Application of bioinformatics in studies of sphingolipid biosynthesis

Sphingolipids are diverse family of molecules found in all eukaryotes and some prokaryotes. They modulate critical functions related to cell survival, apoptosis, migration and membrane structures. As sphingolipids and their biosynthetic pathways are highly complex, studies in sphingolipid metabolism have been challenging. This dissertation addresses this challenge by the novel application of bioinformatics analysis to investigate sphingolipid and glycosphingolipid alterations at the system, as well as molecular levels.;As part of this thesis, updated pathway maps were prepared based on recent reports to visualize differences in gene expression and metabolites in the different branches of sphingolipid biosynthesis. Supporting Perl scripts were written to extract and format gene expression datasets for pathway visualization.;Cancer gene expression datasets were examined for sphingolipid gene expression differences using the pathway maps and compared to metabolites that were either reported or analyzed by mass spectrometry analysis conducted during this study. Comparison of 9 gene expression datasets and corresponding sphingolipid alterations identified a 72.8% correlation between transcript and metabolite pattern at a significance p < 0.0001 using the Fisher's exact test. This study also discovered hydroxyl sphingolipids (phyto-sphingolipids) and short chain ceramides in MCF-7 and MDA-MB-231 cells respectively that were initially predicted by a gene expression pathway diagram.;The next study focuses on the discovery of the transcript that encodes the human 3-Ketosohinganine reductase (3KSR) which was not identified during the preparation of the pathway map. By blast comparison to the yeast enzyme sequence, Follicular lymphoma type 1 (FVT1) was identified as the human homolog of 3KSR. After over expressing FVT1 in HeLa cells and E. coli, its 3KSR activity was verified by an in vitro enzyme activity assay. In HeLa cells we have further discovered that the absence of FOXC1, a putative transcription regulator of FVT1 results in lower 3KSR activity and the accumulation of intermediate sphingolipids.;One of the critical factors influencing de novo sphingolipid biosynthesis is the expression of serine palmitoyltransferase (SPTLC1 and SPTLC2 subunits) and its role has been widely studied using a Chinese hamster ovary cells (LY-B) that lack the enzyme. Because the molecular abnormality of LY-B cells in the cells was unknown, we cloned the SPTLC1 transcript and discovered a point mutation. Homology modeling of the mutant protein suggested that the substitution would cause the protein to misfold and degrade. The use of chemical chaperones, DMSO and glycerol resulted in the partial stabilization of the SPT peptide but no significant SPT activity was determined.;These studies demonstrate the application of bioinformatics to investigate different molecular aspects of the sphingolipid biosynthesis.