RESUMO
For tissue engineering of skeletal muscles, there is a need for biomaterials which do not only allow cell attachment, proliferation, and differentiation, but also support the physiological conditions of the tissue. Next to the chemical nature and structure of the biomaterial, its response to the application of biophysical stimuli, such as mechanical deformation or application of electrical pulses, can impact in vitro tissue culture. In this study, gelatin methacryloyl (GelMA) is modified with hydrophilic 2-acryloxyethyltrimethylammonium chloride (AETA) and 3-sulfopropyl acrylate potassium (SPA) ionic comonomers to obtain a piezoionic hydrogel. Rheology, mass swelling, gel fraction, and mechanical characteristics are determined. The piezoionic properties of the SPA and AETA-modified GelMA are confirmed by a significant increase in ionic conductivity and an electrical response as a function of mechanical stress. Murine myoblasts display a viability of >95% after 1 week on the piezoionic hydrogels, confirming their biocompatibility. The GelMA modifications do not influence the fusion capacity of the seeded myoblasts or myotube width after myotube formation. These results describe a novel functionalization providing new possibilities to exploit piezo-effects in the tissue engineering field.
Assuntos
Gelatina , Hidrogéis , Camundongos , Animais , Hidrogéis/farmacologia , Hidrogéis/química , Gelatina/farmacologia , Gelatina/química , Sobrevivência Celular , Materiais Biocompatíveis/farmacologia , Materiais Biocompatíveis/química , Engenharia Tecidual/métodos , Metacrilatos/farmacologia , Metacrilatos/química , Alicerces Teciduais/químicaRESUMO
Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenitor cells are cocultured with human umbilical vein endothelial cells (HUVECs) in a fibrin hydrogel. The cell-gel mix is cast into silicone molds with end attachment sites and cultured in endothelial growth medium (EGM-2) for 1 week. The passive forces generated in the contracted hydrogel align the myogenic cells parallel to the long axis of the contracted gel such that they fuse into aligned multinucleated myofibers. This results in the formation of a 2 cm long and ~1.5 mm tick human BAM construct with endothelial networks.
Assuntos
Técnicas de Cocultura , Células Endoteliais/metabolismo , Músculo Esquelético/metabolismo , Engenharia Tecidual , Biópsia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Desenvolvimento Muscular , Músculo Esquelético/citologia , Mioblastos/citologia , Mioblastos/metabolismoRESUMO
Membrane-associated glycoprotein neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) play an important role in brain plasticity by regulating cell-cell interactions. Here, we demonstrate that the cytosolic serine protease prolyl endopeptidase (PREP) is able to regulate NCAM and PSA-NCAM. Using a SH-SY5Y neuroblastoma cell line with stable overexpression of PREP, we found a remarkable loss of PSA-NCAM, reduced levels of NCAM180 and NCAM140 protein species, and a significant increase in the NCAM immunoreactive band migrating at an apparent molecular weight of 120â kDa in PREP-overexpressing cells. Moreover, increased levels of NCAM fragments were found in the concentrated medium derived from PREP-overexpressing cells. PREP overexpression selectively induced an activation of matrix metalloproteinase-9 (MMP-9), which could be involved in the observed degradation of NCAM, as MMP-9 neutralization reduced the levels of NCAM fragments in cell culture medium. We propose that increased PREP levels promote epidermal growth factor receptor (EGFR) signaling, which in turn activates MMP-9. In conclusion, our findings provide evidence for newly-discovered roles for PREP in mechanisms regulating cellular plasticity through NCAM and PSA-NCAM.
Assuntos
Moléculas de Adesão de Célula Nervosa/metabolismo , Proteólise , Serina Endopeptidases/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , Meios de Cultura , Receptores ErbB/metabolismo , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Metaloproteinase 9 da Matriz/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Prolil Oligopeptidases , Proteólise/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes/farmacologia , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismoRESUMO
BACKGROUND: Alpha-synuclein (α-SYN) aggregates represent a key feature of Parkinson's disease, but the exact relationship between α-SYN aggregation and neurodegeneration remains incompletely understood. Therefore, the availability of a cellular assay that allows medium-throughput analysis of α-SYN-linked pathology will be of great value for studying the aggregation process and for advancing α-SYN-based therapies. NEW METHOD: Here we describe a high-content neuronal cell assay that simultaneously measures oxidative stress-induced α-SYN aggregation and apoptosis. RESULTS: We optimized an automated and reproducible assay to quantify both α-SYN aggregation and cell death in human SH-SY5Y neuroblastoma cells. COMPARISON WITH EXISTING METHODS: Quantification of α-SYN aggregates in cells has typically relied on manual imaging and counting or cell-free assays, which are time consuming and do not allow a concurrent analysis of cell viability. Our high-content analysis method for quantification of α-SYN aggregation allows simultaneous measurements of multiple cell parameters at a single-cell level in a fast, objective and automated manner. CONCLUSIONS: The presented analysis approach offers a rapid, objective and multiparametric approach for the screening of compounds and genes that might alter α-SYN aggregation and/or toxicity.
Assuntos
Apoptose , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Agregação Patológica de Proteínas , alfa-Sinucleína/química , Benzotiazóis , Western Blotting , Contagem de Células , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Vetores Genéticos , Humanos , Indóis , Lentivirus/genética , Microscopia de Fluorescência/métodos , Estresse Oxidativo , Multimerização Proteica , Software , Tiazóis , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2 × 10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1 × 10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2 × 10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.
Assuntos
Técnicas de Cocultura/métodos , Músculo Esquelético/citologia , Engenharia Tecidual/métodos , Células Cultivadas , Matriz Extracelular/química , Fibrina/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imuno-Histoquímica , Mioblastos Esqueléticos/citologiaRESUMO
Loss-of-function mutations in the PINK1 gene lead to recessive forms of Parkinson's disease. Animal models with depleted PINK1 expression have failed to reproduce significant nigral dopaminergic neurodegeneration and clear alpha-synuclein pathology, main characteristics of the disease. In this study, we investigated whether alpha-synuclein pathology is altered in the absence of PINK1 in cell culture and in vivo. We observed that downregulation of PINK1 enhanced alpha-synuclein aggregation and apoptosis in a neuronal cell culture model for synucleinopathy. Silencing of PINK1 expression in mouse substantia nigra using recombinant adeno-associated viral vectors did not induce dopaminergic neurodegeneration in a long-term study up to 10 months, nor did it enhance or accelerate dopaminergic neurodegeneration after alpha-synuclein overexpression. However, in PINK1 knockout mice, overexpression of alpha-synuclein in the substantia nigra resulted in enhanced dopaminergic neurodegeneration as well as significantly higher levels of alpha-synuclein phosphorylation at serine 129 at 4 weeks postinjection. In conclusion, our results demonstrate that total loss of PINK1 leads to an increased sensitivity to alpha-synuclein-induced neuropathology and cell death in vivo.
Assuntos
Doenças Neurodegenerativas/genética , Doença de Parkinson/genética , Agregação Patológica de Proteínas/genética , Proteínas Quinases/genética , alfa-Sinucleína/metabolismo , Animais , Apoptose/genética , Células Cultivadas , Progressão da Doença , Regulação para Baixo , Expressão Gênica , Humanos , Camundongos Knockout , Mutação , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Doença de Parkinson/patologia , Fosforilação , Agregados Proteicos , Agregação Patológica de Proteínas/patologia , Proteínas Quinases/deficiência , Substância Negra/metabolismoRESUMO
The aggregation of alpha-synuclein (α-SYN) into fibrils is characteristic for several neurodegenerative diseases, including Parkinson's disease (PD). Ninety percent of α-SYN deposited in Lewy Bodies, a pathological hallmark of PD, is phosphorylated on serine129. α-SYN can also be phosphorylated on tyrosine125, which is believed to regulate the membrane binding capacity and thus possibly its normal function. A better understanding of the effect of phosphorylation on the aggregation of α-SYN might shed light on its role in the pathogenesis of PD. In this study we compare the aggregation properties of WT α-SYN with the phospho-dead and phospho-mimic mutants S129A, S129D, Y125F and Y125E and in vitro phosphorylated α-SYN using turbidity, thioflavin T and circular dichroism measurements as well as transmission electron microscopy. We show that the mutants S129A and S129D behave similarly compared to wild type (WT) α-SYN, while the mutants Y125F and Y125E fibrillate significantly slower, although all mutants form fibrillar structures similar to the WT protein. In contrast, in vitro phosphorylation of α-SYN on either S129 or Y125 does not significantly affect the fibrillization kinetics. Moreover, FK506 binding proteins (FKBPs), enzymes with peptidyl-prolyl cis-trans isomerase activity, still accelerate the aggregation of phosphorylated α-SYN in vitro, as was shown previously for WT α-SYN. In conclusion, our results illustrate that phosphorylation mutants can display different aggregation properties compared to the more biologically relevant phosphorylated form of α-SYN.
