RESUMO
Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation of cytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERK phosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of the IL-7Rα transmembrane domain with the PPCL insertion into natural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.
Assuntos
Cisteína , Transdução de Sinais , Cisteína/metabolismo , Cisteína/química , Humanos , Ligantes , Animais , Camundongos , Receptores de Citocinas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/genética , Dimerização , Multimerização ProteicaRESUMO
Therapeutic agents targeting cytokine-cytokine receptor (CK-CKR) interactions lead to the disruption in cellular signaling and are effective in treating many diseases including tumors. However, a lack of universal and quick access to annotated structural surface regions on CK/CKR has limited the progress of a structure-driven approach in developing targeted macromolecular drugs and precision medicine therapeutics. Herein we develop CytoSIP (Single nucleotide polymorphisms (SNPs), Interface, and Phenotype), a rich internet application based on a database of atomic interactions around hotspots in experimentally determined CK/CKR structural complexes. CytoSIP contains: (1) SNPs on CK/CKR; (2) interactions involving CK/CKR domains, including CK/CKR interfaces, oligomeric interfaces, epitopes, or other drug targeting surfaces; and (3) diseases and phenotypes associated with CK/CKR or SNPs. The database framework introduces a unique tri-level SIP data model to bridge genetic variants (atomic level) to disease phenotypes (organism level) using protein structure (complexes) as an underlying framework (molecule level). Customized screening tools are implemented to retrieve relevant CK/CKR subset, which reduces the time and resources needed to interrogate large datasets involving CK/CKR surface hotspots and associated pathologies. CytoSIP portal is publicly accessible at https://CytoSIP.biocloud.top , facilitating the panoramic investigation of the context-dependent crosstalk between CK/CKR and the development of targeted therapeutic agents.
Assuntos
Citocinas , Polimorfismo de Nucleotídeo Único , Receptores de Citocinas , Humanos , Receptores de Citocinas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/genética , Citocinas/metabolismo , Citocinas/genética , Citocinas/química , Bases de Dados de Proteínas , FenótipoRESUMO
Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.
Assuntos
Proteínas de Membrana , Ligação Proteica , Receptores da Eritropoetina , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Receptores da Eritropoetina/metabolismo , Receptores da Eritropoetina/química , Modelos Moleculares , Proliferação de Células/efeitos dos fármacos , Receptores de Citocinas/metabolismo , Receptores de Citocinas/química , Sequência de Aminoácidos , Multimerização Proteica , Animais , Células HEK293RESUMO
Targeted degradation of cell surface and extracellular proteins via lysosomal delivery is an important means to modulate extracellular biology. However, these approaches have limitations due to lack of modularity, ease of development, restricted tissue targeting and applicability to both cell surface and extracellular proteins. We describe a lysosomal degradation strategy, termed cytokine receptor-targeting chimeras (KineTACs), that addresses these limitations. KineTACs are fully genetically encoded bispecific antibodies consisting of a cytokine arm, which binds its cognate cytokine receptor, and a target-binding arm for the protein of interest. We show that KineTACs containing the cytokine CXCL12 can use the decoy recycling receptor, CXCR7, to target a variety of target proteins to the lysosome for degradation. Additional KineTACs were designed to harness other CXCR7-targeting cytokines, CXCL11 and vMIPII, and the interleukin-2 (IL-2) receptor-targeting cytokine IL-2. Thus, KineTACs represent a general, modular, selective and simple genetically encoded strategy for inducing lysosomal delivery of extracellular and cell surface targets with broad or tissue-specific distribution.
Assuntos
Quimera de Direcionamento de Proteólise , Receptores de Citocinas , Membrana Celular , Interleucina-2 , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Transdução de Sinais , Proteólise , Quimiocina CXCL12/químicaRESUMO
Cytokines signal through cell surface receptor dimers to initiate activation of intracellular Janus kinases (JAKs). We report the 3.6-angstrom-resolution cryo-electron microscopy structure of full-length JAK1 complexed with a cytokine receptor intracellular domain Box1 and Box2 regions captured as an activated homodimer bearing the valineâphenylalanine (VF) mutation prevalent in myeloproliferative neoplasms. The seven domains of JAK1 form an extended structural unit, the dimerization of which is mediated by close-packing of the pseudokinase (PK) domains from the monomeric subunits. The oncogenic VF mutation lies within the core of the JAK1 PK interdimer interface, enhancing packing complementarity to facilitate ligand-independent activation. The carboxy-terminal tyrosine kinase domains are poised for transactivation and to phosphorylate the receptor STAT (signal transducer and activator of transcription)-recruiting motifs projecting from the overhanging FERM (four-point-one, ezrin, radixin, moesin)-SH2 (Src homology 2)-domains. Mapping of constitutively active JAK mutants supports a two-step allosteric activation mechanism and reveals opportunities for selective therapeutic targeting of oncogenic JAK signaling.
Assuntos
Janus Quinase 1 , Receptores de Citocinas , Domínios de Homologia de src , Regulação Alostérica , Microscopia Crioeletrônica , Ativação Enzimática , Humanos , Janus Quinase 1/química , Janus Quinase 1/metabolismo , Mutação , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Fosforilação , Multimerização Proteica , Receptores de Citocinas/química , Fatores de Transcrição STAT/metabolismoRESUMO
N-degron pathways are a set of proteolytic systems that target the N-terminal destabilizing residues of substrates for proteasomal degradation. Recently, the Gly/N-degron pathway has been identified as a new branch of the N-degron pathway. The N-terminal glycine degron (Gly/N-degron) is recognized by ZYG11B and ZER1, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we present the crystal structures of ZYG11B and ZER1 bound to various Gly/N-degrons. The structures reveal that ZYG11B and ZER1 utilize their armadillo (ARM) repeats forming a deep and narrow cavity to engage mainly the first four residues of Gly/N-degrons. The α-amino group of the Gly/N-degron is accommodated in an acidic pocket by five conserved hydrogen bonds. These structures, together with biochemical studies, decipher the molecular basis for the specific recognition of the Gly/N-degron by ZYG11B and ZER1, providing key information for future structure-based chemical probe design.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Glicina/química , Conformação Proteica , Receptores de Citocinas/ultraestrutura , Sequência de Aminoácidos/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Glicina/genética , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Ligação Proteica/genética , Domínios Proteicos/genética , Proteólise , Receptores de Citocinas/química , Receptores de Citocinas/genética , Especificidade por Substrato , Ubiquitina/genéticaRESUMO
Fifty years ago, the first landmark structures of antibodies heralded the dawn of structural immunology. Momentum then started to build toward understanding how antibodies could recognize the vast universe of potential antigens and how antibody-combining sites could be tailored to engage antigens with high specificity and affinity through recombination of germline genes (V, D, J) and somatic mutation. Equivalent groundbreaking structures in the cellular immune system appeared some 15 to 20 years later and illustrated how processed protein antigens in the form of peptides are presented by MHC molecules to T cell receptors. Structures of antigen receptors in the innate immune system then explained their inherent specificity for particular microbial antigens including lipids, carbohydrates, nucleic acids, small molecules, and specific proteins. These two sides of the immune system act immediately (innate) to particular microbial antigens or evolve (adaptive) to attain high specificity and affinity to a much wider range of antigens. We also include examples of other key receptors in the immune system (cytokine receptors) that regulate immunity and inflammation. Furthermore, these antigen receptors use a limited set of protein folds to accomplish their various immunological roles. The other main players are the antigens themselves. We focus on surface glycoproteins in enveloped viruses including SARS-CoV-2 that enable entry and egress into host cells and are targets for the antibody response. This review covers what we have learned over the past half century about the structural basis of the immune response to microbial pathogens and how that information can be utilized to design vaccines and therapeutics.
Assuntos
Imunidade Adaptativa , Anticorpos Antivirais/química , Antígenos Virais/química , Imunidade Inata , Receptores de Antígenos de Linfócitos T/química , Receptores de Citocinas/química , SARS-CoV-2/imunologia , Alergia e Imunologia/história , Animais , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Apresentação de Antígeno , Antígenos Virais/genética , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/virologia , Cristalografia/história , Cristalografia/métodos , História do Século XX , História do Século XXI , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , SARS-CoV-2/patogenicidade , Recombinação V(D)JRESUMO
Thymic Stromal Lymphopoietin (TSLP) and Interleukin-7 (IL-7) are widely studied cytokines within distinct branches of immunology. On one hand, TSLP is crucially important for mediating type 2 immunity at barrier surfaces and has been linked to widespread allergic and inflammatory diseases of the airways, skin, and gut. On the other hand, IL-7 operates at the foundations of T-cell and innate lymphoid cell (ILC) development and homeostasis and has been associated with cancer. Yet, TSLP and IL-7 are united by key commonalities in their structure and the structural basis of the receptor assemblies they mediate to initiate cellular signaling, in particular their cross-utilization of IL-7Rα. As therapeutic targeting of TSLP and IL-7 via diverse approaches is reaching advanced stages and in light of the plethora of mechanistic and structural data on receptor signaling mediated by the two cytokines, the time is ripe to provide integrated views of such knowledge. Here, we first discuss the major pathophysiological roles of TSLP and IL-7 in autoimmune diseases, inflammation and cancer. Subsequently, we curate structural and mechanistic knowledge about receptor assemblies mediated by the two cytokines. Finally, we review therapeutic avenues targeting TSLP and IL-7 signaling. We envision that such integrated view of the mechanism, structure, and modulation of signaling assemblies mediated by TSLP and IL-7 will enhance and fine-tune the development of more effective and selective approaches to further interrogate the role of TSLP and IL-7 in physiology and disease.
Assuntos
Doenças Autoimunes/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Interleucina-7/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Animais , Doenças Autoimunes/etiologia , Citocinas/química , Citocinas/genética , Suscetibilidade a Doenças , Variação Genética , Humanos , Inflamação/etiologia , Interleucina-7/química , Interleucina-7/genética , Neoplasias/etiologia , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Receptores de Interleucina-7/química , Receptores de Interleucina-7/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Class 1 cytokine receptors (C1CRs) are single-pass transmembrane proteins responsible for transmitting signals between the outside and the inside of cells. Remarkably, they orchestrate key biological processes such as proliferation, differentiation, immunity and growth through long disordered intracellular domains (ICDs), but without having intrinsic kinase activity. Despite these key roles, their characteristics remain rudimentarily understood. METHODS: The current paper asks the question of why disorder has evolved to govern signaling of C1CRs by reviewing the literature in combination with new sequence and biophysical analyses of chain properties across the family. RESULTS: We uncover that the C1CR-ICDs are fully disordered and brimming with SLiMs. Many of these short linear motifs (SLiMs) are overlapping, jointly signifying a complex regulation of interactions, including network rewiring by isoforms. The C1CR-ICDs have unique properties that distinguish them from most IDPs and we forward the perception that the C1CR-ICDs are far from simple strings with constitutively bound kinases. Rather, they carry both organizational and operational features left uncovered within their disorder, including mechanisms and complexities of regulatory functions. CONCLUSIONS: Critically, the understanding of the fascinating ability of these long, completely disordered chains to orchestrate complex cellular signaling pathways is still in its infancy, and we urge a perceptional shift away from the current simplistic view towards uncovering their full functionalities and potential. Video abstract.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Humanos , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
Controlling of pro-inflammation induced by pro-inflammatory cytokines and anti-inflammatory response induced by M2 macrophages is important for osteogenesis in the process of bone tissue repair. Thus, we fabricated biomimetic anti-inflammatory nano-capsule (BANC) that can block cytokines and promote M2 macrophage polarization, presenting a positive role for bone tissue repair. The BANC is a biomimic nanosystem, coated with lipopolysaccharide-treated macrophage cell membranes with cytokine receptors enveloping gold nanocage (AuNC) as "cytokine blocker", and loaded with resolvin D1 inside into AuNC as "M2 polarization inducer" whose controlled-release could be triggered under near-infrared laser irradiation in sequence, and these chronological events were consistent with the healing process of bone tissue repair. Moreover, in vivo application of femoral bone defects revealed that the BANC composite boron-containing mesoporous bioactive glass scaffolds improved the final effects of bone tissue repair through preventing inflammatory response, promoting M2 polarization in sequence in accord with the in vitro investigation. Hence, cytokine neutralization and M2 macrophage polarization enables the BANC to enhance the bone tissue repair as a biomimetic anti-inflammation effector. Therefore, this study provides potential therapeutic strategies for trauma-mediated or inflammation-related bone defects based on a biomimetic nanomaterial with weakened pro-inflammatory and enhanced anti-inflammatory effects. STATEMENT OF SIGNIFICANCE: Cell membrane-mimic nanomaterials have been popular for blocking natural cell responses for some infection diseases, yet their role in biological process of bone repair is unknown. Here, we fabricated Biomimetic Anti-inflammatory Nano-Capsule (BANC), coated with cell membrane with cytokines receptors on the surface which could neutralize the pro-inflammatory cytokine receptor to block activated pro-inflammation, loaded with Resolvin D1 inside which could be controllably released by NIR irradiation to promote M2 macrophage polarization for the following bone formation during the process of bone repair. Administration of BANC as cytokines blocker and M2 polarization inducer to enhance the bone regeneration, thus presenting a promising potential for the treatment of bone repair and regeneration.
Assuntos
Anti-Inflamatórios/uso terapêutico , Regeneração Óssea/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Inflamação/prevenção & controle , Macrófagos/efeitos dos fármacos , Nanocápsulas/uso terapêutico , Animais , Materiais Biomiméticos/química , Membrana Celular/química , Ácidos Docosa-Hexaenoicos/uso terapêutico , Portadores de Fármacos/química , Portadores de Fármacos/uso terapêutico , Feminino , Fêmur/efeitos dos fármacos , Lipopolissacarídeos/química , Lipopolissacarídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Nanocápsulas/química , Células RAW 264.7 , Receptores de Citocinas/química , Receptores de Citocinas/uso terapêuticoRESUMO
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 SinaisRESUMO
Chronic inflammation is a pivotal event in the pathogenesis of cardiovascular diseases, including atherosclerosis, restenosis, and coronary artery disease. The efficacy of current treatment or preventive strategies for such inflammation is still inadequate. Thus, new anti-inflammatory strategies are needed. In this study, based on molecular docking and structural analysis, a potential peptide KCF18 with amphiphilic properties (positively charged and hydrophobic residues) derived from the receptors of proinflammatory cytokines was designed to inhibit cytokine-induced inflammatory response. Simulations suggested that KCF18 could bind to cytokines simultaneously, and electrostatic interactions were dominant. Surface plasmon resonance detection showed that KCF18 bound to both tumor necrosis factor-α (TNF-α) and interleukin-6, which is consistent with MM/PBSA binding free energy calculations. The cell experiments showed that KCF18 significantly reduced the binding of proinflammatory cytokines to their cognate receptors, suppressed TNF-α mRNA expression and monocyte binding and transmigration, and alleviated the infiltration of white blood cells in a peritonitis mouse model. The designed peptide KCF18 could remarkably diminish the risk of vascular inflammation by decreasing plasma cytokines release and by directly acting on the vascular endothelium. This study demonstrated that a combination of structure-based in silico design calculations, together with experimental measurements can be used to develop potential anti-inflammatory agents.
Assuntos
Inflamação/tratamento farmacológico , Inflamação/metabolismo , Peptídeos/química , Peptídeos/uso terapêutico , Receptores de Citocinas/química , Humanos , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Ligação Proteica , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.
