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Biomedicine & Pharmacotherapy =... Jul 2024The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its...
(Homo-)harringtonine prevents endothelial inflammation through IRF-1 dependent downregulation of VCAM1 mRNA expression and inhibition of cell adhesion molecule protein biosynthesis.
The plant alkaloid homoharringtonine (HHT) is a Food and Drug Administration (FDA)-approved drug for the treatment of hematologic malignancies. In addition to its well-established antitumor activity, accumulating evidence attributes anti-inflammatory effects to HHT, which have mainly been studied in leukocytes to date. However, a potential influence of HHT on inflammatory activation processes in endothelial cells, which are a key feature of inflammation and a prerequisite for the leukocyte-endothelial cell interaction and leukocyte extravasation, remains poorly understood. In this study, the anti-inflammatory potential of HHT and its derivative harringtonine (HT) on the TNF-induced leukocyte-endothelial cell interaction was assessed, and the underlying mechanistic basis of these effects was elucidated. HHT affected inflammation in vivo in a murine peritonitis model by reducing leukocyte infiltration and proinflammatory cytokine expression as well as ameliorating abdominal pain behavior. In vitro, HT and HHT impaired the leukocyte-endothelial cell interaction by decreasing the expression of the endothelial cell adhesion molecules intracellular adhesion molecule -1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). This effect was mediated by a bipartite mechanism. While HHT did not affect the prominent TNF-induced pro-inflammatory NF-ĸB signaling cascade, the compound downregulated the VCAM1 mRNA expression in an IRF-1-dependent manner and diminished active ICAM1 mRNA translation as determined by polysome profiling. This study highlights HHT as an anti-inflammatory compound that efficiently hampers the leukocyte-endothelial cell interaction by targeting endothelial activation processes.
Topics: Animals; Down-Regulation; Vascular Cell Adhesion Molecule-1; Inflammation; RNA, Messenger; Humans; Interferon Regulatory Factor-1; Mice; Homoharringtonine; Male; Human Umbilical Vein Endothelial Cells; Anti-Inflammatory Agents; Intercellular Adhesion Molecule-1; Endothelial Cells; Mice, Inbred C57BL; Cell Adhesion Molecules; Leukocytes
PubMed: 38865849
DOI: 10.1016/j.biopha.2024.116907 -
RNA (New York, N.Y.) Jun 2024In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery....
In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue specific and essential for some tissue development, such as liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by employing a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays more relevant role in small RNA metabolism and ribosome biogenesis.
PubMed: 38844344
DOI: 10.1261/rna.080097.124 -
Circulation Jun 2024Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the...
BACKGROUND
Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms.
METHODS
Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control.
RESULTS
We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo.
CONCLUSIONS
These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.
PubMed: 38836349
DOI: 10.1161/CIRCULATIONAHA.123.067606 -
Journal of Experimental & Clinical... May 2024RNA modifications of transfer RNAs (tRNAs) are critical for tRNA function. Growing evidence has revealed that tRNA modifications are related to various disease...
BACKGROUND
RNA modifications of transfer RNAs (tRNAs) are critical for tRNA function. Growing evidence has revealed that tRNA modifications are related to various disease processes, including malignant tumors. However, the biological functions of methyltransferase-like 1 (METTL1)-regulated mG tRNA modifications in breast cancer (BC) remain largely obscure.
METHODS
The biological role of METTL1 in BC progression were examined by cellular loss- and gain-of-function tests and xenograft models both in vitro and in vivo. To investigate the change of mG tRNA modification and mRNA translation efficiency in BC, mG-methylated tRNA immunoprecipitation sequencing (mG tRNA MeRIP-seq), Ribosome profiling sequencing (Ribo-seq), and polysome-associated mRNA sequencing were performed. Rescue assays were conducted to decipher the underlying molecular mechanisms.
RESULTS
The tRNA mG methyltransferase complex components METTL1 and WD repeat domain 4 (WDR4) were down-regulated in BC tissues at both the mRNA and protein levels. Functionally, METTL1 inhibited BC cell proliferation, and cell cycle progression, relying on its enzymatic activity. Mechanistically, METTL1 increased mG levels of 19 tRNAs to modulate the translation of growth arrest and DNA damage 45 alpha (GADD45A) and retinoblastoma protein 1 (RB1) in a codon-dependent manner associated with mG. Furthermore, in vivo experiments showed that overexpression of METTL1 enhanced the anti-tumor effectiveness of abemaciclib, a cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor.
