Elucidating ryanodine receptor domain interactions in sudden cardiac death : towards the development of novel therapeutic strategies

Interdomain Interactions within the complex three-dimensional architecture of the cardiac ryanodine receptor (RyR2) are pivotal in channel regulation. Acquired or genetic abnormalities that perturb these stabilising intra-molecular interactions are pathogenic. This laboratory identified the interacting- or l-Domain of human RyR2 that mediated interaction between cytoplasmic and transmembrane (TM) assemblies. To further elucidate the precise roles of functional motifs within the l-Domain, three contiguous fragments spanning RyR2 amino acid residues 3722-4610 were synthesised using a cell-free system. One fragment termed IDB (amino acid residues 4353-4499) profoundly modulated cellular Ca2+ cycling and resulted in the remarkable normalisation of intercellular synchrony following its microinjection into ouabain-treated cardiomyocyte monolayers. These phenomena were linked to IDB- mediated stabilisation of RyR2 and were fully corroborated using IDB purified from a bacterial expression system. Bioinformatic analysis revealed striking structural homology between sub-fragments of the RyR2 l-Domain and l-Domain-like regions of inositol 1,4,5- trisphosphate receptors (IP3R). Recombinant expression of l-domain sub-fragments in RyR- null human embryonic kidney (HEK) cells remodelled carbachol-evoked Ca2+-responses and suppressed homeostatic Ca2+ signalling events indicating that IDB also modulated IP3R signalling mechanisms. In both HL-1 and HEK cells, IDB-dependent Ca2+ modulation extended to surrounding cells that were not microinjected with recombinant protein. This so- called 'bystander effect' was mediated by the transfer of signalling molecules via direct cell- to-cell coupling (gap junctions) and also by the extracellular transmission of diffusible effectors. This thesis supports the concept that RyR2 stabilisation rescues pathogenic Ca2+ dysregulation and suggests that there is substantial merit in developing further epitope-targeting strategies for the therapeutic normalisation of Ca2+ cycling in cardiac disease.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Elucidating ryanodine receptor domain interactions in sudden cardiac death: towards the development of novel therapeutic strategies

Interdomain Interactions within the complex three-dimensional architecture of the cardiac ryanodine receptor (RyR2) are pivotal in channel regulation. Acquired or genetic abnormalities that perturb these stabilising intra-molecular interactions are pathogenic. This laboratory identified the interacting- or l-Domain of human RyR2 that mediated interaction between cytoplasmic and transmembrane (TM) assemblies. To further elucidate the precise roles of functional motifs within the l-Domain, three contiguous fragments spanning RyR2 amino acid residues 3722-4610 were synthesised using a cell-free system. One fragment termed IDB (amino acid residues 4353-4499) profoundly modulated cellular Ca2+ cycling and resulted in the remarkable normalisation of intercellular synchrony following its microinjection into ouabain-treated cardiomyocyte monolayers. These phenomena were linked to IDB- mediated stabilisation of RyR2 and were fully corroborated using IDB purified from a bacterial expression system. Bioinformatic analysis revealed striking structural homology between sub-fragments of the RyR2 l-Domain and l-Domain-like regions of inositol 1,4,5- trisphosphate receptors (IP3R). Recombinant expression of l-domain sub-fragments in RyR- null human embryonic kidney (HEK) cells remodelled carbachol-evoked Ca2+-responses and suppressed homeostatic Ca2+ signalling events indicating that IDB also modulated IP3R signalling mechanisms. In both HL-1 and HEK cells, IDB-dependent Ca2+ modulation extended to surrounding cells that were not microinjected with recombinant protein. This so- called 'bystander effect' was mediated by the transfer of signalling molecules via direct cell- to-cell coupling (gap junctions) and also by the extracellular transmission of diffusible effectors. This thesis supports the concept that RyR2 stabilisation rescues pathogenic Ca2+ dysregulation and suggests that there is substantial merit in developing further epitope-targeting strategies for the therapeutic normalisation of Ca2+ cycling in cardiac disease

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca2+ channels that regulate Ca2+ release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca2+-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca2+ handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca2+ phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca2+ handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca2+ release channels

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca2+ channels that regulate Ca2+ release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca2+-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca2+ handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca2+ phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca2+ handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca2+ release channels

Duodenal Adenomas and Cancer in MUTYH-associated Polyposis: An International Cohort Study

Although duodenal adenomas and cancer appear to occur significantly less frequently in autosomal recessive MUTYH-associated polyposis (MAP) than in autosomal dominant familial adenomatous polyposis (FAP),1 current guidelines recommend similar endoscopic surveillance for both disorders.2-4 This involves gastro-duodenoscopy starting at 25 to 35 years of age and repeated at intervals determined by Spigelman staging based on the number, size, histological type and degree of dysplasia of adenomas, and by ampullary staging. Case reports of duodenal cancers in MAP suggest that they may develop in the absence of advanced Spigelman stage benign disease and even without coexisting adenomas.1 Recent molecular analyses suggest thatMAPduodenal adenomashave a higher mutational burden than FAP adenomas and are more likely to harbor oncogenic drivermutations, such as those in KRAS.5 These apparent differences in the biology and natural history of duodenal polyposis in FAP and MAP challenge the assumption that the same surveillance should be applied in both conditions