Resistance training-induced changes in muscle proteolysis and extracellular matrix remodeling biomarkers in the untrained and trained states.

PURPOSE: Resistance training (RT) induces muscle growth at varying rates across RT phases, and evidence suggests that the muscle-molecular responses to training bouts become refined or attenuated in the trained state. This study examined how proteolysis-related biomarkers and extracellular matrix (ECM) remodeling factors respond to a bout of RT in the untrained (UT) and trained (T) state. METHODS: Participants (19 women and 19 men) underwent 10 weeks of RT. Biopsies of vastus lateralis were collected before and after (24 h) the first (UT) and last (T) sessions. Vastus lateralis cross-sectional area (CSA) was assessed before and after the experimental period. RESULTS: There were increases in muscle and type II fiber CSAs. In both the UT and T states, calpain activity was upregulated and calpain-1/-2 protein expression was downregulated from Pre to 24 h. Calpain-2 was higher in the T state. Proteasome activity and 20S proteasome protein expression were upregulated from Pre to 24 h in both the UT and T. However, proteasome activity levels were lower in the T state. The expression of poly-ubiquitinated proteins was unchanged. MMP activity was downregulated, and MMP-9 protein expression was elevated from Pre to 24 h in UT and T. Although MMP-14 protein expression was acutely unchanged, this marker was lower in T state. TIMP-1 protein levels were reduced Pre to 24 h in UT and T, while TIMP-2 protein levels were unchanged. CONCLUSION: Our results are the first to show that RT does not attenuate the acute-induced response of proteolysis and ECM remodeling-related biomarkers.

Capturing the Polarization Response of Solvated Proteins under Constant Electric Fields in Molecular Dynamics Simulations.

We capture and compare the polarization response of a solvated globular protein ubiquitin to static electric (E-fields) using atomistic molecular dynamics simulations. We collectively follow E-field induced changes, electrical and structural, occurring across multiple trajectories using the magnitude of the protein dipole vector (Pp ). E-fields antiparallel to Pp induce faster structural changes and more facile protein unfolding relative to parallel fields of the same strength. While weak E-fields (0.1-0.5 V/nm) do not unfold ubiquitin and produce a reversible polarization, strong E-fields (1-2 V/nm) unfold the protein through a pathway wherein the helix:ß-strand interactions rupture before those for the ß1-ß5 clamp. Independent of E-field direction, high E-field induced structural changes are also reversible if the field is switched off before Pp exceeds 2 times its equilibrium value. We critically examine the dependence of water properties, protein rotational diffusion and E-field induced protein unfolding pathways on the thermostat/barostat parameters used in our simulations.

Protac: redirigiendo los sistemas de degradación de proteínas a nuevos sustratos / Protac: redirecting protein degradation systems to new substrates

Tradicionalmente el diseño de fármacos se ha basado en el diseño de moléculas pequeñas hasta la aparición de terapias basadas en ácidos nucleicos, ya sea por la modificación de ciertos genes o por impedir que las proteínas se transcriban de forma efectiva. Empleando estas nuevas aproximaciones se han podido modificar dianas que hasta el momento se consideraban inmodificables o al menos no lo podían ser por moléculas pequeñas.Sin embargo, estas nuevas aproximaciones no están carentes de limitaciones como la baja biodisponibilidad debido a su limitada estabilidad y dificultad para poder atravesar las barreras celulares. Además, en muchas ocasiones las modificaciones que generan son irreversibles con el consiguiente riesgo de padecer efectos adversos de forma crónica.Como alternativa, han surgido con fuerza una serie de compuestos quiméricos heterobifuncionales denominados PROTACs (Protein Targeting Chimeras). Estos PROTACs son capaces de mantener en la proximidad de la ligasa E3 a una proteína de interés, marcándola con ubiquitina y finalmente, promoviendo su degradación mediada por el proteasoma. Esta aproximación permite la generación de diferentes estructuras de PROTAC por diseño racional o basado en la estructura y, además, permite las modificaciones estructurales necesarias para mejorar su perfil de estabilidad y farmacocinético manteniendo su actividad.Esta revisión pretende dar una visión general de qué son los PROTACs, qué ligasas E3 se emplean por el momento, factores relevantes a la hora de desarrollar un PROTAC y otras aproximaciones similares que no emplean el proteasoma como ruta de degradación. (AU)

The small molecules development has dominated the design of new drugs until the rise of nucleic acid-based therapies, either by modifying a gene or by preventing it from being effectively transcribed. Taking advantage of this new approaches, the pharmacological intervention in therapeutic targets that are considered unmodifiable up to now with small molecules were allowed.However, these new approaches are not devoid of defects such as low bioavailability due to their stability and pharmacokinetic problems, in addition to being irreversible DNA modifications in many cases, with the subsequent risk of suffering chronic adverse effects.Alternatively, a series of chimeric heterobifunctional compounds, called PROTACs (Protein Targeting Chimeras), have emerged with force in recent years. These PROTACs are able to bring E3 ligases closer with proteins of interest in space to label them with ubiquitin. Finally, it was degraded by the proteasome. This approach enables the generation of different PROTACs structures by rational design and, also, allows the chemical structure modification to improve their stability and pharmacokinetic profile keeping their activity.This review aims to give a comprehensive approach of what PROTACs are, what E3 ligases recruit, relevant factors in PROTAC development, and other approaches similar to this but that use non-proteasomal degradation pathways. (AU)

Protac: redirigiendo los sistemas de degradación de proteínas a nuevos sustratos / Protac: redirecting protein degradation systems to new substrates

