Ribosome Function Research Articles

Analysis of the Anticancer Mechanism of OR3 Pigment from Streptomyces coelicolor JUACT03 Against the Human Hepatoma Cell Line Using a Proteomic Approach.

This study assessed OR3 pigment, derived from Streptomyces coelicolor JUACT03, for its anticancer potential on HepG2 liver cancer cells and its safety on HEK293 normal cells. OR3 induced apoptosis and inhibited HepG2 cell proliferation, confirmed by caspase activation, Sub-G1 phase cell cycle arrest, and reduced colony formation. Proteomic analysis revealed altered expression of proteins associated with ribosomal function, mRNA processing, nuclear transport, proteasome activity, carbohydrate metabolism, chaperone function, histone regulation, and vesicle-mediated transport. Downregulation of proteins in MAPKAP kinase1, EIF2, mTOR, and EIF4 pathways contributed to apoptosis and cell cycle arrest. Changes in c-MYC, FUBP1 target proteins and upregulation of Prohibitin-1 (PHB1) were also noted. Western blot analysis supported alterations in eIF2, mTOR, and RAN pathways, including downregulation of RAB 5, c-MYC, p38, MAPK1, and MAPK3. OR3 exhibited significant anti-angiogenic activity in the in ovo CAM assay. In summary, OR3 demonstrated strong anticancer effects, inducing apoptosis, hindering proliferation, and displaying antiangiogenic properties. These findings highlight OR3's potential as an anticancer drug candidate, warranting further in vivo exploration.

HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells

BackgroundHigh-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins.ResultsHere, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis.ConclusionTaken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

Critical steps in the assembly process of the bacterial 50S ribosomal subunit.

During assembly, ribosomal particles in bacteria fold according to energy landscapes comprised of multiple parallel pathways. Cryo-electron microscopy studies have identified a critical maturation step that occurs during the late assembly stages of the 50S subunit in Bacillus subtilis. This step acts as a point of convergency for all the parallel assembly pathways of the subunit,where an assembly intermediate accumulates in a 'locked' state, causing maturation to pause. Assembly factors then act on this critical step to 'unlock' the last maturation steps involving the functional sites. Without these factors, the 50S subunit fails to complete its assembly, causing cells to die due to a lack of functional ribosomes to synthesize proteins. In this review, we analyze these findings in B. subtilis and examine other cryo-EM studies that have visualized assembly intermediates in different bacterial species, to determine if convergency points in the ribosome assembly process are a common theme among bacteria. There are still gaps in our knowledge, as these methodologies have not yet been applied to diverse species. However, identifying and characterizing these convergency points can reveal how different bacterial species implement unique mechanisms to regulate critical steps in the ribosome assembly process.

Sugar-coated cannonballs for effective antibacterial treatments but with negligible cytotoxicity based on silver (copper) -containing carbon dots and their mechanism analysis

With the gradual development of carbon dots (CDs) in the antimicrobic application, efforts have been still devoted to overcome the dilemma that more effective antibacterial treatment would lead to inevitable higher cytotoxicity. In addition, their bactericidal mechanisms are still in urgent need to be clarified. Therefore, silver (copper)-containing carbon dots have been prepared via one microwave-irradiation strategy using glucose as precursors and metal salts as passivators. It was discovered that the as-prepared metal-containing CDs have the comparable or better bactericidal effect while lower cytotoxicity towards mammalian cells compared with most of corresponding commercial metal-containing antibacterial agents and their free metal ions, because of the good permeability towards bacterial cells originating from the excellent metal dispersion and glucose disguiser in/on the CDs. The minimum bactericidal concentration of silver(copper)-containing carbon dots against 1.5 (0.5) × 107 CFU/mL Escherichia coli was less than 25 (75) μg/mL. Different bactericidal mechanisms between the two metal-containing CDs and the free copper/silver ions were revealed in details. It was found that the metal ions could directly inhibit the function of ribosomes in bacterial cells, while the metal-containing CDs showed negligible influence towards ribosome directly, instead, they inhibited bacterial metabolism through more complex pathways such as metabolic disorders, transporter inactivation or inhibition of the nucleic acid synthesis. Since the mammalians cells can switch to other pathways more flexibly than bacterial cells under the disturbance of the same metabolism pathway inhibition, the metal-containing CDs provides excellent biocompatibility.

Molecular mechanisms implicated in protein changes in the Alzheimer’s disease human hippocampus

This study aimed to elucidate the specific biochemical pathways linked to changes in proteins in the Alzheimer's disease (AD) human hippocampus. Our data demonstrate a constant rise in the expression of four proteins (VGF, GFAP, HSPB1, and APP) across all eleven studies. Notably, UBC was the most centrally involved and had increased expression in the hippocampus tissue of individuals with AD. Modified proteins in the hippocampal tissue were found to activate the innate immune system and disrupt communication across chemical synapses. Four hub proteins (CD44, APP, ITGB2, and APOE) are connected to amyloid plaques, whereas two hub proteins (RPL24 and RPS23) are related to neurofibrillary tangles (NFTs). The presence of modified proteins was discovered to trigger the activation of microglia and decrease the functioning of ribosomes and mitochondria in the hippocampus. Three significant microRNAs (hsa-miR-106b-5p, hsa-miR-17–5p, and hsa-miR-16–5p) and transcription factors (MYT1L, PIN1, and CSRNP3) have been discovered to improve our understanding of the alterations in proteins within the hippocampal tissues that lead to the progression of AD. These findings establish a path for possible treatments for AD to employ therapeutic strategies that specifically focus on the proteins or processes linked to the illness.

Abstract 2763: A specific ribomethylome pattern in lung cancer is associated with increased risk of metastasis

Abstract Non-coding RNAs drive cancer phenotypes and are associated with patients’ outcome. SnoRNAs are required for the 2’-O-methylation (2’-O-Me) and pseudouridylation of ribosomal RNA (rRNA) thus being essential for ribosomal biogenesis and function. So far, the role of rRNA methylation (ribomethylation) for non-small cell lung cancer (NSCLC) pathogenesis is unknown. To investigate the role of ribomethylation we included samples from 92 patients with lung adenocarcinoma (pathological stage IA to IIB) for comprehensive multi-omics profiling. Amongst transcriptomics, small RNA sequencing, proteomics, and whole exome sequencing, ribomethylation as epitranscriptomic dimension was analyzed using RiboMethSeq. We focused the analysis on complete tumor resection samples. Here 47 out of 106 2’-O-Me sites were fully methylated in all tumor samples suggesting a crucial role for methylation at this position. Surprisingly, 59 sites had a dynamic methylation pattern. By combining ribomethylation sequencing data of dynamic sites with whole-exome-sequencing as well as transcriptome and proteome analyses using a multi-omics factor analysis (MOFA), we discovered a 2’-O-Me signature in a subset of NSCLC patients with a high risk of metastasis and poor prognosis. We termed the patient subset, with underlying ribomethylation signature, epitranscriptomic pro-metastatic phenotype (EPROMET). This phenotype was not associated with genetic mutations as analyzed by exome sequencing. Analysis of gene sets in both transcriptomics and proteomics showed an upregulation of secreted and extracellular matrix proteins in EPROMET patients. Alongside, the ribomethylation site with major contribution to the EPROMET signature; 18S-Um799, mediated through SNORD105/105B, was found to be the most dynamic within the dataset. It also showed the largest difference between EPROMET and non-EPROMET patients. For functional validation, lung cancer cell lines with knockout of snoRNAs contributing to EPROMET signature were generated using CRISPR/Cas9. Lung cancer cells lacking the corresponding rRNA modifications migrated slower in vitro, failed to grow at a distant site in vivo and were impaired in metastasis. Functionally, it was shown that the modulated ribomethylation signature effected translation of secreted proteins by altered mRNA binding to ribosomes. This study indicates the presence of a ribomethylome-associated phenotype of NSCLC related to metastasis and poor prognosis. Altered ribomethylation affects the ribosome function towards differential expression patterns of secreted proteins. Taken together, an epitranscriptomic pattern of 2’-O-Me is associated with invasion/migration properties of NSCLC cells and with development of metastasis. The ribomethylome may present a suitable biomarker and a potential therapeutic target. Citation Format: Cornelius Pauli, Daniel Heid, Christian Rohde, Nadja Krall, Sylvain Delaunay, Michael Kienhoefer, Christian Tischer, Michael Allgäuer, Maximilian Felix Blank, Fengbiao Zhou, Michael Kardorff, Michael Thomas, Hauke Winter, Sarah Sandmann, Marc Kriegsmann, Marc Schneider, Thomas Muley, Alexander Brobeil, Nicole Bäumer, Sebastian Bäumer, Simon Raffel, Albrecht Stenzinger, Peter Schirmacher, Junyan Lu, Judith Zaugg, Michaela Frye, Carsten Müller-Tidow. A specific ribomethylome pattern in lung cancer is associated with increased risk of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2763.

Abstract 1558: Deciphering the role of ribosomal paralogs RPL39/RPL39L in metaplastic breast cancer

Abstract Metaplastic breast cancer (MpBC) is a rare, highly aggressive, and metastatic malignancy that accounts for <5% of all breast cancer diagnoses. Most MpBCs display a triple-negative breast cancer (TNBC) phenotype (ER-/PR-/HER2-). MpBCs exhibit the most dismal prognosis of all breast cancer subtypes. The standard of care for metastatic MpBC remains systemic chemotherapy, despite known resistance to most cytotoxic drugs. MpBCs are enriched with epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) markers. We previously described a unique molecular signature for breast cancer stem cells (BCSCs) derived from patient biopsies and identified ribosomal protein L39 (RPL39) as a critical driver of treatment resistance, BCSCs self-renewal, and lung metastases in TNBC/MpBC. Growing evidence has also demonstrated aberrant expression of the testis-specific ribosomal protein paralog 39L (RPL39L) in several cancer types. Here, we hypothesize that RPL39 and its paralog RPL39L alter ribosome composition and function to initiate a specific translational landscape that promotes tumor growth and enhanced metastatic potential in MpBC. We first examined the relative expression of RPL39 and RPL39L in TNBC/MpBC cell lines and patient-derived xenografts (PDXs) using real-time qPCR. We detected co-expression of RPL39/RPL39L transcripts, however, RPL39L mRNA levels were significantly lower than RPL39. These results suggest that both RPL39 and RPL39L contribute to ribosome heterogeneity in TNBC/MpBC. To investigate the function of RPL39/RPL39L transcripts, we designed a small interfering RNA (siRNA)-strategy targeting their differential 5' untranslated region (5’ UTR). Selective downregulation of RPL39 and RPL39L mRNA inhibited in vitro cell proliferation in TNBC/MpBC models. Notably, combined targeted silencing of both RPL39 and RPL39L further decreased cell proliferation in comparison to individual siRNAs. We next evaluated the efficacy of RPL39/RPL39L dual blockade in vivo using MpBC PDXs. MpBC PDX4664 was implanted into the mammary fat pad of NSG mice. Once tumors were established (100-150 mm3), mice received phosphate buffered saline (PBS controls) or 0.5 mg/kg RPL39/RPL39L siRNAs encapsulated into liposome-like nanoparticles (NPs) via tail vain (i.v.) injection. Animals were treated thrice a week for 21 days and sacrificed 48 h after the last injection. We found that siRNAs-NP formulation silenced RPL39/RPL39L expression in vivo and significantly reduced tumor growth. Overall, our findings support the notion that RPL39/RPL39L ribosome heterogeneity may confer preferential translation of oncogenic and BCSC-related genes, facilitating tumor progression and metastasis in MpBC. Ongoing studies are directed to validate ribosome heterogeneity by proteomic analysis and evaluate the role of RPL39/RPL39L on global translation by ribosome profiling. Citation Format: Maria F. Chervo, Main li, Chiara Mancino, Karina A. Ortega Martinez, Vrutant V. Shah, Wei Qian, Jianying Zhou, Liliana Guzman-Rojas, Polina Matre, Francesca Taraballi, Kristopher W. Brannan, Jenny C. Chang. Deciphering the role of ribosomal paralogs RPL39/RPL39L in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1558.

Abstract 5446: The role of snoRNAs in GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues

Abstract rRNA modification are a crucial step in ribosome assembly and function. Small nucleolar RNAs select which nucleotides in rRNAs get modified. Typically, C/D box snoRNAs are implicated in 2’-O-ribose methylation (2’Omet) while H/ACA box snoRNAs are responsible for pseudo-uridylations. Alterations in snoRNA expression have been described in multiple tumor types while distinct snoRNAs have been linked to cancer-relevant phenotypes. Our initial study on snoRNA expression using a dedicated Ampliseq platform revealed a specific glioma stem cell (GSC) signature. We propose this snoRNA signature creates a unique rRNA modification pattern that shapes the ribosome, favoring the translation of genes in pathways critical for GSC growth and survival. We will expand our study and characterize the expression profile of snoRNAs and 2’Omethylation in a large panel of GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues. Next, we will select snoRNAs displaying differential expression in GSCs and determine if changes in their levels affect proliferation, viability, response to temozolomide and radiation, and translation. We and others have observed that the integrity of protein complexes that regulate ribosomal biogenesis depends on PolyADP-ribosylation (PARylation)and PAR-binding. In agreement, genomic screenings determined that multiple genes regulating ribosome biogenesis confer sensitivity to PARP inhibitors. These findings offer a therapeutic opportunity. We will investigate the functional impact of PARylation on rRNA modification by determining the effect of PARP inhibitors on snoRNP assembly and 2’Omethylation. Finally, we will test the hypothesis that PARP and ribosome biogenesis inhibitor combinations would be more effective than PARP inhibition alone in treating GBM. Citation Format: Sujash Chatterjee, Scott Kuersten, Christina Middle, Morgan Roos, Pedro A. Galante, Gabriela Guardia, Luiz Penalva. The role of snoRNAs in GBM cells, GSCs, neuronal precursor cells (NPCs), astrocytes, and normal brain tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5446.

Mechanism of the Synergistic Toxicity of Ampicillin and Cefazoline on Selenastrum capricornutum.

Ampicillin (AMP) and cefazolin (CZO) are commonly used β-lactam antibiotics which are extensively globally produced. Additionally, AMP and CZO are known to have relatively high ecotoxicity. Notably, the mix of AMP and CZO creates a synergistic effect that is more harmful to the environment, and how exposure to AMP-CZO can induce synergism in algae remains virtually unknown. To yield comprehensive mechanistic insights into chemical toxicity, including dose-response relationships and variations in species sensitivity, the integration of multiple endpoints with de novo transcriptomics analyses were used in this study. We employed Selenastrum capricornutum to investigate its toxicological responses to AMP and CZO at various biological levels, with the aim of elucidating the underlying mechanisms. Our assessment of multiple endpoints revealed a significant growth inhibition in response to AMP at the relevant concentrations. This inhibition was associated with increased levels of reactive oxygen species (ROS) and perturbations in nitrogen metabolism, carbohydrate metabolism, and energy metabolism. Growth inhibition in the presence of CZO and the AMP-CZO combination was linked to reduced viability levels, elevated ROS production, decreased total soluble protein content, inhibited photosynthesis, and disruptions in the key signaling pathways related to starch and sucrose metabolism, ribosome function, amino acid biosynthesis, and the production of secondary metabolites. It was concluded from the physiological level that the synergistic effect of Chlorophyll a (Chla) and Superoxide dismutase (SOD) activity strengthened the growth inhibition of S. capricornutum in the AMP-CZO synergistic group. According to the results of transcriptomic analysis, the simultaneous down-regulation of LHCA4, LHCA1, LHCA5, and sodA destroyed the functions of the photosynthetic system and the antioxidant system, respectively. Such information is invaluable for environmental risk assessments. The results provided critical knowledge for a better understanding of the potential ecological impacts of these antibiotics on non-target organisms.

A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

(p)ppGpp modifies RNAP function to confer β-lactam resistance in a peptidoglycan-independent manner.

(p)ppGpp is a nucleotide alarmone that controls bacterial response to nutrient deprivation. Since elevated (p)ppGpp levels confer mecillinam resistance and are essential for broad-spectrum β-lactam resistance as mediated by the β-lactam-insensitive transpeptidase YcbB (LdtD), we hypothesized that (p)ppGpp might affect cell wall peptidoglycan metabolism. Here we report that (p)ppGpp-dependent β-lactam resistance does not rely on any modification of peptidoglycan metabolism, as established by analysis of Escherichia coli peptidoglycan structure using high-resolution mass spectrometry. Amino acid substitutions in the β or β' RNA polymerase (RNAP) subunits, alone or in combination with the CRISPR interference-mediated downregulation of three of seven ribosomal RNA operons, were sufficient for resistance, although β-lactams have no known impact on the RNAP or ribosomes. This implies that modifications of RNAP and ribosome functions are critical to prevent downstream effects of the inactivation of peptidoglycan transpeptidases by β-lactams.

Abstract 2257 Quality Control During Ribosome Assembly and Function

Abstract 2257 Quality Control During Ribosome Assembly and Function

Exploring the Role and Prognostic Value of TMEM208 in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the world's eight most common malignancies and a severe hazard to human health. Transmembrane protein 208 (TMEM208) has been reported to be associated with autophagy,which is strongly tied to the onset and development of numerous illnesses, including cancer. For this reason, we investigated the expression and prognostic significance of TMEM208 in HNSCC. To explore the role and molecular mechanism of this gene in HNSCC, we performed a comprehensive analysis of the TMEM208 gene, including gene expression analysis, prognostic analysis, and immune infiltration analysis using the UALCAN, HPA, CVCDAP, DAVID, TIMER, CIBERSORTx, TISIDB, and cBioPortal online databases. It was further validated by in vitro cell culture. Analysis of TCGA data showed that TMEM208 was highly expressed in HNSCC (P < 0.01) and significantly correlated with several clinicopathologic features, and in vitro cellular assays demonstrated that TMEM208 was highly expressed in multiple squamous carcinoma cell lines. Survival analysis showed that high expression of TMEM208 decreased OS (P=0.0088), PFI (P=0.0062), and DSS (P=0.0036) in HNSCC patients. cox regression analysis indicated that high expression of TMEM208 was an independent risk factor for OS in HNSCC patients (P<0.05). In addition, functional enrichment analysis showed that TMEM208 was closely associated with translation, ribosomal and mitochondrial functions, and GSVA analysis showed that TMEM208 was negatively correlated with a variety of immune cell differentiation in HNSCC, with a statistically significant difference. Immunocorrelation analysis showed that TMEM208 could affect immune cell infiltration in HNSCC; in addition, TMEM208 correlated with CD24, CD276, LAG3, and HVEM. In conclusion, TMEM208 holds promise as a prognostic indicator for HNSCC and is closely related to ICI treatment.

Comprehensive map of ribosomal 2'-O-methylation and C/D box snoRNAs in Drosophila melanogaster.

