N-tert-Butoxycarbonyl-N-(2-(tritylthio)ethoxy)glycine as a Building Block for Peptide Ubiquitination.

N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.

A Multifaceted Hit-Finding Approach Reveals Novel LC3 Family Ligands.

Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.

Erratum: Diagnosing and mitigating method-based avidity artifacts that confound polyubiquitin-binding assays.

[This corrects the article DOI: 10.1016/j.bpr.2021.100033.].

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Diagnosing and mitigating method-based avidity artifacts that confound polyubiquitin-binding assays.

Polyubiquitination is a complex form of posttranslational modification responsible for the control of numerous cellular processes. Many ubiquitin-binding proteins recognize distinct polyubiquitin chain types, and these associations help drive ubiquitin-signaling pathways. There is considerable interest in understanding the specificity of ubiquitin-binding proteins; however, because of the multivalent nature of polyubiquitin, affinity measurements of these interactions that rely on affixing ubiquitin-binding proteins to a surface can display artifactual, method-dependent avidity, or "bridging." This artifact, which is distinct from biologically relevant, avid interactions with polyubiquitin, is commonplace in such polyubiquitin-binding measurements and can lead to dramatic overestimations of binding affinities for particular chain types, and thus, incorrect conclusions about specificity. Here, we use surface-based measurements of ubiquitin binding in three model systems to illustrate bridging and lay out practical ways of identifying and mitigating it. Specifically, we describe a simple fitting model that enables researchers to diagnose the severity of bridging artifacts, determine whether they can be minimized, and more accurately evaluate polyubiquitin-binding specificity.

Picomolar zinc binding modulates formation of Bcl10-nucleating assemblies of the caspase recruitment domain (CARD) of CARD9.

The caspase recruitment domain-containing protein 9 (CARD9)-B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)-CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity-a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9-CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9-CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9-CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9-Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Conformational dynamics control ubiquitin-deubiquitinase interactions and influence in vivo signaling.

Ubiquitin is a highly conserved eukaryotic protein that interacts with a diverse set of partners to act as a cellular signaling hub. Ubiquitin's conformational flexibility has been postulated to underlie its multifaceted recognition. Here we use computational and library-based means to interrogate core mutations that modulate the conformational dynamics of human ubiquitin. These ubiquitin variants exhibit increased affinity for the USP14 deubiquitinase, with concomitantly reduced affinity for other deubiquitinases. Strikingly, the kinetics of conformational motion are dramatically slowed in these variants without a detectable change in either the ground state fold or excited state population. These variants can be ligated into substrate-linked chains in vitro and in vivo but cannot solely support growth in eukaryotic cells. Proteomic analyses reveal nearly identical interaction profiles between WT ubiquitin and the variants but identify a small subset of altered interactions. Taken together, these results show that conformational dynamics are critical for ubiquitin-deubiquitinase interactions and imply that the fine tuning of motion has played a key role in the evolution of ubiquitin as a signaling hub.

Recent insights into the complexity of Tank-binding kinase 1 signaling networks: the emerging role of cellular localization in the activation and substrate specificity of TBK1.

Tank-binding kinase 1 (TBK1) serves as an important component of multiple signaling pathways. While the majority of research on TBK1 has focused on its role in innate immunity, critical functions for TBK1 in autophagy and cancer are beginning to emerge. This review highlights recent structural and biochemical studies that provide insights into the molecular mechanism of TBK1 activation and summarizes what is known to date about TBK1 substrate selection. Growing evidence suggests that both processes rely on TBK1 subcellular localization, with a variety of adaptor proteins each directing TBK1 to discrete signaling complexes for different cellular responses. Further study of TBK1-mediated pathways will require careful consideration of TBK1 mechanisms of activation and specificity for proper dissection of these distinct signaling cascades.

Conformational stabilization of ubiquitin yields potent and selective inhibitors of USP7.

Protein conformation and function are often inextricably linked, such that the states a protein adopts define its enzymatic activity or its affinity for various partners. Here we combine computational design with macromolecular display to isolate functional conformations of ubiquitin that tightly bind the catalytic core of the oncogenic ubiquitin-specific protease 7 (USP7) deubiquitinase. Structural and biochemical characterization of these ubiquitin variants suggest that remodeled backbone conformations and core packing poise these molecules for stronger interactions, leading to potent and specific inhibition of enzymatic activity. A ubiquitin variant expressed in human tumor cell lines binds and inhibits endogenous USP7, thereby enhancing Mdm2 proteasomal turnover and stabilizing p53. In sum, we have developed an approach to rationally target macromolecular libraries toward the remodeling of protein conformation, shown that engineering of ubiquitin conformation can greatly increase its interaction with deubiquitinases and developed powerful tools to probe the cellular role of USP7.

Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation.

Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptor-mediated pathogen detection and as a modulator of IFN levels. Efforts to better understand the biology of this key immunological factor have intensified recently as growing evidence implicates aberrant TBK1 activity in a variety of autoimmune diseases and cancers. Nevertheless, key molecular details of TBK1 regulation and substrate selection remain unanswered. Here, structures of phosphorylated and unphosphorylated human TBK1 kinase and ubiquitin-like domains, combined with biochemical studies, indicate a molecular mechanism of activation via transautophosphorylation. These TBK1 structures are consistent with the tripartite architecture observed recently for the related kinase IKKß, but domain contributions toward target recognition appear to differ for the two enzymes. In particular, both TBK1 autoactivation and substrate specificity are likely driven by signal-dependent colocalization events.

Preparation of distinct ubiquitin chain reagents of high purity and yield.

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.

Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7.

Microtubules have pivotal roles in fundamental cellular processes and are targets of antitubulin chemotherapeutics. Microtubule-targeted agents such as Taxol and vincristine are prescribed widely for various malignancies, including ovarian and breast adenocarcinomas, non-small-cell lung cancer, leukaemias and lymphomas. These agents arrest cells in mitosis and subsequently induce cell death through poorly defined mechanisms. The strategies that resistant tumour cells use to evade death induced by antitubulin agents are also unclear. Here we show that the pro-survival protein MCL1 (ref. 3) is a crucial regulator of apoptosis triggered by antitubulin chemotherapeutics. During mitotic arrest, MCL1 protein levels decline markedly, through a post-translational mechanism, potentiating cell death. Phosphorylation of MCL1 directs its interaction with the tumour-suppressor protein FBW7, which is the substrate-binding component of a ubiquitin ligase complex. The polyubiquitylation of MCL1 then targets it for proteasomal degradation. The degradation of MCL1 was blocked in patient-derived tumour cells that lacked FBW7 or had loss-of-function mutations in FBW7, conferring resistance to antitubulin agents and promoting chemotherapeutic-induced polyploidy. Additionally, primary tumour samples were enriched for FBW7 inactivation and elevated MCL1 levels, underscoring the prominent roles of these proteins in oncogenesis. Our findings suggest that profiling the FBW7 and MCL1 status of tumours, in terms of protein levels, messenger RNA levels and genetic status, could be useful to predict the response of patients to antitubulin chemotherapeutics.

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.