Tradicionalmente el diseño de fármacos se ha basado en el diseño de moléculas pequeñas hasta la aparición de terapias basadas en ácidos nucleicos, ya sea por la modificación de ciertos genes o por impedir que las proteínas se transcriban de forma efectiva. Empleando estas nuevas aproximaciones se han podido modificar dianas que hasta el momento se consideraban inmodificables o al menos no lo podían ser por moléculas pequeñas. Sin embargo, estas nuevas aproximaciones no están carentes de limitaciones como la baja biodisponibilidad debido a su limitada estabilidad y dificultad para poder atravesar las barreras celulares. Además, en muchas ocasiones las modificaciones que generan son irreversibles con el consiguiente riesgo de padecer efectos adversos de forma crónica. Como alternativa, han surgido con fuerza una serie de compuestos quiméricos heterobifuncionales denominados PROTACs (Protein Targeting Chimeras). Estos PROTACs son capaces de mantener en la proximidad de la ligasa E3 a una proteína de interés, marcándola con ubiquitina y finalmente, promoviendo su degradación mediada por el proteasoma. Esta aproximación permite la generación de diferentes estructuras de PROTAC por diseño racional o basado en la estructura y, además, permite las modificaciones estructurales necesarias para mejorar su perfil de estabilidad y farmacocinético manteniendo su actividad. Esta revisión pretende dar una visión general de qué son los PROTACs, qué ligasas E3 se emplean por el momento, factores relevantes a la hora de desarrollar un PROTAC y otras aproximaciones similares que no emplean el proteasoma como ruta de degradación.(AU)

The small molecules development has dominated the design of new drugs until the rise of nucleic acid-based therapies, either by modifying a gene or by preventing it from being effectively transcribed. Taking advantage of this new approaches, the pharmacological intervention in therapeutic targets that are considered unmodifiable up to now with small molecules were allowed. However, these new approaches are not devoid of defects such as low bioavailability due to their stability and pharmacokinetic problems, in addition to being irreversible DNA modifications in many cases, with the subsequent risk of suffering chronic adverse effects. Alternatively, a series of chimeric heterobifunctional compounds, called PROTACs (Protein Targeting Chimeras), have emerged with force in recent years. These PROTACs are able to bring E3 ligases closer with proteins of interest in space to label them with ubiquitin. Finally, it was degraded by the proteasome. This approach enables the generation of different PROTACs structures by rational design and, also, allows the chemical structure modification to improve their stability and pharmacokinetic profile keeping their activity. This review aims to give a comprehensive approach of what PROTACs are, what E3 ligases recruit, relevant factors in PROTAC development, and other approaches similar to this but that use non-proteasomal degradation pathways.(AU)

Amelioration of Mitochondrial Quality Control and Proteostasis by Natural Compounds in Parkinson's Disease Models.

Parkinson's disease (PD) is a well-known age-related neurodegenerative disorder associated with longer lifespans and rapidly aging populations. The pathophysiological mechanism is a complex progress involving cellular damage such as mitochondrial dysfunction and protein homeostasis. Age-mediated degenerative neurological disorders can reduce the quality of life and also impose economic burdens. Currently, the common treatment is replacement with levodopa to address low dopamine levels; however, this does not halt the progression of PD and is associated with adverse effects, including dyskinesis. In addition, elderly patients can react negatively to treatment with synthetic neuroprotection agents. Recently, natural compounds such as phytochemicals with fewer side effects have been reported as candidate treatments of age-related neurodegenerative diseases. This review focuses on mitochondrial dysfunction, oxidative stress, hormesis, proteostasis, the ubiquitin‒proteasome system, and autophagy (mitophagy) to explain the neuroprotective effects of using natural products as a therapeutic strategy. We also summarize the efforts to use natural extracts to develop novel pharmacological candidates for treatment of age-related PD.

Proteasoma, subtipos y sus implicaciones en la tolerancia central / The proteasome: subtypes and involvement in central tolerance

El proteasoma es un complejo proteico grande, el cual se encuentra fundamentalmente en todas las células eucariotas ya que juega un rol muy importante en los procesos celulares, tales como: la diferenciación celular, la progresión del ciclo celular, el desarrollo y la apoptosis celular. Existen varios tipos de proteasomas como el constitutivo, los intermedios, el inmunoproteasoma y el timoproteasoma, los cuales están presenten en las células del cuerpo en dependencia de la estructura y función de ellas. Sin embargo, se encuentran en las células del sistema inmune donde no solo juegan un papel muy importante en el procesamiento antigénico para la respuesta inmune, sino en los mecanismos de tolerancia central durante el proceso de ontogenia de los linfocitos T en el timo. Así, las células epiteliales tímicas corticales son células presentadoras de antígenos, las cuales presentan características intrínsecas únicas al presentar el timoproteasoma, la catepsina L y la proteasa serin específica del timo. Además, se ha observado una alta tasa de macroautofagia en comparación a las otras células del cuerpo, por lo que serán esenciales en la obtención de un repertorio de linfocitos T CD4+ y CD8+ que tendrán la capacidad de discriminar lo propio y lo no propio. Por lo que se debería considerar que la tolerancia central no está únicamente definida por el mecanismo de selección negativa, sino que a su vez la selección positiva juega un papel muy importante en la definición del repertorio de clones de linfocitos T no autorreactivos. El objetivo es discutir acerca del proteasoma, los tipos de proteasomas y sus implicaciones en la tolerancia central de los linfocitos T(AU)

The proteasome is a large protein complex mainly located in all eukaryote cells, where it plays a very important role in cellular processes like differentiation, cycle progression, development and apoptosis. There are several types of proteasomes: constitutive, intermediate, immunoproteasome and thymoproteasome, which are present in cells depending on their structure and functions. However, they are found in cells of the immune system, where they play a very important role not only in antigenic processes related to the immune response, but also in central tolerance mechanisms during T lymphocyte ontogeny in the thymus. Thymic cortical epithelial cells are therefore antigen-presenting cells with unique intrinsic characteristics, since they contain thymoproteasome, cathepsin L, and thymus-specific serine protease. Additionally, a high rate of macroautophagy has been observed in comparison with bibr cells of the body. They are thus essential to obtain a repertoire of CD4+ and CD8+ T lymphocytes capable of distinguishing the body's own elements from alien structures. Therefore, central tolerance should not be viewed as only defined by the negative selection mechanism, since positive selection also plays a very important role in defining the repertoire of non-autoreactive T lymphocyte clones. The purpose of the study was to discuss the proteasome, the types of proteasomes and their involvement in T lymphocyte central tolerance(AU)

Bowman‒Birk Inhibitor Suppresses Herpes Simplex Virus Type 2 Infection of Human Cervical Epithelial Cells.

The Bowman‒Birk inhibitor (BBI), a protease inhibitor derived from soybeans, has been extensively studied in anti-tumor and anti-inflammation research. We recently reported that BBI has an anti-HIV-1 property in primary human macrophages. Because HSV-2 infection plays a role in facilitating HIV-1 sexual transmission, we thus examined whether BBI has the ability to inhibit HSV-2 infection. We demonstrated that BBI could potently inhibit HSV-2 replication in human cervical epithelial cells (End1/E6E7). This BBI-mediated HSV-2 inhibition was partially through blocking HSV-2-mediated activation of NF-κB and p38 MAPK pathways. In addition, BBI could activate the JAK/STAT pathway and enhance the expression of several antiviral interferon-stimulated genes (ISGs). Furthermore, BBI treatment of End1/E6E7 cells upregulated the expression of tight junction proteins and reduced HSV-2-mediated cellular ubiquitinated proteins' degradation through suppressing the ubiquitin‒proteasome system. These observations indicate that BBI may have therapeutic potential for the prevention and treatment of HSV-2 infections.

The destruction complex of beta-catenin in colorectal carcinoma and colonic adenoma / O complexo destruidor de betacatenina no carcinoma colorretal e no adenoma cólico

ABSTRACT Objective To evaluate the destruction complex of beta-catenin by the expression of the proteins beta-catetenin, adenomatous polyposis coli, GSK3β, axin and ubiquitin in colorectal carcinoma and colonic adenoma. Methods Tissue samples from 64 patients with colorectal carcinoma and 53 patients with colonic adenoma were analyzed. Tissue microarray blocks and slides were prepared and subjected to immunohistochemistry with polyclonal antibodies in carcinoma, adjacent non-neoplastic mucosa, and adenoma tissues. The immunoreactivity was evaluated by the percentage of positive stained cells and by the intensity assessed through of the stained grade of proteins in the cytoplasm and nucleus of cells. In the statistical analysis, the Spearman correlation coefficient, Student’s t, χ2, Mann-Whitney, and McNemar tests, and univariate logistic regression analysis were used. Results In colorectal carcinoma, the expressions of beta-catenin and adenomatous polyposis coli proteins were significantly higher than in colonic adenomas (p<0.001 and p<0.0001, respectively). The immunoreactivity of GSK3β, axin 1 and ubiquitin proteins was significantly higher (p=0.03, p=0.039 and p=0.03, respectively) in colorectal carcinoma than in the colonic adenoma and adjacent non-neoplastic mucosa. The immunohistochemistry staining of these proteins did not show significant differences with the clinical and pathological characteristics of colorectal cancer and colonic adenoma. Conclusions These results suggest that, in adenomas, the lower expression of the beta-catenin, axin 1 and GSK3β proteins indicated that the destruction complex of beta-catenin was maintained, while in colorectal carcinoma, the increased expression of beta-catenin, GSK3β, axin 1, and ubiquitin proteins indicated that the destruction complex of beta-catenin was disrupted.

RESUMO Objetivo Avaliar o complexo de destruição da betacatenina no carcinoma colorretal e no adenoma do colo pela expressão das proteínas betacatenina, adenomatous polyposis coli, GSK3β, axina e ubiquitina. Métodos Amostras de tecidos de 64 doentes com carcinoma colorretal e de 53 pacientes com adenoma do colo foram analisadas. Blocos de tecidos foram submetidos ao estudo imuno-histoquímico com anticorpos policlonais nos tecidos do carcinoma, mucosa não neoplásica adjacente e adenoma. A imunorreatividade foi avaliada pela porcentagem de positividade de células coradas e pela intensidade do grau de coloração das proteínas no citoplasma e no núcleo das células. Na análise estatística, foram utilizados o coeficiente de correlação de Spearman, os testes t de Student, χ2, Mann-Whitney e de McNemar, e a análise de regressão logística univariada. Resultados No carcinoma colorretal, as expressões da betacatenina e da adenomatous polyposis coli foram significativamente maiores do que em adenomas do colo (p<0,001 e p<0,0001, respectivamente). A imunorreatividade das proteínas GSK3β, axina 1 e ubiquitina foi significativamente maior (p=0,03, p=0,039 e p=0,03, respectivamente) no carcinoma colorretal do que no adenoma e na mucosa não neoplásica adjacente. A coloração imuno-histoquímica dessas proteínas não apresentou diferenças significantes em relação às características clinicopatológicas do câncer colorretal e do adenoma. Conclusões Em adenomas, as menores expressões de betacatenina, axina 1 e GSK3β indicaram que o complexo de destruição da betacatenina estava conservado, enquanto que, no carcinoma colorretal, o aumento das expressões da betacatenina, GSK3β, 1 axina, e ubiquitina indicaram que o complexo de destruição de betacatenina estava alterado.

Loss of Ovarian Function Results in Increased Loss of Skeletal Muscle in Arthritic Rats / Perda da função ovariana resulta em perda de músculo esquelético aumentada em ratos artríticos

Objective We studied the effects of loss of ovarian function (ovariectomy) onmuscle mass of gastrocnemius and themRNA levels of IGF-1, atrogin-1, MuRF-1, andmyostatin in an experimental model of rheumatoid arthritis in rats. Methods We randomly allocated 24 female Wistar rats (9 weeks, 195.3±17.4 grams) into four groups: control (CT-Sham; n = 6); rheumatoid arthritis (RA; n = 6); ovariectomy without rheumatoid arthritis (OV; n = 6); ovariectomy with rheumatoid arthritis (RAOV; n = 6). We performed the ovariectomy (OV and RAOV) or Sham (CTSham or RA) procedures at the same time, fifteen days before the rheumatoid arthritis induction. The RA and RAOV groups were immunized and then were injected with Met- BSA in the tibiotarsal joint. After 15 days of intra-articular injections the animals were euthanized. We evaluated the external manifestations of rheumatoid arthritis (perimeter joint) as well as animal weight, and food intake throughout the study. We also analyzed the cross-sectional areas (CSA) of gastrocnemius muscle fibers in 200 fibers (H&E method). In the gastrocnemius muscle, we analyzed mRNA expression by quantitative real time PCR followed by the Livak method (ΔΔCT). Results The rheumatoid arthritis induced reduction in CSA of gastrocnemius muscle fibers. The RAOV group showed a lower CSA of gastrocnemius muscle fibers compared to RA and CT-Sham groups. Skeletal muscle IGF-1 mRNA increased in arthritics and ovariectomized rats. The increased IGF-1 mRNA was higher in OV groups than in the RA and RAOV groups. Antrogin-1 mRNA also increased in the gastrocnemius muscle of arthritic and ovariectomized rats. However, the increased atrogin-1 mRNA was higher in RAOV groups than in the RA and OV groups. Gastrocnemius muscle MuRF-1 mRNA increased in the OVand RAOVgroups, but not in the RA and Shamgroups. However, the RAOV group showed higher MuRF-1 mRNA than the OV group. The myostatin gene expression was similar in all groups. Conclusion Loss of ovarian function results in increased loss of skeletal musclerelated ubiquitin ligases atrogin-1 and MuRF-1 in arthritic rats.

Objetivo Foram estudados os efeitos da perda da função ovariana (ovariectomia) sobre músculo esquelético e os níveis de RNAm de IGF-1, atrogina-1, MuRF-1, e de miostatina em modelo experimental de artrite reumatóide em ratos. Métodos 24 ratos Wistar (9 semanas, 195,3±17,4 gramas) foram distribuídos aleatoriamente em quatro grupos: controle (CT-Sham, n = 6); artrite reumatóide (RA, n = 6); ovariectomia sem artrite reumatóide (OV; n = 6); ovariectomia com artrite reumatóide (RAOV; n = 6). Os procedimentos da ovariectomia (OV e RAOV) ou simulação da ovariectomia (CT-Shamou RA) foramrealizados aomesmo tempo, quinze dias antes da indução da artrite reumatóide. Os grupos RA e RAOV foramimunizados e, em seguida, foram injetados com Met-BSA na articulação tibiotársica. Após 15 dias das injeções intra-articulares, os animais foram eutanasiados. Foram avaliadas as manifestações externas da artrite reumatóide (perimetria articular), bem como o peso dos animais e a ingestão de alimentos ao longo do estudo. Além disso, as áreas de secção transversa (CSA) do músculo gastrocnêmio foram analisadas em 200 fibras (método H & E). No músculo gastrocnêmio, a expressão de RNAm foi analisada por PCR quantitativo em tempo real, seguido pelo método Livak (ΔΔCT). Resultados A artrite reumatoide reduziu a CSA das fibras do músculo gastrocnêmio. O grupo RAOV mostrou uma CSA menor nas fibras do músculo gastrocnêmio em comparação com os grupos RA e CT-Sham. O RNAm do IGF-1 do músculo esquelético aumentou nos ratos artríticos e ovariectomizados. O RNAm do IGF-1 foi maior nos grupos OV do que nos grupos RA e RAOV. A expressão de antrogina-1 também aumentou no músculo gastrocnêmio dos ratos artríticos e ovariectomizados. No entanto, o aumento do RNAm da atrogina-1 foi maior no grupo RAOV do que nos grupos RA e OV. O RNAm da MuRF-1 aumentou nos grupos OV e RAOV, mas não nos grupos RA e CT-Sham. Porém, o grupo RAOV apresentou maior expressão gênica de MuRF-1 do que o grupo OV. A expressão do gene da miostatina foi semelhante em todos os grupos. Conclusão A perda de função ovariana resulta em perda de músculo esquelético associado às ubiquitina-ligases atrogina-1 e MuRF-1 em ratos artríticos.

Assessment three constitutive promoters for GUS expression in rice (Oryza sativa L., var. J-104) / Evaluación de tres promotores constitutivos para la expresión GUS en arroz (Oryza sativa L., cv. J-104)

This work analyzed the constitutive expression of the ß-Glucuronidase (GUS) reporter gene fused to three promoters: the cauliflower mosaic virus (CaMV) 35S, the chimerical A9 promoter which contains rice Act1, and the Ubiquitine-1 promoter from maize. The activity of the promoters was qualitative and quantitatively obtained in different tissues and various growth stages of rice plants (cv J-104) transformed by biolistic. All the promoters were found to be active, with distinct patterns of relative activity in leaves, stem and roots from in vitro and ex vitro plants, and in plants of T1 progeny. The chimerical A9 promoter increased significantly levels of GUS expression in all the tissues and at all growth stages of the plants.

Se analizó la expresión constitutiva del gen reportero de la ß-Glucuronidasa (GUS) fusionado a tres promotores: el 35S del virus del mosaico de la coliflor (CaMV), el promotor quimérico A9 que contiene la actina-1 de arroz y el promotor ubiquitina-1 de maíz. La actividad de los promotores fue analizada cualitativa y cuantitativamente en diferentes tejidos y estadíos de crecimiento de plantas de arroz (variedad J-104) transformadas mediante biobalística. Se demostró la expresión constitutiva de GUS bajo los promotores estudiados, con distintos patrones de actividad relativa en hojas, tallos y raíces de plantas in vitro y ex vitro, y en plantas de la progenie T1. Bajo el promotor quimérico A9 se lograron los mayores niveles de expresión GUS en todos los tejidos y fases de crecimiento de las plantas.

