Physical and chemical properties of nabak (Zizyphus spina-christi) seed kernel and sweet pepper (Capsicum annuum L.) seed oils.

BACKGROUND: Nabak seed kernels and sweet pepper seeds, which are separated from the fruits and discarded as waste after processing or consumption, contain high levels of oils (30.19% and 19.57%, respectively). The chemical and thermal characteristics of nabak seed kernel oil (NSO) and sweet pepper seed oil (PSO) were investigated in this study. RESULTS: The NSO and PSO contained high levels of unsaturated fatty acids (84.1% and 86.5%, respectively), and the major fatty acid was oleic acid (57.3%) in NSO, but it was linoleic acid (69.4%) in PSO. The triacylglycerol (TAG) profiles show that NSO contained ten TAG species, three of which represented 87.1%, namely C54:3, C52:2 and C54:4, and triolein was the dominant (OOO, 47.0%). Pepper seed oil contained nine TAG molecular species, four of which represented 93.6%, namely C54:6, C52:4, C54:4 and C52:5, and trilinolein was dominant (LLL, 44.0%). The differential scanning calorimetry (DSC) analysis of NSO revealed that three exothermal peaks were detected during cooling, two endothermal peaks were detected during melting, and the major peak occurred at a low temperature. For PSO, three exothermal peaks were detected during cooling, three peaks were detected (one of them was exothermal) during melting, and the major peaks were observed at low temperatures. Fourier transform infrared (FTIR) spectra indicated that NSO and PSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids. CONCLUSION: This study offers a scientific basis for the use of NSO and PSO as new sources of edible oils for food applications. © 2021 Society of Chemical Industry.

Improved preparation of group-specific component (Gc) protein to derive macrophage activating factor.

Cancer immunotherapy has recently attracted attention as an approach for cancer treatment through the activation of the immune system. Group-specific component (Gc) protein is a precursor for macrophage activating factor (GcMAF), which has a promising immunomodulatory effect on the suppression of tumor growth and angiogenesis. In this study, we successfully purified Gc protein from human serum using anion-exchange chromatography combined with affinity chromatography using a 25-OH-D3-immobilized column. The purity of Gc protein reached 95.0% after anion-exchange chromatography. The known allelic variants of Gc protein are classified into three subtypes-Gc1F, Gc1S and Gc2. The fragment sequence of residues 412-424 determined according to their MS/MS spectra is available to evaluate the subtypes of Gc protein. The data showed that the Gc protein purified in this study consisted of the Gc1F and Gc2 subtypes. Our method improved the purity of Gc protein, which was not affected by the treatment to convert it into GcMAF using ß-galactosidase- or neuraminidase-immobilized resin, and will be useful for biological studies and/or advanced clinical uses of GcMAF, such as cancer immunotherapy.

SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity.

The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4-P1 and P1'. In this study, we determined the crystal structure of 3CLpro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3' position [Phe(P3')] is snugly accommodated in the S3' pocket. Mutations of Phe(P3') impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3' subsite is formed by Phe(P2)-induced conformational changes of 3CLpro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3' subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3'). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4-P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3') is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).

A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis.

The Lon AAA+ protease degrades damaged or misfolded proteins in its intramolecular chamber. Its activity must be precisely controlled, but the mechanism by which Lon is regulated in response to different environments is not known. Facultative anaerobes in the Enterobacteriaceae family, mostly symbionts and pathogens, encounter both anaerobic and aerobic environments inside and outside the host's body, respectively. The bacteria characteristically have two cysteine residues on the Lon protease (P) domain surface that unusually form a disulfide bond. Here we show that the cysteine residues act as a redox switch of Lon. Upon disulfide bond reduction, the exit pore of the P-domain ring narrows by ∼30%, thus interrupting product passage and decreasing activity by 80%; disulfide bonding by oxidation restores the pore size and activity. The redox switch (E°' = -227 mV) is appropriately tuned to respond to variation between anaerobic and aerobic conditions, thus optimizing the cellular proteolysis level for each environment.

Crystallization and melting properties studied by DSC and FTIR spectroscopy of goldenberry (Physalis peruviana) oil.

The chemical and thermal characteristics of goldenberry pomace oil (GPO) and goldenberry seed oil (GSO) were investigated. GPO and GSO contained high levels of unsaturated fatty acids (90.1% and 85.1%, respectively), and the major fatty acid was linoleic (62.0% and 72.8%, respectively). Additionally, GPO contained eleven triacylglycerol (TAG) species, three of which represented 82.7%, namely C54:6, C54:4 and C52:4, and trilinolein was the dominant one (35.5%). GSO contained nine TAG species, two of which represented 80.3%, namely C54:6 and C52:4, and trilinolein was dominant (53.3%). The DSC analysis of GPO and GSO revealed that three exothermal peaks were detected during cooling. Three endothermal peaks (one of which is exothermal for GSO) were detected during melting, and the most significant peaks occurred at low temperatures. FTIR spectra indicated that GPO and GSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids.

Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase.

Cyanophycin is a natural biopolymer consisting of equimolar amounts of aspartate and arginine as the backbone and branched sidechain, respectively. It is produced by a single enzyme, cyanophycin synthetase (CphA1), and accumulates as a nitrogen reservoir during N2 fixation by most cyanobacteria. A recent structural study showed that three constituent domains of CphA1 function as two distinct catalytic sites and an oligomerization interface in cyanophycin synthesis. However, it remains unclear how the ATP-dependent addition of aspartate to cyanophycin is initiated at the catalytic site of the glutathione synthetase-like domain. Here, we report the cryogenic electron microscopy structures of CphA1, including a complex with aspartate, cyanophycin primer peptide, and ATP analog. These structures reveal the aspartate binding mode and phosphate-binding loop movement to the active site required for the reaction. Furthermore, structural and mutational data show a potential role of protein dynamics in the catalytic efficiency of the arginine condensation reaction.

A novel method of literature mining to identify candidate COVID-19 drugs.

Summary: COVID-19 is a serious infectious disease that has recently emerged and continues to spread worldwide. Its spreading rate is too high to expect that new specific drugs will be developed in sufficient time. As an alternative, drugs already developed for other diseases have been tested for use in the treatment of COVID-19 (drug repositioning). However, to select candidate drugs from a large number of compounds, numerous inhibition assays involving viral infection of cultured cells are required. For efficiency, it would be useful to narrow the list of candidates down using logical considerations prior to performing these assays. We have developed a powerful tool to predict candidate drugs for the treatment of COVID-19 and other diseases. This tool is based on the concatenation of events/substances, each of which is linked to a KEGG (Kyoto Encyclopedia of Genes and Genomes) code based on a relationship obtained from text mining of the vast literature in the PubMed database. By analyzing 21 589 326 records with abstracts from PubMed, 98 556 KEGG codes with NAME/DEFINITION fields were connected. Among them, 9799 KEGG drug codes were connected to COVID-19, of which 7492 codes had no direct connection to COVID-19. Although this report focuses on COVID-19, the program developed here can be applied to other infectious diseases and used to quickly identify drug candidates when new infectious diseases appear in the future. Availability and implementation: The programs and data underlying this article will be shared on reasonable request to the corresponding authors. Contact: atmuramatsu@g.ecc.u-tokyo.ac.jp, amtanok@mail.ecc.u-tokyo.ac.jp. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

NMR-based metabolic profiling and comparison of Japanese persimmon cultivars.

Persimmons are a traditional, autumnal, and healthy fruit commonly consumed in Japan and East Asia based on the saying, "a persimmon a day keeps the doctor away." The differences in metabolites among five major Japanese persimmon cultivars were investigated using a nuclear magnetic resonance (NMR)-based metabolomics approach. By using a broadband water suppression enhanced through T1 effects (WET) method for the sensitive detection of minor metabolites, better discrimination among cultivars and more informative details regarding their metabolic differences have been achieved compared to those achieved in conventional 1H NMR sequences. Among the nonastringent cultivars analyzed, the Taishu cultivar has the highest abundance of amino acids. The Matsumotowase-Fuyu cultivar contains ethyl-ß-glycosides as characteristic components, which may relate to fruit softening. Citric acid concentration is higher in Maekawa Jiro than in other nonastringent cultivars. Among the two astringent cultivars analyzed, ethanol was significantly higher in Hiratanenashi than in Yotsumizo, which indicates different reactivity during deastringency treatments. The present study proposes an efficient and relatively quantitative metabolomics approach based on broadband WET NMR spectra.

The effects of mutations in the carboxyl-terminal region on the catalytic activity of Escherichia coli signal peptidase I.

Escherichia coli signal peptidase I (SPase I) is a membrane-bound serine endopeptidase that catalyses the cleavage of signal peptides from the pre-forms of membrane or secretory proteins. Our previous studies using chemical modification and site-directed mutagenesis suggested that Trp(300) and Arg(77), Arg(222), Arg(315) and Arg(318) are important for the proper and stable conformation of the active site of SPase I. Interestingly, many of these residues reside in the C-terminal region of the enzyme. As a continuation of these studies, we investigated in the present study the effects of mutations in the C-terminal region including amino acid residues at positions from 319 to 323 by deletions and site-directed mutagenesis. As a result, the deletion of the C-terminal His(323) was shown to scarcely affect the enzyme activity of SPase I, whereas the deletion of Gly(321)-His(323) or Ile(319)-His(323) as well as the point mutation of Ile(322) to alanine was shown to decrease significantly both the activity in vitro and in vivo without a big gross conformational change in the enzyme. These results suggest a significant contribution of Ile(322) to the construction and maintenance of the proper and critical local conformation backing up the active site of SPase I.

The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli.

Leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is an N-end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl-tRNAs to exposed N-terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 A resolution crystal structure of L/F-transferase (JW0868) from Escherichia coli, the first three-dimensional structure of an L/F-transferase. The L/F-transferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N-terminal domain forms a small lobe with a novel fold. The large C-terminal domain has a highly conserved fold, which is observed in the GCN5-related N-acetyltransferase (GNAT) family. Most of the conserved residues of L/F-transferase reside in the central cavity, which exists at the interface between the N-terminal and C-terminal domains. A comparison of the structures of L/F-transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C-terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F-transferase suggest that this region is important for the enzyme catalysis.

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.

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 (M(pro)), which is also called the 3C-like protease (3CL(pro)). 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.

Characterization of genomic sequence coding for bromelain inhibitors in pineapple and expression of its recombinant isoform.

Bromelain inhibitor (BI) is a cysteine proteinase inhibitor isolated from pineapple stem (Reddy, M. N., Keim, P. S., Heinrikson, R. L., and Kézdy, F. J. (1975) J. Biol. Chem. 250, 1741-1750). It consists of eight isoinhibitors, and each isoinhibitor has a two-chain structure. In this study, the genomic DNA has been cloned and found to encode a precursor protein with 246 amino acids (M(r) = approximately 27,500) containing three isoinhibitor domains (BI-III, -VI, and -VII) that are 93% identical to one another in amino acid sequences. The gene structure indicated that these isoinhibitors are produced by removal of the N-terminal pre-peptide (19 residues), 3 interchain peptides (each 5 residues), 2 interdomain peptides (each 19 residues), and the C-terminal pro-peptide (18 residues). Moreover, all the amino acid sequences of bromelain isoinhibitors could be explained by removal of one or two amino acids from BI-III, -VI, and -VII with exopeptidases. A recombinant single-chain BI-VI with and without the interchain peptide showed the same and no bromelain inhibitory activity as compared with the native BI-VI, respectively. These results indicate that the interchain peptide plays an important role of the folding process of the mature isoinhibitors.

The unique sites in SulA protein preferentially cleaved by ATP-dependent Lon protease from Escherichia coli.

SulA protein is known to be one of the physiological substrates of Lon protease, an ATP-dependent protease from Escherichia coli. In this study, we investigated the cleavage specificity of Lon protease toward SulA protein. The enzyme was shown to cleave approximately 27 peptide bonds in the presence of ATP. Among them, six peptide bonds were cleaved preferentially in the early stage of digestion, which represented an apparently unique cleavage sites with mainly Leu and Ser residues at the P1, and P1' positions, respectively, and one or two Gln residues in positions P2-P5. They were located in the central region and partly in the C-terminal region, both of which are known to be important for the function of SulA, such as inhibition of cell growth and interaction with Lon protease, respectively. The other cleavage sites did not represent such consensus sequences, though hydrophobic or noncharged residues appeared to be relatively preferred at the P1 sites. On the other hand, the cleavage in the absence of ATP was very much slower, especially in the central region, than in the presence of ATP. The central region was predicted to be rich in alpha helix and beta sheet structures, suggesting that the enzyme required ATP for disrupting such structures prior to cleavage. Taken together, SulA is thought to contain such unique cleavage sites in its functionally and structurally important regions whose preferential cleavage accelerates the ATP-dependent degradation of the protein by Lon protease.

Identification of arginine residues important for the activity of Escherichia coli signal peptidase I.

Escherichia coil signal peptidase I (leader peptidase, SPase I) is an integral membrane serine protease that catalyzes the cleavage of signal (leader) peptides from pre-forms of membrane or secretory proteins. We previously demonstrated that E. coil SPase I was significantly inactivated by reaction with phenylglyoxal with concomitant modification of three to four of the total 17 arginine residues in the enzyme. This result indicated that several arginine residues are important for the optimal activity of the enzyme. In the present study, we have constructed 17 mutants of the enzyme by site-directed mutagenesis to investigate the role of individual arginine residues in the enzyme. Mutation of Arg127, Arg146, Arg198, Arg199, Arg226, Arg236, Arg275, Arg282, and Arg295 scarcely affected the enzyme activity in vivo and in vitro. However, the enzymatic activity toward a synthetic substrate was significantly decreased by replacements of Arg77, Arg222, Arg315, or Arg318 with alanine/lysine. The kcat values of the R77A, R77K, R222A, R222K, R315A, R318A, and R318K mutant enzymes were about 5.5-fold smaller than that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type. Moreover, the complementing abilities in E. Arg222, Arg315, coil IT41 were lost completely when Arg77, or Arg318 was replaced with alanine/lysine. The circular dichroism spectra and other enzymatic properties of these mutants were comparable to those of the wild-type enzyme, indicating no global conformational changes. However, the thermostability of R222A, R222K, R315A, and R318K was significantly lower compared to the wild type. Therefore, Arg77, Arg222, Arg315, and Arg318 are thought to be important for maintaining the proper and stable conformation of SPase I.