Engineering and cytosolic delivery of a native regulatory protein and its variants for modulation of ERK2 signaling pathway

Abstract

The modulation of a cell signaling process using a molecular binder followed by an analysis of the cellular response is crucial for understanding its role in the cellular function and developing pharmaceuticals. Herein, we present the modulation of the ERK2-mediated signaling pathway through the cytosolic delivery of a native regulatory protein for ERK2, that is, PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa), and its engineered variants using a bacterial toxin-based delivery system. Based on biochemical and structural analyses, PEA-15 variants with different phosphorylation sites and a high affinity for ERK2 were designed. Semi-rational approach led to about an 830-fold increase in the binding affinity of PEA-15, resulting in more effective modulation of the ERK2-mediated signaling. Our approach enabled an understanding of the cellular function of the ERK2-mediated signaling process and the effect of PEA-15 phosphorylation on its action as an ERK2 blocker. We demonstrated the utility and potential of our approach by showing an efficient cytosolic delivery of these PEA-15 variants and the effective suppression of cell proliferation through the inhibition of the ERK2 function. The present approach can be used broadly for modulating the cell signaling processes and understanding their roles in cellular function, as well as for the development of therapeutics.

Nonphosphorylatable PEA15 mutant inhibits epithelial-mesenchymal transition in triple-negative breast cancer partly through the regulation of IL-8 expression

“…The delivery platform is composed of a targeting moiety, translocation domain, and PEA15 derivative. The successfully developed rEgH9 and TDP were utilized by Ryou et al [ 18 ] as an effective tool for cytosolic delivery of cargo proteins in an EGFR-specific manner, implying broad uses for cancer therapy. As seen in Fig.…”

Trapped! A Critical Evaluation of Methods for Measuring Total Cellular Uptake versus Cytosolic Localization

“…Prior to the mid-2000s, cells were often fixed prior to observation by fluorescence microscopy, in part to improve resolution. These fixation conditions ranged from mild (4% paraformaldehyde) to harsh (methanol). ,− A historically relevant example is the discussion of fixation artifacts in the measurement of the delivery efficacy of VP22, a type I herpes simplex virus tegument protein . Results suggested that a greater degree of internalization was observed with methanol fixation as compared to paraformaldehyde fixation.…”

Targeted Protein Degradation through Cytosolic Delivery of Monobody Binders Using Bacterial Toxins

“…The Stx2B-ETAII chimera has been developed and successfully used to deliver EGFP, certain enzymes, and an ERK2 kinase regulator, and has proven to be more stable when fused to cargo proteins than Stx2B alone. 29,30 Here, we describe and validate the receptor-specific cytoplasmic delivery of VHL−monobody fusion proteins to cancer cells using a chimeric toxin delivery system, resulting in targeted degradation and signaling inhibition.…”

“…Following furin protease cleavage within the ETA-II domain, the C-terminal portion reaches the cytosol via the Golgi apparatus and the endoplasmatic reticulum (ER) (see Figure a). The Stx2B-ETAII chimera has been developed and successfully used to deliver EGFP, certain enzymes, and an ERK2 kinase regulator, and has proven to be more stable when fused to cargo proteins than Stx2B alone. , …”