Inhibition Of Protein Biosynthesis Research Articles

Biosynthesis of the benzylpyrrolidine precursor in anisomycin by a unique ThDP-dependent enzyme

Anisomycin (compound 1), a multifunctional pyrrolidine antibiotic, primarily inhibits protein biosynthesis by binding to the ribosome. Upon binding to the ribosome, the para-phenol moiety of anisomycin inserts completely into the hydrophobic crevice of the A-site and blocks the access of the incoming aminoacyl-tRNAs, disrupting peptide bond formation. Hence, the para-methoxyphenyl group serves as a starting point for developing novel anisomycin analogs with potent antifungal and insecticidal properties. However, the activation and condensation mechanism of phenylpyruvic acid has not yet been elucidated. In this study, genetic manipulations of aniP and its homologue siAniP confirmed their indispensable role in 1 biosynthesis. Bioinformatics analysis suggested that AniP and siAniP function as transketolase. siAniP was found to catalyzed condensation between 4-hydroxyphenylpyruvic acid (3) and glyceraldehyde (GA), initiating pyrrolidine synthesis. siAniP was specific for aromatic keto acids and tolerant of aliphatic and aromatic aldehydes, and was able to catalyze the asymmetric intermolecular condensation of two keto acids, leading to the formation of 24 α-hydroxy ketone. To the best of our knowledge, siAniP is the first TK that catalyzes the transfer of a C2 ketol and symmetrical intermolecular coupling using aromatic keto acids as donor substrates. Structural analysis, docking model construction, and site-directed mutagenesis identified that I220, H275, R322 and W391 were crucial for substrate binding. Moreover, sequence similarity network (SSN)-based genome neighborhood network (GNN) analyses of AniP suggested the widespread occurrence of the AniP-like-mediated reaction in the biosynthesis of 1 and its analogs, particularly in the assembly of benzylpyrrolidine. These findings not only expand the repertoire of TKs but also provide a potent biocatalyst that could be used for the structural innovation of 1 and its derivatives.

Citrus sinensis manganese tolerance: Insight from manganese-stimulated secretion of root exudates and rhizosphere alkalization

We used manganese (Mn)-tolerant ‘Xuegan’ (Citrus sinensis) seedlings as materials and examined the characterization of Mn uptake and Mn-activated-release of root exudates under hydroponic conditions. We observed that root and shoot Mn bioaccumulation factor (BCF) reduced with the increase of Mn supply, and that Mn transfer factor (Tf) reduced greatly as Mn supply increased from 0 to 500 μM, beyond which Tf slightly increased with increasing Mn supply, suggesting that Mn supply reduced the ability to absorb and accumulate Mn in roots and shoots, as well as root-to-shoot Mn translocation. Without Mn, roots alkalized the solution pH from 5.0 to above 6.2, while Mn supply reduced root-induced alkalization. As Mn supply increased from 0 to 2000 μM, the secretion of root total phenolics (TPs) increased, while the solution pH decreased. Mn supply did not alter the secretion of root total free amino acids, total soluble sugars, malate, and citrate. Mn-activated-release of TPs was inhibited by low temperature and anion channel inhibitors, but not by protein biosynthesis inhibitor. Using widely targeted metabolome, we detected 48 upregulated [35 upregulated phenolic compounds + 13 other secondary metabolites (SMs)] and three downregulated SMs, and 39 upregulated and eight downregulated primary metabolites (PMs). These findings suggested that reduced ability to absorb and accumulate Mn in roots and shoots and less root-to-shoot Mn translocation in Mn-toxic seedlings, rhizosphere alkalization, and Mn-activated-release of root exudates (especially phenolic compounds) contributed to the high Mn tolerance of C. sinensis seedlings.

Development and Validation of a TaqMan Multiplex Real-Time PCR Assay for High-Throughput Quantification of Fusarium graminearum Biomass in Barley Spikes and Grains

Fusarium head blight (FHB) is a devastating disease primarily caused by Fusarium graminearum that commonly affects North American small grains. Although the disease causes visible symptoms, the more pressing concern is the contamination of grain with trichothecene toxins, especially deoxynivalenol (DON), which can cause toxicity in humans and animals by inhibiting protein biosynthesis. The DON levels are more critical when breeding for resistance, but this does not account for pathogen infection and disease severity. To link DON and Fusarium infection, we optimized and validated a real-time multiplex quantitative PCR protocol for high-throughput quantification of Fusarium biomass based on the Tri5 gene, which encodes trichodiene synthase, the first enzyme involved in trichothecene toxin production. The primers and probes were designed to measure the abundance of Tri5 and normalized to the barley actin gene. The assay is repeatable, robust, and sensitive, up to 0.003 ng/µl of F. graminearum and 0.3 ng/µl of barley DNA. The method is cost-effective and has the potential to quantify Fusarium biomass, monitor disease progression, and determine DON levels in barley spikes and harvested grains. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Cladosporin, A Highly Potent Antimalaria Drug?

Cladosporin, a unique natural product from the fungus Cladosporium cladosporioides, exhibits nanomolar inhibitory activity against Plasmodium falciparum by targeting its cytosolic lysyl-tRNA synthetase (PfKRS) to inhibit protein biosynthesis. Due to its exquisite selectivity towards pathogenic parasites, cladosporin has become a very promising lead compound for developing antiparasitic drugs to treat drug-resistant malaria and cryptosporidiosis infections. Here we review the recent research progress of cladosporin covering aspects of the chemical synthesis, biosynthesis, bioactivity, cellular target and structure-activity relationship.

Biocontrol Potential of Novel Borrelidin-Producing <i>Streptomyces rochei</i> 3IZ-6 Isolated from Izraeli Soil

The soil actinobiota of various climatic zones are a rich source of bioactive natural products, including novel drugs. A complex of soil actinomycetes in the upper horizon of the grumusols (Vertisols) on the western coast of Lake Kinneret in the vicinity of Tiberias (Lower Galilee, Israel) was studied. The screening of the antagonistic activity of 26 isolates using on a dual reporter system revealed the bacterial strain 3IZ-6, which had the ability to inhibit protein synthesis. Strain 3IZ-6 was assigned to Streptomyces rochei by polyphase taxonomy approach. The active substance of S. rochei 3IZ-6 was isolated and purified using solid-phase extraction and HPLC. Toe-print analysis and mass spectrometry data allowed to establish, that active compound is a known inhibitor of protein biosynthesis, borrelidin. S. rochei 3IZ-6 can be used as a producer of borrelidin in biocontrol against phytopathogens and weeds.

Quassinoid analogs exert potent antitumor activity via reversible protein biosynthesis inhibition in human colorectal cancer

Cellular protein synthesis is accelerated in human colorectal cancer (CRC), and high expression of protein synthesis regulators in CRC patients is associated with poor prognosis. Thus, inhibition of protein synthesis may be an effective therapeutic strategy for CRC. We previously demonstrated that the quassinoid bruceantinol (BOL) had antitumor activity against CRC. Herein, potent tumor growth suppression (>80%) and STAT3 inhibition was observed in two different mouse models following BOL administration. Loss of body and spleen weight was observed but was eliminated upon nanoparticle encapsulation while maintaining strong antitumor activity. STAT3 siRNA knockdown exhibited modest suppression of cell proliferation. Surprisingly, STAT3 inhibition using a PROTAC degrader (SD-36) had little effect on cancer cell proliferation suggesting the possibility of additional mechanism(s) of action for quassinoids. BOL-resistant (BR) cell lines, HCT116BR and HCA7BR, were equally sensitive to standard CRC therapeutic agents and known STAT3 inhibitors but resistant to homoharringtonine (HHT), a known protein synthesis inhibitor. The ability of quassinoids to inhibit protein synthesis was dependent on the structure of the C15 sidechain. Of note, BOL did not inhibit protein synthesis in normal human colon epithelial cells whereas HHT and napabucasin remained effective in these normal cells. Novel quassinoids were designed, synthesized, and evaluated in pre-clinical CRC models. Treatment with the most potent analog, 5c, resulted in significant inhibition of cell proliferation and protein synthesis at nanomolar concentrations. These quassinoid analogs may represent a novel class of protein synthesis inhibitors for the treatment of human CRC.

Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction and Autophagy.

Our previous study reported that seminaturally occurring arthrocolins A to C with unprecedented carbon skeletons could restore the antifungal activity of fluconazole against fluconazole-resistant Candida albicans. Here, we showed that arthrocolins synergized with fluconazole, reducing the fluconazole minimum and dramatically augmenting the survivals of 293T human cells and nematode Caenorhabditis elegans infected with fluconazole-resistant C. albicans. Mechanistically, fluconazole can induce fungal membrane permeability to arthrocolins, leading to the intracellular arthrocolins that were critical to the antifungal activity of the combination therapy by inducing abnormal cell membranes and mitochondrial dysfunctions in the fungus. Transcriptomics and reverse transcription-quantitative PCR (qRT-PCR) analysis indicated that the intracellular arthrocolins induced the strongest upregulated genes that were involved in membrane transports while the downregulated genes were responsible for fungal pathogenesis. Moreover, riboflavin metabolism and proteasomes were the most upregulated pathways, which were accompanied by inhibition of protein biosynthesis and increased levels of reactive oxygen species (ROS), lipids, and autophagy. Our results suggested that arthrocolins should be a novel class of synergistic antifungal compounds by inducing mitochondrial dysfunctions in combination with fluconazole and provided a new perspective for the design of new bioactive antifungal compounds with potential pharmacological properties. IMPORTANCE The prevalence of antifungal-resistant Candida albicans, which is a common human fungal pathogen causing life-threatening systemic infections, has become a challenge in the treatment of fungal infections. Arthrocolins are a new type of xanthene obtained from Escherichia coli fed with a key fungal precursor toluquinol. Different from those artificially synthesized xanthenes used as important medications, arthrocolins can synergize with fluconazole against fluconazole-resistant Candida albicans. Fluconazole can induce the fungal permeability of arthrocolins into fungal cells, and then the intracellular arthrocolins exerted detrimental effects on the fungus by inducing fungal mitochondrial dysfunctions, leading to dramatically reduced fungal pathogenicity. Importantly, the combination of arthrocolins and fluconazole are effective against C. albicans in two models, including human cell line 293T and nematode Caenorhabditis elegans. Arthrocolins should be a novel class of antifungal compounds with potential pharmacological properties.

Structural Insights into Pseudomonas aeruginosa Exotoxin A-Elongation Factor 2 Interactions: A Molecular Dynamics Study.

Exotoxin A (ETA) is an extracellular secreted toxin and a single-chain polypeptide with A and B fragments that is produced by Pseudomonas aeruginosa. It catalyzes the ADP-ribosylation of a post-translationally modified histidine (diphthamide) on eukaryotic elongation factor 2 (eEF2), which results in the inactivation of the latter and the inhibition of protein biosynthesis. Studies show that the imidazole ring of diphthamide plays an important role in the ADP-ribosylation catalyzed by the toxin. In this work, we employ different in silico molecular dynamics (MD) simulation approaches to understand the role of diphthamide versus unmodified histidine in eEF2 on the interaction with ETA. Crystal structures of the eEF2-ETA complexes with three different ligands NAD+, ADP-ribose, and βTAD were selected and compared in the diphthamide and histidine containing systems. The study shows that NAD+ bound to ETA remains very stable in comparison with other ligands, enabling the transfer of ADP-ribose to the N3 atom of the diphthamide imidazole ring in eEF2 during ribosylation. We also show that unmodified histidine in eEF2 has a negative impact on ETA binding and is not a suitable target for the attachment of ADP-ribose. Analyzing of radius of gyration and COM distances for NAD+, βTAD, and ADP-ribose complexes revealed that unmodified His affects the structure and destabilizes the complex with all different ligands throughout the MD simulations.

An Undefined Interaction between Polyamines and Heat Shock Proteins Leads to Cellular Protection in Plasmodium falciparum and Proliferating Cells in Various Organisms.

Environmental stimuli can distress the internal reaction of cells and their normal function. To react promptly to sudden environmental changes, a cascade of heat shock proteins (Hsps) functions to protect and act as housekeepers inside the cells. In parallel to the heat shock response, the metabolic polyamine (PA) status changes. Here, we discuss possible ways of putative interactions between Hsps and polyamines in a wide lineage of eukaryotic model organisms with a particular focus on parasitic protozoa such as Plasmodium falciparum (P. falciparum). The supposed interaction between polyamines and Hsps may protect the parasite from the sudden change in temperature during transmission from the female Anopheles mosquito to a human host. Recent experiments performed with the spermidine mimetic inhibitor 15-deoxyspergualine in Plasmodium in vitro cultures show that the drug binds to the C-terminal EEVD motif of Hsp70. This leads to inhibition of protein biosynthesis caused by prevention of eIF5A2 phosphorylation and eukaryotic initiation factor 5A (eIF5A) modification. These observations provide further evidence that PAs are involved in the regulation of protein biosynthesis of Hsps to achieve a protective effect for the parasite during transmission.

Морфофізіологічні реакції мохів на дію абіотичних чинників на посттехногенних територіях видобутку сірки

The morphological structure of turfs and the activity of the main enzymes of antioxidant protection: peroxidase, superoxide dismutase (SOD) and catalase of the dominant species of mosses Barbula unguiculata Hedw,, Bryum caespiticium Hedw., Bryum argenteum Hedw. and Brachythecium salebrosum (Hoffm. eх F.Weber & D. Mohr) Schimp. from different transects of the northern exposure of sulfur mining dump No. 1 in the summer and autumn seasons were studied. It is shown that the morphological structure of the turfs of the mosses Bryum argenteum and Brachythecium salebrosum in the post-technological areas of sulfur mining is important for moisture conservation and depends on the microclimatic conditions of local growth and the life form of the species. It was found that the activity of enzymes depends on the species characteristics of mosses. The dependence of the activity of antioxidant enzymes in the investigated mosses on the intensity of light and temperature on the transects of dump No. 1 was defined. In the summer period, the highest indicators of the activity of antioxidant enzymes were noted in moss samples from the northern top of the dump, where the influence of high temperatures and light intensity, as well as lack of moisture, are particularly noticeable. Under the experimental conditions, in Barbula unguiculata and Bryum caespiticium from the northern top of the dump, under the influence of 24-hour exposure to a temperature of 42 °C, the most significant increase in peroxidase activity by 1.2–1.4 and SOD by 1.6–1.9 times was found. Increasing in peroxidase activity and SOD caused by hyperthermia were reversed by pretreatment with the protein biosynthesis inhibitor cyclohexamide, that may indicate about involvement of the protein synthesizing system in this process. It is likely that the increasing of the activity of antioxidant enzymes is caused primarily by the change in gene expression of stress proteins that control the synthesis of specific adaptogens and protectors, indicating that the extreme conditions of an anthropogenically transformed environment contribute to the development of forms of organisms with the highest potential opportunities.

Bacillus velezensis TSA32-1 as a Promising Agent for Biocontrol of Plant Pathogenic Fungi.