Avaliação do 17-AAG como agente leishmanicida e seu mecanismo de ação na indução da morte de parasitos do gênero leishmania spp / 17-AAG assessment as leishmanicidal agent and its mechanism of action for induction of parasites of the Leishmania genus death spp

A leishmaniose é uma doença endêmica no Brasil causada por parasitos protozoários do gênero Leishmania. A quimioterapia continua sendo a forma mais efetiva de tratamento com os antimoniais pentavalentes sendo usados há mais de 70 anos como a primeira linha de tratamento. O uso deste e de outros fármacos apresenta efeitos adversos graves, os esquemas terapêuticos empregados são desconfortáveis, além de relatos do aumento de casos de resistência. A proteína de choque térmico 90 (HSP90) é um membro da família das chaperonas presente em células eucarióticas e bactérias. Essa proteína é fundamental para o dobramento e estabilização de diferentes proteínas, chamadas genericamente de proteínas cliente. Essa chaperona vem sendo considerada um importante alvo molecular para o tratamento de diferentes doenças parasitárias. Nessa tese, o inibidor específico da atividade ATPásica da HSP90, o 17-allilamino-17-demethoxigeldanamicina (17- AAG) foi testado em parasitos do gênero Leishmania. Inicialmente, avaliamos o efeito em cultura axênica e observamos que o 17-AAG causa a morte desses parasitos em concentrações inferiores às necessárias para causar a morte de macrófagos. Observamos também que o tratamento com 17-AAG promove a morte intracelular dos parasitos em concentrações que variam de 25 a 500 nM nos tempos de 24 e 48 h...

Leishmaniases are endemic disease in Brazil caused by protozoan parasites from the genus Leishmania. Chemotherapy remains the most effective way of treatment and pentavalent antimonials, used for more than 70 years, remaining as first choice drugs for leishmaniasis treatment. The use of this and other drugs causes severe side effects, therapeutic regimens employed for leishmaniasis treatment are unpleasant, besides an increase number of resistance cases. The Heat Shock Protein 90 (HSP90) is a member of the chaperone family present in bacteria and eukaryotic cells. This protein is essential for the folding and stabilization of different proteins, known as client proteins. This chaperone has been considered an important molecular target for the treatment of different parasitic diseases. In this thesis, the specific inhibitors of the ATPase activity from the HSP90, 17-allylamino- 17-demethoxygeldanamycin (17-AAG), were tested against parasites from the genus Leishmania. First we evaluated its effect on axenic culture and observed that 17- AAG induces parasite cell death in lowerconcentrations than those needed to induce macrophage cell death. We also observed that 17-AAG intracellular parasite death in concentrations ranging from 25 to 500 nM after 24 or 48 h...

Lack of association between polymorphisms in the UBASH3A gene and autoimmune thyroid disease: a case control study / Falta de associação entre polimorfismos do gene UBASH3A e doença tiroidiana autoimune: estudo caso controle

Objective: The aim of this study was to investigate UBASH3A gene variation association with autoimmune thyroid disease and clinical features in a Chinese Han population. Subjects and methods: A total of 667 AITD patients (417 GD and 250 HT) and 301 healthy controls were genotyped for two single nucleotide polymorphisms (SNPs) rs11203203, rs3788013 of UBASH3A gene, utilizing the Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometer (MALDI-TOF-MS) Platform. Results: Between the control group and AITD, GD and HT group, no statistically significant difference was observed in the genotypic and allelic frequencies of the two SNPs. There was no significant difference in allelic frequencies of the two SNPs between GD with and without ophthalmopathy. There was no significant difference in haplotype distributions between the control group and AITD, GD or HT group. Conclusion: Rs11203203 and rs3788013 in UBASH3A gene may not be associated with AITD patients in Chinese Han population. .

Objetivo: O objetivo deste estudo foi investigar a variação no gene UBASH3A com a doença tiroidiana autoimune e características clínicas na população chinesa Han. Sujeitos e métodos: Um total de 667 pacientes com DTAI (417 com DG e 250 com TH) e 301 controles saudáveis foi genotipado para dois polimorfismos de nucleotídeo simples (SNPs) rs11203203, rs3788013 do gene UBASH3A, usando-se a plataforma MALDI-TOF-MS (Ionização/Dessorção de Matriz Assistida por Laser – Tempo de Voo/Espectrômetro de Massa). Resultados: Não foram observadas diferenças significativas entre as frequências genotípicas e alélicas dos dois SNPs nos grupos controle e DTAI, DG e TH. Não houve diferenças significativas entre as frequências alélicas dos dois SNPs em pacientes com DG com ou sem olftalmopatia. Não houve diferenças significativas nas distribuições de haplótipos no grupo controle e nos grupos DTAI, DG e TH. Conclusão: Os SNPs rs11203203 e rs3788013 do gene UBASH3A podem não estar associados a pacientes com DTAI na população chinesa Han. .

Preliminary study of the enzyme ubiquitin carboxylterminal hydrolase 14 (UBP6) in Giardia intestinalis: structural bioinformatic analysis and transcriptional profile during encystation / Estudio preliminar de la enzima ubiquitina carboxiterminal hidrolasa 14 (UBP6) en Giardia intestinalis: análisis bioinformático de su estructura y perfil transcripcional durante la enquistación / Estudo preliminar da enzima ubiquitina carboxi-terminal hidrolasa 14 (UBP6) em Giardia intestinalis: análise bioinformática estrutural e perfil transcricional durante encistamento

This paper presents a combined approach with two aims. The first is to analyze the reported sequence of the enzyme ubiquitin carboxyl-terminal hydrolase 14 of Giardia intestinalis (UBP6) through computational methods to find components related with its hypothetical function. The second is to determine if the protein-coding gene is expressed in G. intestinalis and, if such is the case, also determine its transcription pattern along the life cycle of the parasite. It was established that the protein belongs to the family of Cys-dependent deubiquitinases and more specifically to ubiquitin specific proteases (USPs). Moreover, the catalytic center with the complete triad as well as typical features of the USP motif were also identified. Since the computational findings suggest that the enzyme could be functional, reverse transcription coupled to PCR was used as a first approach to establish if in fact the coding gene is expressed in the parasite. Interestingly, it was found not only that the gene is expressed, but also that there is a transcription variation along the life cycle of the parasite. These two findings are the starting point for further studies since they tentatively suggest that this enzyme could be involved in the protein turnover that occurs during parasite encystation. Although preliminary, this study is the first report concerning the study of a specific deubiquitinating enzyme in the parasite G. intestinalis.

