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In-cell isotope-coded labeling and LC-MS/MS analysis probe warfarin inhibition mechanism

ABSTRACT: Warfarin targets human vitamin K epoxide reductase (hVKOR), a redox enzyme in the membrane of endoplasmic reticulum (ER), to prevent the formation of blood clots. Although warfarin has been a popular medication since 1954, its mechanism of action is still unclear. A fundamental issue is the controversial orientation of transmembrane helices (TM) in hVKOR. Stable isotope-coded reagents were usedto label VKOR in free, mutated, and warfarin-binding states directly in the cellular environment, followed by LC-MS/MS bottom-up approach to investigate the warfarin binding mechanism.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Early Embryonic Cell

SUBMITTER: Weidong Cui

LAB HEAD: Michael L. Gross

PROVIDER: PXD003774 | Pride | 2017-03-30

REPOSITORIES: Pride

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			Action	DRS
	010915_VKOR_C132A.raw	Raw
	010915_VKOR_C135A.raw	Raw
	010915_VKOR_C16A.raw	Raw
	010915_VKOR_C43132A.raw	Raw
	010915_VKOR_C43135A.raw	Raw

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Publications

Warfarin traps human vitamin K epoxide reductase in an intermediate state during electron transfer.

Shen Guomin G Cui Weidong W Zhang Hao H Zhou Fengbo F Huang Wei W Liu Qian Q Yang Yihu Y Li Shuang S Bowman Gregory R GR Sadler J Evan JE Gross Michael L ML Li Weikai W

Nature structural & molecular biology 20161205 1

Although warfarin is the most widely used anticoagulant worldwide, the mechanism by which warfarin inhibits its target, human vitamin K epoxide reductase (hVKOR), remains unclear. Here we show that warfarin blocks a dynamic electron-transfer process in hVKOR. A major fraction of cellular hVKOR is in an intermediate redox state containing a Cys51-Cys132 disulfide, a characteristic accommodated by a four-transmembrane-helix structure of hVKOR. Warfarin selectively inhibits this major cellular form ...[more]

PMID: 27918545

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The catalytic mechanism of vitamin K epoxide reduction in a cellular environment

Project description:Vitamin K epoxide reductases (VKOR) constitute a major family of integral membrane thiol oxidoreductases. In humans, VKOR sustains blood coagulation and bone mineralization through the vitamin K cycle. Previous chemical models assumed that the catalysis of human VKOR (hVKOR) starts from a fully reduced active site. This state, however, constitutes only a minor cellular fraction (5.6%). Thus, how hVKOR catalysis is carried out in the cellular environment remains largely unknown. Here we use quantitative mass spectrometry (MS) and electrophoretic mobility analysis to show that KO forms a covalent complex with a cysteine mutant mimicking hVKOR in a partially oxidized state. Trapping of this covalent reaction intermediate suggests that the partially oxidized state is catalytically active in cells. To investigate this activity, we analyze the correlation between the cellular activity and the cellular cysteine status of hVKOR. We find that the partially oxidized hVKOR has a considerably lower activity than hVKOR with a fully reduced active site. Although there are more partially oxidized hVKOR than fully reduced hVKOR in cells, these two reactive states contribute about equally to the overall hVKOR activity, and hVKOR catalysis can initiate from either of these states. Overall, the combination of MS quantification and biochemical analyses reveal the catalytic mechanism of this integral membrane enzyme in a cellular environment.

2020-12-03 | PXD020676 | Pride

Proteome adaptations of AC16 cells different oxygen exposure

Project description:To simulate in vitro different oxidative stress exposures, AC16 cells were cultured either under physioxia (5% oxygen) or normoxia (21% oxygen), and were consequently harvested either under physioxia or normoxia.

