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1.
Methods Mol Biol ; 2383: 257-264, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34766295

RESUMO

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step.


Assuntos
Peptídeos Penetradores de Células/análise , Escherichia coli/genética , Produtos do Gene tat , Humanos , Pirazinas , Proteínas Recombinantes de Fusão/genética , Fatores de Transcrição
2.
PLoS One ; 16(9): e0256715, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34499687

RESUMO

The twin-arginine translocation (Tat) pathway transports folded proteins across energetic membranes. Numerous Tat substrates contain co-factors that are inserted before transport with the assistance of redox enzyme maturation proteins (REMPs), which bind to the signal peptide of precursor proteins. How signal peptides are transferred from a REMP to a binding site on the Tat receptor complex remains unknown. Since the signal peptide mediates both interactions, possibilities include: i) a coordinated hand-off mechanism; or ii) a diffusional search after REMP dissociation. We investigated the binding interaction between substrates containing the TorA signal peptide (spTorA) and its cognate REMP, TorD, and the effect of TorD on the in vitro transport of such substrates. We found that Escherichia coli TorD is predominantly a monomer at low micromolar concentrations (dimerization KD > 50 µM), and this monomer binds reversibly to spTorA (KD ≈ 1 µM). While TorD binds to membranes (KD ≈ 100 nM), it has no apparent affinity for Tat translocons and it inhibits binding of a precursor substrate to the membrane. TorD has a minimal effect on substrate transport by the Tat system, being mildly inhibitory at high concentrations. These data are consistent with a model in which the REMP-bound signal peptide is shielded from recognition by the Tat translocon, and spontaneous dissociation of the REMP allows the substrate to engage the Tat machinery. Thus, the REMP does not assist with targeting to the Tat translocon, but rather temporarily shields the signal peptide.


Assuntos
Proteínas de Escherichia coli/genética , Produtos do Gene tat/genética , Chaperonas Moleculares/genética , Oxirredutases N-Desmetilantes/genética , Sistema de Translocação de Argininas Geminadas/genética , Sítios de Ligação/genética , Escherichia coli/genética , Ligação Proteica/genética , Sinais Direcionadores de Proteínas/genética , Transporte Proteico/genética , Especificidade por Substrato
3.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34204001

RESUMO

Radiodynamic therapy (RDT) is a recent extension of conventional photodynamic therapy, in which visible/near infrared light irradiation is replaced by a well-tolerated dose of high-energy X-rays. This enables greater tissue penetration to allow non-invasive treatment of large, deep-seated tumors. We report here the design and testing of a drug delivery system for RDT that is intended to enhance intra- or peri-nuclear localization of the photosensitizer, leading to DNA damage and resulting clonogenic cell kill. This comprises a photosensitizer (Verteporfin, VP) incorporated into poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) that are surface-functionalized with a cell-penetrating HIV trans-activator of transcription (TAT) peptide. In addition to a series of physical and photophysical characterization studies, cytotoxicity tests in pancreatic (PANC-1) cancer cells in vitro under 4 Gy X-ray exposure from a clinical 6 MV linear accelerator (LINAC) showed that TAT targeting of the nanoparticles markedly enhances the effectiveness of RDT treatment, particularly when assessed by a clonogenic, i.e., DNA damage-mediated, cell kill.


Assuntos
Composição de Medicamentos , Produtos do Gene tat/química , Nanopartículas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Verteporfina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , DNA/metabolismo , Endocitose/efeitos dos fármacos , Humanos , Lipídeos de Membrana/metabolismo , Nanopartículas/ultraestrutura , Oxigênio Singlete/metabolismo
4.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205205

RESUMO

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.


Assuntos
Antioxidantes/farmacologia , Células da Medula Óssea/citologia , Peptídeos Penetradores de Células/genética , Produtos do Gene tat/genética , Proteínas Recombinantes de Fusão/farmacologia , Superóxido Dismutase-1/genética , Animais , Antioxidantes/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Peptídeos Penetradores de Células/metabolismo , Células Cultivadas , Produtos do Gene tat/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase-1/metabolismo , Irradiação Corporal Total
5.
mBio ; 12(3): e0130221, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34154411

RESUMO

The cell envelope of Gram-negative bacteria consists of two membranes surrounding the periplasm and peptidoglycan layer. ß-Lactam antibiotics target the periplasmic penicillin-binding proteins that synthesize peptidoglycan, resulting in cell death. The primary means by which bacterial species resist the effects of ß-lactam drugs is to populate the periplasmic space with ß-lactamases. Resistance to ß-lactam drugs is spread by lateral transfer of genes encoding ß-lactamases from one species of bacteria to another. However, the resistance phenotype depends in turn on these "alien" protein sequences being recognized and exported across the cytoplasmic membrane by either the Sec or Tat protein translocation machinery of the new bacterial host. Here, we examine BKC-1, a carbapenemase from an unknown bacterial source that has been identified in a single clinical isolate of Klebsiella pneumoniae. BKC-1 was shown to be located in the periplasm, and functional in both K. pneumoniae and Escherichia coli. Sequence analysis revealed the presence of an unusual signal peptide with a twin arginine motif and a duplicated hydrophobic region. Biochemical assays showed this signal peptide directs BKC-1 for translocation by both Sec and Tat translocons. This is one of the few descriptions of a periplasmic protein that is functionally translocated by both export pathways in the same organism, and we suggest it represents a snapshot of evolution for a ß-lactamase adapting to functionality in a new host. IMPORTANCE Bacteria can readily acquire plasmids via lateral gene transfer (LGT). These plasmids can carry genes for virulence and antimicrobial resistance (AMR). Of growing concern are LGT events that spread ß-lactamases, particularly carbapenemases, and it is important to understand what limits this spread. This study provides insight into the sequence features of BKC-1 that exemplify the limitations on the successful biogenesis of ß-lactamases, which is one factor limiting the spread of AMR phenotypes by LGT. With a very simple evolutionary adaptation, BKC-1 could become a more effective carbapenemase, underscoring the need to understand the evolution, adaptability, and functional assessment of newly reported ß-lactamases rapidly and thoroughly.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos do Gene tat/genética , Klebsiella pneumoniae/genética , Canais de Translocação SEC/genética , beta-Lactamases/genética , beta-Lactamases/metabolismo , Antibacterianos/farmacologia , Transporte Biológico , Escherichia coli/genética , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/enzimologia , Testes de Sensibilidade Microbiana , Periplasma/metabolismo , beta-Lactamas/farmacologia
6.
Chem Biol Interact ; 344: 109495, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33961834

RESUMO

Cataracts, a clouding of the eye lens, are a leading cause of visual impairment and are responsible for one of the most commonly performed surgical procedures worldwide. Although generally safe and effective, cataract surgery can lead to a secondary lens abnormality due to transition of lens epithelial cells to a mesenchymal phenotype (EMT) and opacification of the posterior lens capsular bag. Occurring in up to 40% of cataract cases over time, posterior capsule opacification (PCO) introduces additional treatment costs and reduced quality of life for patients. Studies have shown that PCO pathogenesis is driven in part by TGF-ß, signaling through the action of the family of Smad coactivators to effect changes in gene transcription. In the present study, we evaluated the ability of Smad-7, a well characterized inhibitor of TGF-ß -mediated Smad signaling, to suppress the EMT response in lens epithelial cells associated with PCO pathogenesis. Treatment of lens epithelial cells with a cell-permeable form of Smad7 variant resulted in suppressed expression of EMT markers such as alpha smooth muscle actin and fibronectin. A single application of cell-permeable Smad7 variant in the capsular bag of a mouse cataract surgery model resulted in suppression of gene transcripts encoding alpha smooth muscle actin and fibronectin. These results point to Smad7 as a promising biotherapeutic agent for prevention or substantial reduction in the incidence of PCO following cataract surgery.


Assuntos
Opacificação da Cápsula/prevenção & controle , Peptídeos Penetradores de Células/uso terapêutico , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Produtos do Gene tat/uso terapêutico , Cristalino/efeitos dos fármacos , Proteína Smad7/uso terapêutico , Actinas/metabolismo , Animais , Opacificação da Cápsula/etiologia , Opacificação da Cápsula/patologia , Catarata/complicações , Catarata/patologia , Células Epiteliais/efeitos dos fármacos , Cristalino/patologia , Camundongos Transgênicos , Domínios Proteicos , Proteínas Recombinantes/uso terapêutico
7.
Cells ; 10(4)2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919804

RESUMO

Recent studies of cerebral hypoxia-ischemia (HI) have highlighted slowly progressive neurodegeneration whose mechanisms remain elusive, but if blocked, could considerably improve long-term neurological function. We previously established that the cytokine transforming growth factor (TGF)ß1 is highly elevated following HI and that delivering an antagonist for TGFß receptor activin-like kinase 5 (ALK5)-SB505124-three days after injury in a rat model of moderate pre-term HI significantly preserved the structural integrity of the thalamus and hippocampus as well as neurological functions associated with those brain structures. To elucidate the mechanism whereby ALK5 inhibition reduces cell death, we assessed levels of autophagy markers in neurons and found that SB505124 increased numbers of autophagosomes and levels of lipidated light chain 3 (LC3), a key protein known to mediate autophagy. However, those studies did not determine whether (1) SB was acting directly on the CNS and (2) whether directly inducing autophagy could decrease cell death and improve outcome. Here we show that administering an ALK5 antagonist three days after HI reduced actively apoptotic cells by ~90% when assessed one week after injury. Ex vivo studies using the lysosomal inhibitor chloroquine confirmed that SB505124 enhanced autophagy flux in the injured hemisphere, with a significant accumulation of the autophagic proteins LC3 and p62 in SB505124 + chloroquine treated brain slices. We independently activated autophagy using the stimulatory peptide Tat-Beclin1 to determine if enhanced autophagy is directly responsible for improved outcomes. Administering Tat-Beclin1 starting three days after injury preserved the structural integrity of the hippocampus and thalamus with improved sensorimotor function. These data support the conclusion that intervening at this phase of injury represents a window of opportunity where stimulating autophagy is beneficial.


Assuntos
Autofagia , Lesões Encefálicas/etiologia , Lesões Encefálicas/patologia , Hipóxia-Isquemia Encefálica/complicações , Animais , Animais Recém-Nascidos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/administração & dosagem , Benzodioxóis/farmacologia , Produtos do Gene tat/administração & dosagem , Hipóxia-Isquemia Encefálica/patologia , Imidazóis/farmacologia , Neocórtex/patologia , Degeneração Neural/patologia , Piridinas/farmacologia , Ratos Wistar
8.
Proc Natl Acad Sci U S A ; 118(12)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33723047

RESUMO

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between "closed" and "opened" states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.


Assuntos
Membrana Celular/metabolismo , Produtos do Gene tat/metabolismo , Ativação do Canal Iônico , Íons/metabolismo , Canais de Translocação SEC/metabolismo , Arginina/metabolismo , Peptídeos Penetradores de Células/metabolismo , Ligação Proteica , Transporte Proteico
9.
Cells ; 10(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33572372

RESUMO

The present study explored the effects of endophilin A1 (SH3GL2) against oxidative damage brought about by H2O2 in HT22 cells and ischemic damage induced upon transient forebrain ischemia in gerbils. Tat-SH3GL2 and its control protein (Control-SH3GL2) were synthesized to deliver it to the cells by penetrating the cell membrane and blood-brain barrier. Tat-SH3GL2, but not Control-SH3GL2, could be delivered into HT22 cells in a concentration- and time-dependent manner and the hippocampus 8 h after treatment in gerbils. Tat-SH3GL2 was stably present in HT22 cells and degraded with time, by 36 h post treatment. Pre-incubation with Tat-SH3GL2, but not Control-SH3GL2, significantly ameliorated H2O2-induced cell death, DNA fragmentation, and reactive oxygen species formation. SH3GL2 immunoreactivity was decreased in the gerbil hippocampal CA1 region with time after ischemia, but it was maintained in the other regions after ischemia. Tat-SH3GL2 treatment in gerbils appreciably improved ischemia-induced hyperactivity 1 day after ischemia and the percentage of NeuN-immunoreactive surviving cells increased 4 days after ischemia. In addition, Tat-SH3GL2 treatment in gerbils alleviated the increase in lipid peroxidation as assessed by the levels of malondialdehyde and 8-iso-prostaglandin F2α and in pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1ß, and interleukin-6; while the reduction of protein levels in markers for synaptic plasticity, such as postsynaptic density 95, synaptophysin, and synaptosome associated protein 25 after transient forebrain ischemia was also observed. These results suggest that Tat-SH3GL2 protects neurons from oxidative and ischemic damage by reducing lipid peroxidation and inflammation and improving synaptic plasticity after ischemia.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Hipocampo/patologia , Peroxidação de Lipídeos , Plasticidade Neuronal , Neurônios/patologia , Fármacos Neuroprotetores/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Animais , Isquemia Encefálica/fisiopatologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Produtos do Gene tat/metabolismo , Gerbillinae , Hipocampo/fisiopatologia , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos/efeitos dos fármacos , Camundongos , Atividade Motora/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Fatores de Tempo
10.
Life Sci ; 266: 118847, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33309720

RESUMO

Immunotherapy methods using potential tumor microenvironment modulators have elicited durable therapeutic responses in cancer treatment. Immune checkpoint molecule programmed cell death-ligand 1 (PD-L1) and oncogenic transcription factor STAT3 (signal transducer and activator of transcription-3) assigned as inhibitory targets of our study and particular delivery system designed to deliver small interfering RNAs (siRNAs) to silence the targeted genes. Generated trimethyl chitosan (TMC) and thiolated chitosan (TC) nanoparticles (NPs) conjugated with HIV-1-derived TAT peptide and HA (hyaluronic acid) exhibited eligible physicochemical characteristics, notable siRNA encapsulation, serum stability, non-toxicity, controlled siRNA release, and extensive cellular uptake by cancer cells. Dual inhibition with STAT3/PD-L1 siRNA-loaded HA-TAT-TMC-TC NPs led to promising results, including significant downregulation of PD-L1 and STAT3 genes, striking suppressive effects on proliferation, migration, and angiogenesis of breast and melanoma cancer cell lines, and restrained tumor growth in vivo. These findings infer the capability of HA-TAT-TMC-TC NPs containing STAT3/PD-L1 siRNAs as a novel tumor-suppressive candidate in cancer treatment.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Neoplasias da Mama/terapia , Melanoma Experimental/terapia , Nanopartículas/administração & dosagem , Nanopartículas/química , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Quitosana/química , Progressão da Doença , Feminino , Produtos do Gene tat/química , Humanos , Ácido Hialurônico/química , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Anim Biotechnol ; 32(1): 100-105, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31476967

RESUMO

Nanotechnology is a fast-growing research technology. Nanoparticles have intensive scientific applications in many fields. Depending on the physical and chemical characteristics of a nanoparticle, it can be used either as a treatment agent to fight disease or as a delivery vehicle to transport the therapeutic drug to a specified biological organ, tissue, and cell. Cytotoxicity evaluation of nanoparticles is one of the primary concerns in clinical practices to avoid unpredicted or undesirable interactions that could worsen the case. Iron oxide nanoparticle (IONP) is the most utilized nanoparticle in medical fields for treatment, diagnostic, and imaging. This paper is designated to investigate the cytotoxicity of IONPs that decorated with Trans-Activator of Transcription (TAT) protein. WST-1 assay and flow cytometry were used to assess the cytotoxicity of TAT-IONPs, which showed no significant cytotoxic effect on mammalian breast cancer cells (MCF-7). Nanoparticles accumulation in the cell's cytoplasm was evaluated from TEM images by measuring the size of the endosome. The results indicate that TAT-IONPs can be used as a safe and non-toxic nanoplatform for targeted delivery at 50 µg/ml or less. Also, they present an approach by which the area of intracellular endosome can be assessed from the TEM images of fixed cells. In this study, the endosome size increased in a time-dependent manner.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Produtos do Gene tat/química , Nanopartículas Magnéticas de Óxido de Ferro , Humanos , Células MCF-7 , Nanopartículas Magnéticas de Óxido de Ferro/química , Nanopartículas Magnéticas de Óxido de Ferro/toxicidade , Sais de Tetrazólio
12.
Neuropharmacology ; 181: 108326, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-32966847

