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1.
J Am Soc Nephrol ; 32(6): 1389-1408, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-33785583

RESUMEN

BACKGROUND: Podocytes embrace the glomerular capillaries with foot processes, which are interconnected by a specialized adherens junction to ultimately form the filtration barrier. Altered adhesion and loss are common features of podocyte injury, which could be mediated by shedding of cell-adhesion molecules through the regulated activity of cell surface-expressed proteases. A Disintegrin and Metalloproteinase 10 (ADAM10) is such a protease known to mediate ectodomain shedding of adhesion molecules, among others. Here we evaluate the involvement of ADAM10 in the process of antibody-induced podocyte injury. METHODS: Membrane proteomics, immunoblotting, high-resolution microscopy, and immunogold electron microscopy were used to analyze human and murine podocyte ADAM10 expression in health and kidney injury. The functionality of ADAM10 ectodomain shedding for podocyte development and injury was analyzed, in vitro and in vivo, in the anti-podocyte nephritis (APN) model in podocyte-specific, ADAM10-deficient mice. RESULTS: ADAM10 is selectively localized at foot processes of murine podocytes and its expression is dispensable for podocyte development. Podocyte ADAM10 expression is induced in the setting of antibody-mediated injury in humans and mice. Podocyte ADAM10 deficiency attenuates the clinical course of APN and preserves the morphologic integrity of podocytes, despite subepithelial immune-deposit formation. Functionally, ADAM10-related ectodomain shedding results in cleavage of the cell-adhesion proteins N- and P-cadherin, thus decreasing their injury-related surface levels. This favors podocyte loss and the activation of downstream signaling events through the Wnt signaling pathway in an ADAM10-dependent manner. CONCLUSIONS: ADAM10-mediated ectodomain shedding of injury-related cadherins drives podocyte injury.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Nefritis/metabolismo , Síndrome Nefrótico/metabolismo , Podocitos/metabolismo , Podocitos/patología , Insuficiencia Renal Crónica/metabolismo , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Autoanticuerpos/efectos adversos , Nitrógeno de la Urea Sanguínea , Cadherinas/metabolismo , Adhesión Celular , Comunicación Celular , Membrana Celular/metabolismo , Células Cultivadas , Creatinina/orina , Modelos Animales de Enfermedad , Femenino , Barrera de Filtración Glomerular/patología , Barrera de Filtración Glomerular/fisiopatología , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis/patología , Síndrome Nefrótico/patología , Podocitos/fisiología , Proteómica , Análisis de Matrices Tisulares , Transcriptoma , Vía de Señalización Wnt
2.
FASEB J ; 33(11): 11925-11940, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31381863

RESUMEN

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.


Asunto(s)
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/metabolismo
3.
Biochim Biophys Acta Mol Cell Res ; 1864(11 Pt B): 2071-2081, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28624438

RESUMEN

Proteolytic cleavage represents a unique and irreversible posttranslational event regulating the function and half-life of many intracellular and extracellular proteins. The metalloproteinase ADAM10 has raised attention since it cleaves an increasing number of protein substrates close to the extracellular membrane leaflet. This "ectodomain shedding" regulates the turnover of a number of transmembrane proteins involved in cell adhesion and receptor signaling. It can initiate intramembrane proteolysis followed by nuclear transport and signaling of the cytoplasmic domain. ADAM10 has also been implicated in human disorders ranging from neurodegeneration to dysfunction of the immune system and cancer. Targeting proteases for therapeutic purposes remains a challenge since these enzymes including ADAM10 have a wide range of substrates. Accelerating or inhibiting a specific protease activity is in most cases associated with unwanted side effects and a therapeutic useful window of application has to be carefully defined. A better understanding of the regulatory mechanisms controlling the expression, subcellular localization and activity of ADAM10 will likely uncover suitable drug targets which will allow a more specific and fine-tuned modulation of its proteolytic activity.


Asunto(s)
Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Proteínas de la Membrana/genética , Terapia Molecular Dirigida , Neoplasias/genética , Degeneración Nerviosa/genética , Proteína ADAM10/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/uso terapéutico , Humanos , Sistema Inmunológico/metabolismo , Sistema Inmunológico/patología , Proteínas de la Membrana/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Procesamiento Proteico-Postraduccional/genética , Proteolisis
4.
Biochim Biophys Acta Mol Cell Res ; 1864(11 Pt B): 2128-2137, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28693923

RESUMEN

Proteolytic processing of the cellular and disease-associated form of the prion protein leads to generation of bioactive soluble prion protein fragments and modifies the structure and function of its cell-bound form. The nature of proteases responsible for shedding, α-, ß-, and γ-cleavage of the prion protein are only partially identified and their regulation is largely unknown. Here, we provide an overview of the increasingly multifaceted picture of prion protein proteolysis and shed light on physiological and pathological roles associated with these cleavages. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.


Asunto(s)
Enfermedades por Prión/genética , Proteínas Priónicas/genética , Proteolisis , Animales , Humanos , Enfermedades por Prión/patología , Proteínas Priónicas/metabolismo , Agregación Patológica de Proteínas/genética
5.
Biochem Biophys Res Commun ; 442(3-4): 234-41, 2013 Dec 13.
Artículo en Inglés | MEDLINE | ID: mdl-24239882

RESUMEN

Adult hematopoiesis requires tightly regulated cell-cell interactions between hematopoietic cells and the bone marrow stromal microenvironment. We addressed the question if the ectodomain sheddase ADAM10 is essential to regulate adult hematopoiesis. Induced ADAM10 deletion in hematopoietic cells resulted in morphological and histological abnormalities that resemble an unclassified myeloproliferative disorder (MPD). The MPD was characterized by an expansion of granulocytic subpopulations and their infiltration of peripheral hematopoietic tissues, the development of hepatosplenomegaly with extramedullary erythropoiesis, lymphnodepathy and death of the mice around 20weeks after induction. ADAM10 expression analysis during the different stages of the MPD revealed that non-targeted hematopoietic cells repopulated the immune system of the ADAM10-deficient mice. Examination of mice with a myeloid- or epidermis-specific deletion of ADAM10 and bone marrow transplantation (BMT) experiments indicated that the development of the MPD can be triggered by non-cell autonomous effects. We found that plasma levels of clinical markers for MPD such as G-CSF, TIMP-1 and IL-16 were significantly elevated in ADAM10-deficient mice. Our findings indicate that a tightly controlled ADAM10 expression is needed to balance hematopoietic cell-fate decisions in adult mice.


Asunto(s)
Proteínas ADAM/fisiología , Secretasas de la Proteína Precursora del Amiloide/fisiología , Desintegrinas/fisiología , Hematopoyesis/fisiología , Proteínas de la Membrana/fisiología , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Médula Ósea/enzimología , Médula Ósea/patología , Desintegrinas/genética , Eccema/genética , Eccema/patología , Femenino , Hematopoyesis/genética , Hepatomegalia/genética , Hepatomegalia/patología , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células Mieloides/enzimología , Células Mieloides/patología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Esplenomegalia/genética , Esplenomegalia/patología , Células del Estroma/enzimología , Células del Estroma/patología , Trombocitosis/genética , Trombocitosis/patología
6.
J Chem Theory Comput ; 18(2): 1122-1128, 2022 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-34995061

RESUMEN

We use voxel deep neural networks to predict energy densities and functional derivatives of electron kinetic energies for the Thomas-Fermi model and Kohn-Sham density functional theory calculations. We show that the ground-state electron density can be found via direct minimization for a graphene lattice without any projection scheme using a voxel deep neural network trained with the Thomas-Fermi model. Additionally, we predict the kinetic energy of a graphene lattice within chemical accuracy after training from only two Kohn-Sham density functional theory (DFT) calculations. We identify an important sampling issue inherent in Kohn-Sham DFT calculations and propose future work to rectify this problem. Furthermore, we demonstrate an alternative, functional derivative-free, Monte Carlo based orbital-free density functional theory algorithm to calculate an accurate two-electron density in a double inverted Gaussian potential with a machine-learned kinetic energy functional.

7.
Mol Neurodegener ; 13(1): 18, 2018 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-29625583

RESUMEN

Background: Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer's and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. Methods: We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. Results: We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. Conclusions: With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.


