RESUMO
Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments-assessing ghrelin ability to inhibit calcium channels-and in vivo studies-evaluating ghrelin orexigenic effects-indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone.
Assuntos
Grelina/metabolismo , Albumina Sérica/metabolismo , Sequência de Aminoácidos , Animais , Calorimetria , Cromatografia em Gel , Grelina/química , Humanos , Camundongos , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Insulin-degrading enzyme (IDE) is central to the turnover of insulin and degrades amyloid beta (Abeta) in the mammalian brain. Biochemical and genetic data support the notion that IDE may play a role in late onset Alzheimer disease (AD), and recent studies suggest an association between AD and diabetes mellitus type 2. Here we show that a natively folded recombinant IDE was capable of forming a stable complex with Abeta that resisted dissociation after treatment with strong denaturants. This interaction was also observed with rat brain IDE and detected in an SDS-soluble fraction from AD cortical tissue. Abeta sequence 17-27, known to be crucial in amyloid assembly, was sufficient to form a stable complex with IDE. Monomeric as opposed to aggregated Abeta was competent to associate irreversibly with IDE following a very slow kinetics (t(1/2) approximately 45 min). Partial denaturation of IDE as well as preincubation with a 10-fold molar excess of insulin prevented complex formation, suggesting that the irreversible interaction of Abeta takes place with at least part of the substrate binding site of the protease. Limited proteolysis showed that Abeta remained bound to a approximately 25-kDa N-terminal fragment of IDE in an SDS-resistant manner. Mass spectrometry after in gel digestion of the IDE .Abeta complex showed that peptides derived from the region that includes the catalytic site of IDE were recovered with Abeta. Taken together, these results are suggestive of an unprecedented mechanism of conformation-dependent substrate binding that may perturb Abeta clearance, insulin turnover, and promote AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Insulisina/química , Doença de Alzheimer/metabolismo , Animais , Sítios de Ligação , Encéfalo/metabolismo , Domínio Catalítico , Humanos , Cinética , Espectrometria de Massas , Modelos Biológicos , Ligação Proteica , Ratos , Espalhamento de Radiação , Especificidade por SubstratoRESUMO
Familial British dementia (FBD) and familial Danish dementia (FDD) are autosomal dominant disorders characterized by cerebrovascular and parenchymal amyloid deposition and neurofibrillary degeneration. In both conditions, the genetic defects cause the loss of the normal stop codon in the precursor BRI, generating novel 34-residue peptides named ABri and ADan in FBD and FDD, respectively. ABri and ADan show a strong tendency to aggregate into non-fibrillar and fibrillar structures at neutral pH and this property seems to be directly related to neurotoxicity. Here we report that a recombinant insulin-degrading enzyme (rIDE) was capable of degrading monomeric ABri and ADan in vitro more efficiently than oligomeric species. These peptides showed high beta-structure content and were more resistant to proteolysis as compared to the BRI wild-type product of 23 amino acids. Specific sites of cleavage within the C-terminal pathogenic extensions raise the possibility that proteolysis of monomeric soluble precursors by IDE may delay ABri and ADan aggregation in vivo.
Assuntos
Amiloide/genética , Amiloide/metabolismo , Demência/genética , Demência/metabolismo , Insulisina/metabolismo , Mutação , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Amiloide/química , Animais , Sítios de Ligação/genética , Cisteína/química , Dinamarca , Genes Dominantes , Humanos , Técnicas In Vitro , Insulisina/genética , Glicoproteínas de Membrana , Proteínas de Membrana , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Reino UnidoRESUMO
The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses.
Assuntos
Peptídeos beta-Amiloides/química , Encéfalo/metabolismo , Angiopatia Amiloide Cerebral/genética , Insulisina/fisiologia , Adulto , Animais , Western Blotting , Encéfalo/patologia , Angiopatia Amiloide Cerebral/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Imunoprecipitação , Insulina/metabolismo , Insulisina/química , Cinética , Espectrometria de Massas , Microcirculação , Microscopia de Fluorescência , Pessoa de Meia-Idade , Mutação , Peptídeos/química , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.
Assuntos
Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Insulisina/metabolismo , Animais , Hemorragia Cerebral/enzimologia , Hemorragia Cerebral/patologia , Demência/genética , Dimerização , Saúde da Família , Variação Genética , Humanos , Fragmentos de Peptídeos/análise , Ratos , Proteínas Recombinantes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Acidente Vascular Cerebral/genéticaRESUMO
The deposition of 4-kDa amyloid beta peptide in the brain is a prominent feature of several human diseases. Such process is heterogeneous in terms of causative factors, biochemical phenotype, localization and clinical manifestations. Amyloid beta accumulates in the neuropil or within the walls of cerebral vessels, and associates with dementia or stroke, both hereditary and sporadic. Amyloid beta is normally released by cells as soluble monomeric-dimeric species yet, under pathological conditions, it self-aggregates as soluble oligomers or insoluble fibrils that may be toxic to neurons and vascular cells. Lowering amyloid beta levels may be achieved by inhibiting its generation from the amyloid beta-precursor protein or by promoting its clearance by transport or degradation. We will summarize recent findings on brain proteases capable of degrading amyloid beta with a special focus on those enzymes for which there is genetic, transgenic or biochemical evidence suggesting that they may participate in the proteolysis of amyloid beta in vivo. We will also put in perspective their possible utilization as therapeutic agents in amyloid beta diseases.