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ePaper created 2013-09-24, 11:56:35 | version 1.25.20
8 0 PHARMACY SYNTHETIC PROTEINS IN HUMAN PATHOGENIC YEASTS Dipl.-Biol. Silke Palzer, Dr. rer. nat. Kai Sohn Virulence factors determine pathogenicity The numbers of infectious diseases caused by human patho- genic yeasts have been continuously increasing over recent years. Their high morbidity and mortality have turned them into a serious public health problem. An efficient treatment is particularly complicated by systemic mycoses, which spread throughout the whole body, or emerging resistance to anti- biotics. The most prevalent causative of systemic mycoses in humans is the pathogenic yeast Candida albicans, which can elicit severe infections if the immune system of its host is sup- pressed by, for example, operations, chemotherapy or dis- eases. Candida albicans features a multitude of mechanisms which lead to pathogenicity. These mechanisms are mediated by virulence factors, proteins with various functions in the cell. Virulence factors are essential for pathogenicity and there- fore appear to be a promising target for the development of therapeutics. However, this requires profound knowledge of the molecular characteristics and physiological interaction net- works of cell proteins. As techniques to study protein inter- action networks in vivo are scarce, especially for C. albicans, scientists at the Fraunhofer IGB developed a new method to analyze protein-protein interactions with artificial amino acids. Analysis of protein-protein interactions with synthetic proteins The protein of interest is modified in only one building block, which is one amino acid that is replaced by an artificial ami- no acid. This artificial amino acid confers new physicochemi- cal properties to the protein of interest. Artificial amino acids belong to the scientific area of synthetic biology and there are currently over 300 artificial amino acids available. These synthetic amino acids offer scientists a variety of applications for proteins, for example, to facilitate analyses or add entirely new properties. The artificial amino acid p-azidophenylalanine (Fig. 2), a derivative of the natural amino acid phenylalanine, is particularly suitable for the study of molecular interactions. The azido-group does not occur naturally in proteins and can be activated by UV light to form a stable covalent bond with molecules in close vicinity. If the modified, synthetic protein interacts with another protein in the cell, the interaction can be captured by UV photo crosslinking under physiological con- ditions and is stable for further purification or identification of the interacting partner. An expanded genetic code In order to conduct in vivo interaction studies, the synthetic amino acid must be incorporated into proteins. A synthetic biology method, the so-called expanded genetic code, can be used to achieve this. The artificial amino acid is incorpo- rated during cellular protein synthesis specifically and with unrivalled efficiency into the protein of interest, at the desired position, mediated by special biomolecules, tRNAs and tRNA synthetases. 1 2 N3 COOHH2N