Investigating cellular stress in industry and medicine

Abstract

Cellular stress involves any external or internal condition which is detrimental to cell survival. Many forms of cellular stress exist, such as heat shock, DNA damage, oxidative stress and the unfolded protein response. In order to study cellular stress, a broad range of techniques have been used. Holistic approaches such as microarray gene expression analysis have shown the potential for global studies to illustrate the effects of cellular stress in cells. These techniques while useful, are to be conducted with caution, and are often considered to be a 'stab in the dark' approach to determining the underlying molecular mechanisms. Therefore, reductionist techniques are employed to support holistic data and narrow down the list of potential causes for stress. Biochemical assays such as enzyme studies, protein expression or the determination of pathway intermediates can help to further characterise the downstream effects of cellular stress. In this study, the importance of cellular stress to both Industry and Medicine was determined. Firstly, hop extracts, both common and novel were analysed using three techniques. Due to the presence of hydrogen peroxide during beer brewing and the potential for further oxidation of important flavour, aroma and appearance compounds, a novel antioxidant assay was developed. The Europium Tetracycline Hydrogen Peroxide Reduction Assay (EHRA) was shown to be a robust, reliable, and reproducible method for the determination of antioxidant activity in a wide range of compounds. Of most significance, hop extracts were tested in order to provide an insight into the ability of a novel extract, such as hop leaves, to reduce hydrogen peroxide. It was found that antioxidant activity is largely dependent on the extraction techniques, and that while fresh 'green' extracts had a large level of antioxidant activity, their use in industry would be limited due to availability. Therefore, it was concluded that hop cone and hop leaf materials, kiln-dried extracted, would provide the best avenue for testing and application. Interestingly, the hop cone and leaf material maintained a similar level of antioxidant activity during further extraction procedures with water or ethanol in both hot and cold environments. Further to the initial study of antioxidant activity of hop cone and leaf material, pilot-scale fermentations were run in order to determine the global gene expression changes on yeast when exposed to hops. Primarily, this study was conducted to validate the use of hop leaf material in brewing. Indeed the results indicated that hop cone and hop leaf material have similar effects on yeast gene expression, although changes in yeast growth kinetics and sugar metabolism genes were detected. Of most significance, hop cone material was shown to increase the expression of several key flocculation genes. FLO 1, 3 and 9 in combination with DAN2, 3 and increased maltose utilisation point to the ability of hop cones to impact flocculation during fermentation. Previously, the ability of certain malts to cause early flocculation have been extensively studied due to the financial impact this has on brewing production. In regards to cellular stress in Medicine, a neurodegenerative disease affecting humans was studied. Hereditary sensory neuropathy type 1 is an autosomal-dominant disease caused by several mutations in the serine palmitoyl transferase sub unit 1 (SPTLC1). SPTLC1 is the primary subunit of the serine palmitoyl transferase (SPT) enzyme, the initial and rate limiting step in the de novo sphingolipid biosynthesis pathway. These mutations in SPTLC1 have been shown to cause axonal degeneration, leading to axon retraction and a range of symptoms including the loss of pain and temperature sensation. To date, the molecular mechanisms behind this disease are unknown, although there is belief that the mutations in SPTLC1 produce two toxic forms of sphingolipids based on a change in affinity to various amino acids of the SPT enzyme. This study has shown through the use of transformed HSN1 lymphocytes, that the mutations in SPTLC1 cause a change in ER homeostasis via altered expression of several key ER stress/UPR response proteins namely BiP, PERK, IRE1I, PDI and ERO1-LI. It is believed that because of these changes in protein expression, the ER and mitochondria bunch in the peri-nuclear region in order to maintain normal cell growth. This close proximity is likely to cause an increased interaction between the two organelles via enhanced connections, called mitochondrial associated membranes or MAM's, which have been shown to be related to ER stress, oxidative stress and the regulation of ER-mitochondrial calcium flux. In addition many of the proteins studied are shown to be enriched in the MAM region further indicating the significance of expression changes to ER homeostasis and mitochondrial function.

Date of Award	2012
Original language	English