An integrative structural study of the human full-length RAD52 at 2.2 Å resolution.

Human RAD52 (RAD52) is a DNA-binding protein involved in many DNA repair mechanisms and genomic stability maintenance. In the last few years, this protein was discovered to be a promising novel pharmacological target for anticancer strategies. Although the interest in RAD52 has exponentially grown in the previous decade, most information about its structure and mechanism still needs to be elucidated. Here, we report the 2.2 Å resolution cryo-EM reconstruction of the full-length RAD52 (FL-RAD52) protein. This allows us to describe the hydration shell of the N-terminal region of FL-RAD52, which is structured in an undecamer ring. Water molecules coordinate with protein residues to promote stabilization inside and among the protomers and within the inner DNA binding cleft to drive protein-DNA recognition. Additionally, through a multidisciplinary approach involving SEC-SAXS and computational methods, we comprehensively describe the highly flexible and dynamic organization of the C-terminal portion of FL-RAD52. This work discloses unprecedented structural details on the FL-RAD52, which will be critical for characterizing its mechanism of action and inhibitor development, particularly in the context of novel approaches to synthetic lethality and anticancer drug discovery.

The supramolecular processing of liposomal doxorubicin hinders its therapeutic efficacy in cells.

The successful trajectory of liposome-encapsulated doxorubicin (e.g., Doxil, which has been approved by the U.S. Food and Drug Administration) as an anticancer nanodrug in clinical applications is contradicted by in vitro cell viability data that highlight its reduced efficacy in promoting cell death compared with non-encapsulated doxorubicin. No reports to date have provided a mechanistic explanation for this apparently discordant evidence. Taking advantage of doxorubicin intrinsic fluorescence and time-resolved optical microscopy, we analyze the uptake and intracellular processing of liposome-encapsulated doxorubicin (L-DOX) in several in vitro cellular models. Cell entry of L-DOX was found to lead to a rapid (seconds to minutes), energy- and temperature-independent release of crystallized doxorubicin nanorods into the cell cytoplasm, which then disassemble into a pool of fibril-shaped derivatives capable of crossing the cellular membrane while simultaneously releasing active drug monomers. Thus, a steady state is rapidly established in which the continuous supply of crystal nanorods from incoming liposomes is counteracted by a concentration-guided efflux in the extracellular medium of fibril-shaped derivatives and active drug monomers. These results demonstrate that liposome-mediated delivery is constitutively less efficient than isolated drug in establishing favorable conditions for drug retention in the cell. In addition to explaining previous contradictory evidence, present results impose careful rethinking of the synthetic identity of encapsulated anticancer drugs.