RESUMO
INTRODUCTION: Insulin-like growth factor (IGF)1 and IGF2 have neuroprotective effects, but less is known regarding how other members of the IGF system, including IGF binding proteins (IGFBPs) and the regulatory proteinase pappalysin-1 (PAPP-A) and its endogenous inhibitor stanniocalcin-2 (STC2) participate in this process. Here, we analyzed whether these members of the IGF system are modified in neurons and astrocytes in response to palmitic acid (PA), a fatty acid that induces cell stress when increased centrally. METHODS: Primary hypothalamic astrocyte cultures from male and female PND2 rats and the pro-opiomelanocortin (POMC) neuronal cell line, mHypoA-POMC/GFP-2, were treated with PA, IGF1 or both. To analyze the role of STC2 in astrocytes, siRNA assays were employed. RESULTS: In astrocytes of both sexes, PA rapidly increased cell stress factors followed by increased Pappa and Stc2 mRNA levels and then a decrease in Igf1, Igf2, and Igfbp2 expression and cell number. Exogenous IGF1 did not revert these effects. In mHypoA-POMC/GFP-2 neurons, PA reduced cell number and Pomc and Igf1 mRNA levels, and increased Igfbp2 and Stc2, again with no effect of exogenous IGF1. PA increased STC2 expression, but no effects of decreasing its levels by interference assays or exogenous STC2 treatment in astrocytes were found. CONCLUSIONS: The response of the IGF system to PA was cell and sex specific, but no protective effects of the IGFs were found. However, the modifications in hypothalamic PAPP-A and STC2 indicate that further studies are required to determine their role in the response to fatty acids and possibly in metabolic control.
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Normal brain development depends on tight temporal and spatial regulation of connections between cells. Mutations in L1cam, a member of the immunoglobulin (Ig) superfamily that mediate cell-cell contacts through homo- and heterophilic interactions, are associated with several developmental abnormalities of the nervous system, including mental retardation, limb spasticity, hydrocephalus, and corpus callosum aplasia. L1cam has been reported to be shed from the cell surface, but the significance of this during different phases of brain development is unknown. We here show that ADAM10-mediated shedding of L1cam is regulated by its fibronectin type III (FNIII) domains. Specifically, the third FNIII domain is important for maintaining a conformation where access to a membrane proximal cleavage site is restricted. To define the role of ADAM10/17/BACE1-mediated shedding of L1cam during brain development, we used a zebrafish model system. Knockdown of the zebrafish, l1camb, caused hydrocephalus, defects in axonal outgrowth, and myelination abnormalities. Rescue experiments with proteinase-resistant and soluble L1cam variants showed that proteolytic cleavage is not required for normal axonal outgrowth and development of the ventricular system. In contrast, metalloproteinase-mediated shedding is required for efficient myelination, and only specific fragments are able to mediate this stimulatory function of the shedded L1cam.
Assuntos
Encéfalo/crescimento & desenvolvimento , Hidrocefalia/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Proteínas ADAM/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Hidrocefalia/metabolismo , Molécula L1 de Adesão de Célula Nervosa/química , Proteólise , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Formation of functional myelin sheaths within the central nervous system depends on expression of myelin basic protein (MBP). Following process extension and wrapping around axonal segments, this highly basic protein is required for compaction of the multi-layered membrane sheath produced by oligodendrocytes. MBP is hypothesized to be targeted to the membrane sheath by mRNA transport and local translation, which ensures that its expression is temporally and spatially restricted. The mechanistic details of how this might be regulated are still largely unknown, in particular because a model system that allows this process to be studied in vivo is lacking. We here show that the expression of the zebrafish MBP orthologs, mbpa and mbpb, is developmentally regulated, and that expression of specific mbpa isoforms is restricted to the peripheral nervous system. By analysis of transgenic zebrafish, which express a fluorescent reporter protein specifically in myelinating oligodendrocytes, we demonstrate that both mbpa and mbpb include a 3'UTR sequence, by which mRNA transport and translation is regulated in vivo. Further functional analysis suggests that: (1) the 3'UTRs delay the onset of protein expression; and that (2) several regulatory elements contribute to targeting of the mbp mRNA to the myelin sheath. Finally, we show that a pharmacological compound known to enhance neuronal activity stimulates the translation of Mbp in zebrafish in a 3'UTR-dependent manner. A similar effect was obtained following stimulation with a TrkB receptor agonist, and cell-based assays further confirmed that the receptor ligand, BDNF, in combination with other signals reversed the inhibitory effect of the 3'UTR on translation.
