Hepatocyte Proteome Alterations Induced by Individual and Combinations of Common Free Fatty Acids.

Non-alcoholic fatty liver disease is a pathology with a hard-to-detect onset and is estimated to be present in a quarter of the adult human population. To improve our understanding of the development of non-alcoholic fatty liver disease, we treated a human hepatoma cell line model, HepG2, with increasing concentrations of common fatty acids, namely myristic, palmitic and oleic acid. To reproduce more physiologically representative conditions, we also included combinations of these fatty acids and monitored the cellular response with an in-depth proteomics approach and imaging techniques. The two saturated fatty acids initially presented a similar phenotype of a dose-dependent decrease in growth rates and impaired lipid droplet formation. Detailed analysis revealed that the drop in the growth rates was due to delayed cell-cycle progression following myristic acid treatment, whereas palmitic acid led to cellular apoptosis. In contrast, oleic acid, as well as saturated fatty acid mixtures with oleic acid, led to a dose-dependent increase in lipid droplet volume without adverse impacts on cell growth. Comparing the effects of harmful single-fatty-acid treatments and the well-tolerated fatty acid mixes on the cellular proteome, we were able to differentiate between fatty-acid-specific cellular responses and likely common lipotoxic denominators.

Transcriptome and proteome of the corm, leaf and flower of Hypoxis hemerocallidea (African potato).

The corm of Hypoxis hemerocallidea, commonly known as the African potato, is used in traditional medicine to treat several medical conditions such as urinary infections, benign prostate hyperplasia, inflammatory conditions and testicular tumours. The metabolites contributing to the medicinal properties of H. hemerocallidea have been identified in several studies and, more recently, the active terpenoids of the plant were profiled. However, the biosynthetic pathways and the enzymes involved in the production of the terpene metabolites in H. hemerocallidea have not been characterised at a transcriptomic or proteomic level. In this study, total RNA extracted from the corm, leaf and flower tissues of H. hemerocallidea was sequenced on the Illumina HiSeq 2500 platform. A total of 143,549 transcripts were assembled de novo using Trinity and 107,131 transcripts were functionally annotated using the nr, GO, COG, KEGG and SWISS-PROT databases. Additionally, the proteome of the three tissues were sequenced using LC-MS/MS, revealing aspects of secondary metabolism and serving as data validation for the transcriptome. Functional annotation led to the identification of numerous terpene synthases such as nerolidol synthase, germacrene D synthase, and cycloartenol synthase amongst others. Annotations also revealed a transcript encoding the terpene synthase phytoalexin momilactone A synthase. Differential expression analysis using edgeR identified 946 transcripts differentially expressed between the three tissues and revealed that the leaf upregulates linalool synthase compared to the corm and the flower tissues. The transcriptome as well as the proteome of Hypoxis hemerocallidea presented here provide a foundation for future research.

Endothelial lipase increases antioxidative capacity of high-density lipoprotein.

Endothelial lipase (EL) is a strong determinant of structural and functional properties of high-density lipoprotein (HDL). We examined whether the antioxidative capacity of HDL is affected by EL. EL-modified HDL (EL-HDL) and control EV-HDL were generated by incubation of HDL with EL- overexpressing or control HepG2 cells. As determined by native gradient gel electrophoresis, electron microscopy, and small-angle X-ray scattering EL-HDL is smaller than EV-HDL. Mass spectrometry revealed an enrichment of EL-HDL with lipolytic products and depletion of phospholipids and triacylglycerol. Kinetics of conjugated diene formation and HPLC-based malondialdehyde quantification revealed that EL-HDL exhibited a significantly higher resistance to copper ion-induced oxidation and a significantly higher capacity to protect low-density lipoprotein (LDL) from copper ion-induced oxidation when compared to EV-HDL. Depletion of the lipolytic products from EL-HDL abolished the capacity of EL-HDL to protect LDL from copper ion-induced oxidation, which could be partially restored by lysophosphatidylcholine enrichment. Proteomics of HDL incubated with oxidized LDL revealed significantly higher levels of methionine 136 sulfoxide in EL-HDL compared to EV-HDL. Chloramine T (oxidizes methionines and modifies free thiols), diminished the difference between EL-HDL and EV-HDL regarding the capacity to protect LDL from oxidation. In absence of LDL small EV-HDL and EL-HDL exhibited higher resistance to copper ion-induced oxidation when compared to respective large particles. In conclusion, the augmented antioxidative capacity of EL-HDL is primarily determined by the enrichment of HDL with EL-generated lipolytic products and to a lesser extent by the decreased HDL particle size and the increased activity of chloramine T-sensitive mechanisms.

Proteomic Analysis of Vocal Fold Fibroblasts Exposed to Cigarette Smoke Extract: Exploring the Pathophysiology of Reinke's Edema.

Reinke's edema is a smoking-associated, benign, mostly bilateral lesion of the vocal folds leading to difficulties in breathing and voice problems. Pronounced histological changes such as damaged microvessels or immune cell infiltration have been described in the vocal fold connective tissue, the lamina propria Thus, vocal fold fibroblasts, the main cell type of the lamina propria, have been postulated to play a critical role in disease mediation. Yet information about the pathophysiology is still scarce and treatment is only surgical, i.e. symptomatic. To explore the pathophysiology of Reinke's edema, we exposed near-primary human vocal fold fibroblasts to medium conditioned with cigarette smoke extract for 24 h as well as 4 days followed by quantitative mass spectrometry.Proteomic analyses after 24 h revealed that cigarette smoke increased proteins previously described to be involved in oxidative stress responses in other contexts. Correspondingly, gene sets linked to metabolism of xenobiotics and reactive oxygen species were significantly enriched among cigarette smoke-induced proteins. Among the proteins most downregulated by cigarette smoke, we identified fibrillar collagens COL1A1 and COL1A2; this reduction was validated by complementary methods. Further, we found a significant increase of UDP-glucose 6-dehydrogenase, generating a building block for biosynthesis of hyaluronan, another crucial component of the vocal fold lamina propria In line with this result, hyaluronan levels were significantly increased because of cigarette smoke exposure. Long term treatment of 4 days did not lead to significant changes.The current findings corroborate previous studies but also reveal new insights in possible disease mechanisms of Reinke's edema. We postulate that changes in the composition of the vocal folds' extracellular matrix -reduction of collagen fibrils, increase of hyaluronan- may lead to the clinical findings. This might ease the identification of better, disease-specific treatment options.