Search Results (101)

Introduction: Bovine leukemia virus is a single-stranded RNA virus that targets B cell CD5⁺ lymphocytes in cattle. Only a tiny percentage of individuals develop malignant lymphoproliferative disorders, while most remain healthy carriers or experience persistent lymphocytosis. The exact mechanisms leading to lymphoma development are complex and not fully understood. RNA-seq analysis of cows’ peripheral blood leukocytes (PBLs) with and without Bovine leukemia virus (BLV) antibodies was conducted to gain a deeper understanding of molecular events beyond BLV infection. Method: Eighteen samples were selected, and their RNA was sequenced. For gene expression analysis and protein–protein network interactions, three groups were selected, including healthy negative samples (CT, n = 7), asymptomatic carriers (AC, n = 5), and persistent lymphocytosis (PL, n = 6), to provide the differentially expressed gene (DEG) and protein–protein interaction network (PPIN) outputs. Results: Our results demonstrated that in comparison to CT, ACs upregulated TLR7 and transcription activation factors. In the CT vs. PL group, MHC class II, transcription activation factors, and anti-inflammatory cytokines increased, while the acute-phase proteins, antiviral receptors, and inflammatory cytokines decreased. Additionally, antiviral receptors, acute-phase proteins, and inflammatory receptors were downregulated in the PL versus the AC groups. Moreover, PPINs analysis suggested that nuclear receptor corepressor 1 (NCOR1), serine/arginine repetitive matrix 2 (SRRM2), LUC7 like 3 pre-mRNA splicing factor (LUC7L3), TWIST neighbor (TWISTNB), U6 small nuclear RNA and mRNA degradation associated (LSM4), eukaryotic translation elongation factor 2 (EEF2), ubiquitin C (UBC), CD74, and heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNP A2B1) are possible hub gene candidates in the PL group. Conclusions: Our results suggest that innate and cellular immune responses are more loose in severe BLV infectious conditions, while the PPINs revealed that new protein interactions are necessary for oncogenesis. Full article

Eukaryotic elongation factor 1A (eEF1A), the second most abundant intracellular protein, not only plays a key role in peptide elongation, but is also capable of numerous moonlighting functions. Within malignant cells, eEF1A is by no means a neutral bystander but instead actively participates in oncogenic transformations via a myriad of molecular pathways. Thus far, a broad range of small-molecule inhibitors have been identified, which, despite their structural diversity, suppress tumor growth by targeting eEF1A. Interestingly, just as eEF1A enables its oncogenic potential far beyond boosting protein translation, these targeted agents disrupt this oncoprotein via multiple axes distinct from mere protein synthesis inhibition. Whereas the oncogenic mechanisms of eEF1A has been well documented, there lacks a systemic survey of the eEF1A-targeting agents in terms of their mechanisms. Accordingly, the present work aims to examine their multifaceted modes of action more than just blocking protein synthesis. By unveiling these insights, our deepened knowledge of these eEF1A-binding inhibitors will inform the development of future eEF1A-targeted drugs for cancer treatment. Full article

Vibrio harveyi, a pathogenic vibrio, is ubiquitous and the most prevalent disease infecting tropical and subtropical mariculture animals in marine and estuarine environments. It presents a major risk to mariculture companies worldwide and can cause serious disease problems in aquaculture. Recent studies have shown that various pathogens employ post-translational modifications (PTMs) to regulate cellular processes. One of the major PTMs is lysine succinylation, which is widespread in eukaryotic and prokaryotic cells. Many basic biological functions of bacteria are associated with the regulation of lysine (K) succinylation (Ksuc). However, little is known about the role of lysine succinylation in V. harveyi pathogenesis. Here, we performed LC-MS/MS analysis of 1271 proteins from V. harveyi to identify 4252 Ksuc modification sites. The modification of S-ribosylhomocysteine lyase (LuxS) and transcription elongation factor GreA proteins by Ksuc was confirmed through immunoprecipitation combined with Western blot, further validating our proteomics results. Bioinformatics study revealed that the identified Ksuc proteins play roles in multiple cellular processes and vital metabolic pathways, including LuxS, biofilm exopolysaccharide biosynthesis protein EpsG, and the general secretory system (Sec systems), and are proteins that regulate bacterial virulence. Generally, this scientific study serves as the basis for additional research on the pathogenic nature of Ksuc in V. harveyi and reveals potential targets that would accelerate the manufacturing of attenuated vaccines. Full article

Translational control is crucial for maintaining cellular homeostasis, yet the distinct features and regulatory requirements governing protein synthesis during erythropoiesis remain unclear. Here, we reveal that erythroid cells exhibit an extraordinarily high demand for protein synthesis, which is required for their differentiation but also implies the need for tight regulation to prevent excessive erythropoiesis. Notably, we identify significant phosphorylation of eukaryotic elongation factor 2 (eEF2) at threonine 56 during erythroid differentiation, which reduces protein synthesis and acts as a molecular brake to limit unchecked erythropoiesis. This is evidenced by elevated red blood cell counts in peripheral blood and increased incidence of blood hyperviscosity and thrombosis in eEF2_T56M mice, which are deficient in eEF2 phosphorylation. Mechanistic studies demonstrate that eEF2 phosphorylation selectively regulates the translation of a subset of proteins, including NFE2, which partially mediates the effects of eEF2 modification. Collectively, our findings highlight a previously unappreciated role for translational control in achieving efficient and balanced erythropoiesis, with eEF2 phosphorylation serving as a critical protective mechanism against hyperactive erythropoiesis and offering a potential therapeutic target for hematologic disorders such as polycythemia vera. Full article

The eukaryotic translation elongation factor 1 (eEF1) family exhibits critical roles in RNA viral infection beyond its canonical function in protein synthesis. This review analyzes the structural characteristics of eEF1A and the eEF1B complex, and their regulatory mechanisms during viral infection. eEF1A impacts viral replication by stabilizing viral RNA-dependent RNA polymerase (RdRp) complexes, modulating genomic RNA synthesis, and facilitating viral assembly through cytoskeletal regulation. eEF1B subunits contribute through enhancing viral mRNA translation, regulating nuclear transport of viral components, and mediating post-translational modifications. The high conservation of eEF1 proteins across species and their involvement in multiple stages of viral replication establish them as promising broad-spectrum antiviral targets. Current eEF1-targeting compounds like plitidepsin demonstrate efficacy against diverse viral families, though therapeutic development faces challenges in balancing antiviral activity with host toxicity. This review provides a theoretical foundation for developing novel antiviral strategies targeting host–virus interaction interfaces and offers insights into addressing emerging infectious diseases. Full article

Functional gene expression is closely linked to an organism’s physiology and can be quantified using Real-Time Quantitative Reverse Transcription PCR (RT-qPCR). However, the stability of reference gene expression is not absolute, which may impact the accuracy of RT-qPCR results. In this study, we evaluated the suitability of nine genes including receptor for activated protein kinase c1 (rack1), ribosomal protein L6 (rpl6), ribosomal protein L9 (rpl9), ribosomal protein S2 (rps2), ribosomal protein S18 (rps18), ribosomal protein lateral stalk subunit P0 (rplp0), eukaryotic translation elongation factor 1β (eef1b), eukaryotic translation initiation factor 4a (eif4a), eukaryotic translation initiation factor 5a (eif5a) analyzed from RNA sequencing (RNA-Seq) data in addition to three genes including eukaryotic elongation factor 1α (eef1a), β-actin (actb), and glyceraldehyde 3-phosphate dehydrogenase (gapdh) selected from the literature to obtain the best internal controls in the RT-qPCR analysis of M. rosenbergii under overmating stress and natural aging. RefFinder was used to comprehensively evaluate the stability of the candidate reference genes. The initial results showed that three genes (eif5a, rps18, and rplp0) from the RNA-Seq data had relatively stable expression levels, which were more stable than those of the three commonly used reference genes. Eif5a and rps18 were the best combination for the RT-qPCR analysis of M. rosenbergii under overmating stress and aging. Further analysis indicated that eif5a might be the best reference gene for the study of M. rosenbergii. Full article

This study utilized MAC-T cells cultured in vitro as a model to investigate the effects of varying concentrations of valine on α-casein synthesis and its underlying regulatory mechanisms. In this experiment, MAC-T cells were subjected to a 12 h starvation period, followed by the addition of valine in a range of concentrations (a total of seven concentrations: 0.000, 1.596, 3.192, 6.384, 12.768, 25.536, and 51.072 mM, as well as in 10% Fetal Bovine Serum). The suitable range of valine concentrations was determined using enzyme-linked immunosorbent assays (ELISAs). Real-time fluorescent quantitative PCR (RT-qPCR) and Western blot analyses were employed to evaluate the expression levels and phosphorylation states of the casein alpha s1 gene (CSN1S1), casein alpha s2 gene (CSN1S2) and mTOR signaling pathway-related genes. The functionality of the mTOR signaling pathway was further validated through rapamycin (100.000 nM) inhibition experiments. Results indicated that 1× Val (6.384 mM), 2× Val (12.768 mM), 4× Val (25.536 mM), and 8× Val (51.072 mM) significantly enhanced α-casein synthesis (p < 0.01). Within this concentration range, valine significantly upregulated the expression of CSN1S1, CSN1S2, and mTOR signaling pathway-related genes including the RagA gene (RRAGA), RagB gene (RRAGB), RagC gene (RRAGC), RagD gene (RRAGD), mTOR, raptor gene (RPTOR), and 4EBP1 gene (EIF4EBP1), eukaryotic initiation factor 4E (EIF4E), and S6 Kinase 1 (S6K1) (p < 0.01). Notably, the expression of the eukaryotic elongation factor 2 (EEF2) gene peaked at 1× Val (6.384 mM), while the expression of other genes reached their maximum at 4× Val (25.536 mM). Additionally, valine significantly increased the phosphorylation levels of mTOR, S6K1, 4E-binding protein-1 (4EBP1), ribosomal protein S6 (RPS6), and eEF2 (p < 0.01), with the highest phosphorylation levels of mTOR, S6K1, and RPS6 observed at 4× Val (25.536 mM). Rapamycin treatment significantly inhibited mTOR phosphorylation and α-casein synthesis (p < 0.01); however, the addition of 4× Val (25.536 mM) partially mitigated this inhibitory effect. In conclusion, valine promotes α-casein synthesis by activating the mTOR signaling pathway, with an optimal concentration of 4× Val (25.536 mM). Full article

Background: Chinese hamster ovary (CHO) cells are extensively used in the pharmaceutical industry for producing complex proteins, primarily because of their ability to perform human-like post-translational modifications. However, the efficiency of high-quality protein production can vary significantly for monoclonal antibody-producing cell lines, within the CHO host cell lines or by extrinsic factors. Methods: To investigate the complex cellular mechanisms underlying this variability, a phosphoproteomics analysis was performed using label-free quantitative liquid chromatography after a phosphopeptide enrichment of recombinant CHO cells producing two different antibodies and a tunicamycin treatment experiment. Using MaxQuant and Perseus for data analysis, we identified 2109 proteins and quantified 4059 phosphosites. Results: Significant phosphorylation dynamics were observed in nuclear proteins of cells producing the difficult-to-produce 2G12 mAb. It suggests that the expression of 2G12 regulates nuclear pathways based on increases and decreases in phosphorylation abundance. Furthermore, a substantial number of changes in the phosphorylation pattern related to tunicamycin treatment have been detected. TM treatment affects, among other phosphoproteins, the eukaryotic elongation factor 2 kinase (Eef2k). Conclusions: The alterations in the phosphorylation landscape of key proteins involved in cellular processes highlight the mechanisms behind stress-induced cellular responses. Full article

Background: Eukaryotic RNA polymerase I consists of 12 or 11 core subunits and three dissociable subunits, Rrn3, A34, and A49. The A34 and A49 subunits exist as a heterodimer. In silico analysis of the A34 family of transcription factors demonstrates a commonly shared domain structure despite a lack of sequence conservation, as well as N–terminal and C-terminal disordered regions. The common structure of A34 has an N–terminal disordered region followed by a dimerization domain that, in conjunction with A49, contributes to a fold that resembles the TFIIF core. This in turn is followed by a short region that cryo-EM demonstrates resembles an arm and intimately interacts with the PolR1A, PolR1B, and PolR1C subunits of Pol I. Analyses: This Pol I–binding domain is then followed by a region that is not resolved in cryo-EM and is predicted to be intrinsically disordered. Interestingly, the size/length of this disordered structure increases from yeast to humans, and is composed of repeats with unique sequence and biochemical features that also increase in number. Further analyses of the A34 CTD (carboxy–terminal domain) indicate that it has a high probability of undergoing liquid–liquid phase separation. Conclusions: We suggest that this intrinsically disordered domain found in the A34 family of Pol I transcription factors serves a function similar to the CTD of the PolR2A subunit in coordinating transcription initiation and elongation and RNA processing. Lastly, we propose that dynamic acetylation of PAF49 may regulate interactions of the intrinsically disordered CTD and thereby specify liquid–liquid phase separations. Overall, we propose a new paradigm for a repeat-containing CTD in Pol I transcription. Full article

