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1.
Cell ; 184(20): 5163-5178.e24, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34559985

RESUMO

Rift Valley fever virus (RVFV) is a zoonotic pathogen with pandemic potential. RVFV entry is mediated by the viral glycoprotein (Gn), but host entry factors remain poorly defined. Our genome-wide CRISPR screen identified low-density lipoprotein receptor-related protein 1 (mouse Lrp1/human LRP1), heat shock protein (Grp94), and receptor-associated protein (RAP) as critical host factors for RVFV infection. RVFV Gn directly binds to specific Lrp1 clusters and is glycosylation independent. Exogenous addition of murine RAP domain 3 (mRAPD3) and anti-Lrp1 antibodies neutralizes RVFV infection in taxonomically diverse cell lines. Mice treated with mRAPD3 and infected with pathogenic RVFV are protected from disease and death. A mutant mRAPD3 that binds Lrp1 weakly failed to protect from RVFV infection. Together, these data support Lrp1 as a host entry factor for RVFV infection and define a new target to limit RVFV infections.


Assuntos
Interações Hospedeiro-Patógeno , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Vírus da Febre do Vale do Rift/fisiologia , Internalização do Vírus , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Encéfalo/patologia , Encéfalo/virologia , Sistemas CRISPR-Cas/genética , Membrana Celular/metabolismo , Células Cultivadas , Glicoproteínas/metabolismo , Glicosaminoglicanos/metabolismo , Glicosilação , Humanos , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Ligantes , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/deficiência , Glicoproteínas de Membrana/metabolismo , Camundongos , Ligação Proteica , Desnaturação Proteica , Febre do Vale de Rift/patologia , Febre do Vale de Rift/prevenção & controle , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/imunologia
2.
Nat Immunol ; 23(7): 1031-1041, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35761082

RESUMO

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.


Assuntos
Antígenos CD/metabolismo , Ativação Linfocitária , Animais , Anticorpos , Fibrinogênio , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunoterapia , Ligantes , Camundongos , Receptores Imunológicos , Proteína do Gene 3 de Ativação de Linfócitos
3.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33649232

RESUMO

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.


Assuntos
Proteína DEAD-box 58 , Helicase IFIH1 Induzida por Interferon , Interferon beta , Receptores Imunológicos , Proteínas não Estruturais Virais , Cristalografia por Raios X , Proteína DEAD-box 58/química , Proteína DEAD-box 58/metabolismo , Medição da Troca de Deutério , Células HEK293 , Humanos , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/metabolismo , Interferon beta/química , Interferon beta/metabolismo , Ligação Proteica , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo
4.
Cells ; 13(12)2024 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-38920634

RESUMO

BACKGROUND: Identifying cells engaged in fundamental cellular processes, such as proliferation or living/death statuses, is pivotal across numerous research fields. However, prevailing methods relying on molecular biomarkers are constrained by high costs, limited specificity, protracted sample preparation, and reliance on fluorescence imaging. METHODS: Based on cellular morphology in phase contrast images, we developed a deep-learning model named Detector of Mitosis, Apoptosis, Interphase, Necrosis, and Senescence (D-MAINS). RESULTS: D-MAINS utilizes machine learning and image processing techniques, enabling swift and label-free categorization of cell death, division, and senescence at a single-cell resolution. Impressively, D-MAINS achieved an accuracy of 96.4 ± 0.5% and was validated with established molecular biomarkers. D-MAINS underwent rigorous testing under varied conditions not initially present in the training dataset. It demonstrated proficiency across diverse scenarios, encompassing additional cell lines, drug treatments, and distinct microscopes with different objective lenses and magnifications, affirming the robustness and adaptability of D-MAINS across multiple experimental setups. CONCLUSIONS: D-MAINS is an example showcasing the feasibility of a low-cost, rapid, and label-free methodology for distinguishing various cellular states. Its versatility makes it a promising tool applicable across a broad spectrum of biomedical research contexts, particularly in cell death and oncology studies.


Assuntos
Apoptose , Senescência Celular , Aprendizado Profundo , Interfase , Mitose , Necrose , Humanos , Linhagem Celular Tumoral , Neoplasias/patologia , Neoplasias/metabolismo , Processamento de Imagem Assistida por Computador/métodos
5.
Cancer Res Commun ; 4(5): 1174-1188, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38626341

RESUMO

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in approximately 50% of all human cancers. In its canonical role, p16 inhibits the G1-S-phase cell cycle progression through suppression of cyclin-dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. However, the broader impact of p16/CDKN2A loss on other nucleotide metabolic pathways and potential therapeutic targets remains unexplored. Using CRISPR knockout libraries in isogenic human and mouse melanoma cell lines, we determined several nucleotide metabolism genes essential for the survival of cells with loss of p16/CDKN2A. Consistently, many of these genes are upregulated in melanoma cells with p16 knockdown or endogenously low CDKN2A expression. We determined that cells with low p16/CDKN2A expression are sensitive to multiple inhibitors of de novo purine synthesis, including antifolates. Finally, tumors with p16 knockdown were more sensitive to the antifolate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2Alow tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents. SIGNIFICANCE: Antimetabolites were the first chemotherapies, yet many have failed in the clinic due to toxicity and poor patient selection. Our data suggest that p16 loss provides a therapeutic window to kill cancer cells with widely-used antifolates with relatively little toxicity.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina , Purinas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Metotrexato/farmacologia , Purinas/metabolismo , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico
6.
bioRxiv ; 2024 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-38370789

RESUMO

Homologous recombination (HR) deficiency enhances sensitivity to DNA damaging agents commonly used to treat cancer. In HR-proficient cancers, metabolic mechanisms driving response or resistance to DNA damaging agents remain unclear. Here we identified that depletion of alpha-ketoglutarate (αKG) sensitizes HR-proficient cells to DNA damaging agents by metabolic regulation of histone acetylation. αKG is required for the activity of αKG-dependent dioxygenases (αKGDDs), and prior work has shown that changes in αKGDD affect demethylases. Using a targeted CRISPR knockout library consisting of 64 αKGDDs, we discovered that Trimethyllysine Hydroxylase Epsilon (TMLHE), the first and rate-limiting enzyme in de novo carnitine synthesis, is necessary for proliferation of HR-proficient cells in the presence of DNA damaging agents. Unexpectedly, αKG-mediated TMLHE-dependent carnitine synthesis was required for histone acetylation, while histone methylation was affected but dispensable. The increase in histone acetylation via αKG-dependent carnitine synthesis promoted HR-mediated DNA repair through site- and substrate-specific histone acetylation. These data demonstrate for the first time that HR-proficiency is mediated through αKG directly influencing histone acetylation via carnitine synthesis and provide a metabolic avenue to induce HR-deficiency and sensitivity to DNA damaging agents.

7.
bioRxiv ; 2023 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-37961201

RESUMO

DNA damage and cellular metabolism are intricately linked with bidirectional feedback. Two of the main effectors of the DNA damage response and control of cellular metabolism are ATR and mTORC1, respectively. Prior work has placed ATR upstream of mTORC1 during replication stress, yet the direct mechanism for how mTORC1 is activated in this context remain unclear. We previously published that p16-low cells have mTORC1 hyperactivation, which in part promotes their proliferation. Using this model, we found that ATR, but not ATM, is upstream of mTORC1 activation via de novo cholesterol synthesis and is associated with increased lanosterol synthase (LSS). Indeed, p16-low cells showed increased cholesterol abundance. Additionally, knockdown of either ATR or LSS decreased mTORC1 activity. Decreased mTORC1 activity due to ATR knockdown was rescued by cholesterol supplementation. Finally, using both LSS inhibitors and multiple FDA-approved de novo cholesterol synthesis inhibitors, we found that the de novo cholesterol biosynthesis pathway is a metabolic vulnerability of p16-low cells. Together, our data provide new evidence coupling the DNA damage response and cholesterol metabolism and demonstrate the feasibility of using FDA-approved cholesterol-lowering drugs in tumors with loss of p16.

