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1.
Mol Cancer ; 22(1): 191, 2023 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-38031106

RESUMO

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.


Assuntos
Interferon Tipo I , Neoplasias , Humanos , Linfócitos T CD8-Positivos , Interferon Tipo I/metabolismo , Microambiente Tumoral , Antígeno B7-H1/metabolismo , Neoplasias/metabolismo , Imunoterapia , Linhagem Celular Tumoral
2.
J Autoimmun ; 97: 70-76, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30467068

RESUMO

Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects. To improve clinical systemic IFN usage, we are developing AcTaferons (Activity-on-Target IFNs = AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting. In experimental autoimmune encephalitis (EAE) in mice, high dose WT mIFNα could delay disease, but caused mortality and severe hematological deficits. In contrast, AFN targeted to dendritic cells (DC, via Clec9A) protected without mortality or hematological consequences. Conversely, CD8-targeted AFN did not protect and exacerbated weight loss, indicating the presence of both protective and unfavorable IFN effects in EAE. Comparing Clec9A-, XCR1-and SiglecH-targeting, we found that targeting AFN to plasmacytoid (p) and conventional (c) DC is superior and non-toxic compared to WT mIFN. DC-targeted AFN increased pDC numbers and their tolerogenic potential, evidenced by increased TGFß and IDO synthesis and regulatory T cell induction. In addition, both regulatory T and B cells produced significantly more immunosuppressive TGFß and IL-10. In conclusion, specific DC-targeting of IFN activity induces a robust in vivo tolerization, efficiently protecting against EAE, without noticeable side effects. Thus, dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer MS therapy and response rates.


Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Tolerância Imunológica , Interferons/metabolismo , Animais , Antígeno B7-H1/metabolismo , Biomarcadores , Antígeno CTLA-4/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/patologia , Masculino , Camundongos , Modelos Biológicos
3.
Cell Mol Life Sci ; 72(3): 629-644, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25098352

RESUMO

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. We here provide genetic and biochemical evidence that the metabolic and immune functions of leptin can be uncoupled at the receptor level. First, homozygous mutant fatt/fatt mice carry a spontaneous splice mutation causing deletion of the leptin receptor (LR) immunoglobulin-like domain (IGD) in all LR isoforms. These mice are hyperphagic and morbidly obese, but display only minimal changes in size and cellularity of the thymus, and cellular immune responses are unaffected. These animals also displayed liver damage in response to concavalin A comparable to wild-type and heterozygous littermates. Second, treatment of healthy mice with a neutralizing nanobody targeting IGD induced weight gain and hyperinsulinaemia, but completely failed to block development of experimentally induced autoimmune diseases. These data indicate that leptin receptor deficiency or antagonism profoundly affects metabolism, with little concomitant effects on immune functions.


Assuntos
Leptina/imunologia , Leptina/metabolismo , Receptores para Leptina/metabolismo , Análise de Variância , Animais , Artrite Experimental/patologia , Sequência de Bases , Western Blotting , Doença Hepática Induzida por Substâncias e Drogas/patologia , Primers do DNA/genética , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/patologia , Citometria de Fluxo , Células HEK293 , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito/toxicidade , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Receptores para Leptina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Deleção de Sequência/genética
4.
J Hepatol ; 60(1): 175-82, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23973929

RESUMO

BACKGROUND & AIMS: Immunometabolism is an emerging field of clinical investigation due to the obesity epidemic worldwide. A reciprocal involvement of immune mediators in the body energy metabolism has been recognized for years, but is only partially understood. We hypothesized that the adipokine leptin could provide an important modulator of iNKT cells. METHODS: The expression of leptin receptor (LR) on resting and activated iNKT cells was measured by flow cytometry. FACS-sorted hepatic iNKT cells were stimulated with anti-CD3/CD28Ab coated beads in the absence or presence of a neutralizing anti-leptin Ab. Furthermore, we evaluated the outcome of LR blocking nanobody treatment in ConA induced hepatitis and towards metabolic parameters in WT and iNKT cell deficient mice. RESULTS: The LR is expressed on iNKT cells and leptin suppresses iNKT cell proliferation and cytokine production in vitro. LR deficient iNKT cells are hyper-responsive further enforcing the role of leptin as an important inhibitor of iNKT cell function. Consistently, in vivo blockade of LR signaling exacerbated ConA hepatitis in wild-type but not in iNKT cell deficient mice, through both Janus kinase (JAK)2 and mitogen-activated protein kinase (MAPK) dependent mechanisms. Moreover, LR inhibition altered fat pad features and was accompanied by insulin resistance, only in wild-type mice. Curiously, this interaction was strictly dependent on MAPK mediated LR signaling in iNKT cells and uncoupled from the more central effects of leptin. CONCLUSIONS: Our data support a new concept of immune regulation by which leptin protects towards T cell mediated hepatitis via modulation of iNKT cells.