In this study, we revealed a critical role of eukaryotic elongation factor-2 kinase (eEF-2K), a negative regulator of protein synthesis, in regulating T cells during vaccinia virus (VACV) infection. We found that eEF-2K-deficient (eEF-2K⁻/⁻) mice exhibited a significantly higher proportion of VACV-specific effector CD8⁺ T cells without compromising the development of VACV-specific memory CD8⁺ T cells. RNA sequencing demonstrated that eEF-2K⁻/⁻ VACV-specific effector CD8⁺ T cells had enhanced functionality, which improves their capacity to combat viral infection during the effector phase. Moreover, we identified tumor necrosis factor receptor-associated factor 3 (TRAF3) as a critical mediator of the stronger antiviral response observed in eEF-2K⁻/⁻ effector CD8⁺ T cells. These findings suggest that targeting eEF-2K may provide a novel strategy to augmenting effector CD8⁺ T cell responses against viral infections. Full article

Objectives: Aminoglycosides are one of the first classes of natural antibiotics which have not lost relevance due to their broad spectrum of action against Gram-positive, Gram-negative bacteria and mycobacteria. The high growth rate of antimicrobial resistance (AMR) together with the severe side effects of aminoglycosides increase the importance of developing improved semisynthetic derivatives. Methods: In this work, we proposed a synthetic route to new tobramycin derivatives modified at the 6″-position with aminoalkylamine or guanidinoalkylamine residues. Results: The antibacterial activity of the new compounds against reference strains of microorganisms was comparable to the parental tobramycin. In striking contrast to tobramycin (resistance index, >256), its 6″-modified derivatives were significantly more potent against resistant clinical isolates of P. aeruginosa strains (resistance index = 4–16) and they demonstrated a promising AMR circumvention in E. coli strains associated with mutations in the fusA gene encoding elongation factor G. All the obtained tobramycin derivatives exhibited reduced cytotoxicity for the eukaryotic HEK293T cells compared to the tobramycin and thereby they potentially may have improved therapeutic index. The proposed modification of the 6″-position of tobramycin does not change the mechanism of aminoglycoside’s antibacterial activity: new compounds induced translation errors which resulted in the inhibition of protein synthesis in bacterial cells. Conclusions: Taken together, we can suggest that further modifications of the 6″-position of tobramycin may be beneficial for circumvention of AMR to aminoglycosides or used for conjugation with other molecules of interest. Full article

The RNA-binding S1 domain is a β-barrel with a highly conserved RNA-binding site on its surface. This domain is an important part of the structures of different bacterial, archaeal, and eukaryotic proteins. A distinctive feature of the S1 domain is multiple presences (structural repeats) in proteins and protein complexes. Here, we have analyzed all available protein sequences in the UniProt database to obtain data on the distribution of bacterial, eukaryotic and archaeal proteins containing the S1 domain. Mainly, the S1 domain is found in bacterial proteins with the number of domains varying from one to eight. Eukaryotic proteins contain from one to fifteen S1 domains, while in archaeal proteins, only one S1 domain is identified. Analysis of eukaryotic proteins containing S1 domains revealed a group of chloroplast S1 ribosomal proteins (ChRpS1) with characteristic properties of bacterial S1 ribosomal proteins (RpS1) from the Cyanobacteria. Also, in a separate group, chloroplast and mitochondrial elongation factor Ts containing two S1 structural domains were assigned. For mitochondrial elongation factor Ts, the features of S1 in comparison with the RpS1 from Cyanobacteria phylum and the Alphaproteobacteria class were revealed. The data obtained allow us to consider the S1 domain as one of the evolutionary markers of the symbiogenesis of bacterial and eukaryotic organisms. Full article

