Ribosomal Protein S6 Research Articles

RNA-binding protein RPS7 promotes hepatocellular carcinoma progression via LOXL2-dependent activation of ITGB1/FAK/SRC signaling

BackgroundMetastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood.MethodsBy performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9–mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis.ResultsGain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155–3375 region of the 3’UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7.ConclusionsTaken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC.

Identification of Candidate Male-Reproduction-Related Genes from the Testis and Androgenic Gland of Macrobrachium nipponense, Regulated by PDHE1, through Transcriptome Profiling Analysis.

The previous publication identified that pyruvate dehydrogenase E1 (PDHE1) positively regulated the process of male reproduction in M. nipponense through affecting the expressions of insulin-like androgenic gland hormone. The present study aimed to identify the potential male-reproduction-related genes that were regulated by PDHE1 through performing the transcriptome profiling analysis in the testis and androgenic gland after the knockdown of the expressions of PDHE1 by the injection of dsPDHE1. Both RNA-Seq and qPCR analysis identified the significant decreases in PDHE1 expressions in the testis and androgenic gland in dsPDHE1-injected prawns compared to those in dsGFP-injected prawns, indicating the efficiency of dsPDHE1 in the present study. Transcriptome profiling analysis identified 56 and 127 differentially expressed genes (DEGs) in the testis and androgenic gland, respectively. KEGG analysis revealed that the energy-metabolism-related pathways represented the main enriched metabolic pathways of DEGs in both the testis and androgenic gland, including pyruvate metabolism, the Citrate cycle (TCA cycle), Glycolysis/Gluconeogenesis, and the Glucagon signaling pathway. Thus, it is predicted that these metabolic pathways and the DEGs from these metabolic pathways regulated by PDHE1 may be involved in the regulation of male reproduction in M. nipponense. Furthermore, four genes were found to be differentially expressed in both the testis and androgenic gland, of which ribosomal protein S3 was down-regulated and uncharacterized protein LOC113829596 was up-regulated in both the testis and androgenic gland in dsPDHE1-injected prawns. The present study provided valuable evidence for the establishment of an artificial technique to regulate the process of male reproduction in M. nipponense.

Expression of PD-L1 and p-RPS6 in epithelial dysplasia and squamous cell carcinoma of the oral cavity.

Oral squamous cell carcinoma (OSCC) is often preceded by oral epithelial dysplasia (OED). The role of ribosomal protein S6 (RPS6) and programmed cell death ligand-1 (PD-L1) in the progression of OED to OSCC remains unclear. This study aimed to investigate the expression of phosphorylated RPS6 (p-RPS6) and PD-L1 in OSCC and OED and to examine its relationship with clinicopathological features. Fifty-two OSCC and 48 OED cases were recruited for immunohistochemical analysis of p-RPS6 and PD-L1 expression. The expression of markers was correlated with clinicopathological features of OSCC and OED. We found p-RPS6 expression in all cases of OSCC and OED, whereas PD-L1 was expressed in 42/48 (87%) OED and in 28/52 (53%) OSCC. The patients with mild OED presented higher expression level of PD-L1 and p-RPS6 significantly, when compared to moderate-differentiated OSCC patients (p < 0.05). Moreover, we found a significant positive correlation between PD-L1 and p-RPS6 expression in OED and OSCC patients (p < 0.01). The PD-L1 expression was significantly related to more than 2 cm tumor size in OSCC patients (p = 0.007). Our findings suggest the upregulation of PD-L1 may be related with activation of the mTOR pathway in the early events of tumor progression and the pathogenesis of OSCC.

Establishing a carcinoembryonic antigen-associated competitive endogenous RNA network and forecasting an important regulatory axis in colon adenocarcinoma patients.

