Differential effects of angiotensin II and aldosterone on human neutrophil adhesion and concomitant secretion of proteins, free amino acids and reactive oxygen and nitrogen species.

Invasion and adhesion of neutrophils into tissues and their concomitant secretion play an important role in the development of vascular pathologies, including abdominal aortic aneurysm (AAA). Chronic administration of angiotensin II is used to initiate AAA formation in mice. The role of aldosterone in this process is being studied. We conducted for the first time a complex comparative study of the effects of angiotensin II and aldosterone on the adhesion of human neutrophils to fibronectin and the concomitant secretion of proteins, free amino acids as well as reactive oxygen (ROS) and nitrogen (NO) species. Neither angiotensin II nor aldosterone affected the attachment of neutrophils to fibronectin and the concomitant production of ROS. We showed for the first time that aldosterone stimulated the release of amino acid hydroxylysine, a product of lysyl hydroxylase, the activity of which is positively correlated with cell invasiveness. Aldosterone also initiates the secretion of matrix metalloproteinase 9 (MMP-9) and cathepsin G, which may reorganize the extracellular matrix and stimulate the recruitment and adhesion of neutrophils to the aortic walls. Angiotensin II did not affect protein secretion. It may contribute to neutrophil-induced vascular injury by inhibiting the production of NO or by increasing the secretion of isoleucine. Our results suggest that it is aldosterone-induced neutrophil secretion that may play a significant role in neutrophil-induced vascular wall destruction in angiotensin II-induced AAA or other vascular complications.

Acrylate Reductase of an Anaerobic Electron Transport Chain of the Marine Bacterium Shewanella woodyi.

Many microorganisms are capable of anaerobic respiration in the absence of oxygen, by using different organic compounds as terminal acceptors in electron transport chain. We identify here an anaerobic respiratory chain protein responsible for acrylate reduction in the marine bacterium Shewanella woodyi. When the periplasmic proteins of S. woodyi were separated by ion exchange chromatography, acrylate reductase activity copurified with an ArdA protein (Swoo_0275). Heterologous expression of S. woodyi ardA gene (swoo_0275) in Shewanella oneidensis MR-1 cells did not result in the appearance in them of periplasmic acrylate reductase activity, but such activity was detected when the ardA gene was co-expressed with an ardB gene (swoo_0276). Together, these genes encode flavocytochrome c ArdAB, which is thus responsible for acrylate reduction in S. woodyi cells. ArdAB was highly specific for acrylate as substrate and reduced only methacrylate (at a 22-fold lower rate) among a series of other tested 2-enoates. In line with these findings, acrylate and methacrylate induced ardA gene expression in S. woodyi under anaerobic conditions, which was accompanied by the appearance of periplasmic acrylate reductase activity. ArdAB-linked acrylate reduction supports dimethylsulfoniopropionate-dependent anaerobic respiration in S. woodyi and, possibly, other marine bacteria.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

Phytaspase Is Capable of Detaching the Endoplasmic Reticulum Retrieval Signal from Tobacco Calreticulin-3.

Soluble chaperones residing in the endoplasmic reticulum (ER) play vitally important roles in folding and quality control of newly synthesized proteins that transiently pass through the ER en route to their final destinations. These soluble residents of the ER are themselves endowed with an ER retrieval signal that enables the cell to bring the escaped residents back from the Golgi. Here, by using purified proteins, we showed that Nicotiana tabacum phytaspase, a plant aspartate-specific protease, introduces two breaks at the C-terminus of the N. tabacum ER resident calreticulin-3. These cleavages resulted in removal of either a dipeptide or a hexapeptide from the C-terminus of calreticulin-3 encompassing part or all of the ER retrieval signal. Consistently, expression of the calreticulin-3 derivative mimicking the phytaspase cleavage product in Nicotiana benthamiana cells demonstrated loss of the ER accumulation of the protein. Notably, upon its escape from the ER, calreticulin-3 was further processed by an unknown protease(s) to generate the free N-terminal (N) domain of calreticulin-3, which was ultimately secreted into the apoplast. Our study thus identified a specific proteolytic enzyme capable of precise detachment of the ER retrieval signal from a plant ER resident protein, with implications for the further fate of the escaped resident.

