Browsing Department of Animal Biology by Author "Adel, Susan"
Results Per Page
ItemConversion of pro-inflammatory murine Alox5 into an anti-inflammatory 15S-lipoxygenating enzyme by multiple mutations of sequence determinants( 2013-02-01) Hofheinz, Katharina ; Kakularam, Kumar Reddy ; Adel, Susan ; Anton, Monika ; Polymarasetty, Aparoy ; Reddanna, Pallu ; Kuhn, Hartmut ; Horn, Thomas5-Lipoxygenase (ALOX5) is a key enzyme in biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Although mammalian LOX-isoforms share a high degree of structural similarity X-ray coordinates indicated that the substrate-binding pocket of ALOX5 is some 20% bigger than that of ALOX15 suggesting the possibility of interconverting the two isoenzymes. To test this "space-based" hypothesis we reduced the volume of the substrate-binding pocket of mouse Alox5 by introducing space-filling amino acids at critical positions and found that multiple mutations at Phe359, Ala424, Asn425 and Ala603 of Alox5 led to gradual increase in 15-HETE formation. The Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant was a major (67 ± 2%) 15-lipoxygenating enzyme and similar data were confirmed for human ALOX5. Structural modeling on the basis of the X-ray coordinates of ALOX5 indicated that the volume of the substrate-binding pocket inversely correlates with the share of 15-HETE biosynthesis for the human (r2 = 0.79, p < 0.05) and the mouse (r2 = 0.59, p < 0.01) enzyme. This data proves the principle possibility of converting pro-inflammatory 5-lipoxygenases to anti-inflammatory 15-lipoxygenases by reducing the volume of the substrate-binding pocket. © 2012 Elsevier Inc. All rights reserved.
ItemLeukotriene signaling in the extinct human subspecies Homo denisovan and Homo neanderthalensis. Structural and functional comparison with Homo sapiens( 2015-01-01) Adel, Susan ; Kakularam, Kumar Reddy ; Horn, Thomas ; Reddanna, Pallu ; Kuhn, Hartmut ; Heydeck, DagmarMammalian lipoxygenases (LOXs) have been implicated in cell differentiation and in the biosynthesis of pro- and anti-inflammatory lipid mediators. The initial draft sequence of the Homo neanderthalensis genome (coverage of 1.3-fold) suggested defective leukotriene signaling in this archaic human subspecies since expression of essential proteins appeared to be corrupted. Meanwhile high quality genomic sequence data became available for two extinct human subspecies (H. neanderthalensis, Homo denisovan) and completion of the human 1000 genome project provided a comprehensive database characterizing the genetic variability of the human genome. For this study we extracted the nucleotide sequences of selected eicosanoid relevant genes (ALOX5, ALOX15, ALOX12, ALOX15B, ALOX12B, ALOXE3, COX1, COX2, LTA4H, LTC4S, ALOX5AP, CYSLTR1, CYSLTR2, BLTR1, BLTR2) from the corresponding databases. Comparison of the deduced amino acid sequences in connection with site-directed mutagenesis studies and structural modeling suggested that the major enzymes and receptors of leukotriene signaling as well as the two cyclooxygenase isoforms were fully functional in these two extinct human subspecies.