Hideshi Inoue

Design and Synthesis of Highly Sensitive Fluorogenic Substrates for Glutathione S-Transferase and Application for Activity Imaging in Living Cells

Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.

Tyrosine-7 is an essential residue for the catalytic activity of human class PI glutathione S-transferase: chemical modification and site-directed mutagenesis studies.

The glutathione (GSH)-conjugating activity of human class Pi glutathione S-transferase (GST pi) toward 1-chloro-2,4-dinitrobenzene (CDNB) was significantly lowered by reaction with N-acetylimidazole, an O-acetylating reagent for tyrosine residues. Further, the replacement of Tyr7 in GST pi, which is conserved in all cytosolic GSTs, with phenylalanine by site-directed mutagenesis also lowered the activities toward CDNB and ethacrynic acid. The Km values of the mutant for both GSH and CDNB were almost equivalent to those of the wild type, while the Vmax of the former was about 55-fold smaller than that of the latter. Therefore, Tyr7 is considered to be an essential residue for the catalytic activity of GST pi.

Non-essentiality of cysteine and histidine residues for the activity of human class PI glutathione S-transferase

In order to examine the roles of cysteine and histidine residues in the activity of human class Pi glutathione S-transferase (GST pi), site-directed mutagenesis was used to replace each of the four cysteine residues (at positions 14, 47, 101 and 169) with serine and each of the two histidine residues (at positions 71 and 162) with asparagine using a cDNA for the enzyme (Kano, T. et al. (1987) Cancer Res., 47, 5626-5630) and an E. coli expression system. The replacements of Cys101, Cys169, His71 and His162 did not affect the GSH-conjugating activity toward 1-chloro-2,4-dinitrobenzene and ethacrynic acid. On the other hand, the activities were partly decreased by the replacements of Cys47 and Cys14. These results indicated that the cysteine and histidine residues in GST pi are not essential for the catalytic activity, although Cys47 and Cys14 may contribute to some extent to the catalytic efficiency.

Tyrosine-7 in human class Pi glutathione S-transferase is important for lowering the pKa of the thiol group of glutathione in the enzyme-glutathione complex

Previously, we reported the importance of Tyr7 for the catalytic activity of human class Pi glutathione S-transferase [Kong et al. (1992) Biochem. Biophys. Res. Comm., 182, 1122]. As an extension of this study, we investigated the pH dependence of kinetic parameters of the wild-type enzyme and the Y7F mutant. The replacement of Tyr7 with phenylalanine was found to alter the pH dependence of Vmax and Vmax/KmCDNB of the enzyme for conjugation of GSH with 1-chloro-2,4-dinitrobenzene (CDNB). The pKa of the thiol of GSH in the wild-type enzyme-GSH complex was estimated to be about 2.4 pK units lower than that in the Y7F-GSH complex. Tyr7 is thus considered to be important for catalytic activity in lowering the pKa of the thiol of GSH in the enzyme-GSH complex.

The nucleotide and deduced amino acid sequences of porcine liver proline-β-naphthylamidase: swEvidence for the identity with carboxylesterase

A cDNA clone for porcine liver proline‐β‐naphthylamidase was isolated and sequenced. The deduced amino acid sequence of 567 residues was highly homologous with those of carboxylesterases (EC 3.1.1.1) previously reported for other species. In addition, proline‐β‐naphthylamidase purified from porcine liver was shown to have strong activity towards p‐nitrophenylacetate, a representative substrate for carboxylesterases. These results suggest that proline‐β‐naphthylamidase is identical with carboxylesterase.

Metalloproteases with EGF, CUB, and thrombospondin-1 domains function in molting of Caenorhabditis elegans.

Abstract Functional analysis using RNAi was performed on eleven genes for metalloproteases of the M12A family in Caenorhabditis elegans and the interference of the C17G1.6 gene (nas-37) was found to cause incomplete molting. The RNAi of the C26C6.3 gene (nas-36) also caused a similar molting defect but not so severely as that of the nas-37 gene. Both the genes encode an astacinlike metalloprotease with an epidermal growth factor (EGF) like domain, a CUB domain, and a thrombospondin-1 domain, in this order. The promoterdriven green fluorescent protein (GFP) expression analysis suggested that they are expressed in hypodermal cells throughout the larval stages and in the vulva of adult animals. In the genetic background of rde-1(ne219), where RNAi does not work, the molting defect caused by the nas-37 interference was observed when the transgenic wildtype rde-1 gene was expressed under the control of the dpy-7 promoter, known to be active in the hypodermal cells, but not under the control of the myo-3 promoter, active in the muscular cells. Therefore these proteases are thought to be secreted by the hypodermal cells and to participate in shedding of old cuticles.

