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1.
EMBO Rep ; 23(6): e53890, 2022 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-35438230

RESUMO

Aggregation of the multifunctional RNA-binding protein TDP-43 defines large subgroups of amyotrophic lateral sclerosis and frontotemporal dementia and correlates with neurodegeneration in both diseases. In disease, characteristic C-terminal fragments of ~25 kDa ("TDP-25") accumulate in cytoplasmic inclusions. Here, we analyze gain-of-function mechanisms of TDP-25 combining cryo-electron tomography, proteomics, and functional assays. In neurons, cytoplasmic TDP-25 inclusions are amorphous, and photobleaching experiments reveal gel-like biophysical properties that are less dynamic than nuclear TDP-43. Compared with full-length TDP-43, the TDP-25 interactome is depleted of low-complexity domain proteins. TDP-25 inclusions are enriched in 26S proteasomes adopting exclusively substrate-processing conformations, suggesting that inclusions sequester proteasomes, which are largely stalled and no longer undergo the cyclic conformational changes required for proteolytic activity. Reporter assays confirm that TDP-25 impairs proteostasis, and this inhibitory function is enhanced by ALS-causing TDP-43 mutations. These findings support a patho-physiological relevance of proteasome dysfunction in ALS/FTD.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas de Ligação a DNA , Demência Frontotemporal , Neurônios , Fragmentos de Peptídeos , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
2.
Nat Rev Drug Discov ; 2022 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-35351998

RESUMO

Proteins are the main targets of most drugs; however, system-wide methods to monitor protein activity and function are still underused in drug discovery. Novel biochemical approaches, in combination with recent developments in mass spectrometry-based proteomics instrumentation and data analysis pipelines, have now enabled the dissection of disease phenotypes and their modulation by bioactive molecules at unprecedented resolution and dimensionality. In this Review, we describe proteomics and chemoproteomics approaches for target identification and validation, as well as for identification of safety hazards. We discuss innovative strategies in early-stage drug discovery in which proteomics approaches generate unique insights, such as targeted protein degradation and the use of reactive fragments, and provide guidance for experimental strategies crucial for success.

3.
Nat Cancer ; 3(3): 318-336, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35122074

RESUMO

KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted and immunotherapies. Among the different PDAC subtypes, basal-like mesenchymal PDAC, which is driven by allelic imbalance, increased gene dosage and subsequent high expression levels of oncogenic KRAS, shows the most aggressive phenotype and strongest therapy resistance. In the present study, we performed a systematic high-throughput combination drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib, which targets KRAS-directed oncogenic signaling in mesenchymal PDAC. This combination treatment induces cell-cycle arrest and cell death, and initiates a context-dependent remodeling of the immunosuppressive cancer cell secretome. Using a combination of single-cell RNA-sequencing, CRISPR screens and immunophenotyping, we show that this combination therapy promotes intratumor infiltration of cytotoxic and effector T cells, which sensitizes mesenchymal PDAC to PD-L1 immune checkpoint inhibition. Overall, our results open new avenues to target this aggressive and therapy-refractory mesenchymal PDAC subtype.


Assuntos
Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Adenocarcinoma/tratamento farmacológico , Carcinoma Ductal Pancreático/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente Tumoral
4.
J Clin Invest ; 132(3)2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-34847081

RESUMO

Ulcerating skin lesions are manifestations of human ISG15 deficiency, a type I interferonopathy. However, chronic inflammation may not be their exclusive cause. We describe two siblings with recurrent skin ulcers that healed with scar formation upon corticosteroid treatment. Both had a homozygous nonsense mutation in the ISG15 gene, leading to unstable ISG15 protein lacking the functional domain. We characterized ISG15-/- dermal fibroblasts, HaCaT keratinocytes, and human induced pluripotent stem cell-derived vascular endothelial cells. ISG15-deficient cells exhibited the expected hyperinflammatory phenotype, but also dysregulated expression of molecules critical for connective tissue and epidermis integrity, including reduced collagens and adhesion molecules, but increased matrix metalloproteinases. ISG15-/- fibroblasts exhibited elevated ROS levels and reduced ROS scavenger expression. As opposed to hyperinflammation, defective collagen and integrin synthesis was not rescued by conjugation-deficient ISG15. Cell migration was retarded in ISG15-/- fibroblasts and HaCaT keratinocytes, but normalized under ruxolitinib treatment. Desmosome density was reduced in an ISG15-/- 3D epidermis model. Additionally, there were loose architecture and reduced collagen and desmoglein expression, which could be reversed by treatment with ruxolitinib/doxycycline/TGF-ß1. These results reveal critical roles of ISG15 in maintaining cell migration and epidermis and connective tissue homeostasis, whereby the latter likely requires its conjugation to yet unidentified targets.


Assuntos
Citocinas/deficiência , Derme/metabolismo , Fibroblastos/metabolismo , Homeostase , Queratinócitos/metabolismo , Ubiquitinas/deficiência , Linhagem Celular Transformada , Citocinas/metabolismo , Humanos , Ubiquitinas/metabolismo
5.
Life Sci Alliance ; 5(3)2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34933920

RESUMO

The autophagy-lysosomal pathway is impaired in many neurodegenerative diseases characterized by protein aggregation, but the link between aggregation and lysosomal dysfunction remains poorly understood. Here, we combine cryo-electron tomography, proteomics, and cell biology studies to investigate the effects of protein aggregates in primary neurons. We use artificial amyloid-like ß-sheet proteins (ß proteins) to focus on the gain-of-function aspect of aggregation. These proteins form fibrillar aggregates and cause neurotoxicity. We show that late stages of autophagy are impaired by the aggregates, resulting in lysosomal alterations reminiscent of lysosomal storage disorders. Mechanistically, ß proteins interact with and sequester AP-3 µ1, a subunit of the AP-3 adaptor complex involved in protein trafficking to lysosomal organelles. This leads to destabilization of the AP-3 complex, missorting of AP-3 cargo, and lysosomal defects. Restoring AP-3µ1 expression ameliorates neurotoxicity caused by ß proteins. Altogether, our results highlight the link between protein aggregation, lysosomal impairments, and neurotoxicity.


Assuntos
Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Mutação com Ganho de Função , Neurônios/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/ultraestrutura , Proteínas Amiloidogênicas/ultraestrutura , Sobrevivência Celular/genética , Expressão Gênica , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Neurônios/ultraestrutura , Agregados Proteicos , Agregação Patológica de Proteínas/metabolismo , Transdução de Sinais
6.
Nucleic Acids Res ; 49(21): 12284-12305, 2021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34850154

RESUMO

Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.


Assuntos
Cromatina/genética , Dano ao DNA , Reparo do DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Neurônios Motores/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Animais , Axônios/metabolismo , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios Motores/citologia , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo
7.
Mol Syst Biol ; 17(7): e10125, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34318608

RESUMO

Cells signal through rearrangements of protein communities governed by covalent modifications and reversible interactions of distinct sets of proteins. A method that identifies those post-transcriptional modifications regulating signaling complex composition and functional phenotypes in one experimental setup would facilitate an efficient identification of novel molecular signaling checkpoints. Here, we devised modifications, interactions and phenotypes by affinity purification mass spectrometry (MIP-APMS), comprising the streamlined cloning and transduction of tagged proteins into functionalized reporter cells as well as affinity chromatography, followed by MS-based quantification. We report the time-resolved interplay of more than 50 previously undescribed modification and hundreds of protein-protein interactions of 19 immune protein complexes in monocytes. Validation of interdependencies between covalent, reversible, and functional protein complex regulations by knockout or site-specific mutation revealed ISGylation and phosphorylation of TRAF2 as well as ARHGEF18 interaction in Toll-like receptor 2 signaling. Moreover, we identify distinct mechanisms of action for small molecule inhibitors of p38 (MAPK14). Our method provides a fast and cost-effective pipeline for the molecular interrogation of protein communities in diverse biological systems and primary cells.


