Role of Commensal Microbes in the γ-Ray Irradiation-Induced Physiological Changes in Drosophila melanogaster.

Ionizing radiation induces biological/physiological changes and affects commensal microbes, but few studies have examined the relationship between the physiological changes induced by irradiation and commensal microbes. This study investigated the role of commensal microbes in the γ-ray irradiation-induced physiological changes in Drosophila melanogaster. The bacterial load was increased in 5 Gy irradiated flies, but irradiation decreased the number of operational taxonomic units. The mean lifespan of conventional flies showed no significant change by irradiation, whereas that of axenic flies was negatively correlated with the radiation dose. γ-Ray irradiation did not change the average number of eggs in both conventional and axenic flies. Locomotion of conventional flies was decreased after 5 Gy radiation exposure, whereas no significant change in locomotion activity was detected in axenic flies after irradiation. γ-Ray irradiation increased the generation of reactive oxygen species in both conventional and axenic flies, but the increase was higher in axenic flies. Similarly, the amounts of mitochondria were increased in irradiated axenic flies but not in conventional flies. These results suggest that axenic flies are more sensitive in their mitochondrial responses to radiation than conventional flies, and increased sensitivity leads to a reduced lifespan and other physiological changes in axenic flies.

JNK activation induced by ribotoxic stress is initiated from 80S monosomes but not polysomes.

Translation is a costly, but inevitable, cell maintenance process. To reduce unnecessary ATP consumption in cells, a fine-tuning mechanism is needed for both ribosome biogenesis and translation. Previous studies have suggested that the ribosome functions as a hub for many cellular signals such as ribotoxic stress response, mammalian target of rapamycin (mTOR), and ribosomal S6 kinase (RSK) signaling. Therefore, we investigated the relationship between ribosomes and mitogen-activated protein kinase (MAPK) activation under ribotoxic stress conditions and found that the activation of c-Jun N-terminal kinases (JNKs) was suppressed by ribosomal protein knockdown but that of p38 was not. In addition, we found that JNK activation is driven by the association of inactive JNK in the 80S monosomes rather than the polysomes. Overall, these data suggest that the activation of JNKs by ribotoxic stress is attributable to 80S monosomes. These 80S monosomes are active ribosomes that are ready to initiate protein translation, rather than polysomes that are already acting ribosomes involved in translation elongation. [BMB Reports 2019; 52(8): 502-507].

RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy.

RACK1, which was first demonstrated as a substrate of PKCß II, functions as a scaffold protein and associates with the 40S small ribosomal subunit. According to previous reports, ribosomal RACK1 was also suggested to control translation depending on the status in translating ribosome. We here show that RACK1 knockdown induces autophagy independent of upstream canonical factors such as Beclin1, Atg7 and Atg5/12 conjugates. We further report that RACK1 knockdown induces the association of mRNAs of LC3 and Bcl-xL with polysomes, indicating increased translation of these proteins. Therefore, we propose that the RACK1 depletion-induced autophagy is distinct from canonical autophagy. Finally, we confirm that cells expressing mutant RACK1 (RACK1R36D/K38E) defective in ribosome binding showed the same result as RACK1-knockdown cells. Altogether, our data clearly show that the depletion of ribosomal RACK1 alters the capacity of the ribosome to translate specific mRNAs, resulting in selective translation of mRNAs of genes for non-canonical autophagy induction.

Ribosomal protein S3 (rpS3) secreted from various cancer cells is N-linked glycosylated.

Ribosomal protein S3 (rpS3) is a 243 amino acid component of the 40S ribosomal small subunit. It has multiple roles in translation and extra-ribosomal functions like apoptosis and DNA repair. RpS3 is secreted only in cancer cell lines. Presently, mass spectrometry analysis revealed rpS3 to be glycosylated at the Asn165 residue. A point mutation at this residue decreased secretion of rpS3 in cancer cell lines. Secretion was also inhibited by the endoplasmic reticulum (ER)-Golgi transport inhibitor Brefeldin A and by Tunicamycin, an inhibitor of N-linked glycosylation. N-linked glycosylation of rpS3 was confirmed as necessary for rpS3 secretion into culture media via the ER-Golgi dependent pathway. RpS3 bound to Concanavalin A, a carbohydrate binding lectin protein, while treatment with peptide-N-glycosidase F shifted the secreted rpS3 to a lower molecular weight band. In addition, the N165G mutant of rpS3 displayed reduced secretion compared to the wild-type. An in vitro binding assay detected rpS3 homodimer formation via the N-terminal region (rpS3:1-85) and a middle region (rpS3:95-158). The results indicate that the Asn 165 residue of rpS3 is a critical site for N-linked glycosylation and passage through the ER-Golgi secretion pathway.

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.

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.