During their maturation, ribosomal RNAs (rRNAs) are decorated by hundreds of chemical modifications that participate in proper folding of rRNA secondary structures and therefore in ribosomal function. Along with pseudouridine, methylation of the 2'-hydroxyl ribose moiety (Nm) is the most abundant modification of rRNAs. The majority of Nm modifications in eukaryotes are placed by Fibrillarin, a conserved methyltransferase belonging to a ribonucleoprotein complex guided by C/D box small nucleolar RNAs (C/D box snoRNAs). These modifications impact interactions between rRNAs, tRNAs and mRNAs, and some are known to fine tune translation rates and efficiency. In this study, we built the first comprehensive map of Nm sites in Drosophila melanogaster rRNAs using two complementary approaches (RiboMethSeq and Nanopore direct RNA sequencing) and identified their corresponding C/D box snoRNAs by whole-transcriptome sequencing. We de novo identified 61 Nm sites, from which 55 are supported by both sequencing methods, we validated the expression of 106 C/D box snoRNAs and we predicted new or alternative rRNA Nm targets for 31 of them. Comparison of methylation level upon different stresses show only slight but specific variations, indicating that this modification is relatively stable in D. melanogaster. This study paves the way to investigate the impact of snoRNA-mediated 2'-O-methylation on translation and proteostasis in a whole organism.

Transcriptomics and metabolomics analysis reveal the dietary copper deficiency and supplementation effects of liver gene expression and metabolite change in grazing sheep.

The appropriate mineral nutrients are essential for sheep growth and reproduction. However, traditional grazing sheep often experience mineral nutrient deficiencies, especially copper (Cu), due to inadequate mineral nutrients from natural pastures. The results indicated that dietary Cu deficiency and supplementation significantly reduced and elevated liver concentration of Cu, respectively (p < 0.05). FOXO3, PLIN1, ACTN2, and GHRHR were identified as critical genes using the weighted gene co-expression network analysis (WGCNA), quantitative real-time polymerase chain reaction (qRT-PCR), and receiver operating characteristic curve (ROC) validation as potential biomarkers for evaluating Cu status in grazing sheep. Combining these critical genes with gene functional enrichment analysis, it was observed that dietary Cu deficiency may impair liver regeneration and compromise ribosomal function. Conversely, dietary Cu supplementation may enhance ribosomal function, promote lipid accumulation, and stimulate growth and metabolism in grazing sheep. Metabolomics analysis indicated that dietary Cu deficiency significantly decreased the abundance of metabolites such as cholic acid (p < 0.05). On the other hand, dietary Cu supplementation significantly increased the abundance of metabolites such as palmitic acid (p < 0.05). Integrative analysis of the transcriptome and metabolome revealed that dietary Cu deficiency may reduce liver lipid metabolism while Cu supplementation may elevate it in grazing sheep. The Cu content in diets may have an impact on hepatic lipid metabolism in grazing sheep. These findings provide new insights into the consequences of dietary Cu deficiency and supplementation on sheep liver and can provide valuable guidance for herders to rationalize the use of mineral supplements.

Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway

BackgroundClear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive.MethodsThe differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa.ResultsCircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway.ConclusionsOur data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

A rationally designed antimicrobial peptide from structural and functional insights of Clostridioides difficile translation initiation factor 1.

A significant increase of hospital-acquired bacterial infections during the COVID-19 pandemic has become an urgent medical problem. Clostridioides difficile is an urgent antibiotic-resistant bacterial pathogen and a leading causative agent of nosocomial infections. The increasing recurrence of C. difficile infection and antibiotic resistance in C. difficile has led to an unmet need for the discovery of new compounds distinctly different from present antimicrobials, while antimicrobial peptides as promising alternatives to conventional antibiotics have attracted growing interest recently. Protein synthesis is an essential metabolic process in all bacteria and a validated antibiotic target. Initiation factor 1 from C. difficile (Cd-IF1) is the smallest of the three initiation factors that acts to establish the 30S initiation complex to initiate translation during protein biosynthesis. Here, we report the solution nuclear magnetic resonance (NMR) structure of Cd-IF1 which adopts a typical β-barrel fold and consists of a five-stranded β-sheet and one short α-helix arranged in the sequential order β1-β2-β3-α1-β4-β5. The interaction of Cd-IF1 with the 30S ribosomal subunit was studied by NMR titration for the construction of a structural model of Cd-IF1 binding with the 30S subunit. The short α-helix in IF1 was found to be critical for IF1 ribosomal binding. A peptide derived from this α-helix was tested and displayed a high ability to inhibit the growth of C. difficile and other bacterial strains. These results provide a clue for the rational design of new antimicrobials.IMPORTANCEBacterial infections continue to represent a major worldwide health hazard due to the emergence of drug-resistant strains. Clostridioides difficile is a common nosocomial pathogen and the causative agent in many infections resulting in an increase in morbidity and mortality. Bacterial protein synthesis is an essential metabolic process and an important target for antibiotic development; however, the precise structural mechanism underlying the process in C. difficile remains unknown. This study reports the solution structure of C. difficile translation initiation factor 1 (IF1) and its interaction with the 30S ribosomal subunit. A short α-helix in IF1 structure was identified as critically important for ribosomal binding and function in regulating the translation initiation, which allowed a rational design of a new peptide. The peptide demonstrated a high ability to inhibit bacterial growth with broad-spectrum antibacterial activity. This study provides a new clue for the rational design of new antimicrobials against bacterial infections.

Effects of sublethal fipronil exposure on cross-generational functional responses and gene expression in Binodoxys communis.

The effective systemic insecticide fipronil is widely used on a variety of crops and in public spaces to control insect pests. Binodoxys communis (Gahan) (Hymenoptera: Braconidae) is the dominant natural enemy of Aphis gossypii Glover (Homoptera: Aphididae), an important cotton pest, and has good efficiency in inhibiting aphid populations. The direct effects of environmental residues of sublethal fipronil doses on adult B. communis have not previously been reported. This study therefore aimed to evaluate the side effects and transcriptomic impacts of sublethal fipronil doses on B. communis. The results showed that exposure to the LC10 dose of fipronil significantly reduced the survival rate and parasitism rate of the F0 generation, but did not affect these indicators in the F1 generation. The LC25 dose did not affect the survival or parasitic rates of the F0 generation, but did significantly reduce the survival rate of F1 generation parasitoids. These results indicated that sublethal doses of fipronil affected B. communis population growth. Transcriptome analysis showed that differentially expressed genes (DEGs) in B. communis at 1h after treatment were primarily enriched in pathways associated with fatty acid elongation, biosynthesis of fatty acids, and fatty acid metabolism. DEGs at 3days after treatment were mainly enriched in ribosomal functions, glycolysis/gluconeogenesis, and tyrosine metabolism. Six DEGs (PY, ELOVL, VLCOAR, MRJP1, ELOVL AAEL008004-like, and RPL13) were selected for validation with real-time fluorescent quantitative PCR. This is the first report of sublethal, trans-generational, and transcriptomic side effects of fipronil on the dominant parasitoid of A. gossypii. The results of this study show that adaptation of parasitoids to high concentrations of pesticides may be at the expense of their offspring. These findings broaden our overall understanding of the intergenerational adjustments used by insects to respond to pesticide stress and call for risk assessments of the long-term impacts and intergenerational effects of other pesticides.

Transcriptomic analysis of the submandibular gland under psychological stress condition.

Psychological stress is associated with changes in salivary flow and composition. However, studies to show the effect of psychological stress on the transcriptome of the salivary gland are limited. This study aims to perform a transcriptomic analysis of the submandibular gland under psychological stress using a chronic restraint stress model of rats. Sprague-Dawley rats were divided into stress groups and control groups. Psychological stress was induced in the stress group rats by enclosing them in a plastic tube for 4 h daily over 6 weeks. RNA sequencing was performed on RNA extracted from the submandibular gland. The differentially expressed genes were identified, and the genes of interest were further validated using qRT-PCR, immunofluorescence, and western blot. A comparison between control and stress groups showed 45 differentially expressed genes. The top five altered genes in RNA sequencing data showed similar gene expression in qRT-PCR validation. The most downregulated gene in the stress group, FosB, was a gene of interest and was further validated for its protein-level expression using immunofluorescence and western blot. The genesets for gene ontology cellular component, molecular function, and KEGG showed that pathways related to ribosome biosynthesis and function were downregulated in the stress group compared to the control. Psychological stress showed transcriptomic alteration in the submandibular gland. The findings may be important in understanding stress-related oral diseases.