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INTRODUCTION: Triggering receptor expressed on myeloid cells 2 (TREM2) agonists are being clinically evaluated as disease-modifying therapeutics for Alzheimer's disease. Clinically translatable pharmacodynamic (PD) biomarkers are needed to confirm drug activity and select the appropriate therapeutic dose in clinical trials. METHODS: We conducted multi-omic analyses on paired non-human primate brain and cerebrospinal fluid (CSF), and stimulation of human induced pluripotent stem cell-derived microglia cultures after TREM2 agonist treatment, followed by validation of candidate fluid PD biomarkers using immunoassays. We immunostained microglia to characterize proliferation and clustering. RESULTS: We report CSF soluble TREM2 (sTREM2) and CSF chitinase-3-like protein 1 (CHI3L1/YKL-40) as PD biomarkers for the TREM2 agonist hPara.09. The respective reduction of sTREM2 and elevation of CHI3L1 in brain and CSF after TREM2 agonist treatment correlated with transient microglia proliferation and clustering. DISCUSSION: CSF CHI3L1 and sTREM2 reflect microglial TREM2 agonism and can be used as clinical PD biomarkers to monitor TREM2 activity in the brain. HIGHLIGHTS: CSF soluble triggering receptor expressed on myeloid cells 2 (sTREM2) reflects brain target engagement for a novel TREM2 agonist, hPara.09. CSF chitinase-3-like protein 1 reflects microglial TREM2 agonism. Both can be used as clinical fluid biomarkers to monitor TREM2 activity in brain.
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Mass spectrometry-based single-cell proteomics has undergone rapid progress and has become an active research area. However, because of the ultralow amount of proteins in single cells, it is still highly challenging to achieve efficient sample preparation and sensitive LC-MS detection. Here, we provide a detailed protocol for isobaric labeling-based single-cell proteomics relying on a microfluidic droplet-based sample processing technology. The protocol allows for processing both single cells and carrier samples in separate microchips using a commercially available platform (cellenONE) with high sample recovery and high throughput. We also provide an optimized LC-MS method for sensitive and robust data collection.
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Proteômica , Análise de Célula Única , Proteômica/métodos , Análise de Célula Única/métodos , Análise de Célula Única/instrumentação , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Microfluídica/instrumentação , Dispositivos Lab-On-A-ChipRESUMO
Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor.
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Proteínas do Domínio Armadillo , Córtex Cerebral , Proteínas do Citoesqueleto , Células-Tronco Pluripotentes Induzidas , Neurônios , Proteínas do Domínio Armadillo/metabolismo , Proteínas do Domínio Armadillo/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Masculino , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Degeneração Neural/patologia , Degeneração Neural/metabolismo , Degeneração Neural/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Estresse Fisiológico/fisiologia , Axônios/metabolismo , Axônios/patologia , Mitocôndrias/metabolismoRESUMO
The identification of VHL-binding proteolysis targeting chimeras (PROTACs) that potently degrade the BRM protein (also known as SMARCA2) in SW1573 cell-based experiments is described. These molecules exhibit between 10- and 100-fold degradation selectivity for BRM over the closely related paralog protein BRG1 (SMARCA4). They also selectively impair the proliferation of the H1944 "BRG1-mutant" NSCLC cell line, which lacks functional BRG1 protein and is thus highly dependent on BRM for growth, relative to the wild-type Calu6 line. In vivo experiments performed with a subset of compounds identified PROTACs that potently and selectively degraded BRM in the Calu6 and/or the HCC2302 BRG1 mutant NSCLC xenograft models and also afforded antitumor efficacy in the latter system. Subsequent PK/PD analysis established a need to achieve strong BRM degradation (>95%) in order to trigger meaningful antitumor activity in vivo. Intratumor quantitation of mRNA associated with two genes whose transcription was controlled by BRM (PLAU and KRT80) also supported this conclusion.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Quimera de Direcionamento de Proteólise , Xenoenxertos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , DNA Helicases/genética , Proteínas Nucleares/genéticaRESUMO
Argonaute (AGO) proteins execute microRNA (miRNA)-mediated gene silencing. However, it is unclear whether all 4 mammalian AGO proteins (AGO1, AGO2, AGO3, and AGO4) are required for miRNA activity. We generate Ago1, Ago3, and Ago4-deficient mice (Ago134Δ) and find AGO1/3/4 to be redundant for miRNA biogenesis, homeostasis, or function, a role that is carried out by AGO2. Instead, AGO1/3/4 regulate the expansion of type 2 immunity via precursor mRNA splicing in CD4+ T helper (Th) lymphocytes. Gain- and loss-of-function experiments demonstrate that nuclear AGO3 interacts directly with SF3B3, a component of the U2 spliceosome complex, to aid global mRNA splicing, and in particular the isoforms of the gene Nisch, resulting in a dysregulated Nisch isoform ratio. This work uncouples AGO1, AGO3, and AGO4 from miRNA-mediated RNA interference, identifies an AGO3:SF3B3 complex in the nucleus, and reveals a mechanism by which AGO proteins regulate inflammatory diseases.
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MicroRNAs , Precursores de RNA , Animais , Camundongos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Receptores de Imidazolinas/genética , Receptores de Imidazolinas/metabolismo , Mamíferos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Microglial reactivity is a pathological hallmark in many neurodegenerative diseases. During stimulation, microglia undergo complex morphological changes, including loss of their characteristic ramified morphology, which is routinely used to detect and quantify inflammation in the brain. However, the underlying molecular mechanisms and the relation between microglial morphology and their pathophysiological function are unknown. Here, proteomic profiling of lipopolysaccharide (LPS)-reactive microglia identifies microtubule remodeling pathways as an early factor that drives the morphological change and subsequently controls cytokine responses. We find that LPS-reactive microglia reorganize their microtubules to form a stable and centrosomally-anchored array to facilitate efficient cytokine trafficking and release. We identify cyclin-dependent kinase 1 (Cdk-1) as a critical upstream regulator of microtubule remodeling and morphological change in-vitro and in-situ. Cdk-1 inhibition also rescues tau and amyloid fibril-induced morphology changes. These results demonstrate a critical role for microtubule dynamics and reorganization in microglial reactivity and modulating cytokine-mediated inflammatory responses.
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Citocinas , Microglia , Citocinas/metabolismo , Microglia/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Proteômica , Microtúbulos/metabolismoRESUMO
The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.
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Neoplasias , Animais , Humanos , Proteólise , Neoplasias/genética , Mutação , Mamíferos , Fatores de Transcrição/genética , DNA Helicases/genética , Proteínas Nucleares/genéticaRESUMO
The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.
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Proteínas de Bactérias/metabolismo , Disenteria Bacilar/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Bactérias/genética , Disenteria Bacilar/genética , Disenteria Bacilar/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteólise , Shigella flexneri/genética , Shigella flexneri/fisiologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Single-cell proteomics by mass spectrometry (SCoPE-MS) is a recently introduced method to quantify multiplexed single-cell proteomes. While this technique has generated great excitement, the underlying technologies (isobaric labeling and mass spectrometry (MS)) have technical limitations with the potential to affect data quality and biological interpretation. These limitations are particularly relevant when a carrier proteome, a sample added at 25-500× the amount of a single-cell proteome, is used to enable peptide identifications. Here we perform controlled experiments with increasing carrier proteome amounts and evaluate quantitative accuracy, as it relates to mass analyzer dynamic range, multiplexing level and number of ions sampled. We demonstrate that an increase in carrier proteome level requires a concomitant increase in the number of ions sampled to maintain quantitative accuracy. Lastly, we introduce Single-Cell Proteomics Companion (SCPCompanion), a software tool that enables rapid evaluation of single-cell proteomic data and recommends instrument and data analysis parameters for improved data quality.
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Fragmentos de Peptídeos/análise , Proteoma/análise , Proteômica/métodos , Análise de Célula Única/métodos , Software , Espectrometria de Massas em Tandem/métodos , Células HeLa , Humanos , Células K562RESUMO
Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.
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Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Tioléster Hidrolases/genética , Fatores de Tempo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Acquisition of drug resistance remains a chief impediment to successful cancer therapy, and we previously described a transient drug-tolerant cancer cell population (DTPs) whose survival is in part dependent on the activities of the histone methyltransferases G9a/EHMT2 and EZH2, the latter being the catalytic component of the polycomb repressive complex 2 (PRC2). Here, we apply multiple proteomic techniques to better understand the role of these histone methyltransferases (HMTs) in the establishment of the DTP state. Proteome-wide comparisons of lysine methylation patterns reveal that DTPs display an increase in methylation on K116 of PRC member Jarid2, an event that helps stabilize and recruit PRC2 to chromatin. We also find that EZH2, in addition to methylating histone H3K27, also can methylate G9a at K185, and that methylated G9a better recruits repressive complexes to chromatin. These complexes are similar to complexes recruited by histone H3 methylated at K9. Finally, a detailed histone post-translational modification (PTM) analysis shows that EZH2, either directly or through its ability to methylate G9a, alters H3K9 methylation in the context of H3 serine 10 phosphorylation, primarily in a cancer cell subpopulation that serves as DTP precursors. We also show that combinations of histone PTMs recruit a different set of complexes to chromatin, shedding light on the temporal mechanisms that contribute to drug tolerance.
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Neoplasias , Proteômica , Tolerância a Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Histonas/metabolismo , Metilação , Complexo Repressor Polycomb 2/genética , Complexo Repressor Polycomb 2/metabolismoRESUMO
The H3K4 demethylase KDM5B is amplified and overexpressed in luminal breast cancer, suggesting it might constitute a potential cancer therapy target. Here, we characterize, in breast cancer cells, the molecular effects of a recently developed small-molecule inhibitor of the KDM5 family of proteins (KDM5i), either alone or in combination with the DNA-demethylating agent 5-aza-2'-deoxycytidine (DAC). KDM5i treatment alone increased expression of a small number of genes, whereas combined treatment with DAC enhanced the effects of the latter for increasing expression of hundreds of DAC-responsive genes. ChIP-seq studies revealed that KDM5i resulted in the broadening of existing H3K4me3 peaks. Furthermore, cells treated with the drug combination exhibited increased promoter and gene body H3K4me3 occupancy at DAC-responsive genes compared with DAC alone. Importantly, treatment with either DAC or DAC+KDM5i induced a dramatic increase in H3K27ac at enhancers with an associated significant increase in target gene expression, suggesting a previously unappreciated effect of DAC on transcriptional regulation. KDM5i synergized with DAC to reduce the viability of luminal breast cancer cells in in vitro assays. Our study provides the first look into the molecular effects of a novel KDM5i compound and suggests that combinatorial inhibition along with DAC represents a new area to explore in translational epigenetics.Significance: This study offers a first look into the molecular effects of a novel KDM5 inhibitory compound, suggesting how its use in combination with DNA methylation inhibitors presents new opportunities to explore in translational cancer epigenetics. Cancer Res; 78(5); 1127-39. ©2017 AACR.
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Neoplasias da Mama/tratamento farmacológico , Metilação de DNA , Decitabina/farmacologia , Inibidores Enzimáticos/farmacologia , Genoma Humano , Histonas/química , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Proteínas Repressoras/antagonistas & inibidores , Apoptose , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proliferação de Células , Epigênese Genética , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Células Tumorais CultivadasRESUMO
Antibody-drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027-37. ©2017 AACR.
