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1.
Cells ; 11(3)2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-35159195

RESUMO

Intracellular peptides (InPeps) generated by proteasomes were previously suggested as putative natural regulators of protein-protein interactions (PPI). Here, the main aim was to investigate the intracellular effects of intracellular peptide VFDVELL (VFD7) and related peptides on PPI. The internalization of the peptides was achieved using a C-terminus covalently bound cell-penetrating peptide (cpp; YGRKKRRQRRR). The possible inhibition of PPI was investigated using a NanoBiT® luciferase structural complementation reporter system, with a pair of plasmids vectors each encoding, simultaneously, either FK506-binding protein (FKBP) or FKBP-binding domain (FRB) of mechanistic target of rapamycin complex 1 (mTORC1). The interaction of FKBP-FRB within cells occurs under rapamycin induction. Results shown that rapamycin-induced interaction between FKBP-FRB within human embryonic kidney 293 (HEK293) cells was inhibited by VFD7-cpp (10-500 nM) and FDVELLYGRKKRRQRRR (VFD6-cpp; 1-500 nM); additional VFD7-cpp derivatives were either less or not effective in inhibiting FKBP-FRB interaction induced by rapamycin. Molecular dynamics simulations suggested that selected peptides, such as VFD7-cpp, VFD6-cpp, VFAVELLYGRKKKRRQRRR (VFA7-cpp), and VFEVELLYGRKKKRRQRRR (VFA7-cpp), bind to FKBP and to FRB protein surfaces. However, only VFD7-cpp and VFD6-cpp induced changes on FKBP structure, which could help with understanding their mechanism of PPI inhibition. InPeps extracted from HEK293 cells were found mainly associated with macromolecular components (i.e., proteins and/or nucleic acids), contributing to understanding InPeps' intracellular proteolytic stability and mechanism of action-inhibiting PPI within cells. In a model of cell death induced by hypoxia-reoxygenation, VFD6-cpp (1 µM) increased the viability of mouse embryonic fibroblasts cells (MEF) expressing mTORC1-regulated autophagy-related gene 5 (Atg5), but not in autophagy-deficient MEF cells lacking the expression of Atg5. These data suggest that VFD6-cpp could have therapeutic applications reducing undesired side effects of rapamycin long-term treatments. In summary, the present report provides further evidence that InPeps have biological significance and could be valuable tools for the rational design of therapeutic molecules targeting intracellular PPI.


Assuntos
Sirolimo , Proteína 1A de Ligação a Tacrolimo , Animais , Autofagia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Peptídeos/farmacologia , Sirolimo/farmacologia , Tacrolimo , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo
2.
J Neurochem ; 150(3): 296-311, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31206169

RESUMO

Temporal lobe epilepsy (TLE) is a chronic disease, characterized by severe and refractory seizures, triggered in the hippocampus and/or amygdala, disrupting the blood-brain barrier. This disruption can sustain, or aggravate, the epileptic condition. The aim of this study was to evaluate the activation of the kallikrein-kinin system in patients with TLE, as it relates to the maintenance of blood-brain barrier. Human hippocampal sclerotic tissues removed after surgery for seizure control, plasma, and serum were used in the following assays: immunostaining for white blood cells in the TLE hippocampus, C-reactive protein in serum, quantification of plasma kallikrein (PKal) and cathepsin B (CatB) activity in serum and plasma, quantification of C1-inhibitor, analysis of high-molecular-weight kininogen (H-kininogen) fragments, and activation of plasma prekallikrein for comparison with healthy controls. Infiltration of white blood cells in the sclerotic hippocampus and a significant increase in the neutrophil/lymphocyte ratio in the blood of TLE patients were observed. High levels of C-reactive protein (TLE = 1.4 ± 0.3 µg/mL), PKal (TLE = 5.4 ± 0.4 U/mL), and CatB (TLE = 4.9 ± 0.4 U/mL) were also evident in the serum of TLE patients comparing to controls. A strong linear correlation was observed between active CatB and PKal in the serum of TLE patients (r = 0.88). High levels of cleaved H-kininogen and free PKal, and low levels of C1-inhibitor (TLE = 188 ± 12 µg/mL) were observed in the serum of TLE patients. Our data demonstrated that the plasma kallikrein-kinin system is activated in patients with TLE. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.


Assuntos
Catepsina B/sangue , Epilepsia do Lobo Temporal/metabolismo , Inflamação/metabolismo , Sistema Calicreína-Cinina/fisiologia , Calicreínas/sangue , Adulto , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade
3.
Mol Biochem Parasitol ; 150(1): 83-95, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16887207

RESUMO

RNA triphosphatases act in the first step of the mRNA capping process, removing the gamma-phosphoryl group from the 5' end of nascent RNA. A metal-dependent catalysis is found in the enzymes from trypanosomes and several other lower eukaryotes. This contrasts with the cysteine-dependent activity of the corresponding enzymes of mammals, a difference that points to these enzymes as potential targets for drug design. This work describes the identification, expression, purification, enzyme kinetics, and the role of divalent metal in the ATPase activity of the RNA triphosphatase from Trypanosoma cruzi, the agent of Chagas' disease, and compares it with the previously characterized enzyme from Trypanosoma brucei. Sequence similarity of the T. cruzi enzyme with the RNA triphosphatase of Saccharomyces cerevisiae indicates that a tunnel domain containing the divalent metal forms its active site. Based on enzyme kinetics, circular dichroism, and intrinsic fluorescence analysis, a kinetic mechanism for the ATPase activity of the T. cruzi tunnel triphosphatase is proposed. A single metal is sufficient to interact with the enzyme through the formation of a productive MnATP-enzyme complex, while free ATP inhibits activity. Manganese is also required for the tunnel stability of the T. cruzi enzyme, while the T. brucei homologue remains stable in the absence of metal, as shown for other triphosphatases. These findings may be useful to devise specific triphosphatase inhibitors to the T. cruzi enzyme.


Assuntos
Hidrolases Anidrido Ácido/química , Hidrolases Anidrido Ácido/metabolismo , Trypanosoma cruzi/enzimologia , Hidrolases Anidrido Ácido/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Catálise , Dicroísmo Circular , Coenzimas/metabolismo , Estabilidade Enzimática , Fluorescência , Temperatura Alta , Cinética , Manganês/metabolismo , Dados de Sequência Molecular , RNA/metabolismo , Alinhamento de Sequência , Trypanosoma brucei brucei/enzimologia
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