Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; García, K. Christopher; Müller, Thomas; Piehler, Jacob

doi:10.1038/ncomms15976

Cited by 32 publications

(35 citation statements)

References 82 publications

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“…Ligand stimulation led to a significant decrease of diffusion mobility, particularly for IL-10Rα (Sup. Figure 3b), which is in line with previous reports on cytokine receptor dimerization (Moraga et al, 2015c;Richter et al, 2017;Wilmes et al, 2015).…”

Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodsupporting

confidence: 92%

“…IL-10 binds with high affinity to IL-10Rα and much lower affinity to IL-10Rβ, in the order of high micromolar/low millimolar range, (Logsdon et al, 2002). Recent studies established that sub-micromolar affinities are required to ensure efficient cytokine receptor dimerization (Richter et al, 2017;Wilmes et al, 2015).Thus, assembly of active IL-10 receptor signaling complexes in the plasma membrane is probably limited by recruitment of IL-10Rβ, making this system exquisitely sensitive to changes in either ligand concentrations and/or receptor densities in the plasma membrane. We therefore hypothesise that IL-10's poor clinical activities result from its weak affinity for the IL-10Rβ subunit.…”

Section: Introductionmentioning

confidence: 99%

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Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Gorby

Wilmes

et al. 2020

Preprint

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Interleukin-10 is a dimeric cytokine with both immune-suppressive and immune-stimulatory activities. Despite its immuno-modulatory potential, IL-10-based therapies have shown only marginal benefits in the clinic. Here we have explored whether the stability of the IL-10-receptor complex contributes to IL-10 immuno-modulatory potency. For that, we have generated an IL-10 mutant with greatly enhanced affinity for its IL-10Rβ receptor via yeast surface display. The affinity enhanced IL-10 variants recruited IL-10Rβ more efficiently into active cell surface signaling complexes than the wild-type cytokine and triggered more potent STAT1 and STAT3 activation in human monocytes and CD8 T cells. This in turn led to more robust induction of IL-10-mediated gene expression programs at a wide range of ligand concentrations in both human cell subsets. IL-10 regulated genes are involved in monocyte energy homeostasis, migration and trafficking, and genes involved in CD8 T cell exhaustion. Interestingly, at non-saturating doses, IL-10 lost key components of its gene-expression program, which may explain its lack of efficacy in clinical settings. Remarkably, our engineered IL-10 variant exhibited a more robust bioactivity profile than IL-10 wt at all the doses tested in monocytes and CD8 T cells. Moreover, CAR-modified T cells expanded with the engineered IL-10 variant displayed superior cytolytic activity than those expanded with IL-10 wt. Our study provides unique insights into how IL-10-receptor complex stability fine-tunes IL-10 biology, and opens new opportunities to revitalize failed IL-10 therapies.

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Section: Enhanced Il-10rβ Binding Affinity Increases Receptor Heterodsupporting

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Gorby

Wilmes

et al. 2020

Preprint

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“…Although truncated soluble interleukin and interferon receptors also exist in vivo, primarily by cleavage between the extracellular and transmembrane segments, the QTY code is still meaningful for designing these single transmembrane receptors for additional studies. While for some receptors the transmembrane α-helix appears to play a role in ligand interaction (Richter et al, 2017), inclusion of the QTY-designed water-soluble transmembrane helical segment in the entire receptors may contribute to the overall understanding of receptor functional mechanism and signal transduction. On the other hand, not all native-form soluble interleukin and interferon receptors can be readily synthesized and purified in a high-throughput low cost E. coli system.…”

Section: Discussionmentioning

confidence: 99%

“…The ligand binding domain is typically comprised of multiple stranded β-sheets that form two connected anti-parallel β-barrels. The β-barrels are connected to the transmembrane α-helix which is responsible for signal transduction, presumably playing a role in ligand interaction (Richter et al, 2017). In order to best mimic a native receptor, we included the transmembrane domain in our design with a few amino acids in the cytoplasmic region to serve as a short linker so as to optimize the binding and structure of the QTY code modified receptors.…”