The use of synthetic fungicides has caused major problems such as soil and water pollution and negatively affects non-target species. Microbial biocontrol agents are needed for crop disease management to reduce agrochemical use. Bacillus and related genera produce secondary metabolites with agricultural applications, such as the pathogen-control agent Bacillus velezensis. We isolated B. velezensis TSA32-1 from soil and identified its characteristics by sequencing its 16S rRNA. B. velezensis TSA32-1 showed enzyme activity and antimicrobial effects against phytopathogenic fungi by inhibiting the growth of Fusarium graminearum, F. fujikuroi, Alternatia alternate, and Diaporthe actinidiae. Additionally, B. velezensis TSA32-1 protected diseases in corn and pepper seeds caused by F. graminearum and Pythium ultimum. The complete genome of B. velezensis TSA32-1 was 4.05 Mb with a G+C content of 46.3 mol % and possessed the bacillaene biosynthesis cluster, a polyketide that inhibits protein biosynthesis. We also detected a surfactin synthesis cluster, known as non-ribosomal peptide synthetases, which biosynthesizes the antibacterial substance lipopeptide. Surfactin, and fengycin family compounds, secondary metabolites known as key factors in biological control, also detected B. velezensis TSA32-1 which shows potential as a biocontrol agent for controlling plant pathogens in agriculture.

The protein biosynthesis inhibitor vioprolide A evokes anti-angiogenic and pro-survival actions by targeting NOP14 and decreasing VEGF receptor 2- and TAZ-signaling.

Angiogenesis contributes to the progression of several diseases including cancer or age-related macular degeneration and is crucially driven by pathologically hyperactive endothelial cells (ECs). Targeting angiogenic processes in ECs thus represents a promising strategy to treat these conditions. Vioprolide A (vioA) is a myxobacterial cyclic depsipeptide that targets the nucleolar protein 14 (NOP14) and possesses strong anti-cancer and anti-inflammatory actions. Here, we present evidence that vioA promotes anti-angiogenic actions in vivo and in ECs in vitro. VioA reduced the choroidal neovascularization after laser-induced photocoagulation in mice in vivo, the sprouting of choroidal explant cultures ex vivo and key angiogenic features of ECs in vitro. Mechanistically, vioA decreased VEGFR2 protein levels and phosphorylation leading to impaired downstream pro-angiogenic signaling. Concurrently, vioA influenced TAZ signaling by diminishing its nuclear translocation and protein level, resulting in a reduced expression of pro-angiogenic target genes and dynamic cytoskeletal remodeling. Surprisingly, vioA induced pro-survival signaling in ECs by activating Akt and inhibiting p53-dependent apoptosis. Knockdown of the cellular target NOP14 further revealed a partial involvement in the anti-angiogenic and pro-survival actions of vioA. Taken together, our study introduces vioA as an interesting anti-angiogenic compound that warrants further investigations in preclinical studies.

Limonin stabilises sirtuin 6 (SIRT6) by activating ubiquitin specific peptidase 10 (USP10) in cardiac hypertrophy.

Limonin, a naturally occurring tetracyclic triterpenoid, has extensive pharmacological effects. Its role in cardiac hypertrophy remains to be elucidated. We investigated its effects on cardiac hypertrophy along with the potential mechanisms involved. The effects of limonin on cardiac hypertrophy in C57/BL6 mice caused by aortic banding, plus neonatal rat cardiac myocytes (NRCMs) stimulated with phenylephrine to induce cardiomyocyte hypertrophy in vitro were investigated. Limonin markedly improved the cardiac function and heart weight in aortic banded mice. Limonin-treated mice and NRCMs also produced fewer cardiac hypertrophy markers than those treated with the vehicle in the hypertrophic groups. Sustained aortic banding- or phenylephrine-stimulation impaired cardiac sirtuin 6 (SIRT6) protein levels, which were partially reversed by limonin associated with enhanced activity of PPARα. Sirt6 siRNA inhibited the anti-hypertrophic effects of limonin in vitro. Interestingly, limonin did not influence Sirt6 mRNA levels, but regulated ubiquitin levels. Thus, the protein biosynthesis inhibitor, cycloheximide and proteasome inhibitor, MG-132, were used to determine SIRT6 protein expression levels. Under phenylephrine stimulation, limonin increased SIRT6 protein levels in the presence of cycloheximide, but it did not influence SIRT6 expression in the presence of MG-132, suggesting that limonin promotes SIRT6 levels by inhibiting its ubiquitination degradation. Furthermore, limonin inhibited the degradation of SIRT6 by activating ubiquitin-specific peptidase 10 (USP10), while Usp10 siRNA prevented the beneficial effects of limonin. Limonin mediates the ubiquitination and degradation of SIRT6 by activating USP10, providing an attractive therapeutic target for cardiac hypertrophy.

