RESUMO
KEAP1 is a substrate adaptor protein for a CUL3-based E3 ubiquitin ligase. Ubiquitylation and degradation of the antioxidant transcription factor NRF2 is considered the primary function of KEAP1; however, few other KEAP1 substrates have been identified. Because KEAP1 is altered in a number of human pathologies and has been proposed as a potential therapeutic target therein, we sought to better understand KEAP1 through systematic identification of its substrates. Toward this goal, we combined parallel affinity capture proteomics and candidate-based approaches. Substrate-trapping proteomics yielded NRF2 and the related transcription factor NRF1 as KEAP1 substrates. Our targeted investigation of KEAP1-interacting proteins revealed MCM3, an essential subunit of the replicative DNA helicase, as a new substrate. We show that MCM3 is ubiquitylated by the KEAP1-CUL3-RBX1 complex in cells and in vitro Using ubiquitin remnant profiling, we identify the sites of KEAP1-dependent ubiquitylation in MCM3, and these sites are on predicted exposed surfaces of the MCM2-7 complex. Unexpectedly, we determined that KEAP1 does not regulate total MCM3 protein stability or subcellular localization. Our analysis of a KEAP1 targeting motif in MCM3 suggests that MCM3 is a point of direct contact between KEAP1 and the MCM hexamer. Moreover, KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to the MCM2-7 complex. KEAP1 is thus poised to affect MCM2-7 dynamics or function rather than MCM3 abundance. Together, these data establish new functions for KEAP1 within the nucleus and identify MCM3 as a novel substrate of the KEAP1-CUL3-RBX1 E3 ligase.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Animais , Autofagia , Proteínas de Transporte/metabolismo , Ciclo Celular , Linhagem Celular , Cromatina/metabolismo , Proteínas Culina/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Modelos Moleculares , Mapas de Interação de Proteínas , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Hormones and neurotransmitters are essential to homeostasis, and their disruptions are connected to diseases ranging from cancer to anxiety. The differential reactivation of endobiotic glucuronides by gut microbial ß-glucuronidase (GUS) enzymes may influence interindividual differences in the onset and treatment of disease. Using multi-omic, in vitro, and in vivo approaches, we show that germ-free mice have reduced levels of active endobiotics and that distinct gut microbial Loop 1 and FMN GUS enzymes drive hormone and neurotransmitter reactivation. We demonstrate that a range of FDA-approved drugs prevent this reactivation by intercepting the catalytic cycle of the enzymes in a conserved fashion. Finally, we find that inhibiting GUS in conventional mice reduces free serotonin and increases its inactive glucuronide in the serum and intestines. Our results illuminate the indispensability of gut microbial enzymes in sustaining endobiotic homeostasis and indicate that therapeutic disruptions of this metabolism promote interindividual response variabilities.
Assuntos
Microbioma Gastrointestinal , Glucuronidase , Homeostase , Animais , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , Glucuronidase/metabolismo , Camundongos Endogâmicos C57BL , Serotonina/metabolismo , Glucuronídeos/metabolismo , Humanos , Intestinos/microbiologia , Masculino , Vida Livre de GermesRESUMO
STING is an innate immune sensor for immune surveillance of viral/bacterial infection and maintenance of an immune-friendly microenvironment to prevent tumorigenesis. However, if and how STING exerts innate immunity-independent function remains elusive. Here, the authors report that STING expression is increased in renal cell carcinoma (RCC) patients and governs tumor growth through non-canonical innate immune signaling involving mitochondrial ROS maintenance and calcium homeostasis. Mitochondrial voltage-dependent anion channel VDAC2 is identified as a new STING binding partner. STING depletion potentiates VDAC2/GRP75-mediated MERC (mitochondria-ER contact) formation to increase mitochondrial ROS/calcium levels, impairs mitochondria function, and suppresses mTORC1/S6K signaling leading to RCC growth retardation. STING interaction with VDAC2 occurs through STING-C88/C91 palmitoylation and inhibiting STING palmitoyl-transferases ZDHHCs by 2-BP significantly impedes RCC cell growth alone or in combination with sorafenib. Together, these studies reveal an innate immunity-independent function of STING in regulating mitochondrial function and growth in RCC, providing a rationale to target the STING/VDAC2 interaction in treating RCC.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/metabolismo , Cálcio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Imunidade Inata , Microambiente Tumoral , Canal de Ânion 2 Dependente de Voltagem/metabolismoRESUMO
Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Carcinógenos/antagonistas & inibidores , Colite/prevenção & controle , Neoplasias Colorretais/prevenção & controle , Glucuronidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Triclosan/antagonistas & inibidores , Animais , Anti-Infecciosos Locais/química , Anti-Infecciosos Locais/metabolismo , Anti-Infecciosos Locais/toxicidade , Anticarcinógenos/química , Anticarcinógenos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotransformação , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinógenos/química , Carcinógenos/metabolismo , Carcinógenos/toxicidade , Colite/induzido quimicamente , Colite/enzimologia , Colite/microbiologia , Colo/efeitos dos fármacos , Colo/microbiologia , Colo/patologia , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Expressão Gênica , Glucuronidase/química , Glucuronidase/genética , Glucuronidase/metabolismo , Inibidores de Glicosídeo Hidrolases/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triclosan/química , Triclosan/metabolismo , Triclosan/toxicidadeRESUMO
Homodimers are the most abundant type of enzyme in cells, and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on a similar event at the second site, the most informative state is the asymmetric singly bound (lig1) form, yet it tends to be thermodynamically elusive. Here we obtain milligram quantities of lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of a highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig1 intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but also should facilitate other structural applications in which specific linkage of proteins is helpful.
