RESUMEN
To maintain protein homeostasis, cells must balance protein synthesis with protein degradation. Accumulation of misfolded or partially degraded proteins can lead to the formation of pathological protein aggregates. Here we report the use of destabilizing domains, proteins whose folding state can be reversibly tuned using a high affinity ligand, as model substrates to interrogate cellular protein quality control mechanisms in mammalian cells using a forward genetic screen. Upon knockdown of UBE3C, an E3 ubiquitin ligase, a reporter protein consisting of a destabilizing domain fused to GFP is degraded more slowly and incompletely by the proteasome. Partial proteolysis is also observed when UBE3C is present but cannot ubiquitinate substrates because its active site has been mutated, it is unable to bind to the proteasome, or the substrate lacks lysine residues. UBE3C knockdown also results in less substrate polyubiquitination. Finally, knockdown renders cells more susceptible to the Hsp90 inhibitor 17-AAG, suggesting that UBE3C protects against the harmful accumulation of protein fragments arising from incompletely degraded proteasome substrates.
Asunto(s)
Pliegue de Proteína , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/genética , Benzoquinonas/farmacología , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Lactamas Macrocíclicas/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
The FKBP-derived destabilizing domains are increasingly being used to confer small molecule-dependent stability to many different proteins. The L106P domain confers instability to yellow fluorescent protein when it is fused to the N-terminus, the C-terminus, or spliced into the middle of yellow fluorescent protein, however multiple copies of L106P do not confer greater instability. These engineered destabilizing domains are not dominant to endogenous degrons that regulate protein stability.
Asunto(s)
Modelos Moleculares , Proteínas de Unión a Tacrolimus/química , Secuencias de Aminoácidos , Ciclo Celular/efectos de los fármacos , Ciclina B/metabolismo , Ciclina B1 , Células HeLa , Humanos , Relación Estructura-Actividad , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called 'destabilizing domains', whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies.