ABSTRACT
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Subject(s)
Cysteine , Neoplasms , Animals , Humans , Mice , Cell Line, Tumor , Cysteine/metabolism , Cysteine/chemistry , Ligands , Melanoma/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , NF-kappa B/chemistry , NF-kappa B/metabolism , Oxidation-Reduction , Signal Transduction , SOXE Transcription Factors/chemistry , SOXE Transcription Factors/metabolismABSTRACT
Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Lysosomes , Animals , Humans , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/ultrastructure , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/ultrastructure , Homeostasis , Longevity , Lysosomes/metabolism , Lysosomes/ultrastructure , Amino Acid Motifs , Microscopy, ElectronABSTRACT
Gasdermin D (GSDMD)-activated inflammatory cell death (pyroptosis) causes mitochondrial damage, but its underlying mechanism and functional consequences are largely unknown. Here, we show that the N-terminal pore-forming GSDMD fragment (GSDMD-NT) rapidly damaged both inner and outer mitochondrial membranes (OMMs) leading to reduced mitochondrial numbers, mitophagy, ROS, loss of transmembrane potential, attenuated oxidative phosphorylation (OXPHOS), and release of mitochondrial proteins and DNA from the matrix and intermembrane space. Mitochondrial damage occurred as soon as GSDMD was cleaved prior to plasma membrane damage. Mitochondrial damage was independent of the B-cell lymphoma 2 family and depended on GSDMD-NT binding to cardiolipin. Canonical and noncanonical inflammasome activation of mitochondrial damage, pyroptosis, and inflammatory cytokine release were suppressed by genetic ablation of cardiolipin synthase (Crls1) or the scramblase (Plscr3) that transfers cardiolipin to the OMM. Phospholipid scramblase-3 (PLSCR3) deficiency in a tumor compromised pyroptosis-triggered anti-tumor immunity. Thus, mitochondrial damage plays a critical role in pyroptosis.
Subject(s)
Gasdermins , Pyroptosis , Neoplasm Proteins/metabolism , Cardiolipins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Inflammasomes/metabolismABSTRACT
The simultaneous extraction of crocin and geniposide from gardenia fruits (Gardenia jasminoides Ellis) was performed by integrating natural deep eutectic solvents (NADES) and ultrasound-assisted extraction (UAE). Among the eight kinds of NADES screened, choline chloride-1,2-propylene glycol was the most suitable extractant. The probe-type ultrasound-assisted NADES extraction system (pr-UAE-NADES) demonstrated higher extraction efficiency compared with plate-type ultrasound-assisted NADES extraction system (pl-UAE-NADES). Orthogonal experimental design and a modified multi-index synthetic weighted scoring method were adopted to optimize pr-UAE-NADES extraction process. The optimal extraction conditions that had a maximum synthetic weighted score of 29.46 were determined to be 25 °C for extraction temperature, 600 W for ultrasonic power, 20 min for extraction time, and 25% (w/w) for water content in NADES, leading to the maximum yields (7.39 ± 0.20 mg/g and 57.99 ± 0.91 mg/g, respectively) of crocin and geniposide. Thirty-three compounds including iridoids, carotenoids, phenolic acids, flavonoids, and triterpenes in the NADES extract were identified by LC-Q-TOF-MS2 coupled with a feature-based molecular networking workflow. The kinetics evaluation of the conjugated dienes generation on Cu2+-induced low density lipoprotein (LDL) oxidation via the four-parameter logistic regression model showed that crocin increased the lag time of LDL oxidation in a concentration-dependent manner (15 µg/mL, 30 µg/mL, 45 µg/mL) by 12.66%, 35.44%, and 73.42%, respectively. The quantitative determination for fluorescence properties alteration of the apolipoprotein B-100 exhibited that crocin effectively inhibited the fluorescence quenching of tryptophan residues and the modification of lysine residues caused by reactive aldehydes and malondialdehydes. The pr-UAE-NADES showed significant efficiency toward the simultaneous extraction of crocin and geniposide from gardenia fruits. And this study demonstrates the potential utility of gardenia fruits in developing anti-atherogenic functional food.
