Characterization of TRAF6 mediated ubiquitination of presenilins and γ-secretase substrates

Post-translational modification of the γ-secretase protease complexes and their substrates has an important role in controlling receptor-initiated signalling events, which are critically important in the pathogenesis of cancer, inflammatory and Alzheimer’s disease. Our lab has previously characterised an interaction between TRAF6 and presenilin-1, which lead to the identification of interleukin-1 (IL-1) receptor type 1 (IL-1R1) and Toll-like receptor-4 (TLR4) as novel γ-secretase substrates. Subsequently our group showed that TRAF6 promoted ubiquitination and γ-secretase cleavage of IL-1R1. The aim of this project is to study the association between TRAF6 and the presenilins, the critical γ-secretase complex components, and to determine the functional importance of TRAF6-mediated ubiquitination of γ-secretase substrates. Firstly, we show that the full-length presenilins are novel substrates of TRAF6-mediated Lysine-63-linked polyubiquitination. Secondly, we show that co-expression of TRAF6 and the presenilins increases the stability and alters the turnover of the presenilins. Thirdly, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate the full-length presenilin functions such as ER Ca2+ signalling. Previously, we have reported IL-1R1 as a novel substrate of TRAF6-mediated ubiquitination. In this study, we identified five lysine residues in the IL-1R1 intracellular domain targeted by TRAF6-mediated polyubiquitination. Furthermore, mutagenesis of these five lysine residues led to decreased IL-1R1 cell surface expression, precluded the ectodomain shedding and attenuated the responsiveness to IL-1β stimulation, demonstrating the critical role of TRAF6 in IL-1R1 trafficking.

Identification and characterization of innate immune receptor substrates of γ-secretase enzyme complex

The γ-secretase protease complexes and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signalling events, which have a central role in Alzheimer’s disease, cancer progression and immune surveillance. It has previously been reported that the Interleukin-1 receptor, type 1, (IL-1R1) is a substrate for regulated intramembrane proteolysis, mediated by presenilin (PS)-dependent γ-secretase activity. The aims of this project were twofold. Firstly, to determine the conservation of regulated intramembrane proteolysis as a physiological occurrence amongst other cytokine receptors. In this regard, similar to IL-1R1, we identified the Tumour necrosis factor receptor type 1 (TNFR1) and the Toll like receptor 4 (TLR4) as novel γ-secretase substrates. Secondly, given that the diversity of signalling events mediated by the IL-1R1, TLR4 and TNFR1 are spatially segregated, we investigated the spatial distribution, subcellular trafficking and subcellular occurrence of regulated intramembrane proteolysis of IL-1R1, TLR4 and TNFR1. Using dynasore an inhibitor of clathrin-dependent receptor endocytosis, both ectodomain shedding and γ-secretase-mediated cleavage of IL-1R1 were observed post-internalization. In contrast, TNFR-1 underwent ectodomain shedding at the cell surface followed by endosomal γ-secretase-mediated cleavage. Furthermore, immortalised fibroblasts from PS1-deficient mice showed impaired γ-secretasemediated cleavage of IL-1R1 and TNFR1, indicating that both are cleaved by PS1-and not PS2-containing γ-secretase complexes. Subcellular fractionation and immunofluorescence studies revealed that the γ-secretase generated IL-1R1 ICD translocates to the nucleus on IL-1β stimulation. These observations further demonstrate the novel PS-dependent means of modulating IL-1β, LPS and TNFα- mediated immune responses by regulating IL-1R1/TLR4/TNFR1 protein levels within the cells.

