Biochimie最新文献

Fibrinolytic enzymes are promising in treating cardiovascular diseases due to their capacity to dissolve blood clots. The fibrinolytic enzyme from Arthrospira platensis (FEAP) was purified by ion exchange chromatography to investigate its ability to activate plasminogen, as well as its thrombolytic and fibrinogenolytic potential. Subsequently, two different cytotoxic assays (MTT and NR) and hemolysis test were performed to evaluate FEAP's safety. Furthermore, cell migration and the genotoxic and hemolytic potential were also investigated. The purified enzyme showed thrombus degradation of 43 % and thrombolytic action directly on fibrin, which can reduce possible side effects, such as hemorrhage. MTT assay was more sensitive to determine the enzyme cytotoxicity, which decreased the viability of breast cancer tumor cells (Sarcoma-180 and MDA-MB-231) and macrophages (J774A.1). In addition, the enzyme also exhibited non-hemolytic, antimetastatic, and non-genotoxic characteristics. These findings are innovative for a fibrinolytic protease and may indicate that it is safe for people undergoing cancer treatment, reducing side effects such as hemorrhage, in addition to inhibiting tumor cells and preventing metastasis, which can help with chemotherapy treatment.

The split trehalase biosensor has potential as a versatile diagnostic technology. Split enzymes are engineered proteins, divided into inactive fragments, which can reassemble and regain activity when brought together by an analyte. The split TreA biosensor requires no sample processing and produces stable signals (in the form of glucose). Split trehalase reagents can function in blood, but periplasmic trehalase of E. coli requires blood acidification for maximal activity. The objective of this study was to obtain split trehalase with near physiological pH optimum. For this purpose, periplasmic trehalases of Cellvibrio spp. with higher activity at neutral pH, were split in analogy with the E. coli TreA into hood and catalytic domains. However, these split trehalases displayed self-complementation due to spontaneous reassembly. In contrast, when catalytic domains of Cellvibrio trehalases were combined with E. coli hood domains, these hybrids displayed conditional complementation capacity when split trehalase fragments fused to immunoglobulin-binding protein G (STIGA) were used to quantify immunoglobulin concentrations. Other hybrid combinations of Cellvibrio spp. had increased activity compared to the cognate pairs, albeit with strong self-complementation. A mutagenesis analysis of residues responsible for self-complementation led to uncoupling of self-complementation from allostery. The Michaelis-Menten kinetics of Cellvibrio enzymes and fragment pairs confirmed improved activity of a mutated hybrid pair of Cellvibrio hood and catalytic domains at physiological pH. In conclusion, the improvements in pH optimum and activity, demonstrated with STIGA, can now be leveraged to enhance other variations of the split trehalase biosensor platform, broadening its utility for testing clinical samples.

Natural products are widely used in different aspects of our lives - from household cleaners and food production, via cosmetics and aromatherapy, to both alternative and traditional medicine. In our research group, we have recently described several monoterpenoids with potential in the antiviral and anticancer therapy by allosteric targeting of aryl hydrocarbon receptor (AhR). Prior to any practical application, biological effects on human organism must be taken in concern. This review article is focused on the biological effects of 5 monoterpenoids on the human health previously identified as AhR antagonists with a therapeutic potential as antiviral and anticancer agents. We have thoroughly described cytotoxic, anti-inflammatory, anti-proliferative, and anticancer effects, as well as known interactions with nuclear receptors. As clearly demonstrated, monoterpenoids in general represent almost an inexhaustible reservoir of natural compounds possessing the ability to influence, modulate and improve human health.

Mycoses infect millions of people annually across the world. The most common mycosis agent, Candida albicans is responsible for a great deal of illness and death. C. albicans infection is becoming more widespread and the current antifungals polyenes, triazoles, and echinocandins are less efficient against it. Investigating antifungal peptides (AFPs) as therapeutic is gaining momentum. Therefore, we used MALDI-TOF/MS analysis to identify AFPs and protein-protein docking to analyze their interactions with the C. albicans target protein. Some microorganisms with strong antifungal action against C. albicans were selected for the isolation of AFPs. Using MALDI-TOF/MS, we identified 3 AFPs Chitin binding protein (ACW83017.1; Bacillus licheniformis), the bifunctional protein GlmU (BBQ13478.1; Stenotrophomonas maltophilia), and zinc metalloproteinase aureolysin (BBA25172.1; Staphylococcus aureus). These AFPs showed robust interactions with C. albicans target protein Sap5. We deciphered some important residues in identified APFs and highlighted interaction with Sap5 through hydrogen bonds, protein-protein interactions, and salt bridges using protein-protein docking and MD simulations. The three discovered AFPs-Sap5 complexes exhibit different levels of stability, as seen by the RMSD analysis and interaction patterns. Among protein-protein interactions, the remarkable stability of the BBQ25172.1-2QZX complex highlights the role of salt bridges and hydrogen bonds. Identified AFPs could be further studied for developing successful antifungal candidates and peptide-based new antifungal therapeutic strategies as fresh insights into addressing antifungal resistance also.

It is widely recognized that developing bi- or multifunctional opioid compounds could offer a valuable approach to pain management with fewer side effects compared to single-target compounds. In this study, we designed and characterized two novel chimeric peptides, EM-1-DLS and EM-2-DLS, incorporating endomorphins (EMs) and the ghrelin receptor antagonist [D-Lys3]-GHRP-6 (DLS). Functional assays demonstrated that EM-1-DLS and EM-2-DLS acted as κ-opioid receptor (κ-OR)-preferring agonists, weak μ-opioid receptors (μ-OR) and ghrelin receptor (GHSR) agonists. Upon intracerebroventricular (i.c.v.) administration in mice, both EM-1-DLS and EM-2-DLS exhibited dose- and time-dependent antinociceptive effects in the tail withdrawal test. EM-1-DLS demonstrated the highest antinociceptive potency among the peptides, with an ED₅₀ approximately 8-fold greater than EM-1, while EM-2-DLS showed comparable effects to EM-2. The antinociceptive actions of EM-1-DLS involved activation of GHS-R1α, μ-OR, and κ-OR, whereas EM-2-DLS acted via GHS-R1α, δ-OR, and κ-OR pathways. Additionally, acute antinociceptive tolerance was investigated, revealing that EM-1-DLS induced a tolerance ratio of 2.33-fold, significantly lower than the 5.19-fold ratio induced by EM-1. Cross-tolerance ratios between the chimeric peptides and EMs ranged from 0.92 to 1.76, indicating reduced tolerance compared to EMs alone. These findings highlight the potential of these chimeric peptides to mitigate pain with diminished tolerance development, suggesting a promising strategy for the development of new analgesic therapies with improved safety profiles.

Proline biosynthesis and catabolism pathways are executed by powerful action of specific enzymes that are subjected to environmental fluctuations at the transcriptional level. Previous researches have demonstrated that osmotic stress-induced upstream events can affect the expression of proline metabolism-related genes, which results in adjustable free proline accumulation to protect plant cells from severe damage. Here, we mainly describe the mechanisms for how some key factors, such as transcription factors, ABA (abscisic acid), Ca²⁺, MAPK cascades, CK (cytokinin) and phospholipase, in a phosphorylated manner, vividly function in the transcriptional regulation of proline metabolism under osmotic stress. These mechanisms reveal that sustaining of proline homeostasis is an efficient way for plants to adapt to osmotic stress.

Plasminogen activator inhibitor 1 (PAI-1) is a crucial serine protease inhibitor that prevents plasminogen activation by inhibiting tissue- and urokinase-type plasminogen activators (tPA, uPA). PAI-1 is well-known for its role in modulating hemocoagulation or extracellular matrix formation by inhibiting plasmin or matrix metalloproteinases, respectively. PAI-1 is induced by pro-inflammatory cytokines across various tissues, yet its regulation by ligand-activated transcription factors is partly disregarded. Therefore, we have attempted to summarize the current knowledge on the transcriptional regulation of PAI-1 expression by the most relevant xenobiotic and endocrine receptors implicated in modulating PAI-1 levels. This review aims to contribute to the understanding of the specific, often tissue-dependent regulation of PAI-1 and provide insights into the modulation of PAI-1 levels beyond its direct inhibition.

Kingella kingae, an emerging pediatric pathogen, secretes the pore-forming toxin RtxA, which has been implicated in the development of various invasive infections. RtxA is synthesized as a protoxin (proRtxA), which gains its biological activity by fatty acylation of two lysine residues (K558 and K689) by the acyltransferase RtxC. The low acylation level of RtxA at K558 (2-23 %) suggests that the complete acylation at K689 is crucial for toxin activity. Using a bacterial two-hybrid system, we show that substitutions of K558, but not K689, partially reduce the interaction of proRtxA with RtxC and that the acyltransferase interacts independently with each acylated site in vivo. While substitutions of K558 had no effect on the acylation of K689, substitutions of K689 resulted in an average 40 % increase in the acylation of K558. RtxA mutants monoacylated at either K558 or K689 irreversibly bound to erythrocyte membranes, with binding efficiency corresponding to the extent of lysine acylation. However, these mutants lysed erythrocytes with similarly low efficiency as nonacylated proRtxA and showed only residual overall membrane activity in planar lipid bilayers. Interestingly, despite forming fewer pores, the monoacylated mutants exhibited single-pore characteristics, such as conductance and lifetime, similar to those of intact RtxA. These findings indicate that the acylation at either K558 or K689 is sufficient for the irreversible insertion of RtxA into the membrane, but not for the efficient formation of membrane pores. Alternatively, K558 and K689 per se may play a crucial structural role in pore formation, regardless of their acylation status.

Bacterial methionine biosynthesis is an attractive target for research due to its central role in cellular metabolism, as most steps of this pathway are missing in mammals. Up to now little is known about sulfur metabolism in pathogenic Clostridia species, making the study of the enzymes of Cys/Met metabolism in Clostridium tetani particularly relevant. Analysis of the C. tetani genome has shown that the bacterium is capable of synthesizing methionine by direct sulfhydration. In this study, we describe purification of recombinant O-acetylhomoserine sulfhydrylase, a member of the Cys/Met metabolism pyridoxal 5'-phosphate-dependent enzyme family, from C. tetani for the first time. The gene encoding O-acetylhomoserine sulfhydrylase was cloned into the pET-28a(+) vector and expressed in Escherichia coli. The expression product was purified and identified as a 462-amino acid protein with a molecular mass of ∼50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the C. tetani enzyme showed a high degree of similarity to O-acetylhomoserine sulfhydrylases from other bacterial sources. We confirmed the O-acetylhomoserine sulfhydrylase activity, and found the enzyme to be optimally active at pH 7.5 and 50 °C. The native enzyme assembles into a homotetramer of approx. 200 kDa as revealed by gel filtration. The obtained enzyme is capable of l-methionine formation using methanethiol as a sulfur source, that has been revealed by ¹H NMR spectral data. These findings broaden the understanding of the role of O-acetylhomoserine sulfhydrylase in C. tetani Cys/Met metabolism and provide a basis for its future investigations and research.

Skeletal muscle has an important role in whole body energy metabolism and various proteases are involved in skeletal muscle functions. We have previously identified the cysteine protease legumain in cultured human skeletal muscle cells. However, the potential role of legumain in regulation of energy metabolism remains unexplored. This study aimed to investigate cellular uptake, processing, and activation of prolegumain in human myotubes. Additionally, we sought to determine the effects of prolegumain on energy substrate metabolism in these cells. During differentiation of human myoblast to myotubes, legumain mRNA expression and activity were upregulated. Interestingly, legumain activity in myotubes was inversely correlated with the body mass index (BMI) of the obese cell donors. Myotubes exposed to conditioned medium enriched in prolegumain during the last two days of differentiation demonstrated the capacity to internalize and process prolegumain into its active form. Pre-treatment with prolegumain induced a metabolic shift towards increased fatty acid uptake in myotubes, as evidenced by elevated oleic acid uptake whereas glucose uptake and oxidation were reduced. The metabolic changes were not reversed by a legumain inhibitor, indicating a different mechanism for this effect. The metabolic alterations were accompanied by increased mRNA expression of the fatty acid transporter CD36, whereas the glucose transporter GLUT1 mRNA level remained unchanged. These findings suggest that legumain may play a regulatory role in skeletal muscle energy metabolism, highlighting its potential as a novel therapeutic target of metabolic disorders.