RESUMO
The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.
Assuntos
Serina Endopeptidases , Serina Endopeptidases/metabolismo , Serina Endopeptidases/química , Humanos , Cristalografia por Raios X , Coronavirus/metabolismo , Coronavirus/química , Precursores Enzimáticos/metabolismo , Precursores Enzimáticos/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Modelos Moleculares , Ligação Proteica , Células HEK293 , Animais , Ativação Enzimática , Internalização do VírusRESUMO
Systematic functional profiling of the gene set that directs embryonic development is an important challenge. To tackle this challenge, we used 4D imaging of C. elegans embryogenesis to capture the effects of 500 gene knockdowns and developed an automated approach to compare developmental phenotypes. The automated approach quantifies features-including germ layer cell numbers, tissue position, and tissue shape-to generate temporal curves whose parameterization yields numerical phenotypic signatures. In conjunction with a new similarity metric that operates across phenotypic space, these signatures enabled the generation of ranked lists of genes predicted to have similar functions, accessible in the PhenoBank web portal, for â¼25% of essential development genes. The approach identified new gene and pathway relationships in cell fate specification and morphogenesis and highlighted the utilization of specialized energy generation pathways during embryogenesis. Collectively, the effort establishes the foundation for comprehensive analysis of the gene set that builds a multicellular organism.
Assuntos
Caenorhabditis elegans , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , FenótipoRESUMO
Induction, production, and release of proinflammatory cytokines are essential steps to establish an effective host defense. Cytokines of the interleukin-1 (IL-1) family induce inflammation and regulate T lymphocyte responses while also displaying homeostatic and metabolic activities. With the exception of the IL-1 receptor antagonist, all IL-1 family cytokines lack a signal peptide and require proteolytic processing into an active molecule. One such unique protease is caspase-1, which is activated by protein platforms called the inflammasomes. However, increasing evidence suggests that inflammasomes and caspase-1 are not the only mechanism for processing IL-1 cytokines. IL-1 cytokines are often released as precursors and require extracellular processing for activity. Here we review the inflammasome-independent enzymatic processes that are able to activate IL-1 cytokines, paying special attention to neutrophil-derived serine proteases, which subsequently induce inflammation and modulate host defense. The inflammasome-independent processing of IL-1 cytokines has important consequences for understanding inflammatory diseases, and it impacts the design of IL-1-based modulatory therapies.
Assuntos
Citocinas/metabolismo , Inflamassomos/metabolismo , Interleucina-1/metabolismo , Animais , Suscetibilidade a Doenças , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.
Assuntos
Neurônios/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Biossíntese de Proteínas , Serina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Idoso , Animais , Axônios/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células , Códon/genética , Feminino , Glicina/metabolismo , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Tecido Nervoso/patologia , Consumo de Oxigênio , Neoplasias Pancreáticas/patologia , Pirazóis , Pirimidinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/genética , RatosRESUMO
Severe asthma patients with low type 2 inflammation derive less clinical benefit from therapies targeting type 2 cytokines and represent an unmet need. We show that mast cell tryptase is elevated in severe asthma patients independent of type 2 biomarker status. Active ß-tryptase allele count correlates with blood tryptase levels, and asthma patients carrying more active alleles benefit less from anti-IgE treatment. We generated a noncompetitive inhibitory antibody against human ß-tryptase, which dissociates active tetramers into inactive monomers. A 2.15 Å crystal structure of a ß-tryptase/antibody complex coupled with biochemical studies reveal the molecular basis for allosteric destabilization of small and large interfaces required for tetramerization. This anti-tryptase antibody potently blocks tryptase enzymatic activity in a humanized mouse model, reducing IgE-mediated systemic anaphylaxis, and inhibits airway tryptase in Ascaris-sensitized cynomolgus monkeys with favorable pharmacokinetics. These data provide a foundation for developing anti-tryptase as a clinical therapy for severe asthma.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Asma/terapia , Mastócitos/enzimologia , Mastócitos/imunologia , Triptases/antagonistas & inibidores , Triptases/imunologia , Adolescente , Regulação Alostérica/imunologia , Animais , Linhagem Celular , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , CoelhosRESUMO
Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Pulmonares/patologia , Bibliotecas de Moléculas Pequenas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Família 4 do Citocromo P450/deficiência , Família 4 do Citocromo P450/genética , Descoberta de Drogas , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Glucocorticoides/farmacologia , Proteínas Facilitadoras de Transporte de Glucose/antagonistas & inibidores , Proteínas Facilitadoras de Transporte de Glucose/genética , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
Serine metabolism is involved in the fate decisions of immune cells; however, whether and how de novo serine synthesis shapes innate immune cell function remain unknown. Here, we first demonstrated that inflammatory macrophages have high expression of phosphoglycerate dehydrogenase (PHGDH, the rate-limiting enzyme of de novo serine synthesis) via nuclear factor κB signaling. Notably, the pharmacological inhibition or genetic modulation of PHGDH limits macrophage interleukin (IL)-1ß production through NAD+ accumulation and subsequent NAD+-dependent SIRT1 and SIRT3 expression and activity. Mechanistically, PHGDH not only sustains IL-1ß expression through H3K9/27 acetylation-mediated transcriptional activation of Toll-like receptor 4 but also supports IL-1ß maturation via NLRP3-K21/22/24/ASC-K21/22/24 acetylation-mediated activation of the NLRP3 inflammasome. Moreover, mice with myeloid-specific depletion of Phgdh show alleviated inflammatory responses in lipopolysaccharide-induced systemic inflammation. This study reveals a network by which a metabolic enzyme, involved in de novo serine synthesis, mediates post-translational modifications and epigenetic regulation to orchestrate IL-1ß production, providing a potential inflammatory disease target.
Assuntos
NAD , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Acetilação , Epigênese Genética , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , NAD/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Processamento de Proteína Pós-Traducional , Serina/metabolismoRESUMO
The glucagon-PKA signal is generally believed to control hepatic gluconeogenesis via the CREB transcription factor. Here we uncovered a distinct function of this signal in directly stimulating histone phosphorylation for gluconeogenic gene regulation in mice. In the fasting state, CREB recruited activated PKA to regions near gluconeogenic genes, where PKA phosphorylated histone H3 serine 28 (H3S28ph). H3S28ph, recognized by 14-3-3ζ, promoted recruitment of RNA polymerase II and transcriptional stimulation of gluconeogenic genes. In contrast, in the fed state, more PP2A was found near gluconeogenic genes, which counteracted PKA by dephosphorylating H3S28ph and repressing transcription. Importantly, ectopic expression of phosphomimic H3S28 efficiently restored gluconeogenic gene expression when liver PKA or CREB was depleted. These results together highlight a different functional scheme in regulating gluconeogenesis by the glucagon-PKA-CREB-H3S28ph cascade, in which the hormone signal is transmitted to chromatin for rapid and efficient gluconeogenic gene activation.
Assuntos
Glucagon , Gluconeogênese , Animais , Camundongos , Gluconeogênese/genética , Glucagon/metabolismo , Histonas/metabolismo , Fosforilação , Proteínas 14-3-3/metabolismo , Fígado/metabolismo , Jejum/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismoRESUMO
Non-covalent complexes of glycolytic enzymes, called metabolons, were postulated in the 1970s, but the concept has been controversial. Here we show that a c-Myc-responsive long noncoding RNA (lncRNA) that we call glycoLINC (gLINC) acts as a backbone for metabolon formation between all four glycolytic payoff phase enzymes (PGK1, PGAM1, ENO1, and PKM2) along with lactate dehydrogenase A (LDHA). The gLINC metabolon enhances glycolytic flux, increases ATP production, and enables cell survival under serine deprivation. Furthermore, gLINC overexpression in cancer cells promotes xenograft growth in mice fed a diet deprived of serine, suggesting that cancer cells employ gLINC during metabolic reprogramming. We propose that gLINC makes a functional contribution to cancer cell adaptation and provide the first example of a lncRNA-facilitated metabolon.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicólise , Proteínas de Membrana/metabolismo , Neoplasias/enzimologia , Fosfoglicerato Quinase/metabolismo , Fosfoglicerato Mutase/metabolismo , Fosfopiruvato Hidratase/metabolismo , RNA Longo não Codificante/metabolismo , Hormônios Tireóideos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Proliferação de Células , Proteínas de Ligação a DNA/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Proteínas de Membrana/genética , Camundongos Nus , Complexos Multienzimáticos , Neoplasias/genética , Neoplasias/patologia , Fosfoglicerato Quinase/genética , Fosfoglicerato Mutase/genética , Fosfopiruvato Hidratase/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Longo não Codificante/genética , Serina/deficiência , Hormônios Tireóideos/genética , Carga Tumoral , Proteínas Supressoras de Tumor/genética , Proteínas de Ligação a Hormônio da TireoideRESUMO
A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.
