCCCAAAAACACAACGCTGATCATATCGCGCATCCCCATCGCC CTCGAG TCGAC L I P K N T T L I I S R I P I A L E S T CTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATATTTTATA TAA ANAGANAA C S Q A N S G R I S Y D L Y F I * Figure 17: Sequence analysis of the pHybLex/Zeo DWNN plasmid construct. The sequence was generated by the automated DNA sequencer ABI prism using pHybLex/Zeo reverse primer 5?-GAG TCA CTT TAA AAT TTG TAT ACA C-3? and the sequence was analysed using DNAMAN. Purple coloured nucleotides are restriction sites used to insert the DWNN encoding region into the vector, green is the stop codon and the underlined sequence is the vector. Blue letters represent the amino acid sequence of DWNN whereas the black letters represent the phyblex/zeo sequence. 44 3.1.1c Preparation of the bait strain S. cerevisiae EGY 48 competent yeast cells containing pSH18-34 reporter plasmid were transformed with the pHybLex/Zeo-DWNN construct. The transformants were selected on YPD rich media supplemented with zeocin. 3.1.1d Transformation of bait strain with cDNA library recombinant plasmids The 0-12 hour Drosophila embryonic cDNA library in pJG 4-5 was used to transform competent S. cerevisiae EGY 48/pSH18-34/phyblex/zeo-DWNN- yeast strain and plated on YNB/glu/zeo/ura-/trp- selective media. Transformants were then frozen and replated to select for all the plasmids. For interaction mating assays the library plasmid was transformed into S. cerevisiae RFY 206 strain and selected on YNB/glu/trp- plates. S. cerevisiae RFY 206 is a MAT a haploid strain of Saccharomyces which can mate with any MAT ? to form a diploid strain. In this case the mating strain was S. cerevisiae EGY48. 3.1.1e Interaction hunt Interaction hunt was performed by plating transformed yeast cells which were selected and shown to carry all the required recombinant plasmids. Transformed yeast cells were replica plated onto YNB selective media lacking uracil, tryptophan and leucine supplemented with galactose (for activation of the GAL1 promoter), raffinose (to encourage yeast growth) and zeocin. Figure 18 shows assays carried out to test for ?-gal reporter activation. Colonies were lifted off the plate onto a filter paper and tested for ?-gal activation by incubating them in an X-gal containing solution. Figure 18a is a positive control picture showing reporter activation caused by interaction between Fos and Jun and figure 18b is a negative control with unaltered phylex/zeo and library plasmid. Transformed cells carrying plasmid which express proteins that interact with DWNN were expected to turn blue when treated with X-gal plates and also to grow on plates lacking leucine. This assay produced no interactors Figure 18 c-f. ba d c fe Figure 18: Yeast tw o hybrid assays. The filter lift assay was carried out on two days old colonies. A, Positive controls with bait (phyblex/Zeo-Fos) and prey (pYestTrp-Jun ) plasmids containing genes of proteins which are known to interact. B, Negative control to test for reporter autoactivation pJG4-5 library plasmid with phyblexzeo. c-f, Experimental results showing assay carried out with pHyblex/Zeo-DWNN (Bait) and pJG4-5 library (prey plasm ids). 3.1.2 Immunoprecipitation assays 3.1.2a Heterologous expression of DWNN in E . coli cells The recombinant pGEX 6P2 DWNN plasmid construct was obtained from Zungu, (2003) and the construct was used to transform E. coli BL-21 (DE3) competent cells. The protein was overexpressed in E. coli BL 21 (DE3) with 0.1 M IPTG for 4 hours and the GST-DWNN fusion was isolated in soluble 45 fraction using the bugbuster reagent (Novagen). Figure 19 shows the SDS- PAGE profile of proteins extracted with the bugbuster reagent. The majority of the fusion protein was obtained in the soluble fraction. M 1 2 3 4 46 118 86 47 GST-DWNN 34 Figure 19 : Large-scale expression of GST-DWNN fusion protein . Proteins were resolved on a 12% SDS PAGE gel. Lane marked M is Prestained protein molecular weight markers (Fermentas). Lane 1, Soluble extract from cells before induction, lane 2, insoluble extract from cells before induction of expression; lane 3 soluble crude extrac ts after induction with 0.1M IPTG at 37oC; lane 4, Insoluble extract after induction. 3.1.2b Immunoprecipitations with the GST-DWNN recombinant protein The crude protein extract from E. coli BL-21(DE3) cells was incubated with anti-GST or anti-SNAMA antibodies for 2 hours at 4oC. Protein A sepharose matrix was then added to the antibody fusion protein reaction and incubated for further 2 hours followed by stringent washes using the RIPA buffer. The eluted protein was analysed on a 12% SDS-P AGE. Figure 20 lanes 1 and 5 depict samples which were immunoprecipitated with anti-GST and anti-SNAMA respectively. To test if DWNN is involved in any protein interactions the adult fly crude protein extract was added to the antibody fusion protein reaction immobilized on a protein A matrix and the samples were also subjected to stringent washes with RIPA buffer. Lane 2 and 3 of figure 20 shows the crude extract from wild type flies and heterozygous SNAMA mutant fly line l(2)rQ313w;GFP;Cyo respectively after im munoprecipitating with the anti-GST and GST-DWNN fusion, whereas lanes 6 and 7 shows products immunoprecipitated with anti-SNAMA. 1 2 3 4 5 6 7 M 160 105 75 50 35 30 25 DWNN GST-DWNN + + + + + + Anti-GST + + + - - - Anti-SNAMA - - - + + + Mutant crude extract - + - - + - Wild type Crude extract - - + - - + Figure 20: The GST-DWNN fusion protein wa s immobilized on a pr otein A sepharose matrix using anti-GST or anti-SNAMA antibodies. In vitro interaction assay was carried with crude protein extract from adult mutant and wild type flies out using fusion protein. The SDS-PAGE profiles obtained from this assay revealed that the GST- DWNN fusion is unstable, especially when using buffers that have detergents. The DWNN band could be seen at the bottom of the gel, thus implying that the fragment might have been cleaved from the GST during the washing steps 47 (Figure 20). Zungu (2003) also reported that the DWNN-GST fusion tends to be unstable which results in the auto-cleavage of DWNN from GST. To counteract the pre-cleavage of the fusion protein the GST-fusion antibody complex immobilised on the protein A matrix was washed with 1 x PBS followed by the addition of the crude protein extract from flies. The proteins eluted from the matrix are shown below (figure 21). Lane 5 (l(2)rQ313w;GFP;Cyo crude protein and GST-DWNN fusion) and lane 7 (Canton S wild type crude protein and GST-DWNN) show proteins immunoprecipitated with anti-SNAMA, whereas lane 6 (l(2)rQ313w;GFP;Cyo crude protein and GST-DWNN fusion) and 8 (Canton S wild type crude protein and GST-DWNN) are proteins immunoprecipitated with anti-GST. GST-DWNN + + + + Anti-GST + - + - Anti-SNAMA - + - + Mutant crude extract + + - - Wild type Crude extract - - + + GST- DWNN X1 X2 IgG?s kDa kDa kDa kDa118 M 1 2 3 4 5 6 7 8 86 47 34 Figure 21: Immunoprecipitations with GST-DWNN. M=Prestain ed molecular weight marker (Fermentas). Lanes 1 preinduction soluble extract and lane 2 preinduction insoluble extract. Lane 3 soluble extract after 4 hours induction with 100mM IPTG and lane 4 is the insoluble extract. Lanes 5 and 7 immunoprecipitations using fusion protein immobilized on the protein A matrix with anti-GST. Lane 6 and 8; Immunopr ecipitation assay using fusion protein immobilized on the protein A matrix with anti-SNAMA. 48 49 Washing the column with PBS buffer did not affect the integrity of the fusion protein. Figure 21 shows some of the proteins which were isolated from the assay. Interestingly two bands at 45 kDa (X2) and 50 kDa (X1) appear reproducibly in three reactions (lanes 6-8). The protein marked X2 might be Dmp53. More experiments are underway to check if the 50 kDa band is Dmp53. 3.1.2c Interactions with Dmp53 Immunoprecipitations were carried out to further verify that SNAMA interact with other proteins as suggested by the primary structure analysis. Homologues of the zinc finger motifs of SNAMA are known to promote protein-protein interactions. The CCHC belongs to a family of zinc finger motifs, which interact with a transcription factor GATA (Fox et al., 1999). The RING finger-like motif also suggests that the protein might be covalently attached to other proteins. Crude protein extracts from adult wild type Cantons S and l(2)rQ313w;GFP;Cyo mutant fly lines was used in the immunoprecipitations with anti-Dmp53, anti-SNAMA and anti?human DWNN. The l(2)rQ313w;GFP;Cyo fly line is a heterozygous mutant where SNAMA has been disrupted. Figure 22 depicts the immunoprecipitation profiles with anti-Dmp53, anti-SNAMA or anti?human DWNN (RBBP6). There was a noticeable difference between the proteins in the mutant and the wild type flies. Anti-humanDWNN-13 - - - + + + - - - Anti-Dmp53 + + + - - - - - - Anti-SNAMA - - - - - - + + + Mutant crude extract - + - - + - - + - Wild type Crude extract - - + - - + - - + Protein A + + + + + + + + + 118 kDa Da Da Da 34 k 47 k 86 k M 1 2 3 4 5 6 7 8 9 Figure 22: Immunoprecipitation assays of crude protein extract from l(2)rQ313/w ; GFP; cyo and Canton S w ild type adult fly extract with anti- Dmp53 anti-SNAMA and anti-Hu manDWNN-13. M is Prestained molecular weight marker (fermentas). Lane 1-3, immunoprecipitations with anti-Dmp53, lanes 4-6 and 7-9 are products immunoprecipitated with