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.

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.

Drosophila Low Temperature Viability Protein 1 (LTV1) Is Required for Ribosome Biogenesis and Cell Growth Downstream of Drosophila Myc (dMyc).

During animal development, various signaling pathways converge to regulate cell growth. In this study, we identified LTV1 as a novel cell growth regulator in Drosophila. LTV1 mutant larvae exhibited developmental delays and lethality at the second larval stage. Using biochemical studies, we discovered that LTV1 interacted with ribosomal protein S3 and co-purified with free 40S ribosome subunits. We further demonstrated that LTV1 is crucial for ribosome biogenesis through 40S ribosome subunit synthesis and preribosomal RNA processing, suggesting that LTV1 is required for cell growth by regulating protein synthesis. We also demonstrated that Drosophila Myc (dMyc) directly regulates LTV1 transcription and requires LTV1 to stimulate ribosome biogenesis. Importantly, the loss of LTV1 blocked the cell growth and endoreplication induced by dMyc. Combined, these results suggest that LTV1 is a key downstream factor of dMyc-induced cell growth by properly maintaining ribosome biogenesis.

Interleukin-4 induces senescence in human renal carcinoma cell lines through STAT6 and p38 MAPK.

Interleukin (IL)-4, originally identified as a lymphocyte growth factor, can directly inhibit growth of certain tumor cell types. We reported previously that IL-4 induced cell cycle arrest in G1 phase through an increase in p21(WAF1/CIP1) expression in human renal cell carcinoma (RCC) cell lines. In the present study, we investigated the underlying mechanism of IL-4-induced growth inhibition. In four of six human RCC cell lines, including Caki-1, A498, SNU482, and SNU228, IL-4 induced cellular senescence as demonstrated by enlarged and flattened morphology, increased granularity, and senescence-associated-ß-galactosidase (SA-ß-gal) staining. Signal tranducer and activator of transcription 6 (STAT6) and p38 MAPK were found to mediate IL-4-induced growth inhibition and cellular senescence. Both of these molecules were activated by 10 min after IL-4 treatment, and inhibition of their activity or expression prevented growth suppression and cellular senescence induced by IL-4. Inhibiting or silencing either STAT6 or p38 MAPK alone partially reduced the effect of IL-4, whereas inhibiting or silencing both molecules exerted an additive effect and almost completely abrogated the effect of IL-4. Thus STAT6 and p38 MAPK appeared to independently mediate IL-4-induced growth inhibition and cellular senescence. The p21(WAF1/CIP1) up-regulation that accompanied growth inhibition and cellular senescence by IL-4 was also attenuated additively when p38 MAPK and STAT6 were silenced. Taken together, these results show that IL-4 induces cellular senescence through independent signaling pathways involving STAT6 and p38 MAPK in some human RCC cell lines.

Cytoplasmic ribosomal protein S3 (rpS3) plays a pivotal role in mitochondrial DNA damage surveillance.

Ribosomal protein S3 (rpS3) is known to play critical roles in ribosome biogenesis and DNA repair. When cellular ROS levels increase, the mitochondrial genes are highly vulnerable to DNA damage. Increased ROS induces rpS3 accumulation in the mitochondria for DNA repair while significantly decreasing the cellular protein synthesis. For the entrance into the mitochondria, the accumulation of rpS3 was regulated by interaction with HSP90, HSP70, and TOM70. Pretreatment with geldanamycin, which binds to the ATP pocket of HSP90, significantly decreased the interaction of rpS3 with HSP90 and stimulated the accumulation of rpS3 in the mitochondria. Furthermore, cellular ROS was decreased and mtDNA damage was rescued when levels of rpS3 were increased in the mitochondria. Therefore, we concluded that when mitochondrial DNA damages accumulate due to increased levels of ROS, rpS3 accumulates in the mitochondria to repair damaged DNA due to the decreased interaction between rpS3 and HSP90 in the cytosol.

Ribosomal protein S3 is secreted as a homodimer in cancer cells.

Protein secretion is a general phenomenon by which cells communicate with the extracellular environment. Secretory proteins, including hormones, enzymes, toxins, and antimicrobial peptides have various functions in extracellular environments. Here, we determined that ribosomal protein S3 (rpS3) is homodimerized and secreted in several cancer cell lines such as HT1080 (human fibrosarcoma) and MPC11 (mouse plasmacytoma). Moreover, we found that the secreted rpS3 protein increased in doxorubicin-resistant MPC11 cells compared to that in MPC11 cells. In addition, we also detected that the level of secreted rpS3 increased in more malignant cells, which were established with continuous exposure of cigarette smoke condensate. These findings suggest that the secreted rpS3 protein is an indicator of malignant tumors.

