RESUMO
Molecular chaperons or amyloid-associated proteins (AAPs) are deposited in vascular and parenchymal amyloid lesions in Alzheimer's disease (AD) and other amyloidoses. AAPs, such as apolipoprotein E (ApoE) or apolipoprotein J (ApoJ) have been strongly implicated in the pathogenesis of AD in vitro and in vivo. Furthermore the possession of the ApoE in4 allele is a well-studied risk factor for AD. In view of the similarities between AD and both familial British dementia (FBD) and familial Danish dementia (FDD), we investigated the presence of AAPs in these two diseases to understand better their role in the general process of amyloidogenesis. Immunohistochemistry for ApoE, ApoJ, serum amyloid P (SAP), alpha-1-antichymotrypsin, cystatin C, heparan sulphate proteoglycans, such as agrin, perlecan, syndecans, glypican-1 and for heparan sulphate glycosaminoglycan (HS GAG) side chains was carried out together with immunohistochemical preparations specific to the amyloid subunits. Significant or extensive staining for ApoE, ApoJ, agrin, glypican-1 and HS GAG side chains was found in both amyloid (fibrillar) and preamyloid (nonfibrillar) deposits in FBD and FDD. The remaining AAPs, including SAP, were predominantly found in amyloid lesions. Only very weak staining was present in a small proportion of the amyloid lesions using perlecan immunohistochemistry. These findings suggest that the deposition patterns of AAPs in FBD and FDD are mostly similar to those in AD. The presence of AAPs in the preamyloid lesions supports the notion that chaperon molecules may play a role in the early steps of fibrillogenesis.
Assuntos
Neuropatias Amiloides/patologia , Demência/genética , Demência/patologia , Chaperonas Moleculares/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/genética , Amiloide/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica , Proteoglicanas/metabolismo , Componente Amiloide P Sérico/metabolismo , Sindecanas , alfa 1-Antiquimotripsina/metabolismoRESUMO
Classic arguments sustaining the importance of amyloid in the pathogenesis of dementia are usually centered on amyloid beta (Abeta) and its role in neuronal loss characteristic of Alzheimer disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias, share many aspects of Alzheimer disease, including the presence of neurofibrillary tangles, parenchymal pre-amyloid and amyloid deposits, cerebral amyloid angiopathy, and a widespread inflammatory response. Both early-onset conditions are linked to specific mutations in the BRI2 gene, causing the generation of longer-than-normal protein products and the release of 2 de novo created peptides ABri and ADan, the main components of amyloid fibrils in these inherited dementias. Although the molecular mechanisms and signal transduction pathways elicited by the amyloid deposits and their relation to cognitive impairment remain to be clarified, new evidence indicates that, independent of the differences in their primary structures, Abeta, ABri, and ADan subunits are able to form morphologically compatible ion-channel-like structures and elicit single ion-channel currents in reconstituted lipid membranes. These findings reaffirm the notion that non-Abeta amyloidosis constitute suitable alternative models to study the role of amyloid deposition in the mechanism of neuronal cell death.
Assuntos
Amiloide/genética , Angiopatia Amiloide Cerebral/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Encéfalo/patologia , Angiopatia Amiloide Cerebral/patologia , Demência/patologia , Dinamarca , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana , Reino UnidoRESUMO
The importance of cerebral amyloid deposition in the mechanism of neurodegeneration is still debatable. Classic arguments are usually centered on amyloid beta(Abeta) and its role in the neuronal loss characteristic of Alzheimer's disease, the most common form of human cerebral amyloidosis. Two non-Abeta cerebral amyloidoses, familial British and Danish dementias (FBD and FDD), share many aspects of Alzheimer's disease, including the presence of neurofibrillary tangles, parenchymal preamyloid and amyloid deposits, cerebral amyloid angiopathy and a variety of amyloid-associated proteins and inflammatory components. Both early-onset conditions are linked to specific mutations at or near the stop codon of the chromosome 13 gene BRI2 that cause generation of longer-than-normal protein products. Furin-like processing of these longer precursors releases two de novo-created peptides, ABri and ADan, which deposit as amyloid fibrils in FBD and FDD, respectively. Due to the similar pathology generated by completely unrelated amyloid subunits, FBD and FDD, collectively referred to as chromosome 13 dementias, constitute alternative models for studying the role of amyloid deposition in the mechanism of neuronal cell death.
Assuntos
Amiloide/metabolismo , Cromossomos Humanos Par 13/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal , Amiloide/genética , Animais , Angiopatia Amiloide Cerebral/genética , Angiopatia Amiloide Cerebral/patologia , Demência/metabolismo , Demência/patologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Glicoproteínas de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutação , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
A point mutation of G to C at codon 693 of the amyloid-beta (Abeta) precursor protein gene results in Glu to Gln substitution at position 22 of the Abeta (AbetaQ22) associated with hereditary cerebrovascular amyloidosis with hemorrhage Dutch type. Factors that regulate AbetaQ22 levels in the central nervous system (CNS) are largely unknown. By using ventriculo-cisternal perfusion technique in guinea pigs, we demonstrated that clearance from the cerebrospinal fluid and transport from the CNS to blood of [(125)I]-AbetaQ22 (1 nM) were reduced by 36% and 52%, respectively, in comparison to the wild type Abeta(1-40) peptide. In contrast to significant uptake and transport of Abeta(1-40) across the brain capillaries and leptomeningeal vessels, AbetaQ22 was not taken up at these CNS vascular transport sites, which was associated with its 53% greater accumulation in the brain. The CNS clearance of Abeta(1-40) was half-saturated at 23.6 nM; AbetaQ22 had about 6.8-fold less affinity for the CNS efflux transporters and its elimination relied mainly on transport across the choroid plexus. Thus, the Dutch mutation impairs elimination of Abeta from brain by reducing its rapid transport across the blood-brain barrier and the vascular drainage pathways, which in turn may result in accumulation of the peptide around the blood vessels and in brain.
Assuntos
Doença de Alzheimer/genética , Substituição de Aminoácidos/genética , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Encéfalo/metabolismo , Circulação Cerebrovascular/genética , Códon/genética , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/líquido cefalorraquidiano , Sequência de Aminoácidos , Animais , Barreira Hematoencefálica , Plexo Corióideo/metabolismo , Feminino , Cobaias , Masculino , Dados de Sequência Molecular , Perfusão , Mutação Puntual , Transporte Proteico/genéticaRESUMO
The term cerebral amyloid angiopathy (CAA) refers to the specific deposition of amyloid fibrils in the walls of leptomeningeal and cortical arteries, arterioles and, although less frequently in capillaries and veins. It is commonly associated with Alzheimers disease, Down's syndrome and normal aging, as well as with a variety of familial conditions related to stroke and/or dementia: hereditary cerebral hemorrhage with amyloidosis of Icelandic type (HCHWA-I), various inherited disorders linked to Abeta mutants (including the Dutch variant of HCHWA), and the recently described chromosome 13 familial dementia in British and Danish kindreds. This review focuses on four different types of hereditary CAA, emphasizing the notion that CAA is not only related to stroke but also to neurodegeneration and dementia of the Alzheimer's type.
Assuntos
Angiopatia Amiloide Cerebral Familiar/etiologia , Demência/etiologia , Acidente Vascular Cerebral/etiologia , Doença de Alzheimer/etiologia , Peptídeos beta-Amiloides/genética , Angiopatia Amiloide Cerebral Familiar/genética , Angiopatia Amiloide Cerebral Familiar/patologia , Cistatina C , Cistatinas/genética , Dinamarca , Genes Dominantes , Humanos , Mutação , Reino UnidoRESUMO
Familial British dementia (FBD) is an early onset inherited disorder that, like familial Alzheimer's disease (FAD), is characterized by progressive dementia, amyloid deposition in the brain, and neurofibrillary degeneration of limbic neurons. The primary structure of the amyloid subunit (ABri) extracted from FBD brain tissues (Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) Nature 399, 776-781) is entirely different and unrelated to any previously known amyloid protein. Patients with FBD have a single nucleotide substitution at codon 267 in the BRI2 gene, resulting in an arginine replacing the stop codon and a longer open reading frame of 277 amino acids instead of 266. The ABri peptide comprises the 34 C-terminal residues of the mutated precursor ABriPP-277 and is generated via furin-like proteolytic processing. Here we report that carriers of the Stop-to-Arg mutation have a soluble form of the amyloid peptide (sABri) in the circulation with an estimated concentration in the range of 20 ng/ml, several fold higher than that of soluble Abeta. In addition, ABri species identical to those identified in the brain were also found as fibrillar components of amyloid deposits predominantly in the blood vessels of several peripheral tissues, including pancreas and myocardium. We hypothesize that the high concentration of the soluble de novo created amyloidogenic peptide and/or the insufficient tissue clearance are the main causative factors for the formation of amyloid deposits outside the brain. Thus, FBD constitutes the first documented cerebral amyloidosis associated with neurodegeneration and dementia in which the amyloid deposition is also systemic.
