Secretase Laboratory
| Contact: | Geneviève Evin |
|---|---|
| Phone: | +61 3 8344 4205 |
| Fax: | +61 3 8344 4004 |
| Email: | g.evin@unimelb.edu.au |
The Secretase Lab studies the biochemical characteristics, enzymatic properties, and subcellular distribution of beta-secretase (BACE) and gamma-secretase, and their expression in human brain. BACE and gamma-secretase are the proteases responsible for the production Aβ amyloid peptide, a toxic protein fragment that accumulates in the brain of patients with Alzheimer’s disease. Therefore, they represent targets for drug development.
Objectives
- To decipher the molecular mechanisms causing an increase in BACE protein and activity in Alzheimer’s disease
- To develop novel strategies to control BACE activity
- To characterize endogenous active gamma-secretase and relate activity to subunit composition
- To study the role of Aph1 subunit in gamma-secretase complexes
Major Achievements
- Demonstration that BACE (beta-secretase) is increased in brain cortex of Alzheimer's patients
- Demonstration that BACE is detectable and elevated in human cerebrospinal fluid
- Demonstration that gamma-secretase has the properties of an aspartyl protease and that the inhibitor, pepstatin binds to presenilins
- To show that some classes of gamma-secretase inhibitors prevent autoproteolysis and activation of presenilins (in collaboration with Merck)
- Identification of APP cytosolic fragments (AICD) produced by gamma-secretase (in collaboration with Dr A. Weidemann, Heidelberg, Germany)
- To establish a whole-cell fluorescence reporter assay for gamma-secretase (with Dr R. Cappai, University of Melbourne)
- To establish an in vitro assay for gamma-secretase based on AICD detection
- To show that gamma-secretase inhibitors associate with a 900 kDa presenilin/nicastrin comple
Techniques
- Tissue homogenization and fractionation by differential centrifugation
- Subcellular fractionation on discontinuous sucrose and iodixanol gradients
- In vitro enzymatic assays based on fluorescence detection
- Antibody production, immunoassays (ELISA) and immunoprecipitation
- SDS-PAGE and Western blotting, including chemiluminescence detection
- Protein purification using chromatographic techniques (size-exclusion, ion-exchange, affinity etc…)
- Aβ assays
- Mammalian cell cultures (SH-SY5Y, COS-7, HEK 293, CHO) and transfections (transient and stable)
- Basic molecular biology techniques: PCR, agarose-gel electrophoresis, DNA sequencing
- Cloning and site-directed mutagenesis
- Expression of recombinant proteins in E. Coli and in various cell systems
Collaborations
Internal:
-
Colin Masters, Kevin Barnham, Janetta Culvenor, Vijaya Kenche, Qiao-Xin Li, Anthony White
MHRI: Ashley Bush, Michael Cater, Brian Dean, Dr Elisabeth Scarr, Dr Ya-Hui Hung
External:
- Elisabeth Coulson (University of Queensland)
- Brett Garner (University of Sydney)
- Brian Smith (WEHI)
- Dr. Javier Saez-Valero and Ximena Silveyra (University Miguel Hernandez, Valencia, Spain)
Funding
- NH&MRC
- ANZ Charitable Trusts
Recent Publications
- G. Evin, R. Cappai, Q-X. Li, J. G. Culvenor, D. H. Small, K. Beyreuther and C. L. Masters. Candidate g-secretases in the generation of the carboxyl-terminus of the Alzheimer's disease βA4 amyloid: possible involvement of cathepsin D. Biochemistry (1995) , 34, 14185-14192
- G. Evin, R. A. Sharples, A. Weidemann, F. B. M. Reinhard, V. Carbone, J. G. Culvenor, R. M. D. Holsinger, M. F. Sernee, K. Beyreuther and C. L. Masters. Aspartyl protease inhibitor pepstatin binds to the presenilins of Alzheimer's disease. Biochemistry (2001) 40, 8359-8368
- D. Beher, J. D. J. Wrigley, A. Nadin, G. Evin, C. L. Masters, T. Harrison, J. L. Castro and M. S. Shearman. Characterisation of novel g-secretase inhibitors blocking presenilin endoproteolysis: implications for presenilin biology. J. Biol. Chem (2001) 276, 45394-45402
- Weidemann, S. Eggert, F. B. M. Reinhard, M. Vogel, K. Paliga, G. Baier, C. L. Masters, K. Beyreuther, and G. Evin. A novel epsilon-Cleavage within the Transmembrane Domain of the Alzheimer Amyloid Precursor Protein demonstrates Homology with Notch Processing. Biochemistry (2002) 41, 2825-2835
- R.M. D. Holsinger, C. A. McLean, K. Beyreuther, C. L. Masters, and G. Evin. Increased expression of the amyloid precursor b-secretase in Alzheimer's disease. Annals of Neurology (2002), 51, 783-786
- M. F. Sernee, G. Evin, J. G. Culvenor, J. A. Villadangos, K. Beyreuther, C. L. Masters, and R. Cappai. Selecting cells with different Alzheimer's disease g-secretase activity using FACS. Eur. J. Biochem. (2003) 270, 495-506
- J. G. Culvenor, N. T. Ilaya, M. T. Ryan, L. Canterford, D, E. Hoke, N. A. Williamson, C. A. McLean, C. L. Masters, and G. Evin. Characterization of presenilin complexes from mouse and human brain using blue native gel electrophoresis reveals high expression in embryonic brain and minimal change in complex mobility with pathogenic presenilin mutations. Eur. J. Biochem. (2004) 271, 375-385
- A. J. George, R. M. D. Holsinger, C. A. McLean, K. M. Laughton, G. Evin, C. L. Masters and Q-X Li. APP intracellular domain is increased and soluble Ab is reduced with diet-induced hypercholesterolemia in a transgenic mouse model of Alzheimer's disease. Neurobiol. Dis. (2004) 16, 124-132
- S. Eggert, K. Paliga, P. Soba, G. Evin, C. L. Masters, A. Weidemann and K. Beyreuther. The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha -, beta -, gamma -, and epsilon -like cleavages. Modulation of APLP-1 processing by N-glycosylation. J. Biol. Chem.( 2004) 279,18146-18156
- R. M. D. Holsinger , C. A. McLean, S. J. Collins, C. L. Masters, and G. Evin. Increased b-secretase activity in cerebrospinal fluid of Alzheimer's disease subjects. Ann. Neurol. (2004) 55, 898-899
- N. T. Ilaya, G. Evin, C. L. Masters and J. G. Culvenor. Nicastrin expression in mouse peripheral tissues is not co-ordinated with presenilin and is high in muscle. J Neurochem. (2004) 91, 230-237
- M. J. Chiocco, L. S. Kulnane, L. Younkin L, S. Younkin , G. Evin, and B. T. Lamb Altered amyloid-b metabolism and deposition in genomic-based b-secretase transgenic mice. J. Biol. Chem. (2004) 279, 52535-42
- G. Evin, L. D. Canterford, D. E. Hoke, J. G. Culvenor, R. A. Sharples and C. L. Masters. Transition-state analogue g-secretase inhibitors stabilize a 900 kDa presenilin/nicastrin complex. Biochemistry(2005) 44, 4332-4341
- G. Evin, M.F. Sernee, and C. L. Masters. Inhibition of g-secretase as a therapeutic intervention for Alzheimer’s disease: prospects, limitations, and strategies. CNS Drugs (2006) 20(5):351-372
- G. Evin, Kenche VJ. BACE Inhibitors as Potential Therapeutics for Alzheimer's disease. Recent Patents on CNS Drug Discovery (2007) 2,188-99. Bentham Science Publishers Ltd
- M-X. Silveyra, G. Evin, M. F. Montenegro, C. J. Vidal, S. Martinez, J. G. Culvenor, J. Sáez-Valero (2008) Presenilin-1 interacts with acetylcholinesterase and alters its enzymatic activity and glycosylation. Mol. Cell. Biol. Published online ahead of print on 25-02-08, doi:10.1128/MCB.02065-07