Search Results (503)

The discovery of melatonin as a multifunctional free radical scavenger and its possible synthesis in the mitochondrial matrix of peripheral eukaryotic somatic cells highlights a critical new perspective on the importance of this indole. Experimental evidence supporting these findings is substantial, but there are still lingering questions whether melatonin is a direct radical scavenger in vivo and whether it is synthesized in the mitochondrial matrix. We systematically analyze the innovative experimental approaches that support melatonin’s radical scavenging actions and assess the compelling data supporting its production in mitochondria. Melatonin concentrations are reportedly higher in this organelle than in other cellular compartments. Proteins for the enzymes required to convert serotonin to melatonin are present in the mitochondrial matrix and purified mitochondria synthesize melatonin. In the mitochondrial matrix, melatonin is likely located within the “damage radius” of highly reactive oxygen species. We also summarize novel actions of melatonin associated with its regulation of membrane fluidity, determine the molecular composition of membrane lipid rafts, and modulate liquid–liquid phase separation and biomolecular condensates intracellularly. If the findings discussed herein continue to be validated, melatonin would be in an optimal position to function as an antioxidant and may be a key driver in the context of preserving mitochondrial redox homeostasis and disease mitigation. Full article

Programmed cell death 4 (PDCD4) protein is a tumour suppressor protein that inhibits mRNA translation by inhibiting RNA helicase, eukaryotic initiation factor 4A (eIF4A). We have previously reported that PDCD4 interacts with the internal ribosome entry site (IRES) element of B-cell lymphoma extra-large (Bcl-xL) mRNA and inhibits its IRES-mediated translation initiation. S6 kinase (S6K)-mediated phosphorylation of PDCD4 activates its degradation and derepresses IRES-mediated translation initiation of Bcl-xL mRNA. eIF3F (one of the subunits of eIF3 complex) was reported to recruit S6K to phosphorylate eIF3G. Therefore, we investigated the possibility of co-regulation of PDCD4 and eIF3F by S6K and the regulation of IRES-mediated translation initiation by PDCD4–eIF3F. Here, we demonstrated that PDCD4 interacts with several subunits of eIF3. Specifically, eIF3F directly interacts with PDCD4 in an RNA-independent manner. Depletion of PDCD4 in glioblastoma (GBM) cells resulted in decreased levels of certain eIF3 subunits, including eIF3F. Additionally, depletion of eIF3F from GBM cells decreased the levels of PDCD4 protein. We also showed that PDCD4 and eIF3F directly interact with Bcl-xL RNA independently of each other. By performing IRES reporter, polysome profiling assays and EMSA we have demonstrated that eIF3F regulates IRES-mediated translation of Bcl-xL mRNA, likely via its interaction with PDCD4. Full article

Euonymus bungeanus is a highly valued ornamental tree/shrub species widely utilized in landscaping and afforestation in Northeast Asia, yet molecular studies on this species remain limited due to the lack of validated reference genes for reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). In this study, 16 candidate reference genes were selected based on classical plant reference genes and our previous transcriptome data. Their expression stability was comprehensively evaluated using 64 samples collected from diverse tissues and plants subjected to various abiotic stress/hormone treatments across multiple time points. Across all samples analyzed, PBG1 (20S proteasome beta subunit G1) exhibited the highest overall expression stability, followed by VAPD (vacuolar ATP synthase subunit D) and EIF4A (eukaryotic translation initiation factor 4A). For tissue-specific analysis, TSR2 (pre-rRNA-processing protein), VAPD, and PBG1 demonstrated the greatest stability. Under specific stress conditions, PBG1 and EIF4A were identified as the most stable genes under low- and high-temperature conditions. PP2A (protein phosphatase 2A) and TUB6 (beta-6 tubulin) were optimal for drought stress, while TSR2, SRP (nuclear speckle splicing regulatory-like protein), and PBG1 exhibited superior stability under salt stress. These findings establish a validated panel of reference genes enabling accurate and reliable gene expression normalization in E. bungeanus, thereby facilitating future functional genomics studies in this economically and ecologically important species. Full article