Assuntos
Amiloide/metabolismo , Demência/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Amiloide/genética , Encéfalo/patologia , Demência/genética , Demência/patologia , Predisposição Genética para Doença , Humanos , Glicoproteínas de Membrana , Proteínas de Membrana , Dados de Sequência Molecular , Fases de Leitura Aberta , Fragmentos de Peptídeos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The BRI3 gene is a member of the BRI gene family, made up of at least three different genes (BRI1-3). Previous studies established the cDNA sequence and structure of the human and mouse BRI1 and BRI2 genes and we recently reported that mutations in the BRI2 isoform, located on chromosome 13, are associated with dementia in humans. In the present work, we determine the complete cDNA sequence and genomic organization of the human BRI3 gene. BRI3 codes for a polypeptide of 267 amino acids, with a Mr of 30 KDa and a pI of 8.47. The amino acid sequence is 43.7% identical to the sequence of the human BRI2, and 38.3% identical to that of human BRI1, with the highest percentage of amino acid identity being concentrated on the C-terminal half of the molecules. In Northern blots, BRI3 cDNA hybridizes only one message of approximately 2.1 kilobases, which is predominantly present in the human brain. The BRI3 gene is localized on chromosome 2 and consists of six exons spanning more than 20 kb. Homology search of EST data banks retrieved a Caenorhabditis briggsae homolog of BRI, indicating that the BRI gene belongs to a strongly conserved gene family. These studies, aimed at characterizing the members of the BRI gene family, may provide valuable clues to the understanding of their normal function and how mutations in BRI2 can cause neurodegeneration and dementia similar to Alzheimer's disease.
Assuntos
Genes/genética , Proteínas de Membrana/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Caenorhabditis/genética , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Éxons , Feminino , Expressão Gênica , Humanos , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , Isoformas de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Distribuição TecidualRESUMO
Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.
Assuntos
Angiopatia Amiloide Cerebral/genética , Hemorragia Cerebral/genética , Cistatinas/química , Proteínas do Tecido Nervoso/química , Substituição de Aminoácidos , Animais , Angiopatia Amiloide Cerebral/metabolismo , Hemorragia Cerebral/metabolismo , Fenômenos Químicos , Físico-Química , Galinhas , Dicroísmo Circular , Códon/genética , Simulação por Computador , Cistatina C , Cistatinas/genética , Dimerização , Glutamina/química , Humanos , Leucina/química , Microscopia Eletrônica , Modelos Moleculares , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Especificidade da Espécie , Espectroscopia de Infravermelho com Transformada de Fourier , TransfecçãoRESUMO
Two hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with amyloid deposition in the central nervous system and neurodegeneration. The two amyloid proteins, ABri and ADan, are degradation products of the same precursor molecule BriPP bearing different genetic defects, namely a Stop-to-Arg mutation in FBD and a ten-nucleotide duplication-insertion immediately before the stop codon in FDD. Both de novo created amyloid peptides have the same length (34 amino acids) and the same post-translational modification (pyroglutamate) at their N-terminus. Neurofibrillary tangles containing the classical paired helical filaments as well as neuritic components in many instances co-localize with the amyloid deposits. In both disorders, the pattern of hyperphosphorylated tau immunoreactivity is almost indistinguishable from that seen in Alzheimer's disease. These issues argue for the primary importance of the amyloid deposits in the mechanism(s) of neuronal cell loss. We propose FBD and FDD, the chromosome 13 dementia syndromes, as models to study the molecular basis of neurofibrillary degeneration, cell death and amyloid formation in the brain.
Assuntos
Cromossomos Humanos Par 13/genética , Demência/genética , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Sequência de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Demência/metabolismo , Demência/patologia , Dinamarca , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Humanos , Modelos Genéticos , Modelos Neurológicos , Dados de Sequência Molecular , Mutação , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Síndrome , Reino UnidoRESUMO
Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.
