Search Results (36)

Caspases orchestrate metazoan apoptosis, regulating processes such as embryogenesis, the death of old and infected cells and immune tolerance. Structural orthologs of caspases have been identified in bacteria, plants, protists and fungi and regulated cell death has been demonstrated in these organisms. This led some researchers to conclude that fungal metacaspases might perform a similar function to caspases. This review discusses regulated cell death, beginning with an account of RCD and the central role of caspases in mammalian RCD. It goes on to give examples of RCD in fungi, compares the structure and activity of caspase orthologs and outlines examples of metacaspase-dependent and metacaspase-independent cell death in fungi, focusing on S. cerevisiae. Finally, it addresses the question “are metacaspases caspases?”, identifies alternative cell death proteases and recommends future research objectives. Full article

Ganoderma lucidum is a medicinal fungus widely utilized in traditional medicine and functional foods. Its primary bioactive constituents are ganoderic acids (GAs), a group of triterpenoid compounds. While chemical-induced apoptosis has previously been shown to enhance GAs production, this study investigates the role of physical stress in this regulatory pathway. We demonstrate that heat-induced apoptosis significantly increases GAs production in G. lucidum. To determine whether apoptosis directly regulates this process, we overexpressed the human anti-apoptotic gene Bcl-2 in G. lucidum, confirming expression via RT-PCR and Western blot analysis. Upon heat-induced apoptosis, these Bcl-2 overexpression mutants exhibited increased mycelial cell viability, accompanied by reduced metacaspase activity and, notably, decreased GAs production. Furthermore, we identified a Type I metacaspase gene in G. lucidum, Glmca1, which contains highly conserved catalytic domains common across fungal species. Silencing of Glmca1 followed by heat-induced apoptosis led to results similar to Bcl-2 overexpression: enhanced cell viability, suppressed metacaspase activity, and a significant reduction in GAs yield. These findings provide compelling evidence that apoptosis functions as a critical regulatory mechanism for secondary metabolite production in G. lucidum. Consequently, modulating apoptotic pathways through physical induction offers a promising strategy for enhancing the production of bioactive ingredients in medicinal fungi. Full article

Oxidative stress is one of the major environmental stresses that the fungal pathogen Candida albicans frequently encounters. In view of the negative regulatory effect of Ume6 on autophagy in Saccharomyces cerevisiae and the close link between autophagy and oxidative stress in mammals, we explored the regulatory effect of Ume6 on autophagy and oxidative stress in C. albicans in this study. Here, we identify the transcriptional regulator Ume6 as a key positive regulator of autophagy under oxidative stress conditions. Deletion of UME6 resulted in reduced autophagy levels under H₂O₂ treatment, correlating with reduced transcriptional expression of core autophagy-related genes. Although UME6 deletion alone did not alter H₂O₂ sensitivity, it significantly exacerbated the sensitivity of a catalase mutant, revealing a functional role for Ume6 in oxidative stress tolerance. Intriguingly, we discovered that 3-methyladenine (3-MA), a canonical autophagy inhibitor in other systems, acts as an autophagy activator in C. albicans, promoting Atg8 transport to the vacuole and enhancing autophagy levels. This 3-MA-induced autophagy alleviated oxidative stress damage, as evidenced by improved growth and protection of vacuolar membrane integrity in H₂O₂-treated cells. Furthermore, deletion of UME6 or nitrogen starvation reduced apoptosis under oxidative stress, including decreased Annexin-V binding, metacaspase activation, mitochondrial membrane depolarization, and mitochondrial cytochrome c release. This study uncovers the critical role of Ume6 in governing oxidative stress, autophagy, and apoptosis. Full article

Fungal pathogens pose a major threat to global agriculture and human health, necessitating alternative antifungal strategies with high efficacy and low resistance potential. Antifungal proteins (AFPs) from filamentous fungi are promising candidates due to their stability, selectivity, and diverse mechanisms of action. Here, we characterize Beauveria brongniartii antifungal protein (BbroAFP), a novel cysteine-rich protein from the entomopathogenic fungus B. brongniartii, and investigate its antifungal activity against Botrytis cinerea. Recombinant BbroAFP was expressed in Pichia pastoris, purified, and verified by liquid chromatography–tandem mass spectroscopy (LC–MS/MS) and in silico modeling. BbroAFP showed potent antifungal activity with minimum inhibitory concentrations (MICs) as low as 1 µM against several phytopathogenic fungi, while exhibiting no significant antibacterial activity. Activity was maintained across a wide range of pH and temperature conditions. Confocal microscopy revealed rapid surface binding followed by cytosolic internalization without major cell wall disruption. BbroAFP induced a rapid, transient burst of reactive oxygen species (ROS), accompanied by nuclear DNA fragmentation. Gene expression analysis revealed a transient increase in aif1, whereas mca1 expression decreased at later time points and mca2 remained largely unchanged, suggesting a metacaspase-independent response. Detached tomato leaf assays showed effective protection against B. cinerea without detectable phytotoxicity. Cytotoxicity assays confirmed a favorable safety profile, supporting further evaluation of BbroAFP for plant protection. Full article

