Rie Fukuda

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

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 γ-secretase, a membrane protease complex that catalyzes the intramembrane cleavage of β-amyloid precursor protein to release Aβ 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 γ-secretase-active state in budded virus particles, whereas nonfunctional PS1 holoproteins massively contaminated the parental Sf9 cell membranes. The relative γ-secretase activity (per molar PS1 fragments) was concentrated by ∼2.5 fold in budded virus particles compared with that in Sf9 membranes. The budded baculovirus system will facilitate structural and functional analyses of γ-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.

A novel method for viral display of ER membrane proteins on budded baculovirus.

The baculovirus expression system has been used to express large quantities of various proteins, including membrane receptors. Here, we reveal a novel property of this expression system to be that certain membrane proteins can be displayed on the budded virus itself. We introduced the genes encoding sterol regulatory element-binding protein-2 (SREBP-2) or SREBP cleavage-activating protein (SCAP), important integral membrane proteins of the endoplasmic reticulum (ER) and/or the Golgi apparatus related to cellular cholesterol regulation, into a baculovirus vector. When insect cells were infected with SREBP-2 or SCAP recombinant viruses, it was found that these ER membrane proteins appeared on the budded baculovirus in addition to the host cell membrane fraction. Compared to proteins expressed on the cell membrane, membrane proteins displayed on virus exhibited both less aggregation and less degradation upon immunoblotting. Using this viral displayed SCAP as the screening antigen, we then generated a new monoclonal antibody specific against SCAP, which was useful for immunological localization studies. This system, which takes advantage of the viral display of membrane proteins, should prove to be a powerful additional tool for postgenomic protein analysis.

Protective effect of the long pentraxin PTX3 against histone-mediated endothelial cell cytotoxicity in sepsis.

Bundling extracellular histone proteins together blocks their cytotoxic effects and protects against sepsis. Smothering Killer Proteins Sepsis is a potentially fatal condition in which inflammation in response to infection goes out of control, and treatment options are limited. Histones are proteins normally found in the nucleus. During sepsis, histones released from dead cells can kill nearby healthy cells. Daigo et al. identified an interaction between pentraxin 3, a protein found in the circulation, and histone proteins. Pentraxin 3 bundled the histones into aggregates that no longer killed healthy cells. When the researchers injected pentraxin 3 into mice with sepsis, the mice had less inflammation and survived. Thus, pentraxin 3 might be an effective therapy against sepsis in humans. Pentraxin 3 (PTX3), a member of the long pentraxin subfamily within the family of pentraxins, is a soluble pattern recognition molecule that functions in the innate immune system. Innate immunity affords the infected host protection against sepsis, a potentially life-threatening inflammatory response to infection. Extracellular histones are considered to be the main cause of septic death because of their cytotoxic effect on endothelial cells, which makes them a potential therapeutic target. We found that PTX3 interacted with histones to form coaggregates, which depended on polyvalent interactions and disorder in the secondary structure of PTX3. PTX3 exerted a protective effect, both in vitro and in vivo, against histone-mediated cytotoxicity toward endothelial cells. Additionally, the intraperitoneal administration of PTX3 reduced mortality in mouse models of sepsis. The amino-terminal domain of PTX3, which was required for coaggregation with histones, was sufficient to protect against cytotoxicity. Our results suggest that the host-protective effects of PTX3 in sepsis are a result of its coaggregation with histones rather than its ability to mediate pattern recognition. This long pentraxin–specific effect provides a potential basis for the treatment of sepsis directed at protecting cells from the toxic effects of extracellular histones.

Functional reconstitution of olfactory receptor complex on baculovirus.

Despite that recent progress in genomics has elucidated the genomic structure of the olfactory receptors (ORs), most of them are still orphan receptors. The low expression level of ORs in heterologous cells has hampered many attempts to establish cell biological OR assay systems. Recently, we demonstrated that certain G protein-coupled receptors, such as the leukotriene B4 receptor or the dopamine D1 receptor, were efficiently reconstituted on baculovirus budding from infected Sf9 cells. The budded virus (BV) was shown to be mostly free of exogenous proteins other than those related to viral infection, resulting in low-noise assay conditions. Taking advantage of these conditions, we attempted to reconstitute OR complexes on BV. Sf9 cells were coinfected with recombinant baculoviruses harboring the cDNAs encoding adenylyl cyclase, trimeric G-protein, and the receptor: mOR-EG or S6. The coexpression of these proteins was detected by western blot, and the agonist- or antagonist-dependent receptor response was confirmed using ligand-dependent cyclic AMP production. These results demonstrated the successful reconstitution of functional OR complex on BV. Additionally, the expression of OR8B3 on BV, one of human orphan ORs, was also confirmed. This BV expression system is expected to be a highly effective tool for screening unknown ligands for ORs.

Characterization of cleavage enzymes for sterol regulatory element binding protein

Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are the key transcription factors for the regulation of the cellular cholesterol level. To identify proteolytic enzymes for SREBPs, a fluorogenic peptide substrate, MOCAc-GRSVLSFK(Dnp)rr-NH2, was synthesized according to the proposed cleavage site of human SREBP-2. In microsome fractions from hamster liver, we found a peptidase activity inhibitable by the synthetic inhibitor Ac-GRSVL-aldehyde with an IC50 of 40 nM. This peptidase separated into three peaks of approximately 400 kDa, 60 kDa, and 30 kDa (Mp400, Mp60 and Mp30 respectively) upon gel permeation chromatography. Mp30 was purified to apparent homogeneity with an Mr of 32 kDa. The partial amino acid sequence of Mp30 possessed homology to cathepsin B (EC 3.4.22.1). A 109 kDa protein band on SDS-PAGE which corresponded to Mp400 exhibited homology to neprilysin (EC 3.4.24.11) in partial amino acid sequence. These findings suggest several degradative pathways for SREBP in liver microsome membranes.