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Anticorpos Monoclonais/metabolismo , Antineoplásicos/metabolismo , Catepsina B/fisiologia , Imunoconjugados/metabolismo , Pró-Fármacos/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Catepsina B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citrulina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Imunoconjugados/uso terapêutico , Oligopeptídeos , Pró-Fármacos/uso terapêutico , Proteólise , Valina/metabolismoRESUMO
A propensity for rewiring genetic and epigenetic regulatory networks, thus enabling sustained cell proliferation, suppression of apoptosis, and the ability to evade the immune system, is vital to cancer cell propagation. An increased understanding of how this is achieved is critical for identifying or improving therapeutic interventions. In this study, using acute myeloid leukemia (AML) human cell lines and a custom CRISPR/Cas9 screening platform, we identify the H3K9 methyltransferase SETDB1 as a novel, negative regulator of innate immunity. SETDB1 is overexpressed in many cancers, and loss of this gene in AML cells triggers desilencing of retrotransposable elements that leads to the production of double-stranded RNAs (dsRNAs). This is coincident with induction of a type I interferon response and apoptosis through the dsRNA-sensing pathway. Collectively, our findings establish a unique gene regulatory axis that cancer cells can exploit to circumvent the immune system.
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Inativação Gênica , Interferon Tipo I/metabolismo , Leucemia Mieloide Aguda/enzimologia , Proteínas Metiltransferases/metabolismo , Retroelementos , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Sobrevivência Celular , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase , Humanos , Imunidade Inata , Interferon Tipo I/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Proteínas Metiltransferases/genética , Interferência de RNA , RNA de Cadeia Dupla/biossíntese , RNA de Cadeia Dupla/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Evasão TumoralRESUMO
UNLABELLED: The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.
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Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Expressão Gênica , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Seleção Genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , VirulênciaRESUMO
The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.
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Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Proteína 2 de Ligação ao Retinoblastoma/antagonistas & inibidores , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Relação Estrutura-AtividadeRESUMO
Manganese plays a central role in cellular detoxification of reactive oxygen species (ROS). Therefore, manganese acquisition is considered to be important for bacterial pathogenesis by counteracting the oxidative burst of phagocytic cells during host infection. However, detailed analysis of the interplay between bacterial manganese acquisition and phagocytic cells and its impact on bacterial pathogenesis has remained elusive for Staphylococcus aureus, a major human pathogen. Here, we show that a mntC mutant, which lacks the functional manganese transporter MntABC, was more sensitive to killing by human neutrophils but not murine macrophages, unless the mntC mutant was pre-exposed to oxidative stress. Notably, the mntC mutant formed strikingly small colonies when recovered from both type of phagocytic cells. We show that this phenotype is a direct consequence of the inability of the mntC mutant to reinitiate growth after exposure to phagocytic oxidative burst. Transcript and quantitative proteomics analyses revealed that the manganese-dependent ribonucleotide reductase complex NrdEF, which is essential for DNA synthesis and repair, was highly induced in the mntC mutant under oxidative stress conditions including after phagocytosis. Since NrdEF proteins are essential for S. aureus viability we hypothesize that cells lacking MntABC might attempt to compensate for the impaired function of NrdEF by increasing their expression. Our data suggest that besides ROS detoxification, functional manganese acquisition is likely crucial for S. aureus pathogenesis by repairing oxidative damages, thereby ensuring efficient bacterial growth after phagocytic oxidative burst, which is an attribute critical for disseminating and establishing infection in the host.
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Proteínas de Bactérias/genética , Replicação do DNA/genética , Manganês/metabolismo , Proteínas de Membrana Transportadoras/genética , Estresse Oxidativo/genética , Explosão Respiratória/genética , Staphylococcus aureus/genética , Animais , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Macrófagos/microbiologia , Camundongos , Neutrófilos/microbiologia , Fagocitose/genética , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/metabolismoRESUMO
Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as 'non-self' neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, â¼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide-MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.
Assuntos
Exoma/genética , Fenômenos Imunogenéticos/genética , Espectrometria de Massas , Mutação , Neoplasias/genética , Animais , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Imunidade Celular/imunologia , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Neoplasias/imunologia , Peptídeos/genética , Estrutura Terciária de ProteínaRESUMO
Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the ß-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.
Assuntos
Anticorpos Neutralizantes/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Evasão da Resposta Imune , Polissacarídeos/imunologia , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Hepacivirus/química , Hepacivirus/genética , Sequenciamento de Nucleotídeos em Larga Escala , Polissacarídeos/metabolismo , Conformação Proteica , RNA Viral/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.