Subject(s)
Deep Eutectic Solvents , Gardenia , Gardenia/chemistry , Fruit/chemistry , Iridoids/pharmacology , Iridoids/analysis , Carotenoids/pharmacology , Carotenoids/analysis , Plant Extracts/pharmacology , Plant Extracts/chemistry , SolventsABSTRACT
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap , an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NFκB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NFκB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.
ABSTRACT
Heightened concentrations of reactive metabolites, including reactive oxygen species (ROS), can damage all macromolecules leading to the erosion of cellular fidelity. In this regard, the control of ROS in the nuclues is essential for cellular homeostasis, and dysregulation of nuclear ROS has been attributed to multiple pathologies and the mechanism of action of certain chemotherapies. How nuclear ROS is generated, detoxified and sensed is poorly understood, and stems in part, from a historical lack of tools that allow for its precise generation and detection. Here, we summarize the latest advances in chemical biology inspired approaches that have been developed to study nuclear ROS and highlight how these tools have led to major breakthroughs in understanding its regulation. The continued development and application of chemical biology approaches to understand nuclear ROS promises to unlock fundamental insights into human physiology and disease.
Subject(s)
Oxidative Stress , Humans , Reactive Oxygen Species/metabolismABSTRACT
Multiple anticancer drugs have been proposed to cause cell death, in part, by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs, exactly how the resultant ROS function and are sensed is poorly understood. It remains unclear which proteins the ROS modify and their roles in drug sensitivity/resistance. To answer these questions, we examined 11 anticancer drugs with an integrated proteogenomic approach identifying not only many unique targets but also shared ones-including ribosomal components, suggesting common mechanisms by which drugs regulate translation. We focus on CHK1 that we find is a nuclear H2O2 sensor that launches a cellular program to dampen ROS. CHK1 phosphorylates the mitochondrial DNA-binding protein SSBP1 to prevent its mitochondrial localization, which in turn decreases nuclear H2O2. Our results reveal a druggable nucleus-to-mitochondria ROS-sensing pathway-required to resolve nuclear H2O2 accumulation and mediate resistance to platinum-based agents in ovarian cancers.
Subject(s)
Antineoplastic Agents , Reactive Oxygen Species , Antineoplastic Agents/pharmacology , Antineoplastic Agents/metabolism , Hydrogen Peroxide/metabolism , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Cell Nucleus/metabolism , HumansABSTRACT
Multiple chemotherapies are proposed to cause cell death in part by increasing the steady-state levels of cellular reactive oxygen species (ROS). However, for most of these drugs exactly how the resultant ROS function and are sensed is poorly understood. In particular, it's unclear which proteins the ROS modify and their roles in chemotherapy sensitivity/resistance. To answer these questions, we examined 11 chemotherapies with an integrated proteogenomic approach identifying many unique targets for these drugs but also shared ones including ribosomal components, suggesting one mechanism by which chemotherapies regulate translation. We focus on CHK1 which we find is a nuclear H 2 O 2 sensor that promotes an anti-ROS cellular program. CHK1 acts by phosphorylating the mitochondrial-DNA binding protein SSBP1, preventing its mitochondrial localization, which in turn decreases nuclear H 2 O 2 . Our results reveal a druggable nucleus-to-mitochondria ROS sensing pathway required to resolve nuclear H 2 O 2 accumulation, which mediates resistance to platinum-based chemotherapies in ovarian cancers.
ABSTRACT
Multiple cancers regulate oxidative stress by activating the transcription factor NRF2 through mutation of its negative regulator, KEAP1. NRF2 has been studied extensively in KEAP1-mutant cancers; however, the role of this pathway in cancers with wild-type KEAP1 remains poorly understood. To answer this question, we induced NRF2 via pharmacological inactivation of KEAP1 in a panel of 50+ non-small cell lung cancer cell lines. Unexpectedly, marked decreases in viability were observed in >13% of the cell lines-an effect that was rescued by NRF2 ablation. Genome-wide and targeted CRISPR screens revealed that NRF2 induces NADH-reductive stress, through the upregulation of the NAD+-consuming enzyme ALDH3A1. Leveraging these findings, we show that cells treated with KEAP1 inhibitors or those with endogenous KEAP1 mutations are selectively vulnerable to Complex I inhibition, which impairs NADH oxidation capacity and potentiates reductive stress. Thus, we identify reductive stress as a metabolic vulnerability in NRF2-activated lung cancers.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , NF-E2-Related Factor 2 , Humans , Carcinoma, Non-Small-Cell Lung/metabolism , Cell Line, Tumor , Kelch-Like ECH-Associated Protein 1/metabolism , Lung Neoplasms/metabolism , NAD/metabolism , NF-E2-Related Factor 2/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/genetics , Signal TransductionABSTRACT
BACKGROUND: Norbixin, a carotenoid extracted from annatto seeds, is widely utilized as a natural pigment in foods, cosmetics and medicines. Its water solubility is relatively high under neutral or alkaline conditions but low under acidic conditions, which limits its application in some food products. RESULTS: This problem was overcome by utilizing liposomes to encapsulate the carotenoids so that they could be easily dispersed within acidic solutions. The norbixin was loaded into the liposomes using the pH-driven method. Liposomes were produced by passing aqueous phospholipid dispersions through a microfluidizer under high pressure. Norbixin was then added to the liposome dispersions at pH 7.0 and then driven into the hydrophobic domains of the phospholipid bilayers by acidifying the system. Measurements of the encapsulation efficiency showed that the norbixin was successfully loaded into the liposomes using the pH-driven method. X-ray diffraction analysis showed that the norbixin was in an amorphous state after incorporation into the liposomes. Encapsulation of norbixin within the liposomes was also shown to increase its water dispersibility and chemical stability under acidic pH conditions. CONCLUSION: The pH-driven method therefore provides a useful means of increasing the application of this bioactive carotenoid within functional foods and other products. © 2021 Society of Chemical Industry.
Subject(s)
Carotenoids , Liposomes , Carotenoids/chemistry , Hydrogen-Ion Concentration , Liposomes/chemistry , SolubilityABSTRACT
Mutant-selective KRASG12C inhibitors, such as MRTX849 (adagrasib) and AMG 510 (sotorasib), have demonstrated efficacy in KRAS G12C-mutant cancers, including non-small cell lung cancer (NSCLC). However, mechanisms underlying clinical acquired resistance to KRASG12C inhibitors remain undetermined. To begin to define the mechanistic spectrum of acquired resistance, we describe a patient with KRAS G12C NSCLC who developed polyclonal acquired resistance to MRTX849 with the emergence of 10 heterogeneous resistance alterations in serial cell-free DNA spanning four genes (KRAS, NRAS, BRAF, MAP2K1), all of which converge to reactivate RAS-MAPK signaling. Notably, a novel KRAS Y96D mutation affecting the switch-II pocket, to which MRTX849 and other inactive-state inhibitors bind, was identified that interferes with key protein-drug interactions and confers resistance to these inhibitors in engineered and patient-derived KRAS G12C cancer models. Interestingly, a novel, functionally distinct tricomplex KRASG12C active-state inhibitor RM-018 retained the ability to bind and inhibit KRASG12C/Y96D and could overcome resistance. SIGNIFICANCE: In one of the first reports of clinical acquired resistance to KRASG12C inhibitors, our data suggest polyclonal RAS-MAPK reactivation as a central resistance mechanism. We also identify a novel KRAS switch-II pocket mutation that impairs binding and drives resistance to inactive-state inhibitors but is surmountable by a functionally distinct KRASG12C inhibitor.See related commentary by Pinnelli and Trusolino, p. 1874.This article is highlighted in the In This Issue feature, p. 1861.
Subject(s)
Acetonitriles/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm/genetics , Lung Neoplasms/drug therapy , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Aged , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/secondary , Female , Humans , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Neoplasm Metastasis , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
In this issue of Cell Chemical Biology, Kang et al. (2019) describe the use of a high-throughput cell-based screen to identify chemical scaffolds that selectively inhibit mTORC1 nutrient sensing. Chemical proteomic-based target identification reveals class I glucose transporters as direct targets for these inhibitors, linking glucose sensing with mTORC1 regulation.
Subject(s)
Glucose Transport Proteins, Facilitative , TOR Serine-Threonine Kinases , Glucose , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , ProteomicsABSTRACT
After endocytosis, transmembrane cargoes such as signaling receptors, channels, and transporters enter endosomes where they are sorted to different destinations. Retromer and ESCRT (endosomal sorting complex required for transport) are functionally distinct protein complexes on endosomes that direct cargo sorting into the recycling retrograde transport pathway and the degradative multivesicular endosome pathway (MVE), respectively. Cargoes destined for degradation in lysosomes are decorated with K63-linked ubiquitin chains, which serve as an efficient sorting signal for entry into the MVE pathway. Defects in K63-linked ubiquitination disrupt MVE sorting and degradation of membrane proteins. Here, we unexpectedly found that UBC-13, the E2 ubiquitin-conjugating enzyme that generates K63-linked ubiquitin chains, is essential for retrograde transport of multiple retromer-dependent cargoes including MIG-14/Wntless. Loss of ubc-13 disrupts MIG-14/Wntless trafficking from endosomes to the Golgi, causing missorting of MIG-14 to lysosomes and impairment of Wnt-dependent processes. We observed that retromer-associated SNX-1 and the ESCRT-0 subunit HGRS-1/Hrs localized to distinct regions on a common endosome in wild type but overlapped on ubc-13(lf) endosomes, indicating that UBC-13 is important for the separation of retromer and ESCRT microdomains on endosomes. Our data suggest that cargo ubiquitination mediated by UBC-13 plays an important role in maintaining the functionally distinct subdomains to ensure efficient cargo segregation on endosomes.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Carrier Proteins/metabolism , Endocytosis , Ubiquitin-Conjugating Enzymes/metabolism , Wnt Signaling Pathway , Animals , Caenorhabditis elegans , Endosomes/metabolism , Intracellular Signaling Peptides and Proteins , Lysosomes/metabolism , Mutation/genetics , Protein Transport , Proteolysis , Ubiquitin/metabolismABSTRACT
Tse1 (Tse is type VI secretion exported), an effector protein produced by Pseudomonas aeruginosa, is an amidase that hydrolyses the γ-D-glutamyl-DAP (γ-D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6-148)-Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1 Å=0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like α+ß fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , N-Acetylmuramoyl-L-alanine Amidase/chemistry , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Pseudomonas aeruginosa/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Catalytic Domain , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Mutagenesis, Site-Directed , N-Acetylmuramoyl-L-alanine Amidase/genetics , Protein Conformation , Protein Interaction Domains and Motifs , Pseudomonas aeruginosa/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
STING functions as both an adaptor protein signaling cytoplasmic double-stranded DNA and a direct immunosensor of cyclic diguanylate monophosphate (c-di-GMP). The crystal structures of the C-terminal domain of human STING (STING(CTD)) and its complex with c-di-GMP reveal how STING recognizes c-di-GMP. In response to c-di-GMP binding, two surface loops, which serve as a gate and latch of the cleft formed by the dimeric STING(CTD), undergo rearrangements to interact with the ligand.
Subject(s)
Membrane Proteins/chemistry , Cyclic GMP/analogs & derivatives , Humans , Membrane Proteins/metabolism , Models, Molecular , Protein ConformationABSTRACT
Apart from inhibiting RecA activity through protein-protein interactions, Deinococcus radiodurans RecX inhibits the expression of RecA and two other anti-oxidant proteins. To identify the repertoire of proteins regulated by RecX, comparative proteomic studies were undertaken on a wild-type strain (R1) and recX null mutant (RecX(-)). Two-dimensional electrophoresis followed by MALDI-TOF identification revealed 35 differentially expressed proteins, including 12 up-regulated and 23 down-regulated proteins in the mutant. The 12 up-regulated proteins are DNA repair proteins, stress response proteins, and metabolism-related proteins. Most of these have been previously characterized as ionizing radiation-induced proteins. The 23 down-regulated proteins are mainly involved in cellular metabolism, and some of these are key enzymes in the metabolic pathway. Thus, RecX is suggested to be involved in the switch between DNA damage response and normal metabolism in D. radiodurans.