RESUMEN
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Asunto(s)
Sistemas CRISPR-Cas , Hepatocitos , Neoplasias Hepáticas , Hígado , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Elementos Transponibles de ADN , Fluorescencia , Hepatocitos/metabolismo , Hepatocitos/citología , Hepatocitos/patología , Factor 4 Similar a Kruppel/metabolismo , Hígado/citología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/secundario , Melanoma/metabolismo , Melanoma/patología , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/prevención & control , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Semaforinas/antagonistas & inhibidores , Semaforinas/metabolismoRESUMEN
Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested â¼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts.
Asunto(s)
Neuronas , Estabilidad del ARN , Neuritas/metabolismo , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transactivadores/metabolismoRESUMEN
Hundreds of RNAs are enriched in the projections of neuronal cells. For the vast majority of them, though, the sequence elements that regulate their localization are unknown. To identify RNA elements capable of directing transcripts to neurites, we deployed a massively parallel reporter assay that tested the localization regulatory ability of thousands of sequence fragments drawn from endogenous mouse 3' UTRs. We identified peaks of regulatory activity within several 3' UTRs and found that sequences derived from these peaks were both necessary and sufficient for RNA localization to neurites in mouse and human neuronal cells. The localization elements were enriched in adenosine and guanosine residues. They were at least tens to hundreds of nucleotides long as shortening of two identified elements led to significantly reduced activity. Using RNA affinity purification and mass spectrometry, we found that the RNA-binding protein Unk was associated with the localization elements. Depletion of Unk in cells reduced the ability of the elements to drive RNAs to neurites, indicating a functional requirement for Unk in their trafficking. These results provide a framework for the unbiased, high-throughput identification of RNA elements and mechanisms that govern transcript localization in neurons.
Asunto(s)
Neuronas , Secuencias Reguladoras de Ácido Ribonucleico , Regiones no Traducidas 3'/genética , Animales , Humanos , Ratones , Neuronas/metabolismo , Nucleótidos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ARNRESUMEN
Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Virus de la Influenza A/genética , Neuraminidasa/metabolismo , Células A549 , Retículo Endoplásmico/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología , Proteínas no Estructurales Virales/genética , Replicación Viral/genéticaRESUMEN
Interferon (IFN) induction of IFN-stimulated genes (ISGs) creates a formidable protective antiviral state. However, loss of appropriate control mechanisms can result in constitutive pathogenic ISG upregulation. Here, we used genome-scale loss-of-function screening to establish genes critical for IFN-induced transcription, identifying all expected members of the JAK-STAT signaling pathway and a previously unappreciated epigenetic reader, bromodomain-containing protein 9 (BRD9), the defining subunit of non-canonical BAF (ncBAF) chromatin-remodeling complexes. Genetic knockout or small-molecule-mediated degradation of BRD9 limits IFN-induced expression of a subset of ISGs in multiple cell types and prevents IFN from exerting full antiviral activity against several RNA and DNA viruses, including influenza virus, human immunodeficiency virus (HIV1), and herpes simplex virus (HSV1). Mechanistically, BRD9 acts at the level of transcription, and its IFN-triggered proximal association with the ISG transcriptional activator, STAT2, suggests a functional localization at selected ISG promoters. Furthermore, BRD9 relies on its intact acetyl-binding bromodomain and unique ncBAF scaffolding interaction with GLTSCR1/1L to promote IFN action. Given its druggability, BRD9 is an attractive target for dampening ISG expression under certain autoinflammatory conditions.
Asunto(s)
Antivirales , Interferones , Antivirales/farmacología , Expresión Génica , Humanos , Factor de Transcripción STAT2/genética , Factor de Transcripción STAT2/metabolismo , Factores de Transcripción/genéticaAsunto(s)
Linfocitos B/inmunología , Linfocitos B/patología , ADN-Topoisomerasas de Tipo II/genética , Hematopoyesis , Síndromes de Inmunodeficiencia/patología , Mutación , Proteínas de Unión a Poli-ADP-Ribosa/genética , Linfocitos B/metabolismo , Femenino , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Masculino , Linaje , Fenotipo , PronósticoRESUMEN
Species' differences in cellular factors limit avian influenza A virus (IAV) zoonoses and human pandemics. The IAV polymerase, vPol, harbors evolutionary sites to overcome restriction and determines virulence. Here, we establish host ANP32A as a critical driver of selection, and identify host-specific ANP32A splicing landscapes that predict viral evolution. We find that avian species differentially express three ANP32A isoforms diverging in a vPol-promoting insert. ANP32As with shorter inserts interact poorly with vPol, are compromised in supporting avian-like IAV replication, and drive selection of mammalian-adaptive vPol sequences with distinct kinetics. By integrating selection data with multi-species ANP32A splice variant profiling, we develop a mathematical model to predict avian species potentially driving (swallow, magpie) or maintaining (goose, swan) mammalian-adaptive vPol signatures. Supporting these predictions, surveillance data confirm enrichment of several mammalian-adaptive vPol substitutions in magpie IAVs. Profiling host ANP32A splicing could enhance surveillance and eradication efforts against IAVs with pandemic potential.
Asunto(s)
Virus de la Influenza A/enzimología , Gripe Aviar/genética , Empalme del ARN , Proteínas de Unión al ARN/genética , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Aves , Pollos , Humanos , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A/química , Virus de la Influenza A/genética , Virus de la Influenza A/fisiología , Gripe Aviar/metabolismo , Gripe Aviar/virología , Gripe Humana/genética , Gripe Humana/metabolismo , Gripe Humana/virología , Proteínas Nucleares , Unión Proteica , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Replicación ViralRESUMEN
Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.
Asunto(s)
Quirópteros/virología , Antígenos de Histocompatibilidad Clase II/metabolismo , Especificidad del Huésped , Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Zoonosis/inmunología , Zoonosis/virología , Animales , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Pollos/genética , Pollos/inmunología , Quirópteros/genética , Quirópteros/inmunología , Quirópteros/metabolismo , Femenino , Perfilación de la Expresión Génica , Antígenos HLA-DR/genética , Antígenos HLA-DR/inmunología , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Especificidad del Huésped/genética , Especificidad del Huésped/inmunología , Humanos , Masculino , Ratones , Ratones Noqueados , Sistema Respiratorio/virología , Porcinos/genética , Porcinos/inmunología , Tropismo Viral/genética , Tropismo Viral/inmunología , Replicación Viral , Zoonosis/genética , Zoonosis/metabolismoRESUMEN
RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.
Asunto(s)
Artritis/genética , Enfermedades Inflamatorias del Intestino/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Inmunodeficiencia Combinada Grave/genética , Alelos , Artritis/inmunología , Citocinas/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Linfopenia/genética , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Linaje , Inmunodeficiencia Combinada Grave/inmunologíaRESUMEN
Mutations in genes encoding components of the immune system cause primary immunodeficiencies. Here, we study a patient with recurrent atypical mycobacterial infection and early-onset metastatic bladder carcinoma. Exome sequencing identified two homozygous missense germline mutations, P733L and P832S, in the JAK1 protein that mediates signalling from multiple cytokine receptors. Cells from this patient exhibit reduced JAK1 and STAT phosphorylation following cytokine stimulations, reduced induction of expression of interferon-regulated genes and dysregulated cytokine production; which are indicative of signalling defects in multiple immune response pathways including Interferon-γ production. Reconstitution experiments in the JAK1-deficient cells demonstrate that the impaired JAK1 function is mainly attributable to the effect of the P733L mutation. Further analyses of the mutant protein reveal a phosphorylation-independent role of JAK1 in signal transduction. These findings clarify JAK1 signalling mechanisms and demonstrate a critical function of JAK1 in protection against mycobacterial infection and possibly the immunological surveillance of cancer.
Asunto(s)
Alelos , Janus Quinasa 1/genética , Mutación/genética , Infecciones por Mycobacterium/enzimología , Infecciones por Mycobacterium/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Sanguíneas/metabolismo , Niño , Preescolar , Citocinas/sangre , Susceptibilidad a Enfermedades , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón-alfa/farmacología , Interferón gamma/farmacología , Janus Quinasa 1/química , Masculino , Linaje , Fosforilación/efectos de los fármacos , Dominios Proteicos , Factores de Transcripción STAT/metabolismo , Transducción de Señal/genética , TYK2 Quinasa/metabolismo , Adulto JovenRESUMEN
IGFs are critical for normal intrauterine and childhood growth and sustaining health throughout life. We showed previously that the production of IGF-1 and IGF-2 requires interaction with the chaperone glucose-regulated protein 94 (GRP94) and that the amount of secreted IGFs is proportional to the GRP94 activity. Therefore, we tested the hypothesis that functional polymorphisms of human GRP94 affect IGF production and thereby human health. We describe a hypomorphic variant of human GRP94, P300L, whose heterozygous carriers have 9% lower circulating IGF-1 concentration. P300L was found first in a child with primary IGF deficiency and was later shown to be a noncommon single-nucleotide polymorphism with frequencies of 1%-4% in various populations. When tested in the grp94(-/-) cell-based complementation assay, P300L supported only approximately 58% of IGF secretion relative to wild-type GRP94. Furthermore, recombinant P300L showed impaired nucleotide binding activity. These in vitro data strongly support a causal relationship between the GRP94 variant and the decreased concentration of circulating IGF-1, as observed in human carriers of P300L. Thus, mutations in GRP94 that affect its IGF chaperone activity represent a novel causal genetic mechanism that limits IGF biosynthesis, quite a distinct mechanism from the known genes in the GH/IGF signaling network.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Proteínas de la Membrana/genética , Polimorfismo de Nucleótido Simple , Somatomedinas/biosíntesis , Alelos , Análisis Mutacional de ADN , Frecuencia de los Genes , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , MutaciónRESUMEN
