Wataru Nishii

Cleavage mechanism of ATP-dependent Lon protease toward ribosomal S2 protein

The Escherichia coli ATP‐dependent protease Lon degrades ribosomal S2 protein in the presence of inorganic polyphosphate (polyP). In this study, the process of the degradation was investigated in detail. During the degradation, 68 peptides with various sizes (4–29 residues) were produced in a processive fashion. Cleavage occurred at 45 sites, whose P1 and P3 positions were dominantly occupied by hydrophobic residues. These cleavage sites were located preferentially at the regions with rigid secondary structures and the P1 residues of the major cleavage sites appeared to be concealed from the surface of the substrate molecule. Furthermore, polyP changed not only the substrate preference but also the oligomeric structure of the enzyme.

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.

Structural and enzymatic characterization of physarolisin (formerly physaropepsin) proves that it is a unique serine-carboxyl proteinase.

Previously, we purified and partially characterized physarolisin, a lysosomal acid proteinase from Physarum polycephalum, which had been suggested to be concerned with the morphological changes of the mold. In this study, a cDNA for the enzyme was cloned and sequenced, and the structural and enzymatic features were investigated. The enzyme shows a sequence similarity to the serine-carboxyl proteinase family (MEROPS S53). Indeed, diisopropylfluorophosphate (DFP) was shown to strongly inhibit the activity of the enzyme. However, the enzyme possesses several unique features distinct from the other members of the family, such as the two-chain structure and inhibition by diazoacetyl-D,L-norleucine methyl ester (DAN). The sites and mode of processing of the precursor to the mature enzyme were deduced, and the major DAN-reactive residue in the enzyme was identified to be Asp529. These features were suggested to be due to the unique local tertiary structure of the enzyme by molecular modeling. We now propose the name physarolisin for the enzyme.

Determination of the cleavage sites in SulA, a cell division inhibitor, by the ATP-dependent HslVU protease from Escherichia coli

HslVU is an ATP‐dependent protease from Escherichia coli and known to degrade SulA, a cell division inhibitor, both in vivo and in vitro, like the ATP‐dependent protease Lon. In this study, the cleavage specificity of HslVU toward SulA was investigated. The enzyme was shown to produce 58 peptides with various sizes (3–31 residues), not following the ‘molecular ruler’ model. Cleavage occurred at 39 peptide bonds preferentially after Leu in an ATP‐dependent manner and in a processive fashion. Interestingly, the central and C‐terminal regions of SulA, which are known to be important for the function of SulA, such as inhibition of cell division and molecular interaction with certain other proteins, were shown to be preferentially cleaved by HslVU, as well as by Lon, despite the fact that the peptide bond specificities of the two enzymes were distinct from each other.

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.

Specific Inhibition and Stabilization of Aspergilloglutamic Peptidase by the Propeptide IDENTIFICATION OF CRITICAL SEQUENCES AND RESIDUES IN THE PROPEPTIDE

Aspergilloglutamic peptidase (formerly called aspergillopepsin II) is an acid endopeptidase produced by Aspergillus niger var. macrosporus, with a novel catalytic dyad of a glutamic acid and a glutamine residue, thus belonging to a novel peptidase family G1. The mature enzyme is generated from its precursor by removal of the putative 41-residue propeptide and an 11-residue intervening peptide through autocatalytic activation. In the present study, the propeptide (Ala1–Asn41) and a series of its truncated peptides were chemically synthesized, and their effects on the enzyme activity and thermal stability were examined to identify the sequences and residues in the propeptide most critical to the inhibition and thermal stabilization. The synthetic propeptide was shown to be a potent competitive inhibitor of the enzyme (Ki = 27 nm at pH 4.0). Various shorter propeptide fragments derived from the central region of the propeptide had significant inhibitory effect, whereas their Ala scan-substituted peptides, especially R19A and H20A, showed only weak inhibition. Substitution of the Pro23-Pro24 sequence near His20 with an Ala-Ala sequence changed the peptide Lys18–Tyr25 to a substrate with His20 as the P1 residue. Furthermore, the propeptide was shown to be able to significantly protect the enzyme from thermal denaturation (ΔTm = ∼19 °C at pH 5.6). The protective potencies of the propeptide as well as truncated propeptides and their Ala scan-substituted peptides are parallel with their inhibitory potencies. These results indicate that the central part, and especially Arg19 and His20 therein, of the propeptide is most critical to the inhibition and thermal stabilization and that His20 interacts with the enzyme at or near the S1 site in a nonproductive fashion.

Substrate Specificities of Porcine and Bovine Enteropeptidases toward the Peptide Val-(Asp)4-Lys-Ile-Val-Gly and Its Analogs

The substrate specificities of porcine and bovine enteropeptidases were investigated using the peptide Val-(Asp)4-Lys-Ile-Val-Gly and its various analogs with mutations in the (Asp)4-Lys sequence as substrates. The results indicated that in addition to P1 Lys, P2 Asp in the substrates is most important, that P3 Asp is additionally important, and that P5 Asp contributes somewhat to the susceptibility, and that P4 Asp is the least important. These results were essentially identical as between porcine and bovine enteropeptidases.

In situ visualization of caspase‐1‐like activity associated with promotion of hippocampal cell death

To clarify the function of caspase‐1‐like proteases in neuronal cell death, it is important to be able to detect the activity in living organs by microscopic visualization. In the present study, we synthesized a novel fluorescent substrate sensitive to the caspase‐1‐like activity, which is easily introduced into cells constituting living organs by extracellular application. As a result, the substrate was shown to be useful in imaging the caspase‐1‐like activity in rat hippocampal slice cultures. After induction of cell death with glutamate, a significant increase in the activity was observed, especially in the pyramidal cells, suggesting the association of the activity with promotion of cell death.

Autoprocessing mechanism of severe acute respiratory syndrome coronavirus 3C‐like protease (SARS‐CoV 3CLpro) from its polyproteins

Like many other RNA viruses, severe acute respiratory syndrome coronavirus (SARS‐CoV) produces polyproteins containing several non‐structural proteins, which are then processed by the viral proteases. These proteases often exist within the polyproteins, and are excised by their own proteolytic activity (‘autoprocessing’). It is important to investigate the autoprocessing mechanism of these proteases from the point of view of anti‐SARS‐CoV drug design. In this paper, we describe a new method for investigating the autoprocessing mechanism of the main protease (Mpro), which is also called the 3C‐like protease (3CLpro). Using our method, we measured the activities, under the same conditions, of the mature form and pro‐forms with the N‐terminal pro‐sequence, the C‐terminal pro‐sequence or both pro‐sequences, toward the pro‐form with both N‐ and C‐terminal pro‐sequences. The data indicate that the pro‐forms of the enzyme have proteolytic activity, and are stimulated by the same proteolytic activity. The stimulation occurs in two steps, with approximately eightfold stimulation by N‐terminal cleavage, approximately fourfold stimulation by C‐terminal cleavage, and 23‐fold stimulation by the cleavage of both termini, compared to the pro‐form with both the N‐ and C‐terminal pro‐sequences. Such cleavage mainly occurs in a trans manner; i.e. the pro‐form dimer cleaves the monomeric form. The stimulation by N‐terminal pro‐sequence removal is due to the cis (intra‐dimer and inter‐protomer) effect of formation of the new N‐terminus, whereas that by C‐terminal cleavage is due to removal of its trans (inter‐dimer) inhibitory effect. A numerical simulation of the maturation pathway is presented.

The Physarum polycephalum php gene encodes a unique cold-adapted serine-carboxyl peptidase, physarolisin II

The php gene from a true slime mold, Physarum polycephalum, is a late‐replicating and transcriptionally active gene. The deduced amino acid sequence of the gene product is homologous to those of the serine‐carboxyl peptidase family, including physarolisin I from the same organism, but lacks the propeptide region. In this study, the protein was expressed in Escherichia coli and shown to possess endopeptidase activity with unique substrate specificity. Thus, we named it physarolisin II. The enzyme was revealed to be a kind of cold‐adapted enzyme since it was maximally active at 16–22°C. The active enzyme was markedly unstable due to rapid autolysis (t 1/2=∼5 min, at 18°C). At higher temperature, the enzyme was less active but more stable, despite the fact that no gross conformational change was observed by circular dichroism spectroscopy.