Chemical biology tools to study Deubiquitinases and Ubl proteases

The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs)for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function ata molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs

Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1-5 (DCNL1-5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain-containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client-substrate ubiquitylation at multiple levels

A single MIU motif of MINDY-1 recognizes K48-linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs

Deregulated Syk inhibits differentiation and induces growth factor–independent proliferation of pre–B cells

The nonreceptor protein spleen tyrosine kinase (Syk) is a key mediator of signal transduction in a variety of cell types, including B lymphocytes. We show that deregulated Syk activity allows growth factor–independent proliferation and transforms bone marrow–derived pre–B cells that are then able to induce leukemia in mice. Syk-transformed pre–B cells show a characteristic pattern of tyrosine phosphorylation, increased c-Myc expression, and defective differentiation. Treatment of Syk-transformed pre–B cells with a novel Syk-specific inhibitor (R406) reduces tyrosine phosphorylation and c-Myc expression. In addition, R406 treatment removes the developmental block and allows the differentiation of the Syk-transformed pre–B cells into immature B cells. Because R406 treatment also prevents the proliferation of c-Myc–transformed pre–B cells, our data indicate that endogenous Syk kinase activity may be required for the survival of pre–B cells transformed by other oncogenes. Collectively, our data suggest that Syk is a protooncogene involved in the transformation of lymphocytes, thus making Syk a potential target for the treatment of leukemia

OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis

Sixteen ovarian tumor (OTU) family deubiquitinases (DUBs) exist in humans, and most members regulate cell-signaling cascades. Several OTU DUBs were reported to be ubiquitin (Ub) chain linkage specific, but comprehensive analyses are missing, and the underlying mechanisms of linkage specificity are unclear. Using Ub chains of all eight linkage types, we reveal that most human OTU enzymes are linkage specific, preferring one, two, or a defined subset of linkage types, including unstudied atypical Ub chains. Biochemical analysis and five crystal structures of OTU DUBs with or without Ub substrates reveal four mechanisms of linkage specificity. Additional Ub-binding domains, the ubiquitinated sequence in the substrate, and defined S1’ and S2 Ub-binding sites on the OTU domain enable OTU DUBs to distinguish linkage types. We introduce Ub chain restriction analysis, in which OTU DUBs are used as restriction enzymes to reveal linkage type and the relative abundance of Ub chains on substrates

Mechanism of activation and regulation of Deubiquitinase activity in MINDY1 and MINDY2

Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2

A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation

Protein UFMylation, i.e., post‐translational modification with ubiquitin‐fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold‐type E3 ligase mechanism that activates the UFM1‐conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex

Comprehensive approach to study branched ubiquitin chains reveals roles for K48-K63 branches in VCP/p97-related processes

Branched ubiquitin (Ub) chains make up a significant proportion of Ub polymers in human cells and are formed when two or more sites on a single Ub molecule are modified with Ub creating bifurcated architectures. Despite their abundance, we have a poor understanding of the cellular functions of branched Ub signals that stems from a lack of facile tools and methods to study them. Here we develop a comprehensive pipeline to define branched Ub function, using K48-K63-branched chains as a case study. We discover branch-specific binders and, by developing a method that monitors cleavage of linkages within complex polyUb, we discover the VCP/p97-associated ATXN3, and MINDY family deubiquitinases to act as debranching enzymes. By engineering and utilizing a branched K48-K63-Ub chain-specific nanobody, we reveal roles for these chains in VCP/p97-related processes. In summary, we provide a blueprint to investigate branched Ub function that can be readily applied to study other branched chain types.<br/