Assuntos
Adipócitos/fisiologia , Comunicação Celular , Hepatite/etiologia , Leptina/fisiologia , Células T Matadoras Naturais/fisiologia , Linfócitos T/imunologia , Tecido Adiposo/metabolismo , Animais , Suscetibilidade a Doenças , Ativação Linfocitária , Sistema de Sinalização das MAP Quinases , Camundongos , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores para Leptina/fisiologia , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/fisiologia
5.
J Biol Chem ; 287(6): 4088-98, 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22139835

RESUMO

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.


Assuntos
Modelos Moleculares , Multimerização Proteica/fisiologia , Receptor 4 Toll-Like/química , Animais , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Fator 88 de Diferenciação Mieloide/química , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Mapeamento de Peptídeos , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Receptor 10 Toll-Like/química , Receptor 10 Toll-Like/genética , Receptor 10 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
6.
Biochem J ; 441(1): 425-34, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-21851341

RESUMO

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.


Assuntos
Anticorpos/farmacologia , Leptina/antagonistas & inibidores , Nanoestruturas/química , Receptores para Leptina/antagonistas & inibidores , Tecido Adiposo , Animais , Anticorpos/química , Camelídeos Americanos , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperinsulinismo , Fígado/anatomia & histologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Tamanho do Órgão , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Aumento de Peso
7.
Nat Struct Mol Biol ; 30(4): 551-563, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36959263

RESUMO

The adipokine Leptin activates its receptor LEP-R in the hypothalamus to regulate body weight and exerts additional pleiotropic functions in immunity, fertility and cancer. However, the structure and mechanism of Leptin-mediated LEP-R assemblies has remained unclear. Intriguingly, the signaling-competent isoform of LEP-R is only lowly abundant amid several inactive short LEP-R isoforms contributing to a mechanistic conundrum. Here we show by X-ray crystallography and cryo-EM that, in contrast to long-standing paradigms, Leptin induces type I cytokine receptor assemblies featuring 3:3 stoichiometry and demonstrate such Leptin-induced trimerization of LEP-R on living cells via single-molecule microscopy. In mediating these assemblies, Leptin undergoes drastic restructuring that activates its site III for binding to the Ig domain of an adjacent LEP-R. These interactions are abolished by mutations linked to obesity. Collectively, our study provides the structural and mechanistic framework for how evolutionarily conserved Leptin:LEP-R assemblies with 3:3 stoichiometry can engage distinct LEP-R isoforms to achieve signaling.


Assuntos
Adipocinas , Leptina , Leptina/genética , Leptina/metabolismo , Leptina/farmacologia , Isoformas de Proteínas/genética , Transdução de Sinais
8.
Nucleic Acids Res ; 38(6): 1902-12, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20015971

RESUMO

The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G-Apobec3G and the Apobec3G-Vif interactions, the mammalian protein-protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G-Apobec3G interaction. All mutations that inhibit Apobec3G-Apobec3G binding also inhibit the Apobec3G-Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G-Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer.


Assuntos
Citidina Desaminase/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Desaminase APOBEC-3G , Sítios de Ligação , Linhagem Celular , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Citosina Desaminase/química , Dimerização , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios e Motivos de Interação entre Proteínas , Homologia Estrutural de Proteína , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo
9.
Microorganisms ; 10(2)2022 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-35208715

RESUMO

Bacteria-host interactions are characterized by the delivery of bacterial virulence factors, i.e., effectors, into host cells where they counteract host immunity and exploit host responses allowing bacterial survival and spreading. These effectors are translocated into host cells by means of dedicated secretion systems such as the type 3 secretion system (T3SS). A comprehensive understanding of effector translocation in a spatio-temporal manner is of critical importance to gain insights into an effector's mode of action. Various approaches have been developed to understand timing and order of effector translocation, quantities of translocated effectors and their subcellular localization upon translocation into host cells. Recently, the existing toolset has been expanded by newly developed state-of-the art methods to monitor bacterial effector translocation and dynamics. In this review, we elaborate on reported methods and discuss recent advances and shortcomings in this area of tracking bacterial effector translocation.

10.
Mol Endocrinol ; 22(4): 965-77, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18165436

RESUMO

Leptin is an adipokine that regulates food intake and energy expenditure by activating its hypothalamic leptin receptor (LR). Members of the insulin receptor substrate (IRS) family serve as adaptor proteins in the signaling pathways of several cytokines and hormones and a role for IRS2 in central leptin physiology is well established. Using mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based two-hybrid method, in the N38 hypothalamic cell line, we here demonstrate that also IRS4 interacts with the LR. This recruitment is leptin dependent and requires phosphorylation of the Y1077 motif of the LR. Domain mapping of IRS4 revealed the critical role of the pleckstrin homology domain for full interaction. In line with its function as an adaptor protein, IRS4 interacted with the regulatory p85 subunit of the phosphatidylinositol 3-kinase, phospholipase Cgamma, and the suppressor of cytokine signaling (SOCS) family members SOCS2, SOCS6, and SOCS7 and thus can modulate LR signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores para Leptina/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/química , Animais , Western Blotting , Células Cultivadas , Cromatografia de Afinidade , Humanos , Imunoprecipitação , Proteínas Substratos do Receptor de Insulina , Leptina/farmacologia , Camundongos , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos
11.
Biochem J ; 401(1): 257-67, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-16961462

RESUMO

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein-protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box.


Assuntos
Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Cromatografia de Afinidade , Primers do DNA , DNA Complementar/genética , Humanos , Rim , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Transfecção
12.
Cancer Res ; 78(2): 463-474, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29187401

RESUMO

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR.


Assuntos
Citocinas/química , Células Dendríticas/imunologia , Imunoterapia , Interferon Tipo I/farmacologia , Neoplasias Mamárias Experimentais/terapia , Melanoma Experimental/terapia , Animais , Apoptose , Proliferação de Células , Terapia Combinada , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/patologia , Feminino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Tumorais Cultivadas
13.
Oncoimmunology ; 7(3): e1398876, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29399401

RESUMO

Despite approval for the treatment of various malignancies, clinical application of cytokines such as type I interferon (IFN) is severely impeded by their systemic toxicity. AcTakines (Activity-on-Target cytokines) are optimized immunocytokines that, when injected in mice, only reveal their activity upon cell-specific impact. We here show that type I IFN-derived AcTaferon targeted to the tumor displays strong antitumor activity without any associated toxicity, in contrast with wild type IFN. Treatment with CD20-targeted AcTaferon of CD20+ lymphoma tumors or melanoma tumors engineered to be CD20+, drastically reduced tumor growth. This antitumor effect was completely lost in IFNAR- or Batf3-deficient mice, and depended on IFN signaling in conventional dendritic cells. Also the presence of, but not the IFN signaling in, CD8+ T lymphocytes was critical for proficient antitumor effects. When combined with immunogenic chemotherapy, low-dose TNF, or immune checkpoint blockade strategies such as anti-PDL1, anti-CTLA4 or anti-LAG3, complete tumor regressions and subsequent immunity (memory) were observed, still without any concomitant morbidity, again in sharp contrast with wild type IFN. Interestingly, the combination therapy of tumor-targeted AcTaferon with checkpoint inhibiting antibodies indicated its ability to convert nonresponding tumors into responders. Collectively, our findings demonstrate that AcTaferon targeted to tumor-specific surface markers may provide a safe and generic addition to cancer (immuno)therapies.

14.
Nucleic Acids Res ; 31(14): e75, 2003 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-12853652

RESUMO

We recently reported a two-hybrid trap for detecting protein-protein interactions in intact mammalian cells (MAPPIT). The bait protein was fused to a STAT recruitment-deficient, homodimeric cytokine receptor and the prey protein to functional STAT recruitment sites. In such a configuration, STAT-dependent responses can be used to monitor a given bait-prey interaction. Using this system, we were able to demonstrate both modification-independent and tyrosine phosphorylation- dependent interactions. Protein modification in this approach is, however, strictly dependent on the receptor-associated JAK tyrosine kinases. We have now extended this concept by using extracellular domains of the heteromeric granulocyte/macrophage colony-stimulating factor receptor (GM-CSFR). Herein, the bait was fused to the (beta)c chain and its modifying enzyme to the GM-CSFRalpha chain (or vice versa). We demonstrate several serine phosphorylation-dependent interactions in the TGFbeta/Smad pathway using the catalytic domains of the ALK4 or ALK6 serine/threonine kinase receptors. In all cases tested, STAT-dependent signaling was completely abolished when mutant baits were used wherein critical serine residues were replaced by alanines. This approach operates both in transient and stable expression systems and may not be limited to serine phosphorylation but has the potential for studying various different types of protein modification-dependent interactions in intact cells.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas Proto-Oncogênicas , Animais , Antígenos de Neoplasias/genética , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Humanos , Janus Quinase 2 , Lectinas Tipo C/genética , Luciferases/genética , Luciferases/metabolismo , Mutação , Proteínas Associadas a Pancreatite , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Tirosina Quinases/metabolismo , Ratos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores para Leptina , Fator de Transcrição STAT3 , Transdução de Sinais , Proteína Smad3 , Proteína Smad4 , Transativadores/genética , Transativadores/metabolismo , Transfecção
15.
PLoS One ; 7(9): e44143, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22970171

RESUMO

The mammalian two-hybrid system MAPPIT allows the detection of protein-protein interactions in intact human cells. We developed a random mutagenesis screening strategy based on MAPPIT to detect mutations that disrupt the interaction of one protein with multiple protein interactors simultaneously. The strategy was used to detect residues of the human cytidine deaminase Apobec3G that are important for its homodimerization and its interaction with the HIV-1 Gag and Vif proteins. The strategy is able to identify the previously described head-to-head homodimerization interface in the N-terminal domain of Apobec3G. Our analysis further detects two new potential interaction surfaces in the N-and C-terminal domain of Apobec3G for interaction with Vif and Gag or for Apobec3G dimerization.


Assuntos
Citidina Desaminase/química , Mutagênese , Técnicas do Sistema de Duplo-Híbrido , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Sítios de Ligação , Citidina Desaminase/metabolismo , Células HEK293 , HIV-1/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína
16.
J Biol Chem ; 283(31): 21334-46, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18508766

RESUMO

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.


Assuntos
Proteínas Supressoras da Sinalização de Citocina/fisiologia , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Elonguina , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/química , Proteínas Supressoras da Sinalização de Citocina/metabolismo
17.
J Biol Chem ; 281(44): 32953-66, 2006 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-16956890

RESUMO

SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct cross-modulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.


Assuntos
Citocinas/metabolismo , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Animais , Linhagem Celular , Elonguina , Humanos , Camundongos , Ligação Proteica , Proteínas Supressoras da Sinalização de Citocina/antagonistas & inibidores , Proteínas Supressoras da Sinalização de Citocina/genética , Fatores de Transcrição/metabolismo
18.
Nat Methods ; 2(6): 427-33, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15908921

RESUMO

Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53.


Assuntos
Mapeamento de Interação de Proteínas/métodos , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Humanos , Janus Quinase 3 , Técnicas do Sistema de Duplo-Híbrido
19.
Blood ; 105(11): 4264-71, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15644415

RESUMO

Signaling via the erythropoietin receptor (EpoR) depends on the interaction of several proteins with phosphorylated tyrosine-containing motifs in its cytosolic domain. Detailed mapping of these interactions is required for an accurate insight into Epo signaling. We recently developed a mammalian protein-protein interaction trap (MAPPIT), a cytokine receptor-based 2-hybrid method that operates in intact Hek293-T mammalian cells. As baits, we used intracellular segments of the EpoR containing 1 or 2 tyrosines. Several known signaling molecules, including cytokine-inducible SH2-containing protein (CIS), suppressor of cytokine signaling-2 (SOCS2), phosphatidylinositol 3'-kinase (PI3-K), phospholipase C-gamma (PLC-gamma), and signal transducer and activator of transcription 5 (STAT5) were used as prey. We also extended the MAPPIT method to enable interaction analysis with wild-type EpoR. In this relay MAPPIT approach, instead of using isolated EpoR fragments as bait, we used the full-length EpoR itself as a "receptor bait." Finally, we introduced MAPPIT in the erythroleukemic TF-1 cell line, which is a more natural setting of the EpoR. With these strategies several known interactions with the EpoR were analyzed and evidence for new interactions was obtained.


Assuntos
Receptores da Eritropoetina/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Tirosina , Motivos de Aminoácidos , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Imediatamente Precoces/metabolismo , Proteínas do Leite/metabolismo , Mapeamento de Peptídeos , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Ligação Proteica , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Proteínas Repressoras , Fator de Transcrição STAT5 , Transdução de Sinais , Proteínas Supressoras da Sinalização de Citocina , Transativadores/metabolismo , Fosfolipases Tipo C/metabolismo
20.
J Biol Chem ; 279(39): 41038-46, 2004 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-15213225

RESUMO

The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo.


Assuntos
Desenho de Fármacos , Leptina/antagonistas & inibidores , Leptina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , Linhagem Celular , Relação Dose-Resposta a Droga , Genes Reporter , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos DBA , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fatores de Tempo , Transfecção
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