Assuntos
Processamento de Proteína Pós-Traducional , Proteômica , Complexo Antígeno-Anticorpo , Espectrometria de Massas , Fenótipo
8.
Cell Rep ; 34(10): 108826, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691121

RESUMO

A major pathway for proinflammatory protein release by macrophages is inflammasome-mediated pyroptotic cell death. As conventional secretion, unconventional secretion, and cell death are executed simultaneously, however, the cellular mechanisms regulating this complex paracrine program remain incompletely understood. Here, we devise a quantitative proteomics strategy to define the cellular exit route for each protein by pharmacological and genetic dissection of cellular checkpoints regulating protein release. We report the release of hundreds of proteins during pyroptosis, predominantly due to cell lysis. They comprise constitutively expressed and transcriptionally induced proteins derived from the cytoplasm and specific intracellular organelles. Many low-molecular-weight proteins including the cytokine interleukin-1ß, alarmins, and lysosomal-cargo proteins exit cells in the absence of cell lysis. Cytokines and alarmins are released in an endoplasmic reticulum (ER)-Golgi-dependent manner as free proteins rather than by extracellular vesicles. Our work provides an experimental framework for the dissection of cellular exit pathways and a resource for pyroptotic protein release.


Assuntos
Alarminas/análise , Citocinas/análise , Proteômica/métodos , Piroptose , Trifosfato de Adenosina/farmacologia , Alarminas/metabolismo , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Citocinas/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nigericina/farmacologia , Espectrometria de Massas em Tandem
9.
Nat Commun ; 12(1): 1278, 2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627647

RESUMO

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.


Assuntos
RNA Longo não Codificante/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN/metabolismo , Animais , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Camundongos , Neurônios Motores/metabolismo , RNA Longo não Codificante/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN/genética , Espectrometria de Massas em Tandem , Transcrição Genética/genética
10.
J Biol Chem ; 295(50): 16931-16948, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-32900848

RESUMO

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in both health and disease.


Assuntos
Colesterol/metabolismo , Hepacivirus/metabolismo , Hepatite C/virologia , Receptores Virais/metabolismo , Tetraspanina 28/metabolismo , Internalização do Vírus , Animais , Linhagem Celular , Cricetinae , Hepacivirus/isolamento & purificação , Hepatite C/metabolismo , Hepatite C/patologia , Humanos , Camundongos , Mutagênese Sítio-Dirigida/métodos , Elementos Estruturais de Proteínas
11.
Nat Immunol ; 21(8): 927-937, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632289

RESUMO

In response to pathogenic threats, naive T cells rapidly transition from a quiescent to an activated state, yet the underlying mechanisms are incompletely understood. Using a pulsed SILAC approach, we investigated the dynamics of mRNA translation kinetics and protein turnover in human naive and activated T cells. Our datasets uncovered that transcription factors maintaining T cell quiescence had constitutively high turnover, which facilitated their depletion following activation. Furthermore, naive T cells maintained a surprisingly large number of idling ribosomes as well as 242 repressed mRNA species and a reservoir of glycolytic enzymes. These components were rapidly engaged following stimulation, promoting an immediate translational and glycolytic switch to ramp up the T cell activation program. Our data elucidate new insights into how T cells maintain a prepared state to mount a rapid immune response, and provide a resource of protein turnover, absolute translation kinetics and protein synthesis rates in T cells ( https://www.immunomics.ch ).


Assuntos
Ativação Linfocitária/fisiologia , Biossíntese de Proteínas/imunologia , Linfócitos T/imunologia , Humanos , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo
12.
Nat Commun ; 11(1): 431, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31969567

RESUMO

Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.


Assuntos
Arginina/metabolismo , Células Gigantes/imunologia , Animais , Artrite/genética , Artrite/metabolismo , Artrite/fisiopatologia , Remodelação Óssea , Ciclo do Ácido Cítrico , Feminino , Células Gigantes/citologia , Humanos , Interleucina-4/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/genética , Ligante RANK/metabolismo
13.
Curr Opin Cell Biol ; 63: 20-30, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31927463

RESUMO

Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)-based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease.


Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional/genética , Proteômica/métodos , Humanos , Transdução de Sinais
14.
Cell Rep ; 30(4): 1260-1270.e5, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995763

RESUMO

The inflammatory functions of the cytokine tumor necrosis factor (TNF) rely on its ability to induce cytokine production and to induce cell death. Caspase-dependent and caspase-independent pathways-apoptosis and necroptosis, respectively-regulate immunogenicity by the release of distinct sets of cellular proteins. To obtain an unbiased, systems-level understanding of this important process, we here applied mass spectrometry-based proteomics to dissect protein release during apoptosis and necroptosis. We report hundreds of proteins released from human myeloid cells in time course experiments. Both cell death types induce receptor shedding, but only apoptotic cells released nucleosome components. Conversely, necroptotic cells release lysosomal components by activating lysosomal exocytosis at early stages of necroptosis-induced membrane permeabilization and show reduced release of conventionally secreted cytokines.