Soil salinisation is threatening crop sustainability worldwide, mainly due to anthropogenic climate change. Molecular mechanisms developed to counteract salinity have been intensely studied in model plants. Nevertheless, the economically relevant olive tree (Olea europaea subsp. europaea L.), being highly exposed to soil salinisation, deserves a specific review to extract the recent genomic advances that support the known morphological and biochemical mechanisms that make it a relative salt-tolerant crop. A comprehensive list of 98 olive cultivars classified by salt tolerance is provided, together with the list of available olive tree genomes and genes known to be involved in salt response. Na⁺ and Cl^– exclusion in leaves and retention in roots seem to be the most prominent adaptations, but cell wall thickening and antioxidant changes are also required for a tolerant response. Several post-translational modifications of proteins are emerging as key factors, together with microbiota amendments, making treatments with biostimulants and chemical compounds a promising approach to enable cultivation in already salinised soils. Low and high-throughput transcriptomics and metagenomics results obtained from salt-sensitive and -tolerant cultivars, and the future advantages of engineering specific metacaspases involved in programmed cell death and autophagy pathways to rapidly raise salt-tolerant cultivars or rootstocks are also discussed. The overview of bioinformatic tools focused on olive tree, combined with machine learning approaches for studying plant stress from a multi-omics perspective, indicates that the development of salt-tolerant cultivars or rootstocks adapted to soil salinisation is progressing. This could pave the way for ‘smart oliviculture’, promoting more productive and sustainable practices under salt stress. Full article

Programmed cell death (PCD) plays a crucial role in the development and homeostasis maintenance of multicellular organisms. Apoptosis is a form of PCD that prevents pathological development by eliminating damaged or useless cells. Despite the complexity of fungal apoptosis mechanisms being similar to those of plants and metazoans, fungal apoptosis lacks the core regulatory elements of animal apoptosis. Apoptosis-like PCD in fungi can be triggered by a variety of internal and external factors, participating in biological processes such as growth, development, and stress response. Although the core regulatory elements are not fully understood, apoptosis-inducing factor and metacaspase have been found to be involved. This article summarizes various proteins closely related to fungal apoptosis, such as apoptosis-inducing factor, metacaspase, and inhibitors of apoptosis proteins, as well as their structures and functions. This research provides new strategies and ideas for the development of natural drugs targeting fungal apoptosis and the control of fungal diseases. Full article

Elucidation of pathways regulating parasite cell death is believed to contribute to identification of novel therapeutic targets for protozoan diseases, and in this context, apoptosis-like cell death has been reported in different groups of protozoa, in which metacaspases seem to play a role. In the genus Plasmodium, apoptotic markers have been detected in P. falciparum and P. berghei, and no study focusing on P. vivax cell death has been reported so far. In the present study, we investigated the susceptibility of P. vivax to undergo apoptotic cell death after incubating mature trophozoites with the classical apoptosis inducer staurosporine. As assessed by flow cytometry assays, staurosporine inhibited parasite intraerythrocytic development, which was accompanied by a decrease in cell viability, evidenced by reduced plasmodial mitochondrial activity. However, typical signs of apoptosis, such as DNA fragmentation, chromatin condensation, and nuclear segregation, were not detected in the parasites induced to cell death, and no significant alteration in metacaspase gene expression (PvMCA1) was observed under cell death stimulus. Interestingly, dying parasites positively modulated cell death (eryptosis) of host erythrocytes, which was marked by externalization of phosphatidylserine and cell shrinkage. Our study shows for the time that P. vivax blood stages may not be susceptible to apoptosis-like processes, while they could trigger eryptosis of parasitized cells by undergoing cell death. Further studies are required to elucidate the cellular machinery involved in cell death of P. vivax parasites as well as in the modulation of host cell death. Full article

