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/metabolismoRESUMO
Inflammatory arthritis, such as rheumatoid arthritis (RA), stands out as one of the main sources of pain and impairment to the quality of life. The use of hemopressin (PVNFKFLSH; Hp), an inverse agonist of type 1 cannabinoid receptor, has proven to be effective in producing analgesia in pain models, but its effect on neuro-inflammatory aspects of RA is limited. In this study, antigen-induced arthritis (AIA) was evoked by the intraarticular (i.art.) injection of methylated bovine serum albumin (mBSA) in male Sprague Dawley rats. Phosphate buffered saline (PBS)-injected ipsilateral knee joints or AIA contralateral were used as control. Nociceptive and inflammatory parameters such as knee joint oedema and leukocyte influx and histopathological changes were carried out in addition to the local measurement of interleukins (IL) IL-6, IL-1ß, tumor necrosis factor-α and the immunoreactivity of the neuropeptides substance P (SP) and calcitonin gene related peptide (CGRP) in the spinal cord (lumbar L3-5 segments) of AIA rats. For 4 days, AIA rats were treated daily with a single administration of saline, Hp injected (10 or 20 µg/day, i.art.), Hp given orally (20 µg/Kg, p.o.) or indomethacin (Indo; 5 mg/Kg, i.p.). In comparison to the PBS control group, the induction of AIA produced a significant and progressive mono-arthritis condition. The degree of AIA severity progressively compromised the normal walking pattern and impaired mobility over the next four days in relation to PBS-injected rats or contralateral knee joints. In AIA rats, the reduction of the distance between footprints and disturbances of gait evidenced signs of nociception. This response worsened at day 4, and a loss of footprint from the ipsilateral hind paw was evident. Daily treatment of the animals with Hp either i.art. (10 and 20 µg/knee) or p.o. (20 µg/Kg) as well as Indo (5 mg/Kg, i.p.) ameliorated the impaired mobility in a time-dependent manner (P < 0.05). In parallel, the AIA-injected ipsilateral knee joints reach a peak of swelling 24 h after AIA induction, which persisted over the next four days in relation to PBS-injected rats or contralateral knee joints. There was a significant but not dose-dependent inhibitory effect produced by all dosages and routes of Hp treatments on AIA-induced knee joint swelling (P < 0.05). In addition, the increased synovial levels of MPO activity, total leukocytes number and IL-6, but not IL-1ß, were significantly reduced by the lower i.art. dose of Hp. In conclusion, these results successfully demonstrate that Hp may represent a novel therapeutic strategy to treat RA, an effect which is unrelated to the proinflammatory actions of the neuropeptides CGRP and SP.
Assuntos
Anti-Inflamatórios/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Hemoglobinas/farmacologia , Dor Nociceptiva/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Administração Oral , Animais , Anti-Inflamatórios/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Citocinas/metabolismo , Edema/tratamento farmacológico , Marcha/efeitos dos fármacos , Hemoglobinas/administração & dosagem , Inflamação/tratamento farmacológico , Injeções Intra-Articulares , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/metabolismo , Articulação do Joelho/patologia , Leucócitos/efeitos dos fármacos , Masculino , Fragmentos de Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Substância P/metabolismoRESUMO
Thimet oligopeptidase (EC 3.4.24.15; EP24.15, THOP1) is a metallopeptidase ubiquitously distributed in mammalian tissues. Beyond its previously well characterized role in major histocompatibility class I (MHC-I) antigen presentation, the recent characterization of the THOP1 C57BL6/N null mice (THOP1-/-) phenotype suggests new key functions for THOP1 in hyperlipidic diet-induced obesity, insulin resistance and non-alcoholic liver steatosis. Distinctive levels of specific intracellular peptides (InPeps), genes and microRNAs were observed when comparing wild type C57BL6/N to THOP1-/- fed either standard or hyperlipidic diets. A possible novel mechanism of action was suggested for InPeps processed by THOP1, which could be modulating protein-protein interactions and microRNA processing, thus affecting the phenotype. Together, research into the biochemical and biomedical significance of THOP1 suggests that degradation by the proteasome is a step in the processing of various proteins, not merely for ending their existence. This allows many functional peptides to be generated by proteasomal degradation in order to, for example, control mRNA translation and the formation of protein complexes.