Assuntos
Antígeno CD47/metabolismo , Cromossomos Humanos Par 10/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Degeneração Macular/genética , Osteopontina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação/fisiologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Olho/patologia , Predisposição Genética para Doença/genética , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Humanos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Transdução de Sinais/genéticaRESUMO
Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Ciclo Celular , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleotídeos/metabolismo , Serina/metabolismo , Fator 3 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Nucleotídeos/genética , Serina/genéticaRESUMO
In tumors, nutrient availability and metabolism are known to be important modulators of growth signaling. However, it remains elusive whether cancer cells that are growing out in the metastatic niche rely on the same nutrients and metabolic pathways to activate growth signaling as cancer cells within the primary tumor. We discovered that breast-cancer-derived lung metastases, but not the corresponding primary breast tumors, use the serine biosynthesis pathway to support mTORC1 growth signaling. Mechanistically, pyruvate uptake through Mct2 supported mTORC1 signaling by fueling serine biosynthesis-derived α-ketoglutarate production in breast-cancer-derived lung metastases. Consequently, expression of the serine biosynthesis enzyme PHGDH was required for sensitivity to the mTORC1 inhibitor rapamycin in breast-cancer-derived lung tumors, but not in primary breast tumors. In summary, we provide in vivo evidence that the metabolic and nutrient requirements to activate growth signaling differ between the lung metastatic niche and the primary breast cancer site.
Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Mamárias Experimentais/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosfoglicerato Desidrogenase/genética , Serina/biossíntese , Animais , Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Ácidos Cetoglutáricos/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Fosfoglicerato Desidrogenase/metabolismo , Ácido Pirúvico/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologiaRESUMO
Cancer cells adapt their metabolism to support elevated energetic and anabolic demands of proliferation. Folate-dependent one-carbon metabolism is a critical metabolic process underpinning cellular proliferation supplying carbons for the synthesis of nucleotides incorporated into DNA and RNA. Recent research has focused on the nutrients that supply one-carbons to the folate cycle, particularly serine. Tryptophan is a theoretical source of one-carbon units through metabolism by IDO1, an enzyme intensively investigated in the context of tumor immune evasion. Using in vitro and in vivo pancreatic cancer models, we show that IDO1 expression is highly context dependent, influenced by attachment-independent growth and the canonical activator IFNγ. In IDO1-expressing cancer cells, tryptophan is a bona fide one-carbon donor for purine nucleotide synthesis in vitro and in vivo. Furthermore, we show that cancer cells release tryptophan-derived formate, which can be used by pancreatic stellate cells to support purine nucleotide synthesis.
Assuntos
Carcinoma Ductal Pancreático/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Neoplasias Pancreáticas/genética , Células Estreladas do Pâncreas/metabolismo , Evasão Tumoral/efeitos dos fármacos , Aloenxertos , Animais , Antineoplásicos/farmacologia , Carbono/imunologia , Carbono/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Formiatos/imunologia , Formiatos/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Interferon gama/genética , Interferon gama/imunologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Oximas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/mortalidade , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Serina/imunologia , Serina/metabolismo , Serina/farmacologia , Transdução de Sinais , Sulfonamidas/farmacologia , Triptofano/imunologia , Triptofano/metabolismo , Triptofano/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
Phosphorylation of α-synuclein protein at serine-129 (Ser129P) is a widely used marker for disease pathology in neurodegenerative disorders termed synucleinopathies. In groundbreaking work by Parra-Rivas, Madhivanan et al., Ser129P was shown to facilitate the normal function of α-synuclein, bearing significant implications for the transition from a physiological to pathological state.
Assuntos
Encéfalo , Neurônios , Serina , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Fosforilação , Humanos , Serina/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , AnimaisRESUMO
The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.
