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1.
Cell Stem Cell ; 2020 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-32818433

RESUMO

Modulation of Wnt signaling has untapped potential in regenerative medicine due to its essential functions in stem cell homeostasis. However, Wnt lipidation and Wnt-Frizzled (Fzd) cross-reactivity have hindered translational Wnt applications. Here, we designed and engineered water-soluble, Fzd subtype-specific "next-generation surrogate" (NGS) Wnts that hetero-dimerize Fzd and Lrp6. NGS Wnt supports long-term expansion of multiple different types of organoids, including kidney, colon, hepatocyte, ovarian, and breast. NGS Wnts are superior to Wnt3a conditioned media in organoid expansion and single-cell organoid outgrowth. Administration of Fzd subtype-specific NGS Wnt in vivo reveals that adult intestinal crypt proliferation can be promoted by agonism of Fzd5 and/or Fzd8 receptors, while a broad spectrum of Fzd receptors can induce liver zonation. Thus, NGS Wnts offer a unified organoid expansion protocol and a laboratory "tool kit" for dissecting the functions of Fzd subtypes in stem cell biology.

2.
Elife ; 92020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32762848

RESUMO

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/ß-catenin signaling in response to Wnt3a versus a 'surrogate agonist' that cross-links Fzd to LRP6, revealed identical structure-activity relationships. Thus, canonical Wnt/ß-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.

3.
Elife ; 92020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32773038

RESUMO

T regulatory (Treg) cells play vital roles in modulating immunity and tissue homeostasis. Their actions depend on TCR recognition of peptide-MHC molecules; yet the degree of peptide specificity of Treg-cell function, and whether Treg ligands can be used to manipulate Treg cell biology are unknown. Here, we developed an Ab-peptide library that enabled unbiased screening of peptides recognized by a bona fide murine Treg cell clone isolated from the visceral adipose tissue (VAT), and identified surrogate agonist peptides, with differing affinities and signaling potencies. The VAT-Treg cells expanded in vivo by one of the surrogate agonists preserved the typical VAT-Treg transcriptional programs. Immunization with this surrogate, especially when coupled with blockade of TNFα signaling, expanded VAT-Treg cells, resulting in protection from inflammation and improved metabolic indices, including promotion of insulin sensitivity. These studies suggest that antigen-specific targeting of VAT-localized Treg cells could eventually be a strategy for improving metabolic disease.

4.
Cell ; 182(4): 1027-1043.e17, 2020 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-32822567

RESUMO

Cell-surface protein-protein interactions (PPIs) mediate cell-cell communication, recognition, and responses. We executed an interactome screen of 564 human cell-surface and secreted proteins, most of which are immunoglobulin superfamily (IgSF) proteins, using a high-throughput, automated ELISA-based screening platform employing a pooled-protein strategy to test all 318,096 PPI combinations. Screen results, augmented by phylogenetic homology analysis, revealed ∼380 previously unreported PPIs. We validated a subset using surface plasmon resonance and cell binding assays. Observed PPIs reveal a large and complex network of interactions both within and across biological systems. We identified new PPIs for receptors with well-characterized ligands and binding partners for "orphan" receptors. New PPIs include proteins expressed on multiple cell types and involved in diverse processes including immune and nervous system development and function, differentiation/proliferation, metabolism, vascularization, and reproduction. These PPIs provide a resource for further biological investigation into their functional relevance and may offer new therapeutic drug targets.

5.
Science ; 369(6500): 161-167, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32646996

RESUMO

Muscarinic toxins (MTs) are natural toxins produced by mamba snakes that primarily bind to muscarinic acetylcholine receptors (MAChRs) and modulate their function. Despite their similar primary and tertiary structures, MTs show distinct binding selectivity toward different MAChRs. The molecular details of how MTs distinguish MAChRs are not well understood. Here, we present the crystal structure of M1AChR in complex with MT7, a subtype-selective anti-M1AChR snake venom toxin. The structure reveals the molecular basis of the extreme subtype specificity of MT7 for M1AChR and the mechanism by which it regulates receptor function. Through in vitro engineering of MT7 finger regions that was guided by the structure, we have converted the selectivity from M1AChR toward M2AChR, suggesting that the three-finger fold is a promising scaffold for developing G protein-coupled receptor modulators.


