An atypical ubiquitin ligase at the heart of neural development and programmed axon degeneration

The degeneration of nerve fibres following injury was first described by Augustus Waller over 170 years ago. Initially assumed to be a passive process, it is now evident that axons respond to insult via regulated cellular signaling events resulting in their programmed degeneration. Pro-survival and pro-degenerative factors have been identified and their regulatory mechanisms are beginning to emerge. The ubiquitin system has been implicated in the pro-degenerative process and a key component is the ubiquitin E3 ligase MYCBP2 (also known as PHR1). Ubiquitin E3 ligases are tasked with the transfer of the small protein modifier ubiquitin to substrates and consist of hundreds of members. They can be classified as single subunit systems or as multi-subunit complexes. Their catalytic domains can also be assigned to three general architectures. Hints that MYCBP2 might not conform to these established formats came to light and it is now clear from biochemical and structural studies that MYCBP2 is indeed an outlier in terms of its modus operandi. Furthermore, the unconventional way in which MYCBP2 transfers ubiquitin to substrates has been linked to neurodevelopmental and pro-degenerative function. Herein, we will summarize these research developments relating to the unusual features of MYCBP2 and postulate therapeutic strategies that prevent Wallerian degeneration. These have exciting potential for providing relief from pathological neuropathies and neurodegenerative diseases

A new dawn beyond lysine ubiquitination

The ubiquitin system has become synonymous with the modification of lysine residues. However, the substrate scope and diversity of the conjugation machinery have been underappreciated, bringing us to an epoch in ubiquitin system research. The striking discoveries of metazoan enzymes dedicated toward serine and threonine ubiquitination have revealed the important role of nonlysine ubiquitination in endoplasmic reticulum-associated degradation, immune signaling and neuronal processes, while reports of nonproteinaceous substrates have extended ubiquitination beyond the proteome. Bacterial effectors that bypass the canonical ubiquitination machinery and form unprecedented linkage chemistry further redefine long-standing dogma. While chemical biology approaches have advanced our understanding of the canonical ubiquitin system, further study of noncanonical ubiquitination has been hampered by a lack of suitable tools. This Perspective aims to consolidate and contextualize recent discoveries and to propose potential applications of chemical biology, which will be instrumental in unraveling this new frontier of ubiquitin research.</p

Photocrosslinking Activity-Based Probes for Ubiquitin RING E3 Ligases

Summary: Activity-based protein profiling is an invaluable technique for studying enzyme biology and facilitating the development of therapeutics. Ubiquitin E3 ligases (E3s) are one of the largest enzyme families and regulate a host of (patho)physiological processes. The largest subtype are the RING E3s of which there are &gt;600 members. RING E3s have adaptor-like activity that can be subject to diverse regulatory mechanisms and have become attractive drug targets. Activity-based probes (ABPs) for measuring RING E3 activity do not exist. Here we re-engineer ubiquitin-charged E2 conjugating enzymes to produce photocrosslinking ABPs. We demonstrate activity-dependent profiling of two divergent cancer-associated RING E3s, RNF4 and c-Cbl, in response to their native activation signals. We also demonstrate profiling of endogenous RING E3 ligase activation in response to epidermal growth factor (EGF) stimulation. These photocrosslinking ABPs should advance E3 ligase research and the development of selective modulators against this important class of enzymes

Selective Inhibition of Cysteine-Dependent Enzymes by Bioorthogonal Tethering

A general approach for the rapid and selective inhibition of enzymes in cells using a common tool compound would be of great value for research and therapeutic development. We previously reported a chemogenetic strategy that addresses this challenge for kinases, relying on bioorthogonal tethering of a pan inhibitor to a target kinase through a genetically encoded non-canonical amino acid. However, pan inhibitors are not available for many enzyme classes. Here, we expand the scope of the chemogenetic strategy to cysteine-dependent enzymes by bioorthogonal tethering of electrophilic warheads. For proof of concept, selective inhibition of two E2 ubiquitin-conjugating enzymes, UBE2L3 and UBE2D1, was demonstrated in biochemical assays. Further development and optimization of this approach should enable its use in cells as well as other cysteine-dependent enzymes, facilitating the investigation of their cellular function and validation as therapeutic targets

An Atypical E3 Ligase Module in UBR4 Mediates Destabilization of N-degron Substrates

UBR4 is an E3 ligase (E3) of the N-degron pathway and is involved in neurodevelopment, age-associated muscular atrophy and cancer progression. The location and mechanistic classification of the E3 module within the 600 kDa protein UBR4 remains unknown. Herein, we identify and characterize, at a biochemical and structural level, a distinct E3 module within human UBR4 consisting of a novel “hemiRING” zinc finger, a helical-rich UBR Zinc-finger Interacting (UZI) subdomain, and a predicted backside interacting N-terminal helix. A structure of an E2 conjugating enzyme (E2)-E3 complex provides atomic level insight into the exquisite specificity of the hemiRING towards the E2s UBE2A/B. The UZI subdomain can be considered a component of the E3 module as it has a modest activating effect on the ubiquitin loaded E2 (E2∼Ub), which is complemented by the intrinsically high lysine reactivity of UBE2A. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3 ligase, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains associated with ubiquitin E3 activity.<br/

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

UBE2A and UBE2B are recruited by an atypical E3 ligase module in UBR4

UBR4 is a 574 kDa E3 ligase (E3) of the N-degron pathway with roles in neurodevelopment, age-associated muscular atrophy and cancer. The catalytic module that carries out ubiquitin (Ub) transfer remains unknown. Here we identify and characterize a distinct E3 module within human UBR4 consisting of a ‘hemiRING’ zinc finger, a helical-rich UBR zinc-finger interacting (UZI) subdomain, and an N-terminal region that can serve as an affinity factor for the E2 conjugating enzyme (E2). The structure of an E2–E3 complex provides atomic-level insight into the specificity determinants of the hemiRING toward the cognate E2s UBE2A/UBE2B. Via an allosteric mechanism, the UZI subdomain modestly activates the Ub-loaded E2 (E2∼Ub). We propose attenuated activation is complemented by the intrinsically high lysine reactivity of UBE2A, and their cooperation imparts a reactivity profile important for substrate specificity and optimal degradation kinetics. These findings reveal the mechanistic underpinnings of a neuronal N-degron E3, its specific recruitment of UBE2A, and highlight the underappreciated architectural diversity of cross-brace domains with Ub E3 activity.</p

Structural basis for RING-Cys-Relay E3 ligase activity and its role in axon integrity.

MYCBP2 is a ubiquitin (Ub) E3 ligase (E3) that is essential for neurodevelopment and regulates axon maintenance. MYCBP2 transfers Ub to nonlysine substrates via a newly discovered RING-Cys-Relay (RCR) mechanism, where Ub is relayed from an upstream cysteine to a downstream substrate esterification site. The molecular bases for E2-E3 Ub transfer and Ub relay are unknown. Whether these activities are linked to the neural phenotypes is also unclear. We describe the crystal structure of a covalently trapped E2~Ub:MYCBP2 transfer intermediate revealing key structural rearrangements upon E2-E3 Ub transfer and Ub relay. Our data suggest that transfer to the dynamic upstream cysteine, whilst mitigating lysine activity, requires a closed-like E2~Ub conjugate with tempered reactivity, and Ub relay is facilitated by a helix-coil transition. Furthermore, neurodevelopmental defects and delayed injury-induced degeneration in RCR-defective knock-in mice suggest its requirement, and that of substrate esterification activity, for normal neural development and programmed axon degeneration

Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability

Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage. </p