Assuntos
Enzimas Desubiquitinantes/metabolismo , Serina/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Células A549 , Proteínas de Bactérias/metabolismo , Domínio Catalítico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Legionella pneumophila/patogenicidade , Doença dos Legionários/metabolismo , Vacúolos/metabolismoRESUMO
Serine phosphorylations on insulin receptor substrate 1 (IRS-1) by diverse kinases aoccur widely during obesity-, stress-, and inflammation-induced conditions in models of insulin resistance and type 2 diabetes. In this study, we define a region within the human IRS-1, which is directly C-terminal to the PTB domain encompassing numerous serine phosphorylation sites including Ser307 (mouse Ser302) and Ser312 (mouse 307) creating a phosphorylation insulin resistance (PIR) domain. We demonstrate that the IRS-1 PTB-PIR with its unphosphorylated serine residues interacts with the insulin receptor (IR) but loses the IR-binding when they are phosphorylated. Surface plasmon resonance studies further confirm that the PTB-PIR binds stronger to IR than just the PTB domain, and that phosphorylations at Ser307, Ser312, Ser315, and Ser323 within the PIR domain result in abrogating the binding. Insulin-responsive cells containing the mutant IRS-1 with all these four serines changed into glutamates to mimic phosphorylations show decreased levels of phosphorylations in IR, IRS-1, and AKT compared to the wild-type IRS-1. Hydrogen-deuterium exchange mass spectrometry experiments indicating the PIR domain interacting with the N-terminal lobe and the hinge regions of the IR kinase domain further suggest the possibility that the IRS-1 PIR domain protects the IR from the PTP1B-mediated dephosphorylation.
Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Camundongos , Humanos , Animais , Fosforilação , Serina/metabolismo , Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Linhagem Celular , Fosfoproteínas/metabolismo , Insulina/metabolismoRESUMO
Delta receptors (GluD1 and GluD2), members of the large ionotropic glutamate receptor (iGluR) family, play a central role in numerous neurodevelopmental and psychiatric disorders. The amino-terminal domain (ATD) of GluD orchestrates synapse formation and maturation processes through its interaction with the Cbln family of synaptic organizers and neurexin (Nrxn). The transsynaptic triad of Nrxn-Cbln-GluD also serves as a potent regulator of synaptic plasticity, at both excitatory and inhibitory synapses. Despite these recognized functions, there is still debate as to whether GluD functions as a "canonical" ion channel, similar to other iGluRs. A recent report proposes that the ATD of GluD2 imposes conformational constraints on channel activity; removal of this constraint by binding to Cbln1 and Nrxn, or removal of the ATD, reveals channel activity in GluD2 upon administration of glycine (Gly) and d-serine (d-Ser), two GluD ligands. We were able to reproduce currents when Gly or d-Ser was administered to clusters of heterologous human embryonic kidney 293 (HEK293) cells expressing Cbln1, GluD2 (or GluD1), and Nrxn. However, Gly or d-Ser, but also l-glutamate (l-Glu), evoked similar currents in naive (i.e., untransfected) HEK293 cells and in GluD2-null Purkinje neurons. Furthermore, no current was detected in isolated HEK293 cells expressing GluD2 lacking the ATD upon administration of Gly. Taken together, these results cast doubt on the previously proposed hypothesis that extracellular ligands directly gate wild-type GluD channels.
Assuntos
Ativação do Canal Iônico , Receptores de Glutamato , Animais , Humanos , Camundongos , Glicina/metabolismo , Células HEK293 , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos de Abertura Ativada por Ligante/metabolismo , Canais Iônicos de Abertura Ativada por Ligante/genética , Ligantes , Receptores de Glutamato/metabolismo , Serina/metabolismoRESUMO
The human malaria parasite Plasmodium falciparum requires exogenous fatty acids to support its growth during the pathogenic, asexual erythrocytic stage. Host serum lysophosphatidylcholine (LPC) is a significant fatty acid source, yet the metabolic processes responsible for the liberation of free fatty acids from exogenous LPC are unknown. Using an assay for LPC hydrolysis in P. falciparum-infected erythrocytes, we have identified small-molecule inhibitors of key in situ lysophospholipase activities. Competitive activity-based profiling and generation of a panel of single-to-quadruple knockout parasite lines revealed that two enzymes of the serine hydrolase superfamily, termed exported lipase (XL) 2 and exported lipase homolog (XLH) 4, constitute the dominant lysophospholipase activities in parasite-infected erythrocytes. The parasite ensures efficient exogenous LPC hydrolysis by directing these two enzymes to distinct locations: XL2 is exported to the erythrocyte, while XLH4 is retained within the parasite. While XL2 and XLH4 were individually dispensable with little effect on LPC hydrolysis in situ, loss of both enzymes resulted in a strong reduction in fatty acid scavenging from LPC, hyperproduction of phosphatidylcholine, and an enhanced sensitivity to LPC toxicity. Notably, growth of XL/XLH-deficient parasites was severely impaired when cultured in media containing LPC as the sole exogenous fatty acid source. Furthermore, when XL2 and XLH4 activities were ablated by genetic or pharmacologic means, parasites were unable to proliferate in human serum, a physiologically relevant fatty acid source, revealing the essentiality of LPC hydrolysis in the host environment and its potential as a target for anti-malarial therapy.
Assuntos
Malária Falciparum , Parasitos , Animais , Humanos , Plasmodium falciparum , Lisofosfatidilcolinas/metabolismo , Lisofosfolipase/genética , Lisofosfolipase/metabolismo , Malária Falciparum/parasitologia , Eritrócitos/metabolismo , Parasitos/metabolismo , Ácidos Graxos/metabolismo , Lipase/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
Congenital tufting enteropathy (CTE) is a life-threatening intestinal disorder resulting from loss-of-function mutations in EPCAM and SPINT2. Mice deficient in Spint2, encoding the protease inhibitor HAI-2, develop CTE-like intestinal failure associated with a progressive loss of the EpCAM protein, which is caused by unchecked activity of the serine protease matriptase (ST14). Here, we show that loss of HAI-2 leads to increased proteolytic processing of EpCAM. Elimination of the reported matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of cleavage-resistant EpCAM failed to prevent intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase (St14) counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM, indicating that matriptase does not drive intestinal dysfunction by excessive proteolysis of EpCAM. Interestingly, mice expressing cleavage-resistant EpCAM developed late-onset intestinal defects and exhibited a shortened lifespan even in the presence of HAI-2, suggesting that EpCAM cleavage is indispensable for EpCAM function. Our findings provide new insights into the role of EpCAM and the etiology of the enteropathies driven by Spint2 deficiency.
Assuntos
Insuficiência Intestinal , Animais , Camundongos , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Intestinos , Proteínas Secretadas Inibidoras de ProteinasesRESUMO
BACKGROUND: Cerebral vascular malformations (CCMs) are primarily found within the brain, where they result in increased risk for stroke, seizures, and focal neurological deficits. The unique feature of the brain vasculature is the blood-brain barrier formed by the brain neurovascular unit. Recent studies suggest that loss of CCM genes causes disruptions of blood-brain barrier integrity as the inciting events for CCM development. CCM lesions are proposed to be initially derived from a single clonal expansion of a subset of angiogenic venous capillary endothelial cells (ECs) and respective resident endothelial progenitor cells (EPCs). However, the critical signaling events in the subclass of brain ECs/EPCs for CCM lesion initiation and progression are unclear. METHODS: Brain EC-specific CCM3-deficient (Pdcd10BECKO) mice were generated by crossing Pdcd10fl/fl mice with Mfsd2a-CreERT2 mice. Single-cell RNA-sequencing analyses were performed by the chromium single-cell platform (10× genomics). Cell clusters were annotated into EC subtypes based on visual inspection and GO analyses. Cerebral vessels were visualized by 2-photon in vivo imaging and tissue immunofluorescence analyses. Regulation of mTOR (mechanistic target of rapamycin) signaling by CCM3 and Cav1 (caveolin-1) was performed by cell biology and biochemical approaches. RESULTS: Single-cell RNA-sequencing analyses from P10 Pdcd10BECKO mice harboring visible CCM lesions identified upregulated CCM lesion signature and mitotic EC clusters but decreased blood-brain barrier-associated EC clusters. However, a unique EPC cluster with high expression levels of stem cell markers enriched with mTOR signaling was identified from early stages of the P6 Pdcd10BECKO brain. Indeed, mTOR signaling was upregulated in both mouse and human CCM lesions. Genetic deficiency of Raptor (regulatory-associated protein of mTOR), but not of Rictor (rapamycin-insensitive companion of mTOR), prevented CCM lesion formation in the Pdcd10BECKO model. Importantly, the mTORC1 (mTOR complex 1) pharmacological inhibitor rapamycin suppressed EPC proliferation and ameliorated CCM pathogenesis in Pdcd10BECKO mice. Mechanistic studies suggested that Cav1/caveolae increased in CCM3-depleted EPC-mediated intracellular trafficking and complex formation of the mTORC1 signaling proteins. CONCLUSIONS: CCM3 is critical for maintaining blood-brain barrier integrity and CCM3 loss-induced mTORC1 signaling in brain EPCs initiates and facilitates CCM pathogenesis.