NO20021956L - Membrane receptors for steroids and sterols - Google Patents
Membrane receptors for steroids and sterols Download PDFInfo
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- NO20021956L NO20021956L NO20021956A NO20021956A NO20021956L NO 20021956 L NO20021956 L NO 20021956L NO 20021956 A NO20021956 A NO 20021956A NO 20021956 A NO20021956 A NO 20021956A NO 20021956 L NO20021956 L NO 20021956L
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- CGPUWJWCVCFERF-UHFFFAOYSA-N staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(OC)O1 CGPUWJWCVCFERF-UHFFFAOYSA-N 0.000 description 1
- 108091008672 sterol hormone receptors Proteins 0.000 description 1
- 230000001300 stimulation of adenylate cyclase Effects 0.000 description 1
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Classifications
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
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- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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Description
Foreliggende oppfinnelse gjelder nukleinsyrer som koder for en membranreseptor for steroider og steroler, polypeptider og anvendelse av disse. The present invention relates to nucleic acids which code for a membrane receptor for steroids and sterols, polypeptides and their use.
Ifølge den klassiske modell for steroidhormonvirkningen binder steroidene seg til intracellulære reseptorer som regulerer genekspresjonen som ligandaktiverte transkripsjonsfaktorer. Det er vanlig å betegne den direkte virkning av steroidhormonene via sine kjernereseptorer på transkripsjonen av målgener som genomiske virkninger. Genomiske virkninger kjennetegnes ved sin tidmessig forsinkede inntreden, dvs. fra mer enn 5 minutter til flere timer etter hormontilførsel (McEwen et al. 1978 i: Reichlin et al. red., The Hypothalamus, Raven Press s. 255ff). According to the classic model of steroid hormone action, the steroids bind to intracellular receptors that regulate gene expression as ligand-activated transcription factors. It is common to designate the direct effect of the steroid hormones via their nuclear receptors on the transcription of target genes as genomic effects. Genomic effects are characterized by their time-delayed onset, i.e. from more than 5 minutes to several hours after hormone administration (McEwen et al. 1978 in: Reichlin et al. ed., The Hypothalamus, Raven Press p. 255ff).
I motsetning til de genomiske virkningene ble det allerede for flere år siden observert "hurtige", ikke-genomiske virkninger som opptrer i løpet av noen få sekunder eller minutter etter tilførsel av steroidhormon og hvis årsak ikke kan skyldes en regulering av transkripsjonen. Biokjemiske indikatorer for disse virkningene er endringer i membranpotensialet og/eller påvirkning på ionekanaler i cellemembranen (Watson et al. 1999, Proe Soc Exp Biol Med 220, 9ff). Disse virkningene kan tenkes å formidles via reseptorer i membranen. Ofte kan steroidvirkniriger som peker på forekomst av en membranreseptor, også utløses av steroidderivater som grunnet kobling til et plasskrevende bærermolekyl (f eks serumalbumin) ikke lenger kan trenge gjennom plasmamembranen og som når de synliggjøres ved fluorescensmikroskopi, viser seg å bindes til celleoverflaten. Disse observasjonene, og likeså bindingsundersøkelser med cellemembraner og immunfarging, tyder på forekomst av en steroidreseptor i membranen. In contrast to the genomic effects, "rapid", non-genomic effects were already observed several years ago, which occur within a few seconds or minutes after administration of steroid hormone and whose cause cannot be due to a regulation of the transcription. Biochemical indicators for these effects are changes in the membrane potential and/or effects on ion channels in the cell membrane (Watson et al. 1999, Proe Soc Exp Biol Med 220, 9ff). These effects can be thought to be mediated via receptors in the membrane. Often, steroid effects that point to the presence of a membrane receptor can also be triggered by steroid derivatives which, due to coupling to a space-consuming carrier molecule (e.g. serum albumin), can no longer penetrate the plasma membrane and which, when visualized by fluorescence microscopy, are shown to be bound to the cell surface. These observations, as well as binding studies with cell membranes and immunostaining, indicate the presence of a steroid receptor in the membrane.
Et eksempel på en ikke-genomisk virkning er modulering av 7-aminosmørsyre (GABA)-reseptorer via progesteron. Denne signalvei er ansvarlig for formidling av den anestetiske virkning av progesteron (Covery DF et al. 2000 J Pharmacol Exp Ther, 1009). Nok et eksempel på en ikke-genomisk progesteronvirkning er gjenopptakelse av meiosen i amfibie-oocytter. Den progesteroninduserte gjenopptakelse av meiosen omfatter aktivering av cAMP-avhengige proteinkinaser og aktivering av MAPK-signalveier (Palmer A et al. 2000 Prog Cell Cycle Res 4, 131 ff), og fører til "germinal vesicle breakdowm", GVBD (Masui Y et al. 1971, J Exp Zool 177, 129ff). Selv om noe taler for at virkningene kan formidles via en membranreseptor for progesteron (mPR), taler de nyeste undersøkelsene for at den kjernelokaliserte PR kan interagere med tyrosinkinasesignalveier (Tian J et al. 2000, Proe Nati Acad Sei USA 97, 14358ff; Bayaa M et al. 2000, Proe Nati Acad Sei USA 97,12607). An example of a non-genomic effect is the modulation of 7-aminobutyric acid (GABA) receptors via progesterone. This signaling pathway is responsible for mediating the anesthetic effect of progesterone (Covery DF et al. 2000 J Pharmacol Exp Ther, 1009). Another example of a non-genomic progesterone action is the resumption of meiosis in amphibian oocytes. The progesterone-induced resumption of meiosis involves activation of cAMP-dependent protein kinases and activation of MAPK signaling pathways (Palmer A et al. 2000 Prog Cell Cycle Res 4, 131 ff), and leads to "germinal vesicle breakdown", GVBD (Masui Y et al .1971, J Exp Zool 177, 129ff). Although some suggest that the effects may be mediated via a membrane progesterone receptor (mPR), recent studies suggest that the nuclear localized PR may interact with tyrosine kinase signaling pathways (Tian J et al. 2000, Proe Nati Acad Sei USA 97, 14358ff; Bayaa M et al 2000, Proe Nati Acad Sei USA 97,12607).
I et stort antall publikasjoner beskrives ikke-genomiske østrogenvirkninger for svært mange vev og celler i kultur. I løpet av noen få minutter utløser tilførsel av østrogen en rekke virkninger: intracellulære Ca<2+->"spikes" (Mendoza C, Soler A., Tesarik J. 1995, Biochem Biophys Res Commun 210: 518-523), stimulering av adenylatsyklase (Aronica SM, Kraus WL, Katzenellenbogen BS 1994, Proe Nati Acad Sei 91: 8517-21), aktivering av NO-syntetase og fosfolipase C (Le Mellay V, Grosse B, Lieberherr M 1997, J Biol Chem 272: 11902-11907). In a large number of publications, non-genomic estrogen effects are described for many tissues and cells in culture. Within a few minutes, administration of estrogen triggers a series of effects: intracellular Ca<2+->"spikes" (Mendoza C, Soler A., Tesarik J. 1995, Biochem Biophys Res Commun 210: 518-523), stimulation of adenylate cyclase (Aronica SM, Kraus WL, Katzenellenbogen BS 1994, Proe Nati Acad Sei 91: 8517-21), activation of NO synthetase and phospholipase C (Le Mellay V, Grosse B, Lieberherr M 1997, J Biol Chem 272: 11902- 11907).
Tross en omfattende litteratur vedrørende ikke-genomiske virkninger av steroidhormoner har en steroidreseptor i membranen til nå ikke entydig blitt påvist i pattedyr. Dersom en slik reseptor eksisterer, ville dette åpne for en helt ny vei for utvikling av medikamenter. Fremstilling av en slik reseptor er derfor foreliggende oppfinnelses oppgave. Despite an extensive literature regarding non-genomic effects of steroid hormones, a steroid receptor in the membrane has not yet been unequivocally demonstrated in mammals. If such a receptor exists, this would open up a completely new path for drug development. The production of such a receptor is therefore the task of the present invention.
Problemet ble løst ved fremstilling av en nukleinsyre som omfatterThe problem was solved by producing a nucleic acid that includes
a. nukleotidsekvensen vist i sekv. ID NO 1, NO 3, NO 5 og NO 7,a. the nucleotide sequence shown in seq. ID NO 1, NO 3, NO 5 and NO 7,
b. en nukleotidsekvens som tilsvarer sekvensene ifølge a. innenfor rammene b. a nucleotide sequence corresponding to the sequences according to a. within the frames
til degenerasjonen av den genetiske kode, ellerto the degeneration of the genetic code, or
c. en nukleotidsekvens som kan hybridisere med sekvensene ifølge a. eller b. c. a nucleotide sequence that can hybridize with the sequences according to a. or b.
under stringente betingelser.under stringent conditions.
Begrepet "hybridisering under stringente betingelser" ifølge foreliggende oppfinnelse defineres i Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). En stringent hybridisering foreligger f eks dersom et hybridiseirngssignal fortsatt observeres etter vask i 1 time med 1 x SSC og 0,1 % SDS ved 50 °C, fortrinnsvis ved 55 °C, spesielt foretrukket ved 62 °C og mest foretrukket ved 68 °C, særlig i 1 time i 0,2 x SSC og 0,1 % SDS ved 55 °C, fortrinnsvis ved 62 °C, og mest foretrukket ved 68 °C. Nukleinsyrene som under disse betingelsene hybridiserer til nukleinsyrene vist i SEQ ID NO 1 og/eller 3, eller en sekvens som tilsvarer disse sekvensene innenfor rammene av degenerasjonen av den genetiske kode, er også gjenstand for foreliggende oppfinnelse. The term "hybridization under stringent conditions" according to the present invention is defined in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989). Stringent hybridization exists, for example, if a hybridization signal is still observed after washing for 1 hour with 1 x SSC and 0.1% SDS at 50 °C, preferably at 55 °C, particularly preferred at 62 °C and most preferred at 68 °C , particularly for 1 hour in 0.2 x SSC and 0.1% SDS at 55°C, preferably at 62°C, and most preferably at 68°C. The nucleic acids which under these conditions hybridize to the nucleic acids shown in SEQ ID NO 1 and/or 3, or a sequence corresponding to these sequences within the framework of the degeneration of the genetic code, are also subject of the present invention.
Nukleinsyrer kan være enkelt- eller dobbelttrådet DNA, f eks cDNA eller RNA, hhv. mRNA, cRNA, pre-mRNA. Nucleic acids can be single- or double-stranded DNA, e.g. cDNA or RNA, respectively. mRNA, cRNA, pre-mRNA.
Nukleinsyrene som vises i SEQ ID NO 1, 3, 5 og 7, koder for en membranreseptor for steroider og steroler. De fire nukleinsyrene er spleisevarianter fra samme gen. Variantene vist i SEQ ID NO 3 og SEQ ID NO 7 har alltid en insersjon i sekvensens 5'-område. The nucleic acids shown in SEQ ID NOs 1, 3, 5 and 7 encode a membrane receptor for steroids and sterols. The four nucleic acids are splice variants from the same gene. The variants shown in SEQ ID NO 3 and SEQ ID NO 7 always have an insertion in the 5' region of the sequence.
Sekvensene vist i SEQ ID NO 5 og 7 inneholder alltid en delesjon mellom to ankyrindomener. The sequences shown in SEQ ID NO 5 and 7 always contain a deletion between two ankyrin domains.
Nukleinsyrer som omfatter et proteinkodende område fra nukleinsyresekvensen vist i SEQ ID NO 1, 3, 5 eller 7, foretrekkes. Et proteinkodende område i sekvensen vist i SEQ ID NO 1 ligger i området fra nukleotid 227 til nukleotid 6271, et kodende område i sekvensen vist i SEQ ID NO 3 ligger i området fra nukleotid 360 til 6482, i sekvensen vist i SEQ ID NO 5 i området fra nukleotid 227-6993, og i sekvensen vist i SEQ ID NO 7 i området fra nukleotid 360-6408. Nucleic acids comprising a protein coding region from the nucleic acid sequence shown in SEQ ID NO 1, 3, 5 or 7 are preferred. A protein coding region in the sequence shown in SEQ ID NO 1 lies in the region from nucleotide 227 to nucleotide 6271, a coding region in the sequence shown in SEQ ID NO 3 lies in the region from nucleotide 360 to 6482, in the sequence shown in SEQ ID NO 5 in the area from nucleotide 227-6993, and in the sequence shown in SEQ ID NO 7 in the area from nucleotide 360-6408.
Også omfattet av oppfinnelsen er nukleinsyrer som koder for et polypeptid med aminosyresekvensen vist i SEQ ID NO 2,4, 6 eller 8. Also covered by the invention are nucleic acids that code for a polypeptide with the amino acid sequence shown in SEQ ID NO 2, 4, 6 or 8.
Nukleinsyrene ifølge oppfinnelsen kan erholdes fra pattedyr, f eks humane celler, eller fra et cDNA-bibliotek eller genomisk bibliotek som f eks er fremstilt fra humane celler. De kan isoleres ifølge kjente teknikker ved benyttelse av korte områder fra nukleotidsekvensen vist i SEQ ID NO 1,3,5 eller 7, som hybridiseringsprober eller amplifiseringsprimere. The nucleic acids according to the invention can be obtained from mammals, e.g. human cells, or from a cDNA library or genomic library which is e.g. prepared from human cells. They can be isolated according to known techniques using short regions from the nucleotide sequence shown in SEQ ID NO 1,3,5 or 7, as hybridization probes or amplification primers.
Oppfinnelsen gjelder videre polypeptider som kodes av en nukleinsyre ifølge oppfinnelsen. Disse polypeptidene har funksjonen til en steroidreseptor. The invention further relates to polypeptides which are encoded by a nucleic acid according to the invention. These polypeptides have the function of a steroid receptor.
Videre omfattes av oppfinnelsen polypeptider som omfatter aminosyresekvensen vist i SEQ ID NO 2, 4, 6 eller 8. Furthermore, the invention covers polypeptides comprising the amino acid sequence shown in SEQ ID NO 2, 4, 6 or 8.
Polypeptidene ifølge oppfinnelsen kan være rekombinante polypeptider, naturlige, isolerte polypeptider eller syntetiske polypeptider. The polypeptides according to the invention can be recombinant polypeptides, natural, isolated polypeptides or synthetic polypeptides.
Polypeptidene ifølge oppfinnelsen inneholder forskjellige domener: 12 såkalte "leucin-rich-repeats" (LRR), 3 ankyrin-domener (ANK) og ett kinasedomene. Disse polypeptidene er membranassosierte. Kinasedomenet fosforylerer etter binding av et steroid eller en sterol serin- og treoninrester i proteiner og setter derved en intracellulær signalkaskade i gang. Polypeptidene ifølge oppfinnelsen inneholder videre et såkalt "Island"-domene som omfatter 600 aminosyrer og som foreligger mellom det 11. og det 12. "leucin-rich-repeat"-domene i polypeptidet ifølge oppfinnelsen. Dette domene deltar i binding av steroid eller sterol. The polypeptides according to the invention contain different domains: 12 so-called "leucine-rich repeats" (LRR), 3 ankyrin domains (ANK) and one kinase domain. These polypeptides are membrane associated. After binding a steroid or a sterol, the kinase domain phosphorylates serine and threonine residues in proteins and thereby sets off an intracellular signaling cascade. The polypeptides according to the invention further contain a so-called "Island" domain which comprises 600 amino acids and which is present between the 11th and the 12th "leucine-rich-repeat" domain in the polypeptide according to the invention. This domain participates in steroid or sterol binding.
Oppfinnelsen gjelder også polypeptider som omfatter et kinasedomene fra polypeptidene ifølge oppfinnelsen eller deler av dette. Kinasedomenet befinner seg i området fra aminosyre 1270 til 1565, hvor nummereringen viser til sekvensen vist i SEQ ID NO 2. The invention also applies to polypeptides which comprise a kinase domain from the polypeptides according to the invention or parts thereof. The kinase domain is located in the area from amino acid 1270 to 1565, where the numbering refers to the sequence shown in SEQ ID NO 2.
Også gjenstand for oppfinnelsen er polypeptider som omfatter Island-domenet fra polypeptidene ifølge oppfinnelsen. Dette inneholder et steroid- hhv. sterolbindingssete. Also subject to the invention are polypeptides comprising the Island domain from the polypeptides according to the invention. This contains a steroid or sterol binding site.
Polypeptidene ifølge oppfinnelsen vist i SEQ ID NO 2, 4, 6 og 8 uttrykkes fortrinnsvis i vev som vites å være steroidhormonsensisitve. Eksempler er ovarier, eggleder, prostata og blod. The polypeptides according to the invention shown in SEQ ID NO 2, 4, 6 and 8 are preferably expressed in tissues known to be steroid hormone sensitive. Examples are ovaries, fallopian tubes, prostate and blood.
Polypeptidene ifølge oppfinnelsen eller delområder fra disse (peptider) kan benyttes for fremstilling av antistoffer. For fremstilling av polyklonale antistoffer kan polypeptidene eller peptidene for eksempel bindes til KLH ("Keyhole Limpet Hemocyanin") og innsprøytes i dyr, f eks kaniner. De kan også anvendes for fremstilling av monoklonale antistoffer. For antistoffremstilling kan et polypeptid eller et peptid ifølge oppfinnelsen eller en blanding av flere peptider ifølge oppfinnelsen benyttes. Fremstilling av antistoffer skjer ifølge standardfremgangsmåter, for eks som beskrevet i Kohler, G og Milstein, C, Nature 1975, 256,495-497 og Nelson, P.N. et al., Mol. Pathol. 2000, 53, 111-117. The polypeptides according to the invention or sub-regions from these (peptides) can be used for the production of antibodies. For the production of polyclonal antibodies, the polypeptides or peptides can, for example, be bound to KLH ("Keyhole Limpet Hemocyanin") and injected into animals, e.g. rabbits. They can also be used for the production of monoclonal antibodies. For antibody preparation, a polypeptide or a peptide according to the invention or a mixture of several peptides according to the invention can be used. Antibodies are prepared according to standard procedures, for example as described in Kohler, G and Milstein, C, Nature 1975, 256,495-497 and Nelson, P.N. et al., Mol. Pathol. 2000, 53, 111-117.
Også gjenstand for oppfinnelsen er antistoffer som er rettet mot et polypeptid ifølge oppfinnelsen. Also subject to the invention are antibodies which are directed against a polypeptide according to the invention.
Antistoffene ifølge oppfinnelsen kan anvendes for påvisning av polypeptidene ifølge oppfinnelsen. Dette kan for eksempel skje ved immunohistokjemisk analyse. Antistoffene ifølge oppfinnelsen kan også benyttes i andre immunanalyser, f eks en ELISA (enzymkoblet immunadsorpsjonsanalyse) eller i en radioimmunanalyse. På denne måten kan konsentrasjonen av et polypeptid ifølge oppfinnelsen i vevs- eller celleekstrakter måles. The antibodies according to the invention can be used for detection of the polypeptides according to the invention. This can be done, for example, by immunohistochemical analysis. The antibodies according to the invention can also be used in other immunoassays, for example an ELISA (enzyme-linked immunoadsorption assay) or in a radioimmunoassay. In this way, the concentration of a polypeptide according to the invention in tissue or cell extracts can be measured.
Påvisning av ekspresjon av polypeptidet ifølge oppfinnelsen kan også skje ved påvisning av mRNA i cellene. Også omfattet av oppfinnelsen er derfor også anvendelse av probe med en nukleotidsekvens som er komplementær til nukleotidsekvensen som koder for polypeptidet ifølge oppfinnelsen for fremstilling av et reagens for påvisning av nærvær av mRNA ifølge oppfinnelsen i celler. En probe er et kort DNA-stykke med minst 14 nukleotider. Probene ifølge oppfinnelsen kan for eks anvendes i en Northern Blot-analyse. Disse fremgangsmåtene beskrives for eksempel i Sambrook, J. et al., 1989, Cold Spring Harbor Laboratory Press. Andre fremgangsmåter for påvisning av RNA er in siYw-hybirdisering, RNAse-beskyttelsesanalyse eller PCR. Detection of expression of the polypeptide according to the invention can also take place by detection of mRNA in the cells. Also covered by the invention is therefore also the use of a probe with a nucleotide sequence that is complementary to the nucleotide sequence that codes for the polypeptide according to the invention for the production of a reagent for detecting the presence of mRNA according to the invention in cells. A probe is a short piece of DNA with at least 14 nucleotides. The probes according to the invention can, for example, be used in a Northern Blot analysis. These methods are described, for example, in Sambrook, J. et al., 1989, Cold Spring Harbor Laboratory Press. Other methods for detecting RNA are in siYw hybridization, RNAse protection assay or PCR.
Videre omfatter oppfinnelsen vektorer som inneholder minst en kopi av en nukleinsyre ifølge oppfinnelsen. Vektorene kan være prokaryote eller eukaryote vektorer. Eksempler på vektorer er pPRO (Clontech), pBAD (Invitrogen), pSG5 (Stratagene), pCl (Promega), pIRES (Clontech), pBAC (Clontech), pMET (Invitrogen) og pBlueBac (Invitrogen). I disse vektorene kan nukleinsyrene ifølge oppfinnelsen innføres ifølge fremgangsmåter som er kjente blant fagfolk. Nukleinsyrene ifølge oppfinnelsen foreligger fortrinnsvis forbundet til ekspresjonssignaler som for eksempel promotere og enhancere i vektoren. Furthermore, the invention includes vectors that contain at least one copy of a nucleic acid according to the invention. The vectors can be prokaryotic or eukaryotic vectors. Examples of vectors are pPRO (Clontech), pBAD (Invitrogen), pSG5 (Stratagene), pCl (Promega), pIRES (Clontech), pBAC (Clontech), pMET (Invitrogen) and pBlueBac (Invitrogen). In these vectors, the nucleic acids according to the invention can be introduced according to methods known to those skilled in the art. The nucleic acids according to the invention are preferably connected to expression signals such as promoters and enhancers in the vector.
Oppfinnelsen omfatter videre celler som er transfektert med en nukleinsyresekvens ifølge oppfinnelsen eller med en vektor ifølge oppfinnelsen. Som celler kan for eksempel E. coli, gjær, Pichia, Sf9, COS, CV-1 eller BHK anvendes. Disse cellene kan anvendes for fremstilling av polypeptider ifølge oppfinnelsen eller som analysesystemer. The invention further comprises cells that have been transfected with a nucleic acid sequence according to the invention or with a vector according to the invention. As cells, for example, E. coli, yeast, Pichia, Sf9, COS, CV-1 or BHK can be used. These cells can be used for the production of polypeptides according to the invention or as analysis systems.
En gjenstand for oppfinnelsen er anvendelse av polypeptidene ifølge oppfinnelsen eller nukleinsyrene som koder for disse som målsubstanser for fremstilling av et middel for behandling av sterol- eller steroidhormonavhengige sykdommer. An object of the invention is the use of the polypeptides according to the invention or the nucleic acids that code for them as target substances for the production of an agent for the treatment of sterol- or steroid hormone-dependent diseases.
Nærmere bestemt omfatter oppfinnelsen anvendelse avMore specifically, the invention includes the use of
a. en nukleinsyre ifølge oppfinnelsen,a. a nucleic acid according to the invention,
b. et polypeptid ifølge oppfinnelsen ellerb. a polypeptide according to the invention or
c. en celle ifølge oppfinnelsen,c. a cell according to the invention,
for identifisering av effektorer for et polypeptid ifølge oppfinnelsen. Effektorer er forbindelser som virker inhiberende eller aktiverende på polypeptidet ifølge oppfinnelsen, og som kan påvirke steroidreseptorfunksjonen til polypeptidet ifølge oppfinnelsen. for the identification of effectors for a polypeptide according to the invention. Effectors are compounds that have an inhibitory or activating effect on the polypeptide according to the invention, and which can affect the steroid receptor function of the polypeptide according to the invention.
Oppfinnelsen omfatter videre et analysesystem for påvisning av effektorer for et polypeptid ifølge oppfinnelsen, hvori et polypeptid ifølge oppfinnelsen, enten hele polypeptidet eller en delsekvens av dette, inkuberes med et steroid, en steroidanalog eller en sterol og mengden bundet steroid, steroidanalog eller sterol bestemmes. Som delsekvens kan f eks anvendes området som omfatter "leucin-rich-repeats"- og Island-domenet, eller kortere deler av dette. Bindingene av effektorene kan måles ved at man benytter merkede forbindelser og måler merkingen (for eks radioaktivitet eller fluorscens). Bindingen kan også måles ved benyttelse av en fortrengningsreaksjon hvor man benytter et bindende, merket steroid og måler fortrengning av merkingen fra reseptoren etter tilsetting av analyseforbindelsene. The invention further comprises an analysis system for the detection of effectors for a polypeptide according to the invention, in which a polypeptide according to the invention, either the entire polypeptide or a partial sequence thereof, is incubated with a steroid, a steroid analogue or a sterol and the amount of bound steroid, steroid analogue or sterol is determined. As a partial sequence, for example, the area comprising the "leucine-rich-repeats" and Island domains, or shorter parts thereof, can be used. The binding of the effectors can be measured by using labeled compounds and measuring the labeling (for example radioactivity or fluorescence). The binding can also be measured using a displacement reaction where a binding, labeled steroid is used and displacement of the labeling from the receptor is measured after addition of the analysis compounds.
Denne bindingsanalyse kan også gjennomføres med en celle ifølge oppfinnelsen som inneholder polypeptidet ifølge oppfinnelsen. I tillegg til binding av analyseforbindelsene kan også en intracellulær virkning måles, for eksempel endring av Ca -konsentrasjonen. Denne variant av analysesystemet gir således også informasjon vedrørende effektorens funksjonelle aktivitet. This binding analysis can also be carried out with a cell according to the invention which contains the polypeptide according to the invention. In addition to binding of the analyte compounds, an intracellular effect can also be measured, for example a change in the Ca concentration. This variant of the analysis system thus also provides information regarding the effector's functional activity.
Også gjenstand for oppfinnelsen er et analysesystem for påvisning av inhibitorer av kinasefunksjonen til polypeptid ifølge oppfinnelsen, hvorved et polypeptid ifølge oppfinnelsen eller kinasedomenet fra dette settes i forbindelse med analyseforbindelsene, og kinaseaktiviteten bestemmes. Kinaseaktiviteten bestemmes med og uten analyseforbindelse, og omfanget av hemmingen bestemmes. Also subject to the invention is an analysis system for the detection of inhibitors of the kinase function of the polypeptide according to the invention, whereby a polypeptide according to the invention or the kinase domain thereof is put in connection with the analysis compounds, and the kinase activity is determined. Kinase activity is determined with and without assay compound, and the extent of inhibition is determined.
Effektorene for sterol- eller steroidreseptorfunksjonen til polypeptidet ifølge oppfinnelsen kan benyttes for beharidling av sterol- eller steroidhormonavhengige sykdommer. Eksempler på slike sykdommer er peri- og postmenopausale lidelser, sykdommer i urogenitalsystemet, ovariesvikt, osteoporose, godartet prostatahyperplasi, hjertekretsløpsykdommer, karsykdommer, neurodegenerative sykdommer, betennelses-sykdommer og sykdommer i immunsystemet. I tillegg kan disse effektorene benyttes for behandling av infertilitet hos kvinner og menn. The effectors for the sterol or steroid receptor function of the polypeptide according to the invention can be used for the treatment of sterol or steroid hormone-dependent diseases. Examples of such diseases are peri- and postmenopausal disorders, diseases of the urogenital system, ovarian failure, osteoporosis, benign prostatic hyperplasia, cardiovascular diseases, vascular diseases, neurodegenerative diseases, inflammatory diseases and diseases of the immune system. In addition, these effectors can be used for the treatment of infertility in women and men.
