Abstract B32: Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties

Abstract

Abstract Previous studies proposed that myosin-Va regulates apoptosis by sequestering pro-apoptotic Bmf to the actin cytoskeleton through dynein light chain-2 (DLC2). Adhesion loss or other cytoskeletal perturbations would unleash Bmf, allowing it to bind and inhibit pro-survival Bcl2 proteins. Recently, our research group has demonstrated that overexpression of a myosin-Va medial tail fragment (MVaf) harboring the binding site for DLC2 dramatically decreased melanoma cell viability. Morphological and molecular changes, including surface blebbing, mitochondrial outer membrane permeabilization, cytochrome-c and Smac release, as well as caspase-9/-3 activation and DNA fragmentation indicated that melanoma cells died of apoptosis. Besides, MVaf expression attenuated B16-F10 solid tumor growth in mice suggesting that this peptide may be useful as an apoptosis-inducing tool for basic cancer research. We suggest that MVaf induces apoptosis by sequestering DLC2 and DLC1, thereby unleashing the pair of sensitizer and activator BH3-only proteins Bmf and Bim. In view of such findings, we have proposed to develop a recombinant Tat-fusion MVaf to be explored in peptide-based therapeutic approaches. The native MVaf sequence was altered through the N-terminal addition of a transducible Tat-HA tag. EGFP was alternatively introduced into recombinant products to assess their subcellular localization. Bacterially expressed His-tagged fusion proteins were purified by immobilized metal chelate affinity chromatography on Ni-NTA beads, and transduction assays were initially performed in HeLa cells. The kinetics of Tat-HA-MVaf (THM, 13.7 kDa) and Tat-HA-EGFP-MVaf (THEM, 41.9 kDa) uptake, at concentrations ranging from 1 to 2 μM, was evaluated by confocal laser scanning microscopy (CLSM). Tat-HA-EGFP (THE, 33.9 kDa) was used as a control. Tat-HA-MVaf displayed a diffuse distribution throughout the cells in the first 5 min incubation, with a clear perinuclear accumulation after 1h incubation. THEM and THE fluorescence were detected in many developing vesicles adjacent to cell membrane, and distributed throughout the cytoplasm and nucleus after 5 min incubation, but became markedly weaker after 1h, suggesting proteolytic loss. Despite intense diffusion and intracellular accumulation of recombinant proteins, no significant changes in morphology and cell viability were detected by means of flow cytometry and MTT assay. Pull-down assay revealed that THEM but not THE binds to immobilized GST-DLC2 and to GST-DLC1 in a lesser extent, which confirms peptide-binding capacity to DLCs. On the other hand, pretreatment of cells with chloroquine, an inhibitor of endosomal acidification, sensitized cells to peptide treatment. We believe that Tat-mediated subcellular localization may have predominated, resulting in mislocalization of the peptide cargo. Also, one major drawback in using pTAT-conjugated peptides is their entrapment in cytoplasmic compartments and nucleus, resulting in limited availability and biological activity. One possible strategy is to produce recombinant peptides conjugated reversibly to Tat by a disulfide bond, which are disrupted upon intracellular delivery. Funding support. FAPESP / CNPq. Citation Format: Denise P. S. Leitão Mazzi, Cleidson P. Alves, Enilza Maria Espreafico. Cell penetrating Myosin-Va fragment covering the binding motif of DLC2: Evaluation of pro-apoptotic properties. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B32.