Presenilins Are Processed by Caspase-type Proteases*
- Hansruedi Loetscher,
- Ulrich Deuschle,
- Manfred Brockhaus,
- Dieter Reinhardt,
- Peter Nelboeck,
- Jan Mous,
- Jürgen Grünberg‡,
- Christian Haass‡ and
- Helmut Jacobsen§
- From Pharma Division, Preclinical Central Nervous System Research-GeneTechnology, F. Hoffmann-La Roche Ltd., Ch-4070 Basel, Switzerland and the ‡Department of Molecular Biology, Central Institue of Mental Health, Mannheim D-68159, Germany
Abstract
Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27–35-kDa N-terminal and 15–24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced. The processing sites identified in PS1 and PS2 (Asp345/Ser346 and Asp329/Ser330, respectively) are typical for caspase-type proteases. Specific caspase inhibitors and cleavage site mutations confirmed the involvement of caspase(s) in PS1 and PS2 processing in cell transfectants. Fluorescent peptide substrates carrying the PS-identified cleavage sites were hydrolyzed by proteolytic activity from mouse brain. The PS2-derived peptide substrate was also cleaved by recombinant human caspase-3. Additional processing of PS2 by non-caspase-type proteases was also observed.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: F. Hoffmann - La Roche Ltd., PRPN-G, Bldg.66–709, Ch-4070 Basel/Switzerland. Tel.: 41-61-688-6403; Fax: 41-61-688-1448; E-mail: helmut.jacobsen{at}roche.com.
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↵1 The abbreviations used are: PS1, presenilin 1; PS2, presenilin 2; FAD, familial Alzheimer’s disease; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BHK, baby hamster kidney; CHO, Chinese hamster ovary; SFV, Semliki Forest virus; PAGE, polyacrylamide gel electrophoresis; AMC, aminomethylcoumarin; Ac-DEVD-AMC, Ac-Asp-Glu-Val-Asp-AMC; Ac-DSYD-AMC, Ac-Asp-Ser-Tyr-Asp-AMC; Ac-AQRD-AMC, Ac-Ala-Gln-Arg-Asp-AMC; b-DEVD-CHO, biotinyl-Asp-Glu-Val-Asp-aldehyde; b-DSYD-CHO, biotinyl-amidocaproyl-Asp-Ser-Tyr-Asp-aldehyde; b-AQRD-CHO, biotinyl-amidocaproyl-Ala-Gln-Arg-Asp-aldehyde; Ac-YVAD-dbmk, Ac-Tyr-Val-Ala-Asp-dimethylbenzoyloxymethyl ketone; TLCK,N α-p-tosyl-l-lysine chloromethyl ketone; ER, endoplasmic reticulum; PCR, polymerase chain reaction.
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↵2 U. Deuschle, N. Holzworth, A. Hayes, and J. Mons, manuscript in preparation.
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↵3 H. Loetscher, unpublished data.
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↵4 Processing of presenilins by caspase-type proteases was also reported by R. E. Tanzi at the 17th Blankenese Conference on Neurodegeneration, Hamburg, Germany, June 29–July 3, 1997.
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- Received June 11, 1997.