Dairy cows with ketosis display immune dysfunction and a high incidence of infectious diseases, which may partly be attributed to excessive endoplasmic reticulum stress (ERS) and apoptosis in macrophages. The objective of the present study was to assess the role of ERS in macrophage apoptosis of ketotic dairy cows. Compared with healthy cows, the apoptosis number of macrophages and the protein abundance of glucose regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), and activating transcription factor 6 (ATF6); the ratio of phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK)/PERK, phosphorylated inositol-requiring enzyme 1 (p-IRE1)/IRE1 and phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α)/eIF2α; and mean fluorescence intensity of C/EBP homology protein (CHOP) were greater in cows with clinically ketosis (CK). Treatment with FFA increased protein abundance of GRP78, CHOP, ATF6 and p-IRE1/IRE1, and mean fluorescence intensity of CHOP. Furthermore, FFA increased the protein abundance of cysteinyl aspartate-specific proteinase-3 (Caspase-3) and mean fluorescence intensity of Caspase-3 but decreased the Bcl-2/Bax protein abundance ratio, which was accompanied by an increase in the number macrophage apoptosis. Inhibition of ERS via TUDCA attenuated the increased macrophage apoptosis and the activated apoptotic pathways induced by Tn or FFA. Thus, hyperphysiological concentrations of FFA induce apoptosis in macrophages by triggering ERS in ketotic dairy cows. Full article

Cell-free protein synthesis has become a powerful tool for producing functional proteins, circumventing many limitations of live-cell systems. Platforms based on wheat germ extract are favored for their high efficiency in translating and folding complex eukaryotic proteins. To overcome the energy limitation common in such systems, we engineered an Escherichia coli strain to function as a self-renewing ATP source. This strain co-expresses a three-enzyme cascade—adenosine kinase, adenylate kinase, and acetate kinase—that efficiently converts adenosine and acetyl phosphate into ATP. Using the lysate from this biocatalyst to energize an optimized wheat germ extract, we established a high-performance cell-free synthesis platform. This integrated system supported the robust production of multiple recombinant proteins. As a key demonstration, we synthesized human vascular endothelial growth factor 165, which exhibited full biological activity. The cell-free-produced VEGF₁₆₅ significantly stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs). It also potently induced angiogenic responses, including the formation of extensive, interconnected capillary-like networks by HUVECs in vitro and accelerated cell migration in scratch-wound assays. Our work establishes a scalable and efficient platform for on-demand production of bioactive eukaryotic proteins, highlighting its considerable potential for advancing regenerative medicine and related therapeutic applications. Full article

African swine fever virus (ASFV) causes a fatal disease in domestic pigs and wild boars (Sus scrofa), leading to nearly 100% mortality during acute infection and significant economic losses in swine production. Unlike other eukaryotic viruses, ASFV encodes a histone-like nucleic acid-binding protein, pA104R, which is highly conserved and present in all described ASFV isolates of different genotypes. Moreover, A104R-like sequences have been identified in the genomes of soft ticks, which can replicate and transmit ASFV. Using a virulent genotype IX field isolate from Kenya, we analyzed the importance of A104R for viral replication in a permissive wild boar cell line (WSL). In this study, we confirmed that A104R is not essential for in vitro replication of ASFV. Loss of A104R did not detectably affect viral DNA replication or RNA transcription but led to a moderate reduction in virus titers and plaque sizes. Substitution of A104R with a similar ASFV-like element derived from the genome of an Ornithodoros moubata soft tick was not capable of rescuing the deletion mutant phenotype. In contrast, reintroduction of the authentic A104R open reading frame (ORF) into the deletion mutant fully restored wild-type virus growth properties. In accompanying studies, we verified the DNA-binding activities of the ASFV- and tick-derived A104R proteins and performed mass spectrometric analyses of the pA104R interactome. These experiments revealed, besides DNA-dependent co-precipitated proteins, specific DNA-independent protein–protein interactions of pA104R with other viral and cellular proteins. Full article