Candidiasis is considered an emerging public health concern because of the occurrence of drug-resistant Candida strains and the lack of an available structurally diverse antifungal drug armamentarium. The indole alkaloid globospiramine from the anticandidal Philippine medicinal plant Voacanga globosa exhibits a variety of biological activities; however, its antifungal properties remain to be explored. In this study, we report the in vitro anticandidal activities of globospiramine against two clinically relevant Candida species (C. albicans and C. tropicalis) and the exploration of its possible target proteins using in silico methods. Thus, the colony-forming unit (CFU) viability assay revealed time- and concentration-dependent anticandidal effects of the alkaloid along with a decrease in the number of viable CFUs by almost 50% at 60 min after treatment. The results of the MIC and MFC assays indicated inhibitory and fungicidal effects of globospiramine against C. albicans (MIC = 8 µg/mL; MFC = 8 µg/mL) and potential fungistatic effects against C. tropicalis at lower concentrations (MIC = 4 µg/mL; MFC > 64 µg/mL). The FAM-FLICA poly-caspase assay showed metacaspase activation in C. albicans cells at concentrations of 16 and 8 µg/mL, which agreed well with the MIC and MFC values. Molecular docking and molecular dynamics simulation experiments suggested globospiramine to bind strongly with 1,3-β-glucan synthase and Als3 adhesin—enzymes indirectly involved in apoptosis-driven candidal inhibition. Full article

YCA1, the only metacaspase in Saccharomyces cerevisiae, plays important roles in the regulation of chronological lifespan, apoptosis, and cytokinesis. YCA1 has protein hydrolase activity and functions by cleaving itself and target proteins. However, there are few reports about the regulation of YCA1 activity. In this study, we observed that reactive sulfane sulfur (RSS) can inhibit the activity of YCA1. In vitro experiments demonstrated that RSS reacted with the Cys₂₇₆ of YCA1, the residue central to its protein hydrolase activity, to form a persulfidation modification (protein-SSH). This modification inhibited both its self-cleavage and the cleavage of its substrate protein, BIR1. To investigate further, we constructed a low-endogenous-RSS mutant of S. cerevisiae, BY4742 Δcys3, in which the RSS-producing enzyme cystathionine-γ-lyase (CYS3) was knocked out. The activity of YCA1 was significantly increased by the deletion of CYS3. Moreover, increased YCA1 activity led to reduced chronological lifespan (CLS) and CLS-driven apoptosis. This study unveils the first endogenous factor that regulates YCA1 activity, introduces a novel mechanism of how yeast cells regulate chronological lifespan, and broadens our understanding of the multifaceted roles played by RSS. Full article

LESION-SIMULATING DISEASE1 (LSD1) is one of the well-known cell death regulatory proteins in Arabidopsis thaliana. The lsd1 mutant exhibits runaway cell death (RCD) in response to various biotic and abiotic stresses. The phenotype of the lsd1 mutant strongly depends on two other proteins, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN-DEFICIENT 4 (PAD4) as well as on the synthesis/metabolism/signaling of salicylic acid (SA) and reactive oxygen species (ROS). However, the most interesting aspect of the lsd1 mutant is its conditional-dependent RCD phenotype, and thus, the defined role and function of LSD1 in the suppression of EDS1 and PAD4 in controlled laboratory conditions is different in comparison to a multivariable field environment. Analysis of the lsd1 mutant transcriptome in ambient laboratory and field conditions indicated that there were some candidate genes and proteins that might be involved in the regulation of the lsd1 conditional-dependent RCD phenotype. One of them is METACASPASE 8 (AT1G16420). This type II metacaspase was described as a cell death-positive regulator induced by UV-C irradiation and ROS accumulation. In the double mc8/lsd1 mutant, we discovered reversion of the lsd1 RCD phenotype in response to UV radiation applied in controlled laboratory conditions. This cell death deregulation observed in the lsd1 mutant was reverted like in double mutants of lsd1/eds1 and lsd1/pad4. To summarize, in this work, we demonstrated that MC8 is positively involved in EDS1 and PAD4 conditional-dependent regulation of cell death when LSD1 function is suppressed in Arabidopsis thaliana. Thus, we identified a new protein compound of the conditional LSD1-EDS1-PAD4 regulatory hub. We proposed a working model of MC8 involvement in the regulation of cell death and we postulated that MC8 is a crucial protein in this regulatory pathway. Full article