2021-09-10 | PXD023620 | Pride

Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies

Project description:Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML samples. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in samples from patient subtypes with FLT3 mutations. These samples also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

2023-05-03 | PXD032104 | Pride

Mass spectrometry-based redox and protein profiling reveals distinct protein expression and oxidation patterns in failing human hearts

Project description:Oxidative stress plays a key role in development and progression of cardiovascular diseases and it is correlated with left ventricular dysfunction and heart failure (HF). Oxidative environments lead to the formation of intra- and intermolecular disulfide bonds, as well as to plethora of other reversible and irreversible oxidative amino acid modifications, affecting the functionality of the proteins. Here we report that heart failure due to ischemic cardiomyopathy (ICM) or dilated cardiomyopathy (DCM) is correlated with increase in oxidative stress compared to non-failing control hearts, manifested through decreased GSH/GSSG ratio in failing heart tissue samples and adaptations of cardiac redox proteome which occur in correlation with two different heart pathologies.

2021-09-09 | PXD021261 | Pride

Quantification of sulfenic acid on model protein

Project description:Aldolase contains eight free cysteine residues without disulfide formation in its native state, which makes it a suitable model to manipulate the oxidative modifications on cysteine. H2O2 was chosen to treat aldolase since the principle product between which is sulfenic acid.To assess the reduction of the newly formed sulfenic acids on aldolase by arsenite, a differential alkylation strategy was adapted.To further investigate the formation of other reversible cysteine oxidations such as disulfide on aldolase, arsenite was replaced by DTT in the differential alkylation method to reduce all the reversible cysteine oxidations.To further validate the selectivity of the reducing ability of arsenite on sulfenic acid but not other oxidative modifications especially disulfide, lysozyme was employed in the differential alkylation method. Lysozyme has 8 cysteine residues all bound into disulfide bonds, thereby was not treated with H2O2 to avoid further oxidation.

2016-10-10 | PXD003348 | Pride

Combining redox proteomics with proximity labeling to monitor localized cysteine oxidation

Project description:Reactive oxygen species (ROS) can modulate protein function through cysteine oxidation. Identifying targets of ROS can provide insight into uncharacterized ROS-regulated pathways. Several redox-proteomic workflows, such as OxICAT, exist to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular compartments and ROS hotspots remains challenging with existing workflows. Here, we present a chemoproteomic platform, PL-OxICAT, which combines proximity labeling (PL) with OxICAT to monitor localized cysteine oxidation events. We show that TurboID-based PL-OxICAT can monitor cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space. Furthermore, we use APEX-based PL-OxICAT to monitor oxidation events within ROS hotspots by using endogenous ROS as the source of peroxide for APEX activation. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS.

2024-05-21 | PXD034118 | Pride

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics

Project description:The nucleus controls cell growth and reproduction by well-orchestrated interactions between nuclear proteins and chromatin. Mutations that result in the dysregulation of these chromatin-associated proteins are known to lead to the onset of numerous diseases. Many of these nuclear proteins have remained recalcitrant to traditional non-covalent small-molecule ligand discovery. Covalent ligands can provide a promising approach for targeting proteins such as transcription factors that lack ordered small-molecule binding pockets. Here, we present a chemoproteomic platform that couples proximity labeling (PL) using a Histone-TurboID (His-TID) construct with competitive isoTOP-ABPP to identify ligandable nuclear cysteines. Application of covalent scout fragments, KB02 and KB05, revealed ligandable cysteine sites on diverse proteins involved in transcriptional regulation, spindle assembly, and DNA repair. To identify functional liganding events that alter target-protein association with chromatin, we monitor the effects of KB02 and KB05 on the chromatin-associated proteome. Importantly, we identify proteins that show both increased and decreased association with chromatin in the presence of the covalent fragments. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and association with chromatin upon covalent liganding at Cys106 by KB02. Together, our PL-assisted nuclear-focused chemoproteomic platform provides us insights into nuclear cysteine ligandability and the functional effects of ligand binding on chromatin association, thereby furthering our understanding of the potentially druggability of the nuclear proteome.

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Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane

Project description:Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.

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Stochiometric Quantification of the Thiol Redox Proteome Reveals Subcellular Compartmentalization and Susceptibility to Oxidative Perturbations

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