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1.
Oncotarget ; 8(33): 54873-54888, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28903388

RESUMO

Transendothelial cell migration (TEM) is crucial for inflammation and metastasis. The adhesion molecule CD99 was shown to be important for correct immune cell extravasation and is highly expressed on certain cancer cells. Recently, we demonstrated that ectodomain shedding of CD99 by the metalloprotease meprin ß promotes TEM in vitro. In this study, we employed an acute inflammation model (air pouch/carrageenan) and found significantly less infiltrated cells in meprin ß knock-out animals validating the previously observed pro-inflammatory activity. To further analyze the impact of meprin ß on CD99 shedding with regard to cell adhesion and proliferation we characterized two lung cancer associated CD99 variants (D92H, D92Y), carrying point mutations at the main cleavage site. Interestingly, ectodomain shedding of these variants by meprin ß was still detectable. However the cleavage site shifted to adjacent positions. Nevertheless, expression of CD99 variants D92H and D92Y revealed partial misfolding and proteasomal degradation. A previously observed influence of CD99 on Src activation and increased proliferation could not be confirmed in this study, independent of wild-type CD99 or the variants D92H and D92Y. However, we identified meprin ß as a potent inducer of Src phosphorylation. Importantly, we found significantly increased cell migration when expressing the cancer-associated CD99 variant D92H compared to the wild-type protein.

2.
Oncotarget ; 8(5): 7839-7851, 2017 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-27999200

RESUMO

Hepatocellular carcinoma is associated with a high rate of intra-hepatic invasion that carries a poor prognosis. Meprin alpha (Mep1A) is a secreted metalloproteinase with many substrates relevant to cancer invasion. We found that Mep1A was a target of Reptin, a protein that is oncogenic in HCC. We studied Mep1A regulation by Reptin, its role in HCC, and whether it mediates Reptin oncogenic effects.MepA and Reptin expression was measured in human HCC by qRT-PCR and in cultured cells by PCR, western blot and enzymatic activity measurements. Cell growth was assessed by counting and MTS assay. Cell migration was measured in Boyden chambers and wound healing assays, and cell invasion in Boyden chambers.Silencing Reptin decreased Mep1A expression and activity, without affecting meprin ß. Mep1A, but not meprin ß, was overexpressed in a series of 242 human HCC (2.04 fold, p < 0.0001), and a high expression correlated with a poor prognosis. Mep1A and Reptin expressions were positively correlated (r = 0.39, p < 0.0001). Silencing Mep1A had little effect on cell proliferation, but decreased cell migration and invasion of HuH7 and Hep3B cells. Conversely, overexpression of Mep1A or addition of recombinant Mep1A increased migration and invasion. Finally, overexpression of Mep1A restored a normal cell migration in cells where Reptin was depleted.Mep1A is overexpressed in most HCC and induces HCC cell migration and invasion. Mep1A expression is regulated by Reptin, and Mep1A mediates Reptin-induced migration. Overall, we suggest that Mep1A may be a useful target in HCC.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Carcinoma Hepatocelular/enzimologia , Proteínas de Transporte/metabolismo , Movimento Celular , DNA Helicases/metabolismo , Neoplasias Hepáticas/enzimologia , Metaloendopeptidases/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Metaloendopeptidases/genética , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção
3.
Biochim Biophys Acta Mol Cell Res ; 1864(1): 217-230, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27818272

RESUMO

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.


Assuntos
Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Endossomos/metabolismo , Proteínas de Membrana/genética , Presenilinas/genética , Tetraspaninas/genética , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Presenilinas/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Técnicas do Sistema de Duplo-Híbrido
4.
FASEB J ; 31(3): 1226-1237, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28003343

RESUMO

The adhesion molecule CD99 is essential for the transendothelial migration of leukocytes. In this study, we used biochemical and cellular assays to show that CD99 undergoes ectodomain shedding by the metalloprotease meprin ß and subsequent intramembrane proteolysis by γ-secretase. The cleavage site in CD99 was identified by mass spectrometry within an acidic region highly conserved through different vertebrate species. This finding fits perfectly to the unique cleavage specificity of meprin ß with a strong preference for aspartate residues and suggests coevolution of protease and substrate. We hypothesized that limited CD99 cleavage by meprin ß would alter cellular transendothelial migration (TEM) behavior in tissue remodeling processes, such as inflammation and cancer. Indeed, meprin ß induced cell migration of Lewis lung carcinoma cells in an in vitro TEM assay. Accordingly, deficiency of meprin ß in Mep1b-/- mice resulted in significantly increased CD99 protein levels in the lung. Therefore, meprin ß could serve as a therapeutic target, given that in a proof-of-concept approach we showed accumulation of CD99 protein in lungs of meprin ß inhibitor-treated mice.-Bedau, T., Peters, F., Prox, J., Arnold, P., Schmidt, F., Finkernagel, M., Köllmann, S., Wichert, R., Otte, A., Ohler, A., Stirnberg, M., Lucius, R., Koudelka, T., Tholey, A., Biasin, V., Pietrzik, C. U., Kwapiszewska, G., Becker-Pauly, C. Ectodomain shedding of CD99 within highly conserved regions is mediated by the metalloprotease meprin ß and promotes transendothelial cell migration.


Assuntos
Antígeno 12E7/metabolismo , Sequência Conservada , Metaloendopeptidases/metabolismo , Proteólise , Migração Transendotelial e Transepitelial , Antígeno 12E7/química , Animais , Carcinoma Pulmonar de Lewis/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL
5.
Biol Chem ; 2016 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-27180357

RESUMO

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It was shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. Since several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified being present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.

6.
Biol Chem ; 397(9): 857-69, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27180358

RESUMO

Meprin ß is a dimeric type I transmembrane protein and acts as an ectodomain sheddase at the cell surface. It has been shown that meprin ß cleaves the amyloid precursor protein (APP), thereby releasing neurotoxic amyloid ß peptides and implicating a role of meprin ß in Alzheimer's disease. In order to identify non-proteolytic regulators of meprin ß, we performed a split ubiquitin yeast two-hybrid screen using a small intestinal cDNA library. In this screen we identified tetraspanin 8 (TSPAN8) as interaction partner for meprin ß. As several members of the tetraspanin family were described to interact with metalloproteases thereby affecting their localization and/or activity, we hypothesized similar functions of TSPAN8 in the regulation of meprin ß. We employed cell biological methods to confirm direct binding of TSPAN8 to meprin ß. Surprisingly, we did not observe an effect of TSPAN8 on the catalytic activity of meprin ß nor on the specific cleavage of its substrate APP. However, both proteins were identified as present in tetraspanin-enriched microdomains. Therefore we hypothesize that TSPAN8 might be important for the orchestration of meprin ß at the cell surface with impact on certain proteolytic processes that have to be further identified.


Assuntos
Metaloendopeptidases/metabolismo , Tetraspaninas/química , Tetraspaninas/metabolismo , Células HEK293 , Humanos , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Especificidade por Substrato
7.
Mol Neurodegener ; 11: 19, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26895626

RESUMO

BACKGROUND: The metalloprotease meprin ß cleaves the Alzheimer's Disease (AD) relevant amyloid precursor protein (APP) as a ß-secretase reminiscent of BACE-1, however, predominantly generating N-terminally truncated Aß2-x variants. RESULTS: Herein, we observed increased endogenous sAPPα levels in the brains of meprin ß knock-out (ko) mice compared to wild-type controls. We further analyzed the cellular interaction of APP and meprin ß and found that cleavage of APP by meprin ß occurs prior to endocytosis. The N-terminally truncated Aß2-40 variant shows increased aggregation propensity compared to Aß1-40 and acts even as a seed for Aß1-40 aggregation. Additionally, we observed that different APP mutants affect the catalytic properties of meprin ß and that, interestingly, meprin ß is unable to generate N-terminally truncated Aß peptides from Swedish mutant APP (APPswe). CONCLUSION: Concluding, we propose that meprin ß may be involved in the generation of N-terminally truncated Aß2-x peptides of APP, but acts independently from BACE-1.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Metaloendopeptidases/metabolismo , Neurônios/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Camundongos Knockout
8.
J Biochem ; 159(3): 305-12, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26491063

RESUMO

Reelin is a secreted glycoprotein whose function is regulated by proteolysis. One of the specific cleavage sites of Reelin, called C-t, is located approximately between the sixth and seventh Reelin repeat but its exact site was unknown. We here show that a metalloprotease present in the culture supernatant of cerebellar granular neurons (CGN) cleaves Reelin between Ala2688 and Asp2689. A Reelin mutant in which Asp2689 is replaced by Lys (Reelin-DK) is resistant to C-t cleavage by culture supernatant of CGN. From biochemical characteristics and the cleavage site preference, meprin α and meprin ß were suggested candidate proteases and both were confirmed to cleave Reelin at the C-t site. Meprin α cleaved Reelin-DK but meprin ß did not. Actinonin, a meprin α and meprin ß inhibitor, did not inhibit the Reelin-cleaving activity of CGN and the amount of Reelin fragments in brains of meprin ß knock-out mice was not significantly different from that of the wild-type, indicating that meprin ß does not play a major role in Reelin cleavage under basal conditions. We propose that meprin α and meprin ß join the modulators of Reelin signalling as they cleave Reelin at a specific site and are upregulated under specific pathological conditions.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/metabolismo , Metaloendopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Proteólise , Serina Endopeptidases/metabolismo , Animais , Células COS , Técnicas de Cultura de Células , Cercopithecus aethiops , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Transdução de Sinais
9.
Biochem J ; 470(1): 91-103, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26251449

RESUMO

Increased expression of metalloprotease meprin ß is associated with fibrotic syndromes and Alzheimer's disease (AD). Hence, regulation of meprin activity might be a suitable strategy for the treatment of these conditions. Meprin ß is a type 1 transmembrane protein, but can be released from the cell surface by ectodomain shedding. The protease is expressed as an inactive zymogen and requires proteolytic maturation by tryptic serine proteases. In the present study, we demonstrate, for the first time, the differences in the activation of soluble and membrane bound meprin ß and suggest transmembrane serine protease 6 [TMPRSS6 or matriptase-2 (MT2)] as a new potent activator, cleaving off the propeptide of meprin ß between Arg(61) and Asn(62) as determined by MS. We show that MT2, but not TMPRSS4 or pancreatic trypsin, is capable of activating full-length meprin ß at the cell surface, analysed by specific fluorogenic peptide cleavage assay, Western blotting and confocal laser scanning microscopy (CLSM). Maturation of full-length meprin ß is required for its activity as a cell surface sheddase, releasing the ectodomains of transmembrane proteins, as previously shown for the amyloid precursor protein (APP).


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Metaloendopeptidases/metabolismo , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Células COS , Cercopithecus aethiops , Células HEK293 , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/genética , Suínos
10.
FASEB J ; 29(8): 3549-57, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25957281

RESUMO

The meprin ß metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin ß substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin ß itself. Analyzing the crystal structures of pro- and mature meprin ß helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D204A and D245A) led to severe differences in proteolytic activity and cellular localization of meprin ß. D245A was almost completely inactive and largely stored into intracellular vesicles, indicating severe misfolding of the protein. Astonishingly, D204A was not transported to the cell surface, but exhibited strong ß-secretase activity, resulting in massive accumulation of Aß-peptides. This could be explained by constitutive maturation of this meprin ß mutant already in the early secretory pathway. We hypothesize that lacking D204 abrogates the capability of binding calcium in the catalytic domain, an important step for proper folding of the propeptide and subsequent inhibition of the protease. This is supported by the inhibition constant of calcium for meprin ß (inhibitory constant 50 = 11 mM), which resembles the physiologic concentrations found in the endoplasmic reticulum. For instance, it was shown for amyotrophic lateral sclerosis that a loss of calcium in the endoplasmic reticulum leads to the misfolding of calcium-dependent proteins, which might also be relevant for proper function of meprin ß.


Assuntos
Cálcio/metabolismo , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sítios de Ligação/fisiologia , Células COS , Linhagem Celular , Cercopithecus aethiops , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Metaloendopeptidases/genética , Metaloproteases/metabolismo , Mutação/genética , Dobramento de Proteína , Alinhamento de Sequência , Especificidade por Substrato/fisiologia
11.
Elife ; 42015 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-25654651

RESUMO

The prion protein (PrP(C)) is highly expressed in the nervous system and critically involved in prion diseases where it misfolds into pathogenic PrP(Sc). Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer's disease. PrP(C) is shed at the plasma membrane by the metalloprotease ADAM10, yet the impact of this on prion disease remains enigmatic. Employing conditional knockout mice, we show that depletion of ADAM10 in forebrain neurons leads to posttranslational increase of PrP(C) levels. Upon prion infection of these mice, clinical, biochemical, and morphological data reveal that lack of ADAM10 significantly reduces incubation times and increases PrP(Sc) formation. In contrast, spatiotemporal analysis indicates that absence of shedding impairs spread of prion pathology. Our data support a dual role for ADAM10-mediated shedding and highlight the role of proteolytic processing in prion disease.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Doenças Priônicas/metabolismo , Proteína ADAM10 , Animais , Calpaína/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas PrPC/metabolismo , Prosencéfalo/metabolismo
12.
Matrix Biol ; 44-46: 7-13, 2015 May-Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25617491

RESUMO

Metalloproteases meprin α and meprin ß were recently discovered as procollagen proteinases, capable of cleaving off the globular C- and N-terminal prodomains of fibrillar collagen type I and type III. This proteolytic process is indeed sufficient to induce collagen fibril assembly as visualized by transmission electron microscopy. The biological relevance was demonstrated with the help of meprin α and meprin ß knock-out mice, which exhibit decreased collagen deposition in skin resulting in impaired tensile strength. On the other hand, overexpression of meprin metalloproteases was found under fibrotic conditions in the skin (keloids) and the lung (pulmonary hypertension). Thus, regulation of meprin activity by specific inhibition to reduce collagen maturation might be a suitable approach for the treatment of certain pathological conditions.


Assuntos
Colágeno Tipo III/metabolismo , Colágeno Tipo I/metabolismo , Metaloendopeptidases/metabolismo , Pró-Colágeno/metabolismo , Animais , Colágeno Tipo I/química , Colágeno Tipo III/química , Regulação Enzimológica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Hipertensão Pulmonar/enzimologia , Queloide/enzimologia , Metaloendopeptidases/genética , Camundongos , Resistência à Tração
13.
Proteomics Clin Appl ; 8(5-6): 382-8, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24470335

RESUMO

The protease web is a synonym for highly regulated molecular networks comprising enzymes, substrates, inhibitors, and other regulatory proteins. Latest high-throughput methods provided huge data sets, revealing an amazing complexity of proteolytic systems important for health and disease. Based on our previous studies, we discuss major problems and questions that have to be solved to gain precise insight into the regulation of the protease web and its impact on pathophysiological conditions. The goal is a combination of different proteomic approaches that help to investigate specific protease function at a glance. Exemplarily, the characterization of the metalloproteases meprin α and meprin ß by proteomic identification of cleavage sites and terminal amine isotopic labeling of substrates demonstrates the power of MS-based techniques. Meprins are rather orphan proteases and could not be assigned to precise biological functions until recently. Proteomics helped to identify meprin α and meprin ß being important for collagen assembly and deposition in skin, which makes them potential therapeutic targets in fibrotic conditions. Additionally, identification of the cleavage site specificity provides the basis for the development of activity-based probes and small compound inhibitors, important for the regulation of meprin activity and subsequent treatment of associated diseases.


Assuntos
Metaloproteases/metabolismo , Terapia de Alvo Molecular/métodos , Proteômica/métodos , Tiopronina/metabolismo , Animais , Humanos , Processamento de Proteína Pós-Traducional , Proteólise
14.
Biochem Biophys Res Commun ; 442(3-4): 234-41, 2013 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-24239882

RESUMO

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.


Assuntos
Proteínas ADAM/fisiologia , Secretases da Proteína Precursora do Amiloide/fisiologia , Desintegrinas/fisiologia , Hematopoese/fisiologia , Proteínas de Membrana/fisiologia , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Medula Óssea/enzimologia , Medula Óssea/patologia , Desintegrinas/genética , Eczema/genética , Eczema/patologia , Feminino , Hematopoese/genética , Hepatomegalia/genética , Hepatomegalia/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Células Mieloides/enzimologia , Células Mieloides/patologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Esplenomegalia/genética , Esplenomegalia/patologia , Células Estromais/enzimologia , Células Estromais/patologia , Trombocitose/genética , Trombocitose/patologia
15.
J Neurosci ; 33(32): 12915-28, 12928a, 2013 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-23926248

RESUMO

The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid ß (Aß) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPß and endogenous Aß peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.


Assuntos
Proteínas ADAM/deficiência , Secretases da Proteína Precursora do Amiloide/deficiência , Encéfalo/ultraestrutura , Espinhas Dendríticas/patologia , Epilepsia/genética , Epilepsia/patologia , Transtornos de Aprendizagem/patologia , Proteínas de Membrana/deficiência , Sinapses/patologia , Proteína ADAM10 , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/patologia , Caderinas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Moléculas de Adesão Celular/metabolismo , Espinhas Dendríticas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Gliose/genética , Transtornos de Aprendizagem/genética , Camundongos , Camundongos Transgênicos , Nectinas , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura
16.
FEBS J ; 280(18): 4338-47, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23413979

RESUMO

The cellular prion protein (PrP(C)) plays important roles in neurodegenerative diseases. First, it is the well-established substrate for the conformational conversion into its pathogenic isoform (PrP(Sc)) giving rise to progressive and fatal prion diseases. Moreover, several recent reports highlight important roles of PrP(C) in other neurodegenerative conditions such as Alzheimer's disease. Since PrP(C) is subject to proteolytic processing, here we discuss the two main cleavage events under physiological conditions, α-cleavage and shedding. We focus on how these cleavages and the resulting fragments may impact prion diseases as well as other neurodegenerative proteinopathies. Finally, we discuss the recently identified sheddase of PrP(C), namely the metalloprotease ADAM10, with regard to therapeutic potential against neurodegenerative diseases.


Assuntos
Proteínas ADAM/metabolismo , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas ADAM/genética , Proteína ADAM10 , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular , Humanos , Proteínas de Membrana/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Conformação Proteica , Dobramento de Proteína , Proteólise , Transdução de Sinais
17.
Neuron ; 76(2): 410-22, 2012 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-23083742

RESUMO

Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to a cognate presynaptic ligand, neurexin. Here we report that neuroligin-1 (NLG1) undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment (CTF) in adult rat brains in vivo as well as in neuronal cultures. Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. NLG1-CTF was subsequently cleaved by presenilin/γ-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Collectively, neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteólise , Proteínas ADAM/deficiência , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Animais Recém-Nascidos , Biotinilação , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Cerebelo/metabolismo , Espinhas Dendríticas/metabolismo , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Agonistas Muscarínicos/toxicidade , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Pilocarpina/toxicidade , Interferência de RNA/fisiologia , Ratos , Ratos Wistar , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , Transfecção
18.
Cell Mol Life Sci ; 69(17): 2919-32, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22446748

RESUMO

A disintegrin and metalloproteinase10 (ADAM10) has been implicated as a major sheddase responsible for the ectodomain shedding of a number of important surface molecules including the amyloid precursor protein and cadherins. Despite a well-documented role of ADAM10 in health and disease, little is known about the regulation of this protease. To address this issue we conducted a split-ubiquitin yeast two-hybrid screen to identify membrane proteins that interact with ADAM10. The yeast experiments and co-immunoprecipitation studies in mammalian cell lines revealed tetraspanin15 (TSPAN15) to specifically associate with ADAM10. Overexpression of TSPAN15 or RNAi-mediated knockdown of TSPAN15 led to significant changes in the maturation process and surface expression of ADAM10. Expression of an endoplasmic reticulum (ER) retention mutant of TSPAN15 demonstrated an interaction with ADAM10 already in the ER. Pulse-chase experiments confirmed that TSPAN15 accelerates the ER-exit of the ADAM10-TSPAN15 complex and stabilizes the active form of ADAM10 at the cell surface. Importantly, TSPAN15 also showed the ability to mediate the regulation of ADAM10 protease activity exemplified by an increased shedding of N-cadherin and the amyloid precursor protein. In conclusion, our data show that TSPAN15 is a central modulator of ADAM10-mediated ectodomain shedding. Therapeutic manipulation of its expression levels may be an additional approach to specifically regulate the activity of the amyloid precursor protein alpha-secretase ADAM10.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Caderinas/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Tetraspaninas/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Western Blotting , Caderinas/genética , Movimento Celular , Células Cultivadas , Retículo Endoplasmático/metabolismo , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Transporte Proteico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspaninas/antagonistas & inibidores , Tetraspaninas/genética , Técnicas do Sistema de Duplo-Híbrido
19.
Exp Brain Res ; 217(3-4): 331-41, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22120156

RESUMO

Alzheimer's disease causing mutations in the amyloid precursor protein (APP) or in the Presenilin 1 (PS1) or Presenilin 2 (PS2) genes increase the production of amyloid peptides (Aß) that precipitate in amyloid plaques. Since amyloid plaques are also a prominent feature of sporadic Alzheimer's disease (AD), abnormal proteolysis of APP and the generation of amyloid beta (Aß) are key events in the pathogenesis of AD. The proteases (secretases) that cleave APP are therefore important therapeutic targets, both for the rare familial forms but likely also for the sporadic forms of AD. The identification and understanding of the (neuro)biological functions of the α-, ß-, and presenilin/γ-secretase (complexes) is important for the development of drugs and the delineation of their associated side effects. The potential impact of this type of research exceeds the AD field since the function of these secretases are also linked to cellular pathways like ectodomain shedding of growth factors and regulated intramembrane proteolysis of receptors in developmental biology, tissue homeostasis, and tumorigenesis. The generation of mice deficient in presenilin 1, presenilin 2, the α-secretase ADAM10, and the ß-secretases BACE1 and BACE2 were instrumental for the elucidation of the physiological functions of these proteases. Using these mouse models understanding how these secretases regulate amyloid peptide formation and how they exert their diverse biological functions could be significantly increased. This review attempts to summarize selected aspects of the current view of the multiple roles such proteases play in health and disease.


Assuntos
Secretases da Proteína Precursora do Amiloide/fisiologia , Proteínas ADAM/fisiologia , Proteína ADAM10 , Animais , Ácido Aspártico Endopeptidases/fisiologia , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Modelos Neurológicos , Vias Neurais/fisiologia , Presenilina-1/fisiologia , Presenilina-2/fisiologia
20.
Mol Neurodegener ; 6: 36, 2011 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-21619641

RESUMO

BACKGROUND: The cellular prion protein (PrPC) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrPSc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrPC generates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrPC. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrPC and its α-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrPC in vivo. RESULTS: Using neuron-specific Adam10 knockout mice, we show that ADAM10 is the sheddase of PrPC and that its absence in vivo leads to increased amounts and accumulation of PrPC in the early secretory pathway by affecting its posttranslational processing. Elevated PrPC levels do not induce apoptotic signalling via p53. Furthermore, we show that ADAM10 is not responsible for the α-cleavage of PrPC. CONCLUSION: Our study elucidates the proteolytic processing of PrPC and proves a role of ADAM10 in shedding of PrPC in vivo. We suggest that ADAM10 is a mediator of PrPC homeostasis at the plasma membrane and, thus, might be a regulator of the multiple functions discussed for PrPC. Furthermore, identification of ADAM10 as the sheddase of PrPC opens the avenue to devising novel approaches for therapeutic interventions against prion diseases.

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