Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C–S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.

DNA damage, a consequence of external factors and inherent metabolic processes, is omnipresent. Nature has devised multiple strategies to safeguard the genetic information and developed intricate repair mechanisms and pathways to reverse an array of different DNA lesions, including mismatches. Failure of the DNA repair systems may result in mutation, premature ageing, and cancer. In this review, we focus on structural and dynamic aspects of detection of lesions in base excision and mismatch repair. A thorough understanding of repair, pathways, and regulation is necessary to develop strategies for targeting DNA-related pathologies.

DNA sequence context has long been known to modulate detection and repair of DNA damage. Recent studies using experimental and computational approaches have sought to provide a basis for this observation. We have previously shown that an α-anomeric adenosine (αA) flanked by cytosines (5′CαAC-3′) resulted in a kinked DNA duplex with an enlarged minor groove. Comparison of different flanking sequences revealed that a DNA duplex containing a 5′CαAG-3′ motif exhibits unique substrate properties. However, this substrate was not distinguished by unusual thermodynamic properties. To understand the structural basis of the altered recognition, we have determined the solution structure of a DNA duplex with a 5′CαAG-3′ core, using an extensive set of restraints including dipolar couplings and backbone torsion angles. The NMR structure exhibits an excellent agreement with the data (total RX <5.3%). The αA base is intrahelical, in a reverse Watson–Crick orientation, and forms a weak base pair with a thymine of the opposite strand. In comparison to the DNA duplex with a 5′CαAC-3′ core, we observe a significant reduction of the local perturbation (backbone, stacking, tilt, roll, and twist), resulting in a straighter DNA with narrower minor groove. Overall, these features result in a less perturbed DNA helix and obscure the presence of the lesion compared to the 5′CαAC-3′ sequence. The improved stacking of the 5′CαAG-3′ core also affects the energetics of the DNA deformation that is required to form a catalytically competent complex. These traits provide a rationale for the modulation of the recognition by endonuclease IV.

Numerous DNA chemistries have been explored to improve oligodeoxynucleotide (ODN) based RNA targeting. The majority of the modifications render the ODN/RNA target insensitive to RNase H1. Borano phosphonate ODN’s are among the few modifications that are tolerated by RNase H1. To understand the effect of the stereochemistry of the BH3 modification on the nucleic acid structure and RNase H1 enzyme activity we have investigated two DNA/RNA hybrids containing either a RP or SP BH3 modification by NMR spectroscopy. TM studies show that the stability of either RP or SP modified DNA/RNA hybrids are essentially identical (313.8 K) and similar to an unmodified control (312.9 K). The similarity is also reflected in the imino proton spectra. In order to characterize such similar structures, a large number of NMR restraints (including dipolar couplings and backbone torsion angles) were used to determine structural features important for RNase H1 activity. The final NMR structures exhibit excellent agreement with the data (total RX values < 6.0) with helical properties between those of an A and B helix. Subtle backbone variations are observed in the DNA near the modification, while the RNA strands are relatively unperturbed. In case of the SP modification, for which more perturbations are recorded, a slightly narrower minor groove is also obtained. Unique NOE base contacts localize the SP -BH3 group in the major groove while the RP -BH3 group points away from the DNA. However, this creates a potential clash of the RP -BH3 groups with important RNase H1 residues in a complex while the SP -BH3 groups could be tolerated. We therefore predict that based on our NMR structures a fully RP BH3 DNA/RNA hybrid would not be a substrate for RNase H1.

Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity.