RESUMO
The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.
Assuntos
Receptor gp130 de Citocina/metabolismo , Citocinas/síntese química , Citocinas/uso terapêutico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ligação Competitiva , Citocinas/química , Diabetes Mellitus Tipo 2/metabolismo , Desenho de Fármacos , Fígado Gorduroso/prevenção & controle , Teste de Tolerância a Glucose , Humanos , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Incretinas/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Obesidade/metabolismo , Pâncreas/metabolismo , Fosfoproteínas/metabolismo , Engenharia de Proteínas , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Fatores de Transcrição , Aumento de Peso/efeitos dos fármacos , Proteínas de Sinalização YAPRESUMO
Interleukin (IL)-12 and IL-23 are composite cytokines consisting of p35/p40 and p19/p40, respectively, which signal via the common IL-12 receptor ß1 (IL-12Rß1) and the cytokine-specific receptors IL-12Rß2 and IL-23R. Previous data showed that the p40 component interacts with IL-12Rß1, whereas p19 and p35 subunits solely bind to IL-23R and IL-12Rß2, resulting in tetrameric signaling complexes. In the absence of p19 and p35, p40 forms homodimers and may induce signaling via IL-12Rß1 homodimers. The critical amino acids of p19 and p35 required for binding to IL-23R and IL-12Rß2 are known, and two regions of p40 critical for binding to IL-12Rß1 have recently been identified. In order to characterize the involvement of the N-terminal region of p40 in binding to IL-12Rß1, we generated deletion variants of the p40-p19 fusion cytokine. We found that an N-terminal deletion variant missing amino acids M23 to P39 failed to induce IL-23-dependent signaling and did not bind to IL-12Rß1, whereas binding to IL-23R was maintained. Amino acid replacements showed that p40W37K largely abolished IL-23-induced signal transduction and binding to IL-12Rß1, but not binding to IL-23R. Combining p40W37K with D36K and T38K mutations eliminated the biological activity of IL-23. Finally, homodimeric p40D36K/W37K/T38K did not interact with IL-12Rß1, indicating binding of homodimeric p40 to IL-12Rß1 is comparable to the interaction of IL-23/IL-12 and IL-12Rß1. In summary, we have defined D36, W37, and T38 as hotspot amino acids for the interaction of IL-12/IL-23 p40 with IL-12Rß1. Structural insights into cytokine-cytokine receptor binding are important to develop novel therapeutic strategies.
Assuntos
Subunidade p40 da Interleucina-12 , Multimerização Proteica , Receptores de Interleucina-12 , Transdução de Sinais , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Camundongos , Ligação Proteica , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , TriptofanoRESUMO
The heterodimeric cytokine interleukin 27 (IL-27) signals through the IL-27Rα subunit of its receptor, combined with gp130, a common receptor chain used by several cytokines, including IL-6. Notably, the IL-27 subunits p28 (IL-27p28) and EBI3 are not always expressed together, which suggests that they may have unique functions. Here we show that IL-27p28, independently of EBI3, antagonized cytokine signaling through gp130 and IL-6-mediated production of IL-17 and IL-10. Similarly, the ability to generate antibody responses was dependent on the activity of gp130-signaling cytokines. Mice transgenic for expression of IL-27p28 showed a substantial defect in the formation of germinal centers and antibody production. Thus, IL-27p28, as a natural antagonist of gp130-mediated signaling, may be useful as a therapeutic for managing inflammation mediated by cytokines that signal through gp130.
Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucinas/metabolismo , Transdução de Sinais/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Separação Celular , Receptor gp130 de Citocina/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Interleucinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Receptores de Citocinas/imunologia , Receptores de Citocinas/metabolismoRESUMO
Interleukin (IL)-12 and IL-23 belong to the IL-12 type family and are composite cytokines, consisting of the common ß subunit p40 and the specific cytokine α subunit p35 and p19, respectively. IL-12 signals via the IL-12Rß1·IL-12Rß2 receptor complex, and IL-23 uses also IL-12Rß1 but engages IL-23R as second receptor. Importantly, binding of IL-12 and IL-23 to IL-12Rß1 is mediated by p40, and binding to IL-12Rß2 and IL-23R is mediated by p35 and p19, respectively. Previously, we have identified a W157A substitution at site 3 of murine IL-23p19 that abrogates binding to murine IL-23R. Here, we demonstrate that the analogous Y185R site 3 substitution in murine and Y189R site 3 substitution in human IL-12p35 abolishes binding to IL-12Rß2 in a cross-species manner. Although Trp157 is conserved between murine and human IL-23p19 (Trp156 in the human ortholog), the site 3 W156A substitution in hIL-23p19 did not affect signaling of cells expressing human IL-12Rß1 and IL-23R, suggesting that the interface of murine IL-23p19 required for binding to IL-23R is different from that in the human ortholog. Hence, we introduced additional hIL-23p19 substitutions within its binding interface to hIL-23R and found that the combined site 3 substitutions of W156A and L160E, which become buried at the complex interface, disrupt binding of hIL-23p19 to hIL-23R. In summary, we have identified substitutions in IL-12p35 and IL-23p19 that disrupt binding to their cognate receptors IL-12Rß2 and IL-23R in a murine/human cross-species manner.
