Ferritin-based fusion protein shows octameric deadlock state of self-assembly.

Ferritin is a universal protein complex responsible for iron perception in almost all living organisms and has applications from fundamental biophysics to drug delivery and structure-based immunogen design. Different platforms based on ferritin share similar technological challenges limiting their development - control of self-assembling processes of ferritin itself as well as ferritin-based chimeric recombinant protein complexes. In our research, we studied self-assembly processes of ferritin-based protein complexes under different expression conditions. We fused a ferritin subunit with a SMT3 protein tag, a homolog of human Small Ubiquitin-like Modifier (SUMO-tag), which was taken to destabilize ferritin 3-fold channel contacts and increase ferritin-SUMO subunits solubility. We first obtained the octameric protein complex of ferritin-SUMO (8xFer-SUMO) and studied its structural organization by small-angle X-ray scattering (SAXS). Obtained SAXS data correspond well with the high-resolution models predicted by AlphaFold and CORAL software of an octameric assembly around the 4-fold channel of ferritin without formation of 3-fold channels. Interestingly, three copies of 8xFer-SUMO do not assemble into 24-meric globules. Thus, we first obtained and structurally characterized ferritin-based self-assembling oligomers in a deadlock state. Deadlock oligomeric states of ferritin extend the known scheme of its self-assembly process, being new potential tools for a number of applications. Finally, our results might open new directions for various biotechnological platforms utilizing ferritin-based tools.

Methionine gamma lyase fused with S3 domain VGF forms octamers and adheres to tumor cells via binding to EGFR.

Methods for targeting enzymes exhibiting anticancer properties, such as methionine γ-lyase (MGL), have not yet been sufficiently developed. Here, we present the data describing the physico-chemical properties and cytotoxic effect of fusion protein MGL-S3 - MGL from Clostridium sporogenes translationally fused to S3 domain of the viral growth factor of smallpox. MGL-S3 has methioninase activity comparable to native MGL. In solution, MGL-S3 protein primarily forms octamers, whereas native MGL, on the contrary, usually forms tetramers. MGL-S3 binds to the surface of the neuroblastoma SH-SY5Y and epidermoid carcinoma A431 cells and, unlike native MGL, remains there and retains its cytotoxic effect after media removal. In HEK293T cells lacking EGFRs, no adhesion was recorded. Confocal fluorescence microscopy confirms the preferential adhesion of MGL-S3 to tumor cells, while it avoids getting into lysosomes. Both MGL and MGL-S3 arrest cell cycle of SH-SY5Y cells mainly in the G1 phase, while only MGL-S3 retains this ability after washing the cells.

Photorhabdus lux-operon heat shock-like regulation.

For decades, transcription of Photorhabdus luminescens lux-operon was considered being constitutive. Therefore, this lux-operon has been used for measurements in non-specific bacterial luminescent biosensors. Here, the expression of Photorhabdus lux-operon under high temperature was studied. The expression was researched in the natural strain Photorhabdus temperata and in the heterologous system of Escherichia coli. P. temperata FV2201 bacterium was isolated from soil in the Moscow region (growth optimum 28 °C). We showed that its luminescence significantly increases when the temperature rises to 34 °C. The increase in luminescence is associated with an increase in the transcription of luxCDABE genes, which was confirmed by RT-PCR. The promoter of the lux-operon of the related bacterium P. luminescens ZM1 from the forests of Moldova, being cloned in the heterologous system of E. coli, is activated when the temperature rises from room temperature to 42 °C. When heat shock is caused by ethanol addition, transcription of lux-operon increases only in the natural strain of P. temperata, but not in the heterologous system of E. coli cells. In addition, the activation of the lux-operon of P. luminescens persists in E. coli strains deficient in both the rpoH and rpoE genes. These results indicate the presence of sigma 32 and sigma 24 independent heat-shock-like mechanism of regulation of the lux-operon of P. luminescens in the heterologous E. coli system.

Methionine Gamma Lyase from Clostridium sporogenes Increases the Anticancer Efficacy of Doxorubicin on A549 Cancer Cells In Vitro and Human Cancer Xenografts.

The anticancer efficacy of methionine γ-lyase (MGL) from Clostridium sporogenes (C. sporogenes) is described. MGL was active against cancer cells in vitro and in vivo. Doxorubicin (DOX) and MGL were more effective on A549 human lung-cancer growth inhibition than either agent alone in vitro and in vivo.

Methionine gamma lyase from Clostridium sporogenes increases the anticancer effect of doxorubicin in A549 cells and human cancer xenografts.

The anti-cancer efficacy of methionine γ-lyase (MGL) from Clostridium sporogenes (C. sporogenes) is described. MGL was active against cancer models in vitro and in vivo. The calculated EC50 values for MGL were 4.4 U/ml for A549, 7.5 U/ml for SK-BR3, 2.4 U/ml for SKOV3, and 0.4 U/ml for MCF7 cells. The combination of doxorubicin (DOX) and MGL was more effective for A549 human lung cancer growth inhibition than either agent alone in vitro and in vivo. MGL reduced the EC50 of doxorubicin from 35.9 µg/mL to 0.01-0.265 µg/mL. The growth inhibitory effect of DOX + MGL on A549 xenografts in vivo was reflective of the results obtained in vitro. The inhibition rate of tumor growth in the combined arm was 57%, significantly higher than that in the doxorubicin (p = 0.033)-alone arm.