Assuntos
Apoptose , Caspase 8/metabolismo , Citocinas/metabolismo , Necroptose , Ácidos Pentanoicos/farmacologia , Proteoma/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Apoptose/efeitos dos fármacos , Inibidores de Caspase/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL24/metabolismo , Dipeptídeos/farmacologia , Exocitose/efeitos dos fármacos , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Indóis/farmacologia , Interleucina-8/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Espectrometria de Massas , Necroptose/efeitos dos fármacos
15.
Sci Rep ; 9(1): 17401, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31758014

RESUMO

Vaccination is the most effective method to prevent infectious diseases. However, approaches to identify novel vaccine candidates are commonly laborious and protracted. While surface proteins are suitable vaccine candidates and can elicit antibacterial antibody responses, systematic approaches to define surfomes from gram-negatives have rarely been successful. Here we developed a combined discovery-driven mass spectrometry and computational strategy to identify bacterial vaccine candidates and validate their immunogenicity using a highly prevalent gram-negative pathogen, Helicobacter pylori, as a model organism. We efficiently isolated surface antigens by enzymatic cleavage, with a design of experiment based strategy to experimentally dissect cell surface-exposed from cytosolic proteins. From a total of 1,153 quantified bacterial proteins, we thereby identified 72 surface exposed antigens and further prioritized candidates by computational homology inference within and across species. We next tested candidate-specific immune responses. All candidates were recognized in sera from infected patients, and readily induced antibody responses after vaccination of mice. The candidate jhp_0775 induced specific B and T cell responses and significantly reduced colonization levels in mouse therapeutic vaccination studies. In infected humans, we further show that jhp_0775 is immunogenic and activates IFNγ secretion from peripheral CD4+ and CD8+ T cells. Our strategy provides a generic preclinical screening, selection and validation process for novel vaccine candidates against gram-negative bacteria, which could be employed to other gram-negative pathogens.


Assuntos
Vacinas Bacterianas , Bactérias Gram-Negativas , Espectrometria de Massas , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Antígenos de Superfície/química , Antígenos de Superfície/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/química , Vacinas Bacterianas/imunologia , Biologia Computacional/métodos , Bactérias Gram-Negativas/imunologia , Proteômica/métodos , Reprodutibilidade dos Testes
16.
Mol Cell Proteomics ; 18(12): 2401-2417, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31570497

RESUMO

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus Uukuniemi/fisiologia , Animais , Antivirais/farmacologia , Infecções por Bunyaviridae/virologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Glicoproteínas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Espectrometria de Massas , Proteômica , Piridinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Vírus de RNA/fisiologia , Vírus Uukuniemi/efeitos dos fármacos , Células Vero , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Replicação Viral
17.
Cancer Cell ; 36(3): 250-267.e9, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31526758

RESUMO

How lymphoma cells (LCs) invade the brain during the development of central nervous system lymphoma (CNSL) is unclear. We found that NF-κB-induced gliosis promotes CNSL in immunocompetent mice. Gliosis elevated cell-adhesion molecules, which increased LCs in the brain but was insufficient to induce CNSL. Astrocyte-derived CCL19 was required for gliosis-induced CNSL. Deleting CCL19 in mice or CCR7 from LCs abrogated CNSL development. Two-photon microscopy revealed LCs transiently entering normal brain parenchyma. Astrocytic CCL19 enhanced parenchymal CNS retention of LCs, thereby promoting CNSL formation. Aged, gliotic wild-type mice were more susceptible to forming CNSL than young wild-type mice, and astrocytic CCL19 was observed in both human gliosis and CNSL. Therefore, CCL19-CCR7 interactions may underlie an increased age-related risk for CNSL.


Assuntos
Envelhecimento/patologia , Neoplasias do Sistema Nervoso Central/patologia , Quimiocina CCL19/metabolismo , Gliose/patologia , Linfoma/patologia , Adolescente , Adulto , Idoso , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/patologia , Linhagem Celular Tumoral/transplante , Neoplasias do Sistema Nervoso Central/diagnóstico por imagem , Neoplasias do Sistema Nervoso Central/cirurgia , Quimiocina CCL19/genética , Quimiocina CXCL12 , Modelos Animais de Doenças , Feminino , Gliose/diagnóstico por imagem , Humanos , Microscopia Intravital , Linfoma/diagnóstico por imagem , Linfoma/cirurgia , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência por Excitação Multifotônica , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Receptores CCR7/genética , Receptores CCR7/metabolismo , Imagem com Lapso de Tempo , Adulto Jovem
18.
Cell ; 178(5): 1102-1114.e17, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442403

RESUMO

Caloric restriction is known to improve inflammatory and autoimmune diseases. However, the mechanisms by which reduced caloric intake modulates inflammation are poorly understood. Here we show that short-term fasting reduced monocyte metabolic and inflammatory activity and drastically reduced the number of circulating monocytes. Regulation of peripheral monocyte numbers was dependent on dietary glucose and protein levels. Specifically, we found that activation of the low-energy sensor 5'-AMP-activated protein kinase (AMPK) in hepatocytes and suppression of systemic CCL2 production by peroxisome proliferator-activator receptor alpha (PPARα) reduced monocyte mobilization from the bone marrow. Importantly, we show that fasting improves chronic inflammatory diseases without compromising monocyte emergency mobilization during acute infectious inflammation and tissue repair. These results reveal that caloric intake and liver energy sensors dictate the blood and tissue immune tone and link dietary habits to inflammatory disease outcome.


Assuntos
Restrição Calórica , Monócitos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Adulto , Animais , Antígenos Ly/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , PPAR alfa/deficiência , PPAR alfa/genética , PPAR alfa/metabolismo
19.
Nat Microbiol ; 4(4): 701-713, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804548

RESUMO

The innate immune system is crucial for eventual control of infections, but may also contribute to pathology. Listeria monocytogenes is an intracellular Gram-positive bacteria and a major cause of food-borne disease. However, important knowledge on the interactions between L. monocytogenes and the immune system is still missing. Here, we report that Listeria DNA is sorted into extracellular vesicles (EVs) in infected cells and delivered to bystander cells to stimulate the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway. This was also observed during infections with Francisella tularensis and Legionella pneumophila. We identify the multivesicular body protein MVB12b as a target for TANK-binding kinase 1 phosphorylation, which is essential for the sorting of DNA into EVs and stimulation of bystander cells. EVs from Listeria-infected cells inhibited T-cell proliferation, and primed T cells for apoptosis. Collectively, we describe a pathway for EV-mediated delivery of foreign DNA to bystander cells, and suggest that intracellular bacteria exploit this pathway to impair antibacterial defence.


Assuntos
Vesículas Extracelulares/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Vesículas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/genética , Listeriose/genética , Listeriose/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Nucleotídeos Cíclicos , Nucleotidiltransferases/genética , Fosforilação , Proteínas de Transporte Vesicular/genética
20.
Life Sci Alliance ; 1(2): e201800070, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-30456350

RESUMO

Frontotemporal dementia and amyotrophic lateral sclerosis patients with C9orf72 mutation show cytoplasmic poly-GR and poly-PR aggregates. Short poly-(Gly-Arg) and poly-(Pro-Arg) (poly-GR/PR) repeats localizing to the nucleolus are toxic in various model systems, but no interactors have been validated in patients. Here, the neuronal interactomes of cytoplasmic GFP-(GR)149 and nucleolar (PR)175-GFP revealed overlapping RNA-binding proteins, including components of stress granules, nucleoli, and ribosomes. Overexpressing the poly-GR/PR interactors STAU1/2 and YBX1 caused cytoplasmic aggregation of poly-GR/PR in large stress granule-like structures, whereas NPM1 recruited poly-GR into the nucleolus. Poly-PR expression reduced ribosome levels and translation consistent with reduction of synaptic proteins detected by proteomics. Surprisingly, truncated GFP-(GR)53, but not GFP-(GR)149, localized to the nucleolus and reduced ribosome levels and translation similar to poly-PR, suggesting that impaired ribosome biogenesis may be driving the acute toxicity observed in vitro. In patients, only ribosomes and STAU2 co-aggregated with poly-GR/PR. Partial sequestration of ribosomes may chronically impair protein synthesis even in the absence of nucleolar localization and contribute to pathogenesis.

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