RNA binding protein RPS3 mediates microglial polarization by activating NLRP3 inflammasome via SIRT1 in ischemic stroke.

BACKGROUND: Ischemic stroke is the obstruction of cerebral blood flow with a high morbidity. Microglial polarization is a contributing factor for ischemic stroke-induced injury. Here, we focused on function and mechanism of RNA binding protein RPS3 in microglial polarization after ischemic stroke. METHODS: Transient middle cerebral artery occlusion (tMCAO) was conducted in SD rats. Infarct area was detected by TTC staining and neurological score was assessed. Fluorescence staining tested neuronal apoptosis and microglial differentiation. Oxygen and glucose deprivation/reoxygenation (OGD/R) was applied for treating microglia. Levels of RPS3, SIRT1, M1 and M2 polarization markers (CD86, iNOS, CD206, Arg-1) were determined by RT-qPCR. Western blot detected RPS3, SIRT1, NLRP3, ASC and Cleaved-caspase-1 expression. RIP assay validated that RPS3 interacted with SIRT1. CCK-8 measured cell viability. Flow cytometry and ELISA assessed M1 and M2 polarization markers. LDH release was detected using colorimetric CytoTox 96 Cytotoxicity kit. RESULTS: RPS3 depletion improved neurological dysfunction and reduced infarction area in rats after tMCAO. Knockdown of RPS3 resulted in increased SIRT1 expression and decreased NLRP3 inflammasome activation, and induced microglia M2 polarization after ischemia-reperfusion (I/R). Besides, RPS3 directly targeted SIRT1 and reduced its expression in microglia. RPS3 silencing suppressed OGD/R-triggered neuronal and microglial cell death through SIRT1. Moreover, RPS3 activated NLRP3 inflammasome and regulated microglial polarization via SIRT1. CONCLUSION: RPS3 regulates microglial polarization and neuronal injury through SIRT1/NLRP3 pathway, suggesting a novel target for ischemic stroke treatment.

Salicylic acid directly binds to ribosomal protein S3 and suppresses CDK4 expression in colorectal cancer cells.

Colorectal cancer is a significant cause of morbidity and represents a serious public health issue in many countries. The development of a breakthrough preventive method for colorectal cancer is urgently needed. Aspirin has recently been attracting attention as a cancer preventive drug, and its inhibitory effects on the development of various cancers have been reported in several large prospective studies. However, the underlying molecular mechanisms have not yet been elucidated in detail. In the present study, we attempted to identify the target proteins of aspirin using a chemical biology technique with salicylic acid, the main metabolite of aspirin. We generated salicylic acid-presenting FG beads and purified salicylic acid-binding proteins from human colorectal cancer HT-29 cells. The results obtained showed the potential of ribosomal protein S3 (RPS3) as one of the target proteins of salicylic acid. The depletion of RPS3 by siRNA reduced CDK4 expression and induced G1 phase arrest in human colorectal cancer cells. These results were consistent with the effects induced by the treatment with sodium salicylate, suggesting that salicylic acid negatively regulates the function of RPS3. Collectively, the present results show the potential of RPS3 as a novel target for salicylic acid in the protective effects of aspirin against colorectal cancer, thereby supporting RPS3 as a target molecule for cancer prevention.

Ribosomal protein S3 associates with the TFIIH complex and positively regulates nucleotide excision repair.

In mammalian cells, the bulky DNA adducts caused by ultraviolet radiation are mainly repaired via the nucleotide excision repair (NER) pathway; some defects in this pathway lead to a genetic disorder known as xeroderma pigmentosum (XP). Ribosomal protein S3 (rpS3), a constituent of the 40S ribosomal subunit, is a multi-functional protein with various extra-ribosomal functions, including a role in the cellular stress response and DNA repair-related activities. We report that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. For optimal repair of UV-induced duplex DNA lesions, the strong helicase activity of the TFIIH complex is required for unwinding damaged DNA around the lesion. Here, we show that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV radiation through XPD and rpS3 interaction. Additionally, the knockdown of rpS3 caused reduced NER efficiency in HeLa cells and the overexpression of rpS3 partially restored helicase activity of the TFIIH complex of XP-D cells in vitro. We also present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We propose that rpS3 is an accessory protein of the NER pathway and its recruitment to the repair machinery augments repair efficiency upon UV damage by enhancing XPD helicase function and increasing its turnover rate.

Ribosomal protein S3 is a novel negative regulator of non-homologous end joining repair of DNA double-strand breaks.

