Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.</p

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.</p

Deubiquitinating enzyme amino acid profiling reveals a class of ubiquitin esterases

The reversibility of ubiquitination by the action of deubiquitinating enzymes (DUBs) serves as an important regulatory layer within the ubiquitin system. Approximately 100 DUBs are encoded by the human genome, and many have been implicated with pathologies, including neurodegeneration and cancer. Non-lysine ubiquitination is chemically distinct, and its physiological importance is emerging. Here, we couple chemically and chemoenzymatically synthesized ubiquitinated lysine and threonine model substrates to a mass spectrometry-based DUB assay. Using this platform, we profile two-thirds of known catalytically active DUBs for threonine esterase and lysine isopeptidase activity and find that most DUBs demonstrate dual selectivity. However, with two anomalous exceptions, the ovarian tumor domain DUB class demonstrates specific (iso)peptidase activity. Strikingly, we find the Machado–Joseph disease (MJD) class to be unappreciated non-lysine DUBs with highly specific ubiquitin esterase activity rivaling the efficiency of the most active isopeptidases. Esterase activity is dependent on the canonical catalytic triad, but proximal hydrophobic residues appear to be general determinants of non-lysine activity. Our findings also suggest that ubiquitin esters have appreciable cellular stability and that non-lysine ubiquitination is an integral component of the ubiquitin system. Its regulatory sophistication is likely to rival that of canonical ubiquitination.We thank Axel Knebel, Richard Ewan, Clare Johnson, and Daniel Fountaine from the Medical Research Council (MRC) Protein Production and Assay Development team, and MRC Reagents and Services, who all contributed to the generation of protein reagents required for the MALDI-TOF DUB assay platform. We thank Ronald Hay for provision of the plasmid encoding the constitutively active RNF4 E3 ligase. This work was funded by the United Kingdom MRC (MC_UU_12016/8), the Biotechnology and Biological Sciences Research Council (BB/P003982/1), and The Michael J. Fox Foundation (12756). We also acknowledge pharmaceutical companies supporting the Division of Signal Transduction Therapy (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA).Peer reviewe

Discovery and characterization of non-canonical E2 conjugating enzymes

E2 conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation is fundamental for maintaining cellular homeostasis and impacts almost all cellular process. By interacting with multiple E3 ligases, E2s direct the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a post-translational modification that specifically targets lysine side chains (canonical ubiquitylation). We used MALDI-TOF Mass Spectrometry to discover and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We employed protein modelling and prediction tools to identify the catalytic determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results join a stream of recent literature that challenges the definition of ubiquitylation as an exquisitely lysine-specific modification and provide crucial insights into the missing E2 element responsible for non-canonical ubiquitylation.<br/

Design and high-throughput implementation of MALDI-TOF/MS-based assays for Parkin E3 ligase activity

Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Loss-of-function mutations in the gene encoding the ubiquitin E3 ligase Parkin are causal for familial and juvenile PD. Among several therapeutic approaches being explored to treat or improve the prognosis of patients with PD, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high-throughput platforms for the robust and accurate quantification of Parkin activity in vitro. Here, we present two different and complementary Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF/MS)-based approaches for the quantification of Parkin E3 ligase activity in vitro. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators

Discovery and characterization of noncanonical E2-conjugating enzymes

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.</p

Discovery and characterization of noncanonical E2-conjugating enzymes

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.</p