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sensitive to stabilization by iminosugars, which bind at the active site to provide the proper conformation. Thus the stabilized protein may escape from degradation processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). In this work the influence of novel derivatives of 1-deoxygalactonojirimycin (DGJ) on the β-Gal activity of cultured GM1 and MBD skin fibroblasts was examined. Furthermore, the effect of selected compounds on natural substrate degradation in GM1 and MBD cells was determined. Several novel iminosugars acting as pharmacological chaperones of β-Gal in specific GM1 fibroblasts were discovered and described in this work. One specific compound, DLHex-DGJ, proved to be a potent competitive inhibitor of β-Gal in vitro, and this work describes its effects on activity, protein expression, maturation and intracellular transport in vivo in 13 fibroblast lines with GLB1 mutations. DLHex-DGJ significantly increased the catalytic activity in six GM1 cell lines, and normalization of transport and lysosomal processing of β-Gal precursors was demonstrated for selected cell lines. Furthermore, DLHex-DGJ and another, similar compound successfully reduced the level of internalized radiolabeled G M1 -gangliosides in a specific GM1 cell line, suggesting that reduction of stored material is possible under certain conditions. Specific antibodies, directed against human β-Gal, were developed with the aid of previously published protocols, and novel approaches to obtain large amounts of the purified human enzyme were tested. Two novel polyclonal anti-β-Gal peptide antibodies were produced and expression of human β-Gal in E. coli cells may provide the basis for further development of antibodies directed against the human enzyme. Large parts of this thesis were carried out at the University hospital under the guidance and supervision of Prof. Pascke. International cooperations Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia Steve Ealick, Cornell University, Ithaca, U.S.A. Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A. Shwu Liaw, National Yang-Ming University, Taipei, Taiwan Matthias Mack, Hochschule Mannheim, Germany Bruce Palfey, University of Michigan, Ann Arbor, U.S.A. Research projects FWF P22361: “Mechanism of redox controlled protein degradation” FWF P19858: “Enzymes of nikkomycin biosynthesis” FWF-Doktoratskolleg “Molecular Enzymology” WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 family” Publications 1) Durchschein, K., Ferreira-da Silva, B., Wallner, S., Macheroux, P., Kroutil, W., Glueck, S. M., Faber, K.: The flavoprotein-catalyzed reduction of aliphatic nitro-compounds represents a biocatalytic equivalent to the Nef-reaction, Green Chemistry, 2010, 12:616- 619. 14
2) Jajcanin-Jozic, N., Deller, S., Pavkov, T., Macheroux, P., Abramic, M.: Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III, Biochimie, 2010, 92:89- 96. Award Dissertation Award of the Austrian Society for Molecular Biosciences and Biotechnology (ÖGMBT) to Andreas Winkler for his dissertation on “Structure-function studies on berberine bridge enzyme (BBE) from the California poppy, Eschscholzia californica”. Dr. Andreas Winkler was a PhD student in the Doktoratskolleg “Molecular Enzymology” and is now a postdoctoral fellow at the Max-Planck Institute for Molecular Medicine in Heidelberg, Germany. 15
Page 1 and 2: Graz University of Technology Austr
Page 3 and 4: Brief History of the Institute of B
Page 5 and 6: Highlights of 2010 In 2010, Peter M
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Page 9 and 10: plant genes that apparently encode
Page 11 and 12: unusual amino acids, i.e. hydroxypy
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