Interleukin-2 druggability is modulated by global conformational transitions controlled by a helical capping switch.

Interleukin-2 (IL-2) is a small α-helical cytokine that regulates immune cell homeostasis through its recruitment to a high-affinity heterotrimeric receptor complex (IL-2Rα/IL-2Rβ/γc). IL-2 has been shown to have therapeutic efficacy for immune diseases by preferentially expanding distinct T cell compartments, and several regulatory T cell (Treg)-biasing anti-IL-2 antibodies have been developed for combination therapies. The conformational plasticity of IL-2 plays an important role in its biological actions by modulating the strength of receptor and drug interactions. Through an NMR analysis of milliseconds-timescale dynamics of free mouse IL-2 (mIL-2), we identify a global transition to a sparse conformation which is regulated by an α-helical capping "switch" at the loop between the A and B helices (AB loop). Binding to either an anti-mouse IL-2 monoclonal antibody (mAb) or a small molecule inhibitor near the loop induces a measurable response at the core of the structure, while locking the switch to a single conformation through a designed point mutation leads to a global quenching of core dynamics accompanied by a pronounced effect in mAb binding. By elucidating key details of the long-range allosteric communication between the receptor binding surfaces and the core of the IL-2 structure, our results offer a direct blueprint for designing precision therapeutics targeting a continuum of conformational states

Leukotoxin Diols from Ground Corncob Bedding Disrupt Estrous Cyclicity in Rats and Stimulate MCF-7 Breast Cancer Cell Proliferation

Previous studies in our laboratory demonstrated that high-performance liquid chromatography (HPLC) analysis of ground corncob bedding extracts characterized two components (peak I and peak II) that disrupted endocrine function in male and female rats and stimulated breast and prostate cancer cell proliferation in vitro and in vivo. The active substances in peak I were identified as an isomeric mixture of 9,12-oxy-10,13-dihydroxyoctadecanoic acid and 10,13-oxy-9,12-dihydroxyoctadecanoic acid, collectively designated tetrahydrofurandiols (THF-diols). Studies presented here describe the purification and identification of the HPLC peak II component as 9,10-dihydroxy-12-octadecenoic acid (leukotoxin diol; LTX-diol), a well-known leukotoxin. A synthetic mixture of LTX-diol and 12,13-dihydroxy-9-octadecenoic acid (isoleukotoxin diol; i-LTX-diol) isomers was separated by HPLC, and each isomer stimulated (p < 0.001) MCF-7 cell proliferation in an equivalent fashion. The LTX-diol isomers failed to compete for [(3)H]estradiol binding to the estrogen receptor or nuclear type II sites, even though oral administration of very low doses of these compounds (>> 0.8 mg/kg body weight/day) disrupted estrous cyclicity in female rats. The LTX-diols did not disrupt male sexual behavior, suggesting that sex differences exist in response to these endocrine-disruptive agents

Structure of human frizzled5 by fiducial-assisted cryo-em supports a heterodimeric mechanism of canonical wnt signaling

Frizzleds (Fzd) are the primary receptors for Wnt morphogens, which are essential regulators of stem cell biology, yet the structural basis of Wnt signaling through Fzd remains poorly understood. Here we report the structure of an unliganded human Fzd5 determined by single-particle cryo-EM at 3.7 Å resolution, with the aid of an antibody chaperone acting as a fiducial marker. We also analyzed the topology of low-resolution XWnt8/Fzd5 complex particles, which revealed extreme flexibility between the Wnt/Fzd-CRD and the Fzd-TM regions. Analysis of Wnt/b-catenin signaling in response to Wnt3a versus a ‘surrogate agonist’ that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/b-catenin signaling appears to be principally reliant on ligand-induced Fzd/LRP6 heterodimerization, versus the allosteric mechanisms seen in structurally analogous class A G protein-coupled receptors, and Smoothened. These findings deepen our mechanistic understanding of Wnt signal transduction, and have implications for harnessing Wnt agonism in regenerative medicine

Immune cells fold and damage fungal hyphae

Acknowledgements We thank Ben Rutter and Alex Brand for providing Mycelia sterilia hyphae, and Gillian Griffiths for insightful advice. We are grateful to the Microscopy and Histology Core Facility at the University of Aberdeen for their help, advice and support. This work was funded by grants from the UK Medical Research Council [www.mrc.ac.uk], to AJPB, NARG, LPE, MN (MR/M026663/1, MR/M026663/2), and from the University of Aberdeen to AP, DL. The work was also supported by Wellcome [www.wellcome.ac.uk]: NARG, GDB, AJPB (097377); NARG (101873, 200208); and GDB (102705). Further support for this work was also provided by the Medical Research Council Centre for Medical Mycology (MR/N006364/1). MGN was supported by an ERC Advanced Grant (#833247) and a Spinoza grant of the Netherlands Organization for Scientific Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Structure of the thrombopoietin-MPL receptor complex is a blueprint for biasing hematopoiesis