RESUMO

We have previously shown that sphingosine kinase 2 (SPK2) interacts with Bcl-2 via its BH3 domain, activating autophagy by inducing the dissociation of Beclin-1/Bcl-2 complexes, and that a TAT-SPK2 peptide containing the BH3 domain of SPK2 protects neurons against ischemic injury. The goals of the present study were to establish the functional significance of these findings, by testing whether TAT-SPK2 was effective in a mouse model of ischemic stroke, and to explore potential underlying mechanisms. Mice were administered with TAT-SPK2 by intraperitoneal injection before or after transient middle cerebral artery occlusion (tMCAO). Infarct volume, neurological deficit and brain water content were assessed 24 h after reperfusion. Mitophagy inhibitor Mdivi-1 and BNIP3 siRNAs were used to examine the involvement of BNIP3-dependent mitophagy in the neuroprotection of TAT-SPK2. Mitophagy was quantified by immunoblotting, immunofluorescence and electron microscopy. The interaction between TAT-SPK2 and Bcl-2, Bcl-2 and BNIP3 was detected by co-immunoprecipitation. In the tMCAO model, pre-treatment with TAT-SPK2 significantly reduced infarct volume, improved neurological function and decreased brain edema. Neuroprotection by TAT-SPK2 was still seen when the peptide was administered 3 h after reperfusion. TAT-SPK2 also significantly improved functional recovery and reduced long-term brain atrophy of the ischemic hemisphere 30 days after administration. Our studies further showed that TAT-SPK2 directly binds to Bcl-2 and disrupts Bcl-2/Beclin-1 or Bcl-2/BNIP3 complexes to induce mitophagy. These results suggest that TAT-SPK2 protects neurons against ischemia reperfusion injury by activating BNIP3-mediated mitophagy. Agents exploiting this molecular mechanism are potential candidates for the treatment of ischemic stroke.


Assuntos
Produtos do Gene tat/farmacologia , Proteínas de Membrana/agonistas , Proteínas Mitocondriais/agonistas , Mitofagia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Autofagia , Proteína Beclina-1 , Edema Encefálico/prevenção & controle , Infarto da Artéria Cerebral Média/tratamento farmacológico , AVC Isquêmico/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia
13.
Exp Eye Res ; 199: 108180, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32777209

RESUMO

PURPOSE: Previously we developed TAT-N24 as a synthetic cell-permeable peptide inhibitor of p55PIK signaling and demonstrated its anti-inflammatory effects. This study aimed to evaluate the potential of TAT-N24 as a new agent for the treatment of ocular inflammatory diseases. METHODS: The endotoxin-induced uveitis (EIU) model was established by intravitreal injection of lipopolysaccharide (LPS) in BALB/c mice and experimental autoimmune uveitis (EAU) model was established by subcutaneous injection of a peptide spanning amino acid residues 161-180 of interphotoreceptor retinoid binding protein (IRBP161-180) with complete Freund's adjuvant (CFA) in B10.RIII mice. TAT-N24 was topically administered in EIU model and intraperitoneally administered in EAU model. The severity levels of uveitis were assessed by clinical and histopathological scores. The mRNA levels of inflammatory cytokines in iris-ciliary body (ICB) and retina were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The protein levels of inflammatory factors were determined by ELISA or Western blotting. RESULTS: The results showed that TAT-N24 alleviated clinical signs, decreased inflammatory cell infiltration and the expression of inflammatory cytokines in both EIU and EAU models. Furthermore, protein levels of tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in aqueous humor and mRNA and protein levels of NF-κB p65 in the ICB significantly decreased in EIU model. In EAU model, TAT-N24 application induced a significant decrease of IFN-gamma (IFN-γ) and interleukin-17 (IL-17) in the retina, which were secreted by Th1 and Th17 cells, respectively. CONCLUSION: In conclusion, TAT-N24 suppressed intraocular inflammation in both EIU and EAU models, and the anti-inflammatory effects were mediated by suppressing the expression of inflammatory cytokines by PI3K/NF-κB signaling pathway. TAT-N24 could be potential candidate for the treatment of ocular inflammatory diseases.