Colorectal cancer is one of the top five malignant tumors in the world in terms of morbidity and mortality. Numerous long non-coding RNAs (lncRNAs) are specifically expressed in tumours and can affect various types of human cancer by participating in competitive endogenous RNA (ceRNA) regulatory networks. However, the specific mechanisms and complex networks of ceRNA regulatory patterns in colon adenocarcinoma (COAD) remain unclear. Using The Cancer Genome Atlas (TCGA) database, we identified the differentially expressed lncRNA, microRNA (miRNA), and messenger RNA (mRNA) between colon cancer and normal tissues, as well as between groups with high and low CEACAM5 expression. Then, we constructed CEACAM5-related ceRNA networks, established the key lncRNA-miRNA-mRNA regulatory axis, and explored the biological mechanisms of this axis and its clinical significance in colon cancer from multiomic aspects. We constructed a ceRNA network of 18 lncRNAs, 177 miRNAs, and 25 mRNAs associated with CEACAM5 and finally established the key LCMT1-AS2/miR-454-3p/ribosomal protein S6 kinase A5 (RPS6KA5) axis associated with overall survival. Subsequent investigations have indicated that this regulatory axis could potentially participate in the progression of COAD and exert influence on the therapeutic outcomes of chemotherapy and immunotherapy. It may be involved in the PI3K-Akt signaling pathway and may modify the tumor immune microenvironment and influence the course of COAD. Additionally, it may be related to ferroptosis, N6-methyladenosine (m6A) methylation, and tumor stemness and interfere with the sensitivity of tumor cells to 5-fluorouracil and immunotherapy. The LCMT1-AS2/RPS6KA5 axis may be instrumental in tumor progression, potentially acting as a prognostic biomarker and therapeutic target.

Effect of Erchen Decoction on liver mitochondrial function by inhibiting mTORC1/SREBP1/CAV1 pathway in mice with high-fat diet

This study aims to investigate the effect of Erchen Decoction(ECD) on liver mitochondrial function in mice with a high-fat diet and its possible mechanism. A total of sixty C57BL/6J mice were randomly divided into a normal group, high-fat group, ECD group, mTORC1 activator(MHY) group, ECD+MHY group, and polyene phosphatidyl choline(PPC) group, with 10 rats in each group. The normal group was given a normal diet, and the other groups were fed a high-fat diet for 20 weeks. At the 17th week, the ECD group and ECD+MHY group were given ECD(8.7 g·kg~(-1)) daily, and the PPC group was given PPC(0.18 g·kg~(-1)) daily, while the remaining groups were given normal saline(0.01 mL·g~(-1)) daily for four weeks. In the 19th week, the MHY group and ECD+MHY group were injected intraperitoneally with MHY(5 mg·kg~(-1)) every other day for two weeks. During the experiment, the general conditions of the mice were observed. The contents of triglyceride(TG) and total cholesterol(TC) in serum were measured. Morphological changes in liver tissue were examined through HE and oil red O staining. The content of adenosine triphosphate(ATP) was determined using chemiluminescence, and mitochondrial membrane potential was assessed using a fluorescence probe(JC-1). Western blot was performed to detect the expression of rapamycin target protein complex 1(mTOR1), ribosomal protein S6 kinase B1(S6K), sterol regulatory element binding protein 1(SREBP1), and caveolin 1(CAV1). RESULTS:: revealed that compared with the normal group, the mice in the high-fat group exhibited significant increases in body weight and abdominal circumference(P&lt;0.01). Additionally, there were significant increases in TG and TC levels(P&lt;0.01). HE and oil red O staining showed that the boundaries of hepatic lobules were unclear; hepatocytes were enlarged, round, and irregularly arranged, with obvious lipid droplet deposition and inflammatory cell infiltration. The liver ATP content and mitochondrial membrane potential decreased significantly(P&lt;0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 increased significantly(P&lt;0.01), while the expression of CAV1 decreased significantly(P&lt;0.01). Compared with the high-fat group, the body weight and TG content of mice in the ECD group and PPC group decreased significantly(P&lt;0.05). Improvements were observed in hepatocyte morphology, lipid deposition, and inflammatory cell infiltration. Furthermore, there were significant increases in ATP content and mitochondrial membrane potential(P&lt;0.05 or P&lt;0.01). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly in the ECD group(P&lt;0.01), while CAV1 expression increased significantly(P&lt;0.01). However, the indices mentioned above did not show improvement in the MHY group. When the ECD+MHY group was compared with the MHY group, there were significant reductions in body weight and TG contents(P&lt;0.05). The morphological changes of hepatocytes, lipid deposition, and inflammatory cell infiltration were recovered. Moreover, there were significant increases in liver ATP content and mitochondrial membrane potential(P&lt;0.05 or P&lt;0.05). The expression of p-mTOR, p-S6K, and n-SREBP1 decreased significantly(P&lt;0.01), while CAV1 expression increased significantly(P&lt;0.01). In conclusion, ECD can improve mitochondrial function by regulating the mTORC1/SREBP1/CAV1 pathway. This mechanism may be involved in the resolution of phlegm syndrome and the regulation of lipid metabolism.

