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].

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.

Significance of EZH2 expression in canine mammary tumors.

BACKGROUND: Current studies report that aberrations in epigenetic regulators or chromatin modifications are related to tumor development and maintenance. EZH2 (Enhancer of zeste homolog 2) is one of the catalytic subunits of Polycomb repressive complex 2, a crucial epigenetic regulator. EZH2 has a master regulatory function in such processes as cell proliferation, stem cell differentiation, and early embryogenesis. In humans, EZH2 is linked to oncogenic function in several carcinomas, including breast cancer, and dysregulation of EZH2 has been particularly associated with loss of differentiation and the development of poorly differentiated breast cancer. In our present study, we were interested in determining whether EZH2 is increased in canine mammary tumors, which show similarities to human breast cancer. RESULTS: Investigation of the expression of EZH2 in canine mammary tumors revealed that EZH2 protein was overexpressed in canine mammary carcinomas, as in human breast cancer. In addition, the immunohistochemical expression level of EZH2 was associated with the degree of malignancy in canine mammary carcinoma. This is the first report to describe EZH2 expression in canine mammary tumors. CONCLUSIONS: Because the expression of EZH2 was similar in canine mammary carcinoma and human breast cancer, spontaneous canine mammary tumors may be a suitable model for studying EZH2 and treatment development.

Optimization of expression conditions for soluble protein by using a robotic system of multi-culture vessels.

We have developed a robotic system for an automated parallel cell cultivation process that enables screening of induction parameters for the soluble expression of recombinant protein. The system is designed for parallelized and simultaneous cultivation of up to 24 different types of cells or a single type of cell at 24 different conditions. Twenty-four culture vessels of about 200 ml are arranged in four columns x six rows. The system is equipped with four independent thermostated waterbaths, each of which accommodates six culture vessels. A two-channel liquid handler is attached in order to distribute medium from the reservoir to the culture vessels, to transfer seed or other reagents, and to take an aliquot from the growing cells. Cells in each vessel are agitated and aerated by sparging filtered air. We tested the system by growing Escherichia coli BL21(DE3) cells harboring a plasmid for a model protein, and used it in optimizing protein expression conditions by varying the induction temperature and the inducer concentration. The results revealed the usefulness of our custom-made cell cultivation robot in screening optimal conditions for the expression of soluble proteins.

Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path.

The Escherichia coli OxyR transcription factor is activated by cellular hydrogen peroxide through the oxidation of reactive cysteines. Although there is substantial evidence for specific disulfide bond formation in the oxidative activation of OxyR, the presence of the disulfide bond has remained controversial. By mass spectrometry analyses and in vivo labeling assays we found that oxidation of OxyR in the formation of a specific disulfide bond between Cys199 and Cys208 in the wild-type protein. In addition, using time-resolved kinetic analyses, we determined that OxyR activation occurs at a rate of 9.7 s(-1). The disulfide bond-mediated conformation switch results in a metastable form that is locally strained by approximately 3 kcal mol(-1). On the basis of these observations we conclude that OxyR activation requires specific disulfide bond formation and that the rapid kinetic reaction path and conformation strain, respectively, drive the oxidation and reduction of OxyR.