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1.
Cell ; 168(1-2): 86-100.e15, 2017 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-27916275

RESUMEN

Type 1 diabetes is characterized by the destruction of pancreatic ß cells, and generating new insulin-producing cells from other cell types is a major aim of regenerative medicine. One promising approach is transdifferentiation of developmentally related pancreatic cell types, including glucagon-producing α cells. In a genetic model, loss of the master regulatory transcription factor Arx is sufficient to induce the conversion of α cells to functional ß-like cells. Here, we identify artemisinins as small molecules that functionally repress Arx by causing its translocation to the cytoplasm. We show that the protein gephyrin is the mammalian target of these antimalarial drugs and that the mechanism of action of these molecules depends on the enhancement of GABAA receptor signaling. Our results in zebrafish, rodents, and primary human pancreatic islets identify gephyrin as a druggable target for the regeneration of pancreatic ß cell mass from α cells.


Asunto(s)
Artemisininas/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Modelos Animales de Enfermedad , Receptores de GABA-A/metabolismo , Transducción de Señal , Animales , Arteméter , Artemisininas/administración & dosificación , Proteínas Portadoras/metabolismo , Transdiferenciación Celular/efectos de los fármacos , Células Cultivadas , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus Tipo 1/patología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Insulina/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones , Estabilidad Proteica/efectos de los fármacos , Ratas , Análisis de la Célula Individual , Factores de Transcripción/metabolismo , Pez Cebra , Ácido gamma-Aminobutírico/metabolismo
2.
Nat Immunol ; 17(12): 1361-1372, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27798618

RESUMEN

Hemolysis drives susceptibility to bacterial infections and predicts poor outcome from sepsis. These detrimental effects are commonly considered to be a consequence of heme-iron serving as a nutrient for bacteria. We employed a Gram-negative sepsis model and found that elevated heme levels impaired the control of bacterial proliferation independently of heme-iron acquisition by pathogens. Heme strongly inhibited phagocytosis and the migration of human and mouse phagocytes by disrupting actin cytoskeletal dynamics via activation of the GTP-binding Rho family protein Cdc42 by the guanine nucleotide exchange factor DOCK8. A chemical screening approach revealed that quinine effectively prevented heme effects on the cytoskeleton, restored phagocytosis and improved survival in sepsis. These mechanistic insights provide potential therapeutic targets for patients with sepsis or hemolytic disorders.


Asunto(s)
Infecciones por Bacterias Gramnegativas/inmunología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemo/metabolismo , Hemólisis/inmunología , Macrófagos/inmunología , Fagocitosis , Sepsis/inmunología , Animales , Antibacterianos/uso terapéutico , Citoesqueleto/metabolismo , Femenino , Infecciones por Bacterias Gramnegativas/tratamiento farmacológico , Factores de Intercambio de Guanina Nucleótido/genética , Hemo-Oxigenasa 1/genética , Hemólisis/efectos de los fármacos , Humanos , Evasión Inmune , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/efectos de los fármacos , Quinina/uso terapéutico , Células RAW 264.7 , Sepsis/tratamiento farmacológico , Proteína de Unión al GTP cdc42/metabolismo
3.
Nat Immunol ; 17(12): 1352-1360, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27776107

RESUMEN

RASGRP1 is an important guanine nucleotide exchange factor and activator of the RAS-MAPK pathway following T cell antigen receptor (TCR) signaling. The consequences of RASGRP1 mutations in humans are unknown. In a patient with recurrent bacterial and viral infections, born to healthy consanguineous parents, we used homozygosity mapping and exome sequencing to identify a biallelic stop-gain variant in RASGRP1. This variant segregated perfectly with the disease and has not been reported in genetic databases. RASGRP1 deficiency was associated in T cells and B cells with decreased phosphorylation of the extracellular-signal-regulated serine kinase ERK, which was restored following expression of wild-type RASGRP1. RASGRP1 deficiency also resulted in defective proliferation, activation and motility of T cells and B cells. RASGRP1-deficient natural killer (NK) cells exhibited impaired cytotoxicity with defective granule convergence and actin accumulation. Interaction proteomics identified the dynein light chain DYNLL1 as interacting with RASGRP1, which links RASGRP1 to cytoskeletal dynamics. RASGRP1-deficient cells showed decreased activation of the GTPase RhoA. Treatment with lenalidomide increased RhoA activity and reversed the migration and activation defects of RASGRP1-deficient lymphocytes.