RESUMO
During development of the central nervous system not all axons are myelinated, and axons may have distinct myelination patterns. Furthermore, the number of myelin sheaths formed by each oligodendrocyte is highly variable. However, our current knowledge about the axo-glia communication that regulates the formation of myelin sheaths spatially and temporally is limited. By using axon-mimicking microfibers and a zebrafish model system, we show that axonal ephrin-A1 inhibits myelination. Ephrin-A1 interacts with EphA4 to activate the ephexin1-RhoA-Rock-myosin 2 signaling cascade and causes inhibition of oligodendrocyte process extension. Both in myelinating co-cultures and in zebrafish larvae, activation of EphA4 decreases myelination, whereas myelination is increased by inhibition of EphA4 signaling at different levels of the pathway, or by receptor knockdown. Mechanistically, the enhanced myelination is a result of a higher number of myelin sheaths formed by each oligodendrocyte, not an increased number of mature cells. Thus, we have identified EphA4 and ephrin-A1 as novel negative regulators of myelination. Our data suggest that activation of an EphA4-RhoA pathway in oligodendrocytes by axonal ephrin-A1 inhibits stable axo-glia interaction required for generating a myelin sheath.
Assuntos
Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Efrina-A1/metabolismo , Bainha de Mielina/metabolismo , Receptor EphA4/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Técnicas de Cocultura , Ratos , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6ß1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active ß1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.
Assuntos
Axônios/fisiologia , Efrinas/metabolismo , Oligodendroglia/fisiologia , Receptores da Família Eph/metabolismo , Animais , Comunicação Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Córtex Cerebral/fisiologia , Técnicas de Cocultura , Gânglios Espinais/crescimento & desenvolvimento , Gânglios Espinais/fisiologia , Integrinas/metabolismo , Bainha de Mielina/fisiologia , Células-Tronco Neurais/fisiologia , Ratos , Transdução de Sinais/fisiologiaRESUMO
Stanniocalcin-1 (STC1) is a disulfide-bound homodimeric glycoprotein, first identified as a hypocalcemic hormone important for maintaining calcium homeostasis in teleost fish. STC1 was later found to be widely expressed in mammals, although it is not believed to function in systemic calcium regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth factor (IGF) signaling through proteolytic cleavage of IGF-binding proteins (IGFBPs). STC1 potently (Ki = 68 pm) inhibits PAPP-A cleavage of IGFBP-4, and we show in a cell-based assay that STC1 effectively antagonizes PAPP-A-mediated type 1 IGF receptor (IGF1R) phosphorylation. It has recently been found that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue. It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity to the pappalysin family of metzincin metalloproteinases. Our data are the first to identify STC1 as a proteinase inhibitor, suggesting a previously unrecognized function of STC1 in the IGF system.
Assuntos
Glicoproteínas/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína Plasmática A Associada à Gravidez/metabolismo , Western Blotting , Glicoproteínas/genética , Glicoproteínas/farmacologia , Células HEK293 , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Plasmática A Associada à Gravidez/antagonistas & inibidores , Proteína Plasmática A Associada à Gravidez/genética , Proteólise/efeitos dos fármacos , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Mammalian stanniocalcin-2 (STC2) is a secreted polypeptide widely expressed in developing and adult tissues. However, although transgenic expression in mice is known to cause severe dwarfism, and targeted deletion of STC2 causes increased postnatal growth, its precise biological role is still unknown. We found that STC2 potently inhibits the proteolytic activity of the growth-promoting metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). Proteolytic inhibition requires covalent binding of STC2 to PAPP-A and is mediated by a disulfide bond, which involves Cys-120 of STC2. Binding of STC2 prevents PAPP-A cleavage of insulin-like growth factor-binding protein (IGFBP)-4 and hence release within tissues of bioactive IGF, required for normal growth. Concordantly, we show that STC2 efficiently inhibits PAPP-A-mediated IGF receptor signaling in vitro and that transgenic mice expressing a mutated variant of STC2, STC2(C120A), which is unable to inhibit PAPP-A, grow like wild-type mice. Our work identifies STC2 as a novel proteinase inhibitor and a previously unrecognized extracellular component of the IGF system.
Assuntos
Glicoproteínas/metabolismo , Crescimento/genética , Proteína Plasmática A Associada à Gravidez/metabolismo , Proteólise , Sequência de Aminoácidos , Animais , Células Cultivadas , Cisteína/química , Cisteína/genética , Feminino , Glicoproteínas/química , Glicoproteínas/genética , Células HEK293 , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptor IGF Tipo 1/metabolismoRESUMO
Pregnancy-associated plasma protein A2 (PAPP-A2, also known as pappalysin-2) is a large metalloproteinase that is known to be required for normal postnatal growth and bone development in mice. We here report the detection of zebrafish papp-a2 mRNA in the chordamesoderm, notochord and lower jaw of zebrafish (Danio rerio) embryos, and that papp-a2-knockdown embryos display broadened axial mesoderm, notochord bends and severely reduced cranial cartilages. Genetic data link these phenotypes to insulin-like growth factor (Igf)-binding protein-3 (Igfbp-3) and bone morphogenetic protein (Bmp) signaling, and biochemical analysis show specific Igfbp-3 proteolysis by Papp-a2, implicating Papp-a2 in the modulation of Bmp signaling by Igfbp-3 proteolysis. Knockdown of papp-a2 additionally resulted in angiogenesis defects, strikingly similar to previous observations in embryos with mutations in components of the Notch system. Accordingly, we find that Notch signaling is modulated by Papp-a2 in vivo, and, furthermore, that human PAPP-A2 is capable of modulating Notch signaling independently of its proteolytic activity in cell culture. Based on these results, we conclude that Papp-a2 modulates Bmp and Notch signaling by independent mechanisms in zebrafish embryos. In conclusion, these data link pappalysin function in zebrafish to two different signaling pathways outside the IGF system.
Assuntos
Cartilagem/enzimologia , Neovascularização Fisiológica , Proteína Plasmática A Associada à Gravidez/metabolismo , Crânio/enzimologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genótipo , Células HEK293 , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Dados de Sequência Molecular , Fenótipo , Proteína Plasmática A Associada à Gravidez/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Crânio/embriologia , Fatores de Tempo , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
In the developing nervous system, abundant synthesis of myelin basic protein (MBP) in oligodendrocytes is required for the formation of compact myelin sheaths around axons. The MBP mRNA is known to be transported into the processes of oligodendrocytes. However, knowledge of the regulatory mechanisms that ensure the tight temporal and spatial control of MBP translation within these processes is limited. Here, we have identified novel regions within the 3'-UTR of the MBP mRNA that are responsible for the regulation of its translation, and we have demonstrated that each of the mRNA-binding proteins heterogeneous nuclear ribonucleoprotein (hnRNP)-A2, hnRNP-K and hnRNP-E1 serve distinct functions to regulate controlled and localized protein synthesis. hnRNP-A2 is responsible for mRNA transport, not for translational inhibition. By contrast, hnRNP-K and hnRNP-E1 play opposing roles in the translational regulation of MBP mRNA. We have identified shared binding sites within the 3'-UTR, and show that translation is promoted by the exchange of inhibitory hnRNP-E1 for stimulatory hnRNP-K. We further show that this molecular switch in the MBP messenger RNA-ribonucleoprotein (mRNP) complex, which regulates the synthesis of MBP, is important for the normal growth and extension of myelin sheets.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína Básica da Mielina/genética , Oligodendroglia/metabolismo , Transporte Biológico , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Proteína Básica da Mielina/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: Pregnancy-associated plasma protein-A (PAPP-A) is a local regulator of insulin-like growth factor (IGF) bioavailability in physiological systems, but many structural and functional aspects of the metzincin metalloproteinase remain to be elucidated. PAPP-A cleaves IGF binding protein (IGFBP)-4 and IGFBP-5. Cleavage of IGFBP-4, but not IGFBP-5, depends on the binding of IGF before proteolysis by PAPP-A can occur. The paralogue PAPP-A2 has two substrates among the six IGFBPs: IGFBP-3 and IGFBP-5. METHODS: Sets of chimeric proteins between IGFBP-4 and -5, and IGFBP-3 and -5 were constructed to investigate the structural requirements for IGF modulation. At the proteinase level, we investigated the importance of individual acidic amino acids positioned in the proteolytic domain of PAPP-A for proteolytic activity against IGFBP-4 and -5. Interaction between PAPP-A and its substrates was analyzed by surface plasmon resonance. RESULTS AND CONCLUSION: We provide data suggesting that the C-terminal domain of the IGFBPs is responsible for IGF-dependent modulation of access to the scissile bond. Loss or reduction of IGFBP proteolysis by PAPP-A was observed upon mutation of residues positioned in the unique 63-residue stretch separating the zinc and Met-turn motifs, and in the short sequence following the Met-turn methionine. A model of the proteolytic domain of PAPP-A suggests the presence of structural calcium ions in the C-terminal subdomain, implicated in IGFBP substrate interactions. GENERAL SIGNIFICANCE: Detailed knowledge of interactions between PAPP-A and its substrates is required to understand the modulatory role of PAPP-A on IGF receptor stimulation.
Assuntos
Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Proteína Plasmática A Associada à Gravidez/química , Somatomedinas/química , Sequência de Aminoácidos , Sítios de Ligação , Feminino , Células HEK293 , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Modelos Moleculares , Dados de Sequência Molecular , Gravidez , Proteína Plasmática A Associada à Gravidez/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Somatomedinas/genética , Especificidade por Substrato , TransfecçãoRESUMO
Myelination in the central nervous system provides a unique example of how cells establish asymmetry. The myelinating cell, the oligodendrocyte, extends processes to and wraps multiple axons of different diameter, keeping the number of wraps proportional to the axon diameter. Local regulation of protein synthesis represents one mechanism used to control the different requirements for myelin sheath at each axo-glia interaction. Prior work has established that ß1-integrins are involved in the axoglial interactions that initiate myelination. Here, we show that integrin activation regulates translation of a key sheath protein, myelin basic protein (MBP), by reversing the inhibitory effect of the mRNA 3'UTR. During oligodendrocyte differentiation and myelination α6ß1-integrin interacts with hnRNP-K, an mRNA-binding protein, which binds to MBP mRNA and translocates from the nucleus to the myelin sheath. Furthermore, knockdown of hnRNP-K inhibits MBP protein synthesis during myelination. Together, these results identify a novel pathway by which axoglial adhesion molecules coordinate MBP synthesis with myelin sheath formation.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/fisiologia , Integrina beta1/fisiologia , Proteína Básica da Mielina/genética , Oligodendroglia/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Transporte Biológico , Diferenciação Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/análise , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Integrina alfa6beta1/metabolismo , Integrina beta1/metabolismo , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Ratos , Alinhamento de Sequência , Transdução de SinaisRESUMO
BACKGROUND: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1). IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker. CONCLUSIONS/SIGNIFICANCE: These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.
Assuntos
Duplicação Gênica , Variação Genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Sequência Conservada , Regulação da Expressão Gênica , Hipóxia/genética , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/classificação , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Peixe-Zebra/classificação , Proteínas de Peixe-Zebra/classificaçãoRESUMO
The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves both insulin-like growth factor (IGF)-binding protein 4 (IGFBP-4) and -5 at a single site in their central domain causing the release of bioactive IGF. Inhibition of IGF signaling is relevant in human disease, and several drugs in development target the IGF receptor. However, inhibition of PAPP-A activity may be a valuable alternative. We have generated monoclonal phage-derived single chain fragment variable (scFv) antibodies which selectively inhibit the cleavage of IGFBP-4 by PAPP-A, relevant under conditions where cleavage of IGFBP-4 represents the final step in the delivery of IGF to the IGF receptor. None of the antibodies inhibited the homologous proteinase PAPP-A2, which allowed mapping of antibody binding by means of chimeras between PAPP-A and PAPP-A2 to the C-terminal Lin12-Notch repeat module, separated from the proteolytic domain by almost 1000 amino acids. Hence, the antibodies define a substrate binding exosite that can be targeted for the selective inhibition of PAPP-A proteolytic activity against IGFBP-4. In addition, we show that the Lin12-Notch repeat module reversibly binds a calcium ion and that bound calcium is required for antibody binding, providing a strategy for the further development of selective inhibitory compounds. To our knowledge these data represent the first example of differential inhibition of cleavage of natural proteinase substrates by exosite targeting. Generally, exosite inhibitors are less likely to affect the activity of related proteolytic enzymes with similar active site environments. In the case of PAPP-A, selective inhibition of IGFBP-4 cleavage by interference with exosite binding is a further advantage, as the activity against other known or unknown PAPP-A substrates, whose cleavage may not depend on binding to the same exosite, is not targeted.
Assuntos
Proteína Plasmática A Associada à Gravidez/metabolismo , Animais , Anticorpos Monoclonais/química , Sítios de Ligação , Linhagem Celular , Galinhas , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/química , Fator de Crescimento Insulin-Like I/metabolismo , Modelos Genéticos , Biblioteca de Peptídeos , Proteína Plasmática A Associada à Gravidez/química , Ligação Proteica , Estrutura Terciária de Proteína , Ressonância de Plasmônio de SuperfícieRESUMO
The biological activity of IGF-I and -II is controlled by six binding proteins (IGFBPs), preventing the IGFs from interacting with the IGF receptor. Proteolytic cleavage of IGFBPs is one mechanism by which IGF can be released to bind the receptor. The IGFBPs are usually studied individually, although the presence of more than one of the IGFBPs in most tissues suggests a cooperative function. Thus, the IGFBPs are part of regulatory networks with proteolytic enzymes in one end and the IGF receptor in the other end. We have established a model system that allows analysis of the dynamics between IGF, IGFBP-4 and -5, the IGF receptor, and the proteolytic enzyme PAPP-A, which specifically cleaves both IGFBP-4 and -5. We demonstrate different mechanisms of IGF release from IGFBP-4 and -5: cooperative binding to IGF is observed for the proteolytic fragments of IGFBP-5, but not fragments of IGFBP-4. Furthermore, we find that PAPP-A-mediated IGF-dependent cleavage of IGFBP-4 is inhibited by IGFBP-5, which sequesters IGF from IGFBP-4, and that cleavage of both IGFBP-4 and -5 is required for the release of bioactive IGF. Finally, we show that cell surface-localized proteolysis of IGFBP-4 represents the final regulatory step of efficient IGF delivery to the receptor. Our data define a regulatory system in which molar ratios between the IGFBPs and IGF and between the different IGFBPs, sequential proteolytic cleavage of the IGFBPs, and surface association of the activating proteinase are key elements in the regulation of IGF receptor stimulation.
Assuntos
Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/fisiologia , Receptores de Somatomedina/fisiologia , Somatomedinas/fisiologia , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Proteína 4 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Rim , Cinética , Peptídeo Hidrolases/metabolismo , FosforilaçãoRESUMO
Myelination is necessary both for rapid salutatory conduction and the long-term survival of the axon. In the CNS the myelin sheath is formed by the oligodendrocytes. Each oligodendrocyte myelinates several axons and, as the number of wraps around each axon is determined precisely by the axon diameter, this requires a close, highly regulated interaction between the axons and each of the oligodendrocyte processes. Adhesion molecules are likely to play an important role in the bi-directional signalling between axon and oligodendrocyte that underlies this interaction. Here we review the current knowledge of the function of adhesion molecules in the different phases of oligodendrocyte differentiation and myelination, and discuss how the properties of these proteins defined by other cell biological systems indicates potential roles in oligodendrocytes. We show how the function of a number of different adhesion and cell-cell interaction molecules such as polysialic acid neural cell adhesion molecule, Lingo-1, Notch, neuregulin, integrins and extracellullar matrix proteins provide negative and positive signals that coordinate the formation of the myelin membrane. Compiling this information from a number of different cell biological and genetic experiments helps us to understand the pathology of multiple sclerosis and direct new areas of research that might eventually lead to potential drug targets to increase remyelination.
RESUMO
The metzincin metalloproteinase pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1) promotes cell growth by proteolytic cleavage of insulin-like growth factor-binding proteins 4 and 5, causing the release of bound insulin-like growth factors. PAPP-A binds an unknown cell-surface heparan sulfate proteoglycan, suggesting that it controls insulin-like growth factor signaling spatially. In human pregnancy, the majority of PAPP-A circulates as a disulfide-bonded complex with its inhibitor, the proform of eosinophil major basic protein (proMBP). Interestingly, Ser-62 of proMBP is substituted with a glycosaminoglycan (GAG) chain, possibly a heparan sulfate type, and the PAPP-A.proMBP complex is unable to bind to the cell surface. We show here that proMBP detaches surface-bound PAPP-A in a process that depends on the proMBP GAG and also on the formation of intermolecular disulfide bonds between PAPP-A and proMBP. Unlike what was expected, we demonstrate that the GAG of proMBP is not required for PAPP-A.proMBP complex formation and that proMBP residues His-137, Ser-178, Arg-179, and Asn-181 are important for the recognition of PAPP-A. Using a mouse model, we find that the half-life of circulating PAPP-A and proMBP in complex is severalfold higher than both of the uncomplexed proteins, further suggesting that the PAPP-A.proMBP complex is formed at the cell surface in vivo rather than in the circulation. Further supporting this, we show that formation of the PAPP-A.proMBP complex at the cell surface proceeds rapidly compared with the slow rate of complex formation in solution. Because both PAPP-A and proMBP are expressed ubiquitously, this model may be applicable to many tissues in which insulin-like growth factor bioavailability is locally regulated.
Assuntos
Membrana Celular/metabolismo , Glicosaminoglicanos/química , Proteína Plasmática A Associada à Gravidez/fisiologia , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Dissulfetos , Feminino , Heparitina Sulfato/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Proteína Plasmática A Associada à Gravidez/química , Somatomedinas/metabolismoRESUMO
Insulin-like growth factor (IGF)-I and -II function in normal physiology to control growth, development, and differentiation, but are also important in pathophysiological conditions, particularly in cancer. The biological effects of the IGFs are mediated by the IGF-I receptor (IGFR), a covalent homodimer composed of two alpha and two beta chains, similar in structure to the insulin receptor (IR). To allow measurement of the stimulation of IGFR in living cells, we developed an assay based on bioluminescence resonance energy transfer (BRET) between a donor molecule, Renilla luciferase, and an acceptor fluorophore, enhanced yellow fluorescent protein (EYFP). Initial attempts based on fusion of the luciferase to IGFR, and EYFP to IGFR, or to downstream signaling molecules, insulin receptor substrate-1 (IRS1) or protein tyrosine phosphatases-1B (PTP-1B), failed. However, similar experiments with IR, carried our in parallel, proved successful. We therefore, constructed assays based on chimeric IGFR/IR proteins, in which the ligand binding site was derived from IGFR. With the most efficient assay, in which the luciferase is fused to a chimeric receptor with the entire intracellular portion derived from IR, and EYFP fused to PTP-1B, IGF activity was measured specifically with sensitivity similar to the corresponding assay for insulin, based on IR. The established system allows efficient evaluation of candidate ligand- or receptor-directed molecules for the modulation of IGF activities. Furthermore, we demonstrate that a set of inhibitory IGF binding proteins (IGFBPs) or activating IGFBP-specific proteinases, unique to the IGF system, may serve as potential targets. In addition to screening, real-time measurement of IGFR stimulation may be important in efforts to understand the kinetics of receptor stimulation, in particular differences between IGFR and IR.
Assuntos
Sistemas Computacionais , Transferência de Energia/genética , Medições Luminescentes/métodos , Proteínas Luminescentes/metabolismo , Somatomedinas/metabolismo , Linhagem Celular , Células Cultivadas , Humanos , Fígado/química , Fígado/citologia , Fígado/metabolismo , Proteínas Luminescentes/genética , Somatomedinas/genética , TransfecçãoRESUMO
The metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) cleaves a subset of insulin-like growth factor binding proteins (IGFBP), which inhibit the activities of insulin-like growth factor (IGF). Through this proteolytic activity, PAPP-A is believed to regulate IGF bioavailability in several biological systems, including the human reproductive system and the cardiovascular system. PAPP-A adheres to mammalian cells by interactions with glycosaminoglycan (GAG), thus targeting the proteolytic activity of PAPP-A to the cell surface. Based on site-directed mutagenesis, we here delineate the PAPP-A GAG-binding site in the C-terminal modules CCP3 and CCP4. Using heparin affinity chromatography, commonly employed in such studies, we define three clusters of arginines and lysines of CCP3, which are important for the interaction of PAPP-A with heparin. In a model of PAPP-A CCP3-CCP4, basic residues of these sequence clusters form a contiguous patch located on one side of the structure. Binding to the unknown, natural cell surface receptor of PAPP-A, assessed by flow cytometry, also depends on residues of these three basic clusters. However, single or double residue substitutions generally have a modest effect on PAPP-A heparin binding assessed by chromatography, but cell surface adhesion was critically reduced by several of these substitutions, emphasizing the relevance of analysis by flow cytometry. The contributions of positively charged residues located in CCP4 were all minor when analyzed by heparin affinity chromatography. However, the mutation of CCP4 residues Arg1459 and Lys1460 to Ala almost abrogated cell surface adhesion. Furthermore, when acidic residues of the homologous proteinase PAPP-A2 (Asp1547, Glu1555 and Glu1567) were introduced into the corresponding positions in the sequence of PAPP-A, located in each of the three basic clusters of CCP3, binding to heparin was strongly impaired and cell surface binding was abrogated. This explains, at least in part, why PAPP-A2 lacks the ability of cell surface adhesion, and further emphasizes the role of the basic clusters defined in PAPP-A.
Assuntos
Aminoácidos Básicos/metabolismo , Adesão Celular/fisiologia , Proteína Plasmática A Associada à Gravidez/química , Proteína Plasmática A Associada à Gravidez/metabolismo , Sequência de Aminoácidos , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Aminoácidos Básicos/química , Aminoácidos Básicos/genética , Sítios de Ligação , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia de Afinidade/métodos , Citometria de Fluxo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteína Plasmática A Associada à Gravidez/genética , Ligação Proteica , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The activities of insulin-like growth factor (IGF)-I and -II are regulated by IGF-binding proteins (IGFBPs). Cleavage of IGFBP-4 by the metalloproteinase pregnancy-associated plasma protein-A (PAPP-A) causes release of bound IGF and has been established in several biological systems including the human reproductive system. Using flow cytometry, we first demonstrate that PAPP-A reversibly binds to the cell surface of several cell types analyzed. Heparin and heparan sulfate, but not dermatan or chondroitin sulfate, effectively compete for PAPP-A surface binding, and because incubation of cells with heparinase abrogated PAPP-A adhesion, binding is probably mediated by a cell surface heparan sulfate proteoglycan. Furthermore, the proteolytic activity of PAPP-A is preserved while bound to cells, suggesting that adhesion functions to target its activity to the vicinity of the IGF receptor, decreasing the probability that released IGF is captured by another IGFBP molecule before receptor binding. This mechanism potentially functions in both autocrine and paracrine regulation, as PAPP-A need not be synthesized in a cell to which it adheres. A truncated PAPP-A variant without the five short consensus repeats in the C-terminal third of the 1547-residue PAPP-A subunit, lacked surface binding. We also show that PAPP-A2, a recently discovered IGFBP-5 proteinase with homology to PAPP-A, does not bind cells. This finding allowed further mapping of the PAPP-A adhesion site to short consensus repeat modules 3 and 4 by the expression and analysis of nine PAPP-A/PAPP-A2 chimeras. Interestingly, the proteolytically inactive, disulfide-bound complex of PAPP-A and the proform of eosinophil major basic protein (proMBP), PAPP-A.proMBP, shows only weak surface binding, probably because the adhesion site of PAPP-A is occupied by heparan sulfate, known to be covalently bound to proMBP. This hypothesis was further substantiated by demonstrating that heparinase treatment of PAPP-A.proMBP restores surface binding. We finally propose a model in which IGF bioactivity is regulated by reversible cell surface binding of PAPP-A, which in turn is regulated by proMBP.
Assuntos
Proteínas Sanguíneas/química , Proteína Plasmática A Associada à Gravidez/química , Ribonucleases , Sítios de Ligação , Proteínas Sanguíneas/metabolismo , Western Blotting , Adesão Celular , Linhagem Celular , Membrana Celular/metabolismo , Cisteína/química , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Proteínas Granulares de Eosinófilos , Citometria de Fluxo , Glicosaminoglicanos/farmacologia , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Modelos Biológicos , Plasmídeos/metabolismo , Proteína Plasmática A Associada à Gravidez/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , TransfecçãoRESUMO
Human pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor (IGF) binding protein-4 (IGFBP-4), causing a dramatic reduction in its affinity for IGF-I and -II. Through this mechanism, PAPP-A is a regulator of IGF bioactivity in several systems, including the human ovary and the cardiovascular system. PAPP-A belongs to the metzincin superfamily of zinc metalloproteinases, and is the founding member of a fifth metzincin family, the pappalysins. Herein, we first determined that PAPP-A cleaves IGFBP-4 at a single site (Met-135/Lys-136), and we analysed the influence of ionic strength, pH and zinc ion concentration on the cleavage reaction. Secondly, we sought to delineate the role of substrate residues in PAPP-A-mediated cleavage by the construction and analysis of 30 IGFBP-4 mutants in which various residues were replaced by alanine, by the analysis of eight mutants of IGFBP-5 (found recently to be a second PAPP-A substrate), and by cleavage analysis of synthetic peptides derived from IGFBP-4. Our data reveal a complex mode of substrate recognition and/or binding, pointing at important roles for several basic residues located up to 16 residues N-terminal to the scissile bond. An unexpected parallel can be drawn with an intracellular enzyme, the mitochondrial processing peptidase, that may help us to understand properties of the pappalysins. Further, proteinase-resistant variants of IGFBP-4 and -5, presented here, will be useful tools for the study of proteolysis in cell-based systems, and our finding that a synthetic peptide can be cleaved by PAPP-A provides the basis for development of quantitative assays for the investigation of PAPP-A enzyme kinetics.