Synergistic action of human RNaseH2 and the RNA helicase-nuclease DDX3X in processing R-loops.

R-loops are three-stranded RNA-DNA hybrid structures that play important regulatory roles, but excessive or deregulated R-loops formation can trigger DNA damage and genome instability. Digestion of R-loops is mainly relying on the action of two specialized ribonucleases: RNaseH1 and RNaseH2. RNaseH2 is the main enzyme carrying out the removal of misincorporated rNMPs during DNA replication or repair, through the Ribonucleotide Excision Repair (RER) pathway. We have recently shown that the human RNA helicase DDX3X possessed RNaseH2-like activity, being able to substitute RNaseH2 in reconstituted RER reactions. Here, using synthetic R-loop mimicking substrates, we could show that human DDX3X alone was able to both displace and degrade the ssRNA strand hybridized to DNA. Moreover, DDX3X was found to physically interact with human RNaseH2. Such interaction suppressed the nuclease and helicase activities of DDX3X, but stimulated severalfold the catalytic activity of the trimeric RNaseH2, but not of RNaseH1. Finally, silencing of DDX3X in human cells caused accumulation of RNA-DNA hybrids and phosphorylated RPA foci. These results support a role of DDX3X as a scaffolding protein and auxiliary factor for RNaseH2 during R-loop degradation.

Integrins Increase Sarcoplasmic Reticulum Activity for Excitation-Contraction Coupling in Human Stem Cell-Derived Cardiomyocytes.

Engagement of the sarcoplasmic reticulum (SR) Ca2+ stores for excitation-contraction (EC)-coupling is a fundamental feature of cardiac muscle cells. Extracellular matrix (ECM) proteins that form the extracellular scaffolding supporting cardiac contractile activity are thought to play an integral role in the modulation of EC-coupling. At baseline, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) show poor utilisation of SR Ca2+ stores, leading to inefficient EC-coupling, like developing or human CMs in cardiac diseases such as heart failure. We hypothesised that integrin ligand-receptor interactions between ECM proteins and CMs recruit the SR to Ca2+ cycling during EC-coupling. hiPSC-CM monolayers were cultured on fibronectin-coated glass before 24 h treatment with fibril-forming peptides containing the integrin-binding tripeptide sequence arginine-glycine-aspartic acid (2 mM). Micropipette application of 40 mM caffeine in standard or Na+/Ca2+-free Tyrode's solutions was used to assess the Ca2+ removal mechanisms. Microelectrode recordings were conducted to analyse action potentials in current-clamp. Confocal images of labelled hiPSC-CMs were analysed to investigate hiPSC-CM morphology and ultrastructural arrangements in Ca2+ release units. This study demonstrates that peptides containing the integrin-binding sequence arginine-glycine-aspartic acid (1) abbreviate hiPSC-CM Ca2+ transient and action potential duration, (2) increase co-localisation between L-type Ca2+ channels and ryanodine receptors involved in EC-coupling, and (3) increase the rate of SR-mediated Ca2+ cycling. We conclude that integrin-binding peptides induce recruitment of the SR for Ca2+ cycling in EC-coupling through functional and structural improvements and demonstrate the importance of the ECM in modulating cardiomyocyte function in physiology.

Self-Assembled Amyloid Peptides with Arg-Gly-Asp (RGD) Motifs As Scaffolds for Tissue Engineering.

Self-assembled peptides gain increasing interest as biocompatible and biodegradable scaffolds for tissue engineering. Rationally designed self-assembling building blocks that carry cell adhesion motifs such as Arg-Gly-Asp (RGD) are especially attractive. We have used a combination of theoretical and experimental approaches toward such rational designs, especially focusing on modular designs that consist of a central ultrashort amphiphilic motif derived from the adenovirus fiber shaft. In this study, we rationally designed RGDSGAITIGC, a bifunctional self-assembling amyloid peptide which encompasses cell adhesion and potential cysteine-mediated functionalization properties through the incorporation of an RGD sequence motif and a cysteine residue at the N- and C- terminal end, respectively. We performed replica exchange MD simulations that suggested that the key factor determining cell adhesion is the total solvent accessibility of the RGD motif and also that the C-terminal cysteine is adequately exposed. The designer peptides self-assembled into fibers that are structurally characterized with Transmission Electron Microscopy, Scanning Electron Microscopy and X-ray fiber diffraction. Furthermore, they supported cell adhesion and proliferation of a model cell line. We consider that the current bifunctional properties of the RGDSGAITIGC fibril-forming peptide can be exploited to fabricate novel biomaterials with promising biomedical applications. Such short self-assembling peptides that are amenable to computational design offer open-ended possibilities toward multifunctional tissue engineering scaffolds of the future.