SPATA2 promotes CYLD activity and regulates TNF‐induced NF‐κB signaling and cell death

K63‐ and Met1‐linked ubiquitylation are crucial posttranslational modifications for TNF receptor signaling. These non‐degradative ubiquitylations are counteracted by deubiquitinases (DUBs), such as the enzyme CYLD, resulting in an appropriate signal strength, but the regulation of this process remains incompletely understood. Here, we describe an interaction partner of CYLD, SPATA2, which we identified by a mass spectrometry screen. We find that SPATA2 interacts via its PUB domain with CYLD, while a PUB interaction motif (PIM) of SPATA2 interacts with the PUB domain of the LUBAC component HOIP. SPATA2 is required for the recruitment of CYLD to the TNF receptor signaling complex upon TNFR stimulation. Moreover, SPATA2 acts as an allosteric activator for the K63‐ and M1‐deubiquitinase activity of CYLD. In consequence, SPATA2 substantially attenuates TNF‐induced NF‐κB and MAPK signaling. Conversely, SPATA2 is required for TNF‐induced complex II formation, caspase activation, and apoptosis. Thus, this study identifies SPATA2 as an important factor in the TNF signaling pathway with a substantial role for the effects mediated by the cytokine

Glucocorticoid receptor Thr524 phosphorylation by MINK1 induces interactions with 14-3-3 protein regulators

The glucocorticoid receptor (GR) is a ligand-dependent transcription factor that plays a central role in inflammation. The GR activity is also modulated via protein-protein interactions, including binding of 14-3-3 proteins induced by GR phosphorylation. However, the specific phosphorylation sites on the GR that trigger these interactions and their functional consequences are less clear. Hence, we sought to examine this system in more detail. We used phosphorylated GR peptides, biophysical studies, and X-ray crystallography to identify key residues within the ligand-binding domain of the GR, T524 and S617, whose phosphorylation results in binding of the representative 14-3-3 protein 14-3-3ζ. A kinase screen identified misshapen-like kinase 1 (MINK1) as responsible for phosphorylating T524 and Rho-associated protein kinase 1 for phosphorylating S617; cell-based approaches confirmed the importance of both GR phosphosites and MINK1 but not Rhoassociated protein kinase 1 alone in inducing GR-14-3-3 binding. Together our results provide molecular-level insight into 14-3-3-mediated regulation of the GR and highlight both MINK1 and the GR-14-3-3 axis as potential targets for future therapeutic intervention

Ligand binding mechanism in steroid receptors; from conserved plasticity to differential evolutionary constraints

Steroid receptor drugs have been available for more than half a century, but details 24 of the ligand binding mechanism has remained elusive. We solved X-ray structures of 25 the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at 26 helix 6-7 region that extend the ligand binding pocket towards the receptor surface. 27 Since none of the endogenous ligands exploit this region, we hypothesized that it 28 constitutes an integral part of the binding event. Extensive all atom unbiased ligand 29 exit and entrance simulations corroborate a ligand binding pathway that gives the 30 observed structural plasticity a key functional role. Kinetic measurements reveal that 31 the receptor residence time correlate with structural rearrangements observed in both 32 structures and simulations. Ultimately, our findings reveal why nature has conserved 33 the capacity to open up this region and highlight how differences in the details of the 34 ligand entry process result in differential evolutionary constraints across the steroid 35 receptors.This study was supported by The European Research Council (2009-Adg25027-535 PELE) to V.G and by the SEV-2011-00067 grant of the Severo Ochoa Program. We 536 would like to acknowledge our AstraZeneca colleagues J. Hartleib, R.Unwin and 537 R.Knöll for helpful discussions. We also thank N. Blomberg (ELIXIR) and R. Neutze 538 (University of Gothenburg) for careful reading of the manuscript.Peer ReviewedPostprint (author's final draft

A cell-active peptide targeting the Nrf2/Keap1 protein-protein interaction

The disruption of the protein-protein interaction (PPI) between Nrf2 and Keap1 is an attractive strategy to counteract the oxidative stress that characterises a variety of severe diseases. Peptides represent a...</jats:p

Mining Natural Products for Macrocycles to Drug Difficult Targets

Lead generation for difficult-to-drug targets that have large, featureless, and highly lipophilic or highly polar and/or flexible binding sites is highly challenging. Here, we describe how cores of macrocyclic natural products can serve as a high-quality in silico screening library that provides leads for difficult-to-drug targets. Two iterative rounds of docking of a carefully selected set of natural-product-derived cores led to the discovery of an uncharged macrocyclic inhibitor of the Keap1-Nrf2 protein- protein interaction, a particularly challenging target due to its highly polar binding site. The inhibitor displays cellular efficacy and is well-positioned for further optimization based on the structure of its complex with Keapl and synthetic access. We believe that our work will spur interest in using macrocyclic cores for in silico-based lead generation and also inspire the design of future macrocycle screening collections

Importance of Binding Site Hydration and Flexibility Revealed When Optimizing a Macrocyclic Inhibitor of the Keap1-Nrf2 Protein-Protein Interaction

Upregulation of the transcription factor Nrf2 by inhibition of the interaction with its negative regulator Keap1 constitutes an opportunity for the treatment of disease caused by oxidative stress. We report a structurally unique series of nanomolar Keap1 inhibitors obtained from a natural product-derived macrocyclic lead. Initial exploration of the structure-derived macrocyclic lead. Initial exploration of the structure-activity relationship of the lead, followed by structure-guided optimization, resulted in a 100-fold improvement in inhibitory potency. The macrocyclic core of the nanomolar inhibitors positions three pharmacophore units for productive interactions with key residues of Keap1, including R415, R483, and Y572. Ligand optimization resulted in the displacement of a coordinated water molecule from the Keap1 binding site and a significantly altered thermodynamic profile. In addition, minor reorganizations of R415 and R483 were accompanied by major differences in affinity between ligands. This study therefore indicates the importance of accounting both for the hydration and flexibility of the Keap1 binding site when designing high-affinity ligands

Combining structural and coevolution information to unveil allosteric sites

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets

Indigenous groups and pandemics

In addition to posing a major threat to global health, pandemics impact economic activity, as witnessed during the spread of COVID-19 around the globe. The disease risks, however, are not uniform for major pandemic threats. For example, risk groups for severe disease during seasonal epidemic influenza, the influenza pandemics of 1918 and 2009, and the ongoing COVID-19 pandemic are different. The 1918 and 2009 influenza pandemics largely killed young adults, while the COVID-19 pandemic has primarily killed the elderly. Indeed, age is the strongest risk factor for severe outcomes of COVID-19. Within age groups, however, persons with underlying medical risk factors, people of lower socioeconomic status, immigrants, ethnic minorities, and Indigenous peoples are at higher risk of infection, hospitalization, and death across these pandemics and epidemics, demonstrating a need for intersectional analyses and preparedness responses