Primary proteolysis of caseins in cheddar cheese

Studies were undertaken to investigate proteolysis of the caseins during the initial stages of maturation of Cheddar cheese. Isolated caseins were hydrolyzed by enzymes thought to be of importance during cheese ripening and the resulting peptides isolated and identified. Large peptides were also isolated from Cheddar cheese and identified, thus enabling the extent to which casein degradation studies could be extrapolated to cheese to be established. The proteolytic specificity of chymosin on bovine αs1- and αs2-caseins and of plasmin on bovine αs1-casein were determined. The action of cathepsin D, the principal indigenous acid milk proteinase, on caseins was studied and its pH optimum and sensitivity to NaCI determined. The action of cathepsin D on αs1-, αs2-, β- and κ-caseins was compared with that of chymosin and was found to be generally similar for the hydrolysis of αs1- and κ-caseins but to differ for αs2-and β- caseins. β-Casein in solution was hydrolyzed by cell wall-associated proteinases from three strains of Lactococcus lactis; comparison of electrophoretograms of the hydrolyzates with those of Cheddar cheese indicated that no peptides produced by cell wall-associated proteinases were detectable in the cheeses. All the major peptides in the water-insoluble fraction of Cheddar cheese were isolated and identified. It was found that β-casein was degraded primarily by plasmin and αs1 -casein by chymosin. Initial chymosin and plasmin cleavage sites in αs1-, and β-casein, respectively, identified in these and other studies corresponded to the peptides isolated from cheese. The importance of non-starter lactic acid bacteria (NSLAB) to the maturation of Cheddar was studied in cheeses manufactured from raw, pasteurized or microfiltered milks. NSLAB were found to strongly influence the quality and patterns of proteolysis. Results presented in this thesis are consistent with the hypothesis that primary proteolysis in Cheddar is catalysed primarily by the action of chymosin and plasmin on intact αs1- and β-caseins, respectively. The resulting large peptides so produced are subsequently degraded by these enzymes and by proteinases and peptidases from the starter and NSLAB.

Variations in total and differential milk somatic cell counts and plasmin levels and their role in proteolysis and quality of milk and cheese

Increased plasmin and plasminogen levels and elevated somatic cell counts (SCC) and polymorphonuclear leucocyte levels (PMN) were evident in late lactation milk. Compositional changes in these milks were associated with increased SCC. The quality of late lactation milks was related to nutritional status of herds, with milks from herds on a high plane of nutrition having composition and clotting properties similar to, or superior to, early-mid lactation milks. Nutritionally-deficient cows had elevated numbers of polymorphonuclear leucocytes (PMNs) in their milk, elevated plasmin levels and increased overall proteolytic activity. The dominant effect of plasmin on proteolysis in milks of low SCC was established. When present in elevated numbers, somatic cells and PMNs in particular had a more significant influence on the proteolysis of both raw and pasteurised milks than plasmin. PMN protease action on the caseins showed proteolysis products of two specific enzymes, cathepsin B and elastase, which were also shown in high SCC milk. Crude extracts of somatic cells had a high specificity on αs1-casein. Cheeses made from late lactation milks had increased breakdown of αs1-casein, suggestive of the action of somatic cell proteinases, which may be linked to textural defects in cheese. Late lactation cheeses also showed decreased production of small peptides and amino acids, the reason for which is unknown. Plasmin, which is elevated in activity in late lactation milk, accelerated the ripening of Gouda-type cheese, but was not associated with defects of texture or flavour. The retention of somatic cell enzymes in cheese curd was confirmed, and a potential role in production of bitter peptides identified. Cheeses made from milks containing high levels of PMNs had accelerated αs1-casein breakdown relative to cheeses made from low PMN milk of the same total SCC, consistent with the demonstrated action of PMN proteinases. The two types of cheese were determined significantly different by blind triangle testing.

Fundamental studies on the application of enzymes when brewing with unmalted oats and sorghum

The use of unmalted oats or sorghum in brewing has great potential for creating new beer types/flavors and saving costs. However, the substitution of barley malt with oat or sorghum adjunct is not only innovative but also challenging due to their specific grain characteristics. The overall objectives of this Ph.D. project were: 1) to investigate the impact of various types and levels of oats or sorghum on the quality/processability of mashes, worts, and beers; 2) to provide solutions as regards the application of industrial enzymes to overcome potential brewing problems. For these purposes, a highly precise rheological method using a controlled stress rheometer was developed and successfully applied as a tool for optimizing enzyme additions and process parameters. Further, eight different oat cultivars were compared in terms of their suitability as brewing adjuncts and two very promising types identified. In another study, the limitations of barley malt enzymes and the benefits of the application of industrial enzymes in high-gravity brewing with oats were determined. It is recommended to add enzymes to high-gravity mashes when substituting 30% or more barley malt with oats in order to prevent filtration and fermentation problems. Pilot-scale brewing trials using 10–40% unmalted oats revealed that the sensory quality of oat beers improved with increasing adjunct level. In addition, commercially available oat and sorghum flours were implemented into brewing. The use of up to 70% oat flour and 50% sorghum flour, respectively, is not only technically feasible but also economically beneficial. In a further study on sorghum was demonstrated that the optimization of industrial mashing enzymes has great potential for reducing beer production costs. A comparison of the brewing performance of red Italian and white Nigerian sorghum clearly showed that European grown sorghum is suitable for brewing purposes; 40% red sorghum beers were even found to be very low in gluten.

Studies on quinoa (Chenopodium quinoa) for novel food and beverage applications

Quinoa (Chenopodium quinoa) is a seed crop native to the Andes, that can be used in a variety of food product in a similar manner to cereals. Unlike most plants, quinoa contains protein with a balanced amino acid profile. This makes it an interesting raw material for e.g. dairy product substitutes, a growing market in Europe and U.S. Quinoa can however have unpleasant off-flavours when processed into formulated products. One means of improving the palatability is seed germination. Also, the increased activities of hydrolytic enzymes can have a beneficial influence in food processing. In this thesis, the germination pattern of quinoa was studied, and the influence of quinoa malt was evaluated in a model product. Additionally, to explore its potential for dairy-type products, quinoa protein was isolated from an embryo-enriched milling fraction of non-germinated quinoa and tested for functional and gelation properties. Quinoa seeds imbibed water very rapidly, and most seeds showed radicle protrusion after 8-9 h. The α-amylase activity was very low, and started to increase only after 24 hours of germination in the starchy perisperm. Proteolytic activity was very high in dry ungerminated seeds, and increased slightly over 24 h. A significant fraction of this activity was located in the micropylar endosperm. The incorporation of germinated quinoa in gluten-free bread had no significant effect on the baking properties due to low α-amylase activity. Upon acidification with glucono-δ-lactone, quinoa milk formed a structured gel. The gelation behaviour was further studied using a quinoa protein isolate (QPI) extracted from an embryoenriched milling fraction. QPI required a heat-denaturation step to form gel structures. The heating pH influenced the properties drastically: heating at pH 10.5 led to a dramatic increase in solubility, emulsifying properties, and a formation of a fine-structured gel with a high storage modulus (G') when acidified. Heating at pH 8.5 varied very little from the unheated protein in terms of functional properties, and only formed a randomly aggregated coagulum with a low G'. Further study of changes over the course of heating showed that the mechanism of heat-denaturation and aggregation indeed varied largely depending on pH. The large difference in gelation behaviour may be related to the nature of aggregates formed during heating. To conclude, germination for increased enzyme activities may not be feasible, but the structure-forming properties of quinoa protein could possibly be exploited in dairy-type products.

Regulation of tail-anchored ubiquitin conjugating enzymes that are localised to the endoplasmic reticulum

The vast majority of secreted and membrane proteins are translated and folded at the endoplasmic reticulum (ER), where a sophisticated quality control mechanism ensures that only correctly folded proteins exit the ER and traffic to their final destinations. On the other hand, proteins that persistently misfold are eliminated through a process known as ER associated degradation (ERAD). This involves retrotranslocation of the misfolded protein through the ER membrane, and ubiquitination in advance of degradation by cytosolic proteasomes. The process of ERAD is best described in yeast where ubiquitin conjugating enzymes Ubc6p and Ubc7p function with a limited number of E3 ubiquitin ligases to ubiquitinate misfolded proteins. Interestingly, although the mechanistic principles of ERAD have been conserved through evolution, there is increasing evidence that homologues of the yeast enzymes have gained divergent roles and novel regulatory functions in higher eukaryotes, meaning that the process in humans is more complex and involves a larger repertoire of participating proteins. Two homologues of Ubc6p have been described in humans, and have been named as Ubc6 (UBE2J2) and Ubc6e (UBE2J1). However, little work has been done on these enzymes and thus our main objective of this study was to progress the functional characterisation of these ERAD E2 conjugating enzymes. Our studies included a detailed analysis of conditions whereby these proteins are stabilised and degraded. We’ve also explored the different molecular signalling pathways that induced changes on their steady state protein levels. Furthermore, Ubc6e has a phosphorylatable serine residue at position 184. Thus, our studies also involved delineating the signalling kinases that phosphorylate Ubc6e and examining its function in ERAD. Our studies confirm that the E2 Ubc enzymes are regulated posttranslationally and may have important implications in the regulation of ERAD.