Assuntos
Venenos Elapídicos/química , Receptor Muscarínico M1/química , Receptor Muscarínico M1/genética , Animais , Atropina/química , Cristalografia por Raios X , Engenharia Genética , Antagonistas Muscarínicos/química , Conformação Proteica , Receptor Muscarínico M1/antagonistas & inibidores , Células Sf9
6.
Cell Stem Cell ; 27(1): 50-63.e5, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619518

RESUMO

Modulating signaling pathways including Wnt and Hippo can induce cardiomyocyte proliferation in vivo. Applying these signaling modulators to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in vitro can expand CMs modestly (<5-fold). Here, we demonstrate massive expansion of hiPSC-CMs in vitro (i.e., 100- to 250-fold) by glycogen synthase kinase-3ß (GSK-3ß) inhibition using CHIR99021 and concurrent removal of cell-cell contact. We show that GSK-3ß inhibition suppresses CM maturation, while contact removal prevents CMs from cell cycle exit. Remarkably, contact removal enabled 10 to 25 times greater expansion beyond GSK-3ß inhibition alone. Mechanistically, persistent CM proliferation required both LEF/TCF activity and AKT phosphorylation but was independent from yes-associated protein (YAP) signaling. Engineered heart tissues from expanded hiPSC-CMs showed comparable contractility to those from unexpanded hiPSC-CMs, demonstrating uncompromised cellular functionality after expansion. In summary, we uncovered a molecular interplay that enables massive hiPSC-CM expansion for large-scale drug screening and tissue engineering applications.

7.
Elife ; 92020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32716298

RESUMO

T cell cross-reactivity ensures that diverse pathogen-derived epitopes encountered during a lifetime are recognized by the available TCR repertoire. A feature of cross-reactivity where previous exposure to one microbe can alter immunity to subsequent, non-related pathogens has been mainly explored for viruses. Yet cross-reactivity to additional microbes is important to consider, especially in HIV infection where gut-intestinal barrier dysfunction could facilitate T cell exposure to commensal/pathogenic microbes. Here we evaluated the cross-reactivity of a 'public', HIV-specific, CD8 T cell-derived TCR (AGA1 TCR) using MHC class I yeast display technology. Via screening of MHC-restricted libraries comprising ~2×108 sequence-diverse peptides, AGA1 TCR specificity was mapped to a central peptide di-motif. Using the top TCR-enriched library peptides to probe the non-redundant protein database, bacterial peptides that elicited functional responses by AGA1-expressing T cells were identified. The possibility that in context-specific settings, MHC class I proteins presenting microbial peptides influence virus-specific T cell populations in vivo is discussed.

8.
Proc Natl Acad Sci U S A ; 117(13): 7183-7192, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32184322

RESUMO

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-2Rß/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.


Assuntos
Interleucina-2/metabolismo , Animais , Linhagem Celular , Interleucina-2/genética , Espectroscopia de Ressonância Magnética , Camundongos , Conformação Proteica
9.
Science ; 367(6478): 643-652, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029621

RESUMO

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.


Assuntos
Carcinogênese/genética , Membrana Celular/química , Janus Quinase 2/química , Janus Quinase 2/genética , Multimerização Proteica , Receptores da Eritropoetina/química , Receptores da Somatotropina/química , Receptores de Trombopoetina/química , Substituição de Aminoácidos/genética , Células HeLa , Humanos , Janus Quinase 2/antagonistas & inibidores , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Fenilalanina/genética , Pirazóis/farmacologia , Transdução de Sinais , Imagem Individual de Molécula , Valina/genética
10.
Nature ; 580(7802): 269-273, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32106218

RESUMO

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Ilhas Genômicas/genética , Mutagênese , Mutação , Técnicas de Cocultura , Estudos de Coortes , Sequência Consenso , Dano ao DNA , Microbioma Gastrointestinal , Humanos , Organoides/citologia , Organoides/metabolismo , Organoides/microbiologia , Peptídeos/genética , Policetídeos
11.
PLoS One ; 15(1): e0226928, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31914456

RESUMO

Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/ß-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.


Assuntos
Colo/crescimento & desenvolvimento , Anticorpos de Cadeia Única/metabolismo , Trombospondinas/metabolismo , Via de Sinalização Wnt , Sítios de Ligação , Colo/metabolismo , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/imunologia
12.
Proc Natl Acad Sci U S A ; 117(1): 522-531, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871169

RESUMO

Interleukin 15 (IL-15) is an essential cytokine for the survival and proliferation of natural killer (NK) cells. IL-15 activates signaling by the ß and common γ (γc) chain heterodimer of the IL-2 receptor through trans-presentation by cells expressing IL-15 bound to the α chain of the IL-15 receptor (IL-15Rα). We show here that membrane-associated IL-15Rα-IL-15 complexes are transferred from presenting cells to NK cells through trans-endocytosis and contribute to the phosphorylation of ribosomal protein S6 and NK cell proliferation. NK cell interaction with soluble or surface-bound IL-15Rα-IL-15 complex resulted in Stat5 phosphorylation and NK cell survival at a concentration or density of the complex much lower than required to stimulate S6 phosphorylation. Despite this efficient response, Stat5 phosphorylation was reduced after inhibition of metalloprotease-induced IL-15Rα-IL-15 shedding from trans-presenting cells, whereas S6 phosphorylation was unaffected. Conversely, inhibition of trans-endocytosis by silencing of the small GTPase TC21 or expression of a dominant-negative TC21 reduced S6 phosphorylation but not Stat5 phosphorylation. Thus, trans-endocytosis of membrane-associated IL-15Rα-IL-15 provides a mode of regulating NK cells that is not afforded to IL-2 and is distinct from activation by soluble IL-15. These results may explain the strict IL-15 dependence of NK cells and illustrate how the cellular compartment in which receptor-ligand interaction occurs can influence functional outcome.


Assuntos
Proliferação de Células , Células Dendríticas/metabolismo , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular , Endocitose/fisiologia , Voluntários Saudáveis , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosforilação/fisiologia , Cultura Primária de Células , Proteína S6 Ribossômica/metabolismo
13.
Nat Biotechnol ; 37(11): 1322-1331, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31570897

RESUMO

The near-infrared-IIb (NIR-IIb) (1,500-1,700 nm) window is ideal for deep-tissue optical imaging in mammals, but lacks bright and biocompatible probes. Here, we developed biocompatible cubic-phase (α-phase) erbium-based rare-earth nanoparticles (ErNPs) exhibiting bright downconversion luminescence at ~1,600 nm for dynamic imaging of cancer immunotherapy in mice. We used ErNPs functionalized with cross-linked hydrophilic polymer layers attached to anti-PD-L1 (programmed cell death-1 ligand-1) antibody for molecular imaging of PD-L1 in a mouse model of colon cancer and achieved tumor-to-normal tissue signal ratios of ~40. The long luminescence lifetime of ErNPs (~4.6 ms) enabled simultaneous imaging of ErNPs and lead sulfide quantum dots emitting in the same ~1,600 nm window. In vivo NIR-IIb molecular imaging of PD-L1 and CD8 revealed cytotoxic T lymphocytes in the tumor microenvironment in response to immunotherapy, and altered CD8 signals in tumor and spleen due to immune activation. The cross-linked functionalization layer facilitated 90% ErNP excretion within 2 weeks without detectable toxicity in mice.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Antígeno B7-H1/imunologia , Neoplasias do Colo/tratamento farmacológico , Érbio/química , Animais , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/farmacologia , Antígenos CD8/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Imunoterapia , Raios Infravermelhos , Camundongos , Nanopartículas , Imagem Óptica , Pontos Quânticos , Linfócitos T Citotóxicos/imunologia , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Cell ; 179(1): 3-7, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31519306

RESUMO

This year's Lasker Basic Medical Research Award honors Max Cooper and Jacques Miller for discoveries that revealed the organizing principles of adaptive immunity. Their collective contributions have had broad clinical impact in the treatment of immune disease.

15.
Nature ; 572(7770): 481-487, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31391585

RESUMO

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Linfócitos T/imunologia , Linfócitos T/patologia , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Doença Celíaca , Células Clonais/citologia , Células Clonais/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Antígenos H-2/imunologia , Humanos , Imunização , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Glicoproteína Associada a Mielina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Adulto Jovem
16.
J Biol Chem ; 294(38): 13876-13886, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31387945

