5 research outputs found
Profiling the Lipidome Hallmarks of Colorectal Cancer
No full text© 2021 Yepy Hardi RustamAltered lipid metabolism is associated with carcinogenesis and metastatic capacity in human cancers, including colorectal cancer (CRC). Elucidating the composition of dysregulated lipids is therefore essential to understanding CRC biology, to identify diagnostic or prognostic biomarkers, and to highlight novel therapeutic targets for CRC. Here, a mass spectrometric and bioinformatic analysis strategy was developed and employed for comprehensive lipidome analysis of a series of established CRC cell lines, and primary tumour tissues.
A lipidomic analysis workflow employing ultra-high resolution and accurate mass spectrometry (UHRAMS) was developed and optimised to enable high-throughput lipid identification and semi-quantitative analysis at the 'sum composition' level from both CRC cell lines and clinical tissue samples. In addition, an ultraviolet photodissociation tandem mass spectrometry (UVPD-MS/MS) fragmentation technique was developed to localise double bond (unsaturation) position(s) in unsaturated fatty acids and fatty acyl chain containing lipid species. Finally, in order to facilitate robust lipid identification, new software and analytical tools were developed to perform lipidome identification, quantification, statistical analysis, and data visualisation.
Systematic lipidomic profiling of 43 CRC cell lines revealed a highly heterogeneous profile of 755 lipid species across four categories, 14 classes, and 36 subclasses. Statistical analyses, including differential abundance analysis and hierarchical clustering, were performed to test for associations of lipidome profiles with molecular and histopathological CRC subtypes, and mutation status of major CRC driver genes including APC, KRAS, BRAF and PIK3CA. Lipidome profiles at the 'sum composition' level were found to be significantly associated with both microsatellite instability (MSI) status and cancer grade. Detailed characterisation of lipid structure revealed additional lipidome remodelling at the fatty acyl chain level including alterations in the chain length and unsaturation, and the isomeric compositions of 18:1(n-7) and 18:1(n-9) fatty acyl containing lipids as a function of CRC subtypes. However, no associations were identified between lipidome profiles and cancer gene mutations after adjustment for cell line MSI status, although power to detect such relationships was limited given our sample size. To further examine the potential correlation between lipid metabolism and cancer gene mutation status, the lipidomes of isogenic DLD1 and HCT116 cell lines with KRAS mutant (pro-oncogenic activation) or KRAS wildtype (pro-oncogenic deactivation) genotypes were investigated. For both sets of isogenic CRC cell lines, significant alteration in lipid composition across multiple classes and subclasses were observed for KRAS mutated cells as compared to wildtype cells. However, the patterns of lipidome deregulation between the two cell lines were inverse, suggesting that these were not a direct output related to oncogenic KRAS, but rather a function of differential cellular responses.
Multi-omic comparisons were also performed to investigate the correlation between lipidome and mRNA levels of each of the CRC cell lines. A significant positive correlation was identified between the levels of triacylglycerides (TG) and Diacylglycerol O-Acyltransferase 2 (DGAT2) transcript, suggesting a potential functional relationship. Moreover, the changes in transcript levels in the DLD1 and HCT116 isogenic cell lines were also reflected in the lipidome profiles alteration due to KRAS mutation.
The established lipidomic analysis workflow was then applied to perform a pilot translational study examining a series of 32 CRC tumour and a subset of 11 patient matched normal tissue samples. These results revealed distinct lipidomic profiles that distinguished tumour from normal tissues, including lipid species within GL, GP, SP and ST categories. Differences in lipid profiles between tumour and normal tissues are not only observed at the lipid class or subclass level, but also in the fatty acyl chain properties (i.e., chain length and unsaturation). Analysis of a larger number of patient samples will be required to validate and expand these findings. A comparison of lipidome profiles between CRC tumour tissues and cell lines relative to normal tissues revealed that CRC cell lines broadly reflect the lipidome patterns of the primary cancers.
This study is the first to provide a systematic and comprehensive overview of the lipidomic landscape of CRC across a wide range of commonly used CRC cell lines, providing novel insights into the role of aberrant lipid metabolism on CRC biology. The pilot lipidomic study on CRC patient tissue samples establishes the foundation for the discovery of lipidomics-based diagnostic and prognostic biomarkers, and potential novel therapeutic targets for the disease
Enhanced Lipase Production in Pichia pastoris via Multiple Copies of Bacterial Lipase Genes and Co-expression of the HAC1 Gene
No full textThe hac1 gene, a key regulator of the untranslated protein response (UPR), was co-expressed in Pichia pastoris GS115 to enhance the production of a lipase from Geobacillus stearothermophilus. Multicopy lipase constructs (1X and 4X) were transformed with pPICZAwbe_hac1, generating GS115/T1.2RQ(1X)_hac1 and GS115/T1.2RQ(4X)_hac1 strains. The GS115/T1.2RQ(1X)_hac1 strain showed an 186% lipase activity after 120 hours versus the control (100%), while the GS115/T1.2RQ(4X)_hac1 strain showed a faster initial increase (38% at 48 hours) and 28% at 120 hours, which was beneficial for efficient enzyme production. Overexpression of the hac1 gene enhances lipase production because it activates UPR genes when the endoplasmic reticulum is stressed due to a large number of recombinant proteins and forms proteins that are not appropriately folded. SDS-PAGE and tributyrin plate assays confirmed extracellular lipase expression (~43 kDa). These results demonstrate that hac1 co-expression significantly (p = 0.01) enhances lipase production in Pichia pastoris, especially in lower-copy constructs. This is the first report of co-expressing hac1 with Geobacillus stearothermophilus lipase genes in yeast. The findings are expected to contribute to developing more efficient microbial cell factories for producing industrial enzymes
Effect of Multiple Gene Copy Number of Bacterial Lipase to Increase Lipase Production in Pichia pastoris
No full textBacterial lipase poses potential challenges when expressed in eukaryotic protein expression systems such as Pichia pastoris. This research aims to increase extracellular T1.2RQ lipase secretion (free lipase) with multiple gene copy number strategies in Pichia pastoris and it was first performed on lipase from Geobacillus stearothermophilus T1.2. In this study, the T1.2RQ lipase gene from Geobacillus stearothermophilus T1.2 was expressed in Pichia pastoris GS115 through a strategy involving multiple copies of lipase, resulting in increased lipase activity. Three copies of the lipase gene in pPIC9K_T1.2RQ(3x) recombinant plasmid were integrated into the genome of Pichia pastoris GS115, and quantitative analysis using qPCR technique confirmed that the GS115 transformant strain contained six copies of T1.2RQ gene, indicating two integration events. Lipase activity measurement showed that the GS115/T1.2RQ(6x) strain exhibited a 111% increase compared to that containing a copy of the T1.2RQ gene. SDS-PAGE and Zymogram results showed a protein band with a size of 43kDa. Qualitative analysis in LA+TBN media of all strains containing the T1.2RQ gene showed clear zones. Lipase production in flask fermentation took at least 120 hours to produce the best lipase activity. Thus, strategies with multiple copy numbers of gene lipase have significantly increased the expression of the bacterial lipase gene in Pichia pastoris GS115
Effect of Expression of Human Glucosylceramidase 2 Isoforms on Lipid Profiles in COS-7 Cells
No full textGlucosylceramide (GlcCer) is a major membrane lipid and the precursor of gangliosides. GlcCer is mainly degraded by two enzymes, lysosomal acid β-glucosidase (GBA) and nonlysosomal β-glucosidase (GBA2), which may have different isoforms because of alternative splicing. To understand which GBA2 isoforms are active and how they affect glycosphingolipid levels in cells, we expressed nine human GBA2 isoforms in COS-7 cells, confirmed their expression by qRT-PCR and Western blotting, and assayed their activity to hydrolyze 4-methylumbelliferyl-β-D-glucopyranoside (4MUG) in cell extracts. Human GBA2 isoform 1 showed high activity, while the other isoforms had activity similar to the background. Comparison of sphingolipid levels by ultra-high resolution/accurate mass spectrometry (UHRAMS) analysis showed that isoform 1 overexpression increased ceramide and decreased hexosylceramide levels. Comparison of ratios of glucosylceramides to the corresponding ceramides in the extracts indicated that GBA2 isoform 1 has broad specificity for the lipid component of glucosylceramide, suggesting that only one GBA2 isoform 1 is active and affects sphingolipid levels in the cell. Our study provides new insights into how increased breakdown of GlcCer affects cellular lipid metabolic networks
A Novel Function of Sphingosine Kinase 2 in the Metabolism of Sphinga-4,14-Diene Lipids
No full textThe number, position, and configuration of double bonds in lipids affect membrane fluidity and the recruitment of signaling proteins. Studies on mammalian sphingolipids have focused on those with a saturated sphinganine or mono-unsaturated sphingosine long chain base. Using high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS), we observed a marked accumulation of lipids containing a di-unsaturated sphingadiene base in the hippocampus of mice lacking the metabolic enzyme sphingosine kinase 2 (SphK2). The double bonds were localized to positions C4–C5 and C14–C15 of sphingadiene using ultraviolet photodissociation-tandem mass spectrometry (UVPD-MS/MS). Phosphorylation of sphingoid bases by sphingosine kinase 1 (SphK1) or SphK2 forms the penultimate step in the lysosomal catabolism of all sphingolipids. Both SphK1 and SphK2 phosphorylated sphinga-4,14-diene as efficiently as sphingosine, however deuterated tracer experiments in an oligodendrocyte cell line demonstrated that ceramides with a sphingosine base are more rapidly metabolized than those with a sphingadiene base. Since SphK2 is the dominant sphingosine kinase in brain, we propose that the accumulation of sphingadiene-based lipids in SphK2-deficient brains results from the slower catabolism of these lipids, combined with a bottleneck in the catabolic pathway created by the absence of SphK2. We have therefore uncovered a previously unappreciated role for SphK2 in lipid quality control