Assuntos
Amiloide/genética , Doença de Gerstmann-Straussler-Scheinker/etiologia , Fragmentos de Peptídeos/isolamento & purificação , Príons/patogenicidade , Precursores de Proteínas/genética , Adulto , Alelos , Córtex Cerebral/patologia , Doença de Gerstmann-Straussler-Scheinker/genética , Heterozigoto , Humanos , Masculino , Metionina/genética , Proteínas Priônicas , Príons/isolamento & purificação , Análise de Sequência de Proteína , Síndrome , Valina/genéticaRESUMO
Apolipoprotein J (clusterin) is a ubiquitous multifunctional glycoprotein capable of interacting with a broad spectrum of molecules. In pathological conditions, it is an amyloid associated protein, co-localizing with fibrillar deposits in systemic and localized amyloid disorders. In Alzheimer's disease, the most frequent form of amyloidosis in humans and the major cause of dementia in the elderly, apoJ is present in amyloid plaques and cerebrovascular deposits but is rarely seen in NFT-containing neurons. ApoJ expression is up-regulated in a wide variety of insults and may represent a defense response against local damage to neurons. Four different mechanisms of action could be postulated to explain the role of apoJ as a neuroprotectant during cellular stress: (1) function as an anti-apoptotic signal, (2) protection against oxidative stress, (3) inhibition of the membrane attack complex of complement proteins locally activated as a result of inflammation, and (4) binding to hydrophobic regions of partially unfolded, stressed proteins, and therefore avoiding aggregation in a chaperone-like manner. This review focuses on the association of apoJ in biological fluids with Alzheimer's soluble Abeta. This interaction prevents Abeta aggregation and fibrillization and modulates its blood-brain barrier transport at the cerebrovascular endothelium.
Assuntos
Doença de Alzheimer/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares , Proteínas do Tecido Nervoso/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Amiloidose/metabolismo , Barreira Hematoencefálica , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Clusterina , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Glicoproteínas/líquido cefalorraquidiano , Humanos , Placa Amiloide/metabolismoRESUMO
Amyloid beta protein deposition in cortical and leptomeningeal vessels, causing the most common type of cerebral amyloid angiopathy, is found in sporadic and familial Alzheimer's disease (AD) and is the principal feature in the hereditary cerebral hemorrhage with amyloidosis, Dutch type. The presence of the Apolipopriotein E (APOE)-epsilon4 allele has been implicated as a risk factor for AD and the development of cerebral amyloid angiopathy in AD. We report clinical, pathological and biochemical studies on two APOE-epsilon4 homozygous subjects, who had senile dementia and whose main neuropathological feature was a severe and diffuse amyloid angiopathy associated with perivascular tau neurofibrillary pathology. Amyloid beta protein and ApoE immunoreactivity were observed in leptomeningeal vessels as well as in medium-sized and small vessels and capillaries in the parenchyma of the neocortex, hippocampus, thalamus, cerebellum, midbrain, pons, and medulla. The predominant peptide form of amyloid beta protein was that terminating at residue Val40, as determined by immunohistochemistry, amino acid sequence and mass spectrometry analysis. A crown of tau-immunopositive cell processes was consistently present around blood vessels. DNA sequence analysis of the Amyloid Precursor Protein gene and Presenilin-1 (PS-1) gene revealed no mutations. In these APOE-epsilon4 homozygous patients, the pathological process differed from that typically seen in AD in that they showed a heavy burden of perivascular tau-immunopositive cell processes associated with severe amyloid beta protein angiopathy, neurofibrillary tangles, some cortical Lewy bodies and an absence of neuritic plaques. These cases emphasize the concept that tau deposits may be pathogenetically related to amyloid beta protein deposition.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Angiopatia Amiloide Cerebral/metabolismo , Angiopatia Amiloide Cerebral/patologia , Proteínas tau/metabolismo , Idoso , Alelos , Doença de Alzheimer/genética , Apolipoproteína E4 , Apolipoproteínas E/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/fisiopatologia , Angiopatia Amiloide Cerebral/genética , Progressão da Doença , Genótipo , Homozigoto , Humanos , Masculino , Neurônios/metabolismo , Neurônios/patologia , Testes Neuropsicológicos , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Desempenho PsicomotorRESUMO
Amyloidosis is a disorder of protein conformation leading to aggregation. The term defines a diverse group of proteins normally present in body fluids as soluble precursors that can be deposited as insoluble amyloid fibrils in different tissues producing organ dysfunction and cell death. These fibrils are composed of self-assembled, low molecular weight mass peptides adopting beta-pleated sheet structure, the conformation responsible for their physicochemical properties and tinctoreal characteristics. So far, 20 different proteins have been identified as subunits of amyloid fibrils (Westermark et al., 1999). Collectively, they are products of normal genes; however, several amyloid precursors contain abnormal amino acid substitutions that can impose an unusual potential for self-aggregation. The molecular mass of the amyloid peptides is within the 4 to 30-kDa range, with heterogeneity at the amino- and carboxyl-terminal portions found in most amyloid proteins. Increased levels of amyloid precursors, either in the circulation or locally in sites of deposition, are usually the result of overexpression or defective clearance, or both. Of the 20 amyloid proteins identified, few of them are known to cause amyloid deposition in the central nervous system, which in turn results in cognitive deficits, dementia, stroke, cerebellar and extrapyramidal signs, or a combination of them.
