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1.
Biochem Biophys Res Commun ; 457(3): 334-40, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25576872

RESUMO

The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase ß-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.


Assuntos
Catepsina F/genética , Catepsina F/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Receptores Depuradores/metabolismo , Animais , Antígenos CD36/química , Antígenos CD36/genética , Antígenos CD36/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Proteínas de Membrana Lisossomal/química , Proteínas de Membrana Lisossomal/genética , Lisossomos/metabolismo , Camundongos , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Proteólise , Receptores Depuradores/química , Receptores Depuradores/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
2.
Cell Mol Life Sci ; 71(2): 331-48, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23760205

RESUMO

Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single "core program" or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5'-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.


Assuntos
Apoptose/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Benzamidas/farmacologia , Calpaína/metabolismo , Catepsinas/metabolismo , Linhagem Celular , Ceramidas/metabolismo , Sequestradores de Radicais Livres/farmacologia , Guanidinas/farmacologia , Células HEK293 , Células HT29 , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , Células MCF-7 , Metanossulfonato de Metila/farmacologia , Camundongos , Necrose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo
3.
Gut ; 62(4): 520-30, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22442160

RESUMO

OBJECTIVE: Cathepsin K is a lysosomal cysteine protease that has pleiotropic roles in bone resorption, arthritis, atherosclerosis, blood pressure regulation, obesity and cancer. Recently, it was demonstrated that cathepsin K-deficient (Ctsk(-/-) ) mice are less susceptible to experimental autoimmune arthritis and encephalomyelitis, which implies a functional role for cathepsin K in chronic inflammatory responses. Here, the authors address the relevance of cathepsin K in the intestinal immune response during chronic intestinal inflammation. DESIGN: Chronic colitis was induced by administration of 2% dextran sodium sulphate (DSS) in distilled water. Mice were assessed for disease severity, histopathology and endoscopic appearance. Furthermore, DSS-exposed Ctsk(-/-) mice were treated by rectal administration of recombinant cathepsin K. Intestinal microflora was assessed by real-time PCR and 16srDNA molecular fingerprinting of ileal and colonic mucosal and faecal samples. RESULTS: Using Ctsk(-/-) mice, the authors demonstrate a protective role of cathepsin K against chronic DSS colitis. Dissecting the underlying mechanisms the authors found cathepsin K to be present in intestinal goblet cells and the mucin layer. Furthermore, a direct cathepsin K-mediated bactericidal activity against intestinal bacteria was demonstrated, which potentially explains the alteration of intestinal microbiota observed in Ctsk(-/-) mice. Rectal administration of recombinant cathepsin K in DSS-treated Ctsk(-/-) mice ameliorates the severity of intestinal inflammation. CONCLUSION: These data identify extracellular cathepsin K as an intestinal antibacterial factor with anti-inflammatory potential and suggest that topical administration of cathepsin K might provide a therapeutic option for patients with inflammatory bowel disease.


Assuntos
Catepsina K/farmacologia , Colite/tratamento farmacológico , Colite/microbiologia , Animais , Western Blotting , Catepsina K/metabolismo , Colite/induzido quimicamente , Colite/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Endoscopia Gastrointestinal , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real
4.
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
5.
J Neurosci ; 30(36): 12210-8, 2010 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-20826683

RESUMO

Tumor necrosis factor alpha (TNF-alpha) is a potent cytokine in neurodegenerative disorders, but its precise role in particular brain disorders is ambiguous. In motor neuron (MN) disease of the mouse, exemplified by the model wobbler (WR), TNF-alpha causes upregulation of the metalloprotease-disintegrin ADAM8 (A8) in affected brain regions, spinal cord, and brainstem. The functional role of A8 during MN degeneration in the wobbler CNS was investigated by crossing WR with A8-deficient mice: a severely aggravated neuropathology was observed for A8-deficient WR compared with WR A8(+/-) mice, judged by drastically reduced survival [7 vs 81% survival at postnatal day 50 (P50)], accelerated force loss in the forelimbs, and terminal akinesis. In vitro protease assays using soluble A8 indicated specific cleavage of a TNF-alpha receptor 1 (p55 TNF-R1) but not a TNF-R2 peptide. Cleavage of TNF-R1 was confirmed in situ, because levels of soluble TNF-R1 were increased in spinal cords of standard WR compared with wild-type mice but not in A8-deficient WR mice. In isolated primary neurons and microglia, TNF-alpha-induced TNF-R1 shedding was dependent on the A8 gene dosage. Furthermore, exogenous TNF-alpha showed higher toxicity for cultured neurons from A8-deficient than for those from wild-type mice, demonstrating that TNF-R1 shedding by A8 is neuroprotective. Our results indicate an essential role for ADAM8 in modulating TNF-alpha signaling in CNS diseases: a feedback loop integrating TNF-alpha, ADAM8, and TNF-R1 shedding as a plausible mechanism for TNF-alpha mediated neuroprotection in situ and a rationale for therapeutic intervention.


Assuntos
Proteínas ADAM/metabolismo , Antígenos CD/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Membrana/metabolismo , Doenças Neurodegenerativas/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Proteínas ADAM/farmacologia , Animais , Animais Recém-Nascidos , Antígenos CD/genética , Antígenos CD/farmacologia , Contagem de Células/métodos , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Estimativa de Kaplan-Meier , Antígenos Comuns de Leucócito/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atrofia Muscular/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/mortalidade , Doenças Neurodegenerativas/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/efeitos dos fármacos , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
6.
Neurobiol Dis ; 33(2): 282-9, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056495

RESUMO

The beta-site APP-cleaving enzyme 1 (BACE1) is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease. Here, we elaborate on the recent finding that auxiliary subunits of voltage-gated sodium channels (beta2 and beta4) are BACE substrates. BACE1 produced complex effects on sodium channel gating that could be only partially explained by beta2/beta4 cleavage. To characterize the unexpected non-proteolytic effect of BACE1, we examined HEK cells co-transfected with only Nav1.2 and either normal or catalytically inactive BACE1. Both BACE1 variants produced virtually identical effects on sodium channel gating, which would lead to enhanced cellular excitability. The non-proteolytic BACE1 effect on Nav1.2 current was confirmed in murine neuroblastoma cells, which express sodium channels endogenously, but lack beta2 and beta4. Our study reveals an important facet of BACE1 function that should help to decipher the role of BACE1 in normal and demented brain.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismo , Animais , Western Blotting , Linhagem Celular , Humanos , Imuno-Histoquímica , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.2 , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Subunidade beta-2 do Canal de Sódio Disparado por Voltagem , Subunidade beta-4 do Canal de Sódio Disparado por Voltagem
7.
Science ; 314(5799): 664-6, 2006 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-16990514

RESUMO

Although BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is essential for the generation of amyloid-b peptide in Alzheimer's disease, its physiological function is unclear. We found that very high levels of BACE1 were expressed at time points when peripheral nerves become myelinated. Deficiency of BACE1 resulted in the accumulation of unprocessed neuregulin 1 (NRG1), an axonally expressed factor required for glial cell development and myelination. BACE1-/- mice displayed hypomyelination of peripheral nerves and aberrant axonal segregation of small-diameter afferent fibers, very similar to that seen in mice with mutations in type III NRG1 or Schwann cell-specific ErbB2 knockouts. Thus, BACE1 is required for myelination and correct bundling of axons by Schwann cells, probably through processing of type III NRG1.


Assuntos
Axônios/fisiologia , Endopeptidases/metabolismo , Bainha de Mielina/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuregulina-1/metabolismo , Células de Schwann/fisiologia , Nervo Isquiático/fisiologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Endopeptidases/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Camundongos , Camundongos Knockout , Neurônios Motores/metabolismo , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neuregulina-1/química , Neuregulina-1/genética , Neurônios Aferentes/metabolismo , Isoformas de Proteínas , Processamento de Proteína Pós-Traducional , Nervo Isquiático/citologia , Medula Espinal/citologia , Medula Espinal/metabolismo
8.
Dev Dyn ; 232(1): 221-31, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15580619

RESUMO

ADAM8 (a disintegrin and metalloprotease 8, also referred to as MS2/CD156a) is a membrane-anchored metalloprotease that was first identified in a macrophage cell line and has been implicated in neurodegenerative diseases. Here, we evaluated the expression of ADAM8 during mouse development and generated mice lacking ADAM8 (Adam8-/- mice). During early mouse development, ADAM8 is expressed by maternal cells in the decidua and by trophoblast derivatives of the embryo but not in the derivatives of the inner cell mass. At later stages, prominent expression of ADAM8 is seen in the embryo proper, in the gonadal ridge, thymus, developing cartilage and bone, brain and spinal cord, and in the mesenchyme in close proximity to the branch point between the jugular vein and developing lymphatic vessels. Examination of Adam8-/- mice, however, revealed no major defects in these or other structures during development or in adult tissues and no evident pathological phenotypes.


Assuntos
Antígenos CD/biossíntese , Antígenos CD/genética , Deleção de Genes , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Metaloendopeptidases/biossíntese , Metaloendopeptidases/genética , Proteínas ADAM , Alelos , Animais , Western Blotting , Desenvolvimento Ósseo , Osso e Ossos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Óperon Lac , Macrófagos/metabolismo , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Mutação , Fatores de Tempo , Distribuição Tecidual , beta-Galactosidase/metabolismo
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