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2.
Antioxidants (Basel) ; 10(9)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34573102

RESUMO

In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1-EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.

3.
Neurochem Int ; 140: 104806, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32758584

RESUMO

Human cystatin C (CysC) is an amyloid forming protein involved in the hereditary cerebral amyloid angiopathy (HCCAA) that affects arteries in the brain and the peripheral nervous system. In this study we measured the influence of several substances on human CysC aggregation and amyloid fibril formation, induced at pH 4 in vitro. The effect of three polyphenols: resveratrol, quercetin and curcumin and of two antioxidants: vitamin C (VitC) and N-acetyl-L-cysteine (NAC) was explored as well as the effect of sulphoraphane (SF) and α-lipoic acid (AL). The formation of amyloid fibrils was followed by Thioflavin T (ThT) fluorescence and by transmission electron microscopy (TEM). Effects on the length of the lag phase were revealed by following the increase of ThT fluorescence intensity with time. The amount and morphology of fibrils in comparison to prefibrillar aggregates and globular oligomers were evaluated by TEM at the plateau stage of the reaction. Thermal stabilization of the CysC monomer by the small compounds was measured by differential scanning fluorimetry (DSF). NAC, VitC and SF exhibited the largest inhibitory effect on amyloid fibril growth. The effects of polyphenols were not significant, apart from resveratrol, which partly inhibited the amyloid fibril growth.


Assuntos
Amiloide/química , Antioxidantes/farmacologia , Cistatina C/química , Polifenóis/farmacologia , Proteínas Recombinantes/química , Dicroísmo Circular/métodos , Relação Dose-Resposta a Droga , Humanos , Estrutura Secundária de Proteína/efeitos dos fármacos
4.
ACS Chem Neurosci ; 10(6): 2730-2740, 2019 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-30924329

RESUMO

Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.


Assuntos
Amiloide/metabolismo , Cistatina B/metabolismo , Prolina/metabolismo , Agregação Patológica de Proteínas/metabolismo , Amiloide/química , Amiloide/genética , Cistatina B/química , Cistatina B/genética , Análise Mutacional de DNA , Humanos , Mutação , Prolina/química , Prolina/genética , Agregação Patológica de Proteínas/genética
5.
Oxid Med Cell Longev ; 2018: 8613209, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29765505

RESUMO

Amyloid fibril formation is a shared property of all proteins; therefore, model proteins can be used to study this process. We measured protein aggregation of the model amyloid-forming protein stefin B in the presence and absence of several antioxidants. Amyloid fibril formation by stefin B was routinely induced at pH 5 and 10% TFE, at room temperature. The effects of antioxidants NAC, vitamin C, vitamin E, and the three polyphenols resveratrol, quercetin, and curcumin on the kinetics of fibril formation were followed using ThT fluorescence. Concomitantly, the morphology and amount of the aggregates and fibrils were checked by transmission electron microscopy (TEM). The concentration of the antioxidants was varied, and it was observed that different modes of action apply at low or high concentrations relative to the binding constant. In order to obtain more insight into the possible mode of binding, docking of NAC, vitamin C, and all three polyphenols was done to the monomeric form of stefin B.


Assuntos
Antioxidantes/uso terapêutico , Agregação Patológica de Proteínas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Antioxidantes/farmacologia , Humanos , Agregação Patológica de Proteínas/patologia
6.
Int J Mol Sci ; 18(3)2017 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-28272335

RESUMO

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to ß2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.


Assuntos
Amiloide/química , Amiloide/metabolismo , Prolina/química , Agregados Proteicos , Agregação Patológica de Proteínas , Conformação Proteica , Sequência de Aminoácidos , Amiloide/genética , Animais , Cistatina B/química , Cistatina B/metabolismo , Humanos , Cinética , Camundongos , Modelos Moleculares , Mutação , Prolina/genética , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Microglobulina beta-2/química , Microglobulina beta-2/metabolismo
7.
Biochem Biophys Res Commun ; 415(2): 337-41, 2011 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-22033403

RESUMO

The role of the aromatic residue at site 75 to protein stability, the mechanism of folding and the mechanism of amyloid-fibril formation were investigated for the human stefin B variant (bearing Y at site 31) and its point mutation H75W. With an aim to reveal the conformation at the cross-road between folding and aggregation, first, the kinetics of folding and oligomer formation by human stefin B(Y31) variant were studied. It was found to fold in three kinetic phases at pH 4.8 and 10% TFE; the pH and solvent conditions that transform the protein into amyloid fibrils at longer times. The same pH leads to the formation of native-like intermediate (known from previous studies of this variant), meaning that the process of folding and amyloid-fibril formation share the same structural intermediate, which is in this case native-like and dimeric. At pH 5.8 and 7.0 stefin B folded to the native state in four kinetic phases over two intermediates. In distinction, the mutant H75W did not fold to completion, ending in intermediate states at all pH values studied: 4.8, 5.8 and 7.0. At pH 4.8 and 5.8, the mutant folded in one kinetic phase to the intermediate of the "molten globule" type, which leads to the conclusion that its mechanism of folding differs from the one of the parent stefin B at the same pH. At pH 7.0 the mutant H75W folded in three kinetic phases to a native-like intermediate, analogous to folding of stefin B at pH 4.8.


Assuntos
Cistatina B/química , Histidina/química , Triptofano/química , Dicroísmo Circular , Cistatina B/genética , Histidina/genética , Humanos , Concentração de Íons de Hidrogênio , Mutação , Dobramento de Proteína , Triptofano/genética
8.
FEBS Lett ; 583(7): 1114-20, 2009 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-19265692

RESUMO

We report that Pro74 in human stefin B is critical for fibril formation and that proline isomerization plays an important role. The stefin B P74S mutant did not fibrillate over the time of observation at 25 degrees C, and it exhibited a prolonged lag phase at 30 degrees C and 37 degrees C. The peptidyl prolyl cis/trans isomerase cyclophilin A, when added to the wild-type protein, exerted two effects: it prolonged the lag phase and increased the yield and length of the fibrils. Addition of the inactive cyclophilin A R55A variant still resulted in a prolonged lag phase but did not mediate the increase of the final fibril yield. These results demonstrate that peptidyl prolyl cis/trans isomerism is rate-limiting in stefin B fibril formation.


Assuntos
Amiloide/química , Ciclofilina A/química , Cistatina B/química , Amiloide/genética , Amiloide/metabolismo , Ciclofilina A/genética , Ciclofilina A/metabolismo , Cistatina B/genética , Cistatina B/metabolismo , Humanos , Mutação , Prolina/genética , Prolina/metabolismo , Estrutura Quaternária de Proteína/genética , Fatores de Tempo
9.
Med Arh ; 61(2 Suppl 1): 7-10, 2007.
Artigo em Bosnio | MEDLINE | ID: mdl-21553440

RESUMO

The retrospective study included 250 patients, treated at Clinic for cardiovascular diseases of Tuzla Clinical center, between 30.08.2003. and 15.11.2004. In the coronary disease group there were 145 men, 55 women, with diagnosed coronary artery stenosis of 50% or more. The control group had 150 patients, 35 men and 15 women, medium age of 58.2. The control group had coronary artery stenosis of 50% or less. Coronarography was done using AXIOM ARTIS DFC (SIEMENS). Lipoproteins were determined on the Clinic for biochemistry of Tuzla Clinical Center using automatic analyser DIMENSION LxR (DADE BOEHRING). In the coronary artery disease (CAD) group elevated triglycerides were found in 38.5%, total cholesterol in 88% and LDL 55.5% of patients. The concentration of HDL cholesterol was elevated in 52.5% of patients. In the control group elevated values of triglycerides were found in 28%, total cholesterol 46%, LDL cholesterol 16%, and lower values of HDL in 10% of patients. Statistically significant differences of lipide profile of CAD patients in relation to the control group was defined. Using regresional analysis it was established that decide elevated values of total and LDL cholesterol, low values of HDL were also significant.


Assuntos
HDL-Colesterol/sangue , LDL-Colesterol/sangue , Doença da Artéria Coronariana/sangue , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
10.
Protein Sci ; 13(1): 63-70, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14691222

RESUMO

We describe expression, purification, and characterization of three site-specific mutants of recombinant human stefin B: H75W, P36G, and P79S. The far- and near-UV CD spectra have shown that they have similar secondary and tertiary structures to the parent protein. The elution on gel-filtration suggests that recombinant human stefin B and the P36G variant are predominantly monomers, whereas the P79S variant is a dimer. ANS dye binding, reflecting exposed hydrophobic patches, is highest for the P36G variant, both at pH 5 and 3. ANS dye binding also is increased for stefin B and the other two variants at pH 3. Under the chosen conditions the highest tendency to form amyloid fibrils has been shown for the recombinant human stefin B. The P79S variant demonstrates a longer lag phase and a lower rate of fibril formation, while the P36G variant is most prone to amorphous aggregation. This was demonstrated by ThT fluorescence as a function of time and by transmission electron microscopy.


Assuntos
Cistatinas/genética , Cistatinas/metabolismo , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Amiloide/metabolismo , Amiloide/ultraestrutura , Naftalenossulfonato de Anilina , Sítios de Ligação , Cromatografia em Gel , Cromatografia por Troca Iônica , Dicroísmo Circular , Cistatina B , Bases de Dados Factuais , Dimerização , Escherichia coli/genética , Corantes Fluorescentes , Variação Genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/isolamento & purificação , Temperatura , Ureia/farmacologia
11.
Bosn J Basic Med Sci ; 2(1-2): 12-5, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16212561

RESUMO

Cystatin C is a natural inhibitor of the cysteine proteinases papain, and mammalian lysosomal cathepsins B, H, L and S. This protein is thought to serve an important physiological role as a local regulator of enzyme activity. The changes of levels of cystatin C in extracellular fluids have shown themselves having potential clinical importance. We have purified cystatin C from urine of patients with chronic renal failure by procedure using affinity chromatography on CM-papain Sepharose, gel filtration on Sephacryl S-200, and ion exchange chromatography on CM-cellulose. After isolation we obtained three inhibitory peaks (pI's from 7.8 to 9.2) which represent isoforms of the same protein. These isoforms are immunologically identical and differ in N-terminal sequence of the molecule. The form with pI 9.2 represents the intact inhibitor form, whereas the form with pI 7.8 is shortened for 8 amino-acid residues at N-terminal end. Purified cystatin C pI 9.2 was used for immunization of rabbits. Polyclonal antibodies, produced in rabbits, were isolated from rabbit sera by affinity chromatography on Protein A Sepharose. Enzyme immunoassay (ELISA) for cystatin C is developed on the basis of purified antibodies. Using ELISA test we determined amount of cystatin C in urine and serum samples of patients with chronic renal failure. The concentration of the inhibitor in the urine of these patients was approximately 100-fold more than in normal urine. In the serum from the same patients we found concentrations of cystatin C to be five times higher in comparison with the serum of healthy individuals.

12.
Med Arh ; 56(5-6): 305-11, 2002.
Artigo em Servo-Croata (Latino) | MEDLINE | ID: mdl-12693335

RESUMO

Spongiform encephalopathies are the fatal diseases, that affect the brain tissue of mammals. They are caused by a conformational changed prion protein. There is no adequate diagnostic test for in vivo identification of prion protein. Disease can be diagnosed only by clinical sings and EEG in new variant of Creutzfeldt-Jakob disease. Post mortem, histopathological examination of brain tissue reveals spongiform changes and immunohistochemistry detects disease-related prion protein. Appropriate diagnostic in vivo tests are not developed yet; therefore extensive researches are ongoing aimed to introduce such methods. This review describes a few promising experimental methods, which may develop into diagnostic tests in the future: detection of prions in urine samples, PMCA (protein misfolding cyclic amplification), DATAS (differential analysis of transcripts with alternative splicing), SELEX (in vitro selection), detection of prions in tonsils and detection of copper and manganese dysbalance in tissues. Current therapy strategy is based on testing of some known drugs (quinacrine, chlorpromazine), and antioxidant and antibody treatments. The detection of NSE (neuron-specific enolase) and cholesterol in meat products reveals the presence of brain and spinal cord tissue. The spreading of spongiform encephalopathies can be diminished by utilising the adequate in vivo diagnostic tests, effective therapy strategy and preventive steps.


Assuntos
Doenças Priônicas , Humanos , Doenças Priônicas/diagnóstico , Doenças Priônicas/prevenção & controle , Doenças Priônicas/terapia
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