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.

Tank-binding kinase (TBK)1 plays a central role in innate immunity: it serves as an integrator of multiple signals induced by receptormediated 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 IKKβ, 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. Phosphorylation promotes the dimerization and nuclear translocation of these transcription factors that stimulate production of type I interferons (IFNs) (1,3,5). Recent studies have identified an additional role for TBK1 in the xenophagic elimination of bacteria (6-9) and better-defined how cross-talk within the IKK family regulates innate immune response (10).Under pathological conditions, IKK-mediated pathways can also be activated inappropriately by endogenous signals, contributing to inflammatory disorders and oncogenesis (11,12). Whereas canonical IKKs have long been recognized as bridges between chronic inflammation and cancer, IKK-related kinases more recently have also been implicated in cell transformation and tumor progression (13). TBK1 has been of particular interest, given its identification both as an activator of the oncogenic AKT kinase (14-18) and as an essential factor in KRAS-driven cancers (19).TBK1 activity is regulated by phosphorylation on S172 within the classical kinase activation loop. Serine-to-alanine substitution at this position abolishes TBK1 activity, whereas the phosphomimetic mutation S172E partially restores activity to within ∼200-fold of the wild-type kinase (20). Genetic and pharmacological inhibition studies have indicated that TBK1 can be activated by IKKβ, as well as by apparent autophosphorylation (10). Additional posttranslational modifications of TBK1 lysine residues by K63-linked polyubiquitin chains have been shown to promote production of IFNs in viral infections (21).TBK1 contains a predicted ubiquitin-like domain (ULD) (22) that is located between the N-terminal kinase domain (KD) and the C-terminal scaffolding/dimerization domain (SDD), a domain arrangement that appears to be shared among the IKK family of kinases (3). Deletion or mutation of the ULD in TBK1 or IKKε severely impairs kinase activation and substrate phosphorylation in cells (22,23). Furthermore, the integrity of the ULD in IKKβ is not only required for kinase activity (24) bu...

An engineered variant of subtilisin BPN', termed subtiligase, which efficiently ligates esterified peptides in aqueous solution, was used for the complete synthesis of ribonuclease (RNase) A that contains unnatural catalytic residues. Fully active RNase A (124 residues long) was produced in milligram quantities by stepwise ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields averaging 70 percent per ligation. Variants of RNase A were produced in which the catalytic histidines at positions 12 and 119 were substituted with the unnatural amino acid 4-fluorohistidine, which has a pKa of 3.5 compared to 6.8 for histidine. Large changes in the profile of the pH as it affects rate occurred for the single and double mutants with surprisingly little change in the kcat for either the RNA cleavage or hydrolysis steps. The data indicate that these imidazoles function as general acids and bases, but that the proton transfer steps are not rate-limiting when the imidazoles are present in their correct protonation states. These studies indicate the potential of subtiligase for the blockwise synthesis of large proteins.

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with Ki values of 28, 14, 17 and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg and volume of distribution was 0.6 ± 0.2 L/kg.

An attractive approach for developing therapeutic peptides is to enhance binding to their targets by stabilizing their α-helical conformation, for example, stabilized BimBH3 peptides (BimSAHB) designed to induce apoptosis. Unexpectedly, we found that such modified peptides have reduced affinity for their targets, the pro-survival Bcl-2 proteins. We attribute this loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. Altering this network may compromise binding affinity, as in the case of the BimBH3 stapled peptide studied here. Moreover, cells exposed to these peptides do not readily undergo apoptosis, strongly indicating that BimSAHB is not inherently cell permeable.

The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP- and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBP- nor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a "conformational selection" model, in which substrate binds to the active conformer.

Peptides that inhibit binding of vascular endothelial growth factor (VEGF) to its receptors, KDR and Flt-1, have been produced using phage display. Libraries of short disulfide-constrained peptides yielded three distinct classes of peptides that bind to the receptor-binding domain of VEGF with micromolar affinities. The highest affinity peptide was also shown to antagonize VEGF-induced proliferation of primary human umbilical vascular endothelial cells. The peptides bind to a region of VEGF known to contain the contact surface for Flt-1 and the functional determinants for KDR binding. This suggests that the receptor-binding region of VEGF is a binding "hot spot" that is readily targeted by selected peptides and supports earlier assertions that phage-derived peptides frequently target protein-protein interaction sites. Such peptides may lead to the development of pharmacologically useful VEGF antagonists.

Background: Therapeutic inhibition of circulating PCSK9 reduces LDL-c levels. Results: A synthetic PCSK9-binding peptide, which restores cellular LDL receptors, was identified. Conclusion: Pep2-8 is the smallest PCSK9 inhibitor with a defined inhibitory mechanism described to date and structurally mimics the EGF(A) domain of the receptor. Significance: This work demonstrates the feasibility of developing a peptide-based inhibitor of PCSK9.