Background/Objectives: The telomerase RNA (TR) is an indispensable part of the telomerase protein complex responsible for telomere elongation in most eukaryotic species. Although the telomere terminal repeat sequence (TTAGGC)_n in Caenorhabditis elegans has been known for years, a telomerase RNA gene was not identified in the entire phylum of Nematoda until recently. Methods: In this exploratory study, we employ a combination of different approaches to identify likely telomerase RNA candidates among putative non-coding transcripts. Results: A detailed analysis of our prime candidate shows compelling evidence that it encodes the missing RNA element of the telomerase complex, which is notably located in an intron of the coding gene nmy-2. Using nmy-2 homologs in other nematodes as anchors, we annotate the conserved TR gene in 21 Caenorhabditis species. We furthermore show that the intronic localization of the TR gene is conserved in two distinct branching groups of the Caenorhabditis phylogeny and demonstrate that this property likely emerged from a single point of origin. Conclusions: While the intronic TR represents a very interesting evolutionary adaption that seems to have been successful in the Elegans and Japonica groups, the question regarding the macroscopic nematode TR evolution remains. Full article

Polyglutamine expansion in Ataxin-2 (ATXN2) is responsible for rare, dominantly inherited Spinocerebellar Ataxia type 2 (SCA2). Together with its paralog Ataxin-2-like (ATXN2L), both proteins have received much interest, since the deletion of their yeast and fly orthologs alleviates TDP-43-triggered neurotoxicity in Amyotrophic Lateral Sclerosis models. Their typical structure across evolution combines LSm with LSm-Associated Domains and a PAM2 motif. To understand the physiological regulation and functions of Ataxin-2 homologs, the phylogenesis of sequences was analyzed. Human ATXN2 harbors multiple alternative start codons, e.g., from an intrinsically disordered sequence (IDR) present since armadillo, or from the polyQ sequence that arose since amphibians, or from the LSm domain since primitive eukaryotes. Multiple smaller isoforms also exist across the C-terminus. Therapeutic knockdown of polyQ expansions in human ATXN2 should selectively target exon 1B. PolyQ repeats developed repeatedly, usually framed and often interrupted by (poly)Pro, originally near PAM2. The LSmAD sequence appeared in algae as the characteristic Ataxin-2 feature with strong conservation. Frequently, Ataxin-2 has added domains, likely due to transcriptional readthrough of neighbor genes during cell stress. These chimerisms show enrichment of rRNA processing; nutrient store mobilization; membrane strengthening via lipid, protein, and glycosylated components; and cell protrusions. Thus, any mutation of Ataxin-2 has complex effects, also affecting membrane resilience. Full article

Nucleic acids are essential dietary components with diverse physiological functions. Numerous studies have focused on the biological functions of nucleotides, nucleosides, and functional RNAs such as microRNAs. However, the nutritional value of ribosomal RNA (rRNA)-derived oligonucleotides, which are likely the predominant nucleic acid-derived components in foods, remains largely unexplored. Here, yeast was used as a food-associated eukaryotic model organism to investigate the uptake and utilization of rRNA-derived oligonucleotides. Yeast efficiently utilized short RNA oligonucleotides (approximately 5–30 nt) as nutrient sources, supporting robust cell growth. Confocal microscopy confirmed rapid uptake of Cy5-labeled RNA oligonucleotides by yeast cells. Proteomic analysis further revealed marked upregulation of proteins involved in endocytosis and autophagy in yeast cultured with RNA oligonucleotides. Collectively, these findings demonstrate that yeast can internalize and metabolize rRNA-derived oligonucleotides as efficient nutrient sources, likely through coordinated endocytic and autophagic pathways. This study highlights the nutritional potential of rRNA-derived oligonucleotides and provides a foundation for their future application in functional foods and fermentation systems. Full article