Tear nanoDSF Denaturation Profile Is Predictive of Glaucoma.

Primary open-angle glaucoma (POAG) is a frequent blindness-causing neurodegenerative disorder characterized by optic nerve and retinal ganglion cell damage most commonly due to a chronic increase in intraocular pressure. The preservation of visual function in patients critically depends on the timeliness of detection and treatment of the disease, which is challenging due to its asymptomatic course at early stages and lack of objective diagnostic approaches. Recent studies revealed that the pathophysiology of glaucoma includes complex metabolomic and proteomic alterations in the eye liquids, including tear fluid (TF). Although TF can be collected by a non-invasive procedure and may serve as a source of the appropriate biomarkers, its multi-omics analysis is technically sophisticated and unsuitable for clinical practice. In this study, we tested a novel concept of glaucoma diagnostics based on the rapid high-performance analysis of the TF proteome by differential scanning fluorimetry (nanoDSF). An examination of the thermal denaturation of TF proteins in a cohort of 311 ophthalmic patients revealed typical profiles, with two peaks exhibiting characteristic shifts in POAG. Clustering of the profiles according to peaks maxima allowed us to identify glaucoma in 70% of cases, while the employment of artificial intelligence (machine learning) algorithms reduced the amount of false-positive diagnoses to 13.5%. The POAG-associated alterations in the core TF proteins included an increase in the concentration of serum albumin, accompanied by a decrease in lysozyme C, lipocalin-1, and lactotransferrin contents. Unexpectedly, these changes were not the only factor affecting the observed denaturation profile shifts, which considerably depended on the presence of low-molecular-weight ligands of tear proteins, such as fatty acids and iron. Overall, we recognized the TF denaturation profile as a novel biomarker of glaucoma, which integrates proteomic, lipidomic, and metallomic alterations in tears, and monitoring of which could be adapted for rapid non-invasive screening of the disease in a clinical setting.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the ß-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and ß-subunits, close to the anionic amino acid residues of the ß-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Synthesis and Biological Evaluation of Novel Dispiro-Indolinones with Anticancer Activity.

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCTwt, and HCT(-/-). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC50 = 1.2-3.5 µM) and a reasonable selectivity index (SI = 3-10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index-estimated as LD50/ED50-for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action.

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

Effect of Dexamethasone on Adhesion of Human Neutrophils and Concomitant Secretion.

Neutrophils play a dual role in protecting the body. They are able to penetrate infected tissues and destroy pathogens there by releasing aggressive bactericidal substances. While into the surrounding tissues, the aggressive products secreted by neutrophils initiate development of inflammatory processes. Invasion of neutrophils into tissues is observed during the development of pneumonia in the patients with lung diseases of various etiologies, including acute respiratory distress syndrome caused by coronavirus disease. Synthetic corticosteroid hormone dexamethasone has a therapeutic effect in treatment of lung diseases, including reducing mortality in the patients with severe COVID-19. The acute (short-term) effect of dexamethasone on neutrophil adhesion to fibrinogen and concomitant secretion was studied. Dexamethasone did not affect either attachment of neutrophils to the substrate or their morphology. Production of reactive oxygen species (ROS) and nitric oxide (NO) by neutrophils during adhesion also did not change in the presence of dexamethasone. Dexamethasone stimulated release of metalloproteinases in addition to the proteins secreted by neutrophils during adhesion under control conditions, and selectively stimulated release of free amino acid hydroxylysine, a product of lysyl hydroxylase. Metalloproteinases play a key role and closely interact with lysyl hydroxylase in the processes of modification of the extracellular matrix. Therapeutic effect of dexamethasone could be associated with its ability to reorganize extracellular matrix in the tissues by changing composition of the neutrophil secretions, which could result in the improved gas exchange in the patients with severe lung diseases.

Ivermectin Affects Neutrophil-Induced Inflammation through Inhibition of Hydroxylysine but Stimulation of Cathepsin G and Phenylalanine Secretion.

The invasion and integrin-dependent adhesion of neutrophils to lung tissues and their secretion lead to the development of pneumonia in various pulmonary pathologies, including acute respiratory distress syndrome in coronavirus disease. We studied the effect of ivermectin, a possible therapeutic agent for inflammation and cancer, on integrin-dependent neutrophil adhesion to fibronectin and the concomitant secretion. Ivermectin did not affect the attachment of neutrophils to the substrate and the reactive oxygen species production but sharply inhibited the adhesion-induced release of hydroxylysine and stimulated the release of phenylalanine and cathepsin G. Hydroxylysine is a product of lysyl hydroxylase, which is overexpressed in tumor cells with an increased ability to invade and metastasize. The inhibition of hydroxylysine release by ivermectin, by analogy, may indicate the suppression of neutrophil invasion into tissue. The increase in the release of phenylalanine in our experiments coincided with the secretion of cathepsin G, which indicates the possible role of this enzyme in the cleavage of phenylalanine. What is the substrate in such a reaction is unknown. We demonstrated that exogenously added angiotensin II (1-8) can serve as a substrate for phenylalanine cleavage. Mass spectrometry revealed the formation of angiotensin II (1-7) in the secretion of neutrophils, which attached to fibronectin in the presence of ivermectin and exogenous angiotensin II (1-8), indicating a possible involvement of ivermectin in the inactivation of angiotensin II.

Nicotiana benthamiana Kunitz peptidase inhibitor-like protein involved in chloroplast-to-nucleus regulatory pathway in plant-virus interaction.

Plant viruses use a variety of strategies to infect their host. During infection, viruses cause symptoms of varying severity, which are often associated with altered leaf pigmentation due to structural and functional damage to chloroplasts that are affected by viral proteins. Here we demonstrate that Nicotiana benthamiana Kunitz peptidase inhibitor-like protein (KPILP) gene is induced in response to potato virus X (PVX) infection. Using reverse genetic approach, we have demonstrated that KPILP downregulates expression of LHCB1 and LHCB2 genes of antenna light-harvesting complex proteins, HEMA1 gene encoding glutamyl-tRNA reductase, which participates in tetrapyrrole biosynthesis, and RBCS1A gene encoding RuBisCO small subunit isoform involved in the antiviral immune response. Thus, KPILP is a regulator of chloroplast retrograde signaling system during developing PVX infection. Moreover, KPILP was demonstrated to affect carbon partitioning: reduced glucose levels during PVX infection were associated with KPILP upregulation. Another KPILP function is associated with plasmodesmata permeability control. Its ability to stimulate intercellular transport of reporter 2xGFP molecules indicates that KPILP is a positive plasmodesmata regulator. Moreover, natural KPILP glycosylation is indispensable for manifestation of this function. During PVX infection KPILP increased expression leads to the reduction of plasmodesmata callose deposition. These results could indicate that KPILP affects plasmodesmata permeability via callose-dependent mechanism. Thus, virus entering a cell and starting reproduction triggers KPILP expression, which leads to downregulation of nuclear-encoded chloroplast genes associated with retrograde signaling, reduction in photoassimilates accumulation and increase in intercellular transport, creating favorable conditions for reproduction and spread of viral infection.

Isolation and Characterization of a Novel Hydrophobin, Sa-HFB1, with Antifungal Activity from an Alkaliphilic Fungus, Sodiomyces alkalinus.

The adaptations that alkaliphilic microorganisms have developed due to their extreme habitats promote the production of active natural compounds with the potential to control microorganisms, causing infections associated with healthcare. The primary purpose of this study was to isolate and identify a hydrophobin, Sa-HFB1, from an alkaliphilic fungus, Sodiomyces alkalinus. A potential antifungal effect against pathogenic and opportunistic fungi strains was determined. The MICs of Sa-HFB1 against opportunistic and clinical fungi ranged from 1 to 8 µg/mL and confirmed its higher activity against both non- and clinical isolates. The highest level of antifungal activity (MIC 1 µg/mL) was demonstrated for the clinical isolate Cryptococcus neoformans 297 m. The hydrophobin Sa-HFB1 may be partly responsible for the reported antifungal activity of S. alkalinus, and may serve as a potential source of lead compounds, meaning that it can be developed as an antifungal drug candidate.

Recombinant Cathepsin L of Tribolium castaneum and Its Potential in the Hydrolysis of Immunogenic Gliadin Peptides.

Wheat gliadins contain a large amount of glutamine- and proline-rich peptides which are not hydrolyzed by human digestive peptidases and can cause autoimmune celiac disease and other forms of gluten intolerance in predisposed people. Peptidases that efficiently cleave such immunogenic peptides can be used in enzyme therapy. The stored product insect pest Tribolium castaneum efficiently hydrolyzes gliadins. The main digestive peptidase of T. castaneum is cathepsin L, which is from the papain C1 family with post-glutamine cleavage activity. We describe the isolation and characterization of T. castaneum recombinant procathepsin L (rpTcCathL1, NP_001164001), which was expressed in Pichia pastoris cells. The activation of the proenzyme was conducted by autocatalytic processing. The effects of pH and proenzyme concentration in the reaction mixture on the processing were studied. The mature enzyme retained high activity in the pH range from 5.0 to 9.0 and displayed high pH-stability from 4.0 to 8.0 at 20 °C. The enzyme was characterized according to electrophoretic mobility under native conditions, activity and stability at various pH values, a sensitivity to various inhibitors, and substrate specificity, and its hydrolytic effect on 8-, 10-, 26-, and 33-mer immunogenic gliadins peptides was demonstrated. Our results show that rTcCathL1 is an effective peptidase that can be used to develop a drug for the enzyme therapy of various types of gluten intolerance.

A Novel, NADH-Dependent Acrylate Reductase in Vibrio harveyi.

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.

S51 Family Peptidases Provide Resistance to Peptidyl-Nucleotide Antibiotic McC.

Microcin C (McC)-like compounds are natural Trojan horse peptide-nucleotide antibiotics produced by diverse bacteria. The ribosomally synthesized peptide parts of these antibiotics are responsible for their facilitated transport into susceptible cells. Once inside the cell, the peptide part is degraded, releasing the toxic payload, an isoaspartyl-nucleotide that inhibits aspartyl-tRNA synthetase, an enzyme essential for protein synthesis. Bacteria that produce microcin C-like compounds have evolved multiple ways to avoid self-intoxication. Here, we describe a new strategy through the action of S51 family peptidases, which we name MccG. MccG cleaves the toxic isoaspartyl-nucleotide, rendering it inactive. While some MccG homologs are encoded by gene clusters responsible for biosynthesis of McC-like compounds, most are encoded by standalone genes whose products may provide a basal level of resistance to peptide-nucleotide antibiotics in phylogenetically distant bacteria. IMPORTANCE Here, we identified a natural substrate for a major phylogenetic clade of poorly characterized S51 family proteases from bacteria. We show that these proteins can contribute to a basal level of resistance to an important class of natural antibiotics.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu.

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

Complex of HIV-1 Integrase with Cellular Ku Protein: Interaction Interface and Search for Inhibitors.

The interaction of HIV-1 integrase and the cellular Ku70 protein is necessary for HIV replication due to its positive effect on post-integration DNA repair. We have previously described in detail the Ku70 binding site within integrase. However, the integrase binding site in Ku70 remained poorly characterized. Here, using a peptide fishing assay and site-directed mutagenesis, we have identified residues I72, S73, and I76 of Ku70 as key for integrase binding. The molecular dynamics studies have revealed a possible way for IN to bind to Ku70, which is consistent with experimental data. According to this model, residues I72 and I76 of Ku70 form a "leucine zipper" with integrase residues, and, therefore, their concealment by low-molecular-weight compounds should impede the Ku70 interaction with integrase. We have identified such compounds by molecular docking and have confirmed their capacity to inhibit the formation of the integrase complex with Ku70. Our data demonstrate that the site of IN binding within Ku70 identified in the present work may be used for further search for inhibitors of the integrase binding to Ku70.