Assuntos
Citocinas/metabolismo , Produtos do Gene tat/farmacologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Uveíte/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Uveíte/tratamento farmacológico , Uveíte/patologia
14.
Cells ; 9(8)2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32756411

RESUMO

Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the C-terminal domain of cannabinoid 1 receptor (CB1R) and regulates CB1R activities. In this study, we made Tat-CRIP1a fusion proteins to enhance CRIP1a penetration into neurons and brain and to evaluate the function of CRIP1a in neuroprotection following oxidative stress in HT22 hippocampal cells and transient forebrain ischemia in gerbils. Purified exogenous Tat-CRIP1a was penetrated into HT22 cells in a time and concentration-dependent manner and prevented H2O2-induced reactive oxygen species formation, DNA fragmentation, and cell damage. Tat-CRIP1a fusion protein also ameliorated the reduction of 14-3-3η expression by H2O2 treatment in HT22 cells. Ischemia-reperfusion damage caused motor hyperactivity in the open field test of gerbils; however, the treatment of Tat-CRIP1a significantly reduced hyperactivity 1 day after ischemia. Four days after ischemia, the administration of Tat-CRIP1a restored the loss of pyramidal neurons and decreased reactive astrocytosis and microgliosis induced by ischemic damage in the hippocampal cornu Ammonis (CA)1 region. Ischemic damage decreased 14-3-3η expression in all hippocampal sub-regions 4 days after ischemia; however, the treatment of Tat-CRIP1 ameliorated the reduction of 14-3-3η expression. These results suggest that Tat-CRIP1a attenuates neuronal damage and hyperactivity induced by ischemic damage, and it restores normal expression levels of 14-3-3η protein in the hippocampus.


Assuntos
Proteínas 14-3-3/genética , Produtos do Gene tat/genética , Isquemia/patologia , Proteínas de Membrana/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Proteínas 14-3-3/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Dano ao DNA , Gerbillinae , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Isquemia/tratamento farmacológico , Isquemia/metabolismo , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Atividade Motora/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
15.
Osteoarthritis Cartilage ; 28(10): 1394-1400, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32683043

RESUMO

OBJECT: Autophagy maintains cartilage homeostasis and is compromised during osteoarthritis (OA), contributing to cartilage degeneration. We sought to determine if D-isomer TAT-Beclin-1, a potent inducer of autophagy, could attenuate post-traumatic OA in mice. METHODS: 10-week-old mice underwent destabilization of the medial meniscus (DMM) surgery to induce post-traumatic OA, or sham surgery (control), and injected intra-articularly with D-isomer TAT-Beclin-1 (0.5-2 mg/kg) or PBS 1 week post-surgery for up to 9 weeks. Mice were sacrificed at 2 or 10 weeks post-surgery. Knee joint sections were evaluated by histopathology for cartilage degeneration and synovitis, and immunostaining for key markers of autophagy (LC3B), cell proliferation (nuclear Ki67), activated fibroblasts (αSMA), and cells of hematopoietic origin (CD45). RESULTS: All D-isomer TAT-Beclin-1-treated DMM mice had no difference in the degree of cartilage degeneration compared to PBS-injected DMM mice. Surprisingly, all D-isomer TAT-Beclin-1-treated mice exhibited substantial synovial hyperplasia, with increased cellularity and ECM deposition (fibrosis-like phenotype), as compared to PBS-injected mice. Synovial effects of D-isomer TAT-Beclin-1 were dose- and injection frequency-dependent. An increased percentage of cells positive for LC3B and nuclear Ki67 were found in the synovial intima early after injection, which persisted after frequent injections. CONCLUSIONS: D-isomer TAT-Beclin-1 did not attenuate cartilage degeneration, but rather induced synovial hyperplasia associated with increased expression of key markers of autophagy and cell proliferation and a fibrosis-like phenotype, independent of markers of fibroblast activation or persistent hematopoietic-origin cell infiltration. These data suggest that, if not tissue-targeted, caution should be taken using autophagy activators due to diverse cellular responses in the joint.


Assuntos
Autofagia/efeitos dos fármacos , Proteína Beclina-1/farmacologia , Cartilagem Articular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Osteoartrite do Joelho/patologia , Membrana Sinovial/efeitos dos fármacos , Animais , Cartilagem Articular/patologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Produtos do Gene tat/farmacologia , Hiperplasia , Injeções Intra-Articulares , Meniscos Tibiais/cirurgia , Camundongos , Membrana Sinovial/patologia , Sinovite/patologia , Lesões do Menisco Tibial
16.
Biochem Pharmacol ; 178: 114055, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32470548

RESUMO

Inflammation, mitochondrial dysfunction and oxidative stress are closely associated with neurological diseases. In this study, Mn-TAT PTD-Ngb, a novel artificial recombinant protein, exerted inhibitory effects on the inflammatory response and inflammasome activation. During the lipopolysaccharide (LPS)-induced inflammatory response, Mn-TAT PTD-Ngb suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and the release of proinflammatory cytokines and attenuated the phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, the recombinant protein blocked reactive oxygen species (ROS) production, abated mitochondrial dysfunction and significantly suppressed the assembly of the inflammasome, which led to the overproduction of proinflammatory cytokines IL-1ß and IL-18. Mn-TAT PTD-Ngb increased the level of nuclear factor-erythroid 2 -related factor 2 (Nrf2), which protected against oxidative stress and improved pyroptosis. Mn-TAT PTD-Ngb might be a promising drug for curing neurological diseases.


Assuntos
Antioxidantes/metabolismo , Mediadores da Inflamação/metabolismo , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes/administração & dosagem , Animais , Linhagem Celular , Produtos do Gene tat/administração & dosagem , Produtos do Gene tat/química , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Manganês/administração & dosagem , Manganês/química , Camundongos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteínas Recombinantes/química , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
17.
Int J Mol Sci ; 21(8)2020 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-32290442

RESUMO

Reactive oxygen species (ROS) is major risk factor in neuronal diseases including ischemia. Although biliverdin reductase A (BLVRA) plays a pivotal role in cell survival via its antioxidant function, its role in hippocampal neuronal (HT-22) cells and animal ischemic injury is not clearly understood yet. In this study, the effects of transducible fusion protein Tat-BLVRA on H2O2-induced HT-22 cell death and in an animal ischemia model were investigated. Transduced Tat-BLVRA markedly inhibited cell death, DNA fragmentation, and generation of ROS. Transduced Tat-BLVRA inhibited the apoptosis and mitogen activated protein kinase (MAPK) signaling pathway and it passed through the blood-brain barrier (BBB) and significantly prevented hippocampal cell death in an ischemic model. These results suggest that Tat-BLVRA provides a possibility as a therapeutic molecule for ischemia.


Assuntos
Apoptose/efeitos dos fármacos , Produtos do Gene tat , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Linhagem Celular , Modelos Animais de Doenças , Produtos do Gene tat/genética , Gerbillinae , Peróxido de Hidrogênio/metabolismo , Masculino , Fármacos Neuroprotetores/farmacologia , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/genética
18.
Sci Rep ; 10(1): 6591, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313258

RESUMO

The paper aims to investigate the cytotoxic effect on tumor cells of irradiated AuNPs in green light and subsequently functionalized with HS-PEG-NH2. The toxicity level of gold conjugates after their functionalization with DOX and TAT peptide was also evaluated. The AuNPs were prepared using the modified Turkevich method and exposed to visible light at a wavelength of 520 nm prior their PEGylation. The optical properties were analyzed by UV-vis spectroscopy, the surface modification was investigated using FTIR and XPS spectroscopies and their sizes and morphologies were evaluated by TEM and DLS techniques. DOX and TAT peptide were linked to the surface of PEGylated AuNPs by reacting their amino groups with glycidyloxypropyl of PEGylated DOX or TAT conjugates under mild conditions at room temperature and in the presence of ethanol as catalyst. The conjugates containing DOX or DOX and TAT have been characterized by fluorescence and FTIR techniques. The changes of electrochemical features were observed using cyclic voltammetry, suggesting a better stability of irradiated nanoparticles. By mass spectrometry it was confirmed that the compounds of interest were obtained. The cell viability test showed that irradiated and non-irradiated nanoparticles coated with PEG are not toxic in normal cells. Tumor cell viability analysis showed that the PEGylated nanoparticles modified with DOX and TAT peptide were more effective than pristine DOX, indicating cytotoxicity up to 10% higher than non-irradiated ones.