The presence of aberrant double-stranded DNA (dsDNA) in the cytoplasm of cells is sensed by unique pattern recognition receptors (PRRs) to trigger innate immune response. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) signaling pathway is activated by the presence of non-self or mislocalized self-dsDNA from nucleus or mitochondria released in response to DNA damage or cellular stress in the cytoplasm. Activation of cGAS leads to the synthesis of the second messenger cyclic GMP–AMP (cGAMP), which binds and activates STING, triggering downstream signaling cascades that result in the production of type I interferons (IFNs) and proinflammatory cytokines. Here, we show that diverse immunostimulatory dsDNA ligands and chemotherapy agents like Doxorubicin and Taxol trigger the integrated stress response (ISR) by activating endoplasmic reticulum (ER) stress kinase, protein kinase RNA-like ER kinase (PERK), in addition to the canonical IFN pathways. PERK-mediated phosphorylation and inactivation of the alpha subunit of eukaryotic translation initiation factor-2 (eIF2α) result in the formation of stress granules (SGs). SG formation by dsDNA was significantly reduced in PERK knockout cells or by inhibiting PERK activity. Transcriptional induction of IFNβ and cytokines, ISR signaling, and SG formation by dsDNA was dampened in cells lacking PERK activity, STING, or key stress-granule nucleating protein, Ras-GAP SH3 domain-binding protein 1 (G3BP1), demonstrating an important role of the signal transduction pathway mediated by STING and SG assembly. Lastly, STING regulates reactive oxygen species (ROS) production in response to DNA damage, highlighting the crosstalk between DNA sensing and oxidative stress pathways. Together, our data identify STING–PERK–G3BP1 signaling axis that couples cytosolic DNA sensing to stress response pathways in maintaining cellular homeostasis. Full article

The described features of dinoflagellate gene expression indicate the predominance of post-transcriptional and translational regulation over transcriptional control. These microorganisms also exhibit extensive RNA editing and distinctive splicing characteristics. This regulatory landscape underscores the central role of RNA-binding proteins in dinoflagellate biology. In this review, we summarize current knowledge on major RNA-binding protein groups identified or bioinformatically annotated in dinoflagellates, including RNA recognition motif domain-containing proteins, Sm and Sm-like family, KH domain-containing proteins, zinc-finger proteins, and Pumilio family proteins, S1 domain-containing and cold shock domain-containing proteins, DEAD/DEAH-box RNA helicases, and pentatricopeptide repeat proteins. We focus on the features of their conserved domains, their functions in eukaryotes, and available data on their presence and putative roles in dinoflagellate cells. Integrating genomic, transcriptomic, and proteomic evidence, and where possible experimental data, we highlight both their overall conservation and potential lineage-specific traits. Our aim is to provide a concise synthesis of current knowledge, identify key uncertainties, and outline promising directions for future research into the evolution and cellular roles of RNA-binding proteins in this ecologically and biologically remarkable group. Full article

G protein-coupled receptors (GPCRs) are transmembrane proteins crucial for signal transduction in eukaryotes, responding to diverse extracellular signals. Researchers have found and systematically summarized 14 distinct types of GPCRs in fungi but their distribution among numerous fungal species remained largely unexamined. Additionally, three families of mammalian homologs (Rhodopsin, Glutamate, and Frizzled) have been found in previous studies, but they are not included in the systematic classification of fungal GPCRs. Our study establishes a unified classification of 17 GPCR classes in fungi, combining 14 fungal and 3 mammalian previously recognized groups, and classifies 28,294 GPCRs across 1357 fungal species, significantly expanding the scale of GPCRs in fungi and demonstrating their broader distribution. We found that mammalian homologs are notably more prevalent in Early Diverging Fungi (EDF), whereas the previous 14 classes are predominantly found in Ascomycota and Basidiomycota. The most abundant class detected in fungi was Pth11-like GPCRs, exclusively found in Pezizomycotina and involved in fungal pathogenicity. Our analysis suggested that Pezizomycotina ancestor possessed an extensive array of Pth11-like GPCRs, but over time, some species underwent considerable reductions in these GPCRs in conjunction with genome contractions. Utilizing a custom-built convolutional neural network (CNN) for the identification of fungal GPCRs, we identified several putative novel fungal GPCRs. Predicted interactions between these prospective new GPCRs and G-alpha proteins, as simulated by AlphaFold Multimer, provided additional support for their functional relevance. In conclusion, our work defines the first large-scale, unified classification of fungal GPCRs, reveals lineage-specific expansions and contractions, and uncovers previously unrecognized GPCR candidates with potential functional roles in fungal signaling. Full article