Exploring Peptaibol's Profile, Antifungal, and Antitumor Activity of Emericellipsin A of Emericellopsis Species from Soda and Saline Soils.

Features of the biochemical adaptations of alkaliphilic fungi to exist in extreme environments could promote the production of active antibiotic compounds with the potential to control microorganisms, causing infections associated with health care. Thirty-eight alkaliphilic and alkalitolerant Emericellopsis strains (E. alkalina, E. cf. maritima, E. cf. terricola, Emericellopsis sp.) isolated from different saline soda soils and belonging to marine, terrestrial, and soda soil ecological clades were investigated for emericellipsin A (EmiA) biosynthesis, an antifungal peptaibol previously described for Emericellopsis alkalina. The analysis of the Emericellopsis sp. strains belonging to marine and terrestrial clades from chloride soils revealed another novel form with a mass of 1032.7 Da, defined by MALDI-TOF Ms/Ms spectrometers, as the EmiA lacked a hydroxyl (dEmiA). EmiA displayed strong inhibitory effects on cell proliferation and viability of HCT 116 cells in a dose- and time-dependent manners and induced apoptosis.

Inhibitor of Hyaluronic Acid Synthesis 4-Methylumbelliferone Suppresses the Secretory Processes That Ensure the Invasion of Neutrophils into Tissues and Induce Inflammation.

Integrin-dependent adhesion of neutrophils to tissue, accompanied by the development of neutrophil-induced inflammation, occurs both in the focus of infection and in the absence of infection in metabolic disorders such as reperfusion after ischemia, diabetes mellitus, or the development of pneumonia in patients with cystic fibrosis or viral diseases. Hyaluronic acid (HA) plays an important role in the recruitment of neutrophils to tissues. 4-methylumbilliferon (4-MU), an inhibitor of HA synthesis, is used to treat inflammation, but its mechanism of action is unknown. We studied the effect of 4-MU on neutrophil adhesion and concomitant secretion using adhesion to fibronectin as a model for integrin-dependent adhesion. 4-MU reduced the spreading of neutrophils on the substrate and the concomitant secretion of granule proteins, including pro-inflammatory components. 4-MU also selectively blocked adhesion-induced release of the free amino acid hydroxylysine, a product of lysyl hydroxylase, which can influence cell invasion by modifying the extracellular matrix. Finally, 4-MU inhibited the formation of cytonemes, the extracellular membrane secretory structures containing the pro-inflammatory bactericides of the primary granules. The anti-inflammatory effect of 4-MU may be associated with the suppression of secretory processes that ensure the neutrophil invasion and initiate inflammation. We suggest that HA, due to the peculiarities of its synthesis, can promote the release of secretory carriers from the cell and 4-MU can block this process.

Identification of Phytaspase Interactors via the Proximity-Dependent Biotin-Based Identification Approach.

Proteolytic enzymes are instrumental in various aspects of plant development, including senescence. This may be due not only to their digestive activity, which enables protein utilization, but also to fulfilling regulatory functions. Indeed, for the largest family of plant serine proteases, subtilisin-like proteases (subtilases), several members of which have been implicated in leaf and plant senescence, both non-specific proteolysis and regulatory protein processing have been documented. Here, we strived to identify the protein partners of phytaspase, a plant subtilase involved in stress-induced programmed cell death that possesses a characteristic aspartate-specific hydrolytic activity and unusual localization dynamics. A proximity-dependent biotin identification approach in Nicotiana benthamiana leaves producing phytaspase fused to a non-specific biotin ligase TurboID was employed. Although the TurboID moiety appeared to be unstable in the apoplast environment, several intracellular candidate protein interactors of phytaspase were identified. These were mainly, though not exclusively, represented by soluble residents of the endoplasmic reticulum, namely endoplasmin, BiP, and calreticulin-3. For calreticultin-3, whose gene is characterized by an enhanced expression in senescing leaves, direct interaction with phytaspase was confirmed in an in vitro binding assay using purified proteins. In addition, an apparent alteration of post-translational modification of calreticultin-3 in phytaspase-overproducing plant cells was observed.