Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases.

Neuraminidase (NA) is a surface glycoprotein produced by the influenza virus. Specific NA mutations that confer resistance to anti-viral drugs have been reported. The aim of this study was to demonstrate quick preparation of the mutated NAs using the yeast surface display and its applicability for screening inhibitors. Plasmids encoding the head domain of wild-type and drug-resistant NAs were constructed and introduced into yeast, and these were successfully displayed on the yeast surface, with biochemical properties similar to the native virus NAs. This system using mutated NAs-displaying yeast provides an efficient and convenient tool for screening novel inhibitors against the drug-resistant influenza virus.

Mutated intramolecular chaperones generate high-activity isomers of mature enzymes.

The propeptide of carboxypeptidase Y precursor (proCPY) acts as an intramolecular chaperone that ensures the correct folding of the mature CPY (mCPY). Here, to further characterize the folding mechanism mediated by the propeptide, folding analysis was performed using a yeast molecular display system. CPYs with mutated propeptides were successfully displayed on yeast cell surface, and the mature enzymes were purified by the selective cleavage of mutated propeptides. Measurement of the activity and kinetics of the displayed CPYs indicated that the propeptide mutation altered the catalytic efficiency of mCPY. Although the mature region of the wild-type and mutant CPYs had identical amino acid sequences, the mCPYs from the mutant proCPYs had higher catalytic efficiency than the wild-type. These results indicate that proteins with identical amino acid sequence can fold into isomeric proteins with conformational microchanges through mutated intramolecular chaperones.

Identification of interaction site of propeptide toward mature carboxypeptidase Y (mCPY) based on the similarity between propeptide and CPY inhibitor (IC).

Both the propeptide in the precursor carboxypeptidase Y (proCPY) and the mature CPY (mCPY)-specific endogenous inhibitor (I(C)) inhibit CPY activity. The N-terminal inhibitory reactive site of I(C) (the N-terminal seven amino acids of I(C)) binds to the substrate-binding site of mCPY and is essential for mCPY inhibition, but the mechanism of mCPY inhibition by the propeptide is poorly understood. In this study, sequence alignment between I(C) and proCPY indicated that a sequence similar to the N-terminal region of I(C) was present in proCPY. In particular, a region including the C-terminus of the propeptide was similar to the N-terminal seven amino acids of I(C). In the presence of peptides identical to the N-terminus of I(C) and the C-terminus of the propeptide, CPY activity was competitively inhibited. The C-terminal region of the propeptide might bind to the substrate-binding site of mCPY.

Purification of inactive precursor of carboxypeptidase Y using selective cleavage method coupled with molecular display.

A novel purification system for inactive precursor proteins without conventional time-consuming purification steps was established. We purified an inactive precursor protein, procarboxypeptidase Y (proCPY), which was displayed on the cell surface of yeast, due to difficulty of purifying it by a system in which it is produced in cells.

Biochemical analysis of the yeast proteinase inhibitor (IC) homolog ICh and its comparison with IC.

Carboxypeptidase Y (CPY) inhibitor (I(C)) and its homologous protein (I(C)h) are thought to be members of the phosphatidylethanolamine-binding protein (PEBP) family of Saccharomyces cerevisiae. The biochemical characterization of I(C) and its inhibition mode toward CPY were recently reported, but I(C)h has not been characterized. The molecular mass of I(C)h was determined to be 22,033.7. The N-terminal Met1 was cleaved and the amino group of Ser2 was acetylated. I(C)h is folded as a monomeric beta-protein and is devoid of disulfide bonds. It has no inhibitory activity toward CPY, and it does not form a complex with CPY. I(C)h was exclusively expressed in the early log phase, whereas I(C) was expressed in the logarithmic and stationary phase. The intracellular localization of I(C)h was different from that of I(C). These findings provide insights into the physiological functions of I(C)h.

Specific membrane binding of the carboxypeptidase Y inhibitor I(C), a phosphatidylethanolamine-binding protein family member.

I(C), an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phosphatidylethanolamine-binding protein family. The binding of I(C) to phospholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements, which revealed that the affinity of this inhibitor was not for phosphatidylethanolamine but for anionic phospholipids, such as phosphatidylserine, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, with K(D) values below 100 nm. The liposome-binding assay and surface plasmon resonance analyses of I(C), when complexed with carboxypeptidase Y, and the mutant forms of I(C) further suggest that the N-terminal segment (Met1-His18) in its carboxypeptidase Y-binding sites is involved in the specific and efficient binding to anionic phospholipid membranes. The binding of I(C) to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged I(C), suggesting that I(C) is specifically targeted to vacuolar membranes rather than cytoplasmic membranes, during the stationary growth phase. The present findings provide novel insights into the membrane-targeting and biological functions of I(C) and phosphatidylethanolamine-binding proteins.