Asunto(s)
Actinas/metabolismo , Linfocitos B/inmunología , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Factores de Intercambio de Guanina Nucleótido/genética , Síndromes de Inmunodeficiencia/genética , Células Asesinas Naturales/inmunología , Linfocitos T/inmunología , Adolescente , Inhibidores de la Angiogénesis/farmacología , Linfocitos B/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/genética , Niño , Citotoxicidad Inmunológica/genética , Análisis Mutacional de ADN , Dineínas/metabolismo , Femenino , Células HEK293 , Humanos , Cambio de Clase de Inmunoglobulina/genética , Síndromes de Inmunodeficiencia/tratamiento farmacológico , Células Jurkat , Células Asesinas Naturales/efectos de los fármacos , Lenalidomida , Masculino , Mutación/genética , Linaje , ARN Interferente Pequeño/genética , Linfocitos T/efectos de los fármacos , Talidomida/análogos & derivados , Talidomida/farmacología
4.
Immunity ; 51(6): 1074-1087.e9, 2019 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-31784108

RESUMEN

Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón Tipo I/inmunología , Hígado/metabolismo , Virus de la Coriomeningitis Linfocítica/inmunología , Receptor de Interferón alfa y beta/metabolismo , Animales , Arginina/sangre , Línea Celular , Chlorocebus aethiops , Cricetinae , Femenino , Hepatocitos/metabolismo , Hígado/inmunología , Hígado/virología , Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ornitina/sangre , Ornitina Carbamoiltransferasa/genética , Transducción de Señal/inmunología , Urea/metabolismo , Células Vero
5.
Nat Immunol ; 15(5): 439-448, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24681565

RESUMEN

Molecular mechanisms that maintain lineage integrity of helper T cells are largely unknown. Here we show histone deacetylases 1 and 2 (HDAC1 and HDAC2) as crucial regulators of this process. Loss of HDAC1 and HDAC2 during late T cell development led to the appearance of major histocompatibility complex (MHC) class II-selected CD4(+) helper T cells that expressed CD8-lineage genes such as Cd8a and Cd8b1. HDAC1 and HDAC2-deficient T helper type 0 (TH0) and TH1 cells further upregulated CD8-lineage genes and acquired a CD8(+) effector T cell program in a manner dependent on Runx-CBFß complexes, whereas TH2 cells repressed features of the CD8(+) lineage independently of HDAC1 and HDAC2. These results demonstrate that HDAC1 and HDAC2 maintain integrity of the CD4 lineage by repressing Runx-CBFß complexes that otherwise induce a CD8(+) effector T cell-like program in CD4(+) T cells.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Células TH1/inmunología , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Cultivadas , Subunidades alfa del Factor de Unión al Sitio Principal/metabolismo , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Citocinas/metabolismo , Citotoxicidad Inmunológica/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica
6.
EMBO Rep ; 23(12): e54978, 2022 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-36321428

RESUMEN

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.


Asunto(s)
Antiportadores , Protones , Antiportadores/genética
7.
Nat Immunol ; 12(7): 624-30, 2011 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-21642987

RESUMEN

Antiviral innate immunity relies on the recognition of microbial structures. One such structure is viral RNA that carries a triphosphate group on its 5' terminus (PPP-RNA). By an affinity proteomics approach with PPP-RNA as the 'bait', we found that the antiviral protein IFIT1 (interferon-induced protein with tetratricopeptide repeats 1) mediated binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-RNA with nanomolar affinity and required the arginine at position 187 in a highly charged carboxy-terminal groove of the protein. In the absence of IFIT1, the growth and pathogenicity of viruses containing PPP-RNA was much greater. In contrast, IFIT proteins were dispensable for the clearance of pathogens that did not generate PPP-RNA. On the basis of this specificity and the great abundance of IFIT proteins after infection, we propose that the IFIT complex antagonizes viruses by sequestering specific viral nucleic acids.


Asunto(s)
Arginina/inmunología , Proteínas Portadoras/inmunología , ARN Viral/inmunología , Virus/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Arginina/química , Arginina/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Femenino , Células HEK293 , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Unión al ARN
8.
Cell ; 135(3): 462-74, 2008 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-18984158

RESUMEN

tRNAs are synthesized as immature precursors, and on their way to functional maturity, extra nucleotides at their 5' ends are removed by an endonuclease called RNase P. All RNase P enzymes characterized so far are composed of an RNA plus one or more proteins, and tRNA 5' end maturation is considered a universal ribozyme-catalyzed process. Using a combinatorial purification/proteomics approach, we identified the components of human mitochondrial RNase P and reconstituted the enzymatic activity from three recombinant proteins. We thereby demonstrate that human mitochondrial RNase P is a protein enzyme that does not require a trans-acting RNA component for catalysis. Moreover, the mitochondrial enzyme turns out to be an unexpected type of patchwork enzyme, composed of a tRNA methyltransferase, a short-chain dehydrogenase/reductase-family member, and a protein of hitherto unknown functional and evolutionary origin, possibly representing the enzyme's metallonuclease moiety. Apparently, animal mitochondria lost the seemingly ubiquitous RNA world remnant after reinventing RNase P from preexisting components.


Asunto(s)
Mitocondrias/enzimología , ARN Catalítico/análisis , Ribonucleasa P/química , Animales , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , ARN de Transferencia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleasa P/genética , Ribonucleasa P/aislamiento & purificación , Ribonucleasa P/metabolismo
9.
Nat Immunol ; 10(3): 266-72, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19158679

RESUMEN

Cytoplasmic DNA triggers activation of the innate immune system. Although 'downstream' signaling components have been characterized, the DNA-sensing components remain elusive. Here we present a systematic proteomics screen for proteins that associate with DNA, 'crossed' to a screen for transcripts induced by interferon-beta, which identified AIM2 as a candidate cytoplasmic DNA sensor. AIM2 showed specificity for double-stranded DNA. It also recruited the inflammasome adaptor ASC and localized to ASC 'speckles'. A decrease in AIM2 expression produced by RNA-mediated interference impaired DNA-induced maturation of interleukin 1beta in THP-1 human monocytic cells, which indicated that endogenous AIM2 is required for DNA recognition. Reconstitution of unresponsive HEK293 cells with AIM2, ASC, caspase-1 and interleukin 1beta showed that AIM2 was sufficient for inflammasome activation. Our data suggest that AIM2 is a cytoplasmic DNA sensor for the inflammasome.


Asunto(s)
ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Caspasa 1/inmunología , Caspasa 1/metabolismo , Citosol/metabolismo , ADN/inmunología , Proteínas de Unión al ADN , Perfilación de la Expresión Génica , Genómica/métodos , Humanos , Inmunidad Innata , Interferón beta/inmunología , Interferón beta/metabolismo , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Ratones , Células 3T3 NIH , Proteínas Nucleares/inmunología , Proteómica/métodos
10.
Nature ; 519(7544): 477-81, 2015 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-25561175

RESUMEN

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. Here we describe the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR.


Asunto(s)
Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Lisosomas/metabolismo , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleótidos/metabolismo
11.
PLoS Pathog ; 14(11): e1007397, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30475900

RESUMEN

DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease.


