Dissertations, Theses, and Capstone Projects

Date of Degree

9-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Shaneen Singh

Committee Members

Shubha Govind

Robert Dottin

Luis Quadri

Evros Vassiliou

Raj Wadgaonka

Subject Categories

Bioinformatics | Computational Biology | Molecular Biology | Structural Biology

Keywords

Cyclooxygenase, Drosophila, Eicosanoid, Homology modeling, Prostaglandin, leukotriene, thromboxane

Abstract

With increased understanding of their roles in signal transduction and metabolism, eicosanoids have emerged as important players in human health and disease. Mammalian prostanoids and related lipid mediators perform varied functions in different tissues and organs. Synthesized through the oxygenation of C20 polyunsaturated fatty acids, mammalian eicosanoids are both pro- and anti-inflammatory. The physiological contexts in which eicosanoid family members act at the cellular level are not well understood. In this study, we examined whether the genome of Drosophila melanogaster, a powerful model for innate immunity and inflammation, codes for the enzymes required for eicosanoid biosynthesis. We report the existence of putative eicosanoid biosynthesis enzymes in Drosophila melanogaster which may function together as a pathway similar to the mammalian eicosanoid synthesis pathway. Standard sequence-based search methods failed to identify high confidence orthologs for a majority of the mammalian eicosanoid synthesis enzymes in D. melanogaster, and in insects generally. Using sensitive sequence analysis techniques, we identified candidate orthologs in the Drosophila genome that share low global sequence identities with their human counterparts. The Drosophila sequences were further scrutinized by modeling and structural analyses. We generated and evaluated full-length models for top-scoring Drosophila candidates corresponding to each human eicosanoid synthesis enzyme and identified potentially equivalent functional residues. This combination of sensitive sequence and structural analyses revealed that the existence of eight high confidence, five mid-range and eight low confidence candidates. Four predicted cyclooxygenases and two potential lipoxygenase activating proteins, highly divergent from their human counterparts, were identified, although similar methods failed to identify putative lipoxygenase enzymes. Tertiary structures of a majority of identified candidate fly proteins are very similar to the corresponding human target enzymes and appear to possess the necessary catalytic residues. These results, in combination with other recent biochemical studies alluding to eicosanoid activity in insects by other groups, suggest that D. melanogaster may indeed possess biosynthesis pathways for eicosanoid or eicosanoid-like biolipids. However, the predominant view in the field is that an eicosanoid synthesis pathway does not exist in Drosophila primarily because to date clear homologs of the enzymes of this pathway have not been identified. Our study challenges this currently held view. Molecular-genetic and biochemical analyses of individual biosynthetic enzymes in D. melanogaster, a model organism with low genetic redundancy will reveal if the fly enzymes are functionally equivalent to their mammalian counterparts; their in vivo interactions will allow construction of pathways and networks in a physiological context. Our findings predict that classical or novel eicosanoids or eicosanoid-like lipid mediators regulate biological functions in insects. Eicosanoids are known to play important roles in insect immunity. The identification of these lipid mediators will therefore provide new insect control measures or the means of improving the health of beneficial insects.

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