Assuntos
Angiopatia Amiloide Cerebral/genética , Demência/etiologia , Precursor de Proteína beta-Amiloide/metabolismo , Angiopatia Amiloide Cerebral/complicações , Angiopatia Amiloide Cerebral/fisiopatologia , Hemorragia Cerebral/etiologia , Transtornos Cognitivos/etiologia , Transtornos Cognitivos/fisiopatologia , Demência/fisiopatologia , HumanosRESUMO
The inheritance of the apolipoprotein E (apoE) epsilon4 allele is a prevailing risk factor for sporadic and familial Alzheimer's disease (AD). ApoE isoforms bind directly to Alzheimer's amyloid beta (Abeta) peptides both in vitro and in vivo. Recent studies suggest that association of apoE with lipids may modulate its interaction with Abeta. We examined the binding of lipid-associated and delipidated apoE3 and apoE4 isoforms to Abeta utilizing a solid-phase binding assay and estimated the dissociation constants for the interaction of various apoE and Abeta species. Using native apoE isoforms from stably transfected RAW 264 and human embryonic kidney 293 cells, apoE3 had greater affinity than apoE4 for both Abeta1-40 and Abeta1-42. Delipidation of apoE decreased its affinity for Abeta peptides by 5-10-fold and abolished the isoform-specificity. Conversely, incorporation of apoE isoforms produced by baculovirus-infected Sf9 cells into reconstituted human high-density-lipoprotein lipoparticles restored the affinity values for Abeta peptides and resulted in preferential binding of apoE3. The data demonstrate that native lipid-associated apoE3 binds to Abeta peptides with 2-3-fold higher affinity than lipid-associated apoE4. Since the isoforms' binding efficiency correlate inversely with the risk of developing late-onset AD, the results suggest a possible involvement of apoE3 in the clearance or routing out of Abeta from the central nervous system as one of the mechanisms underlying the pathology of the disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/metabolismo , Animais , Apolipoproteína E3 , Apolipoproteína E4 , Linhagem Celular , Humanos , Rim , Cinética , Macrófagos , Camundongos , Isoformas de Proteínas/metabolismo , Spodoptera , TransfecçãoRESUMO
Cerebral amyloid angiopathy is commonly associated with normal aging and Alzheimer's disease and it is also the principal feature of hereditary cerebral hemorrhage with amyloidosis Dutch type, a familial condition associated to a point mutation G to C at codon 693 of the amyloid beta (Abeta) precursor protein gene resulting in a Glu to Gln substitution at position 22 of the Abeta (E22Q). The patients carrying the AbetaE22Q variant usually present with lobar cerebral hemorrhages before 50 years of age. A different mutation described in several members of three Italian kindred who presented with recurrent hemorrhagic strokes late in life, between 60 and 70 years of age, also associated with extensive cerebrovascular amyloid deposition has been found at the same position 22, this time resulting in a Glu to Lys substitution (E22K). We have compared the secondary structure, aggregation, and fibrillization properties of the two Abeta40 variants and the wild type peptide. Using flow cytometry analysis after staining with propidium iodide and annexin V, we also evaluated the cytotoxic effects of the peptides on human cerebral endothelial cells in culture. Under the conditions tested, the E22Q peptide exhibited the highest content of beta-sheet conformation and the fastest aggregation/fibrillization properties. The Dutch variant also induced apoptosis of cerebral endothelial cells at a concentration of 25 micrometer, whereas the wild type Abeta and the E22K mutant had no effect. The data suggest that different amino acids at position 22 confer distinct structural properties to the peptides that appear to influence the onset and aggressiveness of the disease rather than the phenotype.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Apoptose/genética , Encéfalo/irrigação sanguínea , Códon , Endotélio Vascular/patologia , Mutação , Idoso , Idoso de 80 Anos ou mais , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Encéfalo/patologia , Dicroísmo Circular , Endotélio Vascular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Conformação ProteicaRESUMO
Typical of sporadic inclusion body myositis muscle biopsies are vacuolated muscle fibers containing intracellular amyloid deposits and accumulations of "Alzheimer-characteristic" proteins. There is no muscle blood vessel or cardiac amyloidosis. We report on a 70-year-old African-American man homozygous for the transthyretin Val122Ile allele who has both sporadic inclusion body myositis and cardiac amyloidosis. His unique pathological features included transthyretin immunoreactivity in prominent muscle blood vessel amyloid and congophilic amyloid deposits within vacuolated muscle fibers.