Assuntos
Doxorrubicina/farmacologia , Produtos do Gene tat/farmacologia , Nanopartículas Metálicas/química , Osteossarcoma/tratamento farmacológico , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/química , Produtos do Gene tat/química , Ouro/química , Humanos , Osteossarcoma/genética , Osteossarcoma/patologia , Peptídeos/química , Peptídeos/farmacologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Espectroscopia de Infravermelho com Transformada de Fourier
19.
Int J Mol Sci ; 21(5)2020 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-32182734

RESUMO

On account of their excellent capacity to significantly improve the bioavailability and solubility of chemotherapy drugs, amphiphilic block copolymer-based micelles have been widely utilized for chemotherapy drug delivery. In order to further improve the antitumor ability and to also reduce undesired side effects of drugs, cell-penetrating peptides have been used to functionalize the surface of polymer micelles endowed with the ability to target tumor tissues. Herein, we first synthesized functional polyethylene glycol-polylactic acid (PEG-PLA) tethered with maleimide at the PEG section of the block polymer, which was further conjugated with a specific peptide, the transactivating transcriptional activator (TAT), with an approved capacity of aiding translocation across the plasma membrane. Then, TAT-conjugated, paclitaxel-loaded nanoparticles were self-assembled into stable nanoparticles with a favorable size of 20 nm, and displayed a significantly increased cytotoxicity, due to their enhanced accumulation via peptide-mediated cellular association in human breast cancer cells (MCF-7) in vitro. But when further used in vivo, TAT-NP-PTX showed an acceleration of the drug's plasma clearance rate compared with NP-PTX, and therefore weakened its antitumor activities in the mice model, because of its positive charge, its elimination by the endoplasmic reticulum system more quickly, and its targeting effect on normal cells leading towards being more toxic. So further modification of TAT-NP-PTX to shield TAT peptide's positive charges may be a hot topic to overcome the present dilemma.


Assuntos
Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/química , Paclitaxel/administração & dosagem , Poliésteres/química , Polietilenoglicóis/química , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Portadores de Fármacos/química , Retículo Endoplasmático/metabolismo , Feminino , Produtos do Gene tat/química , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Micelas , Nanopartículas/química , Tamanho da Partícula , Polímeros/química
20.
Chemistry ; 26(43): 9449-9453, 2020 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-32167218

RESUMO

HIV transactivator of transcription (Tat) protein could interact with amyloid ß (Aß) peptide which cause the growth of Aß plaques in the brain and result in Alzheimer's disease in HIV-infected patients. Herein, we employ high-resolution atomic force microscopy and quantitative nanomechanical mapping to investigate the effects of Tat protein in Aß peptide aggregation. Our results demonstrate that the Tat protein could bind to the Aß fibril surfaces and result in the formation of Tat-Aß multifibrillar structures. The resultant Tat-Aß multifibrillar aggregates represent an increase in stiffness compared with Aß fibrils due to the increase in ß-sheet formation. The identification and characterization of the Tat-Aß intermediate aggregates is important to understanding the interactions between Tat protein and Aß peptide, and the development of novel therapeutic strategy for Alzheimer's disease-like disorder in HIV infected individuals.


Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/química , Amiloide/química , Produtos do Gene tat/química , Microscopia de Força Atômica/métodos , Placa Amiloide/química , Peptídeos beta-Amiloides/análise , Produtos do Gene tat/metabolismo , Humanos , Placa Amiloide/metabolismo
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