Assuntos
Polimerização , alfa-Sinucleína/química , Humanos , Cinética , Mutação de Sentido Incorreto , Fosforilação , Proteínas de Ligação a Tacrolimo/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMO
We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional (15)N-(1)H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.
Assuntos
Marcação por Isótopo/métodos , Proteínas/química , Amidas/química , Carbono/química , Campos Eletromagnéticos , Escherichia coli/química , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética , Isótopos de Nitrogênio , Conformação Proteica , Prótons , alfa-Sinucleína/químicaRESUMO
The human immunodeficiency virus type 1 (HIV-1) and other lentiviruses are capable of infecting non-dividing cells and, therefore, need to be imported into the nucleus before integration into the host cell chromatin. Transportin-SR2 (TRN-SR2, Transportin-3, TNPO3) is a cellular karyopherin implicated in nuclear import of HIV-1. A model in which TRN-SR2 imports the viral preintegration complex into the nucleus is supported by direct interaction between TRN-SR2 and HIV-1 integrase (IN). Residues in the C-terminal domain of HIV-1 IN that mediate binding to TRN-SR2 were recently delineated. As for most nuclear import cargoes, the driving force behind HIV-1 preintegration complex import is likely a gradient of the GDP- and GTP-bound forms of Ran, a small GTPase. In this study we offer biochemical and structural characterization of the interaction between TRN-SR2 and Ran. By size exclusion chromatography we demonstrate stable complex formation of TRN-SR2 and RanGTP in solution. Consistent with the behavior of normal nuclear import cargoes, HIV-1 IN is released from the complex with TRN-SR2 by RanGTP. Although in concentrated solutions TRN-SR2 by itself was predominantly present as a dimer, the TRN-SR2-RanGTP complex was significantly more compact. Further analysis supported a model wherein one monomer of TRN-SR2 is bound to one monomer of RanGTP. Finally, we present a homology model of the TRN-SR2-RanGTP complex that is in excellent agreement with the experimental small angle x-ray scattering data.
Assuntos
Modelos Moleculares , Complexos Multiproteicos/química , Multimerização Proteica , beta Carioferinas/química , Proteína ran de Ligação ao GTP/química , Transporte Ativo do Núcleo Celular/genética , Integrase de HIV/química , Integrase de HIV/genética , Integrase de HIV/metabolismo , HIV-1 , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
The aggregation of the protein alpha-synuclein (α-SYN) is believed to be a critical event in Parkinson's disease (PD). α-SYN is characterized by a remarkable conformational plasticity, adopting different conformations depending on the environment. In vitro, α-SYN lacks a well-defined structure. Therefore, it was classified as an 'intrinsically disordered protein'. A debate has recently begun over how α-SYN behaves in the cell: is it an intrinsically disordered protein or a stable tetramer with a low propensity for aggregation? In this review, we discuss the aggregation of α-SYN and describe factors that influence this process and their potential relevance in PD pathogenesis. We address the ways in which aggregated α-SYN mediates toxicity and might lead to PD, and propose possible therapeutic strategies.
Assuntos
Doença de Parkinson/metabolismo , alfa-Sinucleína/química , Animais , Humanos , Doença de Parkinson/genética , Conformação Proteica , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidadeRESUMO
OBJECTIVE: Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular binding partner of HIV-1 integrase and a crucial cofactor for HIV-1 replication. Here, we study two LEDGF/p75 exonic variants I436S and T473I, identified in HIV-1 long-term nonprogressors, together with Q472L. METHODS: In-vitro binding assays, cell culture complementation, and functional rescue. RESULTS: Binding affinities of wild-type, I436S, T473I, and Q472L LEDGF/p75 for HIV-1 integrase were comparable. All LEDGF/p75 variants bound equally well to LEDGF/p75 interacting partners JPO2 and PogZ. In addition, HIV-1 replication was evaluated in human somatic LEDGF/p75-knockout cells and LEDGF/p75-knockdown cells complemented with either wild-type LEDGF/p75 or the respective LEDGF/p75 variants. All variants rescued HIV-1 replication to wild-type levels, whereas LEDGF/p75 D366N, defective for interaction with HIV-1 integrase, did not. CONCLUSION: Although identified in a cohort of long-term nonprogressors, our study did not indicate that the I436S or T473I mutation in LEDGF/p75 affects the interaction with HIV-1 integrase.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Integrase de HIV/metabolismo , Sobreviventes de Longo Prazo ao HIV , Soropositividade para HIV/genética , Mutação Puntual , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fármacos Anti-HIV/uso terapêutico , Éxons , Soropositividade para HIV/enzimologia , Humanos , Isoleucina , Ligação Proteica , Serina , Treonina , Fatores de Transcrição/metabolismo , Replicação ViralRESUMO
We have investigated the effect of regiospecifically introducing substituents in the P2 part of the typical dipeptide derived basic structure of PREP inhibitors. This hitherto unexplored modification type can be used to improve target affinity, selectivity, and physicochemical parameters in drug discovery programs focusing on PREP inhibitors. Biochemical evaluation of the produced inhibitors identified several substituent types that significantly increase target affinity, thereby reducing the need for an electrophilic "warhead" functionality. Pronounced PREP specificity within the group of Clan SC proteases was generally observed. Omission of the P1 electrophilic function did not affect the overall binding mode of three representative compounds, as studied by X-ray crystallography, while the P2 substituents were demonstrated to be accommodated in a cavity of PREP that, to date, has not been probed by inhibitors. Finally, we report on results of selected inhibitors in a SH-SY5Y cellular model of synucleinopathy and demonstrate a significant antiaggregation effect on α-synuclein.
Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Mitocondriais/antagonistas & inibidores , Neuroblastoma/tratamento farmacológico , Pirrolidinas/química , alfa-Sinucleína/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Humanos , Cinética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Estrutura Molecular , Neuroblastoma/metabolismo , Multimerização Proteica/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Células Tumorais CultivadasRESUMO
Prolyl oligopeptidase (PREP, E.C.3.4.21.26) is a cytosolic serine protease that hydrolyzes small (<3 kDa), proline-containing peptides on the carboxyl terminal side of proline residues, and is widely distributed in the brain. High PREP activity, due to aging or neurodegenerative disease, has been hypothesised to lead to an increased breakdown of neuropeptides, resulting in a decline of cognitive functions and an acceleration of neurodegeneration. Recent data have suggested that PREP involvement in neurodegeneration cannot be explained by its extracellular space proteolytic activity alone, but may involve intracellular PREP activities as well. In order to test this, appropriate methods for measuring PREP intracellular activity must first be developed. In the present study, we developed and validated an in situ PREP intracellular activity assay in primary rat cortical neurons, using nitroblue tetrazolium chloride salt (NBT) and a PREP specific substrate (S)-benzyl 2-(2-(4-hydroxynaphthalen-l-ylcarbanoyl)pyrrolidin-l-yl)-2-oxoethylcarbamate (UAMC-00682). This novel in situ PREP activity assay was further validated in neuroblastoma SH-SY5Y cells, under conditions of PREP overexpression and inhibited PREP expression. Using this assay, we demonstrated that PREP inhibitors, Z-Pro-Pro-aldehyde-dimethylacetal, Boc-Asn-Phe-Pro-aldehyde, and (S)-1-((S)-1-(4-phenylbutanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (KYP-2047), were able to inhibit intracellular PREP activity in primary rat cortical neurons. KYP-2047 was the most potent PREP inhibitor in all assay systems tested. The validated assay enables localization and quantification of in situ PREP activity in primary rat cortical neurons and neuroblastoma SH-SY5Y cells, as well allows testing cell permeability and efficiency of novel PREP inhibitors.
Assuntos
Bioensaio/métodos , Perfilação da Expressão Gênica/métodos , Neurônios/enzimologia , Serina Endopeptidases/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Ativação Enzimática , Prolil Oligopeptidases , Ratos , Ratos Wistar , Serina Endopeptidases/químicaRESUMO
Although LEDGF/p75 is believed to act as a cellular cofactor of lentiviral integration by tethering integrase (IN) to chromatin, there is no good in vitro model to analyze this functionality. We designed an AlphaScreen assay to study how LEDGF/p75 modulates the interaction of human immunodeficiency virus type 1 IN with DNA. IN bound with similar affinity to DNA mimicking the long terminal repeat or to random DNA. While LEDGF/p75 bound DNA strongly, a mutant of LEDGF/p75 with compromised nuclear localization signal (NLS)/AT hook interacted weakly, and the LEDGF/p75 PWWP domain did not interact, corroborating previous reports on the role of NLS and AT hooks in charge-dependent DNA binding. LEDGF/p75 stimulated IN binding to DNA 10-fold to 30-fold. Stimulation of IN-DNA binding required a direct interaction between IN and the C-terminus of LEDGF/p75. Addition of either the C-terminus of LEDGF/p75 (amino acids 325-530) or LEDGF/p75 mutated in the NLS/AT hooks interfered with IN binding to DNA. Our results are consistent with an in vitro model of LEDGF/p75-mediated tethering of IN to DNA. The inhibition of IN-DNA interaction by the LEDGF/p75 C-terminus may provide a novel strategy for the inhibition of HIV IN activity and may explain the potent inhibition of HIV replication observed after the overexpression of C-terminal fragments in cell culture.
Assuntos
DNA/metabolismo , Integrase de HIV/metabolismo , HIV-1/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Sequência Rica em At/genética , Bioensaio , Ensaio de Imunoadsorção Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Cinética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Sinais de Localização Nuclear/metabolismo , Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Solubilidade , Relação Estrutura-AtividadeRESUMO
FK506-binding proteins (FKBPs) are members of the immunophilins, enzymes that assist protein folding with their peptidyl-prolyl isomerase (PPIase) activity. Some non-immunosuppressive inhibitors of these enzymes have neuroregenerative and neuroprotective properties with an unknown mechanism of action. We have previously shown that FKBPs accelerate the aggregation of α-synuclein (α-SYN) in vitro and in a neuronal cell culture model for synucleinopathy. In this study we investigated whether acceleration of α-SYN aggregation is specific for the FKBP or even the PPIase family. Therefore, we studied the effect of several physiologically relevant PPIases, namely FKBP12, FKBP38, FKBP52, FKBP65, Pin1, and cyclophilin A, on α-SYN aggregation in vitro and in neuronal cell culture. Among all PPIases tested in vitro, FKBP12 accelerated α-SYN aggregation the most. Furthermore, only FKBP12 accelerated α-SYN fibril formation at subnanomolar concentrations, pointing toward an enzymatic effect. Although stable overexpression of various FKBPs enhanced the aggregation of α-SYN and cell death in cell culture, they were less potent than FKBP12. When FKBP38, FKBP52, and FKBP65 were overexpressed in a stable FKBP12 knockdown cell line, they could not fully restore the number of α-SYN inclusion-positive cells. Both in vitro and cell culture data provide strong evidence that FKBP12 is the most important PPIase modulating α-SYN aggregation and validate the protein as an interesting drug target for Parkinson disease.
Assuntos
Doença de Parkinson/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo , alfa-Sinucleína/metabolismo , Morte Celular , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Técnicas de Silenciamento de Genes , Humanos , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Doença de Parkinson/patologia , Peptidilprolil Isomerase/genética , Proteína 1A de Ligação a Tacrolimo/genética , alfa-Sinucleína/genéticaRESUMO
Immunophilins are a family of highly conserved proteins with a peptidyl-prolyl isomerase activity that binds immunosuppressive drugs such as FK506, cyclosporin A, and rapamycin. Immunophilins can be divided into two subfamilies, the cyclophilins, and the FK506 binding proteins (FKBPs). Next to the immunophilins, a third group of peptidyl-prolyl isomerases exist, the parvulins, which do not influence the immune system. The beneficial role of immunophilin ligands in neurodegenerative disease models has been known for more than a decade but remains largely unexplained in terms of molecular mechanisms. In this review, we summarize reported effects of parvulins, immunophilins, and their ligands in the context of neurodegeneration. We focus on the role of FKBP12 in Parkinson's disease and propose it as a novel drug target for therapy of Parkinson's disease.
Assuntos
Degeneração Neural/enzimologia , Peptidilprolil Isomerase/metabolismo , Animais , Humanos , Sistema Imunitário/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Transamidation of α-synuclein by the Ca(2+)-dependent enzyme tissue transglutaminase (tTG, EC 2.3.2.13) is implicated in Parkinson's disease (PD). tTG may therefore offer a novel therapeutic target to intervene in PD. Here we first evaluated the potency and efficacy of three recently developed irreversible active-site inhibitors of tTG (B003, Z006 and KCC009) to inhibit tTG activity in vitro and in living cells. In vitro, all compounds were found to be full inhibitors of tTG activity showing a rank order of potency (defined by IC-50 values) of Z006>B003>KCC009. Upon Ca(2+) ionophore (A23187) induced activation of cellular tTG (measured by incorporation of the tTG-specific amine substrate 5-(biotinamido)pentylamine (BAP) into cellular proteins) in neuroblastoma SH-SY5Y cells, only Z006 (0.3-30 µM) retained the capacity to completely inhibit tTG activity. Under these conditions B003 (3-300 µM) only partially blocked tTG activity whereas KCC009 (3-100 µM) failed to affect tTG activity at any of the concentrations used. Z006 (30 µM) also blocked the tTG mediated incorporation of BAP into α-synuclein monomers and SDS-resistant multimers in vitro and in α-synuclein overexpressing SHSY5Y cells exposed to A23187 or the PD mimetic 1-methyl-4-phenylpyridine (MPP(+)). Moreover, Z006 (30 µM) substantially reduced formation of SDS-resistant α-synuclein multimers in SH-SY5Y cells exposed to A23187 or MPP(+) in the absence of BAP. We conclude that α-synuclein is a cellular substrate for tTG under conditions mimicking PD and blockade of tTG activity counteracts α-synuclein transamidation and aggregation in vitro and in living cells. Moreover, our cell model appears an excellent readout to identify candidate inhibitors of intracellular tTG.
Assuntos
Amidas/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Modelos Biológicos , Doença de Parkinson/metabolismo , Transglutaminases/antagonistas & inibidores , alfa-Sinucleína/metabolismo , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Proteínas de Ligação ao GTP/metabolismo , Humanos , Proteína 2 Glutamina gama-Glutamiltransferase , Transglutaminases/metabolismoRESUMO
Tau isoforms constitute a family of microtubule-associated proteins that are mainly expressed in neurons of the central nervous system. They promote the assembly of tubulin monomers into microtubules and modulate their stability, thus playing a key structural role in the distal portion of axons. In Alzheimer's disease and related tauopathies, Tau aggregation into fibrillary tangles contributes to intraneuronal and glial lesions. We report herein the ability of three natural phenolic derivatives obtained from olives and derived food products to prevent such Tau fibrillization in vitro, namely hydroxytyrosol, oleuropein, and oleuropein aglycone. The latter was found to be more active than the reference Tau aggregation inhibitor methylene blue on both wild-type and P301L Tau proteins, inhibiting fibrillization at low micromolar concentrations. These findings might provide further experimental support for the beneficial nutritional properties of olives and olive oil as well as a chemical scaffold for the development of new drugs aiming at neurodegenerative tauopathies.
Assuntos
Olea/química , Piranos/farmacologia , Proteínas tau/antagonistas & inibidores , Humanos , Glucosídeos Iridoides , Iridoides , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Proteínas tau/metabolismoRESUMO
BACKGROUND: Transportin-SR2 (TRN-SR2, TNPO3, transportin 3) was previously identified as an interaction partner of human immunodeficiency virus type 1 (HIV-1) integrase and functions as a nuclear import factor of HIV-1. A possible role of capsid in transportin-SR2-mediated nuclear import was recently suggested by the findings that a chimeric HIV virus, carrying the murine leukemia virus (MLV) capsid and matrix proteins, displayed a transportin-SR2 independent phenotype, and that the HIV-1 N74D capsid mutant proved insensitive to transportin-SR2 knockdown. RESULTS: Our present analysis of viral specificity reveals that TRN-SR2 is not used to the same extent by all lentiviruses. The DNA flap does not determine the TRN-SR2 requirement of HIV-1. We corroborate the TRN-SR2 independent phenotype of the chimeric HIV virus carrying the MLV capsid and matrix proteins. We reanalyzed the HIV-1 N74D capsid mutant in cells transiently or stably depleted of transportin-SR2 and confirm that the N74D capsid mutant is independent of TRN-SR2 when pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Remarkably, although somewhat less dependent on TRN-SR2 than wild type virus, the N74D capsid mutant carrying the wild type HIV-1 envelope required TRN-SR2 for efficient replication. By pseudotyping with envelopes that mediate pH-independent viral uptake including HIV-1, measles virus and amphotropic MLV envelopes, we demonstrate that HIV-1 N74D capsid mutant viruses retain partial dependency on TRN-SR2. However, this dependency on TRN-SR2 is lost when the HIV N74D capsid mutant is pseudotyped with envelopes mediating pH-dependent endocytosis, such as the VSV-G and Ebola virus envelopes. CONCLUSION: Here we discover a link between the viral entry of HIV and its interaction with TRN-SR2. Our data confirm the importance of TRN-SR2 in HIV-1 replication and argue for careful interpretation of experiments performed with VSV-G pseudotyped viruses in studies on early steps of HIV replication including the role of capsid therein.
Assuntos
HIV-1/fisiologia , Internalização do Vírus , beta Carioferinas/metabolismo , Linhagem Celular , HumanosRESUMO
The neuronal protein α-synuclein (α-syn) plays a central role in Parkinson's disease (PD). The pathological features of PD are the loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies. The C-terminal domain of α-syn is characterized by the presence of 15 acidic amino acids and all five proline residues of the protein (P108, P117, P120, P128, and P138). The aggregation of this natively unfolded protein is accelerated in vitro by FK506 binding proteins (FKBPs) showing peptidyl-prolyl cis-trans isomerase activity. These proteins catalyze the cis-trans conformational change of the X-Pro peptide bond, often a rate-limiting step in protein folding. The acceleration of the folding of α-syn by FKBPs may accelerate disease-associated aggregation. To further elucidate the role of the proline residues in the conformation and aggregation of α-syn, we constructed several mutants of α-syn in which one or more proline residues are mutated to alanine via site-directed mutagenesis. For this purpose, we produced and purified His-WT α-syn, a recombinant α-syn with a polyhistidine tag (six His residues) and a linker, and a number of Pro-to-Ala mutants. The aggregation kinetics of these mutants and His-WT α-syn were studied by turbidity, thioflavin T fluorescence, and CD measurements. We can conclude that mutation of the proline residues to alanine accelerates the aggregation kinetics of α-syn while all proline mutants formed fibrils similar to His-WT α-syn, as visualized via transmission electron microscopy. We also demonstrate that the accelerating effect of hFKBP12 is abolished via removal of the proline residues from the C-terminus. Finally, we show that the mutant of His α-syn with all five proline residues mutated to alanine is more structured (more α-helix) than His-WT α-syn, indicating the role of the Pro residues as potential helix breakers in the inhibitory conformation of the C-terminus.