UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN

Abstract

Ring-between-ring (RBR) E3 ligases have been implicated in autoimmune disorders and neurodegenerative diseases. The functions of many RBR E3s are poorly defined, and their regulation is complex, involving post-translational modifications and allosteric regulation with other protein partners. The functional complexity of RBRs, coupled with the complexity of the native ubiquitination reaction that requires ATP and E1 and E2 enzymes, makes it difficult to study these ligases for basic research and therapeutic purposes. To address this challenge, we developed novel chemical probes, ubiquitin C-terminal fluorescein thioesters UbMES and UbFluor, to qualitatively and quantitatively assess the activity of the RBR E3 ligase PARKIN in a simple experimental setup and in real time using fluorescence polarization. First, we confirmed that PARKIN does not require an E2 enzyme for substrate ubiquitination, lysine selection, and polyubiquitin chain formation. Second, we confirmed that UbFluor quantitatively detects naturally occurring activation states of PARKIN caused by Ser phosphorylation (pPARKIN) and phosphorylated ubiquitin (pUb). Third, we showed that both pUb and the ubiquitin-accepting substrate contribute to maximal pPARKIN ubiquitin conjugation turnover. pUb enhances the transthiolation step, whereas the substrate clears the pPARKIN∼Ub thioester intermediate. Finally, we established that UbFluor can quantify activation or inhibition of PARKIN by structural mutations. These results demonstrate the feasibility of using UbFluor for quantitative studies of the biochemistry of RBR E3s and for high-throughput screening of small-molecule activators or inhibitors of PARKIN and other RBR E3 ligases.

Miro proteins prime mitochondria for Parkin translocation and mitophagy

“…Multi-monoubiquitination of Miro1 (Kazlauskaite et al, 2014a;Klosowiak et al, 2016) and an atypical lysine-27-mediated ubiquitin chain (Birsa et al, 2014) suggest additional roles for Miro1 ubiquitination beyond degradation. Furthermore, Park et al (2017) showed recently that Miro1 enhances Parkin catalytic activity.…”

Monitoring PARKIN RBR Ubiquitin Ligase Activation States with UbFluor

“…The ubiquitin thioester probe UbFluor recapitulates the native biochemical mechanisms of E3 ligases that rely on a catalytic cysteine for covalent ubiquitin transfer, allowing continuous monitoring of ubiquitination reactions at low volumes (Krist et al, 2016; Park et al, 2017). This enables measurement of initial reaction rates for multiple PARKIN variants in combination with allosteric effector proteins and substrates.…”

“…PARKIN E3 ligase, pPARKIN, or PARKIN with point mutations (available from Boston Biochem or LifeSensors and obtainable from E.coli expression) (Park et al, 2017)…”

“…The ubiquitin thioester probe UbFluor recapitulates the native biochemical mechanisms of E3 ligases that rely on a catalytic cysteine for covalent ubiquitin transfer ( Fig. 1) (Krist, Park, Boneh, Rice, & Statsyuk, 2016;Park, Foote, Krist, Rice, & Statsyuk, 2017). By mimicking the E2ßUb thioester, UbFluor provides a stable yet reactive substrate for nucleophilic attack by the catalytic cysteine of a HECT or RBR E3 ligase.…”

How to Target Viral and Bacterial Effector Proteins Interfering with Ubiquitin Signaling

“…In these assays, the E1-and E2-functions are bypassed and Ub-thioesters [Ub-MES (Park et al 2015) or fluorescent analogue Ub-FLUOR (Krist et al 2016)] are directly trans-thiolated by the active cysteines in several E3's. The fluorescent thioester allows real-time quantitative monitoring of E3-ligase activity using a fluorescence polarization set up (Park et al 2017) (see Fig. 3d).…”