Truncating mutations in the giant sarcomeric protein Titin result in dilated cardiomyopathy and skeletal myopathy. The most severely affected dilated cardiomyopathy patients harbor Titin truncations in the C-terminal two-thirds of the protein, suggesting that mutation position might influence disease mechanism. Using CRISPR/Cas9 technology, we generated six zebrafish lines with Titin truncations in the N-terminal and C-terminal regions. Although all exons were constitutive, C-terminal mutations caused severe myopathy whereas N-terminal mutations demonstrated mild phenotypes. Surprisingly, neither mutation type acted as a dominant negative. Instead, we found a conserved internal promoter at the precise position where divergence in disease severity occurs, with the resulting protein product partially rescuing N-terminal truncations. In addition to its clinical implications, our work may shed light on a long-standing mystery regarding the architecture of the sarcomere.DOI: http://dx.doi.org/10.7554/eLife.09406.001
Calcium homeostasis is essential to cardiac and skeletal muscle physiology, where contractile function requires tight but dynamic control of calcium levels across different cellular compartments. The major calcium storage protein in muscle tissues is calsequestrin, a highly acidic protein responsible for buffering up to 50 % of total sarcoplasmic reticulum (SR) calcium. Mutations in cardiac calsequestrin cause a highly lethal familial arrhythmia, 1/51 catecholaminergic polymorphic ventricular tachycardia (CPVT), while mutations in skeletal muscle calsequestrin have been linked to myopathies. Calsequestrin's high density calcium storage is facilitated by homomultimerization of the protein into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. This gap in knowledge has limited our understanding of calsequestrin biochemistry, SR calcium storage, and molecular mechanisms of calseqestrin-associated diseases. We report here a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis by an in vitro filamentation assay. We also report and characterize a novel disease-associated mutation, S173I, which localizes to the structure's filament-forming interface. In addition, we show that a previously reported dominant calsequestrin-associated CPVT mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation in vitro, suggesting a model where dominant disease arises from mutations that disrupt multimer formation. Finally, a ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our basic understanding of calsequestrin biochemistry and also provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias.We thank Jason Roberts for expert clinical review. We thank for technical assistance with crystallographic data collection and processing. We thank Lijun Liu and Tarjani Thaker for technical assistance with protein preparation and crystallization screens. We thank Bruk Mensa for technical assistance with plate reader instrumentation. We thank Michael Grabe, Aimee Kao, Dan Minor, and Oren Rosenberg for helpful discussions. Beamline 8.3.
Neoplastic growth is associated with increased polyamine biosynthetic activity and content. Tumor promoter treatment induces the rate-limiting enzymes in polyamine biosynthesis, ornithine decarboxylase (ODC), and S-adenosylmethionine decarboxylase (AdoMetDC), and targeted ODC overexpression is sufficient for tumor promotion in initiated mouse skin. We generated a mouse model with doxycycline (Dox)-regulated AdoMetDC expression to determine the impact of this second rate-limiting enzyme on epithelial carcinogenesis. TetO-AdoMetDC (TAMD) transgenic founders were crossed with transgenic mice (K5-tTA) that express the tetracycline-regulated transcriptional activator within basal keratinocytes of the skin. Transgene expression in TAMD/K5-tTA mice was restricted to keratin 5 (K5) target tissues and silenced upon Dox treatment. AdoMetDC activity and its product, decarboxylated AdoMet, both increased approximately 8-fold in the skin. This enabled a redistribution of the polyamines that led to reduced putrescine, increased spermine, and an elevated spermine:spermidine ratio. Given the positive association between polyamine biosynthetic capacity and neoplastic growth, it was somewhat surprising to find that TAMD/K5-tTA mice developed significantly fewer tumors than controls in response to 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate chemical carcinogenesis. Importantly, tumor counts in TAMD/K5-tTA mice rebounded to nearly equal the levels in the control group upon Dox-mediated transgene silencing at a late stage of tumor promotion, which indicates that latent viable initiated cells remain in AdoMetDC-expressing skin. These results underscore the complexity of polyamine modulation of tumor development and emphasize the critical role of putrescine in tumor promotion. AdoMetDC-expressing mice will enable more refined spatial and temporal manipulation of polyamine biosynthesis during tumorigenesis and in other models of human disease.
A composite cytomegalovirus-immediate early gene enhancer/chicken β-actin promoter (CAG) was utilized to generate transgenic mice that overexpress human spermidine synthase (SpdS) in order to determine the impact of elevated spermidine synthase activity on murine development and physiology. CAG-SpdS mice were viable and fertile and tissue SpdS activity was increased up to 9-fold. This increased SpdS activity did not result in a dramatic elevation of spermidine or spermine levels but did lead to a 1.5 to 2-fold reduction in tissue spermine:spermidine ratio in heart, muscle and liver tissues with the highest levels of SpdS activity. This new mouse model enabled simultaneous overexpression of SpdS and other polyamine biosynthetic enzymes by combining transgenic animals. The combined overexpression of both SpdS and spermine synthase (SpmS) in CAG-SpdS/CAG-SpmS bitransgenic mice did not impair viability or lead to overt developmental abnormalities but instead normalized the elevated tissue spermine:spermidine ratios of CAG-SpmS mice. The CAG-SpdS mice were bred to MHC-AdoMetDC mice with a >100-fold increase in cardiac S-adenosylmethionine decarboxylase (AdoMetDC) activity to determine if elevated dcAdoMet would facilitate greater spermidine accumulation in mice with SpdS overexpression. CAG-SpdS/MHC-AdoMetDC bitransgenic animals were produced at the expected frequency and exhibited cardiac polyamine levels comparable to MHC-AdoMetDC littermates. Taken together these results indicate that SpdS levels are not rate limiting in vivo for polyamine biosynthesis and are unlikely to exert significant regulatory effects on cellular polyamine content and function.
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