This study investigated the potential role of specific single-nucleotide polymorphisms (SNPs) in the genes Astrotactin 1 (ASTN1), EBF Transcription Factor 1 (EBF1), Eukaryotic Elongation Factor, Selenocysteine-tRNA Specific (EEFSEC), Microtubule-Associated Serine/Threonine Kinase 1 (MAST1), and Tumor Necrosis Factor Alpha (TNF-α) to assess whether these genetic variants contribute to the risk of spontaneous preterm birth (sPTB). A case-control study was conducted involving 573 women from Croatia and Slovenia: 248 with sporadic sPTB (positive personal and negative family history of sPTB before 37 weeks’ gestation), 44 with familial sPTB (positive personal and family history of sPTB before 37 weeks’ gestation), and 281 control women. The analysis of ASTN1 rs146756455, EBF1 rs2963463, EBF1 rs2946169, EEFSEC rs201450565, MAST1 rs188343966, and TNF-α rs1800629 SNPs was performed using TaqMan real-time PCR. p-values were Bonferroni-adjusted for multiple comparisons. EBF1 SNP rs2963463 was significantly associated with sPTB (p adj = 0.03). Women carrying the CC genotype had a 3–4-times lower risk of sPTB (p adj < 0.0001). In addition, a significant difference in the frequency of the minor C allele was observed when comparing familial sPTB cases with controls (p adj < 0.0001). All other associations were based on unadjusted p-values. The minor T allele of EBF1 SNP rs2946169 was more frequent in sPTB cases overall than in controls, especially in sporadic sPTB (p = 0.045). Similarly, the CC genotype of ASTN1 SNP rs146756455 was more frequent in sporadic sPTB cases compared to controls (p = 0.019). Finally, the TNF-α SNP rs1800629 minor A allele and AA genotype were more common in the familial sPTB group compared to sporadic sPTB and controls (p < 0.05). The EBF1 SNP rs2963463 polymorphism showed a protective effect in the pathogenesis of sPTB, particularly in women carrying the CC genotype. Moreover, EBF1 SNP rs2946169 and ASTN1 SNP rs146756455, as well as TNF-α SNP rs1800629, were associated with an increased risk of sPTB, representing suggestive potential risk factors for sporadic and familial sPTB, respectively. Full article

Group B Coxsackieviruses (CVB) are one of the causative pathogens of myocarditis, which may progress to cardiomyopathy. The pathogenesis of CVB is not fully understood, and effective antiviral therapy is not available. N-acetylcysteine (NAC), the classic antioxidant, has been used in clinical practice for several decades to treat various medical conditions. In this study, the anti-CVB effect of NAC was investigated. We show that NAC dramatically suppressed viral replication and alleviated cardiac injury induced by CVB3. To further study the antiviral mechanism of NAC, RNA-sequencing was performed for CVB3-infected cells with NAC treatment. We found that eukaryotic elongation factor 1 alpha 1 (EEF1A1) is one of the most upregulated genes in CVB3-infected cells. However, EEF1A2, the highly homologous isoform of EEF1A1, remains unchanged. EEF1A1 expression was significantly suppressed by NAC treatment in CVB3-infected cells, while EEF1A2 was not affected. eEF1A1 knockdown significantly inhibited CVB3 replication, implicating that eEF1A1 facilitates viral replication. Importantly, we show that eEF1A1, which was not expressed in the myocardia of newborn mice, was significantly upregulated by CVB3 infection. NAC markedly downregulated the expression of eEF1A1 but not eEF1A2 in the myocardia of CVB3-infected mice. Furthermore, NAC accelerated eEF1A1 degradation by promoting autophagy in CVB3-infected cells. We show that p62, one of the critical adaptors of autophagic targets, interacts with eEF1A1 and was downregulated in CVB3-infected cells upon NAC treatment. Taken together, this study demonstrated that NAC shows a potent anti-CVB effect through the downregulation of eEF1A1. Full article

Zinc finger proteins (ZFPs) are essential transcription factors in eukaryotes, particularly the extensively studied C2H2 family, which is known for its involvement in various biological processes. This research provides a thorough examination and analysis of the C2H2-ZFP gene family in Flammulina filiformis. Using bioinformatics tools, 58 FfC2H2-ZFP genes spread across 11 chromosomes were identified and scrutinized in detail for their gene structures, protein characteristics, and phylogenetic relationships. The study of phylogenetics and synteny sheds light on the evolutionary relationships among C2H2-ZFPs in F. filiformis and other fungi, revealing a complex evolutionary past. The identification of conserved cis-regulatory elements in the gene promoter regions suggests intricate functionalities, particularly in the developmental and stress response pathways. By utilizing RNA-seq and qRT-PCR techniques, the expression patterns of these genes were explored across different developmental stages and tissues of F. filiformis, unveiling distinct expression profiles. Notably, significant expression variations were observed in the stipe elongation region and pilei of various sizes, indicating potential roles in fruiting body morphogenesis. This study enhances our knowledge of the C2H2-ZFP gene family in F. filiformis and lays the groundwork for future investigations into their regulatory mechanisms and applications in fungal biology and biotechnology. Full article