Search Results (50)

Background: Bovine babesiosis, caused by the tick-borne apicomplexan parasite Babesia spp., is an economically significant disease that threatens the cattle industry worldwide. Babesia bovis is the most pathogenic species, leading to high morbidity and mortality in infected animals. One promising approach to vaccination against bovine babesiosis involves the use of multiple protective antigens, offering advantages over traditional live-attenuated vaccines. Tools such as immunobioinformatics and reverse vaccinology have facilitated the identification of novel antigens. Enolase, a “moonlighting” enzyme of the glycolytic pathway with demonstrated vaccine potential in other pathogens, has not yet been studied in B. bovis. Methods: In this study, the enolase gene from two B. bovis isolates was successfully identified and sequenced. The gene, consisting of 1366 base pairs, encodes a predicted protein of 438 amino acids. Its expression in intraerythrocytic parasites was confirmed by RT-PCR. Two peptides containing predicted B-cell epitopes were synthesized and used to immunize rabbits. Hyperimmune sera were then analyzed by ELISA, confocal microscopy, Western blot, and an in vitro neutralization assay. Results: The hyperimmune sera showed high antibody titers, reaching up to 1:256,000. Specific antibodies recognized intraerythrocytic merozoites by confocal microscopy and bound to a ~47 kDa protein in erythrocytic cultures of B. bovis as detected by Western blot. In the neutralization assay, antibodies raised against peptide 1 had no observable effect, whereas those targeting peptide 2 significantly reduced parasitemia by 71.99%. Conclusions: These results suggest that B. bovis enolase contains B-cell epitopes capable of inducing neutralizing antibodies and may play a role in parasite–host interactions. Enolase is therefore a promising candidate for further exploration as a vaccine antigen. Nonetheless, additional experimental studies are needed to fully elucidate its biological function and validate its vaccine potential. Full article

Oxidative stress is key in immune defense against fungal infections, such as those caused by Sporothrix schenckii, the dimorphic fungus responsible for sporotrichosis. Phagocytic cells utilize oxidative stress as a crucial mechanism to control pathogen spread. During S. schenckii infection, phagocytic cells recognize pathogen-associated molecular patterns (PAMPs) on their surface through conserved transmembrane or soluble receptors, known as pattern recognition receptors (PRRs). This recognition triggers a cascade of immune responses, including the generation reactive oxygen species (ROS) essential for pathogen elimination. However, S. schenckii has developed sophisticated mechanisms to evade and counteract this response, contributing to its persistence in the host. These mechanisms include the production of antioxidant enzymes, alterations to its cell wall (CW), and the production of melanin, which helps neutralize oxidative stress. In addition, S. schenckii modulates the production of other proteins, such as moonlighting proteins, suggested to have roles in immune evasion and stress response, helping its survival in the host. These strategies, along with the modulation of gene expression, allow the fungus to survive and persist inside the immune system’s hostile environment, facilitating the progression of the infection. Understanding these interactions between phagocytic cells and S. schenckii is key to developing more effective therapeutic strategies to combat sporotrichosis. Full article

As a moonlighting protein with multiple enzymatic activities, peroxiredoxin 6 (PRDX6) maintains redox homeostasis, regulates phospholipid metabolism, and mediates intra- and inter-cellular signaling transduction. Its expression and activity can be regulated by diverse stressors. However, the roles and relevant mechanisms of these regulators in various conditions have yet to be comprehensively reviewed. In this study, these stressors were systematically reviewed both in vivo and in vitro and classified into chemical, physical, and biological categories. We found that the regulatory effects of these stressors on PRDX6 expression were primarily mediated via key transcriptional factors (e.g., NRF2, HIF-1α, SP1, and NF-κB), micro-RNAs, and receptor- or kinase-dependent signaling pathways. Additionally, certain stressors, including reactive oxygen species, pH fluctuations, and post-translational modifications, induced the structure-based functional switches in the PRDX6 enzyme. We further reviewed the altered expression of PRDX6 under various disease conditions, with a particular focus on neuropsychiatric disorders and cancers, and proposed the concept of PRDX6-related disorders (PRD), which refers to a spectrum of diseases mediated by or associated with dysregulated PRDX6 expression. Finally, we found that an exogenous supplementation of PRDX6 protein provided preventive and therapeutic potentials for oxidative stress-related injuries in both in vivo and in vitro models. Taken together, this review underscores the critical role of PRDX6 as a cellular orchestrator in response to various stressors, highlighting its clinical potential for disease monitoring and the development of therapeutic strategies. Full article