RESUMO

Ligand-induced dimerization is the predominant mechanism through which secreted proteins activate cell surface receptors to transmit essential biological signals. Cytokines are a large class of soluble proteins that dimerize transmembrane receptors into precise signaling topologies, but there is a need for alternative, engineerable ligand scaffolds that specifically recognize and stabilize these protein interactions. Recombinant antibodies can potentially serve as robust and versatile platforms for cytokine complex stabilization, and their specificity allows for tunable modulation of dimerization equilibrium. Here, we devised an evolutionary strategy to isolate monovalent antibody fragments that bridge together two different receptor subunits in a cytokine-receptor complex, precisely as the receptors are disposed in their natural signaling orientations. To do this, we screened a naive antibody library against a stabilized ligand-receptor ternary complex that acted as a "molecular cast" of the natural receptor dimer conformation. Our selections elicited "stapler" single-chain variable fragments (scFvs) of antibodies that specifically engage the interleukin-4 receptor heterodimer. The 3.1 Å resolution crystal structure of one such stapler revealed that, as intended, this scFv recognizes a composite epitope between the two receptors as they are positioned in the complex. Extending our approach, we evolved a stapler scFv that specifically binds to and stabilizes the interface between the interleukin-2 cytokine and one of its receptor subunits, leading to a 15-fold enhancement in interaction affinity. This demonstration that scFvs can be selected to recognize epitopes that span protein interfaces presents new opportunities to engineer structurally defined antibodies for a broad range of research and therapeutic applications.


Assuntos
Epitopos/química , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única/genética , Sequência de Aminoácidos/genética , Dimerização , Mapeamento de Epitopos/métodos , Humanos , Ligantes , Conformação Molecular , Biblioteca de Peptídeos , Ligação Proteica/genética , Multimerização Proteica/genética
17.
JCI Insight ; 52019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31237864

RESUMO

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

18.
Science ; 364(6442)2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31123111

RESUMO

Although tunable signaling by G protein-coupled receptors can be exploited through medicinal chemistry, a comparable pharmacological approach has been lacking for the modulation of signaling through dimeric receptors, such as those for cytokines. We present a strategy to modulate cytokine receptor signaling output by use of a series of designed C2-symmetric cytokine mimetics, based on the designed ankyrin repeat protein (DARPin) scaffold, that can systematically control erythropoietin receptor (EpoR) dimerization orientation and distance between monomers. We sampled a range of EpoR geometries by varying intermonomer angle and distance, corroborated by several ligand-EpoR complex crystal structures. Across the range, we observed full, partial, and biased agonism as well as stage-selective effects on hematopoiesis. This surrogate ligand strategy opens access to pharmacological modulation of therapeutically important cytokine and growth factor receptor systems.


Assuntos
Repetição de Anquirina , Materiais Biomiméticos/farmacologia , Hematopoese/efeitos dos fármacos , Engenharia de Proteínas/métodos , Receptores de Citocinas/metabolismo , Receptores da Eritropoetina/metabolismo , Linhagem Celular , Citocinas/metabolismo , Humanos , Ligantes , Multimerização Proteica , Receptores de Citocinas/química , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Transdução de Sinais
19.
Nat Struct Mol Biol ; 26(6): 407-414, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31086346

RESUMO

To discriminate between closely related members of a protein family that differ at a limited number of spatially distant positions is a challenge for drug discovery. We describe a combined computational design and experimental selection approach for generating binders targeting functional sites with large, shape complementary interfaces to read out subtle sequence differences for subtype-specific antagonism. Repeat proteins are computationally docked against a functionally relevant region of the target protein surface that varies in the different subtypes, and the interface sequences are optimized for affinity and specificity first computationally and then experimentally. We used this approach to generate a series of human Frizzled (Fz) subtype-selective antagonists with extensive shape complementary interaction surfaces considerably larger than those of repeat proteins selected from random libraries. In vivo administration revealed that Wnt-dependent pericentral liver gene expression involves multiple Fz subtypes, while maintenance of the intestinal crypt stem cell compartment involves only a limited subset.


Assuntos
Receptores Frizzled/antagonistas & inibidores , Receptores Frizzled/metabolismo , Simulação de Acoplamento Molecular , Animais , Anquirinas/química , Anquirinas/metabolismo , Linhagem Celular , Cristalografia por Raios X , Descoberta de Drogas , Duodeno/citologia , Duodeno/metabolismo , Receptores Frizzled/química , Humanos , Camundongos Endogâmicos C57BL , Ligação Proteica , Conformação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo
20.
Nature ; 567(7746): 56-60, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814731

RESUMO

The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.


Assuntos
Desenho de Fármacos , Interferon gama/agonistas , Interferon gama/imunologia , Receptores de Interferon/química , Receptores de Interferon/metabolismo , Antígeno B7-H1/biossíntese , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Agonismo Parcial de Drogas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/biossíntese , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Interferon gama/química , Interferon gama/genética , Ligantes , Modelos Moleculares , Mutação , Infecções por Mycobacterium/genética , Infecções por Mycobacterium/imunologia , Estabilidade Proteica , Receptores de Interferon/genética , Transdução de Sinais , Relação Estrutura-Atividade
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