Nyborg AC, Kornilova AY, Jansen K, Ladd TB, Wolfe MS, Golde TE.
Signal peptide peptidase forms a homodimer that is labeled by an active site-directed gamma-secretase inhibitor.
J Biol Chem. 2004 Apr 9;279(15):15153-60.
PubMed.
The hypothesis we presented last year suggested that γ-secretase is a dimeric enzyme. This receives further support with strong biochemical data demonstrating that, like presenilin 1, signal peptide peptidase (SPP, a closely related aspartyl iCLiP) exists as a dimer, and that this dimer does not simply form during the isolation process. Importantly, the catalytically active form of SPP is found within these dimers, as demonstrated by inhibitor crosslinking studies; this is the case for PS1, as well. Unlike PS1, SPP dimers form rapidly after synthesis and are the predominant form of the protein in extracts prepared using stringent lysis conditions. This may be due to the fact that no other proteins are included in the active complex, whereas presenilin forms rapid complexes with the other obligate partners, APH-1, nicastrin and Pen-2. The dimer interface could serve to create the hydrophilic pocket required for peptide bond hydrolysis.
While the idea that aspartyl iCLiP are dimers receives a strong boost from this study, some important questions remain to be determined. Is the active site composed of aspartyl residues contributed by both molecules or does each subunit contain its own independent site? If the latter, do the sites face toward or away from each other? In the second case, is only one site active in the dimer, like caspase-9 (Renatus et al., 2001) or are both independently active, like caspase-3 (Pop et al., 2001)? If the sites are not in proximity, one PS1 subunit could present substrate (or inhibitor) to the other and this would still result in the NTF-NTF crosslink we obtained with the double-warhead inhibitor (Schroeter et al., 2003). Attempting to solve these questions with PS1 proved difficult; our results favor the first possibility (joined or closely positioned active sites). Demonstrating transinteractions within the SPP dimer would be even more difficult (without catalytically distinct FAD-like mutations available), but perhaps SPP dimers will produce a better substrate for structural studies.
References:
Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS.
Dimer formation drives the activation of the cell death protease caspase 9.
Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14250-5.
PubMed.
Pop C, Chen YR, Smith B, Bose K, Bobay B, Tripathy A, Franzen S, Clark AC.
Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.
Biochemistry. 2001 Nov 27;40(47):14224-35.
PubMed.
Schroeter EH, Ilagan MX, Brunkan AL, Hecimovic S, Li YM, Xu M, Lewis HD, Saxena MT, De Strooper B, Coonrod A, Tomita T, Iwatsubo T, Moore CL, Goate A, Wolfe MS, Shearman M, Kopan R.
A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis.
Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):13075-80.
PubMed.
Comments
University of Cincinnati College of Medicine
The hypothesis we presented last year suggested that γ-secretase is a dimeric enzyme. This receives further support with strong biochemical data demonstrating that, like presenilin 1, signal peptide peptidase (SPP, a closely related aspartyl iCLiP) exists as a dimer, and that this dimer does not simply form during the isolation process. Importantly, the catalytically active form of SPP is found within these dimers, as demonstrated by inhibitor crosslinking studies; this is the case for PS1, as well. Unlike PS1, SPP dimers form rapidly after synthesis and are the predominant form of the protein in extracts prepared using stringent lysis conditions. This may be due to the fact that no other proteins are included in the active complex, whereas presenilin forms rapid complexes with the other obligate partners, APH-1, nicastrin and Pen-2. The dimer interface could serve to create the hydrophilic pocket required for peptide bond hydrolysis.
While the idea that aspartyl iCLiP are dimers receives a strong boost from this study, some important questions remain to be determined. Is the active site composed of aspartyl residues contributed by both molecules or does each subunit contain its own independent site? If the latter, do the sites face toward or away from each other? In the second case, is only one site active in the dimer, like caspase-9 (Renatus et al., 2001) or are both independently active, like caspase-3 (Pop et al., 2001)? If the sites are not in proximity, one PS1 subunit could present substrate (or inhibitor) to the other and this would still result in the NTF-NTF crosslink we obtained with the double-warhead inhibitor (Schroeter et al., 2003). Attempting to solve these questions with PS1 proved difficult; our results favor the first possibility (joined or closely positioned active sites). Demonstrating transinteractions within the SPP dimer would be even more difficult (without catalytically distinct FAD-like mutations available), but perhaps SPP dimers will produce a better substrate for structural studies.
References:
Renatus M, Stennicke HR, Scott FL, Liddington RC, Salvesen GS. Dimer formation drives the activation of the cell death protease caspase 9. Proc Natl Acad Sci U S A. 2001 Dec 4;98(25):14250-5. PubMed.
Pop C, Chen YR, Smith B, Bose K, Bobay B, Tripathy A, Franzen S, Clark AC. Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer. Biochemistry. 2001 Nov 27;40(47):14224-35. PubMed.
Schroeter EH, Ilagan MX, Brunkan AL, Hecimovic S, Li YM, Xu M, Lewis HD, Saxena MT, De Strooper B, Coonrod A, Tomita T, Iwatsubo T, Moore CL, Goate A, Wolfe MS, Shearman M, Kopan R. A presenilin dimer at the core of the gamma-secretase enzyme: insights from parallel analysis of Notch 1 and APP proteolysis. Proc Natl Acad Sci U S A. 2003 Oct 28;100(22):13075-80. PubMed.
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