RESUMEN
GRP170 (Hyou1) is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds nonnative proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of mouse embryonic fibroblasts obtained from mice in which LoxP sites were engineered in the Hyou1 loci (Hyou1LoxP/LoxP). A doxycycline-regulated Cre recombinase was stably introduced into these cells. Induction of Cre resulted in depletion of Grp170 protein which culminated in cell death. As Grp170 levels fell we observed a portion of BiP fractionating with insoluble material, increased binding of BiP to a client with a concomitant reduction in its turnover, and reduced solubility of an aggregation-prone BiP substrate. Consistent with disrupted BiP functions, we observed reactivation of BiP and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and provide hypotheses as to why mutations in the Hyou1 locus are linked to human disease.
Asunto(s)
Desarrollo Embrionario , Chaperón BiP del Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico , Animales , Humanos , Ratones , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
GRP170, a product of the Hyou1 gene, is required for mouse embryonic development, and its ablation in kidney nephrons leads to renal failure. Unlike most chaperones, GRP170 is the lone member of its chaperone family in the ER lumen. However, the cellular requirement for GRP170, which both binds non-native proteins and acts as nucleotide exchange factor for BiP, is poorly understood. Here, we report on the isolation of embryonic fibroblasts from mice in which LoxP sites were engineered in the Hyou1 loci ( Hyou1 LoxP/LoxP ). A doxycycline-regulated Cre recombinase was also stably introduced into these cells. Induction of Cre resulted in excision of Hyou1 and depletion of Grp170 protein, culminating in apoptotic cell death. As Grp170 levels fell we observed increased steady-state binding of BiP to a client, slowed degradation of a misfolded BiP substrate, and BiP accumulation in NP40-insoluble fractions. Consistent with disrupted BiP functions, we observed reactivation of BiP storage pools and induction of the unfolded protein response (UPR) in futile attempts to provide compensatory increases in ER chaperones and folding enzymes. Together, these results provide insights into the cellular consequences of controlled Grp170 loss and insights into mutations in the Hyou1 locus and human disease.
RESUMEN
Antibody monomers are produced from two immunoglobulin heavy chains and two light chains that are folded and assembled in the endoplasmic reticulum This process is assisted and monitored by components of the endoplasmic reticulum quality control machinery; an outcome made more fraught by the unusual genetic machinations employed to produce a seemingly unlimited antibody repertoire. Proper functioning of the adaptive immune system is as dependent on the success of this operation, as it is on the ability to identify and degrade those molecules that fail to reach their native state. In this study, two rate-limiting steps were identified in the degradation of a non-secreted κ light chain. Both focus on the constant domain (CL), which has evolved to fold rapidly and very stably to serve as a catalyst for the folding of the heavy chain CH1 domain. The first hurdle is the reduction of the disulfide bond in the CL domain, which is required for retrotranslocation to the cytosol. In spite of being reduced, the CL domain retains structure, giving rise to the second rate-limiting step, the unfolding of this domain at the proteasome, which results in a stalled degradation intermediate.
RESUMEN
Pediatric osteosarcomas (OS) exhibit extensive genomic instability that has complicated the identification of new targeted therapies. We found the vast majority of 108 patient tumor samples and patient-derived xenografts (PDXs), which display an unusually dilated endoplasmic reticulum (ER), have reduced expression of four COPII vesicle components that trigger aberrant accumulation of procollagen-I protein within the ER. CRISPR activation technology was used to increase the expression of two of these, SAR1A and SEC24D, to physiological levels. This was sufficient to resolve the dilated ER morphology, restore collagen-I secretion, and enhance secretion of some extracellular matrix (ECM) proteins. However, orthotopic xenograft growth was not adversely affected by restoration of only SAR1A and SEC24D. Our studies reveal the mechanism responsible for the dilated ER that is a hallmark characteristic of OS and identify a highly conserved molecular signature for this genetically unstable tumor. Possible relationships of this phenotype to tumorigenesis are discussed.
RESUMEN
BiP co-chaperones ERdj4, ERdj5, and GRP170 associate in cells with peptides predicted to be aggregation prone. Here, extending these findings to a full-length protein, we examine two Interstitial Lung Disease-associated mutants (ILD) of surfactant protein C (SP-C). The TANGO algorithm, which identifies sequences prone to formation of ß strand aggregates, found three such regions in SP-C: the N-terminal transmembrane (TM) domain and two sites in the intermolecular chaperone BRICHOS domain. We show the ILD mutants disrupt di-sulfide bond formation in the BRICHOS domain and expose the aggregation-prone peptides leading to binding of ERdj4, ERdj5, and GRP170. The destabilized mutant BRICHOS domain fails to properly insert its TM region in the ER membrane, exposing part of the N-terminal TM domain site. Our studies with ILD-associated mutant proteins provide insights into the specificity of ERdj4, ERdj5, and GRP170, identify context-dependent differences in their binding, and reveal molecular consequences of disease-associated mutants on folding.