anti-SNAMA and anti- HumanDWNN-13 antibodies respectively. Immunoprecipitations with protein from wild type flies gave a distinct band of approximately 86 kDa with all the three antibodies (Figure 22 lanes 3, 6 and 9). The size of the band did not correspond with the predicted molecular weight of either SNAMA or Dmp53. Even though the size of the product immunoprecipitated by the three antibodies did not correspond to the expected size of either protein (SNAMA or Dmp53) or the predicted molecular weight of the complex between the two; these results suggest that Dmp53 and SNAMA might be interacting. Immunoprecipitations with the mutant flies protein did give two distinct bands in the sample lanes were anti-SNAMA and anti?hum an 50 DWNN antibodies were used lane 5 and 8 respectively. However, no bands could be seen where Dmp53 antibody was used (lane 2). 1st 2nd 3rd 4th 50 40 kDa 0-3 3-6 Adult 0-3 3-6 Adult Adult 0-3 Anti-SNAMA + + + - - - - - Anti-DMp53 - - - + + + - - Goat serum - - - - - - + - ProteinA + + + + + + + - 1 2 3 4 5 6 7 8 M 140kDa 120kDa 110kDa 95kDa 85 kDa 75 kDa 60 kDa 50kDa 30 kDa 25 kDa 20 kDa Figure 23: Immunoprecipitation assay of crude protein extracts from CantonS wild type flies with anti-SNAMA and anti-Dmp5 3. Lanes 1-3 crude protein extracts from 0-3, 3-6 and adult flies immunoprecipitated with anti- SNAMA. Lanes 4-6 crude protein extracts immunoprecipitated with anti-Dmp53. lane 7 goat serum with adult flies crude, whereas lane 8 is the 0-3 hour embryos crude extract. Based on the results obtained using adult flies the search was broadened further by probing the early developmental stages of wild type flies for any interesting interactions. Crude extracts from wild type 0-3 hours and 3-6 hours old embryos were used in immunoprecipitations with anti-SNAMA and anti-Dmp53 antibodies and compared with the results from adult flies. It must be noted that the adult only control was used as the immunoprecipitations with 0-3 and 3-6 hours old embryos were included for comparison with results obtained previously with adult flies (See figure 22) where an 86 kDa band was observed. Interestingly, the 0-3 hour?s crude protein immunoprecipitations with anti-SNAMA and anti-Dmp53 antibodies did show additional protein bands of approximately 75 kDa, 50 kDa 51 52 and 45 kDa (figure 23 lanes 1 and 4). Proteins that were immunoprecipitated were identified using MALDI-TOF mass spectrophotometer at the Institut de Biologie Moleculaire et Cellulaire at Strasbourg Cedex in France. They were identified as heat shock protein 82 (hsp82), LP19893p (HSP70) and CG2985-PA (Pancreatic lipase like) marked 1st, 2nd and 3rd respectively on the gel (figure 23). Identification of these proteins in early embryonic stages suggests that they are crucial in development. Results obtained with 0-3 hour?s embryos thus suggest that SNAMA plays a different in adult flies compared to early developmental stages. Previous studies have already shown that Dmp53 and SNAMA play a role in apoptosis, thus these results further suggest that SNAMA and Dmp53 are involved in similar processes. Even though the controls used were from adult flies the identification of heat shock proteins immunoprecipitated by both anti- SNAMA and anti-Dmp53 suggest that the SNAMA and Dmp53 might be regulating the activity those proteins. Heat shock proteins are known to be involved in protein turnover by stabilizing the 26s proteasome (Kiss et al 2005). Heat shock protein 70 is expressed at high levels in stressed cells and it tends to reduce cell proliferation whenever it is overexpressed (Krebs a nd Feder, 1997). 3.1.2d Western analysis of immunoprecipitated proteins Immunoprecipitations with protein from flies using anti-Dmp53 suggest that Dmp53 might be modified by SNAMA. Crude protein extracts were collected from 0-3, 3-6 hours old embryos and adult flies. Immunoprecipitations with anti-Dmp53 and anti-SNAMA were carried out and the products were resolved and blotted onto a PVDF membrane. Immunoblot analysis of the immunoprecipitated proteins with anti-SNAMA and anti-Dmp53 further confirmed the specificity of the immunoprecipitation. Figure 24 is a blot probed with anti-Dmp53, the 53 kDa band observed on the gel corresponds to the molecular masses of one Dmp53 isoform and the heavy chain of the antibody. Interestingly the band was also detected in the lanes where anti-SNAMA antibodies were used in the immunoprecipitations, thus suggesting that it might be one of the Dmp53 isoforms. Crude extract 0-3 3-6 Adult 0-3 3-6 Adult 0-3 3-6 Adult Protein A + + + + + + - - + ? SNAMA + + + - - - - - - ?