The RNA interference machinery is crucial for regulating the activity of both native and foreign genes across all eukaryotes. The core protein families involved in this process are Dicer-like, Argonaute, and RNA-dependent RNA polymerase. However, plants exhibit remarkable diversity within each family and extensively use RNA interference mechanisms in their intricate immune responses. This review examines the role of RNA interference in plant interactions with various pathogens, including viruses, viroids, fungi, oomycetes, and bacteria. Plant diseases cause an estimated $220 billion in annual damage, with microorganisms accounting for approximately $150 billion. Hence, the focus is on the most severe plant diseases, specifically those caused by fungi and viruses. Additionally, recent biotechnological advancements are discussed, with an emphasis on the application of RNA interference for the development of novel plant defence strategies. Full article

Life is defined by self-governing networks of molecules that change conformation cyclically, converting thermodynamic motion into directional work and structure. A spectrum of scale, from nanoscopic to macroscopic, involves a shift from intracellular thermodynamically driven processes (thermal agitation ultimately rooted in quantum phenomena) to intercellular bulk flows described by classical physics; from short-distance transport involving diffusion and cytoskeletal transport to long-distance pressure fluxes in hydraulic networks. A review of internal transport systems in macroscopic eukaryotes suggests that a key evolutionary step favoring large size and multicellularity involved exploiting molecular-scale stochasticity to generate organized bulk flows (e.g., motor proteins collectively generating mechanical pressures in metazoan tissues such as cardiac muscle; within tracheophytes, active and passive phloem loading/unloading inducing pressure gradients, and active regulation enabling passive xylem function and hydraulic reliability; sieve-like conduction in heterokonts; and peristaltic shuttle streaming in myxogastrian plasmodia). Macroscopic physiologies are underpinned by Brownian molecular thermodynamics and thus quantum mechanics; the apparently disparate physiologies of large organisms share a fundamental operating principle at small scales. However, the specific translocation mechanisms that extend this functioning to larger scales are embroiled in bauplans, representing phylogenetic constraints to body size. Full article

TPPP (tubulin polymerization promoting protein)-like proteins are found throughout the living world. The individual members of this protein family are distinguished according to how many times and how completely their characteristic structural element, the p25alpha domain, is found in them. Phylogenomic occurrences of the members of the family differ from each other. Animals, fungi, algae, and various groups of unicellular organisms have their characteristic proteins. The two phylogenomic multi-supergroups, Opimoda+ and Diphoda+, show very different patterns in the occurrence of TPPP types. By using BLAST search in protein and nucleotide databases, we found that the previously known phylogenomic distribution is not strictly true, e.g., fungal type TPPPs are not only found in fungi. We primarily analyzed the Opisthokonta clade but also examined broader relationships. It was confirmed that the occurrence of TPPPs/genes is linked to the presence of the eukaryotic flagellum. A TPPP that contains the entire p25alpha domain twice and occurs only in Opisthokonta was identified. We also identified a TPPP in choanoflagellates and in the uncertainly classified Opisthokonta Tunicaraptor unikontis, which was previously known only in the Diphoda+ clade. On the other hand, we found an Opisthokonta (Opimoda+)-specific TPPP in a Heterolobosea (Diphoda+). Based on these results, we need to rethink the evolutionary history of TPPPs. Full article