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Bücher und Buchkapitel

Carbonell, A.; Flores, R.; Gago, S.; Hammerhead Ribozymes Against Virus and Viroid RNAs (Erdmann, V. A. & Barciszewski, J., eds.). RNA Technologies 411-427, (2012) ISBN: 978-3-642-27426-8 DOI: 10.1007/978-3-642-27426-8_16

The hammerhead ribozyme, a small catalytic motif that promotes self-cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically in trans other RNAs in the presence of Mg2+. To be really effective, hammerheads need to operate at the low concentration of Mg2+ existing in vivo. Evidence has been gathered along the last years showing that tertiary stabilizing motifs (TSMs), particularly interactions between peripheral loops, are critical for the catalytic activity of hammerheads at physiological levels of Mg2+. These TSMs, in two alternative formats, have been incorporated into a new generation of more efficient trans-cleaving hammerheads, some of which are active in vitro and in planta when targeted against the highly structured RNA of a viroid (a small plant pathogen). This strategy has potential to confer protection against other RNA replicons, like RNA viruses infecting plants and animals.
Publikation

Dorka, R.; Miersch, O.; Wasternack, C.; Weik, P.; Chronobiological phenomena and seasonal changes in jasmonate levels during the course of the year and under constant conditions in mistletoe (Viscum album L.) Phytomedicine 14, 15, (2007) DOI: 10.1016/j.phymed.2007.07.014

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Publikation

Schilling, S.; Hoffmann, T.; Rosche, F.; Manhart, S.; Wasternack, C.; Demuth, H.-U.; Heterologous Expression and Characterization of Human Glutaminyl Cyclase: Evidence for a Disulfide Bond with Importance for Catalytic Activity Biochemistry 41, 10849-10857, (2002) DOI: 10.1021/bi0260381

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high α-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.
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