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Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR‐Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti‐CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry‐directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease‐responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3‐fold). These advancements enable specific proteolysis‐inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR‐Cas tools for controllable gene manipulation and regulation and clinical diagnostics. [ABSTRACT FROM AUTHOR]

Selective modulation of ligand–receptor interaction is essential in targeted therapy. In this study, we design an intelligent "scan and unlock" DNA automaton (SUDA) system to equip a native protein‐ligand with cell‐identity recognition and receptor‐mediated signaling in a cell‐type‐specific manner. Using embedded DNA‐based chemical reaction networks (CRNs) on the cell surface, SUDA scans and evaluates molecular profiles of cell‐surface proteins via Boolean logic circuits. Therefore, it achieves cell‐specific signal modulation by quickly unlocking the protein‐ligand in proximity to the target cell‐surface to activate its cognate receptor. As a proof of concept, we non‐genetically engineered hepatic growth factor (HGF) with distinct logic SUDAs to elicit target cell‐specific HGF signaling and wound healing behaviors in multiple heterogeneous cell types. Furthermore, the versatility of the SUDA strategy was shown by engineering tumor necrotic factor‐α (TNFα) to induce programmed cell death of target cell subpopulations through cell‐specific modulation of TNFR1 signaling. [ABSTRACT FROM AUTHOR]

The comprehensive understanding of the mechanisms underlying the interaction of cells with their membrane microenvironment is of great value for fundamental biological research; however, tracking biomolecules on cell surfaces with high temporal and spatial resolution remains a challenge. Herein, a modular strategy is presented for the construction of cell surface DNA‐based sensors by engineering DNA motifs and synthetic cofactors. In this strategy, a stimuli‐reactive organic molecule is employed as the cofactor for the DNA motif, and the self‐assembly of them forms a FRET‐based holo DNA‐based sensor. With the use of the DNA‐based sensors, the versatility of this modular strategy has been demonstrated in the ratiometric imaging of the cellular extrusion process of endogenous signaling molecules, including sulfur dioxide derivatives and nitric oxide. [ABSTRACT FROM AUTHOR]

Aims: To understand the roles of the RhoA/ROCK and mitogen-activated protein kinase (MAPK) pathways in high glucose (HG)-induced apoptosis and oxidative stress in cardiomyocytes.Materials and Methods: Neonatal rat cardiomyocytes were cultured in Dulbecco's modified Eagle's medium, supplemented with 5.5 or 30 mmol/L D-glucose, in the presence or absence of fasudil (50 or 100 μM), SB203580, SP600125, or PD98059 (10 μM, respectively). The percentage of early apoptotic cardiomyocytes was evaluated using flow cytometry. The superoxide dismutase activity and malondialdehyde contents in the cellular supernatants were measured. The Bax and Bcl-2 mRNA levels were determined by quantitative real-time PCR. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1), p38MAPK, JNK, and ERK as well as the protein levels of Bax, Bcl-2, and cleaved caspase-3 was analysed by Western blot.Results: Fasudil, SB203580, and SP600125 effectively inhibited the HG-induced early apoptosis increase and decreased Bax mRNA expression, the Bax/Bcl-2 protein expression ratio, and cleaved caspase-3 protein levels in the cardiomyocytes; this was accompanied by upregulation of the Bcl-2 mRNA. Moreover, fasudil markedly increased the superoxide dismutase activity level and suppressed the elevation in HG-induced malondialdehyde content and the phosphorylation of MYPT1, p38MAPK and JNK.Conclusions: The RhoA/ROCK pathway mediates HG-induced cardiomyocyte apoptosis via oxidative stress and activation of p38MAPK and JNK in neonatal rats in vitro. Fasudil effectively ameliorates HG-induced cardiomyocyte apoptosis by suppressing oxidative stress and the p38MAPK and JNK pathways. [ABSTRACT FROM AUTHOR]