RESUMEN
We describe a mechanistic model of polyubiquitination by the SCF(beta TrCP2) E3 ubiquitin (Ub) ligase using human I kappaB alpha as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of I kappaB alpha began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to I kappaB alpha K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at I kappaB alpha K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF(beta TrCP2) and the substrate's N terminus. The I kappaB alpha-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF's RING subcomplex. Taken together, these results suggest a role for the multifaceted interactions between the I kappaB alpha K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34 approximately S approximately Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.
Asunto(s)
Proteínas Ligasas SKP Cullina F-box/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Ciclosoma-Complejo Promotor de la Anafase , Biocatálisis , Línea Celular Tumoral , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , ARN Interferente Pequeño/genética , Especificidad por Sustrato , Enzimas Ubiquitina-Conjugadoras/genética , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and ß-catenin by the Skp1-cullin 1-ßTrCP F-box protein (SCF(ßTrCP)) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF(ßTrCP) formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using "elongation intermediates" containing ß-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF(ßTrCP)/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.
Asunto(s)
Lisina/metabolismo , Extensión de la Cadena Peptídica de Translación , Poliubiquitina/biosíntesis , Ubiquitinación , Células HEK293 , Humanos , Proteínas I-kappa B/metabolismo , Lisina/genética , Proteína NEDD8 , Inhibidor NF-kappaB alfa , Poliubiquitina/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitinas/metabolismo , beta Catenina/metabolismoRESUMEN
RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2â¼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34â¼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2â¼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.
Asunto(s)
Proteínas Portadoras/química , Complejos de Ubiquitina-Proteína Ligasa/química , Ubiquitina-Proteína Ligasas/química , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Humanos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Virus-induced gene silencing (VIGS) has become an important reverse genetics tool for functional genomics. VIGS vectors based on Pea early browning virus (PEBV, genus Tobravirus) and Bean pod mottle virus (genus Comovirus) are available for the legume species Pisum sativum and Glycine max, respectively. With the aim of extending the application of the PEBV VIGS vector to other legumes, we examined susceptibility of 99 accessions representing 24 legume species including 21 accessions of Medicago truncatula and 38 accessions Lotus japonicus. Infectivity of PEBV was tested by agro-inoculation with a vector carrying the complete beta-glucuronidase (GUS) coding sequence. In situ histochemical staining analysis indicated that 4 of 21 M. truncatula and three of three Lathyrus odorata accessions were infected systemically by GUS tagged PEBV, while none of 38 L. japonicus accessions displayed GUS staining of either inoculated or uninoculated leaves. Agro-inoculation of plants representing PEBV-GUS susceptible M. truncatula and L. odorata accessions with PEBV carrying a fragment of Phytoene desaturase (PDS) resulted in development of a bleaching phenotype suggesting a down-regulation of PDS expression. In M. truncatula this was supported by quantification of PDS mRNA levels by real-time PCR.
Asunto(s)
Silenciador del Gen , Vectores Genéticos , Glucuronidasa/metabolismo , Lathyrus/virología , Medicago truncatula/virología , Virus de Plantas , Virus ARN , ARN Mensajero/metabolismo , Regulación de la Expresión Génica de las Plantas , Glucuronidasa/genética , Lathyrus/genética , Lathyrus/metabolismo , Lotus/genética , Lotus/metabolismo , Lotus/virología , Medicago truncatula/genética , Medicago truncatula/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Virus de Plantas/genética , Virus de Plantas/metabolismo , Virus de Plantas/patogenicidad , Plantas Modificadas Genéticamente , Virus ARN/genética , Virus ARN/metabolismo , Virus ARN/patogenicidad , ARN Mensajero/genéticaRESUMEN
A key regulatory node in NF-κB signaling is the removal of the IκBα inhibitor, whose levels are tightly controlled by the ubiquitin-proteasome system. In response to signal activation and transmission, ubiquitin E1, E2, and E3 enzymes are employed to generate a lysine 48-linked ubiquitin chain that triggers degradation of IκBα by the proteasome. In this chapter we describe an in vitro biochemical approach to reconstitute the ubiquitination system. To do so, we detail methods for the preparation of the relevant enzymes and substrate, as well as for the execution of the reaction with high efficiency. This sensitive and highly reproducible readout can be applied to the study of proteins, small molecules, and other factors that modulate IκBα ubiquitination, thereby producing outcomes that impact NF-κB signaling to advance the course of improving human health.
Asunto(s)
Proteínas I-kappa B/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Línea Celular , Humanos , Técnicas In Vitro , Inhibidor NF-kappaB alfa , Proteolisis , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB.