Biology subjects

Shirakawa, R.

Publications and source records attributed to Shirakawa, R..

7 recordsLinked to original sources

bioRxiv2026

Deletion of the ribosomal protein gene rpmJ activates zntA transcription through a translation-dependent mechanism in Escherichia coli

Bacteria tightly regulate intracellular zinc homeostasis by coordinating zinc uptake and efflux systems. We previously showed that deletion of the ribosomal protein gene rpmJ confers zinc resistance in Escherichia coli in a manner dependent on the zinc efflux transporter zntA. Here, we analyzed the effect of rpmJ deficiency on zntA expression. Under zinc excess conditions, zntA mRNA levels were markedly higher in the rpmJ mutant than in the wild-type strain. Enhanced zntA expression required the native zntA promoter, the native Shine-Dalgarno sequence, and the N-terminal coding region of zntA, indicating that translation initiated from the native Shine-Dalgarno sequence and extending through the N-terminal coding region is required for enhanced transcription initiation from the native zntA promoter. Furthermore, ectopic expression of ykgO, a paralog of rpmJ that is known to replace RpmJ on the ribosome under zinc-limited conditions, abolished the increased zntA expression and zinc resistance conferred by rpmJ deletion. Collectively, these findings suggest that ribosomes lacking RpmJ or YkgO promote transcription initiation from the native zntA promoter through translation of zntA mRNA. IMPORTANCEBacteria must carefully control the amount of zinc inside their cells. Too little zinc prevents essential cellular processes, whereas too much zinc is toxic. We found that removing a small ribosomal protein called RpmJ allows Escherichia coli to survive high zinc levels by increasing production of the zinc exporter zntA. Surprisingly, this increase depends not only on the zntA promoter but also on translation of the beginning of the zntA coding region. Our findings suggest that changes in ribosome composition can stimulate gene transcription through early translation of the same messenger RNA, revealing a previously unrecognized mechanism linking translation and transcription during bacterial adaptation to zinc stress.

molecular biology↗

bioRxiv2024

Overexpression of Ribosomal Proteins Leads to Zn Resistance in Escherichia coli

Knockout of ribosomal protein bL36 (RpmJ) leads to Zn resistance in Escherichia coli, and the expression of ribosomal protein genes other than RpmJ increase in the rpmJ-knockout strain. In this study, we examined whether the overexpression of ribosomal proteins causes Zn resistance using an E. coli overexpression gene library (ASKA clone library). The overexpression of 48 of the 54 ribosomal proteins led to Zn resistance. However, the overexpression of proteins other than ribosomal proteins did not lead to Zn resistance, suggesting that Zn resistance is a phenomenon specific to the overexpression of ribosomal proteins. In addition, the overexpression of ribosomal proteins did not lead to resistance to metal ions other than Zn (Cu2+, Ni2+, Mn2+, and Ag+), suggesting a Zn-specific resistance mechanism. Deletion of ZntA, a Zn efflux pump, resulted in the loss of Zn resistance in a ribosomal protein-overexpressing strain. Deletion of Lon protease, which is responsible for degrading misfolded proteins, in a ribosomal protein-overexpressing strain resulted in the accumulation of overexpressed ribosomal proteins and loss of Zn resistance. These results suggest that the overexpression of ribosomal proteins leads to Zn resistance in E. coli via ZntA and Lon protease. ImportanceThe ribosome is a complex comprising ribosomal RNAs and more than 50 types of ribosomal proteins. Ribosomal proteins play an important role in ribosomal function responsible for protein translation; however, their involvement in other cellular processes is not fully understood. Based on the finding that ribosomal protein expression increases in a Zn-resistant E. coli mutant, we analyzed 54 ribosomal proteins and found that the overexpression of 48 ribosomal proteins led to Zn resistance. This finding suggests a role for ribosomal proteins in resistance to zinc stress.

microbiology↗

bioRxiv2023

Protective effects of Mkl1/2 against lipodystrophy and muscle atrophy via PI3K/AKT-independent FoxO repression

FoxO transcription factors are involved in the pathogenesis of lipodystrophy and muscle atrophy. FoxO proteins are phosphorylated and inactivated by PI3K/AKT signaling; however, little is known about FoxO repressors other than this pathway. Our study showed that the Srf cofactors Mkl1 and Mkl2 directly repressed FoxO transcriptional activity, independent of the PI3K/AKT pathway. Loss of Mkl1/2 led to the overactivation of FoxO, which impaired the maintenance of both white adipose tissue (WAT) and skeletal muscle. In Mkl2-deficient preadipocytes, Ppar{gamma} was suppressed and white adipogenesis was severely impaired. In myotubes, Mkl1 and Mkl2 suppressed the expression of atrophy-related genes (atrogenes) induced by FoxO. Mkl1 expression was reduced at the onset of muscle atrophy in vivo, and exogenous supplementation of skeletal muscle Mkl1 suppressed atrogene expression and induced muscle hypertrophy. Finally, both Mkl2 nuclear localization and Mkl1/2 expression were upregulated by exercise, suggesting that Mkl1/2 is involved in the inhibitory effect of exercise on muscle atrophy. These findings indicate that Mkl1/2 act as PI3K/AKT signaling-independent repressors of FoxO and are essential for adipose and muscle tissue homeostasis.

cell biology↗

bioRxiv2022

Knockout of ribosomal protein RpmJ leads to zinc resistance in Escherichia coli

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ, a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. RNA sequence analysis revealed that rpmJ knockout decreased the expression of synthetic genes for iron-sulfur cluster proteins, which are toxic targets for zinc. These findings suggest that knocking out RpmJ causes zinc resistance by decreasing zinc targets and lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance.

microbiology↗

bioRxiv2022

Knockout of ykcB, a putative glycosyltransferase, leads to vancomycin resistance in Bacillus subtilis

Vancomycin resistance of gram-positive bacteria poses a serious health concern around the world. In this study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, to elucidate the mechanism of vancomycin resistance. We found that knockout of ykcB, a glycosyltransferase that is expected to utilize C55-P-glucose to glycosylate cell surface components, caused vancomycin resistance in B. subtilis. Knockout of ykcB altered the susceptibility to multiple antibiotics, including sensitization to {beta}-lactams, and increased the pathogenicity to silkworms. Furthermore, the ykcB-knockout mutant had: i) an increased content of diglucosyl diacylglycerol, a glycolipid that shares a precursor with C55-P-glucose, ii) a decreased amount of lipoteichoic acid, and iii) decreased biofilm formation ability. These phenotypes and vancomycin resistance were abolished by knockout of ykcC, a ykcB-operon partner involved in C55-P-glucose synthesis. Overexpression of ykcC enhanced vancomycin resistance in both wild-type B. subtilis and the ykcB-knockout mutant. These findings suggest that ykcB deficiency induces structural changes of cell surface molecules depending on the ykcC function, leading to resistance to vancomycin, decreased biofilm formation ability, and increased pathogenicity to silkworms. IMPORTANCEAlthough vancomycin is effective against gram-positive bacteria, vancomycin-resistant bacteria is a major public health concern. While the vancomycin resistance mechanisms of clinically important bacteria such as Staphylococcus aureus, Enterococcus faecium, and Streptococcus pneumoniae are well-studied, they remain unclear in other gram-positive bacteria. In the present study, we searched for vancomycin-resistant mutants from a gene deletion library of a model gram-positive bacterium, Bacillus subtilis, and found that knockout of a putative glycosyltransferase, ykcB, caused vancomycin resistance in B. subtilis. Notably, unlike the previously reported vancomycin-resistant bacterial strains, ykcB-deficient B. subtilis exhibited increased virulence while maintaining its growth rate. Our results broaden the fundamental understanding of vancomycin-resistance mechanisms in gram-positive bacteria.

microbiology↗

bioRxiv2019

Correlation between increased atrial expression of genes related to fatty acid metabolism and autophag y in patients with chronic atrial fibrillation

Atrial metabolic disturbance contributes to the onset and development of atrial fibrillation (AF). Autophagy plays a role in maintaining the cellular energy balance. We examined whether the altered atrial expression of genes related to fatty acid metabolism is linked to that related to autophagy in chronic AF. Right atrial tissue was obtained during heart surgery from 51 patients with sinus rhythm (SR, n=38) or chronic AF (n=13). Preoperative fasting serum free-fatty-acid levels were significantly higher in the AF patients. The atrial gene expression of fatty acid binding protein 3 (FABP3), which is involved in the cells fatty acid uptake and intracellular fatty acid transport, was significantly increased in AF patients compared to SR patients; in the SR patients it was positively correlated with the right atrial diameter and intra-atrial EMD, parameters of structural and electrical atrial remodeling that was evaluated by an echocardiography. In contrast, the two groups atrial contents of diacylglycerol (DAG), a toxic fatty acid metabolite, were comparable. Importantly, the atrial gene expression of microtubule-associated protein light chain 3 (LC3) was significantly increased in the AF patients, and autophagy-related genes including LC3 were positively correlated with the atrial expression of FABP3. In conclusion, in chronic AF patients, the atrial expression of FABP3 was upregulated in association with autophagy-related genes without altered atrial DAG content. Our findings may support the hypothesis that dysregulated cardiac fatty acid metabolism contributes to the progression of AF and induction of autophagy has a cardioprotective effect against cardiac lipotoxicity in chronic AF.

molecular biology↗

bioRxiv2019

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of A3B alone in cancer cells. To investigate A3B function in myeloma cells easily and comprehensively, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3xFLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced under known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeting drugs, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed the EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage response triggers A3B expression through ATM, ATR and DNA-PK signaling.

cancer biology↗