Assuntos
Citocininas/química , Receptores de Citocinas/antagonistas & inibidores , 4-Cloro-7-nitrobenzofurazano/farmacologia , Adenina/análogos & derivados , Adenina/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Carbocianinas/química , Corantes/química , Citocininas/farmacologia , Corantes Fluorescentes/química , Regulação da Expressão Gênica de Plantas , Isopenteniladenosina/síntese química , Isopenteniladenosina/química , Isopenteniladenosina/farmacologia , Microscopia Confocal , Estrutura Molecular , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Purinas/química , Receptores de Citocinas/química , Rodaminas/química , Plântula/metabolismo , Terpenos/metabolismo , Zea mays/metabolismoRESUMO
Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment and computation have been used to study receptor dynamics. Recent studies on three distinct classes of receptors (G-protein coupled receptors, ligand-gated ion-channels and single-pass receptors) are highlighted to show that conformational changes across a range of time-scales and length-scales are central to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function.
Assuntos
Canais Iônicos de Abertura Ativada por Ligante/química , Receptores Proteína Tirosina Quinases/química , Receptores de Citocinas/química , Receptores Acoplados a Proteínas G/química , Transdução de Sinais/fisiologia , Animais , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Canais Iônicos de Abertura Ativada por Ligante/fisiologia , Ligantes , Simulação de Dinâmica Molecular , Conformação Proteica , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Citocinas/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Relação Estrutura-AtividadeRESUMO
The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) plays a pivotal role in the pathophysiology of various allergy disorders that are mediated by type 2 helper T cell (Th2) responses, such as asthma and atopic dermatitis. TSLP forms a ternary complex with the TSLP receptor (TSLPR) and the interleukin-7-receptor subunit alpha (IL-7Rα), thereby activating a signaling cascade that culminates in the release of pro-inflammatory mediators. In this study, we conducted an in silico characterization of the TSLP:TSLPR complex to investigate the drugability of this complex. Two commercially available fragment libraries were screened computationally for possible inhibitors and a selection of fragments was subsequently tested in vitro. The screening setup consisted of two orthogonal assays measuring TSLP binding to TSLPR: a BLI-based assay and a biochemical assay based on a TSLP:alkaline phosphatase fusion protein. Four fragments pertaining to diverse chemical classes were identified to reduce TSLP:TSLPR complex formation to less than 75% in millimolar concentrations. We have used unbiased molecular dynamics simulations to develop a Markov state model that characterized the binding pathway of the most interesting compound. This work provides a proof-of-principle for use of fragments in the inhibition of TSLP:TSLPR complexation.
Assuntos
Citocinas/metabolismo , Receptores de Citocinas/metabolismo , Citocinas/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptores de Citocinas/química , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Interface Usuário-Computador , Linfopoietina do Estroma do TimoRESUMO
Thymic stromal lymphopoietin (TSLP) is a type II cytokine which is associated with most inflammatory allergic disorders in humans. It is produced mainly by epithelial cells with important role in the development of chronic inflammatory diseases by activating T-helper cell type-2 (TH2) pathways. In this study, a total of 16 peptides were prepared by solid phase peptide synthesis based on amino acid sequences of the interface between TSLP and TSLP receptor. Their TSLP inhibition activities were determined by ELISA assay. Among them, three peptides (6-8) exhibited >50% inhibition at concentration of 0.3mM. They can be used as hit compounds for developing peptide-based TSLP inhibitors.
Assuntos
Citocinas/antagonistas & inibidores , Peptídeos/metabolismo , Sequência de Aminoácidos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Ligantes , Peptídeos/química , Ligação Proteica , Receptores de Citocinas/química , Receptores de Citocinas/metabolismo , Células Th2/citologia , Células Th2/metabolismo , Linfopoietina do Estroma do TimoRESUMO
The complex microenvironment that surrounds hematopoietic stem cells (HSCs) in the bone marrow niche involves different coordinated signaling pathways. The stem cells establish permanent interactions with distinct cell types such as mesenchymal stromal cells, osteoblasts, osteoclasts or endothelial cells and with secreted regulators such as growth factors, cytokines, chemokines and their receptors. These interactions are mediated through adhesion to extracellular matrix compounds also. All these signaling pathways are important for stem cell fates such as self-renewal, proliferation or differentiation, homing and mobilization, as well as for remodeling of the niche. Among these complex molecular cues, this review focuses on heparan sulfate (HS) structures and functions and on the role of enzymes involved in their biosynthesis and turnover. HS associated to core protein, constitute the superfamily of heparan sulfate proteoglycans (HSPGs) present on the cell surface and in the extracellular matrix of all tissues. The key regulatory effects of major medullar HSPGs are described, focusing on their roles in the interactions between hematopoietic stem cells and their endosteal niche, and on their ability to interact with Heparin Binding Proteins (HBPs). Finally, according to the relevance of HS moieties effects on this complex medullar niche, we describe recent data that identify HS mimetics or sulfated HS signatures as new glycanic tools and targets, respectively, for hematopoietic and mesenchymal stem cell based therapeutic applications.
Assuntos
Citocinas/química , Proteínas da Matriz Extracelular/química , Células-Tronco Hematopoéticas/química , Heparitina Sulfato/química , Células-Tronco Mesenquimais/química , Receptores de Citocinas/química , Animais , Materiais Biomiméticos/farmacologia , Medula Óssea/fisiologia , Configuração de Carboidratos , Sequência de Carboidratos , Citocinas/metabolismo , Células Endoteliais/química , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Heparitina Sulfato/classificação , Heparitina Sulfato/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteoblastos/química , Osteoblastos/metabolismo , Osteoclastos/química , Osteoclastos/metabolismo , Ligação Proteica , Receptores de Citocinas/metabolismo , Transdução de Sinais , Nicho de Células-Tronco/fisiologiaRESUMO
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.
Assuntos
Asma/imunologia , Citocinas/antagonistas & inibidores , Citocinas/química , Hipersensibilidade/imunologia , Complexos Multiproteicos/metabolismo , Receptores de Citocinas/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Asma/patologia , Quimiocinas/biossíntese , Cristalografia por Raios X , Células Dendríticas , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Hipersensibilidade/patologia , Modelos Moleculares , Estrutura Secundária de Proteína , Receptores de Citocinas/química , Receptores de Interleucina-7/química , Receptores de Interleucina-7/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Linfopoietina do Estroma do TimoRESUMO
The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFRα to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-ß (gp130/LIFRß) for signaling. Both CLC and CNTFRα are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFRα (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFRα) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFRα and sorLA, CNTFRα first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFRα, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1. The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFRα expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFRα; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFRα.
Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Citocinas/metabolismo , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de Citocinas/metabolismo , Sítios de Ligação , Western Blotting , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Citocinas/química , Regulação para Baixo , Endocitose , Células HEK293 , Humanos , Imuno-Histoquímica , Proteínas Relacionadas a Receptor de LDL/genética , Ligantes , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/enzimologia , Proteínas de Membrana Transportadoras/genética , Fosforilação , Receptores de Citocinas/química , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
The crystal structure of a construct consisting of the FERM and SH2-like domains of the human Janus kinase 1 (JAK1) bound to a fragment of the intracellular domain of the interferon-λ receptor 1 (IFNLR1) has been determined at the nominal resolution of 2.1Å. In this structure, the receptor peptide forms an 85-Å-long extended chain, in which both the previously identified box1 and box2 regions bind simultaneously to the FERM and SH2-like domains of JAK1. Both domains of JAK1 are generally well ordered, with regions not seen in the crystal structure limited to loops located away from the receptor-binding regions. The structure provides a much more complete and accurate picture of the interactions between JAK1 and IFNLR1 than those given in earlier reports, illuminating the molecular basis of the JAK-cytokine receptor association. A glutamate residue adjacent to the box2 region in IFNLR1 mimics the mode of binding of a phosphotyrosine in classical SH2 domains. It was shown here that a deletion of residues within the box1 region of the receptor abolishes stable interactions with JAK1, although it was previously shown that box2 alone is sufficient to stabilize a similar complex of the interferon-α receptor and TYK2.