CONCLUSION
Our study uncovered the crucial tumor-suppressive role of METTL1-mediated tRNA mG modification in BC by promoting the translation of GADD45A and RB1 mRNAs, selectively blocking the G2/M phase of the cell cycle. These findings also provided a promising strategy for improving the therapeutic benefits of CDK4/6 inhibitors in the treatment of BC patients.
Topics: Humans; Breast Neoplasms; Female; Mice; Animals; Methyltransferases; RNA, Transfer; Methylation; Cell Line, Tumor; Cell Proliferation; Carcinogenesis; Cell Cycle Checkpoints; Protein Biosynthesis; Xenograft Model Antitumor Assays; Mice, Nude
PubMed: 38822363
DOI: 10.1186/s13046-024-03076-x -
Life Science Alliance Aug 2024IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in...
IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing , we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.
Topics: Humans; Protein Biosynthesis; Transcription Factors; Stress, Physiological; DNA-Binding Proteins; K562 Cells; Activating Transcription Factor 4; Gene Deletion; Gene Expression Regulation; RNA, Transfer
PubMed: 38803225
DOI: 10.26508/lsa.202302554 -
PSPC1 Binds to HCV IRES and Prevents Ribosomal Protein S5 Binding, Inhibiting Viral RNA Translation.Viruses May 2024Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by...
Hepatitis C virus (HCV) infects the human liver, and its chronic infection is one of the major causes of Hepatocellular carcinoma. Translation of HCV RNA is mediated by an Internal Ribosome Entry Site (IRES) element located in the 5'UTR of viral RNA. Several RNA Binding proteins of the host interact with the HCV IRES and modulate its function. Here, we demonstrate that PSPC1 (Paraspeckle Component 1), an essential paraspeckle component, upon HCV infection is relocalized and interacts with HCV IRES to prevent viral RNA translation. Competition UV-crosslinking experiments showed that PSPC1 interacts explicitly with the SLIV region of the HCV IRES, which is known to play a vital role in ribosomal loading to the HCV IRES via interaction with Ribosomal protein S5 (RPS5). Partial silencing of PSPC1 increased viral RNA translation and, consequently, HCV replication, suggesting a negative regulation by PSPC1. Interestingly, the silencing of PSPC1 protein leads to an increased interaction of RPS5 at the SLIV region, leading to an overall increase in the viral RNA in polysomes. Overall, our results showed how the host counters viral infection by relocalizing nuclear protein to the cytoplasm as a survival strategy.
Topics: Hepacivirus; Humans; Ribosomal Proteins; Internal Ribosome Entry Sites; RNA, Viral; Protein Biosynthesis; Virus Replication; RNA-Binding Proteins; Protein Binding; Hepatitis C; Host-Pathogen Interactions
PubMed: 38793620
DOI: 10.3390/v16050738 -
ELife May 2024Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a...
Despite current antifungal therapy, invasive candidiasis causes >40% mortality in immunocompromised individuals. Therefore, developing an antifungal vaccine is a priority. Here, we could for the first time successfully attenuate the virulence of by treating it with a fungistatic dosage of EDTA and demonstrate it to be a potential live whole cell vaccine by using murine models of systemic candidiasis. EDTA inhibited the growth and biofilm formation of . RNA-seq analyses of EDTA-treated cells (CAET) revealed that genes mostly involved in metal homeostasis and ribosome biogenesis were up- and down-regulated, respectively. Consequently, a bulky cell wall with elevated levels of mannan and β-glucan, and reduced levels of total monosomes and polysomes were observed. CAET was eliminated faster than the untreated strain () as found by differential fungal burden in the vital organs of the mice. Higher monocytes, granulocytes, and platelet counts were detected in CAET-challenged mice. While hyper-inflammation and immunosuppression caused the killing of -challenged mice, a critical balance of pro- and anti-inflammatory cytokines-mediated immune responses are the likely reasons for the protective immunity in CAET-infected mice.
Topics: Animals; Candida albicans; Mice; Candidiasis; Fungal Vaccines; Disease Models, Animal; Virulence; Female; Cytokines; Biofilms
PubMed: 38787374
DOI: 10.7554/eLife.93760 -
Aging Cell May 2024The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted....
The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted. Nucleus pulposus cell (NPC) ferroptosis is one of the causes of IVDD. N6-methyladenosine (m6A) and its reader protein YTHDF1 regulate cellular activities by affecting RNA metabolism. However, the regulation of ferroptosis in NPCs by m6A-modified RNAs under hypoxic conditions has not been as well studied. In this study, through in vitro and in vivo experiments, we explored the underlying mechanism of HIF-1α and YTHDF1 in regulating ferroptosis in NPCs. The results indicated that the overexpression of HIF-1α or YTHDF1 suppressed NPC ferroptosis; conversely, the knockdown of HIF-1α or YTHDF1 increased ferroptosis levels in NPCs. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF-1α regulated YTHDF1 transcription by directly binding to its promoter region. Polysome profiling results showed that YTHDF1 promoted the translation of SLC7A11 and consequently the expression of the anti-ferroptosis protein GPX4 by binding to m6A-modified SLC7A11 mRNA. In conclusion, HIF-1α-induced YTHDF1 expression reduces NPC ferroptosis and delays IVDD by promoting SLC7A11 translation in a m6A-dependent manner.
PubMed: 38783692
DOI: 10.1111/acel.14210 -
NAR Cancer Jun 2024In this review, we explore the transformative impact of next generation sequencing technologies in the realm of translatomics (the study of how translational machinery... (Review)
Review
In this review, we explore the transformative impact of next generation sequencing technologies in the realm of translatomics (the study of how translational machinery acts on a genome-wide scale). Despite the expectation of a direct correlation between mRNA and protein content, the complex regulatory mechanisms that affect this relationship remark the limitations of standard RNA-seq approaches. Then, the review characterizes crucial techniques such as polysome profiling, ribo-seq, trap-seq, proximity-specific ribosome profiling, rnc-seq, tcp-seq, qti-seq and scRibo-seq. All these methods are summarized within the context of cancer research, shedding light on their applications in deciphering aberrant translation in cancer cells. In addition, we encompass databases and bioinformatic tools essential for researchers that want to address translatome analysis in the context of cancer biology.
PubMed: 38751936
DOI: 10.1093/narcan/zcae024 -
Research Square Apr 2024Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive...
BACKGROUND
Human males and females show differences in the incidence of neutrophil-associated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and reactive arthritis, and differences in neutrophil physiological responses such as a faster response to the chemorepellent SLIGKV. Little is known about the basis of sex-based differences in human neutrophils.
METHODS
Starting with human neutrophils from healthy donors, we used RNA-seq to examine total mRNA profiles, mRNAs not associated with ribosomes and thus not being translated, mRNAs in monosomes, and mRNAs in polysomes and thus heavily translated. We used mass spectrometry systems to identify proteins and phosphoproteins.
RESULTS
There were sex-based differences in the translation of 24 mRNAs. There were 132 proteins with higher levels in male neutrophils; these tended to be associated with RNA regulation, ribosome, and phosphoinositide signaling pathways, whereas 30 proteins with higher levels in female neutrophils were associated with metabolic processes, proteosomes, and phosphatase regulatory proteins. Male neutrophils had increased phosphorylation of 32 proteins. After exposure to SLIGKV, male neutrophils showed a faster response in terms of protein phosphorylation compared to female neutrophils.
CONCLUSIONS
Male neutrophils have higher levels of proteins and higher phosphorylation of proteins associated with RNA processing and signaling pathways, while female neutrophils have higher levels of proteins associated with metabolism and proteolytic pathways. This suggests that male neutrophils might be more ready to adapt to a new environment, and female neutrophils might be more effective at responding to pathogens. This may contribute to the observed sex-based differences in neutrophil behavior and neutrophil-associated disease incidence and severity.
PubMed: 38746380
DOI: 10.21203/rs.3.rs-4284171/v1