Anthracnose, induced by Colletotrichum gloeosporioides, poses a substantial economic threat to rubber tree yields and various other tropical crops. Ede1, an endocytic scaffolding protein, plays a crucial role in endocytic site initiation and maturation in yeast. Metacaspases, sharing structural similarities with caspase family proteases, are essential for maintaining cell fitness. To enhance our understanding of the growth and virulence of C. gloeosporioides, we identified a homologue of Ede1 (CgEde1) in C. gloeosporioides. The knockout of CgEde1 led to impairments in vegetative growth, conidiation, and pathogenicity. Furthermore, we characterized a weakly interacted partner of CgEde1 and CgMca (orthologue of metacaspase). Notably, both the single mutant ΔCgMca and the double mutant ΔCgEde1/ΔCgMca exhibited severe defects in conidiation and germination. Polarity establishment and pathogenicity were also disrupted in these mutants. Moreover, a significantly insoluble protein accumulation was observed in ΔCgMca and ΔCgEde1/ΔCgMca strains. These findings elucidate the mechanism by which CgEde1 and CgMca regulates the growth and pathogenicity of C. gloeosporioides. Their regulation involves influencing conidiation, polarity establishment, and maintaining cell fitness, providing valuable insights into the intricate interplay between CgEde1 and CgMca in C. gloeosporioides. Full article

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response—type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms’ adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor. Full article

Pleurotus ostreatus is one of the most cultivated edible mushrooms worldwide, of which the fruiting body development is a highly complex process involving the precise genetic regulatory network and suitable environmental factors. Metacaspases play important roles in developmental processes and programmed cell death (PCD) induced by some environmental stress in many organisms. In this study, a type I metacaspase, PoMCA1, was identified via the analysis of the enzyme domain and alignment with homologous metacaspases. PoMCA1 overexpression and RNAi mutants were generated via Agrobacterium tumefaciens-mediated transformation (ATMT) into the P. ostreatus mycelium. The roles of the PoMCA1 gene in heat stress and fruiting body development were examined. The results show that both of the overexpression transformants were more tolerant to heat stress than the wild-type strain, while the opposite phenomenons were found for the two RNAi strains. Compared with the wild-type strain, the overexpression strain OE-7 had faster formation of the fruiting body, while the two RNAi strains produced significantly more primordia and young fruiting bodies, and presented morphological deformities and slower fruiting body development. All of the results suggest that the PoMCA1 gene is involved in the positive regulation of heat stress tolerance and fruiting body development in P. ostreatus. Full article

The World Health Organization (WHO) recently published a list of fungal priority pathogens, including Candida albicans and C. auris. The increased level of resistance of Candida is raising concern, considering the availability of only four classes of medicine. The WHO is seeking novel agent classes with different targets and mechanisms of action. Targeting Candida metacaspases to control intrinsic cell death could provide new therapeutic opportunities for invasive candidiasis. In this review, we provide the available evidence for Candida cell death, describe Candida metacaspases, and discuss the potential of Candida metacaspases to offer a new specific target. Targeting Candida cell death has good scientific rationale given that the fungicidal activity of many marketed antifungals is mediated, among others, by cell death triggering. But none of the available antifungals are specifically activating Candida metacaspases, making this target a new therapeutic opportunity for non-susceptible isolates. It is expected that antifungals based on the activation of fungi metacaspases will have a broad spectrum of action, as metacaspases have been described in many fungi, including filamentous fungi. Considering this original mechanism of action, it could be of great interest to combine these new antifungal candidates with existing antifungals. This approach would help to avoid the development of antifungal resistance, which is especially increasing in Candida. Full article

Metacaspases are a class of proteases found in plants that have gained attention in recent years due to their involvement in programmed cell death (PCD) and other essential cellular processes. Although structurally homologous to caspases found in animals, metacaspases have distinct properties and functions. There are nine metacaspase genes in the Arabidopsis thaliana genome; these can be type I or type II, and working out the function of each member of the gene family is challenging. In this study, we report the characterisation of one Arabidopsis type II metacaspase, metacaspase-5 (AtMC5; AtMCA-IIb). We detected the expression of AtMC5 only under specific conditions with a strong upregulation by ER stress and oxidative stress at a narrow 6 h time point. Recombinant AtMC5 was successfully purified from E. coli, with the recombinant AtMC5 working optimally at pH 7, using an optimised reaction buffer containing 10 mM calcium chloride together with 15% sucrose. Like other metacaspases, AtMC5 cleaved after arginine residue and demonstrated a substrate preference towards VRPR. Additionally, AtMC5-RFP was shown to be localised in the cytosol and nucleus of transfected cells. We found no evidence of a strong link between AtMC5 and PCD, and the data provide additional insights into the function of metacaspases in plants and will aid in future research toward further understanding their mode of action. Full article