Phosphorylation of rpS3 by Lyn increases translation of Multi-Drug Resistance (MDR1) gene.

Lyn, a tyrosine kinase that is activated by double-stranded DNAdamaging agents, is involved in various signaling pathways, such as proliferation, apoptosis, and DNA repair. Ribosomal protein S3 (RpS3) is involved in protein biosynthesis as a component of the ribosome complex and possesses endonuclease activity to repair damaged DNA. Herein, we demonstrated that rpS3 and Lyn interact with each other, and the phosphorylation of rpS3 by Lyn, causing ribosome heterogeneity, upregulates the translation of p-glycoprotein, which is a gene product of multidrug resistance gene 1. In addition, we found that two different regions of the rpS3 protein are associated with the SH1 and SH3 domains of Lyn. An in vitro immunocomplex kinase assay indicated that the rpS3 protein acts as a substrate for Lyn, which phosphorylates the Y167 residue of rpS3. Furthermore, by adding various kinase inhibitors, we confirmed that the phosphorylation status of rpS3 was regulated by both Lyn and doxorubicin, and the phosphorylation of rpS3 by Lyn increased drug resistance in cells by upregulating p-glycoprotein translation. [BMB Reports 2023; 56(5): 302-307].

Ribosomal protein S3 interacts with TRADD to induce apoptosis through caspase dependent JNK activation.

It has been reported that ribosomal protein S3 (rpS3) functions as a ribosomal protein, a DNA repair endonuclease, a proapoptotic protein, and an essential subunit of the native NF-κB complex. However, it is unknown how rpS3 induces apoptosis in response to extracellular stresses. We report here that rpS3 sensitizes genotoxic stress-induced apoptosis by activating JNK through a caspase dependent manner. This apoptotic effect was shown to result from the physical interaction between rpS3 and TRADD, as assessed by coimmunoprecipitation. Moreover, GFP-rpS3 colocalized with TRADD around the plasma membrane and in the cytoplasm during apoptosis. Thus, rpS3 appears to be recruited to the death-inducing signaling complex (DISC) to induce apoptosis by interacting TRADD in response to extracellular stresses. Based on the findings of this study, we concluded that rpS3 is recruited to the DISC and plays a critical role in both genotoxic stress and cytokine induced apoptosis.

Ribosomal protein S3 localizes on the mitotic spindle and functions as a microtubule associated protein in mitosis.

The human ribosomal protein S3 (rpS3) has multi-functions such as translation, DNA repair and apoptosis. These multiple functions are regulated by post-translational modifications including phosphorylation, methylation and sumoylation. We report here a novel function of rpS3 that is involved in mitosis. When we examined localization of ribosomal proteins in mitosis, we found that rpS3 specifically localizes on the mitotic spindle. Depletion of the rpS3 proteins caused mitotic arrest during the metaphase. Furthermore, the shape of the spindle and chromosome movement in the rpS3 depleted cell was abnormal. Microtubule (MT) polymerization also decreased in rpS3 depleted cells, suggesting that rpS3 is involved in spindle dynamics. Therefore, we concluded that rpS3 acts as a microtubule associated protein (MAP) and regulates spindle dynamics during mitosis.

Phenylbutyric acid induces the cellular senescence through an Akt/p21(WAF1) signaling pathway.

It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21(WAF1) induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21(WAF1) pathway by PERK inhibition.

Ribosomal protein S3 is stabilized by sumoylation.

Human ribosomal protein S3 (rpS3) acts as a DNA repair endonuclease. The multiple functions of this protein are regulated by post-translational modifications including phosphorylation and methylation. Using a yeast-two hybrid screen, we identified small ubiquitin-related modifier-1 (SUMO-1) as a new interacting partner of rpS3. rpS3 interacted with SUMO-1 via the N- and C-terminal regions. We also observed sumoylation of rpS3 in Escherichia coli and mammalian cell systems. Furthermore, we discovered that one of three lysine residues, Lys18, Lys214, or Lys230, was sumoylated in rpS3. Interestingly, sumoylated rpS3 was resistant to proteolytic activity, indicating that SUMO-1 increased the stability of the rpS3 protein. We concluded that rpS3 is covalently modified by SUMO-1 and this post-translational modification regulates rpS3 function by increasing rpS3 protein stability.

Expression, purification, and characterization of putative Candida albicans Rad3, the product of orf19.7119.

Invasive infections of Candida albicans are life-threatening clinical conditions affecting immunosuppressed patients. To maintain genome integrity and diversity, C. albicans utilizes DNA repair systems, such as nucleotide excision repair (NER), to escape from attack by macrophages. Rad3 helicase is a component of the TFIIH complex, which plays a role in transcription and the NER pathway. Accumulated evidence of studies from Archaea to humans has revealed that the conserved structure, including an iron-containing domain, is essential in the function of Rad3 helicase activity. However, no study of the Rad3 protein of C. albicans has yet been reported. In the present study, putative C. albicans Rad3 (CaRad3) has been cloned with orf19.7119 of the Candida genome. CaRad3 proteins were over-expressed and purified from E. coli and S. cerevisiae using a Ni-NTA column and a size exclusion column for physicochemical and functional characterization. Through EMR and spectrometric analysis, we have proven that the purified CaRad3 protein has a Fe-S cluster. We also revealed that CaRad3 protein has a helicase activity on a duplex DNA substrate. Furthermore, we showed that the CaRad3 protein purified from yeasts was N-glycosylated, and that this protein complemented the defects in both the NER pathway and transcription. These data suggest that the Rad3 helicase in C. albicans is the product of the orf19.7119 gene.

Development of Carbazole Derivatives Compounds against Candida albicans: Candidates to Prevent Hyphal Formation via the Ras1-MAPK Pathway.

Morphogenesis contributes to the virulence of the opportunistic human fungal pathogen Candida albicans. Ras1-MAPK pathways play a critical role in the virulence of C. albicans by regulating cell growth, morphogenesis, and biofilm formation. Ume6 acts as a transcription factor, and Nrg1 is a transcriptional repressor for the expression of hyphal-specific genes in morphogenesis. Azoles or echinocandin drugs have been extensively prescribed for C. albicans infections, which has led to the development of drug-resistant strains. Therefore, it is necessary to develop new molecules to effectively treat fungal infections. Here, we showed that Molecule B and Molecule C, which contained a carbazole structure, attenuated the pathogenicity of C. albicans through inhibition of the Ras1/MAPK pathway. We found that Molecule B and Molecule C inhibit morphogenesis through repressing protein and RNA levels of Ras/MAPK-related genes, including UME6 and NRG1. Furthermore, we determined the antifungal effects of Molecule B and Molecule C in vivo using a candidiasis murine model. We anticipate our findings are that Molecule B and Molecule C, which inhibits the Ras1/MAPK pathway, are promising compounds for the development of new antifungal agents for the treatment of systemic candidiasis and possibly for other fungal diseases.

Production, characterization, and epitope mapping of monoclonal antibodies of ribosomal protein S3 (rpS3).

Ribosomal protein S3 (rpS3), a member of 40S small ribosomal subunit, is a multifunctional protein with various extra-ribosomal functions including DNA repair endonuclease activity and is secreted from cancer cells. Therefore, antibodies with high specificity against rpS3 protein could be useful cancer biomarkers. In this study, polyclonal antibody (pAb) and monoclonal antibodies (mAbs) were raised against rpS3 protein and epitope mapping was performed for each antibody; the amino acid residues of rpS3 were scanned from amino acid 185 to 243 through peptide scanning to reveal the epitopes of each mAb. Results showed that pAb R2 has an epitope from amino acid 203 to 230, mAb M7 has an epitope from amino acid 213 to 221, and mAb M8 has an epitope from amino acid 197 to 219. Taken together, novel mAbs and pAb against rpS3 were raised and mapped against rpS3 with different specific epitopes.

PKCdelta-dependent functional switch of rpS3 between translation and DNA repair.

Ribosomal protein S3 (rpS3) is critically involved in translation as a component of the 40S ribosomal subunit and participates in the processing of DNA damage, functioning as a damage DNA endonuclease. However, it is not yet known how the function of rpS3 switches between translation and DNA repair. Here we show that PKCdelta phosphorylates rpS3 resulting in its mobilization in the nucleus to repair damaged DNA. Phosphorylated rpS3 was only detected in non-ribosomal rpS3 and the repair endonuclease activity of rpS3 was increased by its phosphorylation. In addition, rpS3 knock-down cells showed more sensitivity to genotoxic stress than control cells, and this sensitivity was corrected by overexpressed wild-type rpS3 but not by phosphorylation defective rpS3. In conclusion, we propose that the destiny of rpS3 molecules between translation and DNA repair is regulated by PKCdelta-dependent phosphorylation.

RpS3 translation is repressed by interaction with its own mRNA.

Ribosomal protein S3 (RpS3) is a well-known multi-functional protein mainly involved in protein biosynthesis as a member of the small ribosomal subunit. It also plays a role in repairing various DNA damage acting as a repair UV endonuclease. Most of the rpS3 pool is located in the ribosome while the minority exists in free form in the cytoplasm. We here report an additional function of rpS3 in which it represses its own translation by binding to its cognate mRNA. Through RT-PCR of the RNAs co-immunoprecipitated with ectopically expressed rpS3, rpS3 protein was found to interact with various RNAs-endogenous rpS3, 18S rRNA. The S3-C terminal domain was shown to be the major mRNA binding domain of rpS3, independent of the KH domain. This interaction was shown to occur in cytoplasmic fractions rather than ribosomal fractions, and then is involved in its own mRNA translational inhibition by in vitro translation. Furthermore, when Flag-tagged rpS3 was transiently transfected into 293T cells, the level of endogenous rpS3 gradually decreased regardless of transcription. These results suggest that free rpS3 regulates its own translation via a feedback mechanism.

Deleting key autophagy elongation proteins induces acquirement of tumor-associated phenotypes via ISG15.

Autophagy is a cellular catabolic process that maintains intracellular homeostasis using lysosomal degradation systems. We demonstrate that inhibiting autophagy by depleting essential autophagy elongation proteins, Atg5 or Atg7, induces ISG15 expression through STING-mediated cytosolic dsDNA response. Genome stability is impaired in ATG5- or ATG7-depleted cells, and thus, double-strand breakages of DNA increase and cytosolic dsDNA accumulates. Accumulated cytosolic dsDNA induces the STING pathway to activate type I IFN signals which induce STAT1 activity and downregulate ATF3. When depletion of ATG5 or ATG7 inhibits autophagy, ATF3 is downregulated and STAT1 is upregulated. Furthermore, inhibiting autophagy induces ISG15 expression through STAT1 activation, which promotes acquisition of tumor-associated phenotypes such as migration, invasion, and proliferation. In conclusion, it appears that via the STING-mediated cytosolic dsDNA response, the STAT1-ISG15 axis mediates the relationship between autophagy and the immune system in relation to tumor progression. Moreover, combined with autophagy control, regulating ISG15 expression could be a novel strategy for cancer immunotherapy.

Arginine methylation of ribosomal protein S3 affects ribosome assembly.

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.

Regulators affecting the metastasis suppressor activity of Nm23-H1.

Nm23-H1 encodes nucleoside diphosphate kinase A (NDPK-A) and is known to have a metastasis suppressive activity in many tumor cells. However, it has many other functions as well. Recent studies have shown that the interacting proteins with Nm23-H1 which mediate the cell proliferation, may act as modulators of the metastasis suppressor activity. The interacting proteins with Nm23-H1 can be classified into 3 groups. The first group of proteins can be classified as upstream kinases of Nm23-H1 such as CKI and Aurora-A/STK15. The second group of proteins acts as downstream effectors for the regulation of specific gene transcriptions, GTP-binding protein functions, and signal transduction in Erk signal cascade. The third group of proteins can be classified as bi-directionally influencing binding partners of Nm23-H1. As a result, the interactions with Nm23-H1 and binding partners have implications in the biochemical characterization involved in metastasis and tumorigenesis.

Interaction of Hsp90 with ribosomal proteins protects from ubiquitination and proteasome-dependent degradation.

Heat-shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of various transcription factors and protein kinases in signal transduction. Multifunctional ribosomal protein S3 (rpS3), a component of the ribosomal small subunit, is involved in DNA repair and apoptosis. Our data show that Hsp90 binds directly to rpS3 and the functional consequence of Hsp90-rpS3 interaction results in the prevention of the ubiquitination and the proteasome-dependent degradation of rpS3, subsequently retaining the function and the biogenesis of the ribosome. Interference of Hsp90 activity by Hsp90 inhibitors appears to dissociate rpS3 from Hsp90, associate the protein with Hsp70, and induce the degradation of free forms of rpS3. Furthermore, ribosomal protein S6 (rpS6) also interacted with Hsp90 and exhibited a similar effect upon treatment with Hsp90 inhibitors. Therefore, we conclude that Hsp90 regulates the function of ribosomes by maintaining the stability of 40S ribosomal proteins such as rpS3 and rpS6.