Asunto(s)
Chaperonas Moleculares , Proteína C Asociada a Surfactante Pulmonar , Sitios de Unión , Chaperonas Moleculares/metabolismo , Mutación , Unión Proteica , Pliegue de Proteína , Proteína C Asociada a Surfactante Pulmonar/metabolismoRESUMEN
Protein maturation in the endoplasmic reticulum is controlled by multiple chaperones, but how they recognize and determine the fate of their clients remains unclear. We developed an in vivo peptide library covering substrates of the ER Hsp70 system: BiP, Grp170, and three of BiP's DnaJ-family co-factors (ERdj3, ERdj4, and ERdj5). In vivo binding studies revealed that sites for pro-folding chaperones BiP and ERdj3 were frequent and dispersed throughout the clients, whereas Grp170, ERdj4, and ERdj5 specifically recognized a distinct type of rarer sequence with a high predicted aggregation potential. Mutational analyses provided insights into sequence recognition characteristics for these pro-degradation chaperones, which could be readily introduced or disrupted, allowing the consequences for client fates to be determined. Our data reveal unanticipated diversity in recognition sequences for chaperones; establish a sequence-encoded interplay between protein folding, aggregation, and degradation; and highlight the ability of clients to co-evolve with chaperones, ensuring quality control.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Proteínas del Choque Térmico HSP40/química , Proteínas HSP70 de Choque Térmico/química , Proteínas de Choque Térmico/química , Proteínas de la Membrana/química , Chaperonas Moleculares/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Chaperón BiP del Retículo Endoplásmico , Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Biblioteca de Péptidos , Unión Proteica , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Alineación de Secuencia , Transfección , TransgenesRESUMEN
BACKGROUND: The unfolded protein response (UPR) is regulated by three ER-localized, transmembrane signal transducers that control distinct aspects of the UPR. We previously reported that both increased resistance to etoposide and a reduction in Topoisomerase IIα protein levels were a direct response of UPR activation, and the latter occurred independent of changes in Topo IIα mRNA levels. We have now examined the contribution of each of the three up-stream transducers of the UPR, as well as some of their downstream targets in affecting decreased expression of Topo IIα protein and increased drug resistance. PRINCIPAL FINDINGS: Our data revealed that while Ire1 activation led to Topo IIα loss at the protein level it did not contribute to changes in sensitivity to etoposide. The decreased expression of Topo IIα protein was not downstream of XBP-1, in keeping with the fact that Topo IIα transcription was not affected by ER stress. Conversely, PERK activation did not contribute to changes in Topo IIα protein levels, but it did play a significant role in the UPR-induced decreased sensitivity to etoposide. Several cellular responses downstream of PERK were examined for their potential to contribute to resistance. The ATF6 arm of the UPR did not significantly contribute to etoposide resistance within the time frame of our experiments. CONCLUSIONS AND SIGNIFICANCE: In toto, our data demonstrate that UPR-induced changes in Topo IIα protein levels are not responsible for resistance to etoposide as has been previously hypothesized, and instead demonstrate that the PERK branch plays a Topo IIα-independent role in altered sensitivity to this drug.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Etopósido/farmacología , Respuesta de Proteína Desplegada , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Proteínas de Unión al ADN/genética , Resistencia a Medicamentos , Embrión de Mamíferos/citología , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Células 3T3 NIH , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción del Factor Regulador X , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tapsigargina/farmacología , Inhibidores de Topoisomerasa II/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , eIF-2 Quinasa/genéticaRESUMEN
In response to terminal differentiation signals that enable B cells to produce vast quantities of antibodies, a dramatic expansion of the secretory pathway and a corresponding increase in the molecular chaperones and folding enzymes that aid and monitor immunoglobulin synthesis occurs. Recent studies reveal that the unfolded protein response (UPR), which is normally activated by endoplasmic reticulum (ER) stress, plays a critical role in this process. Although B cells activate all three branches of the UPR in response to pharmacological inducers of the pathway, plasma cell differentiation elicits only a partial UPR in which components of the PKR-like ER kinase (PERK) branch are not expressed. This prompted us to further characterize UPR activation during plasma cell differentiation. We found that in response to lipopolysaccharides (LPS)-induced differentiation of the I.29 micro(+) B cell line, Ire1 was activated early, which led to splicing of XBP-1. PERK was partially phosphorylated with similar kinetics, but this was not sufficient to activate its downstream target eIF-2alpha, which initiates translation arrest, or to induce other targets like CHOP or GADD34. Both of these events preceded increased Ig synthesis, arguing this is not the signal for activating these two transducers. Targets of activating transcription factor 6 (ATF6) were up-regulated considerably later, arguing that the ATF6 branch is activated by a distinct signal. Pretreatment with LPS inhibited activation of the PERK branch by pharmacological inducers of the UPR, suggesting that differentiation-induced signals specifically silence this branch. This unique ability to differentially regulate various branches of the UPR allows B cells to accomplish distinct outcomes via the same UPR machinery.
Asunto(s)
Diferenciación Celular/fisiología , Retículo Endoplásmico/fisiología , Células Plasmáticas/citología , Células Plasmáticas/fisiología , Respuesta de Proteína Desplegada/fisiología , eIF-2 Quinasa/metabolismo , Factor de Transcripción Activador 6/genética , Factor de Transcripción Activador 6/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Femenino , Lipopolisacáridos/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Células Plasmáticas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción del Factor Regulador X , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 de Unión a la X-Box , eIF-2 Quinasa/genéticaRESUMEN
A growing body of literature demonstrates that the unfolded protein response (UPR) is activated in a number of tumors. Recent data suggests that the UPR may play a role in protecting transformed cells from the inadequate environment that exists prior to vascularization and therefore contribute to tumor growth and survival. In addition, data from cell culture based studies demonstrate that pharmacological activation of the UPR can alter the sensitivity of cells to chemotherapeutic agents, making them either more sensitive in some cases or more resistant in others. Thus, it will be important to understand and integrate data on the role of the UPR in tumor growth and survival with studies on the effects of UPR activation on chemosensitivity before considering therapeutic interventions into this signaling pathway.