17 research outputs found
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Covalent targeting of the vacuolar H+-ATPase activates autophagy via mTORC1 inhibition.
Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling
Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism
Covalent targeting of the vacuolar H<sup>+</sup>-ATPase activates autophagy via mTORC1 inhibition.
N-Acryloylindole-alkyne (NAIA) enables profiling new ligandable hotspots in chemoproteomics experiments and imaging thiol oxidation
We report a new class of compounds, N-acryloylindole-alkynes (NAIAs), as promising cysteine-reactive probes for proteome-wide cysteine profiling and imaging of thiol oxidative modifications. NAIAs showed superior cysteine reactivity owing to delocalization of π electrons of the acrylamide warhead over the whole indole scaffold, resulting in its activation for faster reaction with cysteines. This allows NAIAs to ligand functional cysteines more effectively than IAA, as well as to image oxidized thiols in cells facing oxidative stress by confocal fluorescence microscopy. In MS-based ABPP experiments, NAIAs successfully captured a new pool of ligandable cysteines and proteins even compared to the current state-of-the-art cysteine profiling data. Competitive ABPP experiments further demonstrate the ability of NAIA to discover hit compounds targeting these new cysteines and proteins. This work should initiate development of new cysteine-reactive probes, particularly those with activated acrylamide, for advancing cysteine imaging and profiling, and covalent ligand screening for drug research
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A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues
Hydrogen peroxide (H2O2) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H2O2 biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity-based sensing probe that responds to H2O2 via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H2O2 levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H2O2 levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3-4 d
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Versatile Histochemical Approach to Detection of Hydrogen Peroxide in Cells and Tissues Based on Puromycin Staining
Hydrogen peroxide
(H<sub>2</sub>O<sub>2</sub>) is a central reactive
oxygen species (ROS) that contributes to diseases from obesity to
cancer to neurodegeneration but is also emerging as an important signaling
molecule. We now report a versatile histochemical approach for detection
of H<sub>2</sub>O<sub>2</sub> that can be employed across a broad
range of cell and tissue specimens in both healthy and disease states.
We have developed a first-generation H<sub>2</sub>O<sub>2</sub>-responsive
analogue named Peroxymycin-1, which is based on the classic cell-staining
molecule puromycin and enables covalent staining of biological samples
and retains its signal after fixation. H<sub>2</sub>O<sub>2</sub>-mediated
boronate cleavage uncages the puromycin aminonucleoside, which leaves
a permanent and dose-dependent mark on treated biological specimens
that can be detected with high sensitivity and precision through a
standard immunofluorescence assay. Peroxymycin-1 is selective and
sensitive enough to image both exogenous and endogenous changes in
cellular H<sub>2</sub>O<sub>2</sub> levels and can be exploited to
profile resting H<sub>2</sub>O<sub>2</sub> levels across a panel of
cell lines to distinguish metastatic, invasive cancer cells from less
invasive cancer and nontumorigenic counterparts, based on correlations
with ROS status. Moreover, we establish that Peroxymycin-1 is an effective
histochemical probe for in vivo H<sub>2</sub>O<sub>2</sub> analysis,
as shown through identification of aberrant elevations in H<sub>2</sub>O<sub>2</sub> levels in liver tissues in a murine model of nonalcoholic
fatty liver disease, thus demonstrating the potential of this approach
for studying disease states and progression associated with H<sub>2</sub>O<sub>2</sub>. This work provides design principles that should
enable development of a broader range of histochemical probes for
biological use that operate via activity-based sensing
Induced self-assembly and disassembly of water-soluble alkynylplatinum( ii ) terpyridyl complexes with switchable near-infrared (NIR) emission modulated by metalmetal interactions over physiological pH: demonstration of pH-responsive NIR luminescent probes in cell-imaging studies
Water-soluble alkynylplatinum( ii ) terpyridine complexes, [Pt{tpy(C 6 H 4 CH 2 NMe 3 -4)-4}(CCAr)](OTf) 2 [Ar = C 6 H 3 (OH) 2 -3,5 ( 1 ), C 6 H 4 OH-4 ( 2 ), C 6 H 3 (OMe) 2 -3,5 ( 3 )], have been synthesized and characterized. The photophysical and electrochemical properties of the complexes have been studied. Complex 1 has been found to undergo aggregation at low pHs, leading to metalmetal and/or interactions and the emergence of a triplet metal-metal-to-ligand charge transfer ( 3 MMLCT) emission in the near-infrared (NIR) region, the intensity of which has been enhanced 1350-fold over that at physiological pH. Such switchable NIR emission of complex 1 was employed in cell-imaging experiments. The pH response of the 3 MMLCT emission of complex 1 in cellular compartments has been studied using experiments with fixed MadinDarby canine kidney (MDCK) cells, while live cell-imaging experiments revealed that complex 1 could function as a NIR luminescent probe for the tracking of the location of acidic organelles such as lysosomes
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Activity-based ratiometric FRET probe reveals oncogene-driven changes in labile copper pools induced by altered glutathione metabolism
Copper is essential for life, and beyond its well-established ability to serve as a tightly bound, redox-active active site cofactor for enzyme function, emerging data suggest that cellular copper also exists in labile pools, defined as loosely bound to low-molecular-weight ligands, which can regulate diverse transition metal signaling processes spanning neural communication and olfaction, lipolysis, rest-activity cycles, and kinase pathways critical for oncogenic signaling. To help decipher this growing biology, we report a first-generation ratiometric fluorescence resonance energy transfer (FRET) copper probe, FCP-1, for activity-based sensing of labile Cu(I) pools in live cells. FCP-1 links fluorescein and rhodamine dyes through a Tris[(2-pyridyl)methyl]amine bridge. Bioinspired Cu(I)-induced oxidative cleavage decreases FRET between fluorescein donor and rhodamine acceptor. FCP-1 responds to Cu(I) with high metal selectivity and oxidation-state specificity and facilitates ratiometric measurements that minimize potential interferences arising from variations in sample thickness, dye concentration, and light intensity. FCP-1 enables imaging of dynamic changes in labile Cu(I) pools in live cells in response to copper supplementation/depletion, differential expression of the copper importer CTR1, and redox stress induced by manipulating intracellular glutathione levels and reduced/oxidized glutathione (GSH/GSSG) ratios. FCP-1 imaging reveals a labile Cu(I) deficiency induced by oncogene-driven cellular transformation that promotes fluctuations in glutathione metabolism, where lower GSH/GSSG ratios decrease labile Cu(I) availability without affecting total copper levels. By connecting copper dysregulation and glutathione stress in cancer, this work provides a valuable starting point to study broader cross-talk between metal and redox pathways in health and disease with activity-based probes