Improved Cell Selectivity of Pseudin-2 via Substitution in the Leucine-Zipper Motif: In Vitro and In Vivo Antifungal Activity.

Several antimicrobial peptides (AMPs) have been discovered, developed, and purified from natural sources and peptide engineering; however, the clinical applications of these AMPs are limited owing to their lack of abundance and side effects related to cytotoxicity, immunogenicity, and hemolytic activity. Accordingly, to improve cell selectivity for pseudin-2, an AMP from skin, in mammalian cells and pathogenic fungi, the sequence of pseudin-2 was modified by alanine or lysine at each position of two amino acids within the leucine-zipper motif. Alanine-substituted variants were highly selective toward fungi over HaCaT and erythrocytes and maintained their antifungal activities and mode of action (membranolysis).

Anti-Biofilm Effects of Synthetic Antimicrobial Peptides Against Drug-Resistant and Planktonic Cells and Biofilm.

Biofilm-associated infections are difficult to manage or treat as biofilms or biofilm-embedded bacteria are difficult to eradicate. Antimicrobial peptides have gained increasing attention as a possible alternative to conventional drugs to combat drug-resistant microorganisms because they inhibit the growth of planktonic bacteria by disrupting the cytoplasmic membrane. The current study investigated the effects of synthetic peptides (PS1-2, PS1-5, and PS1-6) and conventional antibiotics on the growth, biofilm formation, and biofilm reduction of drug-resistant and .

Functional Characterization of a Rice Thioredoxin Protein OsTrxm and Its Cysteine Mutant Variant with Antifungal Activity.

Although there are many antimicrobial proteins in plants, they are not well-explored. Understanding the mechanism of action of plant antifungal proteins (AFPs) may help combat fungal infections that impact crop yields. In this study, we aimed to address this gap by screening leaves to isolate novel AFPs.

Production of recombinant proteins through sequestration in chloroplasts: a strategy based on nuclear transformation and post-translational protein import.

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts.

Hydrophilic Linear Peptide with Histidine and Lysine Residues as a Key Factor Affecting Antifungal Activity.

Increases in the numbers of immunocompromised patients and the emergence of drug-resistance fungal pathogens have led to the need for new, safe, efficacious antifungal agents. In this study, we designed a histidine-lysine-lysine (HKK) motif and synthesized six HKK peptides with repetitions of the motif. These peptides showed length-dependent antifungal activity against drug-susceptible and drug-resistant fungal pathogens via membranolytic or non-membranolytic action.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria.

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, 'XWZX' basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure.

Proteome profiling of heat, oxidative, and salt stress responses in Thermococcus kodakarensis KOD1.

The thermophilic species, Thermococcus kodakarensis KOD1, a model microorganism for studying hyperthermophiles, has adapted to optimal growth under conditions of high temperature and salinity. However, the environmental conditions for the strain are not always stable, and this strain might face different stresses. In the present study, we compared the proteome response of T.

A 1-Cys Peroxiredoxin from a Thermophilic Archaeon Moonlights as a Molecular Chaperone to Protect Protein and DNA against Stress-Induced Damage.

Peroxiredoxins (Prxs) act against hydrogen peroxide (H2O2), organic peroxides, and peroxynitrite. Thermococcus kodakaraensis KOD1, an anaerobic archaeon, contains many antioxidant proteins, including three Prxs (Tk0537, Tk0815, and Tk1055). Only Tk0537 has been found to be induced in response to heat, osmotic, and oxidative stress.

Chaperone-Like Activity of a Bacterioferritin Comigratory Protein from Thermococcus kodakaraensis KOD1.

Peroxiredoxins (Prxs) are ubiquitous and conserved proteins that can catalyze the reduction of inorganic and organic hydroperoxides to protect against damage by reactive oxygen species. In this study, a Prx subfamily member, and specifically a bacterioferritin comigratory protein from hyperthermophilic Thermococcus kodakaraensis KOD1 (TkBcp), was overexpressed, purified and characterized. Based on the conserved cysteine (Cys) residues in its amino acids sequence, TkBcp can be grouped into 1-Cys Prx family.

Architecture and characterization of a thermostable MoxR family AAA(+) ATPase from Thermococcus kodakarensis KOD1.

AAA(+) ATPases are ubiquitous enzymes that can function as molecular chaperones, employing the energy obtained from ATP hydrolysis to remodel macromolecules. In this report, the MoxR enzyme from Thermococcus kodakarensis KOD1 (TkMoxR) was shown to have two native forms: a two-stack hexameric ring and a hexameric structure, under physiological conditions and cold stress, respectively. TkMoxR was altered to a microtubule-like form in the presence of ATP and tightly interacted with dsDNA molecules of various lengths.

Multifunctional enzymes in archaea: promiscuity and moonlight.

Enzymes from many archaea colonizing extreme environments are of great interest because of their potential for various biotechnological processes and scientific value of evolution. Many enzymes from archaea have been reported to catalyze promiscuous reactions or moonlight in different functions. Here, we summarize known archaeal enzymes of both groups that include different kinds of proteins.

High level expression and characterization of a thermostable lysophospholipase from Thermococcus kodakarensis KOD1.