Ribotoxic Proteins, Known as Inhibitors of Protein Synthesis, from Mushrooms and Other Fungi According to Endo's Fragment Detection.

rRNA N-glycosylases (EC 3.2.2.22) remove a specific adenine (A4324, rat 28S rRNA) in the sarcin ricin loop (SRL) involved into ribosome interaction with elongation factors, causing the inhibition of translation, for which they are known as plant ‘ribosome inactivating proteins’ (RIPs). However, protein synthesis inactivation could be the result of other enzymes, which often have rRNA as the target. In this scenario, Endo’s assay is the most used method to detect the enzymes that are able to hydrolyze a phosphodiester bond or cleave a single N-glycosidic bond (rRNA N-glycosylases). Indeed, the detection of a diagnostic fragment from rRNA after enzymatic action, with or without acid aniline, allows one to discriminate between the N-glycosylases or hydrolases, which release the β-fragment after acid aniline treatment or α-fragment without acid aniline treatment, respectively. This assay is of great importance in the mushroom kingdom, considering the presence of enzymes that are able to hydrolyze phosphodiester bonds (e.g., ribonucleases, ribotoxins and ribotoxin-like proteins) or to remove a specific adenine (rRNA N-glycosylases). Thus, here we used the β-fragment experimentally detected by Endo’s assay as a hallmark to revise the literature available on enzymes from mushrooms and other fungi, whose action consists of protein biosynthesis inhibition.

Targeting the translational machinery in gastrointestinal stromal tumors (GIST): a new therapeutic vulnerability

Although KIT-mutant GISTs can be effectively treated with tyrosine kinase inhibitors (TKIs), many patients develop resistance to imatinib mesylate (IM) as well as the FDA-approved later-line agents sunitinib, regorafenib and ripretinib. Resistance mechanisms mainly involve secondary mutations in the KIT receptor tyrosine kinase gene indicating continued dependency on the KIT signaling pathway. The fact that the type of secondary mutation confers either sensitivity or resistance towards TKIs and the notion that secondary mutations exhibit intra- and intertumoral heterogeneity complicates the optimal choice of treatment in the imatinib-resistant setting. Therefore, new strategies that target KIT independently of its underlying mutations are urgently needed. Homoharringtonine (HHT) is a first-in-class inhibitor of protein biosynthesis and is FDA-approved for the treatment of chronic myeloid leukemia (CML) that is resistant to at least two TKIs. HHT has also shown activity in KIT-mutant mastocytosis models, which are intrinsically resistant to imatinib and most other TKIs. We hypothesized that HHT could be effective in GIST through downregulation of KIT expression and subsequent decrease of KIT activation and downstream signaling. Testing several GIST cell line models, HHT led to a significant reduction in nascent protein synthesis and was highly effective in the nanomolar range in IM-sensitive and IM-resistant GIST cell lines. HHT treatment resulted in a rapid and complete abolishment of KIT expression and activation, while KIT mRNA levels were minimally affected. The response to HHT involved induction of apoptosis as well as cell cycle arrest. The antitumor activity of HHT was confirmed in a GIST xenograft model. Taken together, inhibition of protein biosynthesis is a promising strategy to overcome TKI resistance in GIST.

Effect of Amino Acid Substitutions on 70S Ribosomal Binding, Cellular Uptake, and Antimicrobial Activity of Oncocin Onc112.

Proline‐rich antimicrobial peptides (PrAMPs) are promising candidates for the treatment of infections caused by high‐priority human pathogens. Their mode of action consists of (I) passive diffusion across the outer membrane, (II) active transport through the inner membrane, and (III) inhibition of protein biosynthesis by blocking the exit tunnel of the 70S ribosome. We tested whether in vitro data on ribosomal binding and bacterial uptake could predict the antibacterial activity of PrAMPs against Gram‐negative and Gram‐positive bacteria. Ribosomal binding and bacterial uptake rates were measured for 47 derivatives of PrAMP Onc112 and compared to the minimal inhibitory concentrations (MIC) of each peptide. Ribosomal binding was evaluated for ribosome extracts from four Gram‐negative bacteria. Bacterial uptake was assessed by quantifying each peptide in the supernatants of bacterial cultures. Oncocin analogues with a higher net positive charge appeared to be more active, although their ribosome binding and uptake rates were not necessarily better than for Onc112. The data suggest a complex mode of action influenced by further factors improving or reducing the antibacterial activity, including diffusion through membranes, transport mechanism, secondary targets, off‐target binding, intracellular distribution, and membrane effects. Relying only on in vitro binding and uptake data may not be sufficient for the rational development of more active analogues.

Transcriptomic analysis of formic acid stress response in Saccharomyces cerevisiae.

Formic acid is a representative small molecule acid in lignocellulosic hydrolysate that can inhibit the growth of Saccharomyces cerevisiae cells during alcohol fermentation. However, the mechanism of formic acid cytotoxicity remains largely unknown. In this study, RNA-Seq technology was used to study the response of S. cerevisiae to formic acid stress at the transcriptional level. Scanning electron microscopy and Fourier transform infrared spectroscopy were conducted to observe the surface morphology of yeast cells. A total of 1504 genes were identified as being differentially expressed, with 797 upregulated and 707 downregulated genes. Transcriptomic analysis showed that most genes related to glycolysis, glycogen synthesis, protein degradation, the cell cycle, the MAPK signaling pathway, and redox regulation were significantly induced under formic acid stress and were involved in protein translation and synthesis amino acid synthesis genes were significantly suppressed. Formic acid stress can induce oxidative stress, inhibit protein biosynthesis, cause cells to undergo autophagy, and activate the intracellular metabolic pathways of energy production. The increase of glycogen and the decrease of energy consumption metabolism may be important in the adaptation of S. cerevisiae to formic acid. In addition, formic acid can also induce sexual reproduction and spore formation. This study through transcriptome analysis has preliminarily reveal the molecular response mechanism of S. cerevisiae to formic acid stress and has provided a basis for further research on methods used to improve the tolerance to cell inhibitors in lignocellulose hydrolysate.

Oxidative stress in moss Bryum caespiticium (Bryaceae) under the influence of high temperature and light intensity in a technogenically transformed environment

Mosses are pioneer plants in post-technogenic areas. Therefore, the question of adaptive reactions of mosses from these habitats represents a scientific interest. The research is devoted to the study of adaptive changes in the metabolism of the dominant moss species Bryum caespiticium Hedw., collected in the devastated territories of the Novoyavorivsk State Mining and Chemical Enterprise (SMCE) “Sirka (Sulfur)” exposed to hyperthermia and insolation, which cause oxidative stress in plants. The influence of these stressors on the activity and thermal stability of antioxidant enzymes, hydrogen peroxide content, anion radical generation and accumulation of prooxidant components in moss shoots was studied. The activity and thermal stability of peroxidase and superoxide dismutase (SOD) were analysed forB. caespiticium moss from different locations of northern exposure at the sulfur mining dump No 1 in summer and autumn. We established the dependence of the activity of antioxidant enzymes of moss on the intensity of light and temperature on the experimental plots of the dump No 1. In summer, the highest activity and thermal stability rates of peroxidase and SOD were observed. Under the conditions of the experiment in shoots of В. caespiticium from the northern peak of the dump under the influence of 2 hours temperature action (+ 42 ºС) the most significant increase in peroxidase activity was found by 1.78 times and SOD by 1.89 times, as well as increase in its thermal stability by 1.35–1.42 times, respectively. The increase in peroxidase and SOD activity, as well as the increase in their thermal stability caused by hyperthermia were negated by pre-processing with a protein biosynthesis inhibitor cyclohexamide, which may indicate the participation of the protein-synthesizing system in this process. The effect of increasing the thermal stability of enzymes can be considered as a mechanism of adaptation of the protein-synthesizing system to the action of high temperatures. Increase in the activity and thermal stability of antioxidant enzymes is caused primarily by changes in the expression of stress protein genes, which control the synthesis of specific adaptogens and protectors. The obtained results indicate that the extreme conditions of the anthropogenically transformed environment contribute to the development of forms with the highest potential abilities. The mechanism of action of high temperatures is associated with the development of oxidative stress, which is manifested in the intensification of lipid peroxidation and the generation of superoxide anion radical. It was found that temperature stress and high insolation caused an increased generation of superoxide anion radical as the main inducers of protective reactions in the samples of B. caespiticium from the experimental transect of the sulfur mining heap. It is known that the synthesis of Н2О2 occurs under stress and is a signal to start a number of molecular, biochemical and physiological processes of cells, including adaptation of plants to extreme temperatures. It is shown that high temperatures initiate the generation of hydrogen peroxide. Increased reactive oxygen species (ROS) formation, including Н2О2, under the action of extreme temperatures, can cause the activation of signaling systems. Therefore, the increase in the content of Н2О2 as a signaling mediator is a component of the antioxidant protection system. It is determined that adaptive restructuring of the metabolism of the moss В. caespiticium is associated with the accumulation of signaling prooxidant components (diene and triene conjugates and dienketones). The increase in primary lipid peroxidation products, detected by us, under the action of hyperthermia may indicate the intensification of free radical oxidation under adverse climatic conditions in the area of the sulfur production dump, which leads to the intensification of lipid peroxidation processes. The accumulation of radical and molecular lipid peroxidation products are signals for the activation of protective systems, activators of gene expression and processes that lead to increased resistance of plants.

The type-1 ribosome-inactivating protein OsRIP1 triggers caspase-independent apoptotic-like death in HeLa cells

Ribosome-inactivating proteins (RIPs) are capable of removing a specific adenine from 28S ribosomal RNA, thus inhibiting protein biosynthesis in an irreversible manner. In this study, recombinant OsRIP1, a type 1 RIP from rice (Oryza sativa L.), was investigated for its anti-proliferative properties. Human cervical cancer HeLa cells were incubated in the presence of OsRIP1 for 24–72 h. OsRIP1 treatment yielded an anti-proliferation response of the HeLa cells and resulted in apoptotic-like blebbing of the plasma membrane without causing DNA fragmentation. OsRIP1 labeled with FITC accumulated at the cell surface. Pull-down assays identified ASPP1 (Apoptosis-Stimulating Protein of p53 1) and IFITM3 (interferon-induced transmembrane protein 3) as potential interaction partners for OsRIP1. Transcript levels for several critical genes related to different signaling pathways were quantified by RT-qPCR. OsRIP1 provoked HeLa cells to undergo caspase-independent cell death, associated with a significant transcriptional upregulation of the apoptotic gene PUMA, interferon regulatory factor 1 (IRF1) and the autophagy-related marker LC3. No changes in caspase activities were observed. Together, these data suggest that apoptotic-like events were involved in OsRIP1-driven caspase-independent cell death that might trigger the IRF1 signaling pathway and LC3-mediated autophagy.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.