- Dmp53 - - - + + + - - - Goat serum - - - - - - - - + 1 2 3 4 5 6 7 8 9 140 53 35 33 26 Figure 24: Western blot analysis of proteinns immunoprecipitated with anti- SNAMA and anti-Dmp 53. Protein extracts from wild type Canton S flies used in the immunoprecipitations with anti SNAMA(lanes 1-3), anti Dmp53(lanes 4- 6). Lanes 7-8 crude protein extracts and lane 9 Crude proteins extract from adult flies immunoprecipitated with goat serum. The blot was probed with anti-Dmp53 Figure 25 below shows blot probed with anti-SNAMA. The 53 kDa and the 25 kDa bands detected on the blot correspond to the expected molecular weight of the heavy and light chain SNAMA immunoglobulins. The only interesting band is the ?140 kDa at the top. Even though SNAMA has a predicted molecular weight of 148 kDa, all previous western blots with anti-SNAMA 53 antibodies failed to elicit the band of that molecular weight, thus the high molecular weight band in the blot might be SNAMA. 54 Crude extract 0-3 3-6 Adults 0-3 3-6 Adults 0-3 3-6 Adults anti-Dmp53 + + + - - - - - - anti-SNAMA + + + - - - - - - ProteinA + + + + + + - - - Goat serum - - - - - - - - + 1 2 3 4 5 6 7 8 9 140 53 35 33 26 25 Figure 25: Detection of SNAMA by immunobloting. Crude protein extracts from wild type Canton S flies used in the immunoprecipitations with anti SNAMA (lanes 1-3) and anti Dmp53 (lanes 4-6). Lanes 7-8 crude protein extract from 0-3 and 3-6 hours old embryos, lane 9 Crude proteins extract from adult flies immunoprecipitated with goat serum. The blot was probed with anti-SNAMA. SECTION 4 4. DISCUSSION Previous studies have rev ealed that SNAMA plays a ro le in apoptosis (Mather et al . , 2005). The protein is expresse d at ve ry high levels at early develop m e n t a l stages and is crucia l in em bryo n i c devel o p m e n t since d e let i o n resul t s in reces s i v e lethality. S NAMA ortholgues such as PA CT, P2P-R and Mpe1 play diverse roles in cell cycle regulati o n . PACT is known to interac t with p53 and Rb (Simons et al., 1997), whereas the Mpe1 in yeast is involve d in m R NA process i n g . Sakai et al., (1995) also found that RBBP6 interacts with Rb. The ho m o logy tree below depicts how closely SNAMA is related to its ortholo g u e s (figure 26). R B Q 1 P A C T H u m a n _ D W N N _ f u l l _ l e n P 2 P - R S N A M A H u m a n _ D W N N _ D o m a i n M p e 1 1 0 0 % 2 0 %8 0 % 6 0 % 4 0 % Figure 26: Sequence comparison between SNAMA and related proteins. The scale b a r ab ove signif i e s the percen t a g e hom olo g y betwee n the protei n s 55 Even though the overall sequence hom ology between SNAMA a nd its orthol o g u e s from higher e ukary o t e s appear s to be lo w, they share a sim ila r prim ary structu r e . The RING finge r- l i k e m o tif is presen t in all the orthol o g u e s , where a s Mpe1, SNAMA and the hum an hom ol o g u e conta i n the N-ter m i n a l extens i o n which includ e s the unique DW NN dom a in and the zinc finger. The focus of this study was to charac t e r i s e th e unique D W NN dom a in and SNAMA by isola t i n g p r ote i n s whic h inte r a c t with them . It was m e nti o n e d in section 1.1 that struct u r a l prediction studies reveale d that the dom ain has an ubiqu t i n lik e f o ld . This predi c t i o n im pl i e s tha t the pro t e i n m i ght inter a c t with other protei n s . Bioinfo r m a t i c analys i s of SNAMA orthologues revealed that som e have ubiquit i n - l i k e featur e s . Figure 27: Alignment of insects, mammals, yeasts, worms and plants DWNN domains. T h e al i g n m e n t was do ne usi n g DN AM A N v e rs i o n 4. 03 (Ly n n o n B i os o f t ) . Ubiqui t i n re lat e d prote i n s are d i vid e d into two dif f e r e n t c a teg o r i e s te rm e d ubiqui t i n - l i k e m odi fi e r s (ubl) and ubiquiti n - d o m a i n proteins (ubp) (Jentsch and 56 P y r o w o l a k i s , 2000). Ubp are protein s su ch as NIRF, scythe and parkin which posse s s the ubiqui t i n like dom ain (Mori , 2002; Jentsch and Pyrowolakis 2000). Protein s such as SUMO1 and NEDD8 are the best known ubiquiti n - l i k e proteins . Figure 27 shows the alignm e n t of the N-term inal dom ain of SNAM A and its orthologues. The hum a n ( H. sapien ) , zebra f i s h ( D. rerio) and a protozoan ( E. cuniculi ) orthol o g u e s m i ght be classi f i e d as ubiquitin-like proteins. These prote i n s hav e the di-gl y c i n e re sid u e s , which ar e chara c t e r i s t i c of ubiq u i t i n and most ubl. One of the most common featur e s with the s e p r ote i n s is th e proli n e a t posit i o n 76 of SNAMA ? s N-ter m i n a l regio n ; th e am ino acid is high l y co nse r v e d and m i ght be im por t a n t in the biolo g i c a l processi n g of the protein. Gilchrist et al . , (1997) report e d that som e ubiqiu t i n - s p e c i f i c prote a s e s cleav e ubiqui t i n fusion proteins at the ubiquitin pr oline bond. Proteins such as HUB1 covalen t l y m odify other protei n s even though it lacks the C- termi n a l glycine residue s (Yoshir o d a and Tanaka, 2004). 4.1 Analysis of protein- protein interaction using the yeast two-hybrid system D W N N was insert e d in to a yeast ex pres s i o n p l asm i d pHybl e x / z e o as a lex A fusion const r u c t . The recom b i n a n t plas m i d m o le c u l e was confi r m e d by sequen c i n g to be in fra m e with the lex A coding region. The construc t was used to trans f e c t S. cerevis iae EGY 48 yeast cells and chara c t e r i s e d using stan d a r d two hybrid assays. 57 Y e a s t cells contai n i n g the bait plas m i d were further transfor m e d with the library. Approxim a t e l y 4x10 3 transfor m a n t s were obtained per transfor m a t i o n . The m i nim u m a m ount of plas m i d DNA had to be used in these trans f o r m a t i o n s so as to m a ke sure that only one plasm i d molec u l e is inco r p o r a t e d into the yeas t cells . The galac t o s e in duc i b l e syste m in the pJG4-5 librar y plasm i d offers an added advanta g e , as express i o n of the libra r y prote i n s is repr essed by the presence of glucose. Plating the transfor m a n t s on the galactose cont ai n i n g m e dia induce s express i o n of the library pr o t e i n s . All the trans f o r m a n t s that were screen e d showed no noticea b l e activat i o n and express i o n of reporte r protein s . The presen c e of the 20 am ino acid at the c-term i n a l end howeve r , m a y have caused structu r a l change affecti n g interac t i o n with target protein s . Heterelo g o u s expres s i o n of protein s in yeast has alway s been probl e m a t i c becau s e y east cells tend to be highly codon biased (Akash i , 2001), wherea s a strain such as Saccharomyces cer evis iae tends to hypergly c o s y l a te expressed proteins. SNAMA homolog u e Mpe1 in yeasts is invol ved in mRNA s p licing. It form s part of the CPF com p lex which process e s si ngle stranded RNA. The dom ain m i ght also be acting as an in ert s caffo l d , thus a i din g the m o tif assoc i a t e d with it in interactions. 4.2 Analysis of protein-protein i nteraction DWNN was express e d succes s f u l l y as a GST tagged protei n in E. coli BL 21(DE3) cells. Initially, when the protei n was is olat e d from these cells u s ing th e 58 s o n i c a t i o n m e thod, this protei n was expressed in inclus ion bodies. The sonication m e thod was replace d by the bugbust e r reag ent based m e thod that proved to be more ef f i c i e n t . High q u ant i t i e s of this f u sio n prote i n wer e solub l e w ith non e expresse d in the insolubl e extract. In vitro studies with the heterologously expre s s e d DWNN reveal e d that the protei n does interact with other proteins . Figure 21 shows immunopr e c i p i t a t i o n s w ith the GST-DWNN fusion protein. Protei n s of approx i m a t e l y 45 and 50 kDa wh ere isola t e d in this assay . It m a y be signif i c a n t that the 45 kDa band corres p o n d s to the estim a t e d size of one isofor m of Dmp53. Immunoprecipitations also revealed that SNAMA interacts with other protei n s . Crude protei n extrac t s from adult wild type and SNAMA knocko u t mutan t s us e d were f oun d to co-e l u t e with other proteins. Th e 90 kDa band (figure 22) was obtained with the crude protein extract f r om wild type flies. The protein s were imm u no p r e c i p i t a t e d with anti-SNAMA, a n ti-Dm p 5 3 or anti- h u m a n D W N N . The band size did not correspond with either SNAMA or any of the Dm p53 isofor m s . Even though the size of the band did not correspond to the expected sizes of the both proteins, the fact th at it was immunop r e c t i p i t a t e d by both anti- S N A M A and anti-D m p 5 3 im plie s that Dm p53 and SNAMA m i ght be involved in sim ila r pathwa y s . Protei n s from mutant flies gave a ba nd slightly bigger than the one obtai n e d with the w ild typ e f lies . Furthe r immuno p r e c i p i t a t i o n with crude ex trac t s from e m bryo s showed that SNAMA and p53 m i ght be involved with the 26S proteasom e , figure 23 shows 59 that the antibodies against SNAMA and Dmp53 immunoprecipitate sim ilar proteins . T h e proteins identifi e d we re hsp82, Hsp70 and CG2985-PA (Pancreatic lipase like). Hsp82 has been shown to associa t e with the 26S proteaso m e (Kiss et al 2005). Krebs and Feder (1997) report e d that overexp r e s s i o n of Hsp70 causes reductio n in cell prolifer a t i o n . Figure 