Type 1 ryanodine receptor interactions

Excitation-contraction coupling is an essential part of skeletal muscle contraction. It encompasses the sensing of depolarisation of the plasma membrane coupled with the release of Ca2+ from intracellular stores. The channel responsible for this release is called the Ryanodine receptor (RyR), and forms a hub of interacting proteins which work in concert to regulate the release of Ca2+ through this channel. The aim of this work was to characterise possible novel interactions with a proline-rich region of the RyR1, to characterise a monoclonal antibody (mAb VF1c) raised against a junctional sarcoplasmic reticulum protein postulated to interact with RyR1, and to characterise the protein recognised by this antibody in models of skeletal muscle disease such as Duchenne Muscular dystrophy (DMD) and sarcopenia. These experiments were performed using cell culture, protein purification via immunoprecipitation, affinity purification, low pressure chromatography and western blotting techniques. It was found that the RyR1 complex isolated from rat skeletal muscle co-purifies with the Growth factor receptor bound protein 2 (GRB2), very possibly via an interaction between the proline rich region of RyR1 and one of the SH3 domains located on the GRB2 protein. It was also found that Pleiotrophin and Phospholipase Cγ1, suggested interactors of the proline rich region of RyR1, did not co-purify with the RyR1 complex. Characterisation of mAb VF1c determined that this monoclonal antibody interacts with junctophilin 1, and binds to this protein between the region of 369-460, as determined by western blotting of JPH1 fragments expressed in yeast. It was also found that JPH1 and JPH2 are differentially regulated in different muscles of rabbit, where the highest amount of both proteins was found in the extensor digitorum longus (EDL) muscle. JPH1 and 2 levels were also examined in three rodent models of disease: the mdx mouse (a model of DMD), chronic intermittent hypoxia (CIH)-treated rat, and aged and adult mice, a model of sarcopenia. In the EDL and soleus muscle of CIH treated rats, no difference in either JPH1 or JPH2 abundance was detected in either muscle. An examination of JPH1 and 2 expression in mdx and wild type controls diaphragm, vastus lateralis, soleus and gastrocnemius muscle found no major differences in JPH1 abundance, while JPH2 was decreased in mdx gastrocnemius compared to wild type. In a mouse model of sarcopenia, JPH1 abundance was found to be increased in aged soleus but not in aged quadriceps, while in exercised quadriceps, JPH2 abundance was decreased compared to unexercised controls. Taken together, these results have implications for the regulation of RyR1 and JPH1 and 2 in skeletal muscle in both physiological and pathological states, and provide a newly characterised antibody to expand the field of JPH1 research.

Physicochemical characterisation of protein ingredients prepared from milk by ultrafiltration or microfiltration for application in formulated nutritional products

Formulated food systems are becoming more sophisticated as demand grows for the design of structural and nutritional profiles targeted at increasingly specific demographics. Milk protein is an important bio- and techno-functional component of such formulations, which include infant formula, sports supplements, clinical beverages and elderly nutrition products. This thesis outlines research into ingredients that are key to the development of these products, namely milk protein concentrate (MPC), milk protein isolate (MPI), micellar casein concentrate (MCC), β-casein concentrate (BCC) and serum protein concentrate (SPC). MPC powders ranging from 37 to 90% protein (solids basis) were studied for properties of relevance to handling and storage of powders, powder solubilisation and thermal processing of reconstituted MPCs. MPC powders with ≥80% protein were found to have very poor flowability and high compressibility; in addition, these high-protein MPCs exhibited poor wetting and dispersion characteristics during rehydration in water. Heat stability studies on unconcentrated (3.5%, 140°C) and concentrated (8.5%, 120°C) MPC suspensions, showed that suspensions prepared from high-protein MPCs coagulated much more rapidly than lower protein MPCs. β-casein ingredients were developed using membrane processing. Enrichment of β-casein from skim milk was performed at laboratory-scale using ‘cold’ microfiltration (MF) at <4°C with either 1000 kDa molecular weight cut-off or 0.1 µm pore-size membranes. At pilot-scale, a second ‘warm’ MF step at 26°C was incorporated for selective purification of micellised β-casein from whey proteins; using this approach, BCCs with β-casein purity of up to 80% (protein basis) were prepared, with the whey protein purity of the SPC co-product reaching ~90%. The BCC ingredient could prevent supersaturated solutions of calcium phosphate (CaP) from precipitating, although the amorphous CaP formed created large micelles that were less thermo-reversible than those in CaP-free systems. Another co-product of BCC manufacture, MCC powder, was shown to have superior rehydration characteristics compared to traditional MCCs. The findings presented in this thesis constitute a significant advance in the research of milk protein ingredients, in terms of optimising their preparation by membrane filtration, preventing their destabilisation during processing and facilitating their effective incorporation into nutritional formulations designed for consumers of a specific age, lifestyle or health status