Protein analysis under biological conditions is now regarded as indispensable for understanding the structure and function of proteins, in addition to in vitro studies using purified target proteins. ...Because there are many molecules other than the protein-of-interest (POI) under live cell conditions, selective labeling of a POI is critical to distinguish the POI from other proteins for precise analysis. Protein labeling strategies utilizing genetically encoded tags have been used in POI modification in the complex environment of live cells. However, genetic manipulation may often induce overexpression of the POI and/or perturb the cellular context, resulting in unexpected artifacts in the protein analysis. Alternatively, recent progress in chemical biology has produced two major chemical approaches for analyzing endogenous proteins under native conditions. In this review, we summarize these techniques that utilize either protein-selective chemical labeling or proteome-directed chemical modification.
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Protein analysis in a biologically relevant context is now regarded as indispensable for deciphering the genuine structure and function of proteins. In this review, Shiraiwa et al. summarize recent advances in chemical approaches utilizing protein-selective chemical labeling and proteome-directed chemical modification.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Membrane biology studies have revealed that in addition to providing structural support for compartment formation and membrane protein function, subcellular biomembranes are also critically involved ...in many biological events. To facilitate our understanding of the functions, biophysical properties and structural dynamics of organelle membranes, various exciting chemical biology tools have recently emerged. This short review aims to describe the latest molecular probes for organelle membrane studies. In particular, we will feature chemical strategies to visualize and quantitatively analyze the dynamic propeties of organelle membranes and lipids and discuss current limitations and potential future directions of this challenging research area.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Endogenous protein labeling is one of the most invaluable methods for studying the
bona fide
functions of proteins in live cells. However, multi-molecular crowding conditions, such as those that ...occur in live cells, hamper the highly selective chemical labeling of a protein of interest (POI). We herein describe how the efficient coupling of molecular recognition with a chemical reaction is crucial for selective protein labeling. Recognition-driven protein labeling is carried out by a synthetic labeling reagent containing a protein (recognition) ligand, a reporter tag, and a reactive moiety. The molecular recognition of a POI can be used to greatly enhance the reaction kinetics and protein selectivity, even under live cell conditions. In this review, we also briefly discuss how such selective chemical labeling of an endogenous protein can have a variety of applications at the interface of chemistry and biology.
Endogenous protein labeling is one of the most invaluable methods for studying the
bona fide
functions of proteins in live cells.
Because of its critical roles in regulating cellular signal transduction, the molecular chaperone heat-shock protein 90 (Hsp90) has become a novel therapeutic target for various diseases, including ...cancer, inflammation, and neurological diseases. However, the lack of methods that allow us to directly evaluate the binding of small molecule ligands to intracellular Hsp90 makes the inhibitor development more difficult. Here, we report a simple cell-based assay system for the Hsp90 inhibitor in live-cell environments. In this strategy, the binding activity of ligands of interest is evaluated by competitive inhibition of ligand-directed N-acyl-N-alkyl sulfonamide (LDNASA) chemistry-mediated Hsp90 labeling. Using several known Hsp90 inhibitors, we demonstrated that our method could easily detect the ligand-binding event of Hsp90 in live cells. Our system is applicable to high-throughput ligand screening, and we discovered a new small molecule candidate that binds to the N-terminal ATP binding domain of Hsp90. These results demonstrate the use of the competitive LDNASA-based approach to directly evaluate ligand activity in live cells and identify potent drug candidates from chemical libraries.
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IJS, KILJ, NUK, PNG, UL, UM
Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) can inflict damage to biomolecules under oxidative stress and also act as signaling molecules at physiological levels. Here we developed ...a unique chemical tool to elucidate the biological roles of ROS using both fluorescence imaging and conditional proteomics. H2O2-responsive protein labeling reagents (Hyp-L) were designed to selectively tag proteins under the oxidative conditions in living cells and tissues. The Hyp-L signal remained even after sample fixation, which was compatible with conventional immunostaining. Moreover, Hyp-L allowed proteomic profiling of the labeled proteins using a conditional proteomics workflow. The integrative analysis enabled the identification of ROS generation and/or accumulation sites with a subcellular resolution. For the first time, we characterized that autophagosomes were enriched with H2O2 in activated macrophages. Hyp-L was further applied to mouse brain tissues and clearly revealed oxidative stress within mitochondria by the conditional proteomics.
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IJS, KILJ, NUK, PNG, UL, UM
The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling ...the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.
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IJS, KILJ, NUK, PNG, UL, UM
Here we describe a method for the site-selective attachment of synthetic molecules into specific 'endogenous' proteins in vivo using ligand-directed tosyl (LDT) chemistry. This approach was applied ...not only for chemically labeling proteins in living cells, tissues and mice but also for constructing a biosensor directly inside cells without genetic engineering. These data establish LDT chemistry as a new tool for the study and manipulation of biological systems.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Selective modification of native proteins in live cells is one of the central challenges in recent chemical biology. As a unique bioorthogonal approach, ligand-directed chemistry recently emerged, ...but the slow kinetics limits its scope. Here we successfully overcome this obstacle using N-acyl-N-alkyl sulfonamide as a reactive group. Quantitative kinetic analyses reveal that ligand-directed N-acyl-N-alkyl sulfonamide chemistry allows for rapid modification of a lysine residue proximal to the ligand binding site of a target protein, with a rate constant of ~10
M
s
, comparable to the fastest bioorthogonal chemistry. Despite some off-target reactions, this method can selectively label both intracellular and membrane-bound endogenous proteins. Moreover, the unique reactivity of N-acyl-N-alkyl sulfonamide enables the rational design of a lysine-targeted covalent inhibitor that shows durable suppression of the activity of Hsp90 in cancer cells. This work provides possibilities to extend the covalent inhibition approach that is currently being reassessed in drug discovery.
Protein–protein interactions (PPIs) intimately govern various biological processes and disease states and therefore have been identified as attractive therapeutic targets for small-molecule drug ...discovery. However, the development of highly potent inhibitors for PPIs has proven to be extremely challenging with limited clinical success stories. Herein, we report irreversible inhibitors of the human double minute 2 (HDM2)/p53 PPI, which employ a reactive N-acyl-N-alkyl sulfonamide (NASA) group as a warhead. Mass-based analysis successfully revealed the kinetics of covalent inhibition and the modification sites on HDM2 to be the N-terminal α-amine and Tyr67, both rarely seen in traditional covalent inhibitors. Finally, we demonstrated prolonged p53-pathway activation and more effective induction of the p53-mediated cell death in comparison to a noncovalent inhibitor. This study highlights the potential of the NASA warhead as a versatile electrophile for the covalent inhibition of PPIs and opens new avenues for the rational design of potent covalent PPI inhibitors.
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