Generation of a proteolytic signal: E3/E2-mediated polyubiquitination of IκBα.

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.

A snapshot of ubiquitin chain elongation: lysine 48-tetra-ubiquitin slows down ubiquitination.

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.

Selective recruitment of an E2~ubiquitin complex by an E3 ubiquitin ligase.

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 E2∼ubiquitin-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 CDC34∼ubiquitin 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 E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.

Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-kappaB activation in mouse embryonic stem cells.

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.

Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate.

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.

Virus-induced gene silencing in Medicago truncatula and Lathyrus odorata.

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.