TANGO1 inhibitors reduce collagen secretion and limit tissue scarring.

Uncontrolled secretion of ECM proteins, such as collagen, can lead to excessive scarring and fibrosis and compromise tissue function. Despite the widespread occurrence of fibrotic diseases and scarring, effective therapies are lacking. A promising approach would be to limit the amount of collagen released from hyperactive fibroblasts. We have designed membrane permeant peptide inhibitors that specifically target the primary interface between TANGO1 and cTAGE5, an interaction that is required for collagen export from endoplasmic reticulum exit sites (ERES). Application of the peptide inhibitors leads to reduced TANGO1 and cTAGE5 protein levels and a corresponding inhibition in the secretion of several ECM components, including collagens. Peptide inhibitor treatment in zebrafish results in altered tissue architecture and reduced granulation tissue formation during cutaneous wound healing. The inhibitors reduce secretion of several ECM proteins, including collagens, fibrillin and fibronectin in human dermal fibroblasts and in cells obtained from patients with a generalized fibrotic disease (scleroderma). Taken together, targeted interference of the TANGO1-cTAGE5 binding interface could enable therapeutic modulation of ERES function in ECM hypersecretion, during wound healing and fibrotic processes.

In vivo tracking of functionally tagged Rad51 unveils a robust strategy of homology search.

Homologous recombination (HR) is a major pathway to repair DNA double-strand breaks (DSB). HR uses an undamaged homologous DNA sequence as a template for copying the missing information, which requires identifying a homologous sequence among megabases of DNA within the crowded nucleus. In eukaryotes, the conserved Rad51-single-stranded DNA nucleoprotein filament (NPF) performs this homology search. Although NPFs have been extensively studied in vitro by molecular and genetic approaches, their in vivo formation and dynamics could not thus far be assessed due to the lack of functional tagged versions of Rad51. Here we develop and characterize in budding yeast the first fully functional, tagged version of Rad51. Following induction of a unique DSB, we observe Rad51-ssDNA forming exceedingly long filaments, spanning the whole nucleus and eventually contacting the donor sequence. Emerging filaments adopt a variety of shapes not seen in vitro and are modulated by Rad54 and Srs2, shedding new light on the function of these factors. The filaments are also dynamic, undergoing rounds of compaction and extension. Our biophysical models demonstrate that formation of extended filaments, and particularly their compaction-extension dynamics, constitute a robust search strategy, allowing DSB to rapidly explore the nuclear volume and thus enable efficient HR.

Single molecule microscopy reveals key physical features of repair foci in living cells.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Dual effects of exercise in dysferlinopathy.

Dysferlinopathy refers to a group of autosomal recessive muscular dystrophies due to mutations in the dysferlin gene causing deficiency of a membrane-bound protein crucially involved in plasma membrane repair. The condition is characterized by marked clinical heterogeneity, the different phenotypes/modes of presentation being unrelated to the genotype. For unknown reasons, patients are often remarkably active before the onset of symptoms. Dysferlin deficiency-related persistence of mechanically induced sarcolemma disruptions causes myofiber damage and necrosis. We postulate that limited myodamage may initially remain hidden with well-preserved resistance to physical strains. By subjecting dysferlin-deficient B6.A/J-Dysf(prmd) mice to long-term swimming exercise, we observed that concentric/isometric strain improved muscle strength and alleviated muscular dystrophy by limiting the accumulation of membrane lesions. By contrast, eccentric strain induced by long-term running in a wheel worsened the dystrophic process. Myofiber damage induced by eccentric strain increased with age, reflecting the accumulation of non-necrotic membrane lesions up to a critical threshold. This phenomenon was modulated by daily spontaneous activity. Transposed to humans, our results may suggest that the past activity profile shapes the clinical phenotype of the myopathy and that patients with dysferlinopathy should likely benefit from concentric exercise-based physiotherapy.

In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.