Tomáš Podzimek

Antitumor effects and cytotoxicity of recombinant plant nucleases.

Recombinant plant nucleases R-TBN1 and R-HBN1 were isolated to homogeneity and examined for their antitumor effects and cytotoxicity. Although antiproliferative effects of both recombinant nucleases were not significant on the ML-2 cell culture in vitro, the nucleases were strongly cytostatic in vivo after their administration intravenously as stabilized conjugates with polyethylene glycol (PEG). Recombinant nucleases were as effective against melanoma tumors as previously studied pine pollen (PN) and mung bean nucleases and their effects were reached at about 10 times lower concentrations compared to the use of bovine seminal RNase (BS-RNase). Because the recombinant nucleases R-HBN1 and R-TBN1 share only 67.4% amino acid identity and showed only partial immunochemical cross-reactivity, their similar anticancerogenic effects can be mainly explained by their catalytical similarity. Both recombinant nucleases showed lower degree of aspermatogenesis compared to BS-RNAse and PN nuclease. Unlike BS-RNase, aspermatogenesis induced by both recombinant nucleases could not be prevented by the homologous antibody complexes. Owing to relatively low cytotoxicity on the one hand, and high efficiency at low protein levels on the other, recombinant plant nucleases R-HBN1 and R-TBN1 appear to be stable biochemical agents that can be targeted as potential antitumor cytostatics.

Plant multifunctional nuclease TBN1 with unexpected phospholipase activity: structural study and reaction-mechanism analysis.

Type I plant nucleases play an important role in apoptotic processes and cell senescence. Recently, they have also been indicated to be potent anticancer agents in in vivo studies. The first structure of tomato nuclease I (TBN1) has been determined, its oligomerization and activity profiles have been analyzed and its unexpected activity towards phospholipids has been discovered, and conclusions are drawn regarding its catalytic mechanism. The structure-solution process required X-ray diffraction data from two crystal forms. The first form was used for phase determination; the second form was used for model building and refinement. TBN1 is mainly α-helical and is stabilized by four disulfide bridges. Three observed oligosaccharides are crucial for its stability and solubility. The active site is localized at the bottom of the positively charged groove and contains a zinc cluster that is essential for enzymatic activity. An equilibrium between monomers, dimers and higher oligomers of TBN1 was observed in solution. Principles of the reaction mechanism of the phosphodiesterase activity are suggested, with central roles for the zinc cluster, the nucleobase-binding pocket (Phe-site) and Asp70, Arg73 and Asn167. Based on the distribution of surface residues, possible binding sites for dsDNA and other nucleic acids with secondary structure were identified. The phospholipase activity of TBN1, which is reported for the first time for a nuclease, significantly broadens the substrate promiscuity of the enzyme, and the resulting release of diacylglycerol, which is an important second messenger, can be related to the role of TBN1 in apoptosis.

Structure analysis of group I plant nucleases

Structural properties of plant nuclease TBN1 are studied using synchrotron radiation to explain its specificity, role of glycosylation and to contribute to potential application in cancer treatment.

Crystallization of recombinant bifunctional nuclease TBN1 from tomato

The endonuclease TBN1 from Solanum lycopersicum (tomato) was expressed in Nicotiana benthamiana leaves and purified with suitable quality and in suitable quantities for crystallization experiments. Two crystal forms (orthorhombic and rhombohedral) were obtained and X-ray diffraction experiments were performed. The presence of natively bound Zn2+ ions was confirmed by X-ray fluorescence and by an absorption-edge scan. X-ray diffraction data were collected from the orthorhombic (resolution of 5.2 Å) and rhombohedral (best resolution of 3.2 Å) crystal forms. SAD, MAD and MR methods were applied for solution of the phase problem, with partial success. TBN1 contains three Zn2+ ions in a similar spatial arrangement to that observed in nuclease P1 from Penicillium citrinum.

Phosphate binding in the active centre of tomato multifunctional nuclease TBN1 and analysis of superhelix formation by the enzyme

Tomato multifunctional nuclease TBN1 belongs to the type I nuclease family, which plays an important role in apoptotic processes and cell senescence in plants. The newly solved structure of the N211D mutant is reported. Although the main crystal-packing motif (the formation of superhelices) is conserved, the details differ among the known structures. A phosphate ion was localized in the active site of the enzyme. The binding of the surface loop to the active centre is stabilized by the phosphate ion, which correlates with the observed aggregation of TBN1 in phosphate buffer. The conserved binding of the surface loop to the active centre suggests biological relevance of the contact in a regulatory function or in the formation of oligomers.

A 5′P degradation hot spot influences molecular farming of anticancerogenic nuclease TBN1 in tobacco cells

Tomato bifunctional nuclease 1 (TBN1) is a polyfunctional protein with anticancerogenic activity originally isolated as an overexpressed protein from viroid-infected tomato. Its molecular farming in plant cells could be a non-expensive source for its biotechnology preparation. So we analysed TBN1 expression in Agrobacterium-infiltrated leaf sectors of Nicotiana benthamiana and in transformed suspension culture of tobacco BY-2 cells. During its transient expression, TBN1 mRNA was strongly degraded within a hot spot localized in the 3′ region. This early degradation process was inhibited by PTGS suppressors p19 and p38 resulting in increased TBN1 mRNA and protein yield. In parallel to degradation of TBN1 mRNA, high mRNA levels of two RNA-dependent RNA polymerases were detected in infiltrated leaf sectors, as well as in the transformed tobacco suspension culture BY-2, where low expression of the nuclease was stably maintained. Higher TBN1 mRNA and nuclease activity levels were found during its molecular farming in RDR6-deficient N. benthamiana plants. By fluorescent microscopy of infiltrated and transformed plant cells, the nuclease-GFP fusion protein was shown to be organized in filament-like structures.

N-glycosylation of tomato nuclease TBN1 produced in N. benthamiana and its effect on the enzyme activity

A unique analysis of an enzyme activity versus structure modification of the tomato nuclease R-TBN1 is presented. R-TBN1, the non-specific nuclease belonging to the S1-P1 nuclease family, was recombinantly produced in N. benthamiana. The native structure is posttranslationally modified by N-glycosylation at three sites. In this work, it was found that this nuclease is modified by high-mannose type N-glycosylation with a certain degree of macro- and microheterogeneity. To monitor the role of N-glycosylation in its activity, hypo- and hyperglycosylated nuclease mutants, R-TBN1 digested by α-mannosidase, and R-TBN1 deglycosylated by PNGase F were prepared. Deglycosylated R-TBN1 and mutant N94D/N112D were virtually inactive. Compared to R-TBN1 wt, both N94D and N112D mutants showed about 60% and 10% of the activity, respectively, while the N186D, D36S, and D36S/E104 N mutants were equally or even more active than R-TBN1 wt. The partial demannosylation of R-TBN1 did not affect the nuclease activity; moreover, a little shift in substrate specificity was observed. The results show two facts: 1) which sites must be occupied by a glycan for the proper folding and stability and 2) how N. benthamiana glycosylates the foreign nuclease. At the same time, the modifications can be interesting in designing the nuclease activity or specificity through its glycosylation.