Yogesh Kulathu

OTU Deubiquitinases Reveal Mechanisms of Linkage Specificity and Enable Ubiquitin Chain Restriction Analysis

Summary 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.

OTULIN Antagonizes LUBAC Signaling by Specifically Hydrolyzing Met1-Linked Polyubiquitin

Summary The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that specifically assembles Met1-linked (also known as linear) Ub chains that regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been identified. Here, we reveal a previously unannotated human DUB, OTULIN (also known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal structures of the OTULIN catalytic domain in complex with diubiquitin reveal Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis in which the proximal Ub activates the catalytic triad of the protease. Mutation of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates Met1-polyUb signaling.

Two-sided ubiquitin binding explains specificity of the TAB2 NZF domain

The protein kinase TAK1 is activated by binding to Lys63 (K63)-linked ubiquitin chains through its subunit TAB2. Here we analyze crystal structures of the TAB2 NZF domain bound to Lys63-linked di- and triubiquitin, revealing that TAB2 binds adjacent ubiquitin moieties via two distinct binding sites. The conformational constraints imposed by TAB2 on a Lys63 dimer cannot be adopted by linear chains, explaining why TAK1 cannot be activated by linear ubiquitination events.

On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

Regulation of A20 and Other Otu Deubiquitinases by Reversible Oxidation

Protein ubiquitination is a highly versatile posttranslational modification that regulates as diverse processes as protein degradation and kinase activation. Deubiquitinases hydrolyse ubiquitin modifications from proteins and are hence key regulators of the ubiquitin system. Ovarian Tumour (OTU) deubiquitinases comprise a family of fourteen human enzymes, many of which regulate cellular signalling pathways. OTU deubiquitinases are cysteine proteases that cleave polyubiquitin (polyUb) chains in vitro and in cells, but little is currently known about their regulation. Here we show that OTU deubiquitinases are susceptible to reversible oxidation of the catalytic cysteine residue. High-resolution crystal structures of the catalytic domain of A20 in four different oxidation states reveal that the reversible form of A20 oxidation is a cysteine sulphenic acid intermediate, which is stabilised by the architecture of the catalytic centre. Using chemical tools to detect sulphenic acid intermediates, we show that many OTU deubiquitinases undergo reversible oxidation upon treatment with H2O2, revealing a new mechanism to regulate deubiquitinase activity.

The kinase Syk as an adaptor controlling sustained calcium signalling and B‐cell development

Upon B‐cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain‐containing leukocyte protein of 65 kDa (SLP‐65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP‐65 is not only a downstream target and substrate of Syk but also a direct binding‐partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP‐65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP‐65 complex are defective in BCR‐induced extracellular signal‐regulated kinase, nuclear factor κ B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B‐cell development. Furthermore, we show that formation of the Syk/SLP‐65 complex is required for sustained Ca2+ responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane‐bound Syk/SLP‐65 complex controlling sustained signalling and calcium influx.

Autoinhibition and adapter function of Syk

Summary:  Development, survival, and activation of B lymphocytes are controlled by signals emanating from the B‐cell antigen receptor (BCR). The BCR has an autonomous signaling function also known as tonic signaling that allows for long‐term survival of B cells in the immune system. Upon binding of antigen to the BCR, the tonic signal is amplified and diversified, leading to alteration in gene expression and B‐cell activation. The spleen tyrosine kinase (Syk) intimately cooperates with the signaling subunits of the BCR and plays a central role in the amplification and diversification of BCR signals. In this review, we discuss the molecular mechanisms by which Syk activity is inhibited and activated at the BCR. Importantly, Syk acts not only as a kinase that phosphorylates downstream substrates but also as an adapter that can bind to a diverse set of signaling proteins. Depending on its interactions and localization, Syk can signal opposing cell fate decisions such as proliferation or differentiation of B cells.

A leucine zipper in the N terminus confers membrane association to SLP-65

Membrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins.

MINDY-1 Is a Member of an Evolutionarily Conserved and Structurally Distinct New Family of Deubiquitinating Enzymes.

Summary Deubiquitinating enzymes (DUBs) remove ubiquitin (Ub) from Ub-conjugated substrates to regulate the functional outcome of ubiquitylation. Here we report the discovery of a new family of DUBs, which we have named MINDY (motif interacting with Ub-containing novel DUB family). Found in all eukaryotes, MINDY-family DUBs are highly selective at cleaving K48-linked polyUb, a signal that targets proteins for degradation. We identify the catalytic activity to be encoded within a previously unannotated domain, the crystal structure of which reveals a distinct protein fold with no homology to any of the known DUBs. The crystal structure of MINDY-1 (also known as FAM63A) in complex with propargylated Ub reveals conformational changes that realign the active site for catalysis. MINDY-1 prefers cleaving long polyUb chains and works by trimming chains from the distal end. Collectively, our results reveal a new family of DUBs that may have specialized roles in regulating proteostasis.

K29-Selective Ubiquitin Binding Domain Reveals Structural Basis of Specificity and Heterotypic Nature of K29 Polyubiquitin

Summary Polyubiquitin chains regulate diverse cellular processes through the ability of ubiquitin to form chains of eight different linkage types. Although detected in yeast and mammals, little is known about K29-linked polyubiquitin. Here we report the generation of K29 chains in vitro using a ubiquitin chain-editing complex consisting of the HECT E3 ligase UBE3C and the deubiquitinase vOTU. We determined the crystal structure of K29-linked diubiquitin, which adopts an extended conformation with the hydrophobic patches on both ubiquitin moieties exposed and available for binding. Indeed, the crystal structure of the NZF1 domain of TRABID in complex with K29 chains reveals a binding mode that involves the hydrophobic patch on only one of the ubiquitin moieties and exploits the flexibility of K29 chains to achieve linkage selective binding. Further, we establish methods to study K29-linked polyubiquitin and find that K29 linkages exist in cells within mixed or branched chains containing other linkages.