En este trabajo se presenta una estrategia combinada que buscaba, primero, analizar por métodos computacionales la secuencia de la enzima ubiquitina carboxilo-terminal hidrolasa 14 de Giardia intestinalis (UBP6) reportada para buscar componentes relacionados con su función hipotética y segundo, determinar si el gen que codifica para la proteína se expresa en G. intestinalis y si lo hace, cómo es su patrón de transcripción a lo largo del ciclo de vida del parásito. Se encontró que la proteína pertenece a la familia de deubiquitinasas dependientes de cisteína y más específicamente a las proteasas específicas para ubiquitina (USPs por ubiquitin specific proteases). También se identificaron el centro catalítico con la triada completa así como características típicas del motivo USP. Teniendo en cuenta que los resultados computacionales sugieren que la enzima puede ser funcional, se usó la técnica de transcripción reversa acoplada a PCR como un primer acercamiento para establecer si el gen codificante se expresa en el parásito. De manera interesante, se determinó no solo que el gen se expresa sino que existe una variación de su transcripción a lo largo del ciclo de vida del parásito. Estos hallazgos son el punto de partida para posteriores estudios ya que sugieren de manera preliminar que esta enzima podría estar involucrada en el recambio de proteínas que ocurre en el parásito durante el proceso de enquistación. Aunque preliminar, este estudio es el primer reporte acerca de una enzima deubiquitinadora específica en el parásito G. intestinalis.

Este artigo apresenta uma abordagem combinada com dois objetivos. A primeira é analisar a sequência informou da enzima ubiquitina carboxil-terminal hidrolase 14 de Giardia intestinalis (UBP6) através de métodos computacionais para encontrar os componentes relacionados com a sua função hipotética. A segunda é para determinar se o gene de codificação da proteína é expressa em G. intestinalis e, se for o caso, também determinar o seu padrão de transcrição ao longo do ciclo de vida do parasita. Foi estabelecido que a proteína pertence à família de deubiquitinases Cys-dependentes e mais especificamente para proteases específicas de ubiquitina (USPs por ubiquitin specific proteases). Além disso, o centro catalítico com a tríade completo, bem como as características típicas do motivo USP também foram identificados. Uma vez que os resultados computacionais sugerem que a enzima poderia ser funcional, a transcrição reversa acoplada a PCR foi utilizado como uma primeira abordagem para determinar se, de facto, o gene codificante é expressa no parasita. Curiosamente, verificou-se não só que o gene é expresso, mas também que há uma variação de transcrição ao longo do ciclo de vida do parasita. Estes dois elementos são o ponto de partida para estudos posteriores, uma vez que tentativas sugerem que esta enzima pode estar envolvida no refill de proteínas que ocorre durante o parasita encistamento. Embora preliminares, este estudo é o primeiro relatório relativo ao estudo de uma enzima deubiquitinadora específica no parasita intestinalis.

Regulação diferencial da deubiquitinase A20 em tecido adiposo de obesos: potencial envolvimento na regulação da PGC1a / A20 deubiquitinase controls PGC-1a expression in the adipose tissue

A proteína PGC1α é um co-ativador de transcrição gênica que desempenha papel importante na regulação de uma série de fenômenos metabólicos que compreendem desde o controle da termogênese e mitocondriogênese até a regulação da secreção de insulina e a produção hepática de glicose. Como vários dos fenômenos biológicos controlados direta ou indiretamente pela PGC1α tem importância vital, a regulação dos níveis de PGC1α nos tecidos deve ser finamente ajustada. Nos últimos anos, inúmeros estudos exploraram os mecanismos envolvidos com o controle da expressão gênica e tradução da PGC1α. Entretanto, apenas alguns poucos estudos avaliaram a degradação da mesma. Um dos mais importantes mecanismos envolvidos com a regulação funcional e da meia-vida de proteínas é a ubiquitinação, que pode direcionar proteínas alvo ao proteassoma para degradação ou a outras modificações pós-traducionais. O objetivo do presente estudo foi avaliar a participação de uma proteína com atividade deubiquitinase e ubiquitina ligase, a A20, na manutenção da homeostase do tecido adiposo de animais submetidos à dieta rica em gordura e voluntários humanos magros e obesos antes e após cirurgia de redução de peso. Foram utilizados o tecido adiposo branco visceral e subcutâneo e o tecido adiposo marrom de camundongos Swiss machos submetidos a 16 semanas de dieta hiperlipídica e o tecido adiposo subcutâneo de voluntários magros e obesos antes e após a cirurgia bariátrica. Esses tecidos foram avaliados quanto ao conteúdo protéico e expressão gênica da proteína A20, e sua associação com a PGC1α por imunoprecipitação e imunofluorescência, bem como a ubiquitinação desta última. Os resultados obtidos a partir do tecido adiposo de humanos mostram uma diminuição na expressão da proteína A20 nos pacientes antes e após a cirurgia bariátrica com relação aos voluntários magros.

Peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) plays an important role in whole body metabolism and, particularly in glucose homeostasis. Its expression is tightly regulated and, small variations in tissue levels can have a major impact in a number of physiological and pathological conditions. Recent studies have shown that the ubiquitin/proteasome system plays a role in the control of PGC-1α degradation. Here we evaluated the interaction of PGC-1α with the protein A20, which plays a dual-role in the control of the ubiquitin/proteasome system acting as a deubiquitinase and as an E3 ligase. We employed immunoprecipitation, quantitative real-time PCR and immunofluorescence staining to evaluate PGC-1α, A20, PPARγ and ubiquitin in the adipose tissue of humans and mice. Our results show that, in distinct sites of the adipose tissue A20 binds to PGC-1α. At least in the subcutaneous fat of humans and mice the levels of PGC-1α decrease during obesity, while its physical association with A20 increases. The inhibition of A20 leads to a reduction of PGC-1α and PPARγ expression, suggesting that A20 acts as a protective factor against PGC-1α disposal. Thus, we provide evidence that mechanisms regulating PGC-1α ubiquitination are potentially involved in the control of the function of this transcriptional co-activator.

La vía ubiquitina-proteasoma ¿destruir o construir? ese es el dilema / Ubiquitin-proteasome pathway to built or to destroy? that is the question

Introducción: la construcción de nuevas células es un proceso complejo y para lograr que sea unidireccional e irreversible la célula utiliza el mecanismo de destruir proteínas que se oponen al paso de una etapa a la siguiente. Objetivo: demostrar que la destrucción de proteínas contribuye a la reproducción celular. Método: se analizaron más de 500 artículos de los últimos 5 años publicados en revistas nacionales y de circulación internacional, disponibles en las bases de datos HINARI, PubMed y Perii y localizados mediante el sitio infomed. Desarrollo: primero, se hizo una exposición sobre la vía ubiquitina-proteasoma. Después, se analizó la participación en el ciclo celular de los dos grandes complejos con actividad de ubiquitina-ligasa que son los encargados de marcar las proteínas que deben ser destruidas. Estos complejos actúan consecutiva y coordinadamente, y sus acciones determinan el progreso en un solo sentido del ciclo de vida de la célula. Conclusiones: la destrucción selectiva de proteínas mediante la vía ubiquitina proteasoma permite la formación de nuevas células y con ello la perpetuación de la vida.

Introduction: the generation of new cells is an extremely complex process. To become this process unidirectional and irreversible cells destroy proteins which actions are oppose to the transition from one phase to the next one. Objective: to show that protein destruction is essential for cell reproduction. Material and Methods: more than 500 papers published during the last five years in national and international journals were analyzed. These articles are available in HINARI, PubMed, and Perii databases and were localized through infomed. Development: first, the ubiquitin-proteasome pathway is presented. Next, the contribution of the complexes SCF an anaphase promoting complex to the progression of the cell cycle is analysed. These complexes act consecutively and coordinately and their actions determine de progression of the cell´s life. Conclusions: the selective destroy of specific proteins by mean of ubiquitin-proteasome pathway, allow new cells formation, and ensure the continuity of life.

La vía ubiquitina-proteasoma ¿destruir o construir? ese es el dilema / Ubiquitin-proteasome pathway to built or to destroy? that is the question

Introducción: la construcción de nuevas células es un proceso complejo y para lograr que sea unidireccional e irreversible la célula utiliza el mecanismo de destruir proteínas que se oponen al paso de una etapa a la siguiente. Objetivo: demostrar que la destrucción de proteínas contribuye a la reproducción celular. Método: se analizaron más de 500 artículos de los últimos 5 años publicados en revistas nacionales y de circulación internacional, disponibles en las bases de datos HINARI, PubMed y Perii y localizados mediante el sitio infomed.Desarrollo: primero, se hizo una exposición sobre la vía ubiquitina-proteasoma. Después, se analizó la participación en el ciclo celular de los dos grandes complejos con actividad de ubiquitina-ligasa que son los encargados de marcar las proteínas que deben ser destruidas. Estos complejos actúan consecutiva y coordinadamente, y sus acciones determinan el progreso en un solo sentido del ciclo de vida de la célula.Conclusiones: la destrucción selectiva de proteínas mediante la vía ubiquitina proteasoma permite la formación de nuevas células y con ello la perpetuación de la vida(AU)

Introduction: the generation of new cells is an extremely complex process. To become this process unidirectional and irreversible cells destroy proteins which actions are oppose to the transition from one phase to the next one. Objective: to show that protein destruction is essential for cell reproduction. Material and Methods: more than 500 papers published during the last five years in national and international journals were analyzed. These articles are available in HINARI, PubMed, and Perii databases and were localized through infomed. Development: first, the ubiquitin-proteasome pathway is presented. Next, the contribution of the complexes SCF an anaphase promoting complex to the progression of the cell cycle is analysed. These complexes act consecutively and coordinately and their actions determine de progression of the cell´s life. Conclusions: the selective destroy of specific proteins by mean of ubiquitin-proteasome pathway, allow new cells formation, and ensure the continuity of life(AU)

HSP90 is a target protein for ubiquitination in Giardia intestinalis / HSP90 es una proteina blanco de ubiquitinación en Giardia intestinalis / HSP90 é uma proteína alvo de ubiquitinação em Giardia intestinalis

Previous studies have demonstrated the existence and expression of genes essential to the process of protein ubiquitination in Giardia intestinalis, indicating that the ubiquitin-proteasome system may be involved in the degradation of proteins during its life cycle of the parasite. In this study, purification of ubiquitin was conducted from protein extracts of G. intestinalis trophozoites. Then, an anti-ubiquitin specific antibody was obtained to standardize an assay for the detection and evaluation of ubiquitination patterns. Finally, HSP90 was identified as an ubiquitinated protein in this protozoan. This post-translational modification could have regulatory effects associated with the functionality of the protein or its turnover to regulate key molecular events during the parasite’s life cycle.

Estudios previos han demostrado la existencia y expresión de genes esenciales para el proceso de ubiquitinación de proteínas en Giardia intestinalis, indicando que el sistema ubiquitina-proteosoma puede estar involucrado en el proceso de degradación de proteínas de este parásito durante su ciclo de vida. En el presente trabajo se realizó la purificación de ubiquitina a partir de extractos proteicos de trofozoítos de G. intestinalis, se produjo un anticuerpo anti-ubiquitina específico que permitió la estandarización de un ensayo para la detección y evaluación de los patrones de ubiquitinación, y se identificó la HSP90 como una proteína ubiquitinada en este protozoario. Esta modificación post-transduccional puede tener efectos regulatorios asociados con la funcionalidad de la proteína o con el recambio para regular eventos moleculares claves durante el ciclo de vida del parásito.

Estudos anteriores demonstraram a existência e expressão de genes essenciais para o processo de ubiquitinação de proteínas em Giardia intestinalis, indicando que o sistema ubiquitina-proteassoma podem estar envolvidos na degradação de proteínas do parasita durante seu ciclo de vida. Neste trabalho, foi realizada a purificação da proteína ubiquitina de extratos de trofozoítos de G. intestinalis, foi produzido um antiicorpo anti-ubiquitina específico que permitiu a padronização de um ensaio para a detecção e avaliação de padrões de ubiquitinação e foi identificado a HSP90 como uma proteína ubiquitinada no protozoário. Esta modificação pós-transducional pode ter efeitos regulamentares associados com a funcionalidade das proteínas ou com o a substituição para regular eventos moleculares importantes durante o ciclo de vida do parasita.

Detección de b-Amiloide, proteína TAU hiperfosforilada y ubiquitina por técnica de inmunohistoquímica en cerebros de caninos mayores de 10 años / Detection b-Amiloid, hyperphosphorylate TAU protein and ubiquitin for technique the immunohistochemistry in brains in dogs over 10 years

El interés en el estudio del proceso de envejecimiento cerebral y los cambios comportamentales relacionados con la edad en caninos y felinos geriátricos ha aumentado en la última década (Mentzel, 2005a; Ingram & Williams, 2010). Las alteraciones del comportamiento canino que responden a cambios fisiopatológicos relacionados con la edad y que involucran distintas esferas conductuales y cognitivas, se engloban bajo la denominación de Síndrome de Disfunción Cognoscitiva (SDC) (Mentzel, 2005b). El cual fue descrito en 1997 y se observó en perros de más de 9 años (Ingram & Williams). El Síndrome de Disfunción Cognoscitivo (SDC) es también llamado Alzheimer canino por las similitudes histopatológicas cerebrales y conductuales relacionadas. En el presente estudio, los caninos geriátricos presentaron los mismos depósitos proteicos anormales presentes en la enfermedad de Alzheimer, como las placas seniles, los cuerpos ubiquitinados y la angiopatía amiloide, pero no presentaron los ovillos neurofibrilares, los cuales son los responsables de la demencia que se padece en la enfermedad. Las únicas herramientas de evaluación prácticas en clínica para esta patología son las encuestas y formularios relacionados con aspectos comportamentales. Además existen exámenes diagnósticos postmortem que permiten identificar de una manera eficaz la presencia de elementos anormales involucrados en la neuropatología, uno de ellos es la inmunohistoquímica. En este estudio se utilizaron los anticuerpos monoclonales de uso humano anti-b-amiloide, anti-proteína tau y anti-ubiquitina en muestras de cerebros de perros mayores de 10 años. El modelo canino hoy en día está constituyendo un significado indispensable para el estudio de los procesos de neurodegeneración porque ha permitido un acercamiento a la teoría del problema desde nuevas perspectivas (Dimakopoulos & Mayer, 2002).

The interest in the study of the process of cerebral aging and the changes in the behaviour related to age in geriatric canine and feline has increased in the last decade (Mentzel, 2005a; Ingram & Williams, 2010). The alterations of the canine behaviour that respond to physiopathological changes related to the age and which involve different conducts and cognitive spheres are included under the denomination of Canine Cognitive Dysfunction Syndrome (CDS) called canine Alzheimer, considering the cerebral and histopathologic behavioural similarities. In the present study the geriatric patients displayed the same abnormal protein deposits present in Alzheimer disease, such as senile plaques ubiquitinate bodies and the amyloid angiopathy. However they did not display the balls of neurofibrillary tangles which are characteristic of the disease. The only practical tools of evaluation in clinic for this pathology are the surveys and forms related to behavioural aspects. In addition there are post mortem exams such as those in inmunohistochemistry, that allow an effective identification of abnormal elements present in the neuropathology. In this study the monoclonal antibodies were used anti-b-amyloid, anti-protein tau and anti-ubiquitin in samples of brains in dogs over 10 years of age. The canine model constitutes an indispensable meaning for the study of the neurodegeneration processes because it has allowed an approach to the theory of the problem from a new perspective (Dimakopoulos & Mayer, 2002).

Fundamentos moleculares de la enfermedad de von Hippel Lindau / Molecular bases of von Hipperl Lindau's disease

La enfermedad de von Hippel Lindau es una rara entidad genética que se caracteriza por la predisposición al cáncer, especialmente angiomas de la retina, hemangioblastomas del sistema nervioso central y carcinoma renal de células claras. Los productos del gen presentan un mecanismo de acción peculiar, pues está relacionado con los procesos de adaptación del organismo a la hipoxia. En este trabajo se presenta una panorámica actualizada de esta enfermedad, con énfasis en los aspectos moleculares

The von Hippel Lindau's disease is uncommon genetic entity characterized by a predisposition to cancer, specially the retina angiomas, hemangioblastomas of central nervous system and the clear cells renal carcinoma. Gene products have a typical action mechanism since it is related to organism adaptation processes to hypoxia. In present paper an updated panorama of this disease emphasizing in molecular processes

Fundamentos moleculares de la enfermedad de von Hippel Lindau / Molecular bases of von Hipperl Lindau's disease

La enfermedad de von Hippel Lindau es una rara entidad genética que se caracteriza por la predisposición al cáncer, especialmente angiomas de la retina, hemangioblastomas del sistema nervioso central y carcinoma renal de células claras. Los productos del gen presentan un mecanismo de acción peculiar, pues está relacionado con los procesos de adaptación del organismo a la hipoxia. En este trabajo se presenta una panorámica actualizada de esta enfermedad, con énfasis en los aspectos moleculares (AU)

The von Hippel Lindau's disease is uncommon genetic entity characterized by a predisposition to cancer, specially the retina angiomas, hemangioblastomas of central nervous system and the clear cells renal carcinoma. Gene products have a typical action mechanism since it is related to organism adaptation processes to hypoxia. In present paper an updated panorama of this disease emphasizing in molecular processes (AU)