Pharmaceutical Study

Source text – English

A convergence of evidence indicates that the gamma secretase complex, comprised of the presenilin subunits, mediates the intra-membrane cleavage of Amyloid precursor protein (APP), and the Notch family of proteins (De Strooper, B., et al.. “Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.” Nature 391 (6665): 387-90 (1998); De Strooper, B ct al. “A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.” Nature 398 (6727): 518-22 (1999); Mumm, J. S., et al “A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch 1.” Mo/ Cell 5 (2): 197-206 (2000); Zhang, Z et al. “Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1.” Nat Cell Biol 2(7): 463-5 (2000)). Cleavage of APP by gamma secretase leads to beta-amyloid synthesis.

Cleavage of Notchl by gamma secretase results in release of the Notch intracellular domain (NICD), which translocates to the nucleus and activates gene expression (Jarriault, S., et al “Signalling downstream of activated mammalian Notch.” Nature 311 (6547): 355-8 (1995).; Kopan, R., et al.. “Signal transduction by activated Notch: importance of proteolytic processing and its regulation by the extracellular domain.” Proc Natl Acad Sci USA 93 (4): 1683-8 (1996); Schroeter, E. et al “Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain” Nature 393 (6683): 382-6 (1998)).

In particular, Notch signaling activates transcription of the mammalian homolog of the Drosophila transcription factor hairy-enhancer of split (Hes). Transcriptional activation of Hesl is mediated by de-repression of CBFl/RBPJk upon binding by NICD in the nucleus. These facts have been exploited to develop a reporter gene assay for Notch Signaling Hsieh, J. J. et al. “Truncated mammalian Notchl activates CBFl/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.” Mol Cell Biol 16(3): 952-9 (1996) and Lu, F. M. et al. “Constitutively active human Notchl binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1 -responsive element” Proc Natl Acad Sci USA 93 (11): 5663-7 (1996).
Gamma secretase inhibitors have been observed to block NICD formation, and inhibit Notch signaling (De Strooper, B., et al. “A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.” Nature 398(6727): 518-22 (1999)). Due to the importance of Notch signaling in cell fate determination, and tissue differentiation during both development and in the adult, inhibition of Notch signaling by gamma secretase inhibitors is thought to be a limiting factor in some of its therapeutic utilities. In order to identify gamma secretase inhibitors with some degree of selectivity over Notch, we have employed a reporter gene based Notch signaling assay using a constitutively active rat Notch 1 construct (ZEDN1) provided by Dr Gerry Weinmaster, University of California at Los Angeles (UCLA) as described in Shawber, C, D. Nofziger, J. J. Hsieh, C. Lindsell, O. Boglcr, D. Hayward and G. Weinmaster “Notch signaling inhibits muscle cell differentiation through a CBF1 -independent pathway.” Development 122 (12): 3765-73 (1996) in combination with the CBF1 repressible Luciferase reporter gene 4xwtCBFlLuc (Hsieh, J. J., ct al. “Truncated mammalian Notchl activates CBFl/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.” Mol Cell Biol 16(3): 952-9 (1996).(Pharmaceutical patent)

Translation – Italian

Una serie di prove convergenti indica che il complesso gamma-secretasi, composto dalle sottounità di presenilina, media il cleavage intramembranico della Proteina precursore dell’amiloide (APP), e la famiglia Notch di proteine (De Strooper, B., et al. “Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.” Nature 391 (6665): 387-90 (1998); De Strooper, B et al. “A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.” Nature 398 (6727): 518-22 (1999); Mumm, J. S., et al “A lige-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch 1.” Mol Cell 5 (2): 197-206 (2000); Zhang, Z et al. “Presenilins are required for gamma-secretase cleavage of beta-APP e transmembrane cleavage of Notch-1.” Nat Cell Biol 2(7): 463-5 (2000)).

Il cleavage della APP da parte della gamma-secretasi conduce alla sintesi beta-amiloide. Il cleavage di Notch da parte della gamma-secretasi ha come conseguenza il rilascio del Dominio intracellulare Notch (NICD), che trasloca al nucleo e attiva l’espressione genetica (Jarriault, S., et al. “Signalling downstream of activated mammalian Notch.” Nature 311 (6547): 355-8 (1995); Kopan, R., et al.. “Signal transduction by activated Notch: importance of proteolytic processing e its regulation by the extracellular domain.” Proc Natl Acad Sci USA 93 (4): 1683-8 (1996); Schroeter, E. et al “Notch-1 signalling requires lige-induced proteolytic release of intracellular domain” Nature 393 (6683): 382-6 (1998)).

In particolare, la segnalazione Notch attiva la trascrizione dell’omologo dei mammiferi del fattore di trascrizione Drosophila detto HES (hairy-enhancer of split). L’attivazione trascrizionale di Hesl è mediata dalla derepressione di CBFl/RBPJk in seguito al legame da parte del NICD nel nucleo. Questi fatti sono stati sfruttati per sviluppare un saggio dei geni reporter per la segnalazione Notch, Hsieh, J. J. et al. “Truncated mammalian Notchl activates CBFl/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.” Mol Cell Biol 16(3): 952-9 (1996) e Lu, F. M. et al. “Constitutively active human Notchl binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1 -responsive element” Proc Natl Acad Sci USA 93 (11): 5663-7 (1996).
È stato osservato che gli inibitori della gamma-secretasi bloccano la formazione del NICD e inibiscono la segnalazione Notch (De Strooper, B., et al. “A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain.” Nature 398(6727): 518-22 (1999)). A causa dell’importanza della segnalazione Notch nella determinazione del fato della cellula, e della differenziazione tissutale durante lo sviluppo e nell’adulto, si pensa che l’inibizione della segnalazione Notch da parte degli inibitori della gamma-secretasi sia un fattore limitante in alcuni dei suoi utilizzi terapeutici. Al fine di identificare gli inibitori della gamma-secretasi con un qualche grado di selettività su Notch, abbiamo utilizzato un saggio di segnalazione Notch basato sui geni reporter usando un costrutto Notch 1 di ratto costitutivamente attivo (ZEDN1) fornito dal Dr. Gerry Weinmaster, della University of California di Los Angeles (UCLA), così come descritto in Shawber, C, D. Nofziger, J. J. Hsieh, C. Lindsell, O. Boglcr, D. Hayward e G. Weinmaster “Notch signaling inhibits muscle cell differentiation through a CBF1 -independent pathway.” Development 122 (12): 3765-73 (1996) in combinazione con il gene reporter della luciferasi reprimibile CBF1 4xwtCBFlLuc (Hsieh, J. J., et al. “Truncated mammalian Notchl activates CBFl/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2.” Mol Cell Biol 16(3): 952-9 (1996).

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