The small GTase Arf6 has several important functions in intracellular vesicular trafficking and regulates the recycling of different types of cargo internalized via clathrin-dependent or -independent ...endocytosis. It activates the lipid modifying enzymes PIP 5-kinase and phospholipase D, promotes actin polymerization, and affects several functionally distinct processes in the cell. Arf6 is used for the phagocytosis of pathogens and can be directly or indirectly targeted by various pathogens to block phagocytosis or induce the uptake of intracellular pathogens. Arf6 is also used in the signaling of Toll-like receptors and in the activation of NADPH oxidases. In this review, we first give an overview of the different roles and mechanisms of action of Arf6 and then focus on its role in innate immunity and host-pathogen interactions.
The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma ...and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.
Members of the Toll-like receptor and interleukin-1 (IL-1) receptor families all signal via Toll/IL-1R (TIR) domain-driven assemblies with adaptors such as MyD88. We here combine the mammalian ...two-hybrid system MAPPIT and saturation mutagenesis to complement and extend crystallographic and nuclear magnetic resonance data, and reveal how TIR domains interact. We fully delineate the interaction sites on the MyD88 TIR domain for homo-oligomerization and for interaction with Mal and TLR4. Interactions between three sites drive MyD88 homo-oligomerization. The BB-loop interacts with the αE-helix, explaining how BB-loop mimetics inhibit MyD88 signaling. The αC′-helix interacts symmetrically. The MyD88 TIR domains thus assemble into a left-handed helix, compatible with the Myddosome death domain crystal structure. This assembly explains activation of MyD88 by Mal and by an oncogenic mutation, and regulation by phosphorylation. These findings provide a paradigm for the interaction of mammalian TIR domains.
•We fully delineate four interaction sites in the MyD88 TIR domain•Three binding sites lead to a symmetrical and an asymmetrical MyD88 TIR homodimer•The symmetrical dimer is regulated by phosphorylation and binding to Mal•MyD88 TIR assembles into a helical oligomer compatible with the Myddosome structure
Vyncke et al. fully delineate four interaction sites in the MyD88 TIR domain. Three sites lead to a helical MyD88 TIR complex, regulated by phosphorylation. A symmetrical MyD88 TIR homodimer forms a composite fourth binding site for Mal.
The MAPPIT (mammalian protein protein interaction trap) method allows high-throughput detection of protein interactions by very simple co-transfection of three plasmids in HEK293T cells, followed by ...a luciferase readout. MAPPIT detects a large percentage of all protein interactions, including those requiring posttranslational modifications and endogenous or exogenous ligands. Here, we present a straightforward method that allows detailed mapping of interaction interfaces via MAPPIT. The method provides insight into the interaction mechanism and reveals how this is affected by disease-associated mutations. By combining error-prone polymerase chain reaction (PCR) for random mutagenesis, 96-well DNA prepping, Sanger sequencing, and MAPPIT via 384-well transfections, we test the effects of a large number of mutations of a selected protein on its protein interactions. The entire screen takes less than three months and interactions with multiple partners can be studied in parallel. The effect of mutations on the MAPPIT readout is mapped on the protein structure, allowing unbiased identification of all putative interaction sites. We have thus far analysed 6 proteins and mapped their interfaces for 16 different interaction partners. Our method is broadly applicable as the required tools are simple and widely available.
After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of ...evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential.
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Protein mutagenesis is essential for unveiling the molecular mechanisms underlying protein function in health, disease, and evolution. In the past decade, deep mutational scanning methods have ...evolved to support the functional analysis of nearly all possible single-amino acid changes in a protein of interest. While historically these methods were developed in lower organisms such as E. coli and yeast, recent technological advancements have resulted in the increased use of mammalian cells, particularly for studying proteins involved in human disease. These advancements will aid significantly in the classification and interpretation of variants of unknown significance, which are being discovered at large scale due to the current surge in the use of whole-genome sequencing in clinical contexts. Here, we explore the experimental aspects of deep mutational scanning studies in mammalian cells and report the different methods used in each step of the workflow, ultimately providing a useful guide toward the design of such studies.
The mechanisms controlling the steady-state cell surface levels of cytokine receptors, and consequently the cellular response to cytokines, remain poorly understood. The number of surface-exposed ...receptors is a dynamic balance of de novo synthesis, transport to the plasma membrane, internalization, recycling, degradation and ectodomain shedding. We previously reported that the E3 ubiquitin ligase RING finger protein 41 (RNF41) inhibits basal lysosomal degradation and enhances ectodomain shedding of JAK2-associated cytokine receptors. Ubiquitin-specific protease 8 (USP8), an RNF41-interacting deubiquitylating enzyme (DUB) stabilizes RNF41 and is involved in trafficking of various transmembrane proteins. The present study identifies USP8 as a substrate of RNF41 and reveals that loss of USP8 explains the aforementioned RNF41 effects. RNF41 redistributes and ubiquitylates USP8, and reduces USP8 levels. In addition, USP8 knockdown functionally matches the effects of RNF41 ectopic expression on the model leptin and leukemia inhibitory factor (LIF) receptors. Moreover, RNF41 indirectly destabilizes the ESCRT-0 complex through suppression of USP8. Collectively, our findings demonstrate that RNF41 controls JAK2-associated cytokine receptor trafficking by acting as a key regulator of USP8 and ESCRT-0 stability. Balanced reciprocal cross-regulation of RNF41 and USP8 thus determines whether receptors are sorted for lysosomal degradation or recycling, this way regulating basal cytokine receptor levels.
Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity‐on‐Target cytokines (AcTakines) are a novel class of immunocytokines ...with improved therapeutic index. A TNF‐based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T‐cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8‐targeted type I interferon AcTakine. Co‐treatment with a CD13‐targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.
Synopsis
TNF and IFN‐γ are cytokines with great anticancer potential but with limited clinical application due to side‐effects. In the present study, the tumor endothelium is identified as target cell for their antitumor activity and new biologics that allow their safe targeting are developed.
By using transgenic mouse technologies the tumor endothelium was identified as target for the antitumor effect of TNF and IFN‐γ.
TNF and IFN‐γ AcTakines were developed by fusing inactivated cytokine mutants to a CD13 single domain antibody (resp. CD13‐AFR and CD13‐AFN‐II), allowing selective targeting of cytokine activity to the tumor endothelium.
CD13‐AFR synergized with immunotherapies (CD8‐AFN or CAR T‐cells) for immune‐mediated killing of solid tumors, without side‐effects.
Combined treatment with CD13‐AFR and CD13‐AFN‐II resulted in selective apoptosis of the tumor endothelium and complete tumor destruction, without side‐effects.
TNF and IFN‐γ are cytokines with great anticancer potential but with limited clinical application due to side‐effects. In the present study, the tumor endothelium is identified as target cell for their antitumor activity and new biologics that allow their safe targeting are developed.
RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ...ErbB3, IL-3, EPO and RARα receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies.
The glucocorticoid receptor α (GRα) is a member of the nuclear receptor superfamily and functions as a glucocorticoid (GC)-responsive transcription factor. GR can halt inflammation and kill off ...cancer cells, thus explaining the widespread use of glucocorticoids in the clinic. However, side effects and therapy resistance limit GR's therapeutic potential, emphasizing the importance of resolving all of GR's context-specific action mechanisms. Fortunately, the understanding of GR structure, conformation, and stoichiometry in the different GR-controlled biological pathways is now gradually increasing. This information will be crucial to close knowledge gaps on GR function. In this review, we focus on the various domains and mechanisms of action of GR, all from a structural perspective.
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