24 and 25 show a W e stern blot analysis of the protein s obtaine d with the immunop r e c i p i t a t i o n s . Immunobl ot probed with anti-S N A M A and anti-Dm p 5 3 was used to confirm t h e specifi c i t y of the immunop r e c i p i t a t i o n s . The immunob l o t probed with anti-SNAMA antibody (figur e 25) shows a high m o lecu l a r weight band which corres p o n d s with a predicte d molecula r weight of SNAMA ( 135 kDa). The presence of this high molecula r weight band in the immunobl o t s is highly interesting as it is not appa r e n t in the imm u n o p r e c i p i t a t i o n gel (f igure 23). Thus the presence of the protein m i ght have led to th e precipit a t i o n s of the pr oteins identified above. These results thus suggest that SNAMA m i ght be playing a role in stress relate d cell cycle regul a t i o n . 60 SECTION 5 5. CONCLUSION AND FUTURE PROSPECTS T h i s study was undertak e n to investig a t e the association between SNAMA and other protei n s . The major hypoth e s i s was derived from previous studies with SNAMA ortholog u e s such as P2P-R, PA CT a nd RBBP6 which were shown to interac t with other protein s . Immunopr ecipitation assays with antibodies against SNAMA have shown that the protein doe s associate with other proteins. Inter e s t i n g l y sim il a r exper i m e n t with anti- D m p 5 3 gave a sim il a r prof i l e to th a t obtain e d with anti-S N A M A . The fact that SNAMA and Dm p53 m i ght intera c t with sim ila r protei n s make s this study very interesting and m i ght contribute further to the growing b ody of scientific knowledge about p53. Even though p53 has been widely studied its exact role in carcinogene sis is not clearly known. This study thus presents another dim e nsio n in the study of p53 and SNAMA as som e of the protei n s which were identi f i e d are known to be involve d in cell cycle. Hsp82 plays a role in the st abili z a t i o n of th e 26S prote a s o m e , where a s Hsp70 tend to be involved in cell cycle regulati o n by slowing down prolifer a t i o n of the affected cells. The association of th e Hsp70 with Dm p53 appear s to be highly significant as the Dm p53 in flies does not pl ay a role in cell cycle regulation. The protein bands which were identi f i e d with the immuno p r e c i p i t a t i o n s were observed in em bry o s only and not adult fly extra c t . Even though the contro l s shown in the m a nusc r i p t were done with crude extrac t fr om adult flies the results obtained are still sign i f i c a n t . The prote i n s whic h were ident i f i e d par ti c i p a t e in the proce s s e s 61 s u c h as cell cycle regu la t i o n and p r ot ei n turnov e r . Dm p53 and SNAMA are involv e d in apopto s i s , wherea s the predic te d structur e of SNAMA?s N-term in a l dom a i n sugge s t s that it is ubiquitin like. Assays with the yeast two hybrid syst em using the conser v e d DWNN did not produce any interac t i n g protei n s . T h e assa y is prone to lim it a t i o n s as it relie s heavil y on physic a l intera c t i o n s betwee n two proteins, SNAMA could interact with other protei n s throug h covale n t in teractions involving the DWNN dom a i n these could be observed on the immunoprecipitation ex per i m e n t s . Furth e r experi m e n t s with the f u ll length SNAM A m i ght help in elucid a t i n g the characteristics of the protein even though results obtained with the immunoprecipitations suggest that SNAM A m i ght be part of a regulat o r y com p l e x . 62 63 SECTION 6 6. APPENDIX 1 Media and Supplements LB Agar: Tryptone 10 g/l, NaCl 10 g/l, yeast extra ct 5 g/l, agar 28 g/l LB Broth: Compon e n t s of this medi a were the same as the LBagar above with the exceptio n of agar. Yeast pepto n e dextr o s e : 10 g/l yeast extr act, 20 g/l of peptone, 0.1 g/ladeni n e , 2% gluco s e . Yeast nitrog e n base media: 6.7 g/l yeast nitro g e n base w/o amino acids, 0.6 g/l amino acid dropout mix, 20g/l glucose or (20 g/lgala c t o s e + 10 g/l raffino s e ) IPTG: the stock was made at 0.1 M in water and the solutio n was filte r steri l i ze d and stored at 4 o C X-gal: the stock was made at 50 mg/ ml in N, N, Di methy l for ma mi d e . The working concen t r a t i o n was 25 ?g / ml Ampi c i l l i n: the stock solu t i o n was made at 100 mg/ml in water and steri l i z ed by filtr at i on . Tetra c y cl i n e: the stock was ma de at 12 mg/ ml in 70% ethano l . 64 General stock buffers solutions and kits components TE buffer (10x): 100 mM Tris, 1 mM EDTA adjust e d to pH 7.5 with conce n t ra t e d HCI. This stock solut i on was dilut ed 10 fold with steril e water to make the workin g solut i o n . TAE (50x): 2 M Tris, 5.7% glac i a l aceti c acid, 0.5 M EDTA pH 8.0 Transfor ma t i o n buffer (TFB): 10 mM K-Mes (pH 6.2), 100 mM RbCl, 45 mM MnCl 2 .4H 2 O , 10 mM CaCl.2H 2 O, 3 mM Hexa mine cobalt chlorid e Ethanol acetate : 96% (v/v) ab solu t e ethano l and 4% (v/v) 3M Sodiu m aceta t e LiPEG: 100 mM Lithium acetate pH 7.5, 40% PEG-3350 , 10 mM Tris-HCI pH 7.5. LiTE : 1x Lithium acetat e pH 7.5, 0.5x TE Solutio n 1: 50 mM glucose , 25 mM Tris, 10mM EDTA pH 8.0 Solution 2: 0.2 M NaOH, 1% SDS (w/v) Soluti o n 3: 3 M sodium aceta t e Cell lysis buffer: 25 mM Tr is, 150 mM Na Cl, 0.2% SDS, 0.5 mM PMSF (sigma), 1 ?g / ml aproti n i n , 4.0 mM Pefablo c (Roch e ) TBS: 10 mM Tris pH 8.0, 150 mM NaCl 65 PBS: 150 mM Na Cl, 9.1 mM K 2 HPO 4 , 1.7 mM KH 2 P O 4 , adjusted to pH 7.4 with NaOH SDS-PAG E sample buffer: 0.225 M Tris -Cl (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromophe n o l blue, 0.25 mM DTT SDS PAGE Running buffer: 0.5 M Tr is base, 1.92 M glycine, 0.5% SDS Electro b l o t t i n g buffer: 25 mM Tris , 192 mM Glycine , 3.5 mM SDS, 20% (v/v) meth a n o l Cooma s s i e blue stain : 0.1% Cooma s i e blue R-250 (w/v), 10% acetic acid, 40% metha n o l Destain solution: 40% methan o l , 10% acetic acid RIPA buffer: 1x PBS, 1% Nonidet P-40, 0.5% sodium dioxycholate, 0.1% SDS Z Buffer: 60 mM Na 2 HPO 4 , 40 mM NaH 2 PO 4 , 10 mM KCI, 1 mM MgSO 4 - 7 H 2 O pH 7.0 Glycerol solution : 65% glycerol, 0.1 M MgSO 4 , 25 mM Tris-HCl, pH 8.0 GST purific a t i o n kit (Cat no:7079 4 - 3 ) : BugBust e r pr ote i n extra c t i o n reage n t (cat no: 70584-3 ) , and 10, 000 units Benzona s e nucleas e (cat no: 70746-3), GST Bind resin (cat no: 70541- 3 ) GST bind wash buffer (10x): 43 mM Na 2 HPO 4 , 14.7 mM KH 2 PO 4 , 1.37 M NaCl, 27 mM KCl pH 7.3 66 GST elution buffer: 500 mM Tr is-HC l pH 8.0, 100 mM glutath i o n e GST mag agarose beads: 50% sl urry, 0.05 M phosphat e buffer pH 7.5, 0.15 M NaCl, 0.1 M NaN 3 Membra n e stripi n g buffer : 2% (w/v ) SDS, 62.5 mM Tris-HCl pH 6.8, 100 mM ? - me c a p t o e t h a n o l Protea s e inhibi t o r stocks : 10 mg/ m l PMSF (Sigma , cat no: P 7626), 20 mg/ml Pefabloc (Roche, cat no: 1873601), 1mg/ ml Aprotini n (Roche, cat no: 981532) 67 APPENDIX 2 1 GCATTTCACATCTCTGGGGCTTTGGCGTCACGTTCGCATCTCTAGTAAATTGGAAAAAAA 1 61 TAAAATCGTCGGAGTTTTTATGTGCTTGCAGAGTAGTATTTCTTTCATATGCAACTATGT 21 M S 121 CGGTACACTATAAATTTAAGAGTACACTCAACTTTGATACAATTACTTTTGATGGACTTC 41 V H Y K F K S T L N F D T I T F D G L H 181 ACATTTCTGTCGGGGACTTAAAAAGGGAGATTGTGCAGCAGAAGCGACTGGGCAAAATCA 61 I S V G D L K R E I V Q Q K R L G K I I 241 TCGACTTTGATCTCCAAATAACAAATGCGCAGAGTAAAGAAGAATACAAGGACGATGGGT 81 D F D L Q I T N A Q S K E E Y K D D G F 301 TCCTTATTCCCAAAAACACAACGCTGATCATATCGCGCATCCCCATCGCCCATCCCACAA 101 L I P K N T T L I I S R I P I A H P T K 361 AAAAGGGCTGGGAGCCACCAGCAGCAGAAAATGCCTTTTCGGCGGCGCCTGCCAAGCAGG 121 K G W E P P A A E N A F S A A P A K Q D 421 ACAACTTCAACATGGACCTGTCCAAAATGCAAGGCACGGAGGAGGACAAAATCCAGGCCA 141 N F N M D L S K M Q G T E E D K I Q A M 481 TGATGATGCAGAGCACAGTCGACTATGATCCTAAGACGTACCATCGTATTAAAGGACAAT 161 M M Q S T V D Y D P K T Y H R I K G Q S 541 CGCAAGTGGGAGAAGTTCCCGCATCCTACCGATGCAACAAATGCAAGAAAAGCGGACACT 181 Q V G E V P A S Y R C N K C K K S G H W 601 GGATCAAAAACTGTCCCTTTGTGGGGGGAAAGGACCAGCAAGAGGTCAAACGGAATACTG 201 I K N C P F V G G K D Q Q E V K R N T G 661 GTATTCCGCGGTCTTTCCGCGACAAGCCAGATGCGGCTGAGAACGAATCAGCCGATTTTG 221 I P R S F R D K P D A A E N E S A D F V 721 TGCTGCCTGCTGTACAAAACCAAGAGATACCGGAGGATCTGATATGCGGCATATGCCGAG 241 L P A V Q N Q E I P E D L I C G I C R D 781 ATATATTCGTCGATGCTGTCATGATACCCTGCTGCGGAAGTTCCTTTTGTGACGACTGTG 261 I F V D A V M I P C C G S S F C D D C V 841 TGCGAACCTCCTTATTGGAGTCAGAGGATAGTGAGTGCCCCGATTGCAAGGAGAAGAACT 281 R T S L L E S E D S E C P D C K E K N C 901 GTTCGCCTGGCTCCCTGATACCTAATCGGTTCTTGAGGAATTCGGTGAACGCCTTTAAAA 301 S P G S L I P N R F L R N S V N A F K N 961 ATGAGACTGGGTATAACAAAAGCGCGGCTAAGCCAGCTGCAGTAAAAAATGAGGAAAAAC 321 E T G Y N K S A A K P A A V K N E E K P 1021 CTCCTGTTGAAAAAGAAGTGGAGAAAAAGCCAGTCGCGGAGGTGGAACCCGAAGAGACTG 341 P V E K E V E K K P V A E V E P E E T E 1081 AGGTGAAACCTGAAAAGCAAAAAGAATCCGAAACCAATGGCAGTAATCCGCCAAAATCGG 361 V K P E K Q K E S E T N G S N P P K S E 1141 AATCTCCAGAGCCTCCCGCAACCACAGAACCATCACAGAAGGAGAAAGATAAATATGATT 381 S P E P P A T T E P S Q K E K D K Y D S 1201 CAGACTACGAGGATAACATTACCATAAAAATGCCCCAGCCTGCAGCTGATTCTACAACAG 401 D Y E D N I T I K M P Q P A A D S T T V 1261 TGCCCAGCAAAAGATCCCCCAGTTATTCCCACAGAAGTGAATCCTCTCATCGACGGGACA 421 P S K R S P S Y S H R S E S S H R R D R 1321 GGTCGGATTATGTTTCCGATCACGATCACAAGCACCAACGTCCATCAAAATCGGAGTCTG 441 S D Y V S D H D H K H Q R P S K S E S V 1381 TTAACAAGGATCGCAGTCTCCTGCCCTTGCCCATTGGCACCCTGCCTAGCTACCAGGGCC 461 N K D R S L L P L P I G T L P S Y Q G H 1441 ACATGATGGCCGAATCAGAAGAAGCTCGTCGATCGAGTGCCTATAAGCCCCCTTATATGC 481 M M A E S E E A R R S S A Y K P P Y M Q 1501 AAATGCAGCGGGGCCCACCTCCTATGCACATGATGAGTCACCACATGCCAGCCTACAACA 501 M Q R G P P P M H M M S H H M P A Y N N 1561 ACGGGTTTAACAACATGGGACAGAGGCCTCCCCTCAGCTATGTGCCGTATCAAAACCAAT 521 G F N N M G Q R P P L S Y V P Y Q N Q S 1621 CCGTACACCCAATGCGTGCGCCGTACGGATCTGCAGGCGGAGGTATGAATATGAATATGT 541 V H P M R A P Y G S A G G G M N M N M S 1681 CACAACCATTTCAGTCCCCAAATTTAGCCTCGATATACCAAGGGGTGGCAGCGAAGGTCG 561 Q P F Q S P N L A S I Y Q G V A A K V G 68 1741 GTTCCGGTCCCATTGACGATCCGTTGGAGGCCTTCAATCGCATCATGAAGGAGAAGGAGC 581 S G P I D D P L E A F N R I M K E K E R 1801 GGAAGAAGGTGGACCGCTTTCGAAGCTCTGACCGCCACAGGTCAAGGTCCCCGGATAGAC 601 K K V D R F R S S D R H R S R S P D R Q 1861 AAAGGCACCGCTTTAAGTCTCCCATGTACGAAAAGGACAACTCCAGGGATAATCTCAAGG 621 R H R F K S P M Y E K D N S R D N L K D 1921 ACAAAAGACCGCGATCCCGGGAAAGGAAGCGAGAACATAGCTACGAACGGCATATACGCC 641 K R P R S R E R K R E H S Y E R H I R H 1981 ACCCTCGTTCTAGTCGCCAGCCGAATGATGGCTCTAAGTCCCCAGGTGGCAGAATCAAAA 661 P R S S R Q P N D G S K S P G G R I K R 2041 GATCTGGACATCGTCGCTCTGCATCTCCAAAGCCGGGCTACAAGAGTGATTACAGAGACA 681 S G H R R S A S P K P G Y K S D Y R D K 2101 AGCCGTACAACAAGCCTAGTGCTCCCAAAACGGAGGCAGTTGAGCCTCCTCCCCCCGGAT 701 P Y N K P S A P K T E A V E P P P P G F 2161 TCGAGCCGTTGCAGCTGACGGATGAAGACGGCTACAGGAACAAGCACCCGACCAGTTCGG 721 E P L Q L T D E D G Y R N K H P T S S E 2221 AAGCATCACAAAGCAGCAAGGGTGATAGCAGCAAGAAGAGAGGGGAAAACAGGCACGAAG 741 A S Q S S K G D S S K K R G E N R H E E 2281 AGGCGCCACGAAAGAGGCACAGGTCTCGCAGCATTAGCAAGGAACCGAAGCCGAATGACA 761 A P R K R H R S R S I S K E P K P N D S 2341 GCAACTACAGGAGCCTGACTCCACCAGCAAAGATCACCACACCGAAAATGACTGCTGCCC 781 N Y R S L T P P A K I T T P K M T A A Q 2401 AGTTGAGGCAACGCGAAAGTTCACCGAAGACGCCGGAAAAGAGTCACGACGATTATCTGA 801 L R Q R E S S P K T P E K S H D D Y L T 2461 CCGCGAAGGCCAGAATTATGGCCTCCCAGCCCGTCATCAACGACACGGAAATGGAGACCA 821 A K A R I M A S Q P V I N D T E M E T N 2521 ATGTGGGCAAGGAGAACAAGGCCAAGAGTCCGTTGTCAAAAGATCGCAAGAAGAAGAAGA 841 V G K E N K A K S P L S K D R K K K K K 2581 AGGACAAGGACAAGGCTGAGCGCAAGAAAAACAAGAAGGACAAGCGCGCTAAGAAGGAGA 861 D K D K A E R K K N K K D K R A K K E K 2641 AAGGGGATCGCCAGAAGAAGAGCTCCTCAGTTAATCGATCTGACTCGGATATTAACAACA 881 G D R Q K K S S S V N R S D S D I N N S 2701 GCTCACTAATGAACGAGTCAAATTATAAAGTATTGTCTCCCAGGGCTCAAAGTCCCAGCA 901 S L M N E S N Y K V L S P R A Q S P S I 2761 TTGAGATCAATGCGGCTCAACTTTCCCCTACTCACAACGCTACTGAAAACGTTAATCCGA 921 E I N A A Q L S P T H N A T E N V N P K 2821 AGAGTCATTCCATCCTTACTGTGGGTGCTGCTAGCGACGATAATCTTGGCCCAAGAAGCA 941 S H S I L T V G A A S D D N L G P R S K 2881 AACTCAGCGAGGCTAATTCTGTCAATCTATCCAAATGGGAAATCGACGAGAATATCTTAG 961 L S E A N S V N L S K W E I D E N I L G 2941 GTTTGGAGGATTCCTCCAAAAAAGCTGCCGGGGCCTCCGACGATCCGTCGGAAATAACTT 981 L E D S S K K A A G A S D D P S E I T S 3001 CAGACGTCCTGCGAAAGGCTGAGAACGCAATATTTGCAAAGGCTATTAATGCCATCAGGC 1001 D V L R K A E N A I F A K A I N A I R P 3061 CTATGGAGTTTCAAGTTATTATCAATTCCAAGGACAACAGCAAGGACCGCTCCGTAGTTC 1021 M E F Q V I I N S K D N S K D R S V V R 3121 GAAGTGACAAGGATCGCTCCTCCTCACCCAGGCGTAACAACAGCAGCAGGTCGGTAAAGG 1041 S D K D R S S S P R R N N S S R S V K D 3181 ATAGGCTGGGCACCAAGATTTCCAATGATAGAAGCCGTTCGCGAGACAAGTCGAAGGGCA 1061 R L G T K I S N D R S R S R D K S K G R 3241 GGCGCCGGGCCGCAAGGAGCTCCGACGACGATGCGAACCGCGGCAGGTCGGATCGTCATG 1081 R R A A R S S D D D A N R G R S D R H G 3301 GCAGCCGGAAGAGGGACAACAGATCCCGCGACAGGGCGGCGCCTTCAGAGAAGAGGCAGG 1101 S R K R D N R S R D R A A P S E K R Q E 3361 AGCGTTCGTACAAGCGAAGCTCGCCGGAGGACGACAAGCTGAGGCGCCAGAACAAGGAAC 1121 R S Y K R S S P E D D K L R R Q N K E Q 3421 AGTCCGAATCCAAGCACGGAAAGCATGATCAAAACAATAGCGACGACTCGGATCGGAGGG 1141 S E S K H G K H D Q N N S D D S D R R A 3481 CGGCCAAAAACACCAAGTCCAGCGACAGCCGAGTGGTCTCCTCTGTAACAGCCGTGGTTG 1161 A K N T K S S D S R V V S S V T A V V A 3541 CTCCTCCCAAACCCTGTCGTCCAGACAACCCGTTCCGCAAGTTCGTCGACACCAGTTCGT 1181 P P K P C R P D N P F R K F V D T S S S 3601 CGAGCAGCTTAGTTGTAAAATATGATAACACGATACAGAAGGAGGGCGCGTCCTCGGACA 1201 S S L V V K Y D N T I Q K E G A S S D N 69 3661 ACGGCATGGAGCACAGGAAGCAGAGGGATAAGAAGCTGAAGAAACATTCAAAATATTCGT 1221 G M E H R K Q R D K K L K K H S K Y S S 3721 CAACCGATTCGTTGAAGAGCGAGAAGCGCAAGGATCCGAAGAGCAAAAAGAAGAGCAAGA 1241 T D S L K S E K R K D P K S K K K S K I 3781 TTTTGAAGAAGAAGAAAAAATCAAAGAAGTAGGTTACGGTAGGCTACGAGATAAGAATGA 1261 L K K K K K S K K * 3841 TATAAATATTGAAGATTAATGTGTACAAATCAAAGATTTTTAATGTATGTATTTATCATG 1281 3901 CAACTATAAGTATACAAATAAAACAGAACTACTCAAGGA 1301 Nucleic acid and corresponding amino acid sequence of SNAMA. 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