Asunto(s)
Proteína ADAM10/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Proteínas Priónicas/metabolismo , Animales , Ratones
8.
Phys Rev E ; 96(2-1): 022140, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28950564

RESUMEN

We examine unsupervised machine learning techniques to learn features that best describe configurations of the two-dimensional Ising model and the three-dimensional XY model. The methods range from principal component analysis over manifold and clustering methods to artificial neural-network-based variational autoencoders. They are applied to Monte Carlo-sampled configurations and have, a priori, no knowledge about the Hamiltonian or the order parameter. We find that the most promising algorithms are principal component analysis and variational autoencoders. Their predicted latent parameters correspond to the known order parameters. The latent representations of the models in question are clustered, which makes it possible to identify phases without prior knowledge of their existence. Furthermore, we find that the reconstruction loss function can be used as a universal identifier for phase transitions.

9.
Elife ; 52016 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-26802628

RESUMEN

Metzincin metalloproteases have major roles in intercellular communication by modulating the function of membrane proteins. One of the proteases is the a-disintegrin-and-metalloprotease 10 (ADAM10) which acts as alpha-secretase of the Alzheimer's disease amyloid precursor protein. ADAM10 is also required for neuronal network functions in murine brain, but neuronal ADAM10 substrates are only partly known. With a proteomic analysis of Adam10-deficient neurons we identified 91, mostly novel ADAM10 substrate candidates, making ADAM10 a major protease for membrane proteins in the nervous system. Several novel substrates, including the neuronal cell adhesion protein NrCAM, are involved in brain development. Indeed, we detected mistargeted axons in the olfactory bulb of conditional ADAM10-/- mice, which correlate with reduced cleavage of NrCAM, NCAM and other ADAM10 substrates. In summary, the novel ADAM10 substrates provide a molecular basis for neuronal network dysfunctions in conditional ADAM10-/- mice and demonstrate a fundamental function of ADAM10 in the brain.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Axones/enzimología , Proteínas de la Membrana/metabolismo , Sinapsis/enzimología , Proteína ADAM10/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Animales , Axones/química , Encéfalo/embriología , Encéfalo/enzimología , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Proteínas de la Membrana/deficiencia , Ratones , Proteoma/análisis , Especificidad por Sustrato
10.
Oncotarget ; 7(14): 17431-41, 2016 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-26942887

RESUMEN

UNLABELLED: A Disintegrin And Metalloprotease (ADAM) 10 exerts essential roles during organ development and tissue integrity in different organs, mainly through activation of the Notch pathway. However, only little is known about its implication in liver tissue physiology. Here we show that in contrast to its role in other tissues, ADAM10 is dispensable for the Notch2-dependent biliary tree formation. However, we demonstrate that expression of bile acid transporters is dependent on ADAM10. Consequently, mice deficient for Adam10 in hepatocytes, cholangiocytes and liver progenitor cells develop spontaneous hepatocyte necrosis and concomitant liver fibrosis. We furthermore observed a strongly augmented ductular reaction in 15-week old ADAM10(Δhep/Δch) mice and demonstrate that c-Met dependent liver progenitor cell activation is enhanced. Additionally, liver progenitor cells are primed to hepatocyte differentiation in the absence of ADAM10. These findings show that ADAM10 is a novel central node controlling liver tissue homeostasis. HIGHLIGHTS: Loss of ADAM10 in murine liver results in hepatocyte necrosis and concomitant liver fibrosis. ADAM10 directly regulates expression of bile acid transporters but is dispensable for Notch2-dependent formation of the biliary system. Activation of liver progenitor cells is enhanced through increased c-Met signalling, in the absence of ADAM10. Differentiation of liver progenitor cells to hepatocytes is augmented in the absence of ADAM10.


Asunto(s)
Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/metabolismo , Proteína ADAM10/deficiencia , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Regulación hacia Abajo , Hepatocitos/metabolismo , Hepatocitos/patología , Homeostasis , Hígado/citología , Hígado/patología , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Necrosis , Receptor Notch2/metabolismo , Transducción de Señal
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