Structural and Biochemical Investigation of the Heterodimeric Murine tRNA-Guanine Transglycosylase.

In tRNAAsp, tRNAAsn, tRNATyr, and tRNAHis of most bacteria and eukaryotes, the anticodon wobble position may be occupied by the modified nucleoside queuosine, which affects the speed and the accuracy of translation. Since eukaryotes are not able to synthesize queuosine de novo, they have to salvage queuine (the queuosine base) as a micronutrient from food and/or the gut microbiome. The heterodimeric Zn2+ containing enzyme tRNA-guanine transglycosylase (TGT) catalyzes the insertion of queuine into the above-named tRNAs in exchange for the genetically encoded guanine. This enzyme has attracted medical interest since it was shown to be potentially useful for the treatment of multiple sclerosis. In addition, TGT inactivation via gene knockout leads to the suppressed cell proliferation and migration of certain breast cancer cells, which may render this enzyme a potential target for the design of compounds supporting breast cancer therapy. As a prerequisite to fully exploit the medical potential of eukaryotic TGT, we have determined and analyzed a number of crystal structures of the functional murine TGT with and without bound queuine. In addition, we have investigated the importance of two residues of its non-catalytic subunit on dimer stability and determined the Michaelis-Menten parameters of murine TGT with respect to tRNA and several natural and artificial nucleobase substrates. Ultimately, on the basis of available TGT crystal structures, we provide an entirely conclusive reaction mechanism for this enzyme, which in detail explains why the TGT-catalyzed insertion of some nucleobases into tRNA occurs reversibly while that of others is irreversible.

Development of a system of heparin multilayers on titanium surfaces for dual growth factor release.

The aim of the present study was to establish a modular platform of poly-L-lysine-heparin (PLL-Hep) polyelectrolyte multilayer (PEM) coatings on titanium surfaces for dual growth factor delivery of recombinant human bone morphogenic protein 2 (rhBMP2) and recombinant human vascular endothelial growth factor 165 (rhVEGF165) in clinically relevant quantities. Release characteristics for both growth factors differed significantly depending on film architecture. rhBMP2 induced activation of alkaline phosphatase in C2C12 cells and proliferation of human mesenchymal stem cells (hMSCs). rhVEGF mediated induction of von Willebrand factor (vWF) in hMSCs and proliferation of human umbilical vein endothelial cells. Osteogenic and angiogenic effects were modified by variation in cross-linking and architecture of the PEMs. By creating multilayer films with distinct zones, release characteristics and proportion of both growth factor delivery could be tuned and surface-activity modified to enhance angiogenic or osteogenic function in various ways. In summary, the system provides a modular platform for growth factor delivery that allows for individual composition and accentuation of angiogenic and osteogenic surface properties.

Cellular prion protein mediates early apoptotic proteome alternation and phospho-modification in human neuroblastoma cells.

Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrPc) under stress conditions are well documented. Yet, detrimental effects of elevated PrPc levels under stress conditions, such as exposure to staurosporine (STS) have also been described. In the present study, we focused on discerning early apoptotic STS-induced proteome and phospho-proteome changes in SH-SY5Y human neuroblastoma cells stably transfected either with an empty or PRNP-containing vector, expressing physiological or supraphysiological levels of PrPc, respectively. PrPc-overexpression per se appears to stress the cells under STS-free conditions as indicated by diminished cell viability of PrPc-overexpressing versus control cells. However, PrPc-overexpression becomes advantageous following exposure to STS. Thus, only a short exposure (2 h) to 1 µM STS results in lower survival rates and significantly higher caspase-3 activity in control versus PrPc-overexpressing cells. Hence, by exposing both experimental groups to the same apoptotic conditions we were able to induce apoptosis in control, but not in PrPc-overexpressing cells (as assessed by caspase-3 activity), which allowed for filtering out proteins possibly contributing to protection against STS-induced apoptosis in PrPc-overexpressing cells. Among other proteins regulated by different PrPc levels following exposure to STS, those involved in maintenance of cytoskeleton integrity caught our attention. In particular, the finding that elevated PrPc levels significantly reduce profilin-1 (PFN-1) expression. PFN-1 is known to facilitate STS-induced apoptosis. Silencing of PFN-1 expression by siRNA significantly increased viability of PrPc-overexpressing versus control cells, under STS treatment. In addition, PrPc-overexpressing cells depleted of PFN-1 exhibited increased viability versus PrPc-overexpressing cells with preserved PFN-1 expression, both subjected to STS. Concomitant increase in caspase-3 activity was observed in control versus PrPc-overexpressing cells after treatment with siRNA- PFN-1 and STS. We suggest that reduction of PFN-1 expression by elevated levels of PrPc may contribute to protective effects PrPc-overexpressing SH-SY5Y cells confer against STS-induced apoptosis.

Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival.

The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival.