Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
Mais filtros











Base de dados
Intervalo de ano de publicação
1.
Molecules ; 29(15)2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39124852

RESUMO

A phospholipid bilayer is a typical structure that serves crucial functions in various cells and organelles. However, it is not unusual for it to take part in pathological processes. The cell membrane may be a binding target for amyloid-forming proteins, becoming a factor modulating the oligomerization process leading to amyloid deposition-a hallmark of amyloidogenic diseases-e.g., Alzheimer's disease. The information on the mechanisms governing the oligomerization influenced by the protein-membrane interactions is scarce. Therefore, our study aims to describe the interactions between DPPA, a cell membrane mimetic, and amyloidogenic protein human cystatin C. Circular dichroism spectroscopy and differential scanning calorimetry were used to monitor (i) the secondary structure of the human cystatin C and (ii) the phase transition temperature of the DPPA, during the protein-membrane interactions. NMR techniques were used to determine the protein fragments responsible for the interactions, and molecular dynamics simulations were applied to provide a molecular structure representing the interaction. The obtained data indicate that the protein interacts with DPPA, submerging itself into the bilayer via the AS region. Additionally, the interaction increases the content of α-helix within the protein's secondary structure and stabilizes the whole molecule against denaturation.


Assuntos
Membrana Celular , Cistatina C , Ligação Proteica , Cistatina C/química , Cistatina C/metabolismo , Humanos , Membrana Celular/metabolismo , Membrana Celular/química , Simulação de Dinâmica Molecular , Dicroísmo Circular , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/metabolismo , Estrutura Secundária de Proteína , Varredura Diferencial de Calorimetria
2.
Int J Biol Macromol ; : 134889, 2024 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-39168225

RESUMO

A cell membrane is an essential cellular component providing protection against the outer environment. It is also a host for proteins and carbohydrates responsible for, e.g. transporter, receptor, or enzymatic functions. In parallel, the membrane may also be implicated in pathological processes leading, e.g. to the oligomerization of amyloid-forming proteins, a hallmark of i.a. Alzheimer's disease. The increasing need for detailed information on mechanisms driving the amyloid formation and the potential role of cell membranes in the process proves the research on protein-membrane interactions biologically relevant. Considering the potential and limitations of the relatively well established and newly developed methods, this study focused on selecting methods that allow a broad and comprehensive description of interactions between amyloidogenic protein human cystatin C and lipid bilayers. In the first step, dot-blot and ELISA tests were selected as techniques allowing fast screening for protein-ligand interactions. Next, surface plasmon resonance, spectral shift, biolayer interferometry, and switchSENSE® technology were used to determine kinetic parameters and binding constants for interactions between human cystatin C and the selected lipid bilayers. Based on the obtained results we have proposed the most promising candidates for monitoring of interactions and determining affinity between amyloidogenic proteins and membrane mimetics.

3.
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
4.
Biochim Biophys Acta Biomembr ; 1866(3): 184285, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38237885

RESUMO

A biological membrane is a structure characteristic for various cells and organelles present in almost all living organisms. Even though, it is one of the most common structures in organisms, where it serves crucial functions, a phospholipid bilayer may also take part in pathological processes leading to severe diseases. Research indicates that biological membranes have a profound impact on the pathological processes of oligomerization of amyloid-forming proteins. These processes are a hallmark of amyloid diseases, a group of pathological states involving, e.g., Parkinson's or Alzheimer's disease. Even though amyloidogenic diseases reap the harvest in modern societies, especially in elderly patients, the mechanisms governing the amyloid deposition are not clearly described. Therefore, the presented study focuses on the description of interactions between a model biological membrane (POPG) and one of amyloid forming proteins - human cystatin C. For the purpose of the study molecular dynamics simulations were applied to confirm interactions between the protein and POPG membrane. Next the NMR techniques were used to verify how the data obtained in solution compared to MD simulations and determine fragments of the protein responsible for interactions with POPG. Finally, circular dichroism was used to monitor the changes in secondary structure of the protein and size exclusion chromatography was used to monitor its oligomerization process. Obtained data indicates that the protein interacts with POPG submerging itself into the bilayer with the AS region. However, the presence of POPG bilayer does not significantly affect the structure or oligomerization process of human cystatin C.


Assuntos
Bicamadas Lipídicas , Fosfolipídeos , Humanos , Idoso , Fosfolipídeos/metabolismo , Bicamadas Lipídicas/química , Proteínas Amiloidogênicas/análise , Proteínas Amiloidogênicas/metabolismo , Cistatina C/análise , Cistatina C/metabolismo , Membrana Celular/metabolismo , Amiloide
5.
Biochim Biophys Acta Biomembr ; 1866(3): 184266, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38151198

RESUMO

This work describes the electrochemical studies on the interactions between V57G mutant of human cystatin C (hCC V57G) and membrane bilayer immobilized on the surface of a gold electrode. The electrode was modified with 6-mercaptohexan-1-ol (MCH) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). DMPC was used as a membrane mimetic for monitoring electrochemical changes resulting from the interactions between the functionalized electrode surface and human cystatin C. The interactions between the modified electrode and hCC V57G were investigated by cyclic voltammetry and electrochemical impedance spectroscopy in a phosphate buffered saline (PBS) containing Fe(CN)63-/4- as a redox probe. The electrochemical measurements confirm that fabricated electrode is sensitive to hCC V57G at the concentration of 1 × 10-14 M. The incubation studies carried out at higher concentrations resulted in insignificant changes observed in cyclic voltammetry and electrochemical impedance spectroscopy measurements. The calculated values of surface coverage θR confirm that the electrode is equally covered at higher concentrations of hCC V57G. Measurements of wettability and surface free energy made it possible to determine the influence of individual structural elements of the modified gold electrode on its properties, and thus allowed to understand the nature of the interactions. Contact angle values confirmed the results obtained during electrochemical measurements, indicating the sensitivity of the electrode towards hCC V57G at the concentration of 1 × 10-14 M. In addition, the XPS spectra confirmed the successful anchoring of hCC V57G to the DMPC-functionalized surface.


Assuntos
Bicamadas Lipídicas , Fosfolipídeos , Humanos , Bicamadas Lipídicas/química , Dimiristoilfosfatidilcolina/química , Ouro/química , Cistatina C , Eletrodos
6.
Int J Mol Sci ; 22(24)2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34948248

RESUMO

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.


Assuntos
Oxirredutases/metabolismo , Proteínas Periplásmicas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Sequência de Aminoácidos , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Campylobacter jejuni/patogenicidade , Campylobacter jejuni/fisiologia , Dissulfetos/metabolismo , Oxirredução , Oxirredutases/genética , Periplasma/metabolismo , Proteínas Periplásmicas/genética , Homologia de Sequência de Aminoácidos
7.
Eur J Med Chem ; 224: 113694, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34273660

RESUMO

The glycosylphosphatidylinositol-anchored transmembrane glycoprotein CD160 (cluster of differentiation 160) is a member of the immunoglobulin superfamily. Four isoforms, which differ by the presence or absence of an immunoglobulin-like domain and the mode of anchoring in the cell membrane, have been identified. CD160 has a significant impact on the proper functioning of the immune system by activating natural killer cells and inhibiting T cells. CD160 is a natural ligand for herpes virus entry mediator (HVEM), a member of the tumor necrosis factor superfamily. The CD160-HVEM complex is a rare example of direct interaction between the two different superfamilies. The interaction of these two proteins leads to the inhibition of CD4+ T cells which, in consequence, leads to the inhibition of the correct response of the immune system. Available research articles indicate that CD160 plays a role in various types of cancer, chronic viral diseases, malaria, paroxysmal nocturnal hemoglobinuria, atherosclerosis, autoimmune diseases, skin inflammation, acute liver damage and retinal vascular disease. We present here an overview of the CD160 protein, the general characteristics of the receptor and its isoforms, details of structural studies of CD160 and the CD160-HVEM complex, as well as a description of the role of this protein in selected human diseases.


Assuntos
Antígenos CD/imunologia , Doenças Autoimunes/imunologia , Infecções/imunologia , Neoplasias/imunologia , Receptores Imunológicos/imunologia , Proteínas Ligadas por GPI/imunologia , Humanos
8.
Int J Mol Sci ; 21(22)2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33238640

RESUMO

One of the major current trends in cancer immunotherapy is the blockade of immune checkpoint proteins that negatively regulate the immune response. This has been achieved through antibodies blocking PD-1/PD-L1 and CTLA-4/CD80/CD86 interactions. Such antibodies have revolutionized oncological therapy and shown a new way to fight cancer. Additional (negative) immune checkpoints are also promising targets in cancer therapy and there is a demand for inhibitors for these molecules. Our studies are focused on BTLA/HVEM complex, which inhibits T-cell proliferation and cytokine production and therefore has great potential as a new target for cancer treatment. The goal of the presented studies was the design and synthesis of compounds able to block BTLA/HVEM interactions. For that purpose, the N-terminal fragment of glycoprotein D (gD), which interacts with HVEM, was used. Based on the crystal structure of the gD/HVEM complex and MM/GBSA analysis performed on it, several peptides were designed and synthesized as potential inhibitors of the BTLA/HVEM interaction. Affinity tests, ELISA tests, and cellular-based reporter assays were performed on these compounds to check their ability to bind to HVEM and to inhibit BTLA/HVEM complex formation. For leading peptides candidates, all-atom and subsequent docking simulations with a coarse-grained force field were performed to determine their binding modes. To further evaluate their potential as drug candidates, their stability in plasma and their cytotoxicity effects on PBMCs were assessed. Our data indicate that the peptide gD(1-36)(K10C-T29C) is the best candidate as a future drug. It interacts with HVEM protein, blocks the BTLA/HVEM interaction, and is nontoxic to cells. The present study provides a new perspective on the development of BTLA/HVEM inhibitors that disrupt protein interactions.


Assuntos
Glicoproteínas/farmacologia , Neoplasias/terapia , Peptídeos/farmacologia , Receptores Imunológicos/antagonistas & inibidores , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Glicoproteínas/genética , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia , Ativação Linfocitária/efeitos dos fármacos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/genética , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral/antagonistas & inibidores , Membro 14 de Receptores do Fator de Necrose Tumoral/imunologia
9.
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
10.
Protein Expr Purif ; 164: 105450, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31299214

RESUMO

Herpesvirus entry mediator (HVEM), a member of the TNF-receptor superfamily, plays an important role in the regulation of the immune system. It forms a complex with ligands and can either activate or inhibit the response of the immune system. Furthermore, HVEM can exhibit pro-inflammatory or anti-inflammatory effects in many human diseases. Therefore, understanding the mechanism underlying the interaction of HVEM with other receptors is extremely important to design small therapeutic molecules that can stimulate the response of the immune system. In this study, we attempted to develop the most efficient method for the expression and purification of the extracellular domain of HVEM using Escherichia coli. The soluble fraction constituted only a small portion of the E. coli-expressed protein, whereas majority of the protein was found to be accumulated in the insoluble fraction. Three different protein refolding methods were analyzed: dialysis, dilution, and using chromatographic column. The oligomeric state of the protein was determined by characterizing the obtained fractions using analytical size exclusion chromatography. All the obtained fractions were tested for their ability to form a complex with B- and T-lymphocyte attenuator using enzyme-linked immunosorbent assay. The results of this study provide crucial information regarding the production of HVEM protein in a robust, well-established, and convenient heterologous expression system using E. coli as a host. In addition, it allows for the selection of the most effective method for appropriate refolding of HVEM protein, which gets accumulated in the insoluble fraction.


Assuntos
Escherichia coli/genética , Redobramento de Proteína , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Expressão Gênica , Humanos , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Solubilidade
11.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 4): 577-86, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23519666

RESUMO

Wild-type human cystatin C (hCC wt) is a low-molecular-mass protein (120 amino-acid residues, 13,343 Da) that is found in all nucleated cells. Physiologically, it functions as a potent regulator of cysteine protease activity. While the biologically active hCC wt is a monomeric protein, all crystallization efforts to date have resulted in a three-dimensional domain-swapped dimeric structure. In the recently published structure of a mutated hCC, the monomeric fold was preserved by a stabilization of the conformationally constrained loop L1 caused by a single amino-acid substitution: Val57Asn. Additional hCC mutants were obtained in order to elucidate the relationship between the stability of the L1 loop and the propensity of human cystatin C to dimerize. In one mutant Val57 was substituted by an aspartic acid residue, which is favoured in ß-turns, and in the second mutant proline, a residue known for broadening turns, was substituted for the same Val57. Here, 2.26 and 3.0 Å resolution crystal structures of the V57D andV57P mutants of hCC are reported and their dimeric architecture is discussed in terms of the stabilization and destabilization effects of the introduced mutations.


Assuntos
Substituição de Aminoácidos/genética , Proteínas Amiloidogênicas/química , Proteínas Amiloidogênicas/genética , Cistatina C/química , Cistatina C/genética , Ácido Aspártico/genética , Cristalografia por Raios X , Humanos , Mutagênese Sítio-Dirigida , Prolina/genética , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Valina/genética
12.
Front Mol Neurosci ; 5: 82, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22866027

RESUMO

Human cystatin C (hCC) is a small but very intriguing protein. Produced by all nucleated cells is found in almost all tissues and body fluids where, at physiological conditions, plays a role of a very potent inhibitor of cysteine proteases. Biologically active hCC is a monomeric protein but during cellular trafficking it forms dimers, transiently losing its inhibitory activity. In vitro, dimerization of cystatin C was observed for the mature protein during crystallization trials, revealing that the mechanism of this process is based on the three dimensional swapping of the protein domains. In our work we have focused on the impact of two proposed "hot spots" in cystatin C structure on its conformational stability. Encouraged by promising results of the theoretical calculations, we designed and produced several hCC hinge region point mutation variants that display a variety of conformational stability and propensity for dimerization and aggregation. A similar approach, i.e., rational mutagenesis, has been also applied to study the amyloidogenic L68Q variant to determine the contribution of hydrophobic interactions and steric effect on the stability of monomeric cystatin C. In this overview we would like to summarize the results of our studies. The impact of a particular mutation on the properties of the studied proteins will be presented in the context of their thermal and mechanical stability, in vitro dimerization tendency as well as the outcome of crystallization. Better understanding of the mechanism and, especially, factors affecting conformational stability of cystatin C and access to stable monomeric and dimeric versions of the protein opens new perspectives in explaining the role of dimers and the domain swapping process in hCC oligomerization, as well as designing potential inhibitors of this process.

13.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 67(Pt 12): 1608-11, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22139178

RESUMO

Human cystatin C (hCC) is a low-molecular-mass protein (120 amino-acid residues, 13 343 Da) found in all nucleated cells. Its main physiological role is regulation of the activity of cysteine proteases. Biologically active hCC is a monomeric protein, but all crystallization efforts have resulted in a dimeric domain-swapped structure. Recently, two monomeric structures were reported for cystatin C variants. In one of them stabilization was achieved by abolishing the possibility of domain swapping by the introduction of an additional disulfide bridge connecting the two protein domains (Cys47-Cys69). In the second structure, reported by this group, the monomeric hCC fold was preserved by stabilization of the conformationally constrained loop (L1) by a single-amino-acid substitution (V57N). To further assess the influence of changes in the sequence and properties of loop L1 on the dimerization propensity of cystatin C, two additional hCC mutants were obtained: one with a residue favoured in ß-turns (V57D) and another with proline (V57P), a residue that is known to be a structural element that can rigidify but also broaden turns. Here, the expression, purification and crystallization of V57D and V57P variants of recombinant human cystatin C are described. Crystals were grown by the vapour-diffusion method. Several diffraction data sets were collected using a synchrotron source at the Advanced Photon Source, Argonne National Laboratory, Chicago, USA.


Assuntos
Cistatina C/química , Mutação , Cristalização , Cristalografia por Raios X , Cistatina C/genética , Cistatina C/metabolismo , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Valina/genética , Valina/metabolismo
14.
J Struct Biol ; 173(2): 406-13, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21074623

RESUMO

Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a ß-turn into a part of a long ß-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the ß-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a ß-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel ß-sheet wrapped around an α-helix. Here we report a 2.04 Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.


Assuntos
Cistatina C/química , Cistatina C/metabolismo , Animais , Cistatina C/genética , Cistatinas/química , Cistatinas/genética , Cistatinas/metabolismo , Humanos , Multimerização Proteica , Estrutura Secundária de Proteína
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA