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1.
FEBS J ; 291(9): 1974-1991, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38349797

RESUMO

Human cystatin C (hCC), a small secretory protein, has gained attention beyond its classical role as a cysteine protease inhibitor owing to its potential involvement in neurodegenerative disorders. This study investigates the interaction between copper(II) ions [Cu(II)] and hCC, specifically targeting histidine residues known to participate in metal binding. Through various analytical techniques, including mutagenesis, circular dichroism, fluorescence assays, gel filtration chromatography, and electron microscopy, we evaluated the impact of Cu(II) ions on the structure and oligomerization of hCC. The results show that Cu(II) does not influence the secondary and tertiary structure of the studied hCC variants but affects their stability. To explore the Cu(II)-binding site, nuclear magnetic resonance (NMR) and X-ray studies were conducted. NMR experiments revealed notable changes in signal intensities and linewidths within the region 86His-Asp-Gln-Pro-His90, suggesting its involvement in Cu(II) coordination. Both histidine residues from this fragment were found to serve as a primary anchor of Cu(II) in solution, depending on the structural context and the presence of other Cu(II)-binding agents. The presence of Cu(II) led to significant destabilization and altered thermal stability of the wild-type and H90A variant, confirming differentiation between His residues in Cu(II) binding. In conclusion, this study provides valuable insights into the interaction between Cu(II) and hCC, elucidating the impact of copper ions on protein stability and identifying potential Cu(II)-binding residues. Understanding these interactions enhances our knowledge of the role of copper in neurodegenerative disorders and may facilitate the development of therapeutic strategies targeting copper-mediated processes in protein aggregation and associated pathologies.


Assuntos
Cobre , Cistatina C , Ligação Proteica , Multimerização Proteica , Cobre/metabolismo , Cobre/química , Humanos , Cistatina C/química , Cistatina C/metabolismo , Cistatina C/genética , Sítios de Ligação , Modelos Moleculares , Cristalografia por Raios X , Estabilidade Proteica , Dicroísmo Circular , Histidina/química , Histidina/metabolismo , Conformação Proteica
2.
Diabetes Metab Syndr ; 17(11): 102882, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37898064

RESUMO

BACKGROUND: The association between serum cystatin C level and vascular outcomes has not been fully elucidated in diabetes and is unclear in prediabetes. We aim to evaluate whether cystatin C level predicts future risk for mortality and vascular outcomes in prediabetes and diabetes. METHODS: A total of 85,371 participants with prediabetes and diabetes, and available baseline cystatin C in the UK biobank were included with a 14-year follow-up. Cox hazards models were used to calculate the associations between cystatin C level, mortality (all-cause, cause-specfic mortality) and vascular outcomes (myocardial infarction [MI], stroke, end-stage renal disease [ESRD] and diabetic retinopathy [DR]). The 1136 diabetes subjects in Guangzhou Diabetic Eye Study (GDES) were included for examing the impact of cystatin C on in vivo retinal degeneration and microvascular changes by using SS-OCT and OCTA. RESULTS: The highest cystatin C quartile had increased risks of all-cause (hazard ratio [HR], 2.02; 95% confidence interval [CI] 1.86-2.19), cardiovascular (HR, 2.29; 95% CI 1.97-2.67), cancer (HR, 1.86; 95% CI 1.65-2.10) and other-cause mortality (HR, 2.24; 95% CI 1.90-2.64), MI (HR, 1.40; 95% CI 1.26-1.55), stroke (HR, 1.88; 95% CI, 1.57-2.26), ESRD (HR, 7.33; 95% CI, 5.02-10.71), DR (HR, 1.17; 95% CI 1.03-1.32) than those in the lowest quartile. Adding cystatin C to the conventional model improved C-statistic for all-cause (0.699-0.724), cardiovascular (0.762-0.789), cancer (0.661-0.674) and other-cause mortality (0.675-0.715), MI (0.748-0.750), stroke (0.712-0.718), and ESRD (0.808-0.827). The GDES analysis identified a strong association between increased cystatin C levels and diminished retinal neural layers, as well as microvascular rarefaction in both macular and optic disc regions (all P < 0.05). CONCLUSIONS: Serum cystatin C refines the risk stratification for mortality and vascular outcomes among patients with prediabetes or diabetes.


Assuntos
Diabetes Mellitus , Falência Renal Crônica , Infarto do Miocárdio , Neoplasias , Estado Pré-Diabético , Humanos , Cistatina C/sangue , Cistatina C/química , Diabetes Mellitus/sangue , Diabetes Mellitus/diagnóstico , Estado Pré-Diabético/sangue , Estado Pré-Diabético/diagnóstico , Medição de Risco , Fatores de Risco , Acidente Vascular Cerebral
3.
J Chromatogr Sci ; 61(3): 296-301, 2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-35905461

RESUMO

Accurate quantification of low-abundance protein Cystatin-C (CysC) in serum by liquid chromatography tandem mass spectrometry (LC-MS/MS) method is very difficult. After sample processing and tryptic digestion, the matrix of CysC surrogate peptides is very complexity, and the concentrations of them are very low, so solid-phase extraction (SPE) must be used to make the surrogate peptides purification and enrichment. In this paper, we used C18 reversed-phase SPE (RP-SPE) and mixed-mode SPE as SPE cartridges. We quantitatively assessed and compared the CysC surrogate peptides recoveries and matrix effects by different SPE cartridges. The sequence of CysC surrogate peptide is ALDFAVGEYNK, and sequence-specific subions y6 (VGEYNK+, m/z 709.3) and y9 (DFAVGEYNK+, m/z 1042.4) were selected for quantification of CysC, because the two fragment ions showed the highest sensitivity. In neat solution, the highest recoveries were similarly for y9 and y6 when used RP-SPE and mixed-mode SPE. However, in serum matrix, the recoveries were significantly higher when used mixed-mode SPE than RP-SPE, which was caused by matrix effects. Results showed that both RP-SPE and mixed-mode SPE were resulted in ion enhancement for CysC surrogate peptides quantification by LC-MS/MS, but mixed-mode SPE reduced more matrix effects. So mixed-mode SPE was more suitable SPE type for purification and enrichment of CysC surrogate peptides.


Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/métodos , Peptídeos/química , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Cistatina C/química
4.
Int J Mol Sci ; 23(21)2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36362228

RESUMO

Human cystatin C (HCC), an amyloidogenic protein, forms dimers and higher oligomers (trimers, tetramers and donut like large oligomers) via a domain-swapping mechanism. The aim of this study was the characterization of the HCC oligomeric states observed within the pH range from 2.2 to 10.0 and also in conditions promoting oligomerization. The HCC oligomeric forms obtained in different conditions were characterized using size exclusion chromatography, dynamic light scattering and small-angle X-ray scattering. The marked ability of HCC to form tetramers at low pH (2.3 or 3.0) and dimers at pH 4.0-5.0 was observed. HCC remains monomeric at pH levels above 6.0. Based on the SAXS data, the structure of the HCC tetramer was proposed. Changes in the environment (from acid to neutral) induced a breakdown of the HCC tetramers to dimers. The tetrameric forms of human cystatin C are formed by the association of the dimers without a domain-swapping mechanism. These observations were confirmed by their dissociation to dimers at pH 7.4.


Assuntos
Proteínas Amiloidogênicas , Cistatina C , Humanos , Cistatina C/química , Proteínas Amiloidogênicas/metabolismo , Espalhamento a Baixo Ângulo , Dimerização , Difração de Raios X
5.
Lancet Glob Health ; 10(8): e1159-e1169, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35839814

RESUMO

BACKGROUND: The burden of kidney disease in many African countries is unknown. Equations used to estimate kidney function from serum creatinine have limited regional validation. We sought to determine the most accurate way to measure kidney function and thus estimate the prevalence of impaired kidney function in African populations. METHODS: We measured serum creatinine, cystatin C, and glomerular filtration rate (GFR) using the slope-intercept method for iohexol plasma clearance (mGFR) in population cohorts from Malawi, Uganda, and South Africa. We compared performance of creatinine and cystatin C-based estimating equations to mGFR, modelled and validated a new creatinine-based equation, and developed a multiple imputation model trained on the mGFR sample using age, sex, and creatinine as the variables to predict the population prevalence of impaired kidney function in west, east, and southern Africa. FINDINGS: Of 3025 people who underwent measured GFR testing (Malawi n=1020, South Africa n=986, and Uganda n=1019), we analysed data for 2578 participants who had complete data and adequate quality measurements. Among 2578 included participants, creatinine-based equations overestimated kidney function compared with mGFR, worsened by use of ethnicity coefficients. The greatest bias occurred at low kidney function, such that the proportion with GFR of less than 60 mL/min per 1·73 m2 either directly measured or estimated by cystatin C was more than double that estimated from creatinine. A new creatinine-based equation did not outperform existing equations, and no equation, including the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) 2021 race-neutral equation, estimated GFR within plus or minus 30% of mGFR for 75% or more of the participants. Using a model to impute kidney function based on mGFR, the estimated prevalence of impaired kidney function was more than two-times higher than creatinine-based estimates in populations across six countries in Africa. INTERPRETATION: Estimating GFR using serum creatinine substantially underestimates the individual and population-level burden of impaired kidney function in Africa with implications for understanding disease progression and complications, clinical care, and service provision. Scalable and affordable ways to accurately identify impaired kidney function in Africa are urgently needed. FUNDING: The GSK Africa Non-Communicable Disease Open Lab. TRANSLATIONS: For the Luganda, Chichewa and Xitsonga translations of the abstract see Supplementary Materials section.


Assuntos
Rim , Insuficiência Renal Crônica , Estudos de Coortes , Creatinina/química , Cistatina C/química , Taxa de Filtração Glomerular , Humanos , Rim/metabolismo , Rim/patologia , Malaui/epidemiologia , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/epidemiologia , África do Sul/epidemiologia , Uganda/epidemiologia
6.
Int J Mol Sci ; 23(10)2022 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-35628610

RESUMO

Amyloid fibrils have been known for many years. Unfortunately, their fame stems from negative aspects related to amyloid diseases. Nevertheless, due to their properties, they can be used as interesting nanomaterials. Apart from their remarkable stability, amyloid fibrils may be regarded as a kind of a storage medium and as a source of active peptides. In many cases, their structure may guarantee a controlled and slow release of peptides in their active form; therefore, they can be used as a potential nanomaterial in drug delivery systems. In addition, amyloid fibrils display controllable stiffness, flexibility, and satisfactory mechanical strength. In addition, they can be modified and functionalized very easily. Understanding the structure and genesis of amyloid assemblies derived from a broad range of amyloidogenic proteins could help to better understand and use this unique material. One of the factors responsible for amyloid aggregation is the steric zipper. Here, we report the discovery of steric zipper-forming peptides in the sequence of the amyloidogenic protein, human cystatin C (HCC). The ability of short peptides derived from this fragment of HCC to form fibrillar structures with defined self-association characteristics and the factors influencing this aggregation are also presented in this paper.


Assuntos
Amiloide , Amiloidose , Amiloide/química , Proteínas Amiloidogênicas/química , Cistatina C/química , Humanos , Peptídeos/química
7.
Nat Commun ; 12(1): 1827, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758187

RESUMO

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disease caused by a leucine to glutamine variant of human cystatin C (hCC). L68Q-hCC forms amyloid deposits in brain arteries associated with micro-infarcts, leading ultimately to paralysis, dementia and death in young adults. To evaluate the ability of molecules to interfere with aggregation of hCC while informing about cellular toxicity, we generated cells that produce and secrete WT and L68Q-hCC and have detected high-molecular weight complexes formed from the mutant protein. Incubations of either lysate or supernatant containing L68Q-hCC with reducing agents glutathione or N-acetyl-cysteine (NAC) breaks oligomers into monomers. Six L68Q-hCC carriers taking NAC had skin biopsies obtained to determine if hCC deposits were reduced following NAC treatment. Remarkably, ~50-90% reduction of L68Q-hCC staining was observed in five of the treated carriers suggesting that L68Q-hCC is a clinical target for reducing agents.


Assuntos
Acetilcisteína/farmacologia , Proteínas Amiloidogênicas/metabolismo , Angiopatia Amiloide Cerebral Familiar/dietoterapia , Cistatina C/metabolismo , Cistatinas/metabolismo , Acetilcisteína/administração & dosagem , Acetilcisteína/análogos & derivados , Acetilcisteína/química , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Biópsia , Angiopatia Amiloide Cerebral Familiar/tratamento farmacológico , Angiopatia Amiloide Cerebral Familiar/genética , Cistatina C/química , Cistatina C/genética , Cistatinas/química , Cistatinas/genética , Expressão Gênica , Glutationa/química , Glutationa/farmacologia , Células HEK293 , Humanos , Pele/efeitos dos fármacos , Pele/metabolismo , Adulto Jovem
8.
ACS Appl Mater Interfaces ; 12(52): 58149-58160, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-33326226

RESUMO

Lateral flow immunoassay (LFIA), as a prominent point-of-care (POC) test platform, has been extensively adopted for rapid, on-site, and facile diagnosis of pathogen infections and disease biomarkers. Exploring novel structured optical labels of LFIA with amplified signal and complementary detection modes favors the sensitive and flexible POC diagnosis. Here, bimodal labels with both colorimetric and fluorescent readout were fabricated via a layered sequential assembly strategy based on affinity templates and hydrophobic metal-containing nanounits. High-quality colorimetric and fluorescent nanoparticles were densely incorporated into the colloidal supports and confined in separated regions, without interfering with each other. The hierarchical integration of gold nanoparticles and quantum dots with high loading density and good optical preservation realized dual readout and amplified signals from the assemblies of individual single nanoparticles. The "all-in-one" optical labels allowed both colorimetric and fluorescent detection of cystatin C (Cys C) after surface conjugation with antibodies. The LFIA strips revealed noninterfering dual signals for both visual inspection and quantitative detection of Cys C via the naked eye and portable devices, respectively. The limits of detection by colorimetric and fluorescent modes were 0.61 and 0.24 ng mL-1, respectively. The novel LFIA platform demonstrated sensitive, specific, and reproducible POC testing of biomarkers with flexible detection modes and was reliable for clinical diagnosis.


Assuntos
Corantes Fluorescentes/química , Imunoensaio/métodos , Limite de Detecção , Cistatina C/análise , Cistatina C/química , Modelos Moleculares , Conformação Molecular
9.
FEBS Open Bio ; 10(11): 2464-2477, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33030263

RESUMO

Infection of rat pulmonary microvascular endothelial cells with the bacterium Pseudomonas aeruginosa induces the production and release of cytotoxic oligomeric tau and beta amyloid (Aß). Here, we characterized these cytotoxic amyloids. Cytotoxic behavior and oligomeric tau were partially resistant to digestion with proteinase K, but cytotoxicity was abolished by various denaturants including phenol, diethylpyrocarbonate (DEPC), and 1,1,1,3,3,3-hexafluoro-2-isopropanol (HFIP). Ultracentrifugation for 8 h at 150 000 g was required to remove cytotoxic activity from the supernatant. Ultracentrifugation, DEPC treatment, and immunodepletion using antibodies against Aß also demonstrated that cytoprotective protein(s) are released from endothelial cells during P. aeruginosa infection. Mass spectrometry of endothelial cell culture media following P. aeruginosa infection allowed identification of multiple potential secreted modulators of Aß, including cystatin C, gelsolin, and ApoJ/clusterin. Immunodepletion, co-immunoprecipitation, and ultracentrifugation determined that the cytoprotective factor released during infection of endothelial cells by P. aeruginosa is cystatin C, which appears to be in a complex with Aß. Cytoprotective cystatin C may provide a novel therapeutic avenue for protection against the long-term consequences of infection with P. aeruginosa.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Cistatina C/metabolismo , Células Endoteliais/metabolismo , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/fisiologia , Sequência de Aminoácidos , Animais , Morte Celular , Cistatina C/química , Citoproteção , Endopeptidase K/metabolismo , Ratos
10.
Mikrochim Acta ; 187(10): 585, 2020 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-32997226

RESUMO

The fabrication of a nanointerfaced electrochemical immunosensor is described for the rapid determination of cystatin C, a biomarker that is elevated in diabetic retinopathy. A dispersion of graphene oxide-chitosan (GO-Chit) nanocomposite was used to modify the carbon working electrode, allowing for a high conjugation of anti-cystatin C antibody. This modified sensor was characterized both morphologically and electrochemically, and the sensor performance was evaluated towards selective quantification of cystatin C in simulated as well as serum samples using cyclic voltammetry and differential pulse voltammetry. The sensor was able to detect cystatin C in the concentration range1 - 10 mg/L with a detection limit of 0.0078 mg/L. The preparation time of the sensor was 420 s, which was faster than that of conventional ELISA and other electrochemical sensors reported in literature. The clinical applicability of the proposed electrochemical biosensor was demonstrated through quantification of cystatin C in human serum samples and identification of diabetic retinopathy. A cutoff value of 1.2 mg/L of cystatin C was used beyond which the samples were classified as positive for diabetic retinopathy. Two different working electrodes, namely a glassy carbon electrode (GCE) and paper electrodes, were used in the study. The working potential was set to 0.25 V vs. Ag/AgCl for experiments with the GCE and 0.15 V for the paper electrodes. The prediction was validated by clinical diagnosis wherein the prediction accuracy of the sensor exceeded 85%. The sensor platform was translated onto a paper substrate and characterized for achieving an optimum sensing performance. This work is the first attempt to employ an electrochemical cystatin C sensor for the diagnosis of diabetic retinopathy from serum samples. Graphical abstract.


Assuntos
Cistatina C/química , Técnicas Eletroquímicas/métodos , Microfluídica/métodos , Humanos
11.
Int J Mol Sci ; 21(16)2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32824145

RESUMO

Human cystatin C (HCC), a cysteine-protease inhibitor, exists as a folded monomer under physiological conditions but has the ability to self-assemble via domain swapping into multimeric states, including oligomers with a doughnut-like structure. The structure of the monomeric HCC has been solved by X-ray crystallography, and a covalently linked version of HCC (stab-1 HCC) is able to form stable oligomeric species containing 10-12 monomeric subunits. We have performed molecular modeling, and in conjunction with experimental parameters obtained from atomic force microscopy (AFM), transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) measurements, we observe that the structures are essentially flat, with a height of about 2 nm, and the distance between the outer edge of the ring and the edge of the central cavity is ~5.1 nm. These dimensions correspond to the height and diameter of one stab-1 HCC subunit and we present a dodecamer model for stabilized cystatin C oligomers using molecular dynamics simulations and experimentally measured parameters. Given that oligomeric species in protein aggregation reactions are often transient and very highly heterogeneous, the structural information presented here on these isolated stab-1 HCC oligomers may be useful to further explore the physiological relevance of different structural species of cystatin C in relation to protein misfolding disease.


Assuntos
Cistatina C/química , Simulação de Dinâmica Molecular , Humanos , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica
12.
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
13.
FEBS J ; 287(2): 361-376, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31330077

RESUMO

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. This small protein, in addition to its physiological function, is involved in various diseases, including cerebral amyloid angiopathy, cerebral hemorrhage, stroke, and dementia. Physiologically active hCC is a monomer. However, all structural studies based on crystallization led to the dimeric structure formed as a result of a three-dimensional exchange of the protein domains (3D domain swapping). The monomeric structure was obtained only for hCC variant V57N and for the protein stabilized by an additional disulfide bridge. With this study, we extend the number of models of monomeric hCC by an additional hCC variant with a single amino acid substitution in the flexible loop L1. The V57G variant was chosen for the X-ray and NMR structural analysis due to its exceptional conformational stability in solution. In this work, we show for the first time the structural and dynamics studies of human cystatin C variant in solution. We were also able to compare these data with the crystal structure of the hCC V57G and with other cystatins. The overall cystatin fold is retained in the solute form. Additionally, structural information concerning the N terminus was obtained during our studies and presented for the first time. DATABASE: Crystallographic structure: structural data are available in PDB databases under the accession number 6ROA. NMR structure: structural data are available in PDB and BMRB databases under the accession numbers 6RPV and 34399, respectively.


Assuntos
Cistatina C/química , Simulação de Dinâmica Molecular , Substituição de Aminoácidos , Cristalografia por Raios X , Cistatina C/genética , Humanos , Espectroscopia de Ressonância Magnética , Estabilidade Proteica
14.
J Pharm Biomed Anal ; 177: 112889, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31568966

RESUMO

This work presents the development of a methodology for the accurate and precise quantification of the renal biomarker Cystatin C in human urine by Isotope Dilution Mass Spectrometry (IDMS). The procedure is based on the addition of a known quantity of the proteotypic peptide ALDFAVG*EYNK labelled with 13C2-glycine to the urine sample followed by protein hydrolysis using trypsin. Then, preconcentration and purification of the isotope diluted peptide was carried out by a selective monoclonal antibody bound to magnetic beads and final measurement was done after injection of the sample in a HPLC-MS/MS triple quadrupole instrument. The isotopic distribution of the isotope diluted proteotypic peptide was measured by low resolution selected reaction monitoring. Using this aquisition mode, the bandpass of the first quadrupole was widened (FWHM =13 u) so the whole isotopic clusters for both the natural abundance and the labelled peptides entered the collision cell. The proposed acquisition mode provided similar accuracy and precision than the regular SRM mode (FWHM =0.7 u) but a higher sensitivity was observed. The purification of the sample by antibody based enrichment of the target peptide was shown to remove interfering compounds more efficiently in comparison with a sample purification based on semipreparative liquid chromatography. Using 5 ng of the labelled peptide it was possible to quantify Cystatin C in human urine in patients with normal and impaired renal function. Recoveries from 100 to 104% were obtained in samples containing from 90 to 700 µg L-1 of Cystatin C with relative standard deviations from 0.5 to 6%. The stability of Cystatin C in urine samples was evaluated under different storage conditions showing that only when the urine samples were stored at room temperature during more than 10 days, a significant degradation of Cystatin C was observed.


Assuntos
Cistatina C/urina , Nefropatias/diagnóstico , Manejo de Espécimes/efeitos adversos , Espectrometria de Massas em Tandem/métodos , Biomarcadores/química , Biomarcadores/urina , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Cistatina C/química , Taxa de Filtração Glomerular/fisiologia , Humanos , Técnicas de Diluição do Indicador , Rim/fisiopatologia , Nefropatias/fisiopatologia , Nefropatias/urina , Estabilidade Proteica , Manejo de Espécimes/métodos , Temperatura , Fatores de Tempo
15.
Sci Rep ; 9(1): 8548, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189973

RESUMO

Domain swapping is observed for many proteins with flexible conformations. This phenomenon is often associated with the development of conformational diseases. Importantly, domain swapping has been observed for human cystatin C (HCC), a protein capable of forming amyloid deposits in brain arteries. In this study, the ability of short exposure to high-intensity X-ray radiation to induce domain swapping in solutions of several HCC variants (wild-type HCC and V57G, V57D, V57N, V57P, and L68V mutants) was determined. The study was conducted using time-resolved small-angle X-ray scattering (TR-SAXS) synchrotron radiation. The protein samples were also analysed using small-angle neutron scattering and NMR diffusometry. Exposing HCC to synchrotron radiation (over 50 ms) led to a gradual increase in the dimeric fraction, and for exposures longer than 150 ms, the oligomer fraction was dominant. In contrast, the non-irradiated protein solutions, apart from the V57P variant, were predominantly monomeric (e.g., V57G) or in monomer/dimer equilibrium. This work might represent the first observation of domain swapping induced by high-intensity X-rays.


Assuntos
Cistatina C/química , Síncrotrons , Raios X , Humanos , Difração de Nêutrons , Domínios Proteicos , Espalhamento a Baixo Ângulo
16.
Protein J ; 38(5): 598-607, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31119598

RESUMO

Human cystatin C (HCC) binds and inhibits all types of cysteine proteases from the papain family, including cathepsins (a group of enzymes that participate in a variety of physiological processes), which are some of its natural targets. The affinities of diverse proteases for HCC, expressed as equilibrium binding constants (Kb), range from 106 to 1014 M-1. Isothermal titration calorimetry (ITC) is one of the most useful techniques to characterize the thermodynamics of molecular associations, making it possible to dissect the binding free energy into its enthalpic and entropic components. This information, together with the structural changes that occur during the different associations, could enable better understanding of the molecular basis of affinity. Notwithstanding the high sensitivity of modern calorimeters, ITC requires protein concentrations in at least the 10-100 µM range to obtain reliable data, and it is known that HCC forms oligomers in this concentration range. We present herein a comparative study of the structural, thermal stability, and oligomerization properties of HCC and its stabilized variant (sHCC) L47C/G69C (which possesses an additional disulfide bridge) as well as their binding thermodynamics to the protease chymopapain, analyzed by ITC. The results show that, because sHCC remains monomeric, it is a better reporter than wild-type HCC to characterize the thermodynamics of binding to cysteine proteases.


Assuntos
Cistatina C/química , Cistatina C/metabolismo , Cisteína Proteases/metabolismo , Cistatina C/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica , Termodinâmica
17.
Protein Pept Lett ; 26(6): 423-434, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-30864495

RESUMO

BACKGROUND: Antibacterial peptidyl derivative - Cystapep 1, was previously found to be active both against antibiotic-resistant staphylococci and streptococci as well as antibioticsusceptible strains of these species. Therefore, it is a promising lead compound to search for new antimicrobial peptidomimetics. OBJECTIVES: We focused on identifying structural elements that are responsible for the biological activity of Cystapep 1 and its five analogues. We tried to find an answer to the question about the mechanism of action of the tested compounds. Therefore, we have investigated in details the possibility of interacting these compounds with biological membrane mimetics. METHODS: The subject compounds were synthesized in solution, purified and characterized by HPLC and mass spectrometry. Then, the staphylococci susceptibility tests were performed and their cytotoxicity was established. The results of Cystapep 1 and its analogues interactions with model target were examined using the DSC and ITC techniques. At the end the spatial structures of the tested peptidomimetics using NMR technique were obtained. RESULTS: Antimicrobial and cytotoxicity tests show that Cystapep 1 and its peptidomimetic V are good drug candidates. DSC and ITC studies indicate that disruption of membrane is not the only possible mechanism of action of Cystapep 1-like compounds. For Cystapep 1 itself, a multi-step mechanism of interaction with a negatively charged membrane is observed, which indicates other processes occurring alongside the binding process. The conformational analysis indicated the presence of a hydrophobic cluster, composed of certain side chains, only in the structures of active peptidomimetics. This can facilitate the anchoring of the peptidyl derivatives to the bacterial membrane. CONCLUSION: An increase in hydrophobicity of the peptidomimetics improved the antimicrobial activity against S. aureus, however there is no simple correlation between the biological activity and the strength of interactions of the peptidyl with bacterial membrane.


Assuntos
Antibacterianos/química , Cistatina C/química , Inibidores de Cisteína Proteinase/química , Dipeptídeos/química , Peptidomiméticos/química , Animais , Antibacterianos/farmacologia , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Modelos Moleculares , Peptidomiméticos/farmacologia , Conformação Proteica , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade
19.
J Pept Sci ; 24(4-5): e3073, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29573035

RESUMO

Human cystatin C (hCC) is a low molecular mass protein that belongs to the cystatin superfamily. It is an inhibitor of extracellular cysteine proteinases, present in all human body fluids. At physiological conditions, hCC is a monomer, but it has a tendency to dimerization. Naturally occurring hCC mutant, with leucine in position 68 substituted by glutamine (L68Q), is directly involved in the formation of amyloid deposits, independently of other proteins. This process is the primary cause of hereditary cerebral amyloid angiopathy, observed mainly in the Icelandic population. Oligomerization and fibrillization processes of hCC are not explained equally well, but it is proposed that domain swapping is involved in both of them. Research carried out on the fibrillization process led to new hypothesis about the existence of a steric zipper motif in amyloidogenic proteins. In the hCC sequence, there are 2 fragments which may play the role of a steric zipper: the loop L1 region and the C-terminal fragment. In this work, we focused on the first of these. Nine hexapeptides covering studied hCC fragment were synthesized, and their fibrillogenic potential was assessed using an array of biophysical methods. The obtained results showed that the studied hCC fragment has strong profibrillogenic propensities because it contains 2 fragments fulfilling the requirements for an effective steric zipper located next to each other, forming 1 super-steric zipper motif. This hCC fragment might therefore be responsible for the enhanced amyloidogenic properties of dimeric or partially unfolded hCC.


Assuntos
Amiloide/síntese química , Cistatina C/química , Oligopeptídeos/síntese química , Amiloide/química , Cistatina C/genética , Dimerização , Humanos , Modelos Moleculares , Mutação , Oligopeptídeos/química , Conformação Proteica , Domínios Proteicos
20.
Biosens Bioelectron ; 105: 90-94, 2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29412950

RESUMO

A facile label free, ultrasensitive platform for a rapid detection of chronic kidney disease has been fabricated. Early intervention in patients with chronic kidney disease has the potential to delay, or even prevent, the development of end stage renal disease and complications, leading to a marked impact on life expectancy and quality of life. Thus, a potable electrochemical diagnostic biosensor has become an attractive option as electrochemical analysis is feasible to use for on-site detection of samples. In human, Cystatin C present in human body fluids is freely filtered by the glomerulus, but reabsorbed and catabolised by the renal tubules. Trace detectable amount is eliminated in urine, giving this molecular marker an edge over serum creatinine's disadvantages. A carboxyl functionalized multiwalled carbon nanotubes screen printed electrode was immobilized with papain (cysteine protease) where amino group of papain covalently bound carboxyl group on electrode surface by EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and NHS (N-hydroxysuccinimide) chemistry. The modifications on sensor surface were characterized by field emission scanning electron microscopy. Interaction between papain and chronic kidney disease specific biomarker, Cystatin C was detected by cyclic voltammetry and differential pulse voltammetry within 10min. The sensor is highly specific to Cystatin C and showed negligible response to non-specific macromolecules present in urine. The sensitivity of the sensor was 1583.49µAcm-2µg-1 and lower limit of detection of Cystatin C was found 0.58ngL-1 which presents as a promising platform for designing potable kidney disease detector.


Assuntos
Técnicas Biossensoriais/métodos , Cistatina C/urina , Insuficiência Renal Crônica/urina , Técnicas Biossensoriais/instrumentação , Cistatina C/química , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Humanos , Limite de Detecção , Nanotubos de Carbono/química , Papaína/química , Sistemas Automatizados de Assistência Junto ao Leito , Insuficiência Renal Crônica/diagnóstico
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