Assuntos
Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno , Domínio Catalítico , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Metabolismo Energético , Feminino , Estudos de Associação Genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Neuropeptídeos/metabolismo , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
Overweight and obesity are among the most prominent health problems in the modern world, mostly because they are either associated with or increase the risk of other diseases such as type 2 diabetes, hypertension, and/or cancer. Most professional organizations define overweight and obesity according to individual body-mass index (BMI, weight in kilograms divided by height squared in meters). Overweight is defined as individuals with BMI from 25 to 29, and obesity as individuals with BMI ≥30. Obesity is the result of genetic, behavioral, environmental, physiological, social, and cultural factors that result in energy imbalance and promote excessive fat deposition. Despite all the knowledge concerning the pathophysiology of obesity, which is considered a disease, none of the existing treatments alone or in combination can normalize blood glucose concentration and prevent debilitating complications from obesity. This review discusses some new perspectives for overweight and obesity treatments, including the use of the new orally active cannabinoid peptide Pep19, the advantage of which is the absence of undesired central nervous system effects usually experienced with other cannabinoids.
Assuntos
Índice de Massa Corporal , Canabinoides/uso terapêutico , Diabetes Mellitus Tipo 2 , Proteínas do Tecido Nervoso/uso terapêutico , Obesidade , Peptídeos/uso terapêutico , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/fisiopatologia , Humanos , Obesidade/sangue , Obesidade/tratamento farmacológico , Obesidade/fisiopatologiaRESUMO
Thimet oligopeptidase (EC 3.4.24.15; EP24.15; THOP1) is a potential therapeutic target, as it plays key biological functions in processing biologically functional peptides. The structural conformation of THOP1 provides a unique restriction regarding substrate size, in that it only hydrolyzes peptides (optimally, those ranging from eight to 12 amino acids) and not proteins. The proteasome activity of hydrolyzing proteins releases a large number of intracellular peptides, providing THOP1 substrates within cells. The present study aimed to investigate the possible function of THOP1 in the development of diet-induced obesity (DIO) and insulin resistance by utilizing a murine model of hyperlipidic DIO with both C57BL6 wild-type (WT) and THOP1 null (THOP1-/-) mice. After 24 weeks of being fed a hyperlipidic diet (HD), THOP1-/- and WT mice ingested similar chow and calories; however, the THOP1-/- mice gained 75% less body weight and showed neither insulin resistance nor non-alcoholic fatty liver steatosis when compared to WT mice. THOP1-/- mice had increased adrenergic-stimulated adipose tissue lipolysis as well as a balanced level of expression of genes and microRNAs associated with energy metabolism, adipogenesis, or inflammation. Altogether, these differences converge to a healthy phenotype of THOP1-/- fed a HD. The molecular mechanism that links THOP1 to energy metabolism is suggested herein to involve intracellular peptides, of which the relative levels were identified to change in the adipose tissue of WT and THOP1-/- mice. Intracellular peptides were observed by molecular modeling to interact with both pre-miR-143 and pre-miR-222, suggesting a possible novel regulatory mechanism for gene expression. Therefore, we successfully demonstrated the previously unanticipated relevance of THOP1 in energy metabolism regulation. It was suggested that intracellular peptides were responsible for mediating the phenotypic differences that are described herein by a yet unknown mechanism of action.
Assuntos
Metabolismo Energético , Metaloendopeptidases/metabolismo , Obesidade/metabolismo , Adipogenia , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Deleção de Genes , Resistência à Insulina , Lipólise , Masculino , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/genéticaRESUMO
Previous studies suggested the pharmacological potential of rat hemopressin (PVNFKFLSH) and its shorter synthetic peptide NFKF, to protect from pilocarpine-induced seizures in mice. Orally administered NFKF was shown to be hundred times more potent than cannabidiol in delaying the first seizure induced by pilocarpine in mice. Here, using an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis we have shown that C57BL/6 J mice orally administrated with NFKF (500 µg/kg) presented better EAE clinical scores and improved locomotor activity compared to saline administrated control mice. NFKF blocked the production of IL-1beta and IL-6, and has high scores binding cannabinoid type 2 receptors. Therefore, NFKF is an exciting new possibility to neurodegenerative diseases therapeutics.
Assuntos
Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/prevenção & controle , Hemoglobinas/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Animais , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Hemoglobinas/química , Hemoglobinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular/métodos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , RatosRESUMO
Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.