Enhancement of Haloperidol-Induced Catalepsy by GPR143, an L-Dopa Receptor, in Striatal Cholinergic Interneurons.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y ) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y ) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre;Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs.

Ribosomal protein S6 kinase 2 (RPS6KB2) is a potential immunotherapeutic target for cancer that upregulates proinflammatory cytokines.

Cancer is still a leading cause of mortality. Over the years, cancer therapy has undergone significant advances driven by advancements in science and technology. A promising area of drug discovery in this field involves the development of therapeutic targets for cancer treatment. The urgent need to identify new pharmacological targets arises from the impact of tumor resistance on the effectiveness of current medications. Specifically, the RPS6KB2 gene on chromosome 11 has been implicated in cell cycle regulation and exhibits higher expression levels in tumor tissue. Given this association, there is a potential for this gene to serve as a target for cancer treatment. We conducted an analysis using the GTEx, TCGA, and CCLE databases to explore the relationship between RPS6KB2 and immune infiltration, the tumor microenvironment (TME), microsatellite instability (MSI), and more. Cell proliferation was assessed using EDU detection, while cell invasion and migration were evaluated via wound healing and Transwell assays. Additionally, western blot analysis was employed to measure expression of Bax, Bcl-2, MMP2, MMP9, PCNA, and proinflammatory factors. Through data analysis and molecular biology methods, our study carefully examined the potential role of RPS6KB2 in cancer therapy. The data revealed that RPS6KB2 is aberrantly expressed in most cancers and is associated with poor prognosis. Further analysis indicated its involvement in cancer cell apoptosis and migration, as well as its role in cancer immune processes. We validated the significance of RPS6KB2 in hepatocellular carcinoma (HCC), highlighting its capacity to upregulate proinflammatory cytokines. Our research indicates that RPS6KB2 is a prognostic biomarker associated with immune infiltration in cancer that can affect antitumor immunity by increasing secretion of proinflammatory factors, providing a potential drug target for cancer treatment.

Autophagy-dependent alternative splicing of ribosomal protein S24 produces a more stable isoform that aids in hypoxic cell survival.

Cells remodel splicing and translation machineries to mount specialized gene expression responses to stress. Here, we show that hypoxic human cells in 2D and 3D culture models increase the relative abundance of a longer mRNA variant of ribosomal protein S24 (RPS24L) compared to a shorter mRNA variant (RPS24S) by favoring the inclusion of a 22 bp cassette exon. Mechanistically, RPS24L and RPS24S are induced and repressed, respectively, by distinct pathways in hypoxia: RPS24L is induced in an autophagy-dependent manner, while RPS24S is reduced by mTORC1 repression in a hypoxia-inducible factor-dependent manner. RPS24L produces a more stable protein isoform that aids in hypoxic cell survival and growth, which could be exploited by cancer cells in the tumor microenvironment.

The Anti-Diabetic Drug Metformin Suppresses Pathological Retinal Angiogenesis via Blocking the mTORC1 Signaling Pathway in Mice (Metformin Suppresses Pathological Angiogenesis)

Purpose Metformin, a biguanide antihyperglycemic drug, can exert various beneficial effects in addition to its glucose-lowering effect. The effects of metformin are mainly mediated by AMP-activated protein kinase (AMPK)-dependent pathway. AMPK activation interferes with the action of the mammalian target of rapamycin complex 1 (mTORC1), and blockade of mTORC1 pathway suppresses pathological retinal angiogenesis. Therefore, in this study, we examined the effects of metformin on pathological angiogenesis and mTORC1 activity in the retinas of mice with oxygen-induced retinopathy (OIR). Methods OIR was induced by exposing the mice to 80% oxygen from postnatal day (P) 7 to P10. The OIR mice were treated with metformin, rapamycin (an inhibitor of mTORC1), or the vehicle from P10 to P12 or P14. The formation of neovascular tufts, revascularization in the central avascular areas, expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) 2, and phosphorylated ribosomal protein S6 (pS6), a downstream indicator of mTORC1 activity, were evaluated at P10, P13, or P15. Results Neovascular tufts and vascular growth in the central avascular areas were observed in the retinas of P15 OIR mice. The formation of neovascular tufts, but not the revascularization in the central avascular areas, was attenuated by metformin administration from P10 to P14. Metformin had no significant inhibitory effect on the expression of VEGF and VEGFR2, but it reduced the pS6 immunoreactivity in vascular cells at the sites of angiogenesis. Rapamycin completely blocked the phosphorylation of ribosomal protein S6 and markedly reduced the formation of neovascular tufts. Conclusions These results suggest that metformin partially suppresses the formation of neovascular tufts on the retinal surface by blocking the mTORC1 signaling pathway. Metformin may exert beneficial effects against the progression of ocular diseases in which abnormal angiogenesis is associated with the pathogenesis.

In-silico investigation of RPS6KB1 associated cancer inhibitor: a drug repurposing study

The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments. Communicated by Ramaswamy H. Sarma

Lactate-induced autophagy activation: unraveling the therapeutic impact of high-intensity interval training on insulin resistance in type 2 diabetic rats

Impaired autophagy is a hallmark of diabetes. The current study proposed to investigate if high intensity interval training (HIIT) induced lactate accumulation could stimulate autophagy in type 2 diabetic male rats. 28 male Wistar rats were randomly assigned into four groups: Healthy Control (CO), Diabetes Control (T2D), Exercise (EX), and Diabetes + Exercise (T2D + EX). Diabetes was induced by feeding high-fat diet and administrating single dose of streptozotocin (35 mg/kg). After becoming diabetic, the animals in the exercise groups (EX and T2D + EX) performed an eight-week HIIT (4–10 interval, 80–100% Vmax, 5 days per week). Serum levels of lactate, glucose and insulin as well as the levels of lactate, pyruvate, lactate transporter monocarboxylate transporter 1 (MCT1), phosphorylated mitogen-activated protein kinases (p-MAP 1 and 2), phosphorylated extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1 and 2), mammalian target of rapamycin (p-mTOR), ribosomal protein S6 kinase beta-1 (p-70S6k), p90 ribosomal S6 kinases (p-90RSK), autophagy related 7 (ATG7), Beclin-1, microtubule-associated protein 1A/1B, and 2A/2B -light chain 3 levels (LC3-I), (LC3- II), (LC3I/LC3II) in soleus muscle were measured. Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and serum glucose was lower in T2D + EX compared to T2D group (P < 0.0001). While serum and soleus muscle levels of lactate was not different between T2D and T2D + Ex, the levels of Pyruvate (P < 0.01), MCT1, p-ERK1/2, p-mTOR, p70S6k, P-90RSK, ATG7, LC3-II, and LC3-II/LC3I ratios were higher in T2D + EX compared to T2D group (P < 0.0001). We concluded that eight weeks of high-intensity interval training could activated ERK/P90SRK while inhibiting mTOR/P70S6K signaling pathway in lactate dependent manner. It means increased autophagy which resulted in improve insulin resistance (IR) and reduce blood glucose.

FKBP38 suppresses endometrial cancer cell proliferation and metastasis by inhibiting the mTOR pathway

Endometrial cancer (EC) is a common gynecological malignancy, and advanced-stage or recurrent EC is associated with a high mortality rate owing to the ineffectiveness of currently available treatments. FK506-binding protein 38 (FKBP38) is a member of the immunophilin family and inhibits melanoma and breast cancer cell metastasis. However, the functions of FKBP38 and its potential mechanism in EC remain unclear. Herein, we analyzed the expression levels of FKBP38 in EC cells and found that the FKBP38 expression was high in Ishikawa cells, and low in AN3CA cells, traditionally considered a low grade and a high grade cell line, respectively, in pathology classification. Moreover, FKBP38 inhibited cell proliferation, migration and invasion in EC cells, FKBP38 knockdown significantly promoted tumor growth of Ishikawa cells in a subcutaneous xenograft model and increased the number of lung metastases of Hec-1-A cells in a metastatic mouse model. Furthermore, FKBP38 suppressed several target proteins of epithelial-to-mesenchymal transition (EMT) and reduced the phosphorylation of ribosomal S6 protein (S6), eukaryotic initiation factor 4E-binding protein 1 (4EBP-1), indicating the potent inhibition of the mammalian target of rapamycin (mTOR) pathway. Meanwhile, the inhibition of mTOR neutralized the elevation of EC cell proliferation, migration and invasion after FKBP38 knockdown. In summary, FKBP38 would exert a tumor-suppressing role by modulating the mTOR pathway. Our results indicate that FKBP38 may be considered as a factor of EC metastasis and a new target for EC therapeutic intervention.

Maize requires Embryo defective27 for embryogenesis and seedling development.

The essential role of plastid translation in embryogenesis has been established in many plants, but a retrograde signal triggered by defective plastid translation machinery that may leads to embryogenesis arrest remains unknown. In this study, we characterized an embryo defective27 (emb27) mutant in maize (Zea mays), and cloning indicates that Emb27 encodes the plastid ribosomal protein S13. The null mutant emb27-1 conditions an emb phenotype with arrested embryogenesis; however, the leaky mutant emb27-2 exhibits normal embryogenesis but an albino seedling-lethal phenotype. The emb27-1/emb27-2 trans-heterozygotes display varying phenotypes from emb to normal seeds but albino seedlings. Analysis of the Emb27 transcription levels in these mutants revealed that the Emb27 expression level in the embryo corresponds with the phenotypic expression of the emb27 mutants. In the W22 genetic background, an Emb27 transcription level higher than 6% of the wild-type level renders normal embryogenesis, whereas lower than that arrests embryogenesis. Mutation of Emb27 reduces the level of plastid 16S rRNA and the accumulation of the plastid-encoded proteins. As a secondary effect, splicing of several plastid introns was impaired in emb27-1 and 2 other plastid translation-defective mutants, emb15 and emb16, suggesting that plastome-encoded factors are required for the splicing of these introns, such as Maturase K (MatK). Our results indicate that EMB27 is essential for plastid protein translation, embryogenesis, and seedling development in maize and reveal an expression threshold of Emb27 for maize embryogenesis.

Modulations in the host cell proteome by the hantavirus nucleocapsid protein.

Hantaviruses have evolved a unique translation strategy to boost the translation of viral mRNA in infected cells. Hantavirus nucleocapsid protein (NP) binds to the viral mRNA 5' UTR and the 40S ribosomal subunit via the ribosomal protein S19. NP associated ribosomes are selectively loaded on viral transcripts to boost their translation. Here we demonstrate that NP expression upregulated the steady-state levels of a subset of host cell factors primarily involved in protein processing in the endoplasmic reticulum. Detailed investigation of Valosin-containing protein (VCP/p97), one of the upregulated host factors, in both transfected and virus infected cells revealed that NP with the assistance of VCP mRNA 5' UTR facilitates the translation of downstream VCP ORF. The VCP mRNA contains a 5' UTR of 987 nucleotides harboring six unusual start codons upstream of the correct start codon for VCP which is located at 988th position from the 5' cap. In vitro translation of a GFP reporter transcript harboring the VCP mRNA 5' UTR generated both GFP and a short polypeptide of ~14 KDa by translation initiation from start codon located in the 5' UTR at 542nd position from the 5' cap. The translation initiation from 542nd AUG in the UTR sequence was confirmed in cells using a dual reporter construct expressing mCherry and GFP. The synthesis of 14KDa polypeptide dramatically inhibited the translation of the ORF from the downstream correct start codon at 988th position from the 5' cap. We report that purified NP binds to the VCP mRNA 5' UTR with high affinity and NP binding site is located close to the 542ndAUG. NP binding shuts down the translation of 14KDa polypeptide which then facilitates the translation initiation at the correct AUG codon. Knockdown of VCP generated lower levels of poorly infectious hantavirus particle in the cellular cytoplasm whose egress was dramatically inhibited in human umbilical vein endothelial cells. We demonstrated that VCP binds to the hantavirus glycoprotein Gn before its incorporation into assembled virions and facilitates viral spread to neighboring cells during infection. Our results suggest that ribosome engagement at the 542nd AUG codon in the 5' UTR likely regulates the endogenous steady state levels of VCP in cells. Hantaviruses interrupt this regulatory mechanism to enhance the steady state levels of VCP in virus infected cells. This augmentation facilitates virus replication, supports the transmission of the virus to adjacent cells, and promotes the release of infectious virus particles from the host cell.

Unraveling the Role of RNA-Binding Proteins, with a Focus on RPS5, in the Malignant Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) represents a major global health concern, demanding a thorough understanding of its molecular mechanisms for effective therapeutic strategies. RNA-binding proteins (RBPs) play critical roles in post-transcriptional gene regulation, with their dysregulation increasingly recognized as a hallmark of various cancers. However, the specific contributions of RBPs to HCC pathogenesis and prevention remain incompletely understood. In this study, we systematically conducted an examination of the expression profiles and clinical relevance of RBPs in 556 clinical samples from well-established cohorts. Through comprehensive analyses, a subset of RBPs exhibiting significant overexpression in HCC was identified, establishing a noteworthy correlation between their aberrant expression and HCC progression. Furthermore, 40S ribosomal protein S5 (RPS5), a ribosomal protein, emerged as a potential key contributor in HCC progression. Rigorous analyses established a correlation between elevated RPS5 expression and advanced clinicopathological features, suggesting its potential as a prognostic biomarker. Experiments further confirmed the impact of RPS5 on pivotal cellular processes implicated in cancer progression, including cell proliferation and metastasis. Further mechanistic studies unveiled the potential of RPS5 to activate the cell cycle by binding to key molecules involved in the pathway, thereby promoting the malignant progression of HCC. Additionally, our analysis of the etiology behind RPS5 overexpression in HCC posited it as an outcome of transcriptional regulation by the transcription factors Nuclear Respiratory Factor 1 (NRF1) and MYC-associated zinc finger protein (MAZ). In conclusion, our study contributes to the growing evidence elucidating the intricate involvement of RBPs, exemplified by RPS5, in the malignant progression of HCC. The integration of genomic, transcriptomic, and functional analyses provides a comprehensive understanding of the regulatory mechanisms associated with RPS5 in HCC. This comprehensive analysis not only advances our knowledge of the molecular drivers behind HCC but also highlights the potential therapeutic relevance of targeting RBPs and their regulatory network for the development of more effective treatment strategies.

S6K1 deficiency in tumor stroma impairs lung metastasis of melanoma in mice

Accumulating data suggest that ribosomal protein S6 kinase 1 (S6K1), an effector in the mammalian target of rapamycin (mTOR) pathway, plays pleiotropic roles in tumor progression. However, to date, while the tumorigenic function of S6K1 in tumor cells has been well elucidated, its role in the tumor stroma remains poorly understood. We recently showed that S6K1 mediates vascular endothelial growth factor A (VEGF-A) production in macrophages, thereby supporting tumor angiogenesis and growth. As macrophage-derived VEGF-A is crucial for both tumor cell intravasation and extravasation across the vascular endothelium, our previous findings suggest that stromal S6K1 signaling is required for tumor metastatic spread. Therefore, we aimed to determine the impact of host S6K1 depletion on tumor metastasis using a murine model of pulmonary metastasis (S6k1−/− mice implanted with B16F10 melanoma). The ablation of S6K1 in the host microenvironment significantly reduced the metastasized B16F10 melanoma cells on the lung surface in both spontaneous and intravenous lung metastasis mouse models without affecting the incidence of metastasis to distant lymph nodes. In addition, stromal S6K1 loss decreased the number of tumor cells circulating in the peripheral blood of mice bearing B16F10 xenografts without affecting the vascular leakage induced by VEGF-A in vivo. These observations demonstrate that S6K1 signaling in host cells other than endothelial cells is required to modulate the host microenvironment to facilitate the metastatic spread of tumors via blood circulation, thus revealing its novel role in the tumor stroma during tumor progression.

Dietary glycine supplementation activates mechanistic target of rapamycin signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

Characterisation of common hypothetical surface peptides between protozoan parasites (Perkinsus olseni) originating from different geographical locations.

Perkinsus olseni and P. marinus are classified as notifiable pathogens by the World Organisation for Animal Health and are known to cause perkinsosis in a variety of molluscs globally. Mass mortalities due to these parasites in farms and in the wild have been a recurrent issue. Diagnosis for these protozoans is currently done using Ray's fluid thioglycollate medium method followed by optical microscopy or molecular assays. Both require a high level of skill and are time-consuming. An immunoassay method would make the diagnosis of perkinsosis quicker and cheaper. The present study used mass spectrometry-based proteomics to investigate common hypothetical surface peptides between different geographical isolates of P. olseni, which could be used to develop immunoassays in the future. Two peptides were identified: POLS_08089, which is a 42.7 kDa peptide corresponding to the 60S ribosomal subunit protein L4; and POLS_15916, which is a conserved hypothetical protein of 55.6 kDa. The identification of peptides may allow the development of immunoassays through a more targeted approach.

Sestrin 2 protects human lens epithelial cells from oxidative stress and apoptosis induced by hydrogen peroxide by regulating the mTOR/Nrf2 pathway.

We aimed to explore the effect and potential mechanism of Sestrin 2 (SESN2) in human lens epithelial cells (HLECs). To mimic the oxidative stress environment, SAR01/04 cells were treated with 200μM hydrogen peroxide (H2O2) for 24h. Cell viability and apoptosis were checked by cell counting kit-8 and flow cytometry. Western blot was taken to check the protein changes of SESN2, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax), mechanistic target of rapamycin (mTOR), phosphorylated (p)-mTOR, ribosomal protein S6 kinase B1 (p70S6K), p-p70S6K, and nuclear factor erythroid 2-related factor 2 (Nrf2). Superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and reactive oxygen species (ROS) were detected via the corresponding reagent kit. The levels of interleukin (IL)-1β, IL-18, and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay. SESN2 was down-regulated in cataract lens tissue and up-regulated in SAR01/04 cells treated with H2O2. Under treatment of H2O2, up-regulation of SESN2 improved cell viability, enhanced the activity of SOD and CAT, inhibited cell apoptosis, and reduced the levels of MDA, ROS, IL-1β, IL-18, and TNF-α, while down-regulation of SESN2 caused the contrary effects. Further bioinformatics analysis suggested that SESN2 regulated the mTOR signaling pathway. Treatment of H2O2 inhibited p-mTOR and p-p70S6K protein expression, while overexpression of SESN2 increased p-mTOR and p-p70S6K protein expression in the H2O2 group and down-regulation of SESN2 further decreased p-mTOR and p-p70S6K protein expression in the H2O2 group. Additionally, H2O2 increased Nrf2 protein expression, and overexpression of SESN2 further increased Nrf2 protein expression in the H2O2 group. Importantly, rapamycin (an inhibitor of mTOR signaling pathway) and knockdown of Nrf2 reversed the promotive effects of SESN2 on cell viability and the inhibitive effects of SESN2 on cell apoptosis, oxidative stress, and inflammatory reaction. SESN2 protected HLECs damage induced by H2O2, which was related to the activation of mTOR/Nrf2 pathway.

Duloxetine enhances the sensitivity of non-small cell lung cancer cells to EGFR inhibitors by REDD1-induced mTORC1/S6K1 suppression.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been effective targeted therapies for non-small cell lung cancer (NSCLC), most advanced NSCLC inevitably develop resistance to these therapies. Combination therapies emerge as valuable approach to preventing, delaying, or overcoming disease progression. Duloxetine, an antidepressant known as a serotonin-noradrenaline reuptake inhibitor, is commonly prescribed for the treatment of chemotherapy-induced peripheral neuropathy. In the present study, we investigated the combined effects of duloxetine and EGFR-TKIs and their possible mechanism in NSCLC cells. Compared with either monotherapy, the combination of duloxetine and EGFR-TKIs leads to synergistic cell death. Mechanistically, duloxetine suppresses 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) activity through mechanistic target of rapamycin complex 1 (mTORC1), and this effect is associated with the synergistic induction of cell death of duloxetine combined with EGFR-TKIs. More importantly, activating transcription factor 4 (ATF4)-induced regulated in development and DNA damage response 1 (REDD1) is responsible for the suppression of mTORC1/S6K1 activation. Additionally, we found that the combination effect was significantly attenuated in REDD1 knockout NSCLC cells. Taken together, our findings reveal that the ATF4/REDD1/mTORC1/S6K1 signaling axis, as a novel mechanism, is responsible for the synergistic therapeutic effect of duloxetine with EGFR-TKIs. These results suggest that combining EGFR-TKIs with duloxetine appears to be a promising way to improve EGFR-TKI efficacy against NSCLC.