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation

Facile discovery of surrogate cytokine agonists

Cytokines are powerful immune modulators that initiate signaling through receptor dimerization, but natural cytokines have structural limitations as therapeutics. We present a strategy to discover cytokine surrogate agonists by using modular ligands that exploit induced proximity and receptor dimer geometry as pharmacological metrics amenable to high-throughput screening. Using VHH and scFv to human interleukin-2/15, type-I interferon, and interleukin-10 receptors, we generated combinatorial matrices of single-chain bispecific ligands that exhibited diverse spectrums of functional activities, including potent inhibition of SARS-CoV-2 by surrogate interferons. Crystal structures of IL-2R:VHH complexes revealed that variation in receptor dimer geometries resulted in functionally diverse signaling outputs. This modular platform enabled engineering of surrogate ligands that compelled assembly of an IL-2R/IL-10R heterodimer, which does not naturally exist, that signaled through pSTAT5 on T and natural killer (NK) cells. This “cytokine med-chem” approach, rooted in principles of induced proximity, is generalizable for discovery of diversified agonists for many ligand-receptor systems

Pediatric colloid cysts: a multinational, multicenter study. An IFNE-ISPN-ESPN collaboration

OBJECTIVE Colloid cysts (CCs) are rare at all ages, and particularly among children. The current literature on pediatric CC is limited, and often included in mixed adult/pediatric series. The goal of this multinational, multicenter study was to combine forces among centers and investigate the clinical course of pediatric CCs. METHODS A multinational, multicenter retrospective study was performed to attain a large sample size, focusing on CC diagnosis in patients younger than 18 years of age. Collected data included clinical presentation, radiological characteristics, treatment, and outcome. RESULTS One hundred thirty-four children with CCs were included. Patient age at diagnosis ranged from 2.4 to 18 years (mean 12.8 ± 3.4 years, median 13.2 years, interquartile range 10.3–15.4 years; 22% were \u3c 10 years of age). Twenty-two cases (16%) were diagnosed incidentally, including 48% of those younger than 10 years of age. Most of the other patients had symptoms related to increased intracranial pressure and hydrocephalus. The average follow-up duration for the entire group was 49.5 ± 45.8 months. Fifty-nine patients were initially followed, of whom 28 were eventually operated on at a mean of 19 ± 32 months later due to cyst growth, increasing hydrocephalus, and/or new symptoms. There was a clear correlation between larger cysts and symptomatology, acuteness of symptoms, hydrocephalus, and need for surgery. Older age was also associated with the need for surgery. One hundred three children (77%) underwent cyst resection, 60% using a purely endoscopic approach. There was 1 death related to acute hydrocephalus at presentation. Ten percent of operated patients had some form of complication, and 7.7% of operated cases required a shunt at some point during follow-up. Functional outcome was good; however, the need for immediate surgery was associated with educational limitations. Twenty operated cases (20%) experienced a recurrence of their CC at a mean of 38 ± 46 months after the primary surgery. The CC recurrence rate was 24% following endoscopic resection and 15% following open resections (p = 0.28). CONCLUSIONS CCs may present in all pediatric age groups, although most that are symptomatic present after the age of 10 years. Incidentally discovered cysts should be closely followed, as many may grow, leading to hydrocephalus and other new symptoms. Presentation of CC may be acute and may cause life-threatening conditions related to hydrocephalus, necessitating urgent treatment. The outcome of treated children with CCs is favorable

Runx1 Loss Minimally Impacts Long-Term Hematopoietic Stem Cells

RUNX1 encodes a DNA binding subunit of the core-binding transcription factors and is frequently mutated in acute leukemia, therapy-related leukemia, myelodysplastic syndrome, and chronic myelomonocytic leukemia. Mutations in RUNX1 are thought to confer upon hematopoietic stem cells (HSCs) a pre-leukemic state, but the fundamental properties of Runx1 deficient pre-leukemic HSCs are not well defined. Here we show that Runx1 deficiency decreases both apoptosis and proliferation, but only minimally impacts the frequency of long term repopulating HSCs (LT-HSCs). It has been variously reported that Runx1 loss increases LT-HSC numbers, decreases LT-HSC numbers, or causes age-related HSC exhaustion. We attempt to resolve these discrepancies by showing that Runx1 deficiency alters the expression of several key HSC markers, and that the number of functional LT-HSCs varies depending on the criteria used to score them. Finally, we identify genes and pathways, including the cell cycle and p53 pathways that are dysregulated in Runx1 deficient HSCs

Isolation of a Structural Mechanism for Uncoupling T Cell Receptor Signaling from Peptide-MHC Binding

TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy