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Infecciones por Coronavirus/sangre , Dipeptidil Peptidasa 4/sangre , Neumonía Viral/sangre , Anciano , Betacoronavirus , COVID-19 , Estudios de Casos y Controles , Infecciones por Coronavirus/epidemiología , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Neumonía Viral/epidemiología , SARS-CoV-2 , Sepsis/sangre , Sepsis/epidemiologíaRESUMEN
PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B-subunits. Only a limited number of PP2A-regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site-specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A-B56 and PP2A-B55 holoenzymes. Strikingly, we find that B-subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A-B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A-B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.
Asunto(s)
Proteína ADAM17/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteína ADAM17/genética , Secuencias de Aminoácidos , Animales , Sitios de Unión , Células HeLa , Humanos , Ratones , FosforilaciónRESUMEN
Meprin ß is a membrane-bound metalloprotease involved in extracellular matrix assembly and inflammatory processes in health and disease. A disintegrin and metalloproteinase (ADAM)10 and ADAM17 are physiologic relevant sheddases of inactive promeprin ß, which influences its substrate repertoire and subsequent biologic functions. Proteomic analysis also revealed several ADAMs as putative meprin ß substrates. Here, we demonstrate specific N-terminal processing of ADAM9, 10, and 17 by meprin ß and identify cleavage sites within their prodomains. Because ADAM prodomains can act as specific inhibitors, we postulate a role for meprin ß in the regulation of ADAM activities. Indeed, prodomain cleavage by meprin ß caused increased ADAM protease activities, as observed by peptide-based cleavage assays and demonstrated by increased ectodomain shedding activity. Direct interaction of meprin ß and ADAM proteases could be shown by immunofluorescence microscopy and immunoprecipitation experiments. As demonstrated by a bacterial activator of meprin ß and additional measurement of TNF-α shedding on bone marrow-derived macrophages, meprin ß/ADAM protease interactions likely influence inflammatory conditions. Thus, we identified a novel proteolytic pathway of meprin ß with ADAM proteases to control protease activities at the cell surface as part of the protease web.-Wichert, R., Scharfenberg, F., Colmorgen, C., Koudelka, T., Schwarz, J., Wetzel, S., Potempa, B., Potempa, J., Bartsch, J. W., Sagi, I., Tholey, A., Saftig, P., Rose-John, S., Becker-Pauly, C. Meprin ß induces activities of A disintegrin and metalloproteinases 9, 10, and 17 by specific prodomain cleavage.
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Proteínas ADAM/metabolismo , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAM10/química , Proteína ADAM10/genética , Proteína ADAM17/química , Proteína ADAM17/genética , Animales , Membrana Celular/metabolismo , Células Cultivadas , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Metaloendopeptidasas/genética , Ratones Endogámicos C57BL , Dominios Proteicos , Proteolisis , Proteómica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.
Asunto(s)
Proteína ADAM12/metabolismo , Basigina/metabolismo , Proteína ADAM12/química , Proteína ADAM12/genética , Secuencia de Aminoácidos , Basigina/química , Basigina/genética , Sistemas CRISPR-Cas , Línea Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Mutación , Especificidad por SustratoRESUMEN
The significance of human biorepositories for modern medical research, particularly for comprehensive population-based genetic analyses, is constantly growing. While large and centralized institutions are usually considered best suited to meet the increasing demand for high-quality "biobanks," most medical research institutions still host rather heterogeneous and fragmented biobanking activities, undertaken by clinical departments with oftentimes rather different scientific scope. Undoubtedly, most clinicians and medical researchers would appreciate infrastructural support in terms of the storage and handling of their biosamples, but they are also likely to expect access to their samples avoiding extensive formal requirements. We report on the establishment of the PopGen 2.0 Network (P2N), an overarching alliance of initially seven biobanks from Northern Germany which adopted a joint but lean governance structure and use-and-access policy for their samples and data. In addition, the members of P2N have pursued an intense collaboration on ethical, legal and social issues and maintain a common IT infrastructure. The implementation of P2N has substantially improved the prospects of biobank-based research at the participating institutions. The network may thus serve as a role model for similar initiatives geared at linking pre-existing biorepositories for the benefit of research quality, efficiency, and transparency.
RESUMEN
A disintegrin and metalloproteinase (ADAM) 17 has been implicated in many shedding processes. Major substrates of ADAM17 are TNF-α, IL-6R, and ligands of the epidermal growth factor receptor. The essential role of the protease is emphasized by the fact that ADAM17 deficiency is lethal in mice. To study ADAM17 function in vivo, we generated viable hypomorphic ADAM17 mice called ADAM17ex/ex mice. Recent studies indicated regulation of proteolytic ADAM17 activity by cellular processes such as cytoplasmic phosphorylation and removal of the prodomain by furin cleavage. Maturation and thus activation of ADAM17 is not fully understood. So far, studies of ADAM17 maturation have been mainly limited to mouse embryonic fibroblasts or transfected cell lines relying on nonphysiologic stimuli such as phorbol esters, thus making interpretation of the results difficult in a physiologic context. In this article, we present a robust cell system to study ADAM17 maturation and function in primary cells of the immune system. To this end, HoxB8 conditionally immortalized macrophage precursor cell lines were derived from bone marrow of wild-type and hypomorphic ADAM17ex/ex mice, which are devoid of measurable ADAM17 activity. ADAM17 mutants were stably expressed in macrophage precursor cells, differentiated to macrophages under different growth factor conditions (M-CSF versus GM-CSF), and analyzed for cellular localization, proteolytic activity, and podosome disassembly. Our study reveals maturation and activity of ADAM17 in a more physiological-immune cell system. We show that this cell system can be further exploited for genetic modifications of ADAM17 and for studying its function in immune cells.
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Proteína ADAM17/química , Proteína ADAM17/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Dendríticas/enzimología , Macrófagos/enzimología , Animales , Línea Celular , Proteínas de Homeodominio , RatonesRESUMEN
ADAM9 is an active member of the family of transmembrane ADAMs (a disintegrin and metalloproteases). It plays a role in processes such as bone formation and retinal neovascularization, and importantly, its expression in human cancers correlates with disease stage and poor prognosis. Functionally, ADAM9 can cleave several transmembrane proteins, thereby shedding their ectodomains from the cell surface. Moreover, ADAM9 regulates cell behavior by binding cell-surface receptors such as integrin and membrane-type matrix metalloproteases. Because these functions are mainly restricted to the cell surface, understanding the mechanisms regulating ADAM9 localization and activity at this site is highly important. To this end, we here investigated how intracellular trafficking regulates ADAM9 availability at the cell surface. We found that ADAM9 undergoes constitutive clathrin-dependent internalization and subsequent degradation or recycling to the plasma membrane. We confirmed previous findings of an interaction between ADAM9 and the intracellular sorting protein, sorting nexin 9 (SNX9), as well as its close homolog SNX18. Knockdown of either SNX9 or SNX18 had no apparent effects on ADAM9 internalization or recycling. However, double knockdown of SNX9 and SNX18 decreased ADAM9 internalization significantly, demonstrating a redundant role in this process. Moreover, SNX9 knockdown revealed a nonredundant effect on overall ADAM9 protein levels, resulting in increased ADAM9 levels at the cell surface, and a corresponding increase in the shedding of Ephrin receptor B4, a well-known ADAM9 substrate. Together, our findings demonstrate that intracellular SNX9-mediated trafficking constitutes an important ADAM9 regulatory pathway.
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Proteínas ADAM/genética , Neoplasias de la Mama/metabolismo , Membrana Celular/metabolismo , Endocitosis , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/genética , Nexinas de Clasificación/metabolismo , Proteínas ADAM/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica , Transporte de Proteínas , Nexinas de Clasificación/genética , Células Tumorales CultivadasRESUMEN
Colorectal cancer is treated with antibodies blocking epidermal growth factor receptor (EGF-R), but therapeutic success is limited. EGF-R is stimulated by soluble ligands, which are derived from transmembrane precursors by ADAM17-mediated proteolytic cleavage. In mouse intestinal cancer models in the absence of ADAM17, tumorigenesis was almost completely inhibited, and the few remaining tumors were of low-grade dysplasia. RNA sequencing analysis demonstrated down-regulation of STAT3 and Wnt pathway components. Because EGF-R on myeloid cells, but not on intestinal epithelial cells, is required for intestinal cancer and because IL-6 is induced via EGF-R stimulation, we analyzed the role of IL-6 signaling. Tumor formation was equally impaired in IL-6-/- mice and sgp130Fc transgenic mice, in which only trans-signaling via soluble IL-6R is abrogated. ADAM17 is needed for EGF-R-mediated induction of IL-6 synthesis, which via IL-6 trans-signaling induces ß-catenin-dependent tumorigenesis. Our data reveal the possibility of a novel strategy for treatment of colorectal cancer that could circumvent intrinsic and acquired resistance to EGF-R blockade.
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Proteína ADAM17/metabolismo , Receptores ErbB/metabolismo , Interleucina-6/metabolismo , Neoplasias Intestinales/metabolismo , Neoplasias Intestinales/patología , Transducción de Señal , Proteína ADAM17/deficiencia , Poliposis Adenomatosa del Colon/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Núcleo Celular/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Microbioma Gastrointestinal , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Intestinales/genética , Intestino Delgado/patología , Antígeno Ki-67/metabolismo , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Estadificación de Neoplasias , Organoides/patología , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/metabolismo , Carga Tumoral , beta Catenina/metabolismoRESUMEN
The transmembrane protease ADAM9 is frequently upregulated in human cancers, and it promotes tumour progression in mice. In vitro, ADAM9 regulates cancer cell adhesion and migration by interacting with integrins. However, how ADAM9 modulates integrin functions is not known. We here show that ADAM9 knockdown increases ß1 integrin levels through mechanisms that are independent of its protease activity. In ADAM9-silenced cells, adhesion to collagen and fibronectin is reduced, suggesting an altered function of the accumulated integrins. Mechanistically, ADAM9 co-immunoprecipitates with ß1 integrin, and both internalization and subsequent degradation of ß1 integrin are significantly decreased in ADAM9-silenced cells, with no effect on ß1 integrin recycling. Accordingly, the formation of focal adhesions and actin stress fibres in ADAM9-silenced cells is altered, possibly explaining the reduction in cell adhesion and migration in these cells. Taken together, our data provide mechanistic insight into the ADAM9-integrin interaction, demonstrating that ADAM9 regulates ß1 integrin endocytosis. Moreover, our findings indicate that the reduced migration of ADAM9-silenced cells is, at least in part, caused by the accumulation and altered activity of ß1 integrin at the cell surface.
Asunto(s)
Proteínas ADAM/metabolismo , Movimiento Celular , Endocitosis , Adhesiones Focales/metabolismo , Integrina beta1/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Proteínas ADAM/genética , Actinas/metabolismo , Adhesión Celular , Membrana Celular/metabolismo , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Proteínas de la Membrana/genética , Células PC-3RESUMEN
PACS-2 is a multifunctional sorting protein that mediates cell homeostasis. We recently identified PACS-2 in a functional genome-wide siRNA screen for novel regulators of the metalloproteinase ADAM17, the main sheddase for ligands of the ErbB receptor family. Of note, we showed that Pacs2-/- mice have significantly reduced EGFR activity and proliferative index in the intestinal epithelium. As EGFR signaling is highly mitogenic for intestinal epithelial stem cells, and plays essential roles in intestinal epithelial regeneration and tumor development, we have now examined the role of PACS-2 in these processes. Specifically, we analyzed the role of Pacs2-deficiency in a DSS-induced colitis model as well as in the genetic ApcMin colon cancer model. We now report that loss of PACS-2 delays tissue regeneration after colonic injury with little effect on key inflammatory parameters. We did however not observe any apparent effects on tumor formation driven by excessive proliferative signaling downstream from APC-deficiency. Our findings reveal that the role of PACS-2 in regulating ADAM17-mediated shedding is not an obligate requirement for the epithelium to respond to the strong inflammatory or tumorigenic inducers in the models assessed here.
RESUMEN
Neutrophil and mononuclear cell infiltration during inflammatory processes is highly regulated. The first cells at the site of infection or inflammation are neutrophils, followed by mononuclear cells. IL-6 plays an important role during inflammatory states. It has been shown in several models that the soluble form of IL-6R (sIL-6R) is involved in the recruitment of mononuclear cells by a mechanism called IL-6 trans-signaling. It had been speculated that sIL-6R was generated at the site of inflammation by shedding from neutrophils via activation of the metalloprotease ADAM17. Attempts to genetically delete the floxed ADAM17 gene selectively in myeloid cells infiltrating an air pouch cavity upon injection of carrageenan failed because in transgenic mice, LysMcre did not lead to appreciable loss of the ADAM17 protein in these cells. We therefore used ADAM17 hypomorphic mice, which only express â¼5% of ADAM17 wild-type levels in all tissues and show virtually no shedding of all tested ADAM17 substrates, to clarify the role of ADAM17 during local inflammation in the murine air pouch model. In the present study, we demonstrate that although IL-6 and the trans-signaling mechanism is mandatory for cellular infiltration in this model, it is not ADAM17-mediated shedding of IL-6R within the pouch that orchestrates this inflammatory process. Instead, we demonstrate that sIL-6R is infiltrating from the circulation in an ADAM17-independent process. Our data suggest that this infiltrating sIL-6R, which is needed for IL-6 trans-signaling, is involved in the controlled resolution of an acute inflammatory episode.
Asunto(s)
Proteína ADAM17/metabolismo , Inflamación/inmunología , Macrófagos/inmunología , Neutrófilos/inmunología , Receptores de Interleucina-6/metabolismo , Proteína ADAM17/genética , Animales , Carragenina , Movimiento Celular , Células Cultivadas , Inflamación/inducido químicamente , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de SeñalRESUMEN
The cytokine IL-6 is part of a regulatory signaling network that controls immune responses. IL-6 binds either to the membrane-bound IL-6 receptor-α (classic signaling) or to the soluble IL-6 receptor-α (trans-signaling) to initiate signal transduction via gp130 activation. Because classic and trans-signaling of IL-6 fulfill different tasks during immune responses, controlled shedding of the membrane-bound IL-6 receptor-α from the surface of immune cells can be considered a central regulator of IL-6 function. The results from cell culture-based experiments have implicated both a disintegrin and metalloprotease 10 and a disintegrin and metalloprotease 17 in IL-6 receptor-α shedding. However, the nature of the protease mediating IL-6 receptor-α release in vivo is not yet known. We used hypomorphic a disintegrin and metalloprotease 17 mice and conditional a disintegrin and metalloprotease 10 knock-out mice to identify the natural protease of the murine IL-6 receptor-α. Circulating homeostatic soluble IL-6 receptor-α levels are not dependent on a disintegrin and metalloprotease 10 or 17 activity. However, during Listeria monocytogenes infection, IL-6 receptor-α cleavage by the α-secretase a disintegrin and metalloprotease 17 is rapidly induced from the surface of different leukocyte populations. In contrast, CD4-Cre-driven a disintegrin and metalloprotease 10 deletion in T cells did not influence IL-6 receptor-α shedding from these cells after L. monocytogenes infection. A disintegrin and metalloprotease 17 was also required for IL-6 receptor-α ectodomain cleavage and release during endotoxemia. These results demonstrate a novel physiologic role for a disintegrin and metalloprotease 17 in regulating murine IL-6 signals during inflammatory processes.
Asunto(s)
Proteína ADAM17/metabolismo , Membrana Celular/metabolismo , Inflamación/metabolismo , Inflamación/patología , Interleucina-6/metabolismo , Leucocitos/metabolismo , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Proteína ADAM10/metabolismo , Animales , Células Cultivadas , Endotoxemia/inmunología , Endotoxemia/patología , Fibroblastos/metabolismo , Inflamación/sangre , Interleucina-6/sangre , Listeria monocytogenes/fisiología , Listeriosis/sangre , Listeriosis/inmunología , Listeriosis/patología , Ratones Endogámicos C57BL , Receptores de Interleucina-6/sangre , Linfocitos T/metabolismoRESUMEN
Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.
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Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Receptores de Interleucina-6/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Animales , Línea Celular , Humanos , Lipopolisacáridos/farmacología , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Proteolisis , Empalme del ARN , Receptores de Interleucina-6/genética , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17(ex/ex)) mice, in which ADAM17 expression is reduced by 90-95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17(ex/ex) mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.
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Proteínas ADAM/inmunología , Linfocitos B/inmunología , Inmunidad Humoral , Ligando Coestimulador de Linfocitos T Inducibles/inmunología , Proteínas ADAM/genética , Proteína ADAM17 , Animales , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos/inmunología , Linfocitos B/trasplante , Antígenos CD40/inmunología , Células Cultivadas , Femenino , Cambio de Clase de Inmunoglobulina , Isotipos de Inmunoglobulinas/biosíntesis , Isotipos de Inmunoglobulinas/inmunología , Ligando Coestimulador de Linfocitos T Inducibles/biosíntesis , Ganglios Linfáticos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Poli I-C/inmunología , Bazo/patologíaRESUMEN
ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.
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Proteínas ADAM/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Células HEK293 , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Factor de Necrosis Tumoral alfa/química , Técnicas del Sistema de Dos HíbridosRESUMEN
The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primates.
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Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Inflamación/metabolismo , Macrófagos/metabolismo , Receptores de Superficie Celular/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM17 , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Antígenos de Diferenciación Mielomonocítica/genética , Sitios de Unión/genética , Células Cultivadas , Endotoxemia/genética , Endotoxemia/metabolismo , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Células HEK293 , Humanos , Immunoblotting , Inflamación/genética , Lipopolisacáridos/farmacología , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Proteolisis/efectos de los fármacos , Interferencia de ARN , Receptores de Superficie Celular/genética , Homología de Secuencia de Aminoácido , Acetato de Tetradecanoilforbol/farmacología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.
Asunto(s)
Proteínas ADAM/metabolismo , Citoplasma/metabolismo , Furina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas ADAM/química , Proteína ADAM17 , Animales , Línea Celular , Humanos , Ratones , Proteínas Mutantes/metabolismo , Fosforilación , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Transporte de Proteínas , ProteolisisRESUMEN
Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.