Phospholipases can catalyze the hydrolysis of one or more ester and phosphodiester bonds and have a considerable interest in the food, oil leather and pharmaceutical industries. In this report, a lysophospholipase gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (LysoPL-tk) was cloned. The gene of 783 bp encodes a 260-amino acid protein with a molecular mass of 29 kDa.

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1.

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and L: -proline dehydrogenase activity.

Biochemical characterization of deblocking aminopeptidases from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1.

Deblocking aminopeptidase (DAP) is an exoprotease that can release N-terminal amino acids from blocked peptides. Three DAP homologous (TkDAP1, TkDAP2, and TkDAP3) are annotated in the genome data base of Thermococcus kodakarensis KOD1. TkDAP2 and TkDAP3 were identified as proteins that are overexpressed in response to heat and oxidative stress by two-dimensional electrophoresis.

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from Thermococcus kodakarensis KOD1.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of D-glyceraldehyde 3-phosphate (D-G3P) to 1,3-diphosphoglycerate using NAD(+) as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa.

Oxidized NADH oxidase inhibits activity of an ATP/NAD kinase from a Thermophilic archaeon.

NADH oxidases (NOXs) are important enzymes in detoxifying oxidative stress and regenerating oxidized pyridine nucleotides. In the present study, a NOX from Thermococcus kodakarensis KOD1 (NOXtk) was recombinantly expressed in Escherichia coli and purified to homogeneity. NOXtk displayed NADH oxidase activity that was inhibited by oxidization.

An archaeal NADH oxidase causes damage to both proteins and nucleic acids under oxidative stress.

NADH oxidases (NOXs) catalyze the two-electron reduction of oxygen to H2O2 or four-electron reduction of oxygen to H2O. In this report, we show that an NADH oxidase from Thermococcus profundus (NOXtp) displays two forms: a native dimeric protein under physiological conditions and an oxidized hexameric form under oxidative stress. Native NOXtp displays high NADH oxidase activity, and oxidized NOXtp can accelerate the aggregation of partially unfolded proteins.

In vivo effects of NbSiR silencing on chloroplast development in Nicotiana benthamiana.

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation.

Heat-shock dependent oligomeric status alters the function of a plant-specific thioredoxin-like protein, AtTDX.

We found that Arabidopsis AtTDX, a heat-stable and plant-specific thioredoxin (Trx)-like protein, exhibits multiple functions, acting as a disulfide reductase, foldase chaperone, and holdase chaperone. The activity of AtTDX, which contains 3 tetratricopeptide repeat (TPR) domains and a Trx motif, depends on its oligomeric status. The disulfide reductase and foldase chaperone functions predominate when AtTDX occurs in the low molecular weight (LMW) form, whereas the holdase chaperone function predominates in the high molecular weight (HMW) complexes.

Hexameric ring structure of a thermophilic archaeon NADH oxidase that produces predominantly H2O.

An NADH oxidase (NOX) was cloned from the genome of Thermococcus profundus (NOXtp) by genome walking, and the encoded protein was purified to homogeneity after expression in Escherichia coli. Subsequent analyses showed that it is an FAD-containing protein with a subunit molecular mass of 49 kDa that exists as a hexamer with a native molecular mass of 300 kDa. A ring-shaped hexameric form was revealed by electron microscopic and image processing analyses.

Molecular modeling study of CodX reveals importance of N-terminal and C-terminal domain in the CodWX complex structure of Bacillus subtilis.

In Bacillus subtilis, CodW peptidase and CodX ATPase function together as a distinctive ATP-dependent protease called CodWX, which participates in protein degradation and regulates cell division. The molecular structure of CodX and the assembly structure of CodW-CodX have not yet been resolved. Here we present the first three-dimensional structure of CodX N-terminal (N) and C-terminal (C) domain including possible structure of intermediate (I) domain based on the crystal structure of homologous Escherichia coli HslU ATPase.

Structural and functional characterization of osmotically inducible protein C (OsmC) from Thermococcus kodakaraensis KOD1.

Osmotically inducible protein C (OsmC) is involved in the cellular defense mechanism against oxidative stress caused by exposure to hyperoxides or elevated osmolarity. OsmC was identified by two-dimensional electrophoresis (2DE) analysis as a protein that is overexpressed in response to osmotic stress, but not under heat and oxidative stress. Here, an OsmC gene from T.

Nucleotide triphosphates inhibit the degradation of unfolded proteins by HslV peptidase.

Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms.

Molecular and functional characterization of a cyclophilin with antifungal activity from Chinese cabbage.

An antifungal protein that inhibits the growth of filamentous fungal pathogens was isolated from Chinese cabbage (Brassica campestris L. ssp. pekinensis) by affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose.

Chemical composition and antimicrobial activity of Chamaecyparis obtusa leaf essential oil.

The essential oil obtained from the leaves of Chamaecyparis obtusa was analyzed by GC and GC-MS. alpha-Terpinyl acetate, sabinene, isobornyl acetate and limonene were found to be the major components. The oil showed relatively strong antibacterial activities against Gram (+) bacteria and some fungi.