Assuntos
Subunidade p40 da Interleucina-12 , Subunidade p19 da Interleucina-23 , Receptores de Interleucina-12 , Receptores de Interleucina , Substituição de Aminoácidos , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetulus , Células HEK293 , Humanos , Subunidade p40 da Interleucina-12/química , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Subunidade p19 da Interleucina-23/química , Subunidade p19 da Interleucina-23/genética , Subunidade p19 da Interleucina-23/metabolismo , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Receptores de Interleucina/química , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismoRESUMO
Proteolytic processing of membrane proteins by A disintegrin and metalloprotease-17 (ADAM17) is a key regulatory step in many physiological and pathophysiological processes. This so-called shedding is essential for development, regeneration and immune defense. An uncontrolled ADAM17 activity promotes cancer development, chronic inflammation and autoimmune diseases. Consequently, the ADAM17 activity is tightly regulated. As a final trigger for the shedding event a phosphatidylserine (PS) flip to the outer leaflet of the cell membrane was recently described. PS interacts with the extracellular part of ADAM17, which results in the shedding event by shifting the catalytic domain towards the membrane close to the cleavage sites within ADAM17 substrates. Our data indicate that the intrinsic proteolytic activity of the catalytic domain is prerequisite for the shedding activity and constantly present. However, the accessibility for substrate cleavage sites is controlled on several levels. In this report, we demonstrate that the positioning of the catalytic domain towards the cleavage sites is a crucial part of the shedding process. This finding contributes to the understanding of the complex and multilayered regulation of ADAM17 at the cell surface.
Assuntos
Proteína ADAM17/metabolismo , Receptores de Interleucina-6/metabolismo , Proteína ADAM17/química , Sequência de Aminoácidos , Domínio Catalítico , Células HEK293 , Humanos , Mutação , Fosfatidilserinas/metabolismo , Proteólise , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genéticaRESUMO
Signaling of the cytokine interleukin-6 (IL-6) via its soluble IL-6 receptor (sIL-6R) is responsible for the proinflammatory properties of IL-6 and constitutes an attractive therapeutic target, but how the sIL-6R is generated in vivo remains largely unclear. Here, we use liquid chromatography-mass spectrometry to identify an sIL-6R form in human serum that originates from proteolytic cleavage, map its cleavage site between Pro-355 and Val-356, and determine the occupancy of all O- and N-glycosylation sites of the human sIL-6R. The metalloprotease a disintegrin and metalloproteinase 17 (ADAM17) uses this cleavage site in vitro, and mutation of Val-356 is sufficient to completely abrogate IL-6R proteolysis. N- and O-glycosylation were dispensable for signaling of the IL-6R, but proteolysis was orchestrated by an N- and O-glycosylated sequon near the cleavage site and an N-glycan exosite in domain D1. Proteolysis of an IL-6R completely devoid of glycans is significantly impaired. Thus, glycosylation is an important regulator for sIL-6R generation.
Assuntos
Proteólise , Receptores de Interleucina-6/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Processamento Alternativo/genética , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Glicosilação , Humanos , Espaço Intracelular/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Mutação/genética , Polissacarídeos/metabolismo , Prolina/metabolismo , Domínios Proteicos , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Transdução de Sinais , Solubilidade , Valina/metabolismoRESUMO
Height is a complex human phenotype that is influenced by variations in a high number of genes. Recently, a single nucleotide polymorphism (SNP) within IL11 (rs4252548) has been described to be associated with height in adults of European ancestry. This coding SNP leads to the exchange of Arg-112 to His-112 within the cytokine Interleukin-11 (IL-11), which has a well-established role in osteoclast development and bone turnover. The functional consequences of the R112H mutation are unknown so far. In this study, we show by molecular replacement that Arg-112 does not participate in binding of IL-11 to its receptors IL-11R and glycoprotein 130 (gp130). Recombinant IL-11 R112H expressed in E. coli displays a correct four-helix-bundle folding topology, and binds with similar affinity to IL-11R and the IL-11/IL-11R/gp130 complex. IL-11 R112H induces cell proliferation and phosphorylation of the downstream transcription factor STAT3 indistinguishable from IL-11. However, IL-11 R112H fails to support the survival of osteoclast progenitor cells and is less thermally stable, which is caused by the loss of the positive charge on the protein surface since protonation of the histidine side chain recovers stability.
