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1.
Methods Mol Biol ; 2043: 93-104, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463905

RESUMO

Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.

2.
Methods Mol Biol ; 2043: 105-111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31463906

RESUMO

Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.

3.
Mol Cell Neurosci ; 100: 103401, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31491533

RESUMO

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

4.
Biochem Biophys Res Commun ; 514(3): 815-820, 2019 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-31079931

RESUMO

Reelin is a large secreted protein that is essential for the development and function of the central nervous system. Dimerization and/or oligomerization is required for its biological activity, but the underlying mechanism is not fully understood. There are several widely used anti-Reelin antibodies and we noticed that their reactivity to monomeric or dimeric Reelin protein is different. We also found that their reactivity to Reelin in the solution or in fixed brain tissues also differs. Our results provide the information regarding how the N-terminal region of Reelin folds and contributes to the formation of higher order structure. We also provide a caveat that appropriate use of anti-Reelin antibody is necessary for quantitative analyses.

5.
Biol Pharm Bull ; 42(3): 354-356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30828067

RESUMO

Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.


Assuntos
Proteínas ADAMTS/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas ADAMTS/antagonistas & inibidores , Proteínas ADAMTS/genética , Doença de Alzheimer , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Modelos Animais de Doenças , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
6.
Biochem Biophys Res Commun ; 505(1): 81-86, 2018 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-30241938

RESUMO

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.

7.
Sci Rep ; 8(1): 13046, 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30158644

RESUMO

Reelin protein (RELN), an extracellular matrix protein, plays multiple roles that range from embryonic neuronal migration to spine formation in the adult brain. Results from genetic studies have suggested that RELN is associated with the risk of psychiatric disorders, including schizophrenia (SCZ). We previously identified a novel exonic deletion of RELN in a patient with SCZ. High-resolution copy number variation analysis revealed that this deletion included exons 52 to 58, which truncated the RELN in a similar manner to the Reln Orleans mutation (Relnrl-Orl). We examined the clinical features of this patient and confirmed a decreased serum level of RELN. To elucidate the pathophysiological role of the exonic deletion of RELN in SCZ, we conducted behavioral and neurochemical analyses using heterozygous Relnrl-Orl/+ mice. These mice exhibited abnormalities in anxiety, social behavior, and motor learning; the deficits in motor learning were ameliorated by antipsychotics. Methamphetamine-induced hyperactivity and dopamine release were significantly reduced in the Relnrl-Orl/+ mice. In addition, the levels of GABAergic markers were decreased in the brain of these mice. Taken together, our results suggest that the exonic deletion of RELN plays a pathological role, implicating functional changes in the dopaminergic and GABAergic systems, in the pathophysiology of SCZ.

8.
Yakugaku Zasshi ; 137(10): 1233-1240, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-28966264

RESUMO

Reelin is a large secreted glycoprotein that regulates embryonic neuronal lamination and adult synaptic function. Secreted Reelin binds to lipoprotein receptors expressed on neurons. The Reelin-receptor interaction induces phosphorylation of an intracellular adaptor protein Dab1, which is required for normal embryonic brain development and adult brain functions. It has been suggested that Reelin hypofunction plays a role in the pathogenesis of several neuropsychiatric diseases, such as schizophrenia, autism, and Alzheimer's disease. Thus upregulation of Reelin activity may ameliorate the symptoms of neuropsychiatric diseases. However, the regulatory mechanism underlying the functions of Reelin is largely unknown and there are no good animal models of Reelin malfunction; thus, causal relations between Reelin and neuropsychiatric diseases remain unclear. Recently, our studies have shown that proteolytic cleavage of the Reelin protein regulates its activity. Herein, we will review recent findings about relations between Reelin and Alzheimer's disease, and the mechanism underlying the regulation of Reelin function by proteolytic cleavage. Also, we will discuss the prospect of treating neuropsychiatric diseases by upregulation of Reelin activity.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Serina Endopeptidases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/genética , Animais , Encéfalo/embriologia , Encéfalo/fisiologia , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Camundongos , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Proteólise , Receptores de Superfície Celular/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Regulação para Cima
9.
J Neurosci ; 37(12): 3181-3191, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213441

RESUMO

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.


Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Ligação Proteica
10.
Neuroscience ; 336: 20-29, 2016 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-27586054

RESUMO

In the normal cerebellum, Purkinje cells (PCs) are generated in a zone along the ventricular surface, migrate radially, and align to form a single-cell layer. However, in mice lacking the secreted protein Reelin or its downstream adaptor protein Dab1, the majority of PCs are located ectopically in the deep cerebellar mass. Nonetheless, how Reelin regulates migration and alignment of PCs remains incompletely understood. Reelin has a highly-conserved C-terminal region (CTR), which is required for its full activity. Here, we report an abnormality of the cerebellum in Reelin CTR-lacking knock-in (ΔC-KI) mice. In the ΔC-KI mice, cerebellar formation was largely normal, but some PCs in selected regions were found to be located ectopically and to frequently form clusters. Ectopic PCs contained a higher amount of Dab1 protein and functional Reelin receptors, including mainly very low-density lipoprotein receptor than correctly-aligned PCs. Decreasing Dab1 gene dosage exacerbated mislocalization of PCs and the cerebellar structure in Reelin ΔC-KI mice. These results indicate that ectopic PCs in ΔC-KI mice failed to receive sufficient Reelin signaling en route to their final destinations. Further, we also found that Reelin protein with intact CTR binds preferentially to PCs. Thus, it was suggested that the extent or quality of Reelin/Dab1 signaling that PCs require for correct positioning vary and that Reelin with intact CTR is required for that of a certain subset of PCs.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Cerebelo/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células de Purkinje/citologia , Receptores de Superfície Celular/metabolismo , Serina Endopeptidases/metabolismo , Animais , Cerebelo/crescimento & desenvolvimento , Camundongos Transgênicos , Fosforilação , Receptores de LDL/metabolismo
11.
Sci Rep ; 6: 28636, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27346785

RESUMO

The secreted glycoprotein Reelin is believed to play critical roles in the pathogenesis of several neuropsychiatric disorders. The highly basic C-terminal region (CTR) of Reelin is necessary for efficient activation of its downstream signaling, and the brain structure of knock-in mice that lack the CTR (ΔC-KI mice) is impaired. Here, we performed a comprehensive behavioral test battery on ΔC-KI mice, in order to evaluate the effects of partial loss-of-function of Reelin on brain functions. The ΔC-KI mice were hyperactive and exhibited reduced anxiety-like and social behaviors. The working memory in ΔC-KI mice was impaired in a T-maze test. There was little difference in spatial reference memory, depression-like behavior, prepulse inhibition, or fear memory between ΔC-KI and wild-type mice. These results suggest that CTR-dependent Reelin functions are required for some specific normal brain functions and that ΔC-KI mice recapitulate some aspects of neuropsychiatric disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder.


Assuntos
Comportamento Animal , Encéfalo , Moléculas de Adesão Celular Neuronais , Proteínas da Matriz Extracelular , Transtornos Mentais , Proteínas do Tecido Nervoso , Serina Endopeptidases , Comportamento Social , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Transtornos Mentais/genética , Transtornos Mentais/metabolismo , Transtornos Mentais/fisiopatologia , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Domínios Proteicos , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo
12.
Neurosci Res ; 110: 11-7, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27033969

RESUMO

CUB and Sushi multiple domains 3 (CSMD3) is a large protein expressed in fetal and adult brain. Recently, mutations of the CSMD3 gene were identified in schizophrenia and autism patients. However, biochemical properties and functions of the CSMD3 protein remain unknown. Here, we demonstrate that CSMD3 is an oligomeric type I transmembrane protein localized in the apical dendrites of hippocampal pyramidal neurons in the postnatal brain. In cultured hippocampal neurons, CSMD3 is expressed only after 7 days in vitro. Overexpression of CSMD3 induced dendritic branching in hippocampal neurons. Regulation of dendritic morphology by CSMD3 depended on the presence of its extracellular region, while CSMD3 intracellular region was dispensable for this activity. These results suggest that CSMD3 acts as a co-receptor of an unidentified membrane protein to regulate dendrite development. Therefore, malfunctions of CSMD3 may be one of the factors in the pathogenesis of psychiatric disorders.


Assuntos
Dendritos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Animais , Células Cultivadas , Dendritos/ultraestrutura , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Proteínas de Membrana/fisiologia , Camundongos Endogâmicos ICR , Neurônios/metabolismo , Neurônios/ultraestrutura
14.
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
15.
J Neurosci ; 35(11): 4776-87, 2015 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-25788693

RESUMO

During brain development, Reelin exerts a variety of effects in a context-dependent manner, whereas its underlying molecular mechanisms remain poorly understood. We previously showed that the C-terminal region (CTR) of Reelin is required for efficient induction of phosphorylation of Dab1, an essential adaptor protein for canonical Reelin signaling. However, the physiological significance of the Reelin CTR in vivo remains unexplored. To dissect out Reelin functions, we made a knock-in (KI) mouse in which the Reelin CTR is deleted. The amount of Dab1, an indication of canonical Reelin signaling strength, is increased in the KI mouse, indicating that the CTR is necessary for efficient induction of Dab1 phosphorylation in vivo. Formation of layer structures during embryonic development is normal in the KI mouse. Intriguingly, the marginal zone (MZ) of the cerebral cortex becomes narrower at postnatal stages because upper-layer neurons invade the MZ and their apical dendrites are misoriented and poorly branched. Furthermore, Reelin undergoes proteolytic cleavage by proprotein convertases at a site located 6 residues from the C terminus, and it was suggested that this cleavage abrogates the Reelin binding to the neuronal cell membrane. Results from ectopic expression of mutant Reelin proteins in utero suggest that the dendrite development and maintenance of the MZ require Reelin protein with an intact CTR. These results provide a novel model regarding Reelin functions involving its CTR, which is not required for neuronal migration during embryonic stages but is required for the development and maintenance of the MZ in the postnatal cerebral cortex.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Células COS , Cercopithecus aethiops , Técnicas de Introdução de Genes/métodos , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Proteólise
16.
Neurosci Res ; 88: 23-7, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25149914

RESUMO

The Reelin-Dab1 pathway plays important roles in the development of central nervous system. In the autosomal recessive mutant mouse yotari, there is a replacement of a part of Dab1 gene with a long interspersed nuclear element fragment, and it was previously suggested that no protein derived from this gene was present. We here show that an aberrant fragment of Dab1 protein (p64/60) is present in the brain of yotari mouse. The amount of p64/60 is relatively abundant in the embryonic stages and decreased in the postnatal ones. Unlike wild-type Dab1 protein, p64/60 is not phosphorylated by Fyn kinase and localizes considerably to the nucleus. These data suggested that some phenotypes of yotari may be attributable to the presence of p64/60. It also raises a caveat that a tissue from yotari is not a perfect control for immunostaining of Dab1 protein.


Assuntos
Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Fenótipo , Serina Endopeptidases/metabolismo , Tirosina/metabolismo
17.
J Biol Chem ; 289(18): 12922-30, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24644294

RESUMO

Reelin is a secreted glycoprotein that plays essential roles in the brain. Reelin is specifically cleaved at two distinct sites, called N-t and C-t, with the former being the major one. N-t cleavage can occur both in the extracellular space and in the endosomes, although the physiological importance of endosomal N-t cleavage has not been investigated. In this study, we first determined the exact N-t cleavage site catalyzed by a protease secreted by cerebral cortical neurons. Cleavage occurred between Pro-1244 and Ala-1245 within Reelin repeat 3. A Reelin mutant in which Pro-1244 was replaced with aspartate (Reelin-PD) was resistant to a protease secreted by cultured cerebral cortical neurons, and its biological activity stayed active longer than that of wild-type Reelin. Interestingly, Reelin-PD remained in the intracellular compartments longer than wild-type Reelin and persistently activated downstream signaling. Therefore, N-t cleavage of Reelin is required for halting the signaling machinery in the extracellular space as well as within endosomes of target neurons. We established a monoclonal antibody specific to uncleaved Reelin protein and found that it is localized in the vicinity of Reelin-producing cells, whereas the N-terminal fragment diffuses, or is transported, to distant regions. These data demonstrate that N-t cleavage of Reelin plays critical roles in regulating the duration and range of Reelin functions both in the extracellular milieu and in the intracellular compartments.


Assuntos
Ácido Aspártico/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Mutação , Proteínas do Tecido Nervoso/genética , Prolina/genética , Serina Endopeptidases/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Western Blotting , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Endossomos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Espaço Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeo Hidrolases/metabolismo , Prolina/metabolismo , Proteólise , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo
18.
FEBS Lett ; 586(19): 3349-53, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22819337

RESUMO

Reelin is a glycoprotein essential for brain development and functions. Reelin is subject to specific proteolysis at two distinct (N-t and C-t) sites, and these cleavages significantly diminish Reelin activity. The decrease of Reelin activity is detrimental for brain function, but the protease that catalyzes specific cleavage of Reelin remains elusive. Here we found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-specific manner. Among ADAMTS-4 isoforms, p50 cleaves the N-t site only, while p75 cleaves both sites. This is the first report identifying a protease that can specifically cleave Reelin.


Assuntos
Proteínas ADAM/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAMTS4 , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Primers do DNA/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Gravidez , Pró-Colágeno N-Endopeptidase/química , Pró-Colágeno N-Endopeptidase/genética , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Especificidade por Substrato , Distribuição Tecidual
19.
Biochem Biophys Res Commun ; 417(3): 1014-7, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22209789

RESUMO

Sphingomyelin (SM) plays important roles in regulating structure and function of plasma membrane, but how intracellular localization of SM is regulated in neuronal cells is not understood. Here we show that two isoforms of SM synthase (SMS) are differentially expressed in neuronal subtypes and that only SMS2 proteins localize in neurites of hippocampal neurons. Moreover, SMS proteins induce Lysenin-binding SM clusters exclusively in their vicinity although neurons hardly contain such cluster under control condition. These findings indicate three important notions about SM metabolism in neurons. First, the activity of SMS is the rate-limiting step of SM cluster formation. Second, the SM content or clustering can be modulated by SMS activity. Third, SMS1 and SMS2 play distinct roles in regulating local SM clustering. Particularly, SMS2, rather than SMS1, is likely to be the major enzyme that is important for SM synthesis in the long neurites and its tip, the growth cone.


Assuntos
Hipocampo/enzimologia , Neurônios/enzimologia , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Células Cultivadas , Isoenzimas/metabolismo , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/metabolismo , Camundongos
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