RESUMO
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.
Assuntos
Antineoplásicos , Neuroblastoma , Humanos , Células HEK293 , Liases de Carbono-Enxofre/metabolismo , Receptores ErbB/genética , Metionina/metabolismo , Fatores de Crescimento NeuralRESUMO
This work provides the first characteristics of the rhodopsin SpaR from Sphingomonas paucimobilis, aerobic bacteria associated with opportunistic infections. The sequence analysis of SpaR has shown that this protein has unusual DTS motif which has never reported in rhodopsins from Proteobacteria. We report that SpaR operates as an outward proton pump at low pH; however, proton pumping is almost absent at neutral and alkaline pH. The photocycle of this rhodopsin in detergent micelles slows down with an increase in pH because of longer Schiff base reprotonation. Our results show that the novel microbial ion transporter SpaR of interest both as an object for basic research of membrane proteins and as a promising optogenetic tool.
Assuntos
Bombas de Próton/metabolismo , Rodopsina/metabolismo , Rodopsinas Microbianas/metabolismo , Sphingomonas/metabolismo , Concentração de Íons de Hidrogênio , Luz , Optogenética/métodos , Bombas de Próton/genética , Rodopsina/genética , Rodopsinas Microbianas/genética , Sphingomonas/genéticaRESUMO
The antirestriction ArdB protein inhibits the endonuclease activity of type I restriction/modification (RM) systems in vivo; however, the mechanism of inhibition remains unknown. In this study, we showed that recombinant ArdB from Escherichia coli cells co-purified with DNA. When overexpressed in E. coli cells, a portion of ArdB protein formed insoluble DNA-free aggregates. Only native ArdB, but not the ArdBΔD141 mutant lacking the antirestriction activity, co-purified with DNA upon anion-exchange and affinity chromatography or total DNA isolation from formaldehyde-treated cells. These observations confirm the hypothesis that ArdB blocks DNA translocation via the R subunits of the R2M2S complex of type I RM enzymes.
Assuntos
DNA Bacteriano/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Cromatografia por Troca Iônica , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Formaldeído/química , Espectrometria de Massas , Mutação , Ligação Proteica , Proteínas Recombinantes/metabolismoRESUMO
Membrane integral ATP synthases produce adenosine triphosphate, the universal "energy currency" of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Proteínas de Plantas/ultraestrutura , Estrutura Quaternária de Proteína , Trifosfato de Adenosina/biossíntese , Cloroplastos/enzimologia , Coenzimas/metabolismo , Cristalografia por Raios X , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Moleculares , Proteínas de Plantas/metabolismo , Conformação Proteica , Subunidades Proteicas/metabolismo , Spinacia oleracea/enzimologia , Ubiquinona/metabolismoRESUMO
In humans, two endothelin receptors, ETa and ETb, are activated by three endogenous 21-mer cyclic peptides, ET-1, ET-2, and ET-3, which control various physiological processes, including vasoconstriction, vasodilation, and stimulation of cell proliferation. The first stage of this study it to produce a stable solubilized and purified receptor in a monodisperse state. This article is focused on the engineering, expression, purification, and characterization of the endothelin receptor B for subsequent structural and functional studies.