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1.
Int J Mol Sci ; 25(16)2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39201619

RESUMO

Aggregation of α-synuclein (αSyn) and its accumulation as Lewy bodies play a central role in the pathogenesis of Parkinson's disease (PD). However, the mechanism by which αSyn aggregates in the brain remains unclear. Biochemical studies have demonstrated that αSyn interacts with lipids, and these interactions affect the aggregation process of αSyn. Furthermore, genetic studies have identified mutations in lipid metabolism-associated genes such as glucocerebrosidase 1 (GBA1) and synaptojanin 1 (SYNJ1) in sporadic and familial forms of PD, respectively. In this review, we focus on the role of lipids in triggering αSyn aggregation in the pathogenesis of PD and propose the possibility of modulating the interaction of lipids with αSyn as a potential therapy for PD.


Assuntos
Metabolismo dos Lipídeos , Doença de Parkinson , alfa-Sinucleína , Humanos , Doença de Parkinson/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/etiologia , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genética , Animais , Lipídeos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Mutação
2.
J Biol Chem ; 297(5): 101284, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34624313

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid-liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.


Assuntos
Esclerose Lateral Amiotrófica/genética , Quadruplex G , Mutação de Sentido Incorreto , Proteína FUS de Ligação a RNA , Substituição de Aminoácidos , Humanos , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/genética
3.
Brain ; 143(6): 1811-1825, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32436573

RESUMO

The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal ß-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic ß-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.


Assuntos
Arginina/metabolismo , Arginina/farmacologia , Peptídeos/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Drosophila/metabolismo , Feminino , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Doença de Huntington/genética , Masculino , Camundongos , Camundongos Endogâmicos , Chaperonas Moleculares/genética , Peptídeos/genética , Agregação Patológica de Proteínas , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Ataxias Espinocerebelares/genética
4.
Pathol Int ; 66(4): 193-201, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26969800

RESUMO

Amyloidosis refers to a group of diseases with amyloid fibrils deposited in various organs and is classified into more than 30 diseases in humans based on the kind of amyloid protein. In order to elucidate the molecular pathogenesis of human amyloidosis, we studied the molecular mechanism of amyloid fibril formation in vitro. We first developed a novel fluorometric method to determine amyloid fibrils in vitro based on the unique characteristics of thioflavin T. We next proposed a nucleation-dependent polymerization model to explain the general mechanism of amyloid fibril formation in vitro. Based on this model, we characterized the biological molecular interactions that promote or inhibit amyloid fibril formation in vitro and developed models of pathological molecular environment for inducing human ß2-microglobulin-related amyloidosis in long-term hemodialysis patients. We also proposed a novel and attractive cytotoxic mechanism of ß2-microglobulin amyloid fibrils, that is, the disruption of endosomal/lysosomal membranes by endocytosed amyloid fibrils. These findings may be useful to elucidate the molecular pathogenesis of other kinds of human amyloidosis.


Assuntos
Amiloide/metabolismo , Amiloidose/patologia , Modelos Moleculares , Microglobulina beta-2/metabolismo , Amiloide/análise , Amiloide/ultraestrutura , Amiloidose/classificação , Amiloidose/metabolismo , Benzotiazóis , Fluorometria , Humanos , Polimerização , Tiazóis
5.
Biochim Biophys Acta ; 1834(8): 1624-31, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23608949

RESUMO

Cerebral amyloid angiopathy is caused by deposition of the amyloid ß-peptide which consists of mainly 39-40 residues to the cortical and leptomeningeal vessel walls. There are no definite in vitro systems to support the hypothesis that the vascular basement membrane may act as a scaffold of amyloid ß-peptide carried by perivascular drainage flow and accelerate its amyloid fibril formation in vivo. We previously reported the critical roles of interfaces and agitation on the nucleation of amyloid fibrils at low concentrations of amyloid ß-peptide monomers. Here, we reproduced the perivascular drainage flow in vitro by using N-hydroxysuccinimide-Sepharose 4 Fast flow beads as an inert stirrer in air-free wells rotated at 1rpm. We then reproduced the basement membranes in the media of cerebral arteries in vitro by conjugating Matrigel and other proteins on the surface of Sepharose beads. These beads were incubated with 5µM amyloid ß(1-40) at 37°C without air, where amyloid ß(1-40) alone does not form amyloid fibrils. Using the initiation time of fibril growth kinetics (i.e., the lag time of fibril growth during which nuclei, on-pathway oligomers and protofibrils are successively formed) as a parameter of the efficiency of biological molecules to induce amyloid fibril formation, we found that basement membrane components including Matrigel, laminin, fibronectin, collagen type IV and fibrinogen accelerate the initiation of amyloid ß-peptide fibril growth in vitro. These data support the essential role of vascular basement membranes in the development of cerebral amyloid angiopathy.


Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Membrana Basal/metabolismo , Angiopatia Amiloide Cerebral , Matriz Extracelular/metabolismo , Colágeno/metabolismo , Colágeno Tipo IV/metabolismo , Combinação de Medicamentos , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Humanos , Técnicas In Vitro , Laminina/metabolismo , Modelos Moleculares , Proteoglicanas/metabolismo , Succinimidas/química
6.
Elife ; 122023 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-37461319

RESUMO

Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Demência Frontotemporal/patologia , RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Drosophila/genética
7.
J Biol Chem ; 286(12): 10856-63, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21300800

RESUMO

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of ß(2)-microglobulin K3 fragments and amyloid ß by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.


Assuntos
Amiloide/química , Proteínas da Matriz Extracelular/química , Lasers , Peptídeos/química , Tiazóis/química , Fator de Crescimento Transformador beta/química , Amiloide/metabolismo , Benzotiazóis , Distrofias Hereditárias da Córnea/metabolismo , Distrofias Hereditárias da Córnea/terapia , Proteínas da Matriz Extracelular/metabolismo , Humanos , Terapia a Laser , Microscopia de Fluorescência , Peptídeos/metabolismo , Fator de Crescimento Transformador beta/metabolismo
8.
J Biol Chem ; 286(11): 9668-76, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21216953

RESUMO

The relationship between various amyloidoses and chaperones is gathering attention. In patients with dialysis-related amyloidosis, α(2)-macroglobulin (α2M), an extracellular chaperone, forms a complex with ß(2)-microglobulin (ß2-m), a major component of amyloid fibrils, but the molecular mechanisms and biological implications of the complex formation remain unclear. Here, we found that α2M substoichiometrically inhibited the ß2-m fibril formation at a neutral pH in the presence of SDS, a model for anionic lipids. Binding analysis showed that the binding affinity between α2M and ß2-m in the presence of SDS was higher than that in the absence of SDS. Importantly, SDS dissociated tetrameric α2M into dimers with increased surface hydrophobicity. Western blot analysis revealed that both tetrameric and dimeric α2M interacted with SDS-denatured ß2-m. At a physiologically relevant acidic pH and in the presence of heparin, α2M was also dissociated into dimers, and both tetrameric and dimeric α2M interacted with ß2-m, resulting in the inhibition of fibril growth reaction. These results suggest that under conditions where native ß2-m is denatured, tetrameric α2M is also converted to dimeric form with exposed hydrophobic surfaces to favor the hydrophobic interaction with denatured ß2-m, thus dimeric α2M as well as tetrameric α2M may play an important role in controlling ß2-m amyloid fibril formation.


Assuntos
alfa-Globulinas/química , Amiloide/química , Chaperonas Moleculares/química , Complexos Multiproteicos/química , Multimerização Proteica , Microglobulina beta-2/química , alfa-Globulinas/metabolismo , Amiloide/metabolismo , Amiloidose/metabolismo , Animais , Estrona/análogos & derivados , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Desnaturação Proteica , Estrutura Quaternária de Proteína , Microglobulina beta-2/metabolismo
9.
J Biol Chem ; 285(25): 19660-7, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20406822

RESUMO

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.


Assuntos
Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/química , Amiloide/química , Lasers , Doença de Alzheimer/metabolismo , Benzotiazóis , Humanos , Cinética , Modelos Biológicos , Fragmentos de Peptídeos/química , Peptídeos/química , Fotoquimioterapia/métodos , Dobramento de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tiazóis/química , Ultracentrifugação , Microglobulina beta-2/química
10.
Biochim Biophys Acta ; 1804(4): 986-95, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20100601

RESUMO

Amyloid deposits are pathological hallmarks of various neurodegenerative diseases including Alzheimer's disease (AD), where amyloid beta-peptide (Abeta) polymerizes into amyloid fibrils by a nucleation-dependent polymerization mechanism. The biological membranes or other interfaces as well as the convection of the extracellular fluids in the brain may influence Abeta amyloid fibril formation in vivo. Here, we examined the polymerization kinetics of 2.5, 5, 10 and 20 microM Abeta in the presence or absence of air-water interface (AWI) using fluorescence spectroscopy and fluorescence microscopy with the amyloid specific dye, thioflavin T. When the solutions were incubated with AWI and in quiescence, amyloid fibril formation was observed at all Abeta concentrations examined. In contrast, when incubated without AWI, amyloid fibril formation was observed only at higher Abeta concentrations (10 and 20 microM). Importantly, when the 5 microM Abeta solution was incubated with AWI, a ThT-reactive film was first observed at AWI without any other ThT-reactive aggregates in the bulk. When 5 microM Abeta solutions were voltexed or rotated with AWI, amyloid fibril formation was considerably accelerated, where a ThT-reactive film was first observed at AWI before ThT-reactive aggregates were observed throughout the mixture. When 5 microM Abeta solutions containing a polypropylene disc were rotated without AWI, amyloid fibril formation was also considerably accelerated, where fine ThT-reactive aggregates were first found attached at the edge of the disc. These results indicate the critical roles of interfaces and agitation for amyloid fibril formation. Furthermore, elimination of AWI may be essential for proper evaluation of the roles of various biological molecules in the amyloid formation studies in vitro.


Assuntos
Peptídeos beta-Amiloides/química , Amiloide/química , Fragmentos de Peptídeos/química , Ar , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Amiloide/ultraestrutura , Benzotiazóis , Química Encefálica , Corantes Fluorescentes , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Soluções , Espectrometria de Fluorescência , Tiazóis , Água
11.
ACS Nano ; 13(8): 8766-8783, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31310506

RESUMO

Complex amyloid aggregation of amyloid-ß (1-40) (Aß1-40) in terms of monomer structures has not been fully understood. Herein, we report the microscopic mechanism and pathways of Aß1-40 aggregation with macroscopic viewpoints through tuning its initial structure and solubility. Partial helical structures of Aß1-40 induced by low solvent polarity accelerated cytotoxic Aß1-40 amyloid fibrillation, while predominantly helical folds did not aggregate. Changes in the solvent polarity caused a rapid formation of ß-structure-rich protofibrils or oligomers via aggregation-prone helical structures. Modulation of the pH and salt concentration transformed oligomers to protofibrils, which proceeded to amyloid formation. We reveal diverse molecular mechanisms underlying Aß1-40 aggregation with conceptual energy diagrams and propose that aggregation-prone partial helical structures are key to inducing amyloidogenesis. We demonstrate that context-dependent protein aggregation is comprehensively understood using the macroscopic phase diagram, which provides general insights into differentiation of amyloid formation and phase separation from unfolded and folded structures.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/ultraestrutura , Fragmentos de Peptídeos/ultraestrutura , Agregação Patológica de Proteínas/genética , Conformação Proteica em alfa-Hélice/genética , Doença de Alzheimer/patologia , Amiloide/química , Amiloide/genética , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de Peptídeos/química , Conformação Proteica em Folha beta/genética , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Solubilidade
12.
Sci Rep ; 6: 29077, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27380955

RESUMO

C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer's amyloid ß (Aß) (1-40) and on that from D76N ß2-microglobulin (ß2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aß(1-40) and D76N ß2-m fibril formation in a Ca(2+)-independent manner. CRP and SAP interacted with fresh and aggregated Aß(1-40) and D76N ß2-m on the fibril-forming pathway. Interestingly, in the presence of Ca(2+), SAP first inhibited, then significantly accelerated D76N ß2-m fibril formation. Electron microscopically, the surface of the D76N ß2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed.


Assuntos
Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Amiloidose/sangue , Proteína C-Reativa/metabolismo , Componente Amiloide P Sérico/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Amiloide/sangue , Peptídeos beta-Amiloides/genética , Amiloidose/genética , Amiloidose/patologia , Proteína C-Reativa/genética , Cálcio/metabolismo , Glutationa Transferase/sangue , Glutationa Transferase/genética , Humanos , Imunidade Inata/genética , Mutação de Sentido Incorreto , Agregação Patológica de Proteínas/sangue , Agregação Patológica de Proteínas/genética , Dobramento de Proteína , Componente Amiloide P Sérico/genética , Microglobulina beta-2/sangue , Microglobulina beta-2/genética
13.
PLoS One ; 10(9): e0139330, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26421922

RESUMO

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, ß2-microglobulin (ß2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of ß2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with ß2-m monomers and vehicle buffer exhibited no morphological changes. As compared to ß2-m monomers and vehicle buffer, ß2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. ß2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed ß2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.


Assuntos
Amiloide/metabolismo , Amiloidose/patologia , Apoptose/efeitos dos fármacos , Fibroblastos/patologia , Membranas Intracelulares/efeitos dos fármacos , Diálise Renal/efeitos adversos , Microglobulina beta-2/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Endocitose , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fibroblastos/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Necrose/metabolismo , Necrose/patologia , Coelhos , Proteínas Recombinantes/metabolismo
16.
J Biol Chem ; 284(2): 1009-17, 2009 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-19010783

RESUMO

To understand the mechanism by which amyloid fibrils form, we have been making real-time observations of the growth of individual fibrils, using total internal fluorescence microscopy combined with an amyloid-specific fluorescence dye, thioflavin T (ThT). At neutral pH, irradiation at 442 nm with a laser beam to excite ThT inhibited the fibril growth of beta(2)-microglobulin (beta2-m), a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. Examination with a 22-residue K3 fragment of beta2-m showed that the inhibition of fibril growth and moreover the destruction of preformed fibrils were coupled with the excitation of ThT. Several pieces of evidence suggest that the excited ThT transfers energy to ground state molecular oxygen, producing active oxygen, which causes various types of chemical modifications. The results imply a novel strategy for preventing the deposition of amyloid fibrils and for destroying preformed amyloid deposits.


Assuntos
Amiloide/metabolismo , Microglobulina beta-2/metabolismo , Amiloide/ultraestrutura , Humanos , Lasers , Microscopia de Força Atômica , Soluções , Fatores de Tempo , Microglobulina beta-2/genética , Microglobulina beta-2/ultraestrutura
17.
J Mol Biol ; 376(1): 258-68, 2008 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-18155723

RESUMO

Dialysis-related amyloidosis frequently develops in patients undergoing long-term hemodialysis, in which the major component of fibrils is beta(2)-microglobulin (beta2-m). To prevent the disease, it is important to stop the formation of fibrils. beta2-m has one disulfide bond, which stabilizes the native structure, and amyloid fibrils. Here, the effects of reductants (i.e., dithiothreitol and cysteine) on the formation of beta2-m amyloid fibrils were examined at neutral pH. Fibrils were generated by three methods: seed-dependent, ultrasonication-induced, and salt-and-heat-induced fibrillation. Thioflavin T fluorescence, electron microscopy, and far-UV circular dichroism revealed that the addition of reductants significantly inhibits seed-dependent and ultrasonication-induced fibrillation. For salt-and-heat-induced fibrillation, where the solution of beta2-m was strongly agitated, formation of amyloid fibrils was markedly reduced in the presence of reductants, although a small number of fibrils formed even after the reduction of the disulfide bond. The results suggest that reductants such as cysteine and dithiothreitol would be useful for preventing the formation of beta2-m amyloid fibrils under physiological conditions.


Assuntos
Amiloide/antagonistas & inibidores , Substâncias Redutoras/farmacologia , Compostos de Sulfidrila/metabolismo , Compostos de Sulfidrila/farmacologia , Microglobulina beta-2/metabolismo , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Espectrometria de Fluorescência
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