Selective Reconstitution and Recovery of Functional -Secretase Complex on Budded Baculovirus Particles

Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2004; 279(36):38040-6. DOI: 10.1074/jbc.M405597200
Source: PubMed


In vitro reconstitution of functions of membrane proteins is often hampered by aggregation, misfolding, or lack of post-translational modifications of the proteins attributable to overexpression. To overcome this technical obstacle, we have developed a method to express multimeric integral membrane proteins in extracellular (budded) baculovirus particles that are released from Sf9 cells co-infected with multiple transmembrane proteins. We applied this method to the reconstitution of gamma-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of beta-amyloid precursor protein to release Abeta peptides, the major component of amyloid deposits in Alzheimer brains as well as of Notch. When we co-infected Sf9 cells with human presenilin 1 (PS1), nicastrin, APH-1a, and PEN-2, a high-molecular-weight membrane protein complex that contained PS1 exclusively in its fragment form associated with three other cofactor proteins was reconstituted and recovered in a highly gamma-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative gamma-secretase activity (per molar PS1 fragments) was concentrated by approximately 2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of gamma-secretase, as well as screening of its binding molecules or inhibitors, and will also provide a versatile methodology for the characterization of a variety of membrane protein complexes.

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    • "For detection of the g-secretase activity in solubilized condition, 1% CHAPSO-solubilized membranes were co-incubated with APP-based recombinant substrate under 0.25% CHAPSO condition (Takahashi et al, 2003) and then analysed by immunoblot analysis. Recombinant g-secretase complex as well as SPP was purified from baculovirus-infected Sf9 cells as previously described (Hayashi et al, 2004; Kakuda et al, 2006; Ogura et al, 2006; Fuwa et al, 2007). CHO cells expressing C99 and APP-based recombinant substrate were kindly provided from Drs S Funamoto and Y Ihara (Doshisha University). "
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    ABSTRACT: Amyloid-β peptide ending at the 42nd residue (Aβ42) is implicated in the pathogenesis of Alzheimer's disease (AD). Small compounds that exhibit selective lowering effects on Aβ42 production are termed γ-secretase modulators (GSMs) and are deemed as promising therapeutic agents against AD, although the molecular target as well as the mechanism of action remains controversial. Here, we show that a phenylpiperidine-type compound GSM-1 directly targets the transmembrane domain (TMD) 1 of presenilin 1 (PS1) by photoaffinity labelling experiments combined with limited digestion. Binding of GSM-1 affected the structure of the initial substrate binding and the catalytic sites of the γ-secretase, thereby decreasing production of Aβ42, possibly by enhancing its conversion to Aβ38. These data indicate an allosteric action of GSM-1 by directly binding to the TMD1 of PS1, pinpointing the target structure of the phenylpiperidine-type GSMs.
    The EMBO Journal 11/2011; 30(23):4815-24. DOI:10.1038/emboj.2011.372 · 10.43 Impact Factor
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    • "In this point, the BV system has extremely superior features. G protein and G protein-coupled receptors have been functionally reconstituted in BV [20], [34], and functional γ-secretase complexes have also been reconstituted on BV [35]. In the near future, the reconstituted CL system on BV will be developed and used for the screening of CL binders and modulators, hopefully leading to breakthroughs in pharmaceutical therapies that target CLs. "
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    ABSTRACT: Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.
    PLoS ONE 02/2011; 6(2):e16611. DOI:10.1371/journal.pone.0016611 · 3.23 Impact Factor
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    • "Genetic and biochemical studies have provided strong evidence that γ-secretase is a multiprotein complex, comprised of PS (either PS1 or PS2) containing the catalytic site (De Strooper et al., 1998; Wolfe et al., 1999; Li et al., 2000), NCT, thought to contain a substrate binding site (Shah et al., 2005), Pen2 and Aph1 (1a, 1b or 1c in rodents) of unclear contribution (De Strooper, 2003; Iwatsubo, 2004). Loss of any of these 4 proteins seemed to abolish γ-secretase activity (Francis et al., 2002; Takasugi et al., 2003), and only co-expression of the four components together reconstitutes γ-secretase activity in yeast (Edbauer et al., 2003) or SF9 cells (Hayashi et al., 2004), both of which lack endogenous γ-secretase activity. The active enzyme contains 4 components, consistent with the existence of several distinct γ-secretases, each with potentially unique properties (Serneels et al., 2009). "
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    ABSTRACT: Gamma-secretase is a multiprotein, intramembrane-cleaving protease with a growing list of protein substrates, including the Notch receptors and the amyloid precursor protein. The four components of gamma-secretase complex--presenilin (PS), nicastrin (NCT), Pen2, and Aph1--are all thought to be essential for activity. The catalytic domain resides within PS proteins, NCT has been suggested to be critical for substrate recognition, and the contributions of Pen2 and Aph1 remain unclear. The role of NCT has been challenged recently by the observation that a critical residue (E332) in NCT, which had been thought to be essential for gamma-secretase activity, is instead involved in complex maturation. Here, we report that NCT is dispensable for gamma-secretase activity. NCT-independent gamma-secretase activity can be detected in two independent NCT-deficient mouse embryonic fibroblast lines and blocked by the gamma-secretase inhibitors N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester and L-685,458. This catalytic activity requires prior ectodomain shedding of the substrate and can cleave ligand-activated endogenous Notch receptors, indicating presence of this activity at the plasma membrane. Small interfering RNA knockdown experiments demonstrated that NCT-independent gamma-secretase activity requires the presence of PS1, Pen2, and Aph1a but can tolerate knockdown of PS2 or Aph1b. We conclude that a PS1/Pen2/Aph1a trimeric complex is an active enzyme, displaying biochemical properties similar to those of gamma-secretase and roughly 50% of its activity when normalized to PS1 N-terminal fragment levels. This PS1/Pen2/Aph1a complex, however, is highly unstable. Thus, NCT acts to stabilize gamma-secretase but is not required for substrate recognition.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2010; 30(5):1648-56. DOI:10.1523/JNEUROSCI.3826-09.2010 · 6.34 Impact Factor
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