Assuntos
Amiloidose/patologia , Corpos de Inclusão/patologia , Miocárdio/patologia , Miosite/patologia , Mutação Puntual , Pré-Albumina/genética , Idoso , Alelos , Biópsia , Vasos Coronários , Homozigoto , Humanos , Masculino , Microscopia Eletrônica , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/patologia , Miosite/genética , Condução Nervosa , Polimorfismo GenéticoRESUMO
The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.
Assuntos
Endopeptidases/metabolismo , Fasciola hepatica/enzimologia , Fragmentos de Imunoglobulinas/análise , Imunoglobulina G/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Fasciola hepatica/imunologia , Humanos , Concentração de Íons de Hidrogênio , Imunoglobulina G/química , Imunoglobulina G/classificaçãoRESUMO
Disorders of immunoglobulin (Ig) synthesis that occur in malignant plasma-cell proliferation may result in either granular (LCDD) or fibrillar (AL) tissue deposition of light-chain monoclonal components. The structural features that govern the transition from soluble polypeptides to either fibrillar or granular conformational states remain undefined. Among the many factors presumed to play a role in these transitions the net charge of the molecule has been associated with folding conformation changes. The majority of the proteins involved in AL amyloidosis show acidic isoelectric points (pI 3.8-5.2), whereas most L chains with basic pIs deposit in granular patterns. In our studies a 12 kD VkappaIII fragment was purified as the main component of the fibrils isolated from myocardium and adipose tissue of the pericardium obtained post-mortem from an individual with systemic AL amyloidosis. An apparently identical 12 kD VL fragment with the same N-terminal sequence constituted the BJ protein present in the urine. This urinary protein exhibited strikingly cathodic electrophoretic mobility on agarose gels and lacked retention by anionic exchange chromatography matrices, indicative of a highly basic pI (>10). When it was subjected to in vitro fibril-formation experiments, the BJ protein adopted a fibrillar conformation only at acidic pHs, remaining aggregated but not fibrillar at physiological pH. The data indicate that a specific tissue deposition pattern involves not only structural properties of the protein but rather more complex mechanisms in which acidic micro-environments may contribute to the stabilization of amyloidogenic conformations.
Assuntos
Proteína de Bence Jones/metabolismo , Mieloma Múltiplo/metabolismo , Idoso , Amiloide/química , Amiloidose/metabolismo , Cardiomegalia/metabolismo , Evolução Fatal , Humanos , Concentração de Íons de Hidrogênio , Cadeias kappa de Imunoglobulina/metabolismo , MasculinoRESUMO
Light chain deposition disease (LCDD) and light chain amyloidosis (AL) are disorders of monoclonal immunoglobulin deposition in which normally soluble serum precursors form insoluble deposits in tissues. A common feature in both is the clonal proliferation of B-cells that produce pathogenic light chains. However, the deposits in LCDD differ from those in AL in that they are ultrastructurally granular rather than fibrillar and do not bind Congo red or colocalize with amyloid P component or apolipoprotein E. The reason(s) for their differences are unknown but are likely multifactorial and related to their protein conformation and their interaction with other molecules and tissue factors in the microenvironment. Knowledge of the primary structure of the light chains in LCDD is very limited. In the present study two new kappa(1) light chains from patients with LCDD are described and compared to seven other reported kappa-LCDD proteins. The N-terminal amino acid sequences of light chain GLA extracted from the renal biopsy and light chain CHO from myocardial tissue were each identical to the respective light chains isolated from the urines and to the V-region amino acid sequences translated from the cloned cDNAs obtained from bone marrow cells. The germline V-region sequences, determined from the genomic DNA in both and in MCM, a previously reported kappa(1) LCDD light chain, were identical and related to the L12a germline gene. The expressed light chains in all three exhibit amino acid substitutions that arise from somatic mutation and result in increased hydrophobicity with the potential for protein destabilization and disordered conformation.