Asunto(s)
Listeriosis/inmunología , ARN Helicasas/inmunología , Animales , ARN Helicasas DEAD-box/metabolismo , Resistencia a la Enfermedad/inmunología , Femenino , Fibroblastos/inmunología , Fibroblastos/patología , Células HEK293 , Hematopoyesis/inmunología , Humanos , Inmunidad Innata , Células Asesinas Naturales/inmunología , Listeria monocytogenes/inmunología , Listeriosis/patología , Linfocitos/inmunología , Masculino , Ratones , Ratones Noqueados , FN-kappa B/inmunología , ARN Helicasas/deficiencia , ARN Helicasas/genética , Factores Sexuales , Transducción de Señal
12.
Nature ; 508(7495): 222-7, 2014 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-24695225

RESUMEN

Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.


Asunto(s)
Antineoplásicos/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Enzimas Reparadoras del ADN/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Monoéster Fosfórico Hidrolasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Piridinas/farmacología , Aminoquinolinas/farmacología , Animales , Antineoplásicos/química , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Crizotinib , Cristalización , Roturas del ADN de Cadena Simple/efectos de los fármacos , Reparación del ADN , Enzimas Reparadoras del ADN/biosíntesis , Enzimas Reparadoras del ADN/química , Modelos Animales de Enfermedad , Femenino , Homeostasis/efectos de los fármacos , Humanos , Ratones , Ratones SCID , Modelos Moleculares , Nucleótidos/metabolismo , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/química , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Proteómica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Pirazoles/química , Piridinas/química , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/genética
13.
Mol Cell ; 45(4): 553-66, 2012 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-22365832

RESUMEN

Plk1 activation is required for progression through mitotic entry to cytokinesis. Here we show that at mitotic entry, Plk1 phosphorylates Optineurin (Optn) at serine 177 and that this dissociates Optn from the Golgi-localized GTPase Rab8, inducing its translocation into the nucleus. Mass spectrometry analysis revealed that Optn is associated with a myosin phosphatase complex (MP), which antagonizes the mitotic function of Plk1. Our data also indicate that Optn functionally connects this complex to Plk1 by promoting phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). Accordingly, silencing Optn expression increases Plk1 activity and induces abscission failure and multinucleation, which were rescued upon expression of wild-type (WT) Optn, but not a phospho-deficient mutant (S177A) that cannot translocate into the nucleus during mitosis. Overall, these results highlight an important role of Optn in the spatial and temporal coordination of Plk1 activity.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Factor de Transcripción TFIIIA/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Retroalimentación Fisiológica , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte de Membrana , Fosforilación , Factor de Transcripción TFIIIA/química , Factor de Transcripción TFIIIA/fisiología , Quinasa Tipo Polo 1
14.
Mol Cell Proteomics ; 17(3): 516-532, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29208752

RESUMEN

Peritoneal dialysis (PD) is a modality of renal replacement therapy in which the high volumes of available PD effluent (PDE) represents a rich source of biomarkers for monitoring disease and therapy. Although this information could help guide the management of PD patients, little is known about the potential of PDE to define pathomechanism-associated molecular signatures in PD.We therefore subjected PDE to a high-performance multiplex proteomic analysis after depletion of highly-abundant plasma proteins and enrichment of low-abundance proteins. A combination of label-free and isobaric labeling strategies was applied to PDE samples from PD patients (n = 20) treated in an open-label, randomized, two-period, cross-over clinical trial with standard PD fluid or with a novel PD fluid supplemented with alanyl-glutamine (AlaGln).With this workflow we identified 2506 unique proteins in the PDE proteome, greatly increasing coverage beyond the 171 previously-reported proteins. The proteins identified range from high abundance plasma proteins to low abundance cellular proteins, and are linked to larger numbers of biological processes and pathways, some of which are novel for PDE. Interestingly, proteins linked to membrane remodeling and fibrosis are overrepresented in PDE compared with plasma, whereas the proteins underrepresented in PDE suggest decreases in host defense, immune-competence and response to stress. Treatment with AlaGln-supplemented PD fluid is associated with reduced activity of membrane injury-associated mechanisms and with restoration of biological processes involved in stress responses and host defense.Our study represents the first application of the PDE proteome in a randomized controlled prospective clinical trial of PD. This novel proteomic workflow allowed detection of low abundance biomarkers to define pathomechanism-associated molecular signatures in PD and their alterations by a novel therapeutic intervention.


Asunto(s)
Dipéptidos/farmacología , Diálisis Peritoneal , Proteoma , Proteínas Sanguíneas/metabolismo , Estudios Cruzados , Femenino , Humanos , Masculino
15.
PLoS Pathog ; 13(12): e1006758, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29261807

RESUMEN

RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.


Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus de la Coriomeningitis Linfocítica/enzimología , Modelos Moleculares , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Represoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virales/metabolismo , Animales , Sistemas CRISPR-Cas , Biología Computacional , Cruzamientos Genéticos , ARN Helicasas DEAD-box/química , Femenino , Células HEK293 , Humanos , Inmunoprecipitación , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/veterinaria , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Dominios y Motivos de Interacción de Proteínas , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Organismos Libres de Patógenos Específicos , Proteínas Virales/química , Proteínas Virales/genética
16.
Nat Chem Biol ; 13(7): 771-778, 2017 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-28530711

RESUMEN

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.


Asunto(s)
Evaluación Preclínica de Medicamentos , Receptores Androgénicos/metabolismo , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Flutamida/farmacología , Humanos , Masculino , Estructura Molecular , Fenprocumón/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad
17.
J Am Soc Nephrol ; 29(1): 268-282, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29046343

RESUMEN

Cardiovascular disease (CVD) is the leading cause of increased mortality in patients with CKD and is further aggravated by peritoneal dialysis (PD). Children are devoid of preexisting CVD and provide unique insight into specific uremia- and PD-induced pathomechanisms of CVD. We obtained peritoneal specimens from children with stage 5 CKD at time of PD catheter insertion (CKD5 group), children with established PD (PD group), and age-matched nonuremic controls (n=6/group). We microdissected omental arterioles from tissue layers not directly exposed to PD fluid and used adjacent sections of four arterioles per patient for transcriptomic and proteomic analyses. Findings were validated in omental and parietal arterioles from independent pediatric control (n=5), CKD5 (n=15), and PD (n=15) cohorts. Transcriptomic analysis revealed differential gene expression in control versus CKD5 arterioles and in CKD5 versus PD arterioles. Gene ontology analyses revealed activation of metabolic processes in CKD5 arterioles and of inflammatory, immunologic, and stress-response cascades in PD arterioles. PD arterioles exhibited particular upregulation of the complement system and respective regulatory pathways, with concordant findings at the proteomic level. In the validation cohorts, PD specimens had the highest abundance of omental and parietal arteriolar C1q, C3d, terminal complement complex, and phosphorylated SMAD2/3, a downstream effector of TGF-ß Furthermore, in the PD parietal arterioles, C1q and terminal complement complex abundance correlated with the level of dialytic glucose exposure, abundance of phosphorylated SMAD2/3, and degree of vasculopathy. We conclude that PD fluids activate arteriolar complement and TGF-ß signaling, which quantitatively correlate with the severity of arteriolar vasculopathy.


Asunto(s)
Arteriolas/metabolismo , Activación de Complemento , Proteínas del Sistema Complemento/metabolismo , Fallo Renal Crónico/terapia , Diálisis Peritoneal/efectos adversos , Enfermedades Vasculares/metabolismo , Adolescente , Estudios de Casos y Controles , Niño , Preescolar , Complemento C1q/metabolismo , Complemento C3d/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Femenino , Ontología de Genes , Humanos , Lactante , Recién Nacido , Fallo Renal Crónico/complicaciones , Masculino , Epiplón/irrigación sanguínea , Fosforilación , Proteoma , Índice de Severidad de la Enfermedad , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Transcriptoma , Factor de Crecimiento Transformador beta/metabolismo , Uremia/etiología , Enfermedades Vasculares/etiología , Factor A de Crecimiento Endotelial Vascular/metabolismo
18.
Proteomics ; 18(8): e1700386, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29474001

RESUMEN

Chromosome-centric Human Proteome Project aims at identifying and characterizing protein products encoded from all human protein-coding genes. As of early 2017, 19 837 protein-coding genes have been annotated in the neXtProt database including 2691 missing proteins that have never been identified by mass spectrometry. Missing proteins may be low abundant in many cell types or expressed only in a few cell types in human body such as sperms in testis. In this study, we performed expression proteomics of two near-haploid cell types such as HAP1 and KBM-7 to hunt for missing proteins. Proteomes from the two haploid cell lines were analyzed on an LTQ Orbitrap Velos, producing a total of 200 raw mass spectrometry files. After applying 1% false discovery rates at both levels of peptide-spectrum matches and proteins, more than 10 000 proteins were identified from HAP1 and KBM-7, resulting in the identification of nine missing proteins. Next, unmatched spectra were searched against protein databases translated in three frames from noncoding RNAs derived from RNA-Seq data, resulting in six novel protein-coding regions after careful manual inspection. This study demonstrates that expression proteomics coupled to proteogenomic analysis can be employed to identify many annotated and unannotated missing proteins.


Asunto(s)
Haploidia , Proteogenómica/métodos , Proteoma/genética , Transcriptoma , Secuencia de Aminoácidos , Línea Celular , Humanos , Proteoma/análisis , ARN no Traducido/genética , Análisis de Secuencia de ARN/métodos , Espectrometría de Masas en Tándem/métodos
19.
Nat Methods ; 12(11): 1055-7, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26389571

RESUMEN

Thermal stabilization of proteins after ligand binding provides an efficient means to assess the binding of small molecules to proteins. We show here that in combination with quantitative mass spectrometry, the approach allows for the systematic survey of protein engagement by cellular metabolites and drugs. We profiled the targets of the drugs methotrexate and (S)-crizotinib and the metabolite 2'3'-cGAMP in intact cells and identified the 2'3'-cGAMP cognate transmembrane receptor STING, involved in immune signaling.


Asunto(s)
Proteoma/metabolismo , Pirazoles/química , Piridinas/química , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Biología Computacional , Crizotinib , Diseño de Fármacos , Humanos , Sistema Inmunológico , Células K562 , Ligandos , Espectrometría de Masas , Metotrexato/química , Ratones , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Proteómica , Transducción de Señal , Biología de Sistemas , Temperatura
20.
Hepatology ; 65(4): 1181-1195, 2017 04.
Artículo en Inglés | MEDLINE | ID: mdl-27981604

RESUMEN

Diet-related health issues such as nonalcoholic fatty liver disease and cardiovascular disorders are known to have a major inflammatory component. However, the exact pathways linking diet-induced changes (e.g., hyperlipidemia) and the ensuing inflammation have remained elusive so far. We identified biological processes related to innate immunity and oxidative stress as prime response pathways in livers of low-density lipoprotein receptor-deficient mice on a Western-type diet using RNA sequencing and in silico functional analyses of transcriptome data. The observed changes were independent of the presence of microbiota and thus indicative of a role for sterile triggers. We further show that malondialdehyde (MDA) epitopes, products of lipid peroxidation and markers for enhanced oxidative stress, are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. MDA-induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated MDA epitopes by intravenous injection of a specific MDA antibody results in decreased hepatic inflammation in low-density lipoprotein receptor-deficient mice on a Western-type diet. CONCLUSION: Accumulation of MDA epitopes plays a major role during diet-induced hepatic inflammation and can be ameliorated by administration of an anti-MDA antibody. (Hepatology 2017;65:1181-1195).


Asunto(s)
Dieta Occidental , Epítopos/metabolismo , Hígado Graso/metabolismo , Hígado Graso/patología , Hipercolesterolemia/patología , Malondialdehído/metabolismo , Análisis de Varianza , Animales , Biopsia con Aguja , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epítopos/inmunología , Hígado Graso/inmunología , Femenino , Hipercolesterolemia/fisiopatología , Inmunidad Innata , Inmunohistoquímica , Mediadores de Inflamación/metabolismo , Peroxidación de Lípido , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Microbiota , Estrés Oxidativo , Distribución Aleatoria
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