Section: Design Of Fc-fusion Qty Variant Cytokine Receptorsmentioning

confidence: 99%

QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands

Hao

Jin²,

Zhang

et al. 2020

QRB Discovery

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Cytokine release syndrome (CRS), or ‘cytokine storm’, is the leading side effect during chimeric antigen receptor (CAR)-T therapy that is potentially life-threatening. It also plays a critical role in viral infections such as Coronavirus Disease 2019 (COVID-19). Therefore, efficient removal of excessive cytokines is essential for treatment. We previously reported a novel protein modification tool called the QTY code, through which hydrophobic amino acids Leu, Ile, Val and Phe are replaced by Gln (Q), Thr (T) and Tyr (Y). Thus, the functional detergent-free equivalents of membrane proteins can be designed. Here, we report the application of the QTY code on six variants of cytokine receptors, including interleukin receptors IL4Rα and IL10Rα, chemokine receptors CCR9 and CXCR2, as well as interferon receptors IFNγR1 and IFNλR1. QTY-variant cytokine receptors exhibit physiological properties similar to those of native receptors without the presence of hydrophobic segments. The receptors were fused to the Fc region of immunoglobulin G (IgG) protein to form an antibody-like structure. These QTY code-designed Fc-fusion receptors were expressed in Escherichia coli and purified. The resulting water-soluble fusion receptors bind to their respective ligands with Kd values affinity similar to isolated native receptors. Our cytokine receptor–Fc-fusion proteins potentially serve as an antibody-like decoy to dampen the excessive cytokine levels associated with CRS and COVID-19 infection.

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“…Tracking of single molecules as well as additional data analysis was carried out with well-established localization and tracking algorithms (Jaqaman et al, 2008;Serge et al, 2008), implemented in a software package called 'SLIMfast' (kindly provided by C.P. Richter) written in Matlab (Appelhans et al, 2018;Richter et al, 2017). Localization precision was typically about 20-25 nm.…”

Section: Single Molecule Trackingmentioning

confidence: 99%

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

Gohrbandt

Lipski

Grimshaw

et al. 2019

Preprint

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Important physicochemical properties of cell membranes such as fluidity sensitively depend on fluctuating environmental factors including temperature, pH or diet. To counteract these disturbances, living cells universally adapt their lipid composition in return. In contrast to eukaryotic cells, bacteria tolerate surprisingly drastic changes in their lipid composition while retaining viability, thus making them a more tractable model to study this process. Using the model organisms Escherichia coli and Bacillus subtilis, which regulate their membrane fluidity with different fatty acid types, we show here that inadequate membrane fluidity interferes with essential cellular processes such as morphogenesis and maintenance of membrane potential, and triggers large-scale lipid phase separation that drives protein segregation into the fluid phase. These findings illustrate why lipid homeostasis is such a critical cellular process. Finally, our results provide direct in vivo support for current in vitro and in silico models regarding lipid phase separation and associated protein segregation.Keywords: Lipid phase separation, lipid domains, protein partitioning, membrane fluidity, homeoviscous adaptation, Escherichia coli, Bacillus subtilis, WALP, FOF1 ATP synthase liquid-disordered phase characterized by low packing density and high diffusion rates that forms the regular state of biological membranes, (ii) the more ordered, cholesterol/hopanoid-dependent liquidordered phase found in biological membranes in form of lipid rafts, and (iii) the gel phase characterized by dense lipid packing with little lateral or rotational diffusion, which is generally assumed to be absent in biologically active membranes (Schmid, 2017;Veatch, 2007). In fact, the temperature associated with gel phase formation has been postulated to define the lower end of the temperature range able to support vital cell functions (Burns et al., 2017;Drobnis et al., 1993;Ghetler et al., 2005). At last, the lipid phases can co-exist, resulting in separated membrane areas exhibiting distinctly different composition and characteristics (Baumgart et al., 2007;Elson et al., 2010;Heberle and Feigenson, 2011). This principal mechanism of lipid domain formation is best studied in the context of lipid rafts (Lingwood and Simons, 2010).While in vitro and in silico approaches with simplified lipid models have provided detailed insights into the complex physicochemical behavior of lipid bilayers, testing the formed hypotheses and models in the context of protein-rich biological membranes is now crucial. Bacteria tolerate surprisingly drastic changes in their lipid composition and only possess one or two membrane layers as part of their cell envelope. Consequently, bacteria are both a suitable and a more tractable model to study the fundamental biological process linked to membrane fluidity and phase separation in vivo.We analyzed the biological importance of membrane homeoviscous adaptation in Escherichia coli (phylum Proteobacteria) and Bacillus subtilis (phylum Firmic...

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Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

Cited by 32 publications

References 82 publications

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

Engineered IL-10 variants elicit potent immuno-modulatory activities at therapeutic low ligand doses

QTY Code-designed Water-soluble Fc-fusion Cytokine Receptors Bind to their Respective Ligands

Low membrane fluidity triggers lipid phase separation and protein segregation in vivo

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