Proteolytic activity and cleavage specificity of cathepsin E at the physiological pH as examined towards the B chain of oxidized insulin

Proteolytic activity and cleavage specificity of cathepsin E were investigated in a wide range of pHs from 3.0 to 10.5 using the B chain of oxidized insulin as substrate. Contrary to the previous notion that cathepsin E is virtually inactive above pH 6, significant proteolytic activity was observed at pH 7.4 and above. Further, cleavage specificity appeared to change significantly with pH and rather specific cleavage occurred at pH 7.4 and above as compared to pH 5.5 and 3.0. These results suggest that cathepsin E may function in vivo at the physiological pH with a rather restricted specificity.

Reverse genetic analysis of the Caenorhabditis elegans 26S proteasome subunits by RNA interference.

Abstract Reverse genetic analysis was performed on the Caenorhabditis elegans 26S proteasome subunit genes by doublestranded RNAmediated interference (RNAi). Embryonic and postembryonic lethality was caused by interference of all of the eight tested 20S core subunits and all of the 19S regulatory particle subunits except for CeRpn9, CeRpn10, and Ce Rpn12, where RNAi caused no abnormality. However, synthetic suppression of CeRpn10 and CeRpn12 was lethal, whereas neither the combination of Ce Rpn9 with CeRpn10 nor with CeRpn12 resulted in abnormalities in RNAi. These results indicate that the 26S proteasome is indispensable for embryogenesis and postembryonic development, although Ce Rpn9, CeRpn10, and CeRpn12 are not essential, at least under the conditions used. CeRpn10 and Ce Rpn12 are considered to compensate for the suppression of each other.

Purification and Characterization of a Major Collagenase from Streptomyces parvulus

A major collagenase was purified about 96-fold from a crude enzyme sample of Streptomyces parvulus by chromatography on Q-Sepharose, Sephacryl S-200, and butyl-Toyopearl. The purified enzyme showed a relative molecular mass of approximately 52,000 on SDS–PAGE and a pH optimum at about 9.0, and was strongly inhibited by metal-chelating agents. It also cleaved 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg specifically at the Leu-Gly bond, with a K m value of 0.60 mM at pH 9.0 at 37 °C. Based on the amino acid sequences of the N-terminal region and internal tryptic peptides, the corresponding gene was cloned. The DNA sequence of the cloned gene indicated that the enzyme is produced as an 864-residue precursor protein with a 408-residue prepro sequence followed by a 456-residue mature enzyme moiety. The enzyme is most homologous with the collagenase from S. coelicolor, the identity being 73%, and it is thought to be a member of the Vibrio collagenase subfamily.

Identification of a glutamine residue essential for catalytic activity of aspergilloglutamic peptidase by site-directed mutagenesis

Aspergilloglutamic peptidase (AGP, formerly called aspergillopepsin II) from Aspergillus niger var. macrosporus is a unique acid protease recently classified to the peptidase family G1. Our previous study using site‐directed mutagenesis on the glutamic and aspartic acid residues of AGP conserved among the G1 family suggested that Glu219 and Asp123 (numbering in the preproform) are important for catalytic activity. However, the Asn mutant of Asp123 retained weak but significant activity and therefore it was unclear whether it is an active site residue. In this study, we performed site‐directed mutagenesis on all the other hydrophilic residues including Gln, Asn, Ser, Thr, and Tyr, conserved in this family to screen other residues that might be essential for catalytic function, and found that mutations of only Gln133 resulted in almost complete loss of enzymatic activity without change in the native conformation of the enzyme. Meanwhile, the 3D structure of scytalidoglutamic peptidase, a homologue from Scytalidium lignicolum, has been reported, indicating that Glu136 and Gln53 (the counterparts of Glu219 and Gln133 in AGP) form a catalytic dyad. Therefore, the results obtained in this and our previous studies provide with complementary evidence for the definitive conclusion on the catalytic function of the Glu/Gln dyad in glutamic peptidases.