Det ble funnet at polypeptidet ifølge oppfinnelsen bindes til den endogene forbindelse FF-MAS (meioseaktiverende forbindelse fra follikelvæske). FF-MAS induserer meiose i kvinnelige kjønnsceller og er derfor en vesentlig faktor for fertilitet hos kvinner. Ved hjelp av et analysesystem ifølge oppfinnelsen kan det påvises forbindelser som hemmer virkningen av FF-MAS på reseptoren. Disse forbindelsene hemmer fertiliteten og kan derfor benyttes som befruktningshindrende midler. Ved hjelp av analysesystemet ifølge oppfinnelsen kan det også påvises forbindelser som aktiverer reseptoren og som derved virker analogt med FF-MAS. Disse forbindelsene virker fertilitetsfremmende og kan enten tilføres til kvinner som lider av fertilitetsforstyrrelser, eller tilføres in vitro til en oocyttkultur. De således fremstilte oocytter anvendes så for in vjVrø-fertilisering. It was found that the polypeptide according to the invention binds to the endogenous compound FF-MAS (meiosis-activating compound from follicle fluid). FF-MAS induces meiosis in female germ cells and is therefore an essential factor for female fertility. Using an analysis system according to the invention, compounds can be detected which inhibit the effect of FF-MAS on the receptor. These compounds inhibit fertility and can therefore be used as contraceptives. With the aid of the analysis system according to the invention, it is also possible to detect compounds which activate the receptor and thereby act analogously to FF-MAS. These compounds have a fertility-promoting effect and can either be administered to women suffering from fertility disorders, or administered in vitro to an oocyte culture. The oocytes produced in this way are then used for in vitro fertilisation.
Videre omfatter oppfinnelsen en fremgangsmåte for fremstilling av et farmasøytisk preparat hvori Furthermore, the invention includes a method for producing a pharmaceutical preparation in which
a. forbindelser settes i forbindelse med et analysesystem ifølge oppfinnelsen, b. virkningen av forbindelsene på analysesystemet måles sammenlignet med a. compounds are connected to an analysis system according to the invention, b. the effect of the compounds on the analysis system is measured compared to
kontroller,controls,
c. en forbindelse som viser en modulering av aktiviteten til polypeptidet ifølge oppfinnelsen i trinn b., påvises, og d. forbindelsen påvist i trinn c. blandes med formuleringsstoffer som vanligvis c. a compound showing a modulation of the activity of the polypeptide according to the invention in step b. is detected, and d. the compound detected in step c. is mixed with formulation substances which are usually
benyttes i farmasien.used in pharmacy.
Med aktiviteten til polypeptidet ifølge oppfinnelsen menes den steroidbindende egenskap og/eller kinaseaktiviteten. By the activity of the polypeptide according to the invention is meant the steroid-binding property and/or the kinase activity.
En forbindelse som påvises ved å benytte en fremgangsmåte ifølge oppfinnelsen, kan eventuelt optimaliseres når det gjelder metabolsk stabilitet, aktivitet i et analysesystem ifølge oppfinnelsen og/eller biotilgjengelighet. For dette kan vanlige kjemiske fremgangsmåter benyttes. A compound detected by using a method according to the invention can optionally be optimized in terms of metabolic stability, activity in an analysis system according to the invention and/or bioavailability. For this, normal chemical methods can be used.
Beskrivelse av figureneDescription of the figures
FIG. 1 viser domenestrukturen til polypeptidene ifølge oppfinnelsen. Strukturen begynner til venstre med N-enden. FIG. 1 shows the domain structure of the polypeptides according to the invention. The structure begins on the left with the N terminus.
FIG. 2. viser resultatet fra en elektronisk Northern blot-analyse.FIG. 2. shows the result from an electronic Northern blot analysis.
FIG. 3. viser en sekvenssammenligning mellom DNA-et fra de 4 spleisevariantene. I denne tilsvarer betegnelsene FIG. 3. shows a sequence comparison between the DNA from the 4 splice variants. In this, the designations correspond
SINGER_KINASE_SP 1B SEQ ID1 NO 5,SINGER_KINASE_SP 1B SEQ ID1 NO 5,
SINGERKINASE SP2B SEQ ID NO 7,SINGER KINASE SP2B SEQ ID NO 7,
spliceVariant_2 SEQ ID NO 3 ogspliceVariant_2 SEQ ID NO 3 and
singerKinase_spla SEQ ID NO 1.singerKinase_spla SEQ ID NO 1.
FIG. 4 viser en sammenligning mellom aminosyresekvensen til de 4 spleisevariantene. Betegnelsene er forklart under FIG. 3. FIG. 5 viser fordelingen av RNA ifølge foreliggende oppfinnelse i forskjellige humane vev. For amplifiseringen ble følgende primere benyttet: 5' gtatgaactgtgctgtgggaag 3' (4928) og 5' gatgtaccactccaccacctacc 3' (5532). EtNorthern-blot vises. FIG. 4 shows a comparison between the amino acid sequence of the 4 splice variants. The designations are explained under FIG. 3. FIG. 5 shows the distribution of RNA according to the present invention in different human tissues. For the amplification, the following primers were used: 5' gtatgaactgtgctgtgggaag 3' (4928) and 5' gatgtaccactccaccacctacc 3' (5532). A Northern blot appears.
I venstre bilde betyr 1: milt, 2: brissel, 3: prostata, 4: testikler, 5: ovarie, 6: tynntarm, 7: tykktarm, 8: leukocytter fra perifert blod. I høyre bilde betyr 1: hjerne, 2: hjerte, 3: skjelettmuskel, 4: tykktarm, 5: brissel, 6: milt, 7: nyre, 8: lever, 9: tynntarm, 10: placenta, 11: lunge, 12: leukocytter fra perifert blod. Pilen viser posisjonen for båndet som tilsvarer RNA ifølge oppfinnelsen. FIG. 6 viser ekspresjon og autofosforylering av kinasedomenet. I bildet 1 vises en polyakrylamidgel etter proteinfarging med Coomassie G-250, i bilde 2 det tilsvarende autoradiogram (<33>P) og i bilde 3 et Western-blot. Betegnelsene betyr: a = glutationeluat med bundne proteiner (kontroll), b = glutationeluat (GST-SIN2), c = glutation-sefarose med bundne proteiner (GST-SIN2), e + f = molekylvektsmarkør (SeaBlue), g = SDS-eluat fra glutation-sefarose (kontroll), h = SDS-eluat fra glutation-sefarose (GST-SIN2). Forsøksbetingelsene er beskrevet i eksempel 3 og 4. Western-blottet ble fremstilt med anti-GST-antistoff (geiteserum) som primærantistoff og anti-geite-igG-alkalisk fosfata-sekonjugat som sekundærantistoff. Pilen angir posisjonen for GST-kinasefusjons-proteinet. FIG. 7 viser aminosyresekvensen for fusjonsproteinet mellom glutation-S-transferase og aminosyrene Ala 1267-Leu 1570. In the left picture, 1 means: spleen, 2: pancreas, 3: prostate, 4: testes, 5: ovary, 6: small intestine, 7: large intestine, 8: leukocytes from peripheral blood. In the right picture, 1: brain, 2: heart, 3: skeletal muscle, 4: large intestine, 5: pancreas, 6: spleen, 7: kidney, 8: liver, 9: small intestine, 10: placenta, 11: lung, 12: leukocytes from peripheral blood. The arrow shows the position of the band corresponding to the RNA according to the invention. FIG. 6 shows expression and autophosphorylation of the kinase domain. Picture 1 shows a polyacrylamide gel after protein staining with Coomassie G-250, picture 2 the corresponding autoradiogram (<33>P) and picture 3 a Western blot. The designations mean: a = glutathione eluate with bound proteins (control), b = glutathione eluate (GST-SIN2), c = glutathione-sepharose with bound proteins (GST-SIN2), e + f = molecular weight marker (SeaBlue), g = SDS eluate from glutathione-sepharose (control), h = SDS eluate from glutathione-sepharose (GST-SIN2). The experimental conditions are described in examples 3 and 4. The Western blot was prepared with anti-GST antibody (goat serum) as primary antibody and anti-goat IgG-alkaline phosphatase conjugate as secondary antibody. The arrow indicates the position of the GST-kinase fusion protein. FIG. 7 shows the amino acid sequence of the fusion protein between glutathione-S-transferase and the amino acids Ala 1267-Leu 1570.
EksemplerExamples
De molekylærbiologiske fremgangsmåter som benyttes i eksemplene, for eksempel polymerase-kjedereaksjonen (PCR), fremstilling av cDNA, kloning av DNA og sekvensering av DNA, ble utført som beskrevet i kjente lærebøker, for eksempel i Molecular Cloning, A Laboratory Manual (Sambrook, J. et al., 1989, Cold Spring Harbor Laboratory Press). The molecular biological methods used in the examples, for example the polymerase chain reaction (PCR), production of cDNA, cloning of DNA and sequencing of DNA, were carried out as described in known textbooks, for example in Molecular Cloning, A Laboratory Manual (Sambrook, J .et al., 1989, Cold Spring Harbor Laboratory Press).
Eksempel 1: Kloning av den membranbundne steroidreseptorExample 1: Cloning of the membrane-bound steroid receptor
For kloningen ble det benyttet et cDNA-bibliotek fra human melkekjertel (Clontech). cDNA for spleisevariantene ble ifølge oppfinnelsen fremstilt ved hjelp av PCR. For dette ble følgende primere benyttet: A cDNA library from human mammary gland (Clontech) was used for the cloning. According to the invention, cDNA for the splice variants was produced by PCR. For this, the following primers were used:
N-terminalt område 5 ATGGAGACGCTTAACGGTGC -3" N-terminal region 5 ATGGAGACGCTTAACGGTGC -3"
C-terminal primer 5' G AG AACTCTG CTCC AG AG AAC 3'. C-terminal primer 5' G AG AACTCTG CTCC AG AG AAC 3'.
For amplifisering av spleisevariantene vist i SEQ ID NO 1 og 3 ble også primeren 5 '-CGG ATG AC AACCC AGCCGTGGTGG-3* (579-602) benyttet. Nummereringen angitt i parentes viser til sekvensen vist i SEQ ID NO 1. For amplification of the splice variants shown in SEQ ID NO 1 and 3, the primer 5'-CGG ATG AC AACCC AGCCGTGGTGG-3* (579-602) was also used. The numbering indicated in parentheses refers to the sequence shown in SEQ ID NO 1.
Eksempel 2: Ekspresjon av den membranbundne steroidreseptorExample 2: Expression of the membrane-bound steroid receptor
For ekspresjonen ble det kodende området amplifisert ved hjelp av PCR og innført i Baculovirusekspresjonvektoren pBlueBac4.5/V5-His-TOPO (Invitrogen), hhv. den eukaryote ekspresjonsvektor pcDNA3.1/V5/His-TOPO (Invitrogen). For å forenkle påvisning og rensing ble det laget en fusjon med en His-merkelapp. Etter kotransfek-sjonen av insektceller med Bac-N-Blue-DNA ble det dannet rekombinante virus som ble påvist ved en PCR-fremgangsmåte. En bakteriofagstandard ble så fremstilt og anvendt for videre transfeksjon og fremstilling av reseptoren i større mengder. Rensing av de His-merkede proteinene skjedde ved hjelp av en nikkelaffinitetskonolonne. For the expression, the coding region was amplified by PCR and introduced into the Baculovirus expression vector pBlueBac4.5/V5-His-TOPO (Invitrogen), respectively. the eukaryotic expression vector pcDNA3.1/V5/His-TOPO (Invitrogen). To simplify detection and purification, a fusion with a His-tag was made. After the cotransfection of insect cells with Bac-N-Blue DNA, recombinant viruses were formed which were detected by a PCR method. A bacteriophage standard was then prepared and used for further transfection and production of the receptor in larger quantities. Purification of the His-tagged proteins occurred using a nickel affinity column.
Eksempel 3: Kloning og ekspresjon av kinasedomenetExample 3: Cloning and expression of the kinase domain
Kinasedomenet ble amplifisert fra en lengre sekvens fra et cDNA-bibliotek (brystkjertel og lunge, Clontech) ved PCR og klonet i TOPO-vektoren. Fra denne vektoren amplifiseres bare kinasedomenet. The kinase domain was amplified from a longer sequence from a cDNA library (breast and lung, Clontech) by PCR and cloned into the TOPO vector. From this vector, only the kinase domain is amplified.
For ekspresjon av det rekombinante kinasedomenet som et glutation-S-transferasefusjonsprotein anvendes vektoren pGex-KT (som inneholder et trombinkuttesete mellom proteinene), for fusjon med MBP-vektoren blir pMal-TBVp3 (modifisert også med trombin-kuttesetet) benyttet. For kloning transformeres plasmidene i E. co/i-stamme XL-1, og for påfølgende ekspresjon av proteinene i E. co/i-stamme BL21. Transforma-sjonen skjer alltid ved varmesjokk. Cellene dyrkes i LB-medium med 200 ug/ml, hvor det for ekspresjon av MBP-ekspresjonsproteiner alltid må foreligge 0,1 % glukose i mediet. For ekspresjon av proteinene dyrkes cellene til en OD60opå 0,8-0,9, hvoretter en induksjon med 0,5 mM IPTG i 3 timer ved 35 °C utføres. Oppslutning av cellene skjer i en French-presse. Oppslutningsbufferen inneholder 20 mM Tris pH 7,4,150 mM NaCl, ImM DTT, 0,1 mM EDTA og proteaseinhibitor. For separasjon av uløselige bestanddeler gjennomføres etter oppslutning av cellene, en ultrasentirfugering ved 35 000 x g. MBP-fusjonsproteinene renses deretter over et amylose-resin i buffer (20 mM Tris ph 7,4, 150 mM NaCl, lmM DTT, 0,1 mM EDTA og proteaseinhibitor. GST-fusjonsproteinene renses ved hjelp av sefarose-kuler (buffer som ovenfor). For expression of the recombinant kinase domain as a glutathione-S-transferase fusion protein, the vector pGex-KT (containing a thrombin cut site between the proteins) is used, for fusion with the MBP vector, pMal-TBVp3 (modified also with the thrombin cut site) is used. For cloning, the plasmids are transformed into E. co/i strain XL-1, and for subsequent expression of the proteins into E. co/i strain BL21. The transformation always occurs by heat shock. The cells are grown in LB medium with 200 ug/ml, where for the expression of MBP expression proteins there must always be 0.1% glucose in the medium. For expression of the proteins, the cells are grown to an OD60o of 0.8-0.9, after which an induction with 0.5 mM IPTG for 3 hours at 35°C is performed. Aggregation of the cells takes place in a French press. The digestion buffer contains 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA and protease inhibitor. For the separation of insoluble components, after lysing the cells, an ultracentrifugation at 35,000 x g is carried out. The MBP fusion proteins are then purified over an amylose resin in buffer (20 mM Tris ph 7.4, 150 mM NaCl, 1 mM DTT, 0.1 mM EDTA and protease inhibitor The GST fusion proteins are purified using sepharose beads (buffer as above).
I ytterligere et forsøk på ekspresjon av det rekombinante kinasedomene som et glutation-S-transferasefusjonsprotein benyttes den eukaryote ekspresjonsvektor pDEST27 (Invitrogen 11812-013). In a further attempt to express the recombinant kinase domain as a glutathione-S-transferase fusion protein, the eukaryotic expression vector pDEST27 (Invitrogen 11812-013) is used.
Denne vektor bærer cDNA som koder for kinasedomenet ifølge oppfinnelsen i samme leseramme som N-terminalt foreliggende glutation-S-transferase-cDNA. Dette fører til ekspresjon av et fusjonsprotein med glutation-S-transferase og aminosyrene Ala 1267-Leu 1570 (aminosyrenummerering ifølge SEQ ID NO 2). This vector carries the cDNA that codes for the kinase domain according to the invention in the same reading frame as the N-terminally present glutathione-S-transferase cDNA. This leads to the expression of a fusion protein with glutathione-S-transferase and the amino acids Ala 1267-Leu 1570 (amino acid numbering according to SEQ ID NO 2).
For ekspresjon av det rekombinante kinasedomene benyttes HEK293EBNA-celler (Invitrogen) som er forbigående transfektert med den kinasedomenekodende vektor pDEST27. For dette dyrkes HEK293EBNA-celler i DMEM-medium (pluss 10 % FCS pluss Glutamax) til subsammenflytning. Cellene transfekteres i en kolbe ved hjelp av Lipo-fectamin-reagens (Invitrogen) ifølge produsentens standardfremgangsmåte. Til hver celle på 150 cm benyttes 250 ul reagens og 75 ug pDEST27-kinasedomene-DNA for transfeksjonen (=GST-SIN2). Cellene i en andre kolbe transfekteres ikke (= kontroll). For expression of the recombinant kinase domain, HEK293EBNA cells (Invitrogen) are used which have been transiently transfected with the kinase domain-encoding vector pDEST27. For this, HEK293EBNA cells are grown in DMEM medium (plus 10% FCS plus Glutamax) to subconfluence. The cells are transfected in a flask using Lipofectamine reagent (Invitrogen) according to the manufacturer's standard procedure. For each cell of 150 cm, 250 ul of reagent and 75 ug of pDEST27 kinase domain DNA are used for the transfection (=GST-SIN2). The cells in a second flask are not transfected (= control).
Cellene nedsentrifugeres etter 48 timers inkubering. Det kondisjonerte medium fjernes. Cellene lyseres på is (25 mM HEPES/NaOH pH 7,3 + 1 % NP-40 + 2 mM EGTA + 2 mM EDTA + 10 mM Ø-glyserofosfat + 2 mM DTT + fosfataseinhibitorblanding nr. 1 (SIGMA) + fosfataseinhibitorblanding nr. 2 (SIGMA) + Completemjnj-EDTA (Roche) + 200 mM NaCl), nedfryses, opptines og sentrifugeres. Supernatanten benyttes videre. The cells are centrifuged down after 48 hours of incubation. The conditioned medium is removed. The cells are lysed on ice (25 mM HEPES/NaOH pH 7.3 + 1% NP-40 + 2 mM EGTA + 2 mM EDTA + 10 mM Ø-glycerophosphate + 2 mM DTT + phosphatase inhibitor mixture no. 1 (SIGMA) + phosphatase inhibitor mixture no. 2 (SIGMA) + Completemjnj-EDTA (Roche) + 200 mM NaCl), freeze, thaw and centrifuge. The supernatant is used further.
For binding av GST-fusjonsproteinet til glutation-sefarose tilsettes supernatan-tene til glutation-sefarose, inkuberes og vaskes. 20 ul glutation-sefarose med bundne proteiner benyttes direkte i kinasearialysen som beskrevet i eksempel 4 ^glutation-sefarose med bundne proteiner). Resten av glutation-sefarosen elueres med en buffer som inneholder redusert glutation (=glutationeluat). For binding of the GST fusion protein to glutathione-sepharose, the supernatants are added to glutathione-sepharose, incubated and washed. 20 ul of glutathione-sepharose with bound proteins is used directly in the kinase analysis as described in example 4 (glutathione-sepharose with bound proteins). The rest of the glutathione sepharose is eluted with a buffer containing reduced glutathione (=glutathione eluate).
Eksempel 4: Påvisning av kinasedomenets aktivitet (autofosforylering)Example 4: Detection of the activity of the kinase domain (autophosphorylation)
Det benyttes 10 ul av den rensede proteinløsning (se eksempel 3, glutation-sefarose med bundne proteiner og glutationeluat) med de 10 (il kinaseanalysebuffer (25 mM HEPES/NaOH pH 7,3 + 10 mM MgCl2+ 10 mM MnCl2+ 2 mM DTT + fosfataseinhibitorblanding nr. 1 og 2 (SIGMA) + 200 mM NaCl). Fosforyleringsreaksjonen startes ved tilsetning av 1 ul 100 uM ATP + 4 uCi y[<33>P]-ATP. Reaksjonen skjer i 60 min ved 30 °C. Prøvene fraksjoneres i en SDS-PAGE, og gelen farges med en Coomassie G-250-løsning. Etter tørking av gelen utføres en autoradiografi. 10 ul of the purified protein solution (see example 3, glutathione-sepharose with bound proteins and glutathione eluate) is used with the 10 ul of kinase assay buffer (25 mM HEPES/NaOH pH 7.3 + 10 mM MgCl2+ 10 mM MnCl2+ 2 mM DTT + phosphatase inhibitor mixture Nos. 1 and 2 (SIGMA) + 200 mM NaCl). The phosphorylation reaction is started by adding 1 ul of 100 uM ATP + 4 uCi y[<33>P]-ATP. The reaction takes place for 60 min at 30 °C. The samples are fractionated in an SDS-PAGE, and the gel is stained with a Coomassie G-250 solution After drying the gel, an autoradiography is performed.
Eksempel 5: Analysesystem for påvisning av effektorer (bindingsanalyse) Homogenisering Example 5: Analysis system for detection of effectors (binding analysis) Homogenization
Celler og vev som uttrykker proteinet ifølge oppfinnelse, homogeniseres i buffer 1 ved 4 °C. Buffer 1: 0,02 mmol/1 Tris-buffer, pH 7,5, 5,0 mmol/EDTA, 2mmol/DTT, 20 % glyserol, 0,4 M KCL, 20 mmol/1 molybdat, 0,3 umol/1 aprotinin, 1 umol/1 pepstatin, 10 umol/1 leupeptin. Cells and tissues expressing the protein according to the invention are homogenized in buffer 1 at 4 °C. Buffer 1: 0.02 mmol/1 Tris buffer, pH 7.5, 5.0 mmol/EDTA, 2 mmol/DTT, 20% glycerol, 0.4 M KCL, 20 mmol/1 molybdate, 0.3 umol/ 1 aprotinin, 1 umol/1 pepstatin, 10 umol/1 leupeptin.
ReseptoranalyserReceptor assays
Reseptorbindningsanalysene ble utført med råekstrakter i buffer 1, cytosol-preparater (supernatant etter sentrifugering av råekstrakten ved 105 000 x g, 90 min) og mikrosomfraksjoner fra celler og vev som uttrykker proteiner ifølge oppfinnelsen. The receptor binding assays were performed with crude extracts in buffer 1, cytosol preparations (supernatant after centrifugation of the crude extract at 105,000 x g, 90 min) and microsome fractions from cells and tissues expressing proteins according to the invention.
Råekstrakter og cytosolfraksjoner (0,04 ml) ble inkubert ved 4 °C i 2 timer med 10 ul radioaktivt steroid eller sterol (sluttkonsentrasjon 10 nmol/1), enten i fravær eller nærvær av et 100 gangers overskudd av ikke-radioaktiv forbindelse (for bestemmelse av uspesifikk binding). Etter inkubering av råekstrakten eller cystosolfraksjonen med radioaktivt steroid eller radioaktiv sterol adsorberes ubundet steroid eller sterol til aktivt kull ved inkubering med 0,25 ml av en suspensjon av dekstranbelagt trekull i buffer i 5 minutter ved 4 °C. Etter sentrifugering (5 min, 15 000 x g) uttas en prøve av supernatanten, og radioaktiveten måles ved scintillasjonstelling. Den spesifikke reseptorbinding bestemmes som differansen mellom bundet radioaktivitet i fravær og i nærvær av et 100 gangers overskudd av umerket forbindelse. Som radioaktivt merkede steroider og steroler ble<3>[H]-østradiol,<3>[H]-progesteron,<3>[H]-kortikosteron,<3>[H]-kortisol, [H]-aldosteron, [H]-dihydrotestosteron, [H]-testosteron, [H]-pregnolon, [H]-kolesterol,<3>[H]-androsteron,<3>[H]-dihydroandrosteron,<3>[H]-kalcitriol og<3>[H]-FF-MAS benyttet. Crude extracts and cytosolic fractions (0.04 ml) were incubated at 4 °C for 2 h with 10 µl of radioactive steroid or sterol (final concentration 10 nmol/1), either in the absence or presence of a 100-fold excess of non-radioactive compound (for determination of nonspecific binding). After incubation of the crude extract or cystosol fraction with radioactive steroid or radioactive sterol, unbound steroid or sterol is adsorbed to activated charcoal by incubation with 0.25 ml of a suspension of dextran-coated charcoal in buffer for 5 minutes at 4 °C. After centrifugation (5 min, 15,000 x g), a sample of the supernatant is taken, and the radioactivity is measured by scintillation counting. The specific receptor binding is determined as the difference between bound radioactivity in the absence and in the presence of a 100-fold excess of unlabeled compound. As radioactively labeled steroids and sterols, <3>[H]-estradiol,<3>[H]-progesterone,<3>[H]-corticosterone,<3>[H]-cortisol, [H]-aldosterone, [ H]-dihydrotestosterone, [H]-testosterone, [H]-pregnolone, [H]-cholesterol,<3>[H]-androsterone,<3>[H]-dihydroandrosterone,<3>[H]-calcitriol and <3>[H]-FF-MAS used.
Eksempel 6: KinaseanalyseExample 6: Kinase assay
Peptidet som inneholder kinasedomenet, uttrykkes i SF9- hhv. COS-celler og renses ved affinitetskromatografi (ved hjelp av merkelappene). Det rensede peptid inkuberes i 20 mM Mops pH 7,5, 25 mM Ø-glyserofosfat; 5 mM EGTA; 0,04 % NP40 og 0,2 % BSA i 30 min med<33->ATP i MgCl2og substratet Bio-Lys-Lys-Leu-Asn-Arg-Thr-Leu-Ser-Val-Ala-OH. Dette substratet fosforyleres i nærvær av ATP på serin, hhv. treonin og kan påvises ved hjelp av streptavidinkuler. Reaksjonen stanses med 50 uM ATP, 50 uM EDTA, 0,1 % triton X-100 (vol/vol) og tilsettes samtidig streptavidinkuler. Reaksjonsblandingen må stå i minst 10 min og deretter sentrifugeres i 10 min. Bestemmelse av fosforyleringen skjer ved målingen av radioaktivitet. Som referanse-forbindelse for inhibering av peptidets kinaseaktivitet benyttes staurosporin. The peptide containing the kinase domain is expressed in SF9 or COS cells and purified by affinity chromatography (using the tags). The purified peptide is incubated in 20 mM Mops pH 7.5, 25 mM Ø-glycerophosphate; 5 mM EGTA; 0.04% NP40 and 0.2% BSA for 30 min with <33->ATP in MgCl2 and the substrate Bio-Lys-Lys-Leu-Asn-Arg-Thr-Leu-Ser-Val-Ala-OH. This substrate is phosphorylated in the presence of ATP on serine, resp. threonine and can be detected using streptavidin beads. The reaction is stopped with 50 uM ATP, 50 uM EDTA, 0.1% triton X-100 (vol/vol) and streptavidin beads are added at the same time. The reaction mixture must stand for at least 10 min and then centrifuged for 10 min. Phosphorylation is determined by measuring radioactivity. Staurosporine is used as a reference compound for inhibiting the peptide's kinase activity.
Eksempel 7: Elektronisk Northern-BlotExample 7: Electronic Northern Blot
Et elektronisk northern-blot gir, basert på antall EST fra et gitt vev, et mål på ekspresjonsgraden av et gitt gen i dette vev. For dette ble antall EST for et vev i en database påvist (betegnet totalstørrelsen), og deretter antall EST fra dette vev som tilsvarer nukleinsyren ifølge SEQ ID NO 1 bestemt (betegnet treff). Som fremgangsmåte ble et såkalt "blast-søk" benyttet (NCBI BLAST v. 2.10.10; Altschul et al., Nucleic Acid Res. 1997, 25, 3389-3402). Det ble søkt i en EST-database (LifeSeqGold fra Incyte). An electronic northern blot provides, based on the number of ESTs from a given tissue, a measure of the degree of expression of a given gene in this tissue. For this, the number of ESTs for a tissue in a database was detected (denoted the total size), and then the number of ESTs from this tissue corresponding to the nucleic acid according to SEQ ID NO 1 was determined (denoted hits). As a method, a so-called "blast search" was used (NCBI BLAST v. 2.10.10; Altschul et al., Nucleic Acid Res. 1997, 25, 3389-3402). An EST database (LifeSeqGold from Incyte) was searched.
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| DE2001156597 DE10156597A1 (en) | 2001-11-13 | 2001-11-13 | Nucleic acids encoding membrane receptors for steroids or sterols, useful for identifying modulators of a pgk polypeptide, as a target substance for the production of an agent for steroid hormone-dependent diseases, e.g. infertility |
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