Assuntos
Estatura/genética , Receptor gp130 de Citocina/genética , Interleucina-11/genética , Receptores de Interleucina-11/genética , Arginina/química , Arginina/genética , Linhagem Celular , Proliferação de Células/genética , Receptor gp130 de Citocina/química , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Interleucina-11/química , Polimorfismo de Nucleotídeo Único , Receptores de Interleucina-11/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND/AIMS: Endoplasmic reticulum (ER)-resident proteins with a C-terminal KDEL ERretention sequence are captured in the Golgi apparatus by KDEL receptors (KDELRs). The binding of such proteins to these receptors induces their retrograde transport. Nevertheless, some KDEL proteins, such as Protein Disulfide Isomerases (PDIs), are found at the cell surface. PDIs target disulfide bridges in the extracellular domains of proteins, such as integrins or A Disintegrin And Metalloprotease 17 (ADAM17) leading to changes in the structure and function of these molecules. Integrins become activated and ADAM17 inactivated upon disulfide isomerization. The way that PDIs escape from retrograde transport and reach the plasma membrane remains far from clear. Various mechanisms might exist, depending on whether a local cell surface association or a more global secretion is required. METHODS: To get a more detailed insight in the transport of PDIs to the cell surface, methods such as cell surface biotinylation, flow cytometric analysis, immunoprecipitation, fluorescence microscopy as well as labeling of cells with fluorescence labled recombinant PDIA6 was performed. RESULTS: Here, we show that the C-terminal KDEL ER retention sequence is sufficient to prevent secretion of PDIA6 into the extracellular space but is mandatory for its association with the cell surface. The cell surface trafficking of PDIA1, PDIA3, and PDIA6 is dependent on KDELR1, which travels in a dynamic manner to the cell surface. This transport is assumed to result in PDI cell surface association, which differs from PDI inducible secretion into the extracellular space. Distinct PDIs differ in their trafficking properties. Endogenous KDELR1, detectable at the cell surface, might be involved not only in the transport of cell-surface-associated PDIs, but also in their retrieval and internalization from the extracellular space. CONCLUSION: Beside their ER retention motive PDIs travel to the cell surface. Here they target different proteins to render their function. To escape the ER PDIs travel via various pathways. One of them depends on the KDELR1, which can transport its target to the cell surface, where it is to be expected to release its cargo in close vicinity to its target molecules. Hence, the KDEL sequence is needed for cell surface association of PDIs, such as PDIA6.
Assuntos
Proteína ADAM17/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Peptídeos/metabolismo , Proteína ADAM17/genética , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Isomerases de Dissulfetos de Proteínas/genética , Transporte Proteico/fisiologia , Receptores de Peptídeos/genéticaRESUMO
The expression of nearly all smooth muscle genes are controlled by serum response factor binding sites in their promoter regions. However, SRF alone is not sufficient for regulating smooth muscle cell development. It associates with other cardiovascular specific cofactors to regulate smooth muscle gene expression. Previously, we showed that the transcription co-factor CRP2 was a regulator of smooth muscle gene expression. Here, we report that CSRP2BP, a coactivator for CRP2, is a histone acetyltransferase and a driver of smooth muscle gene expression. CSRP2BP directly interacted with SRF, CRP2 and myocardin. CSRP2BP synergistically activated smooth muscle gene promoters in an SRF-dependent manner. A combination of SRF, GATA6 and CRP2 required CSRP2BP for robust smooth muscle gene promoter activity. Knock-down of Csrp2bp in smooth muscle cells resulted in reduced smooth muscle gene expression. We conclude that the CSRP2BP histone acetyltransferase is a coactivator for CRP2 that works synergistically with SRF and myocardin to regulate smooth muscle gene expression.
Assuntos
Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Miócitos de Músculo Liso/metabolismo , Acetilação , Animais , Linhagem Celular , Núcleo Celular/enzimologia , Células Cultivadas , Cromatina/enzimologia , Expressão Gênica , Histonas/metabolismo , Humanos , Camundongos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ratos , Transativadores/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In contrast to many other signalling mechanisms shedding of membrane-anchored proteins is an irreversible process. A Disintegrin And Metalloproteinase (ADAM) 17 is one of the major sheddases involved in a variety of physiological and pathophysiological processes including regeneration, differentiation, and cancer progression. Due to its central role in signalling the shedding activity of ADAM17 is tightly regulated, especially on the cell surface, where shedding events take place. The activity of ADAM17 can be subdivided into a catalytic activity and the actual shedding activity. Whereas the catalytic activity is constitutively present, the shedding activity has to be induced and is tightly controlled to prevent pathological situations induced by the release of its substrates. The regulation of the shedding activity of ADAM17 is multilayered and different regions of the protease are involved. Intriguingly, its extracellular domains play crucial roles in different regulatory mechanisms. We will discuss the role of these domains in the control of ADAM17 activity. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.
Assuntos
Proteína ADAM17/genética , Membrana Celular/genética , Proteínas de Membrana/genética , Proteólise , Membrana Celular/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND/AIMS: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. METHODS: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. RESULTS: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. CONCLUSION: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.
Assuntos
Receptores de Interleucina-11/metabolismo , Proteína ADAM10/metabolismo , Sequência de Aminoácidos , Animais , Proliferação de Células , Retículo Endoplasmático/metabolismo , Glicosilação , Células HEK293 , Células HeLa , Humanos , Interleucina-11/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Domínios Proteicos , Transporte Proteico , Proteólise , Receptores de Interleucina-11/química , Receptores de Interleucina-11/genética , Fator de Transcrição STAT3/metabolismoRESUMO
The unexpected resistance of psoriasis lesions to fungal infections suggests local production of an antifungal factor. We purified Trichophyton rubrum-inhibiting activity from lesional psoriasis scale extracts and identified the Cys-reduced form of S100A7/psoriasin (redS100A7) as a principal antifungal factor. redS100A7 inhibits various filamentous fungi, including the mold Aspergillus fumigatus, but not Candida albicans. Antifungal activity was inhibited by Zn(2+), suggesting that redS100A7 interferes with fungal zinc homeostasis. Because S100A7-mutants lacking a single cysteine are no longer antifungals, we hypothesized that redS100A7 is acting as a Zn(2+)-chelator. Immunogold electron microscopy studies revealed that it penetrates fungal cells, implicating possible intracellular actions. In support with our hypothesis, the cell-penetrating Zn(2+)-chelator TPEN was found to function as a broad-spectrum antifungal. Ultrastructural analyses of redS100A7-treated T. rubrum revealed marked signs of apoptosis, suggesting that its mode of action is induction of programmed cell death. TUNEL, SYTOX-green analyses, and caspase-inhibition studies supported this for both T. rubrum and A. fumigatus. Whereas redS100A7 can be generated from oxidized S100A7 by action of thioredoxin or glutathione, elevated redS100A7 levels in fungal skin infection indicate induction of both S100A7 and its reducing agent in vivo. To investigate whether redS100A7 and TPEN are antifungals in vivo, we used a guinea pig tinea pedes model for fungal skin infections and a lethal mouse Aspergillus infection model for lung infection and found antifungal activity in both in vivo animal systems. Thus, selective fungal cell-penetrating Zn(2+)-chelators could be useful as an urgently needed novel antifungal therapeutic, which induces programmed cell death in numerous fungi.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Dissulfetos/química , Proteínas S100/farmacologia , Animais , Aspergilose/tratamento farmacológico , Aspergillus fumigatus/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Modelos Animais de Doenças , Cobaias , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Oxirredução , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/química , Proteínas S100/uso terapêuticoRESUMO
By mediating proteolytic shedding on the cell surface the disintegrin and metalloproteinases ADAM10 and ADAM17 function as critical regulators of growth factors, cytokines and adhesion molecules. We here report that stimulation of lung epithelial A549 tumor cells with phorbol-12-myristate-13-acetate (PMA) leads to the downregulation of the surface expressed mature form of ADAM17 without affecting ADAM10 expression. This reduction could not be sufficiently explained by metalloproteinase-mediated degradation, dynamin-mediated internalization or microdomain redistribution of ADAM17. Instead, surface downregulation of ADAM17 was correlated with the presence of its mature form in exosomes. Exosomal ADAM17 release was also observed in monocytic and primary endothelial cells where it could be induced by stimulation with lipopolysaccharide. Antibody-mediated surface labelling of ADAM17 revealed that at least part of exosomal ADAM17 was oriented with the metalloproteinase domain outside and had been expressed on the cell surface. Suppression of iRHOM2-mediated ADAM17 maturation prevented surface expression and exosomal release of ADAM17. Further, deletion of the protease's C-terminus or cell treatment with a calcium chelator diminished exosomal release as well as surface downregulation of ADAM17, underlining that both processes are closely associated. Co-incubation of ADAM17 containing exosomes with cells expressing the ADAM17 substrates TGFα or amphiregulin lead to increased shedding of both substrates. This was prevented when exosomes were prepared from cells with shRNA-mediated ADAM17 knockdown. These data indicate that cell stimulation can downregulate expression of mature ADAM17 from the cell surface and induce release of exosomal ADAM17, which can then distribute and contribute to substrate shedding on more distant cells.
Assuntos
Proteína ADAM17/metabolismo , Exossomos/enzimologia , Células A549 , Proteína ADAM10/metabolismo , Proteína ADAM17/genética , Anfirregulina/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Sinalização do Cálcio , Proteínas de Transporte/metabolismo , Células Endoteliais/enzimologia , Ativação Enzimática , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos/farmacologia , Microdomínios da Membrana/enzimologia , Proteínas de Membrana/metabolismo , Monócitos/enzimologia , Transporte Proteico , Interferência de RNA , Especificidade por Substrato , Acetato de Tetradecanoilforbol/farmacologia , Transfecção , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Antimicrobial peptides are an important part of the innate immune defense in the central nervous system (CNS). The expression of the antimicrobial peptides psoriasin (S100A7) is up-regulated during bacterial meningitis. However, the exact mechanisms induced by psoriasin to modulate glial cell activity are not yet fully understood. Our hypothesis is that psoriasin induced pro- and anti-inflammatory signaling pathways as well as regenerative factors to contribute in total to a balanced immune response. Therefore, we used psoriasin-stimulated glial cells and analyzed the translocation of the pro-inflammatory transcription factor nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) in murine glial cells and the expression of pro- and anti-inflammatory mediators by real time RT-PCR, ELISA technique, and western blotting. Furthermore, the relationship between psoriasin and the antioxidative stress transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) was investigated. Stimulation with psoriasin not only enhanced NFκB translocation and increased the expression of the pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) but also neurotrophin expression. Evidence for functional interactions between psoriasin and Nrf2 were detected in the form of increased antioxidant response element (ARE) activity and induction of Nrf2/ARE-dependent heme oxygenase 1 (HO-1) expression in psoriasin-treated microglia and astrocytes. The results illustrate the ability of psoriasin to induce immunological functions in glia cells where psoriasin exerts divergent effects on the innate immune response.
Assuntos
Imunidade Inata/fisiologia , Neuroglia/imunologia , Neuroglia/metabolismo , Proteínas S100/imunologia , Proteínas S100/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Feminino , Células HEK293 , Humanos , Imunidade Inata/efeitos dos fármacos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Proteína A7 Ligante de Cálcio S100 , Proteínas S100/biossínteseRESUMO
IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting of the IL-23 receptor (IL-23R) and the IL-12 receptor ß1 (IL-12Rß1). Complex formation was hypothesized to follow the "site I-II-III" architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate binding to IL-12Rß1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rß1 is independent of site II in p19. Instead, binding of IL-23 to the cytokine binding module of IL-12Rß1 is mediated by domains 1 and 2 of p40 via corresponding site II amino acids of IL-12Rß1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but, at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rß1. On the basis of our experimental validation, we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rß1 complex. Furthermore, our data help to explain why p40 is an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.
Assuntos
Subunidade beta 1 de Receptor de Interleucina-12/química , Subunidade p40 da Interleucina-12/química , Interleucina-23/química , Receptores de Interleucina-12/química , Receptores de Interleucina/química , Animais , Sítios de Ligação , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Expressão Gênica , Humanos , Subunidade beta 1 de Receptor de Interleucina-12/genética , Subunidade beta 1 de Receptor de Interleucina-12/metabolismo , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/genética , Receptores de Interleucina-12/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the serine protease inhibitor of Kazal-type (SPINK) family. SPINK6 was shown to be expressed in human stratum corneum and is able to inhibit several KLKs such as KLK4, -5, -12, -13 and -14. In order to understand the structural traits of the specific inhibition we solved the structure of SPINK6 in solution by NMR-spectroscopy and studied its interaction with KLKs. Thereby, beside the conserved binding mode, we identified an alternate binding mode which has so far not been observed for SPINK inhibitors.
Assuntos
Modelos Químicos , Modelos Moleculares , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Ativação Enzimática , Humanos , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Análise de Sequência de Proteína/métodos , Inibidores de Serinopeptidase do Tipo KazalRESUMO
To avoid malformation and disease, tissue development and homoeostasis are co-ordinated precisely in time and space. Secreted Frizzled-related protein 3 (sFRP3), encoded by the Frizzled-related protein gene (FRZB), acts as an antagonist of Wnt signalling in bone development by delaying the maturation of proliferative chondrocytes into hypertrophic chondrocytes. A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protease that is essential for developmental processes and promotes cartilage maturation into bone. sFRP3 is chondroprotective and is expressed in chondrocytes of healthy articular cartilage. Upon damage to cartilage, sFRP3 is down-regulated. Rare variants of sFRP3 are associated with osteoarthritis. The present study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R (sIL-6R) is linked to inflammation, cartilage degeneration and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also suggests a new regulatory mechanism by which the substrate is protected against shedding.
Assuntos
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Condrócitos/metabolismo , Osteoartrite do Quadril/metabolismo , Proteínas/metabolismo , Receptores de Interleucina-6/metabolismo , Regulação para Cima , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM17 , Substituição de Aminoácidos , Linhagem Celular Tumoral , Regulação para Baixo , Predisposição Genética para Doença , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Osteoartrite do Quadril/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/genética , Receptores de Interleucina-6/química , Receptores de Interleucina-6/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
A wide variety of biological processes including differentiation, regeneration, and cancer progression are regulated by shedding of membrane-anchored proteins. One of the major sheddases is A Disintegrin And Metalloprotease-17 (ADAM17) whose extracellular region consists of a pro-, a catalytic, a disintegrin-, and a membrane-proximal domain (MPD) as well as a short juxtamembrane segment of 17 amino acid residues that has been named "Conserved ADAM-seventeeN Dynamic Interaction Sequence" (CANDIS). This segment is involved in substrate recognition. Key mediators of inflammation including interleukin-6 receptor (IL-6R) and tumor necrosis factor (TNF-α) are substrates of ADAM17. The shedding activity of ADAM17 is regulated by the conformation of the membrane-proximal domain preceding the CANDIS segment. Here, we show that CANDIS, besides being involved in substrate recognition, is able to interact with lipid bilayers in vitro and that this property could be involved in regulating ADAM17 shedding activity.
Assuntos
Proteínas ADAM/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Dados de Sequência Molecular , Mutação , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
A disintegrin and metalloprotease 17 (ADAM17) is a major sheddase involved in the regulation of a wide range of biological processes. Key substrates of ADAM17 are the IL-6 receptor (IL-6R) and TNF-α. The extracellular region of ADAM17 consists of a prodomain, a catalytic domain, a disintegrin domain, and a membrane-proximal domain as well as a small stalk region. This study demonstrates that this juxtamembrane segment is highly conserved, α-helical, and involved in IL-6R binding. This process is regulated by the structure of the preceding membrane-proximal domain, which acts as molecular switch of ADAM17 activity operated by a protein-disulfide isomerase. Hence, we have termed the conserved stalk region "Conserved ADAM seventeen dynamic interaction sequence" (CANDIS). Finally, we identified the region in IL-6R that binds to CANDIS. In contrast to the type I transmembrane proteins, the IL-6R, and IL-1RII, CANDIS does not bind the type II transmembrane protein TNF-α, demonstrating fundamental differences in the respective shedding by ADAM17.
Assuntos
Proteínas ADAM/metabolismo , Receptores de Interleucina-6/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Dicroísmo Circular , Sequência Conservada , Primers do DNA , Células HEK293 , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de AminoácidosRESUMO
Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR·gp130·LIFR or IL-6R·gp130·LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. Arg(28) is in close proximity to the CNTFR binding site. Using molecular modeling, we hypothesized that Arg(28) might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR-binding site from cardiotrophin-like cytokine to CNTF. Cardiotrophin-like cytokine selectively signals via the CNTFR·gp130·LIFR complex, albeit with a much lower affinity compared with CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2, and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR·gp130·LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R·gp130·LIFR. Quantification of CNTF-dependent proliferation of CNTFR·gp130·LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.