DNA double-strand breaks (DSBs) are one of the most serious types of DNA damage. However, multiple repair pathways are present in cells to ensure rapid and appropriate repair of DSBs. Pathway selection depends on several factors including cell type, cell cycle phase, and damage severity. Ribosomal protein S3 (rpS3), a component of the 40S small ribosomal subunit, is a multi-functional protein primarily involved in protein synthesis. rpS3 is also involved in the mediation of various extra-ribosomal pathways, including DNA damage processing and the stress response. Here, we report that rpS3 is a novel negative regulator of non-homologous end joining (NHEJ)-mediated repair of DSBs. We found that rpS3 interacts with the Ku heterodimers of the DNA-dependent protein kinase (DNA-PK) complex and slows down NHEJ ligation reactions, ultimately triggering p53-dependent cell death following treatment with high-dose ionizing radiation. After DSB formation, DNA-PK phosphorylates rpS3, which consequently reduces the binding of rpS3 to the Ku complex. We hypothesized that rpS3 may play a role in DSB repair by repressing NHEJ, while inducing other repair pathways, and by initiating DSB-induced cell death in response to severe DNA damage.

A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the ß-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus.

Ribosomal protein S3-derived repair domain peptides regulate UV-induced matrix metalloproteinase-1.

Ultraviolet (UV) radiation is a major factor that causes wrinkle formation by affecting the collagen level in the skin. Here, we show that a short peptide (A8) derived from the repair domain of the ribosomal protein S3 (rpS3) reduces UV irradiation-induced increase in matrix metalloproteinase-1 (MMP-1) and prevents collagen degradation by reducing the activation of the mitogen-activated protein kinase (MAPK) signaling proteins (extracellular signal-regulated kinase [ERK], p38, and c-Jun N-terminal kinases [JNK]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in cells. Furthermore, A8 also prevents the increase in the levels of inflammatory modulators such as tumor necrosis factor-alpha (TNF-α) or interleukin-6 (IL-6) in UV-irradiated cells. Collectively, our study suggests that the A8 peptide, derived from yeast or human, has anti-photoaging potential as it prevents UV-induced wrinkle formation.

ASC1 and RPS3: new actors in 18S nonfunctional rRNA decay.

In budding yeast, inactivating mutations within the 40S ribosomal subunit decoding center lead to 18S rRNA clearance by a quality control mechanism known as nonfunctional 18S rRNA decay (18S NRD). We previously showed that 18S NRD is functionally related to No-Go mRNA Decay (NGD), a pathway for clearing translation complexes stalled on aberrant mRNAs. Whereas the NGD factors Dom34p and Hbs1p contribute to 18S NRD, their genetic deletion (either singly or in combination) only partially stabilizes mutant 18S rRNA. Here we identify Asc1p (aka RACK1) and Rps3p, both stable 40S subunit components, as additional 18S NRD factors. Complete stabilization of mutant 18S rRNA in dom34Δ;asc1Δ and hbs1Δ;asc1Δ strains indicates the existence of two genetically separable 18S NRD pathways. A small region of the Rps3p C-terminal tail known to be subject to post-translational modification is also crucial for 18S NRD. We combine these findings with the effects of mutations in the 5' → 3' and 3' → 5' decay machinery to propose a model wherein multiple targeting and decay pathways kinetically contribute to 18S NRD.

Natural allelic variations provide insights into host adaptation of Phytophthora avirulence effector PsAvr3c.

Filamentous pathogens, such as fungi and oomycetes, secrete avirulence (AVR) effectors that trigger plant immune responses and provide striking examples of host adaptations. Avr effector genes display different types of allelic variations, including deletions, epigenetic silencing and sequence polymorphisms, to avoid detection. However, how effector sequence polymorphisms enable pathogens to dodge host immune surveillance remains largely unknown. PsAvr3c is a Phytophthora AVR gene that is recognized by soybean carrying Rps3c. PsAvr3c natural alleles display a rich diversity of single nucleotide polymorphisms in field isolates. We combined both site-directed mutagenesis and population sequence surveys to identify a serine substitution of glycine at position 174 in PsAvr3c that resulted in evasion of Rps3c-mediated soybean immunity. The S174G substitution did not affect the nuclear localization of PsAvr3c in planta, which is required to activate Rps3c, but it significantly impaired the binding affinity of PsAvr3c with a previously identified spliceosome-associated protein GmSKRPs. Silencing GmSKRPs specifically impaired PsAvr3c-triggered cell death in Rps3c soybean. This study uncovered a plant Phytophthora pathogen that adapted to a resistant plant through a key amino acid mutation and subsequently reduced the binding affinity with a plant immune regulator to evade host resistance.

Mt-rps3 is an ancient gene which provides insight into the evolution of fungal mitochondrial genomes.

The nuclear ribosomal protein S3 (Rps3) is implicated in the assembly of the ribosomal small subunit. Fungi and plants present a gene copy in their mitochondrial (mt) genomes. An analysis of 303 complete fungal mt genomes showed that, when rps3 is found, it is either a free-standing gene or an anchored gene within the omega intron of the rnl gene. Early divergent fungi, Basidiomycota and all yeasts but the CTG group belong to the first case, and Pezizomycotina to the second. Its position, size and genetic code employed are conserved within species of the same Order. Size variability is attributed to different number of repeats. These repeats consist of AT-rich sequences. MtRps3 proteins lack the KH domain, necessary for binding to rRNA, in their N-terminal region. Their C-terminal region is conserved in all Domains of life. Phylogenetic analysis showed that nuclear and mtRps3 proteins are descendants of archaeal and a-proteobacterial homologues, respectively. Thus, fungal mt-rps3 gene is an ancient gene which evolved within the endosymbiotic model and presents different evolutionary routes: (a) coming from a-proteobacteria, it was relocated to another region of the mt genome, (b) via its insertion to the omega intron, it was transferred to the nucleus and/or got lost, and (c) it was re-routed to the mt genome again. Today, Basidiomycota and Saccharomycetales seem to follow the first evolutionary route and almost all Pezizomycotina support the second scenario with their exceptions being the result of the third scenario, i.e., the gene's re-entry to the mt genome.

Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3.

In addition to its role in ribosome biogenesis, ribosomal protein S3 (RPS3), a component of the 40S ribosomal subunit, has been suggested to possess several extraribosomal functions, including an apoptotic function. In this study, we demonstrated that in the mouse brain, the protein levels of RPS3 were altered by the degree of nutritional starvation and correlated with neuronal apoptosis. After endurable short-term starvation, the apoptotic function of RPS3 was suppressed by Akt activation and Akt-mediated T70 phosphorylation, whereas after prolonged starvation, the protein levels of RPS3 notably increased, and abundant neuronal death occurred. These events coincided with ubiquitination and subsequent degradation of RPS3, controlled by HSP70 and the cochaperone E3 ligase: carboxy terminus of heat shock protein 70-interacting protein (CHIP). Thus, our study points to an extraribosomal role of RPS3 in balancing neuronal survival or death depending on the degree of starvation through CHIP-mediated polyubiquitination and degradation.

Mismatch of morphological and molecular identifications in native and invasive subspecies of Codium fragile (Bryopsidophyceae, Chlorophyta).

Several subspecies are defined within Codium fragile, including the invasive C. fragile ssp. fragile, first reported in New Zealand in 1973. An endemic subspecies, C. fragile ssp. novae-zelandiae, is also found throughout New Zealand. The two subspecies exhibit morphological and molecular variation, although these have never been evaluated together. We compared variation between subspecies at locations in Auckland, identifying subspecies using rps3-rpl16 DNA sequence data, and assessing gross morphological differences, anatomical utricle characters and morphometrics. The taxonomic utility of the morphometric data sets was assessed by linear discriminant analysis. Utricle characters and measurements varied within individual thalli and between different preservation methods. The phenotypes of both subspecies were highly variable and influenced by environment. Accurate subspecies delimitation using morphological data was not possible; the discriminant analyses performed no better than chance for all combinations of the morphological data. Specimens from New Zealand, Canada, Australia and Ireland were sequenced using both the rps3-rpl16 and tufA plastid markers. The tufA elongation factor was shown to be a good candidate for differentiating subspecies of C. fragile. This marker is twice the length of the rps3-rpl16 spacer, shows greater variation between ssp. fragile and novae-zelandiae, and is less prone to sequencing error. A simple restriction enzyme digest of the tufA amplicon can distinguish ssp. fragile and ssp. novae-zelandiae. Our study expands the known range of the ssp. fragile in New Zealand, including the first record of this subspecies from the west coast of Auckland, and points to a need to re-evaluate morphological and molecular criteria for subspecies currently defined within C. fragile.

Dissociation of MIF-rpS3 complex and sequential NF-κB activation is involved in IR-induced metastatic conversion of NSCLC.

Frequent relapse and spreading of tumors during radiotherapy are principal obstacles to treatment of non-small cell lung cancer (NSCLC). In this study, we aimed to investigate how macrophage migration inhibitory factor (MIF) which is expressed at high levels in metastatic and primary lung cancer cells could regulate NSCLC metastasis in response to ionizing radiation (IR). The results indicated that MIF and ribosomal protein S3 (rpS3) were shown to be connected to inflammation, proliferation, and metastasis of NSCLC via IR-induced activation of the NF-κB pathway. Under unirradiated conditions, MIF physically established a complex with rpS3. MIF-rpS3 dissociation induced by IR activated NF-κB and made the expression of target genes of this factor transactivated in two NSCLC cell lines, A549, and NCI-H358. We also found that IR-induced dissociation of this complex led to increased secretion of pro-inflammatory cytokines and modulated the expression of epithelial-mesenchymal transition marker proteins. Finally, the effects of IR-induced dissociation of the MIF-rpS3 complex on tumor metastasis were confirmed by in vivo xenograft studies. Taken together, the present study revealed that dissociation of the MIF-rpS3 complex and subsequent activation of NF-κB is a critical post-IR exposure event that accounts for IR-induced metastatic conversion of NSCLC.

PELI1: key players in the oncogenic characteristics of pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) is a highly malignant gastrointestinal tumor, which is characterized by difficulties in early diagnosis, early metastasis, limited therapeutic response and a grim prognosis. Therefore, it is imperative to explore potential therapeutic targets for PC. Currently, although the involvement of the Pellino E3 Ubiquitin Protein Ligase 1 (PELI1) in the human growth of some malignant tumors has been demonstrated, its association with PC remains uncertain. METHODS: Bioinformatics, qRT-PCR, Western blot and IHC were used to detect the expression of PELI1 in pancreas or PC tissues and cells at mRNA and protein levels. The effects of PELI1 on the proliferation and metastatic ability of pancreatic cancer in vitro and in vivo were investigated using CCK8, cloning formation, EdU, flow cytometry, IHC, Transwell assay, wound healing, nude mice subcutaneous tumorigenesis and intrasplenic injection to construct a liver metastasis model. The interactions of PELI1 with proteins as well as the main functions and pathways were investigated by protein profiling, Co-IP, GST-pull down, Immunofluorescence techniques, immunohistochemical co-localization and enrichment analysis. The rescue experiment verified the above experimental results. RESULTS: The mRNA and protein expression levels of PELI1 in PC tissues were upregulated and were associated with poor prognosis of patients, in vitro and in vivo experiments confirmed that PELI1 can affect the proliferation and metastatic ability of PC cells. Co-IP, GST-pull down, and other experiments found that PELI1 interacted with Ribosomal Protein S3 (RPS3) through the FHA structural domain and promoted the polyubiquitination of RPS3 in the K48 chain, thereby activates the PI3K/Akt/GSK3ß signaling pathway. Moreover, ubiquitinated degradation of RPS3 further reduces Tumor Protein P53 (p53) protein stability and increases p53 degradation by MDM2 Proto-Oncogene (MDM2). CONCLUSION: PELI1 is overexpressed in PC, which increased ubiquitination of RPS3 proteins and activates the PI3K/Akt/GSK3ß signaling pathway, as well as reduces the protective effect of RPS3 on p53 and promotes the degradation of the p53 protein, which facilitates the progression of PC and leads to a poor prognosis for patients. Therefore, PELI1 is a potential target for the treatment of PC.

Quantitative trait locus mapping identifies REME2, a PPR-DYW protein required for editing of specific C targets in Arabidopsis mitochondria.

Targeted RNA editing by C-to-U alteration occurs at hundreds of sites in the mitochondrial transcriptome of flowering plants. By using natural variation and positional cloning on a population of Arabidopsis recombinant inbred lines between the ecotypes Col and Ler, we found that two of these occurrences involve the Arabidopsis PPR-DYW protein REME2 (Required for Efficiency of Mitochondrial Editing2). The analysis of a knockdown mutant along with silenced tissues confirms the specificity of REME2 for both sites located in mitochondrial ribosomal protein genes (rps3-1534 and rps4-175). The conservation level of both cis elements is relatively high, as is the amino acid conservation among flowering plants for both genes in that location, underlining the importance of these editing events and REME2.

RpS3 Is Required for Spermatogenesis of Drosophila melanogaster.

Ribosomal proteins (RPs) constitute the ribosome, thus participating in the protein biosynthesis process. Emerging studies have suggested that many RPs exhibit different expression levels across various tissues and function in a context-dependent manner for animal development. Drosophila melanogaster RpS3 encodes the ribosomal protein S3, one component of the 40S subunit of ribosomes. We found that RpS3 is highly expressed in the reproductive organs of adult flies and its depletion in male germline cells led to severe defects in sperm production and male fertility. Immunofluorescence staining showed that RpS3 knockdown had little effect on early germ cell differentiation, but strongly disrupted the spermatid elongation and individualization processes. Furthermore, we observed abnormal morphology and activity of mitochondrial derivatives in the elongating spermatids of RpS3-knockdown testes, which could cause the failure of axoneme elongation. We also found that RpS3 RNAi inhibited the formation of the individualization complex that takes charge of disassociating the spermatid bundle. In addition, excessive apoptotic cells were detected in the RpS3-knockdown testes, possibly to clean the defective spermatids. Together, our data demonstrated that RpS3 plays an important role in regulating spermatid elongation and individualization processes and, therefore, is required for normal Drosophila spermatogenesis.

Lnc-SNHG5 Promoted Hepatocellular Carcinoma Progression Through the RPS3-NFκB Pathway.

Background: We planned to explore the underlying mechanism and clinical significance of lnc-SNHG5 and RPS3 in hepatocellular carcinoma in this current study. Methods: The expression of Lnc-SNHG5 and RPS3 in HCC tissues and several cell lines were affirmed, respectively, using UALCAN, TIMER, TCGA and RT-qPCR assay. Cell proliferation ability was detected by colony formation assay and CCK8 assay. Cell apoptosis was monitored by flow cytometry assay. Next, the RPS3 expression levels and the related proteins in NFκB pathway were examined using Western blot analysis. The role of lnc-SNHG5 and RPS3 in vivo was identified by subcutaneous tumor bearing experiment. Results: Lnc-SNHG5 was significantly increased in hepatocellular carcinoma tissues and in hepatocellular carcinoma cells. Further investigation showed that up-regulated lnc-SNHG5 promoted cell viability and cell proliferation ability of SMMC-7721 cells by regulating the cell apoptosis, while down-regulation of lnc-SNHG5 revealed opposite results in QGY-7703 cells. In terms of mechanism, we found that lnc-SNHG5 interacted with RPS3. Lnc-SNHG5 regulated the NFκB pathway through RPS3 in vitro and in vivo. Conclusion: This study suggested that lnc-SNHG5 expression was signally up-regulated in hepatocellular carcinoma, and lnc-SNHG5 promoted the malignant phenotypes in vitro and in vivo via directly regulating RPS3-NFκB pathway. Lnc-SNHG5 might be a target for molecular targeted therapy, a potential and novel diagnostic marker for HCC patients.

BTN3A3 inhibits clear cell renal cell carcinoma progression by regulating the ROS/MAPK pathway via interacting with RPS3A.

Butyrophilin subfamily 3 member A3 (BTN3A3) is a member of the immunoglobulin superfamily and functions as a tumor suppressor in multiple cancer types. Our study has revealed that in clear cell renal cell carcinoma (ccRCC), patients who express high levels of BTN3A3 experience longer survival times than those with lower expression. Further, we have observed that BTN3A3 inhibits the proliferation, migration, and invasion of ccRCC cells. Through the utilization of an immunoprecipitation assay followed by mass spectrometry, we have discovered that BTN3A3 binds directly to RPS3A. Knockdown of BTN3A3 led to increased cell proliferation, migration, and invasion. However, this effect was significantly reduced when RPS3A was simultaneously overexpressed. Previous reports have demonstrated that RPS3A positively regulates mitochondrial function and reactive oxygen species (ROS) levels. Our study has shown that overexpression of both BTN3A3 and RPS3A can increase cellular oxygen consumption rate (OCR) and ROS levels. Furthermore, we have observed that the addition of H2O2 can reverse the effects of BTN3A3 knockdown on cell proliferation and migration by increasing the cellular ROS level. ROS play a crucial role in regulating the MAPK pathway and tumor cell growth. To further explore this relationship, we examined RNA-Seq and immunoblotting data and found that BTN3A3 can negatively regulate the degree of activation of the MAPK signaling pathway. This finding suggests that the BTN3A3/RPS3A complex may regulate ccRCC progression by modulating MAPK pathways. Therefore, BTN3A3 could serve as both a prognostic marker and a potential therapeutic target for ccRCC patients.

SIAH1-mediated RPS3 ubiquitination contributes to chemosensitivity in epithelial ovarian cancer.

The E3 ligase SIAH1 is deregulated in human cancers and correlated with poor prognosis, but its contributions to chemoresistance in epithelial ovarian cancer (EOC) are not evident. Herein we found that SIAH1 was decreased in EOC tumour tissues and cell lines and negatively correlated with the RPS3 levels. SIAH1 overexpression suppressed tumour cell growth, colony formation, invasion, metastasis, and cisplatin resistance in vivo and in vitro. SIAH1 promoted RPS3 ubiquitination and degradation using the RING-finger domain, and these steps were required for RPS3 localization to the cytoplasm, which led to subsequent NF-κB inactivation and thereby conferred chemosensitivity. Moreover, ectopic expression of RPS3 or depletion of RPS3 ubiquitination mediated by SIAH1 via the K214R mutant significantly impaired cisplatin-induced tumour suppression in cells stably expressing SIAH1. Together, our findings reveal a tumour suppressor function of SIAH1 and provide evidence showing that the SIAH1-RPS3-NF-κB axis may act as an appealing strategy for tackling treatment resistance in EOC.

PRRSV Induces HMGB1 Phosphorylation at Threonine-51 Residue to Enhance Its Secretion.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces secretion of high mobility group box 1 (HMGB1) to mediate inflammatory response that is involved in the pulmonary injury of infected pigs. Our previous study indicates that protein kinase C-delta (PKC-delta) is essential for HMGB1 secretion in PRRSV-infected cells. However, the underlying mechanism in HMGB1 secretion induced by PRRSV infection is still unclear. Here, we discovered that the phosphorylation level of HMGB1 in threonine residues increased in PRRSV-infected cells. A site-directed mutagenesis study showed that HMGB1 phosphorylation at threonine-51 was associated with HMGB1 secretion induced by PRRSV infection. Co-immunoprecipitation (co-IP) of HMGB1 failed to precipitate PKC-delta, but interestingly, mass spectrometry analysis of the HMGB1 co-IP product showed that PRRSV infection enhanced HMGB1 binding to ribosomal protein S3 (RPS3), which has various extra-ribosomal functions. The silencing of RPS3 by siRNA blocked HMGB1 secretion induced by PRRSV infection. Moreover, the phosphorylation of HMGB1 at threonine-51 was correlated with the interaction between HMGB1 and RPS3. In vivo, PRRSV infection also increased RPS3 levels and nuclear accumulation in pulmonary alveolar macrophages. These results demonstrate that PRRSV may induce HMGB1 phosphorylation at threonine-51 and increase its interaction with RPS3 to enhance HMGB1 secretion. This finding provides insights into the pathogenesis of PRRSV infection.

Cisplatin-Resistant Gastric Cancer Cells Promote the Chemoresistance of Cisplatin-Sensitive Cells via the Exosomal RPS3-Mediated PI3K-Akt-Cofilin-1 Signaling Axis.

Cisplatin is an important agent in first-line chemotherapy against gastric cancer (GC). However, consequential drug resistance limits its effectiveness for the treatment of GC. In this study, a cisplatin resistant gastric cancer cell line SGC7901R was determined by LC-MS/MS with increased exosomal levels of RPS3 protein. SGC7901R cell-derived exosomes were readily taken up by cisplatin-sensitive SGC7901S cells, thus triggering off a phenotype of chemoresistance in the receptor cells. Subsequently, it was demonstrated that exosomal RPS3 was essential for inducing chemoresistance of receptor cells as shown by the acquisition of this phenotype in SGC7901S cells with enforced expression of RPS3. Further mechanism study demonstrated that cisplatin-resistant gastric cancer cell-derived exosomal RPS3 enhanced the chemoresistance of cisplatin-sensitive gastric cancer cells through the PI3K-Akt-cofilin-1 signaling pathway. All these findings demonstrated that cisplatin-resistant gastric cancer cells communicate with sensitive cells through the intercellular delivery of exosomal RPS3 and activation of the PI3K-Akt-cofilin-1 signaling pathway. Targeting exosomal RPS3 protein in cisplatin-resistant gastric cancer cells may thus be a promising strategy to overcome cisplatin resistance in gastric cancer.