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1.
Elife ; 92020 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-32614325

RESUMEN

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.


Asunto(s)
Retículo Endoplásmico/metabolismo , Inmunidad Innata , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Proteómica
2.
Elife ; 82019 03 14.
Artículo en Inglés | MEDLINE | ID: mdl-30869076

RESUMEN

How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (µs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem.


Asunto(s)
Estrés del Retículo Endoplásmico , Células Epiteliales/fisiología , Proteínas de Choque Térmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Degradación Asociada con el Retículo Endoplásmico , Células HeLa , Humanos , Proteostasis , Respuesta de Proteína Desplegada
3.
EMBO J ; 37(4)2018 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-29378775

RESUMEN

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of ß-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions.


Asunto(s)
Adenocarcinoma/metabolismo , Colon/metabolismo , Neoplasias del Colon/metabolismo , Degradación Asociada con el Retículo Endoplásmico , Regulación de la Expresión Génica , Proteínas Wnt/metabolismo , Aciltransferasas/genética , Aciltransferasas/metabolismo , Adenocarcinoma/genética , Células Cultivadas , Neoplasias del Colon/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Proteína que Contiene Valosina/genética , Proteína que Contiene Valosina/metabolismo , Proteínas Wnt/genética
4.
Elife ; 62017 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-29251598

RESUMEN

Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µs), is well-tolerated in HeLa cells. Upon µs expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µs model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.


Asunto(s)
Proteínas de Choque Térmico/metabolismo , Inmunoglobulina M/metabolismo , Transducción de Señal , Respuesta de Proteína Desplegada , Chaperón BiP del Retículo Endoplásmico , Células Epiteliales/metabolismo , Células HeLa , Humanos
5.
Elife ; 3: e05031, 2014 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-25549299

RESUMEN

Insufficient protein-folding capacity in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). In the ER lumen, accumulation of unfolded proteins activates the transmembrane ER-stress sensor Ire1 and drives its oligomerization. In the cytosol, Ire1 recruits HAC1 mRNA, mediating its non-conventional splicing. The spliced mRNA is translated into Hac1, the key transcription activator of UPR target genes that mitigate ER-stress. In this study, we report that oligomeric assembly of the ER-lumenal domain is sufficient to drive Ire1 clustering. Clustering facilitates Ire1's cytosolic oligomeric assembly and HAC1 mRNA docking onto a positively charged motif in Ire1's cytosolic linker domain that tethers the kinase/RNase to the transmembrane domain. By the use of a synthetic bypass, we demonstrate that mRNA docking per se is a pre-requisite for initiating Ire1's RNase activity and, hence, splicing. We posit that such step-wise engagement between Ire1 and its mRNA substrate contributes to selectivity and efficiency in UPR signaling.


Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Estrés del Retículo Endoplásmico , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Represoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arginina/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Análisis por Conglomerados , Secuencia Conservada , Citosol/metabolismo , Glicoproteínas de Membrana/química , Modelos Biológicos , Datos de Secuencia Molecular , Multimerización de Proteína , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Proteínas Represoras/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Relación Estructura-Actividad , Respuesta de Proteína Desplegada
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