Protein disulfide isomerase A6 (PDIA6) interacts with protein kinase RNA-like endoplasmic reticulum kinase (PERK) and inositol requiring enzyme (IRE)-1 and inhibits their unfolded protein response signaling. In this study, shRNA silencing of PDIA6 expression in insulin-producing mouse cells reduced insulin production (5-fold) and, consequently, glucose-stimulated insulin secretion (3-4-fold). This inhibition of insulin release was independent of the PDIA6-PERK interaction or PERK activity. Acute inhibition of PERK did not change the short-term response of ß cells to glucose. Rather, PDIA6 affected insulin secretion by modulating one of the activities of IRE1. At 11 mM glucose and lower, the regulated IRE1-dependent decay (RIDD) of the mRNA activity of IRE1 was activated, but not its X-box binding protein (XBP)-1 splicing activity. In the absence of PDIA6, RIDD activity toward insulin transcripts was enhanced up to 4-fold, as shown by molecular assays in cultured cells and the use of a fluorescent reporter in intact islets. Such physiologic activation of IRE1 by glucose contrasted with IRE1 activation by chemical stress, when both IRE1 activities were induced. Thus, whereas the stimulus determines the quality of IRE1 signaling, PDIA6 attenuates multiple enzymatic activities of IRE1, maintaining its signaling within a physiologically tolerable range.
Asunto(s)
Endorribonucleasas/metabolismo , Insulina/metabolismo , Proteínas de la Membrana/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/genética , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteína Disulfuro Isomerasas/genética , Proteínas Serina-Treonina Quinasas/genética , Empalme del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción del Factor Regulador X , Tapsigargina/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , eIF-2 Quinasa/genética , eIF-2 Quinasa/metabolismoRESUMEN
In many physiological contexts, intracellular reduction-oxidation (redox) conditions and the unfolded protein response (UPR) are important for the control of cell life and death decisions. UPR is triggered by the disruption of endoplasmic reticulum (ER) homeostasis, also known as ER stress. Depending on the duration and severity of the disruption, this leads to cell adaptation or demise. In this Commentary, we review reductive and oxidative activation mechanisms of the UPR, which include direct interactions of dedicated protein disulfide isomerases with ER stress sensors, protein S-nitrosylation and ER Ca(2+) efflux that is promoted by reactive oxygen species. Furthermore, we discuss how cellular oxidant and antioxidant capacities are extensively remodeled downstream of UPR signals. Aside from activation of NADPH oxidases, mitogen-activated protein kinases and transcriptional antioxidant responses, such remodeling prominently relies on ER-mitochondrial crosstalk. Specific redox cues therefore operate both as triggers and effectors of ER stress, thus enabling amplification loops. We propose that redox-based amplification loops critically contribute to the switch from adaptive to fatal UPR.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/metabolismo , Mitocondrias/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , HumanosRESUMEN
The tight coupling of protein folding pathways with disposal mechanisms promotes the efficacy of protein production in the endoplasmic reticulum (ER). It has been hypothesized that the ER-resident molecular chaperone glucose-regulated protein 94 (GRP94) is part of this quality control coupling because it supports folding of select client proteins yet also robustly associates with the lectin osteosarcoma amplified 9 (OS-9), a component involved in ER-associated degradation (ERAD). To explore this possibility, we investigated potential functions for the GRP94/OS-9 complex in ER quality control. Unexpectedly, GRP94 does not collaborate with OS-9 in ERAD of misfolded substrates, nor is the chaperone required directly for OS-9 folding. Instead, OS-9 binds preferentially to a subpopulation of GRP94 that is hyperglycosylated on cryptic N-linked glycan acceptor sites. Hyperglycosylated GRP94 forms have nonnative conformations and are less active. As a result, these species are degraded much faster than the major, monoglycosylated form of GRP94 in an OS-9-mediated, ERAD-independent, lysosomal-like mechanism. This study therefore clarifies the role of the GRP94/OS-9 complex and describes a novel pathway by which glycosylation of cryptic acceptor sites influences the function and fate of an ER-resident chaperone.
Asunto(s)
Lectinas/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/fisiología , Procesamiento Proteico-Postraduccional , Proteolisis , Adenosina Trifosfato/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Glicosilación , Células HEK293 , Humanos , Cinética , Lectinas/química , Lisosomas/metabolismo , Glicoproteínas de Membrana/química , Proteínas de Neoplasias/química , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
The response to endoplasmic reticulum (ER) stress relies on activation of unfolded protein response (UPR) sensors, and the outcome of the UPR depends on the duration and strength of signal. Here, we demonstrate a mechanism that attenuates the activity of the UPR sensor inositol-requiring enzyme 1α (IRE1α). A resident ER protein disulfide isomerase, PDIA6, limits the duration of IRE1α activity by direct binding to cysteine 148 in the lumenal domain of the sensor, which is oxidized when IRE1 is activated. PDIA6-deficient cells hyperrespond to ER stress with sustained autophosphorylation of IRE1α and splicing of XBP1 mRNA, resulting in exaggerated upregulation of UPR target genes and increased apoptosis. In vivo, PDIA6-deficient C. elegans exhibits constitutive UPR and fails to complete larval development, a program that normally requires the UPR. Thus, PDIA6 activity provides a mechanism that limits UPR signaling and maintains it within a physiologically appropriate range.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Regulación Enzimológica de la Expresión Génica , Proteína Disulfuro Isomerasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Proliferación Celular , Cisteína/química , Disulfuros/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Inositol/química , Ratones , Datos de Secuencia Molecular , Oxígeno/química , Fosforilación , Desnaturalización Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses.
Asunto(s)
Proteínas de Choque Térmico , Glicoproteínas de Membrana , Pliegue de Proteína , Respuesta de Proteína Desplegada/genética , Animales , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/genética , Silenciador del Gen , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Células 3T3 NIH , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Transducción de SeñalRESUMEN
Insulin-like growth factors (IGFs) are critical for development and growth of skeletal muscles, but because several tissues produce IGFs, it is not clear which source is necessary or sufficient for muscle growth. Because it is critical for production of both IGF-I and IGF-II, we ablated glucose-regulated protein 94 (GRP94) in murine striated muscle to test the necessity of local IGFs for normal muscle growth. These mice exhibited smaller skeletal muscles with diminished IGF contents but with normal contractile function and no apparent endoplasmic reticulum stress response. This result shows that muscles rely on GRP94 primarily to support local production of IGFs, a pool that is necessary for normal muscle growth. In addition, body weights were â¼30% smaller than those of littermate controls, and circulating IGF-I also decreased significantly, yet glucose homeostasis was maintained with little disruption to the growth hormone pathway. The growth defect was complemented on administration of recombinant IGF-I. Thus, unlike liver production of IGF-I, muscle IGF-I is necessary not only locally but also globally for whole-body growth.
Asunto(s)
Crecimiento , Glicoproteínas de Membrana/fisiología , Músculo Esquelético/crecimiento & desarrollo , Somatomedinas/antagonistas & inhibidores , Animales , Glucemia/análisis , Células Cultivadas , Inmunohistoquímica , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Somatomedinas/biosíntesisRESUMEN
Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.
Asunto(s)
Diseño de Fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Drosophila/efectos de los fármacos , Drosophila/crecimiento & desarrollo , Células HEK293 , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Proteínas de la Membrana/química , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptores Toll-Like/metabolismoRESUMEN
Glucose-regulated protein 94 is the HSP90-like protein in the lumen of the endoplasmic reticulum and therefore it chaperones secreted and membrane proteins. It has essential functions in development and physiology of multicellular organisms, at least in part because of this unique clientele. GRP94 shares many biochemical features with other HSP90 proteins, in particular its domain structure and ATPase activity, but also displays distinct activities, such as calcium binding, necessitated by the conditions in the endoplasmic reticulum. GRP94's mode of action varies from the general HSP90 theme in the conformational changes induced by nucleotide binding, and in its interactions with co-chaperones, which are very different from known cytosolic co-chaperones. GRP94 is more selective than many of the ER chaperones and the basis for this selectivity remains obscure. Recent development of molecular tools and functional assays has expanded the spectrum of clients that rely on GRP94 activity, but it is still not clear how the chaperone binds them, or what aspect of folding it impacts. These mechanistic questions and the regulation of GRP94 activity by other proteins and by post-translational modification differences pose new questions and present future research avenues. This article is part of a Special Issue entitled: Heat Shock Protein 90 (HSP90).
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Pliegue de ProteínaRESUMEN
A system of endoplasmic reticulum (ER) chaperones has evolved to optimize the output of properly folded secretory and membrane proteins. An important player in this network is Glucose Regulated Protein 94 (GRP94). Over the last decade, new structural and functional data have begun to delineate the unique characteristics of GRP94 and have solidified its importance in ER quality control pathways. This review describes our current understanding of GRP94 and the four ways in which it contributes to the ER quality control: (1) chaperoning the folding of proteins; (2) interacting with other components of the ER protein folding machinery; (3) storing calcium; and (4) assisting in the targeting of malfolded proteins to ER-associated degradation (ERAD).