Molecular Mechanics of the α-Actinin Rod Domain: Bending, Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain

1169Interim analysis of data from a long-term, extension trial of tafamidis meglumine in patients with transthyretin amyloid cardiomyopathy

Abstract Background Transthyretin amyloid cardiomyopathy (ATTR-CM), is an underdiagnosed, fatal disease caused by the deposition of transthyretin amyloid fibrils in the heart leading to heart failure. The Transthyretin Cardiomyopathy Clinical Trial (ATTR-ACT), an international, multi-center, double-blind, placebo-controlled, randomized study, demonstrated the efficacy and safety of tafamidis treatment for patients with ATTR-CM due to variant (ATTRm) or wild-type (ATTRwt) TTR. Purpose This is a pooled analysis of data from ATTR-ACT and interim data from the ongoing, long-term, extension study to evaluate longer term data on the efficacy of tafamidis in patients with ATTR-CM. Methods Patients who completed ATTR-ACT (which had a duration of 30 months) were eligible to be enrolled in a long-term, extension study in which patients either continued to receive tafamidis meglumine at the same dose (the tafamidis/tafamidis [T/T] group) or, for patients previously treated with placebo, were randomised (in a 1:2 ratio) to tafamidis meglumine 20 mg or 80 mg (the placebo/tafamidis [P/T] group) for up to 60 months. The primary efficacy outcome was all-cause mortality. This analysis combined data from the completed ATTR-ACT with interim data from the extension study (cut-off date: 15 Feb, 2018), and included patients treated with tafamidis meglumine across the two studies with a median follow up of 36 months. Results All-cause mortality was significantly lower in the T/T group (n=264; 88 events, 33.3%) compared with the P/T group (n=177; 88 events, 50.3%); hazard ratio (95% CI), 0.64 (0.47, 0.85); P=0.001. In the subgroup of ATTRwt patients, all-cause mortality was significantly reduced in the T/T group (55/201; 27.4%) compared with the P/T group (60/134; 44.8%); 0.64 (0.44, 0.92); P=0.002. In the 106 (24.0%) ATTRm patients, there was a trend towards a reduction in all-cause mortality in the T/T group (33/63; 52.4%) compared with the P/T group (29/43; 67.4%); 0.66 (0.39, 1.09); P=0.17. In patients who were NYHA Class I or II at baseline, all-cause mortality was significantly reduced in the T/T group (38/186; 20.4%) compared with the P/T group (45/114; 39.5%); 0.49 (0.32, 0.75); P=0.001. In those patients with more severe symptoms at baseline (NYHA Class III), there were fewer deaths in the T/T group (50/78; 64.1%) compared with the P/T group (44/63; 69.8%); 0.80 (0.53, 1.21), but this difference was not statistically significant (P=0.50). Conclusions In ATTR-ACT, tafamidis was shown to significantly improve survival, functional capacity, and quality of life in patients with ATTR-CM. This pooled analysis with data from the ongoing extension study further supports the efficacy of tafamidis in patients over a longer period of time and the importance of early diagnosis and treatment. Acknowledgement/Funding This study was sponsored by Pfizer

A genome-wide RNAi screen for Wnt/β-catenin pathway components identifies unexpected roles for TCF transcription factors in cancer

The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/β-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth

Coiled-Coil Nanomechanics and Uncoiling and Unfolding of the Superhelix and α-Helices of Myosin

The nanomechanical properties of the coiled-coils of myosin are fundamentally important in understanding muscle assembly and contraction. Force spectra of single molecules of double-headed myosin, single-headed myosin, and coiled-coil tail fragments were acquired with an atomic force microscope and displayed characteristic triphasic force-distance responses to stretch: a rise phase (R) and a plateau phase (P) and an exponential phase (E). The R and P phases arise mainly from the stretching of the coiled-coils, with the hinge region being the main contributor to the rise phase at low force. Only the E phase was analyzable by the worm-like chain model of polymer elasticity. Restrained molecular mechanics simulations on an existing x-ray structure of scallop S2 yielded force spectra with either two or three phases, depending on the mode of stretch. It revealed that coiled-coil chains separate completely near the end of the P phase and the stretching of the unfolded chains gives rise to the E phase. Extensive conformational searching yielded a P phase force near 40 pN that agreed well with the experimental value. We suggest that the flexible and elastic S2 region, particularly the hinge region, may undergo force-induced unfolding and extend reversibly during actomyosin powerstroke