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K⁺ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K⁺ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding. Full article

Dysfunction of some mitochondrial aminoacyl-tRNA synthetases (encoded by the KARS1, HARS2, LARS2 and NARS2 genes) results in a great variety of phenotypes ranging from non-syndromic hearing impairment (NSHI) to very complex syndromes, with a predominance of neurological signs. The diversity of roles that are played by these moonlighting enzymes and the fact that most pathogenic variants are missense and affect different domains of these proteins in diverse compound heterozygous combinations make it difficult to establish genotype–phenotype correlations. We used a targeted gene-sequencing panel to investigate the presence of pathogenic variants in those four genes in cohorts of 175 Spanish and 18 Colombian familial cases with non-DFNB1 autosomal recessive NSHI. Disease-associated variants were found in five cases. Five mutations were novel as follows: c.766C>T in KARS1, c.475C>T, c.728A>C and c.1012G>A in HARS2, and c.795A>G in LARS2. We provide audiograms from patients at different ages to document the evolution of the hearing loss, which is mostly prelingual and progresses from moderate/severe to profound, the middle frequencies being more severely affected. No additional clinical sign was observed in any affected subject. Our results confirm the involvement of KARS1 in DFNB89 NSHI, for which until now there was limited evidence. Full article

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy. Full article

Moonlighting proteins combine multiple functions in one polypeptide chain. An increasing number of moonlighting proteins are being found in diverse fungal taxa that vary in morphology, life cycle, and ecological niche. In this mini-review we discuss examples of moonlighting proteins in fungi that illustrate their roles in transcription and DNA metabolism, translation and RNA metabolism, protein folding, and regulation of protein function, and their interaction with other cell types and host proteins. Full article

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins’ enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment. Full article

Molybdenum-containing enzymes of the xanthine oxidase (XO) family are well known to catalyse oxygen atom transfer reactions, with the great majority of the characterised enzymes catalysing the insertion of an oxygen atom into the substrate. Although some family members are known to catalyse the “reverse” reaction, the capability to abstract an oxygen atom from the substrate molecule is not generally recognised for these enzymes. Hence, it was with surprise and scepticism that the “molybdenum community” noticed the reports on the mammalian XO capability to catalyse the oxygen atom abstraction of nitrite to form nitric oxide (NO). The lack of precedent for a molybdenum- (or tungsten) containing nitrite reductase on the nitrogen biogeochemical cycle contributed also to the scepticism. It took several kinetic, spectroscopic and mechanistic studies on enzymes of the XO family and also of sulfite oxidase and DMSO reductase families to finally have wide recognition of the molybdoenzymes’ ability to form NO from nitrite. Herein, integrated in a collection of “personal views” edited by Professor Ralf Mendel, is an overview of my personal journey on the XO and aldehyde oxidase-catalysed nitrite reduction to NO. The main research findings and the path followed to establish XO and AO as competent nitrite reductases are reviewed. The evidence suggesting that these enzymes are probable players of the mammalian NO metabolism is also discussed. Full article

Intrinsically disordered proteins (IDPs) are multifunctional due to their ability to adopt different structures depending on the local conditions. The intrinsically disordered regions of methyl-CpG-binding domain (MBD) proteins play important roles in regulating growth and development by interpreting DNA methylation patterns. However, whether MBDs have a stress-protective function is far from clear. In this paper, soybean GmMBD10c protein, which contains an MBD and is conserved in Leguminosae, was predicted to be located in the nucleus. It was found to be partially disordered by bioinformatic prediction, circular dichroism and a nuclear magnetic resonance spectral analysis. The enzyme activity assay and SDS-PAGE results showed that GmMBD10c can protect lactate dehydrogenase and a broad range of other proteins from misfolding and aggregation induced by the freeze–thaw process and heat stress, respectively. Furthermore, overexpression of GmMBD10c enhanced the salt tolerance of Escherichia coli. These data validate that GmMBD10c is a moonlighting protein with multiple functions. Full article

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein–protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321. Full article

The glycolytic enzyme PykA has been reported to drive the metabolic control of replication through a mechanism involving PykA moonlighting functions on the essential DnaE polymerase, the DnaC helicase and regulatory determinants of PykA catalytic activity in Bacillus subtilis. The mutants of this control suffer from critical replication and cell cycle defects, showing that the metabolic control of replication plays important functions in the overall rate of replication. Using biochemical approaches, we demonstrate here that PykA interacts with DnaE for modulating its activity when the replication enzyme is bound to a primed DNA template. This interaction is mediated by the CAT domain of PykA and possibly allosterically regulated by its PEPut domain, which also operates as a potent regulator of PykA catalytic activity. Furthermore, using fluorescence microscopy we show that the CAT and PEPut domains are important for the spatial localization of origins and replication forks, independently of their function in PykA catalytic activity. Collectively, our data suggest that the metabolic control of replication depends on the recruitment of PykA by DnaE at sites of DNA synthesis. This recruitment is likely highly dynamic, as DnaE is frequently recruited to and released from replication machineries to extend the several thousand RNA primers generated from replication initiation to termination. This implies that PykA and DnaE continuously associate and dissociate at replication machineries for ensuring a highly dynamic coordination of the replication rate with metabolism. Full article

Bacterial and fungal adhesins mediate microbial aggregation, biofilm formation, and adhesion to host. We divide these proteins into two major classes: professional adhesins and moonlighting adhesins that have a non-adhesive activity that is evolutionarily conserved. A fundamental difference between the two classes is the dissociation rate. Whereas moonlighters, including cytoplasmic enzymes and chaperones, can bind with high affinity, they usually dissociate quickly. Professional adhesins often have unusually long dissociation rates: minutes or hours. Each adhesin has at least three activities: cell surface association, binding to a ligand or adhesive partner protein, and as a microbial surface pattern for host recognition. We briefly discuss Bacillus subtilis TasA, pilin adhesins, gram positive MSCRAMMs, and yeast mating adhesins, lectins and flocculins, and Candida Awp and Als families. For these professional adhesins, multiple activities include binding to diverse ligands and binding partners, assembly into molecular complexes, maintenance of cell wall integrity, signaling for cellular differentiation in biofilms and in mating, surface amyloid formation, and anchorage of moonlighting adhesins. We summarize the structural features that lead to these diverse activities. We conclude that adhesins resemble other proteins with multiple activities, but they have unique structural features to facilitate multifunctionality. Full article

Predatory outer membrane vesicles (OMVs) secreted by myxobacteria fuse readily with the outer membranes of Gram-negative bacteria, introducing toxic cargo into their prey. Here we used a strain of the myxobacterium Myxococcus xanthus that produces fluorescent OMVs to assay the uptake of OMVs by a panel of Gram-negative bacteria. M. xanthus strains took up significantly less OMV material than the tested prey strains, suggesting that re-fusion of OMVs with producing organisms is somehow inhibited. The OMV killing activity against different prey correlated strongly with the predatory activity of myxobacterial cells, however, there was no correlation between OMV killing activity and their propensity to fuse with different prey. It has previously been proposed that M. xanthus GAPDH stimulates the predatory activity of OMVs by enhancing OMV fusion with prey cells. Therefore, we expressed and purified active fusion proteins of M. xanthus glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase (GAPDH and PGK; moonlighting enzymes with additional activities beyond their roles in glycolysis/gluconeogenesis) to investigate any involvement in OMV-mediated predation. Neither GAPDH nor PGK caused lysis of prey cells or enhanced OMV-mediated lysis of prey cells. However, both enzymes were found to inhibit the growth of Escherichia coli, even in the absence of OMVs. Our results suggest that fusion efficiency is not a determinant of prey killing, but instead resistance to the cargo of OMVs and co-secreted enzymes dictates whether organisms can be preyed upon by myxobacteria. Full article

Coronaviruses interact with protein or carbohydrate receptors through their spike proteins to infect cells. Even if the known protein receptors for these viruses have no evolutionary relationships, they do share ontological commonalities that the virus might leverage to exacerbate the pathophysiology. ANPEP/CD13, DPP IV/CD26, and ACE2 are the three protein receptors that are known to be exploited by several human coronaviruses. These receptors are moonlighting enzymes involved in several physiological processes such as digestion, metabolism, and blood pressure regulation; moreover, the three proteins are expressed in kidney, intestine, endothelium, and other tissues/cell types. Here, we spot the commonalities between the three enzymes, the physiological functions of the enzymes are outlined, and how blocking either enzyme results in systemic deregulations and multi-organ failures via viral infection or therapeutic interventions is addressed. It can be difficult to pinpoint any coronavirus as the target when creating a medication to fight them, due to the multiple processes that receptors are linked to and their extensive expression. Full article