Assuntos
Cobre/química , Compostos Organometálicos/química , Alcinos/química , Sítio Alostérico , Azidas/química , Catálise , Reação de Cicloadição , Dimerização , Espectroscopia de Ressonância Magnética , Ligação Proteica , TermodinâmicaRESUMO
TRIM9 and TRIM67 are neuronally enriched E3 ubiquitin ligases essential for appropriate morphogenesis of cortical and hippocampal neurons and fidelitous responses to the axon guidance cue netrin-1. Deletion of murine Trim9 or Trim67 results in neuroanatomical defects and striking behavioral deficits, particularly in spatial learning and memory. TRIM9 and TRIM67 interact with cytoskeletal and exocytic proteins, but the full interactome is not known. Here we performed the unbiased proximity-dependent biotin identification (BioID) approach to define TRIM9 and TRIM67 protein-protein proximity network in developing cortical neurons and identified putative neuronal TRIM interaction partners. Candidates included cytoskeletal regulators, cytosolic protein transporters, exocytosis and endocytosis regulators, and proteins necessary for synaptic regulation. A subset of high-priority candidates was validated, including Myo16, Coro1A, MAP1B, ExoC1, GRIP1, PRG-1, and KIF1A. For a subset of validated candidates, we utilized total internal reflection fluorescence microscopy to demonstrate dynamic colocalization with TRIM proteins at the axonal periphery, including at the tips of filopodia. Further analysis demonstrated that the RNA interference-based knockdown of the unconventional myosin Myo16 in cortical neurons altered growth cone filopodia density and axonal branching patterns in a TRIM9- and netrin-1-dependent manner. Future analysis of other validated candidates will likely identify novel proteins and mechanisms by which TRIM9 and TRIM67 regulate neuronal form and function. [Media: see text].
Assuntos
Proteínas do Citoesqueleto/metabolismo , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Axônios/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Feminino , Cones de Crescimento/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfogênese/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Pseudópodes/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.
Assuntos
COVID-19/imunologia , COVID-19/metabolismo , Receptores Virais , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Ciclo Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Perfilação da Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Interferon Tipo I/metabolismo , Helicase IFIH1 Induzida por Interferon/metabolismo , Modelos Biológicos , Ligação Proteica , Domínios Proteicos , Proteômica , Receptores Virais/metabolismo , SARS-CoV-2 , Serina Endopeptidases/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Internalização do Vírus , Replicação ViralRESUMO
It is increasingly clear that interindividual variability in human gut microbial composition contributes to differential drug responses. For example, gastrointestinal (GI) toxicity is not observed in all patients treated with the anticancer drug irinotecan, and it has been suggested that this variability is a result of differences in the types and levels of gut bacterial ß-glucuronidases (GUSs). GUS enzymes promote drug toxicity by hydrolyzing the inactive drug-glucuronide conjugate back to the active drug, which damages the GI epithelium. Proteomics-based identification of the exact GUS enzymes responsible for drug reactivation from the complexity of the human microbiota has not been accomplished, however. Here, we discover the specific bacterial GUS enzymes that generate SN-38, the active and toxic metabolite of irinotecan, from human fecal samples using a unique activity-based protein profiling (ABPP) platform. We identify and quantify gut bacterial GUS enzymes from human feces with an ABPP-enabled proteomics pipeline and then integrate this information with ex vivo kinetics to pinpoint the specific GUS enzymes responsible for SN-38 reactivation. Furthermore, the same approach also reveals the molecular basis for differential gut bacterial GUS inhibition observed between human fecal samples. Taken together, this work provides an unprecedented technical and bioinformatics pipeline to discover the microbial enzymes responsible for specific reactions from the complexity of human feces. Identifying such microbial enzymes may lead to precision biomarkers and novel drug targets to advance the promise of personalized medicine.
Assuntos
Proteínas de Bactérias/metabolismo , Cicloexanóis/química , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Inibidores Enzimáticos/química , Microbioma Gastrointestinal/fisiologia , Glucuronidase/metabolismo , Irinotecano/química , Animais , Biomarcadores/metabolismo , Biologia Computacional , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/microbiologia , Inibidores Enzimáticos/metabolismo , Fezes/química , Feminino , Glucuronídeos/metabolismo , Humanos , Hidrólise , Irinotecano/metabolismo , Cinética , Masculino , Metaboloma , Camundongos , Modelos Moleculares , Medicina de Precisão , Ligação Proteica , Conformação ProteicaRESUMO
The Cancer Genome Atlas catalogued alterations in the Kelch-like ECH-associated protein 1 and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway in 6.3% of patient samples across 226 studies, with significant enrichment in lung and upper airway cancers. These alterations constitutively activate NRF2-dependent gene transcription to promote many of the cancer hallmarks, including cellular resistance to oxidative stress, xenobiotic efflux, proliferation, and metabolic reprogramming. Almost universally, NRF2 activity strongly associates with poor patient prognosis and chemo- and radioresistance. Yet to date, FDA-approved drugs targeting NRF2 activity in cancer have not been realized. Here, we review various mechanisms that contribute to NRF2 activation in cancer, organized around the central dogma of molecular biology (i) at the DNA level with genomic and epigenetic alterations, (ii) at the RNA level including differential mRNA splicing and stability, and (iii) at the protein level comprising altered posttranslational modifications and protein-protein interactions. Ultimately, defining and understanding the mechanisms responsible for NRF2 activation in cancer may lead to novel targets for therapeutic intervention.
Assuntos
DNA/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Animais , DNA/genética , Epigênese Genética , Humanos , Fator 2 Relacionado a NF-E2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In its basal state, KEAP1 binds the transcription factor NRF2 (Kd = 5 nM) and promotes its degradation by ubiquitylation. Changes in the redox environment lead to modification of key cysteines within KEAP1, resulting in NRF2 protein accumulation and the transcription of genes important for restoring the cellular redox state. Using phage display and a computational loop grafting protocol, we engineered a monobody (R1) that is a potent competitive inhibitor of the KEAP1-NRF2 interaction. R1 bound to KEAP1 with a Kd of 300 pM and in human cells freed NRF2 from KEAP1 resulting in activation of the NRF2 promoter. Unlike cysteine-reactive small molecules that lack protein specificity, R1 is a genetically encoded, reversible inhibitor designed specifically for KEAP1. R1 should prove useful for studying the role of the KEAP1-NRF2 interaction in several disease states. The structure-based phage display strategy employed here is a general approach for engineering high-affinity binders that compete with naturally occurring interactions.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Modelos Moleculares , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Engenharia de Proteínas/métodos , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Técnicas de Visualização da Superfície Celular , Células HEK293 , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
NRF2 is a transcription factor that mediates stress responses. Oncogenic mutations in NRF2 localize to one of its two binding interfaces with KEAP1, an E3 ubiquitin ligase that promotes proteasome-dependent degradation of NRF2. Somatic mutations in KEAP1 occur commonly in human cancer, where KEAP1 may function as a tumor suppressor. These mutations distribute throughout the KEAP1 protein but little is known about their functional impact. In this study, we characterized 18 KEAP1 mutations defined in a lung squamous cell carcinoma tumor set. Four mutations behaved as wild-type KEAP1, thus are likely passenger events. R554Q, W544C, N469fs, P318fs, and G333C mutations attenuated binding and suppression of NRF2 activity. The remaining mutations exhibited hypomorphic suppression of NRF2, binding both NRF2 and CUL3. Proteomic analysis revealed that the R320Q, R470C, G423V, D422N, G186R, S243C, and V155F mutations augmented the binding of KEAP1 and NRF2. Intriguingly, these "super-binder" mutants exhibited reduced degradation of NRF2. Cell-based and in vitro biochemical analyses demonstrated that despite its inability to suppress NRF2 activity, the R320Q "superbinder" mutant maintained the ability to ubiquitinate NRF2. These data strengthen the genetic interactions between KEAP1 and NRF2 in cancer and provide new insight into KEAP1 mechanics.