8.
J Cell Biol ; 222(1)2023 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-36399181

RESUMO

Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Neoplasias , Pinocitose , Humanos , Adaptação Fisiológica , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reprogramação Celular , Neoplasias/metabolismo , Microambiente Tumoral , Aminoácidos de Cadeia Ramificada/metabolismo , Metabolômica , Animais , Camundongos , Linhagem Celular Tumoral
9.
bioRxiv ; 2023 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-37503050

RESUMO

p16 is a tumor suppressor encoded by the CDKN2A gene whose expression is lost in ~50% of all human cancers. In its canonical role, p16 inhibits the G1-S phase cell cycle progression through suppression of cyclin dependent kinases. Interestingly, p16 also has roles in metabolic reprogramming, and we previously published that loss of p16 promotes nucleotide synthesis via the pentose phosphate pathway. Whether other nucleotide metabolic genes and pathways are affected by p16/CDKN2A loss and if these can be specifically targeted in p16/CDKN2A-low tumors has not been previously explored. Using CRISPR KO libraries in multiple isogenic human and mouse melanoma cell lines, we determined that many nucleotide metabolism genes are negatively enriched in p16/CDKN2A knockdown cells compared to controls. Indeed, many of the genes that are required for survival in the context of low p16/CDKN2A expression based on our CRISPR screens are upregulated in p16 knockdown melanoma cells and those with endogenously low CDKN2A expression. We determined that cells with low p16/Cdkn2a expression are sensitive to multiple inhibitors of de novo purine synthesis, including anti-folates. Tumors with p16 knockdown were more sensitive to the anti-folate methotrexate in vivo than control tumors. Together, our data provide evidence to reevaluate the utility of these drugs in patients with p16/CDKN2A-low tumors as loss of p16/CDKN2A may provide a therapeutic window for these agents.

10.
iScience ; 24(6): 102681, 2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34095780

RESUMO

Nucleocapsid (N) encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays key roles in the replication cycle and is a critical serological marker. Here, we characterize essential biochemical properties of N and describe the utility of these insights in serological studies. We define N domains important for oligomerization and RNA binding and show that N oligomerization provides a high-affinity RNA-binding platform. We also map the RNA-binding interface, showing protection in the N-terminal domain and linker region. In addition, phosphorylation causes reduction of RNA binding and redistribution of N from liquid droplets to loose coils, showing how N-RNA accessibility and assembly may be regulated by phosphorylation. Finally, we find that the C-terminal domain of N is the most immunogenic, based on antibody binding to patient samples. Together, we provide a biochemical description of SARS-CoV-2 N and highlight the value of using N domains as highly specific and sensitive diagnostic markers.

11.
STAR Protoc ; 2(4): 100906, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34642671

RESUMO

Nucleocapsid proteins are essential for SARS-CoV-2 life cycle. Here, we describe protocols to gather domain-specific insights about essential properties of nucleocapsids. These assays include dynamic light scattering to characterize oligomerization, fluorescence polarization to quantify RNA binding, hydrogen-deuterium exchange mass spectrometry to map RNA binding regions, negative-stain electron microscopy to visualize oligomeric species, interferon reporter assay to evaluate interferon signaling modulation, and a serology assay to reveal insights for improved sensitivity and specificity. These assays are broadly applicable to RNA-encapsidated nucleocapsids. For complete details on the use and execution of this protocol, please refer to Wu et al. (2021).


Assuntos
COVID-19/sangue , Proteínas do Nucleocapsídeo de Coronavírus/sangue , Interferons/metabolismo , Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/isolamento & purificação , Antivirais/metabolismo , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Nucleocapsídeo/genética , Fosfoproteínas/sangue , Fosfoproteínas/genética , Ligação Proteica , RNA Viral/genética
12.
bioRxiv ; 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-33269347

RESUMO

Nucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

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