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.

Mutations in the calcium-binding protein calsequestrin cause the highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but an atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a human cardiac calsequestrin filament with supporting mutational analysis and in vitro filamentation assays. We identify and characterize a novel disease-associated calsequestrin mutation, S173I, that is located at the filament-forming interface, and further show that a previously reported dominant disease mutation, K180R, maps to the same surface. Both mutations disrupt filamentation, suggesting that disease pathology is due to defects in multimer formation. An ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. Our study thus provides a unifying molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias.

Animal coloration is a powerful model for studying the genetic mechanisms that determine animal phenotypes. But, there has not been comprehensive characterization of the molecular basis of the complex patterns of coat color phenotype variation in wild boars. This study results indicated that the wild-type allele E+ of the MC1R gene was a dominant allele in wild boars and was not responsible for black, brown or other coat color phenotypes. A novel mutation c.695 T > C was identified in the 3¢-UTR of the ASIP gene. The association analysis showed that the C mutation allele was highly significantly associated with wild-type coat colors between wild boars and Western pig breeds (P=1.35E-33). A non-synonymous g.2254 G > A substitution was found in exon 2 of the TYRP1 gene (p.143His>Arg). The association analysis demonstrated that the G mutation allele was also significantly associated with wild-type coat colors between wild boars and Western pig breeds (P = 5.09E-10). In short, a few mutation sites in MC1R, ASIP, and TYRP1 genes were identified and surveyed several polymorphisms molecular variations in Chinese wild boars. In our identified mutations have caused the morphological diversity in wild boars, but did not influence coat color phenotype variation in some domesticated pig breeds. The conclusion was obtained that some mutations in color-associated genes were associated with wild-type coat colors in wild boar population, and that similar coat colorations observed in domesticated pig and wild boars can be the product of underlying differences in the genetic basis of color variants.

As the most critical part of compressive sensing theory, reconstruction algorithm has an impact on the quality and speed of image reconstruction. After studying some existing convex optimization algorithms and greedy algorithms, we find that convex optimization algorithms should possess higher complexity to achieve higher reconstruction quality. Also, fixed atomic numbers used in most greedy algorithms increase the complexity of reconstruction. In this context, we propose a novel algorithm, called variable atomic number matching pursuit, which can improve the accuracy and speed of reconstruction. Simulation results show that variable atomic number matching pursuit is a fast and stable reconstruction algorithm and better than the other reconstruction algorithms under the same conditions.

To evaluate the role of S-adenosylmethionine decarboxylase (AdoMetDC) and altered polyamine levels in mouse skin carcinogenesis, we utilized the tetracycline-regulated system to produce conditional expression of AdoMetDC in basal keratinocytes. AdoMetDC is a key enzyme in polyamine biosynthesis and it catalyzes the decarboxylation reaction converting AdoMet to dcAdoMet. dcAdoMet provides the aminopropyl groups for the synthesis of the higher polyamines spermidine and spermine from the precursor putrescine. Therefore, elevated AdoMetDC activity may promote the synthesis of higher polyamines at the expense of putrescine, and increased putrescine levels are tightly linked to tumor promotion in mouse skin. AdoMetDC activity is also known to be induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). To make the DNA construct for microinjection, human AdoMetDC cDNA was inserted into the TetO vector followed by an internal ribosome entry site and luciferase coding sequence. Potential founder mice were crossed to mice with skin-specific expression of tetracycline-regulated transcriptional activator (tTA) directed by bovine keratin 5 (K5) promoter elements to generate K5-tTA/TetO-AdoMetDC bitransgenic mice. Transgene expression has been characterized by in vivo bioluminescent imaging of luciferase activity, in vitro luciferase activity as well as AdoMetDC activity assay and Western blotting in 7 week old mice. In bitransgenic mice, AdoMetDC is constitutively expressed in the skin and expression can be repressed by exposure to the tetracycline analogue doxycycline (Dox). In untreated bitransgenic mice generated from two founder lines, we saw a massive increase in luciferase activity and these mice have a thin fur phenotype. Minimal transgene expression is detected in either single transgenic TetO-AdoMetDC or Dox-treated bitransgenic mice. Untreated bitransgenic mice from one founder line exhibit a 7.2- fold increase in AdoMetDC activity in epidermis, a 7.8-fold increase in dermis, and dcAdoMet levels are also elevated by 8.0-fold in dermis. Effects of AdoMetDC overexpression on TPA induced polyamine biosynthesis and susceptibility to 7,12-dimethylbenz(a)anthracene/TPA carcinogenesis are now being investigated. These mice with regulated AdoMetDC expression in the skin provide a valuable model system to evaluate the role of AdoMetDC and polyamines in skin carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2483.