GAPDH binders as potential drugs for the therapy of polyglutamine diseases: Design of a new screening assay

AbstractProteins with long polyglutamine repeats form a complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which enhances aggregation and cytotoxicity in models of Huntington disease. The aim of this study was to develop a novel assay for the screening of anti-aggregation compounds with a focus on the aggregation-promoting capacity of GAPDH. The assay includes a pure Q58 polyglutamine fragment, GAPDH, and a transglutaminase that links the two proteins. The feasibility of the new assay was verified using two GAPDH binders, hydroxynonenal and −(−)deprenyl, and the benzothiazole derivative PGL-135 which exhibits anti-aggregation effect. All three substances were shown to reduce aggregation and cytotoxicity in the cell and in the fly model of Spinocerebellar ataxia

ВЗАИМОСВЯЗЬ КЛИНИКО-МОРФОЛОГИЧЕСКИХ ПАРАМЕТРОВ И ВЫЖИВАЕМОСТИ ПРИ АДЕНОКАРЦИНОМЕ ЛЕГКОГО С АКТИВНОСТЬЮ ЯДРЫШКОВЫХ ОРГАНИЗАТОРОВ В MIB-1 ПОЗИТИВНЫХ КЛЕТКАХ

Aim: To investigate argyrophilic proteins associated with nucleolar organizer regions (Ag-NOR proteins) in proliferating cells in connection with clinical and morphological parameters and survival in patients with adenocarcinoma of the lung.Materials and methods: Specimens of 94 operated patients with lung adenocarcinoma were investigated using double staining for antigen Ki-67 (Clone MIB-1, DAKO) by immunohistochemistry and Ag-NOR proteins staining with silver nitrate.Results: In the tumor tissue Ag-NOR proteins in the MIB-1 positive cells correlated with clinical and morphological parameters on TNM-system: T-index, the tumor size, N-index, disease stage and differentiation. Survival of patients with a small area of Ag-NOR proteins in the MIB-1 positive cells was better than in patients with large area. The area of Ag-NOR proteins in the MIB-1 positive cells – is an independent prognostic factor in patients with adenocarcinoma of the lung.Conclusion: The area of Ag-NOR proteins in the MIB-1 positive cells correlated with clinical and morphological parameters of the TNM-system and survival in patients with adenocarcinoma of the lung.Цель:  Исследовать аргирофильные белки, ассоциированные с ядрышкообразующими районами  (Ag-ЯОР-белки) в пролиферирующих клетках во взаимосвязи  с клинико-морфологическими параметрами и выживаемостью при аденокарциноме легкого.Материал и методы. Исследованы 94 операционных материалов аденокарциномы легкого с помощью двойного окрашивания на антиген Ki-67 (клон  MIB-1,  DAKO) методом иммуногистохимии и на Ag-ЯОР-белки азотнокислым серебром.Результаты: В аденокарциноме  легкого площадь Ag-ЯОР-белков в MIB-1 позитивных клетках взаимосвязана с клинико-морфологическими параметрами по системе TNM: показателем Т, наибольшим размером  опухоли, показателем N, стадией заболевания  и дифференцировкой. Выживаемость больных с небольшой площадью Ag-ЯОР-белков в MIB-1 позитивных клетках лучше по сравнению с большой. Площадь Ag-ЯОР-белков в MIB-1 позитивных клетках – независимый фактор прогноза при аденокарциноме легкого.Заключение: Площадь Ag-ЯОР-белков в MIB-1 позитивных клетках взаимосвязана  с клинико-морфологическими параметрами по системе TNM и выживаемостью при аденокарциноме легкого

Possible Function of Molecular Chaperones in Diseases Caused by Propagating Amyloid Aggregates

The vast majority of neurodegenerative pathologies stem from the formation of toxic oligomers and aggregates composed of wrongly folded proteins. These protein complexes can be released from pathogenic cells and enthralled by other cells, causing the formation of new aggregates in a prion-like manner. By this mechanism, migrating complexes can transmit a disorder to distant regions of the brain and promote gradually transmitting degenerative processes. Molecular chaperones can counteract the toxicity of misfolded proteins. In this review, we discuss recent data on the possible cytoprotective functions of chaperones in horizontally transmitting neurological disorders

Protein Interactome of Amyloid-β as a Therapeutic Target

The amyloid concept of Alzheimer’s disease (AD) assumes the β-amyloid peptide (Aβ) as the main pathogenic factor, which injures neural and other brain cells, causing their malfunction and death. Although Aβ has been documented to exert its cytotoxic effect in a solitary manner, there is much evidence to claim that its toxicity can be modulated by other proteins. The list of such Aβ co-factors or interactors includes tau, APOE, transthyretin, and others. These molecules interact with the peptide and affect the ability of Aβ to form oligomers or aggregates, modulating its toxicity. Thus, the list of potential substances able to reduce the harmful effects of the peptide should include ones that can prevent the pathogenic interactions by specifically binding Aβ and/or its partners. In the present review, we discuss the data on Aβ-based complexes in AD pathogenesis and on the compounds directly targeting Aβ or the destructors of its complexes with other polypeptides

Hydrocortisone 21-hemisuccinate did not prevent exogenous GAPDH-induced apoptosis in human neuroblastoma cells

These data are related to our paper “GAPDH-targeted therapy – a new approach for secondary damage after traumatic brain injury on rats” (Lazarev et al., In press), in which we explore the role of exogenous GAPDH in traumatic brain injury-induced neuron death, and the therapeutic application of small molecules that bind to the enzyme. The current article demonstrates the induction of apoptosis by exogenous GAPDH and the effectiveness of the hydrocortisone derivative for suppressing the pathogenic action of the enzyme

Disruption of the Complex between GAPDH and Hsp70 Sensitizes C6 Glioblastoma Cells to Hypoxic Stress

Hypoxia, which commonly accompanies tumor growth, depending on its strength may cause the enhancement of tumorigenicity of cancer cells or their death. One of the proteins targeted by hypoxia is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and we demonstrated here that hypoxia mimicked by treating C6 rat glioblastoma cells with cobalt chloride caused an up-regulation of the enzyme expression, while further elevation of hypoxic stress caused the enzyme aggregation concomitantly with cell death. Reduction or elevation of GAPDH performed with the aid of specific shRNAs resulted in the augmentation of the tumorigenicity of C6 cells or their sensitization to hypoxic stress. Another hypoxia-regulated protein, Hsp70 chaperone, was shown to prevent the aggregation of oxidized GAPDH and to reduce hypoxia-mediated cell death. In order to release the enzyme molecules from the chaperone, we employed its inhibitor, derivative of colchicine. The compound was found to substantially increase aggregation of GAPDH and to sensitize C6 cells to hypoxia both in vitro and in animals bearing tumors with distinct levels of the enzyme expression. In conclusion, blocking the chaperonic activity of Hsp70 and its interaction with GAPDH may become a promising strategy to overcome tumor resistance to multiple environmental stresses and enhance existing therapeutic tools

Dynamics of the O. felineus Infestation Intensity and Egg Production in Carcinogenesis and Partial Hepatectomy in the Setting of Superinvasive Opisthorchiasis

Clinical and experimental studies have shown that opisthorchii tend to evade tumour growth foci to colonize more distant areas of the liver. When modelling tumours with various carcinogens in the setting of superinvasive opisthorchiasis, the intensity of invasion is reduced both before the formation of neoplasms (>120 days) and after the development of tumours of various histogeneses (liver, pancreas, and stomach) (>240 days). Egg production was observed to increase with the decrease in the number of parasites in the liver. The smallest changes in the infestation intensity indicators and egg production were observed in the experimental stomach tumours (p>0.05). A partial hepatectomy in the setting of opisthorchiasis did not affect the number of parasites in the ecological niche (liver) or the production of eggs by the helminth. With the deterioration of the vegetation state, parasite clumps of opisthorchii increase egg production under the conditions of distress

Etoposide-Induced Apoptosis in Cancer Cells Can Be Reinforced by an Uncoupled Link between Hsp70 and Caspase-3

The Hsp70 chaperone binds and inhibits proteins implicated in apoptotic signaling including Caspase-3. Induction of apoptosis is an important mechanism of anti-cancer drugs, therefore Hsp70 can act as a protective system in tumor cells against therapeutic agents. In this study we present an assessment of candidate compounds that are able to dissociate the complex of Hsp70 with Caspase-3, and thus sensitize cells to drug-induced apoptosis. Using the PASS program for prediction of biological activity we selected a derivative of benzodioxol (BT44) that is known to affect molecular chaperones and caspases. Drug affinity responsive target stability and microscale thermophoresis assays indicated that BT44 bound to Hsp70 and reduced the chaperone activity. When etoposide was administered, heat shock accompanied with an accumulation of Hsp70 led to an inhibition of etoposide-induced apoptosis. The number of apoptotic cells increased following BT44 administration, and forced Caspase-3 processing. Competitive protein–protein interaction and immunoprecipitation assays showed that BT44 caused dissociation of the Hsp70–Caspase-3 complex, thus augmenting the anti-tumor activity of etoposide and highlighting the potential role of molecular separators in cancer therapy

Spectral F-Test power evaluation in the EEG during intermittent photic stimulaton Avaliação do teste-F espectral do EEG durante fotoestimulação intermitente

Intermittent photic stimulation (IPS) is an important functional test, which can induce the photic driving in the electroencephalogram (EEG). It is capable of enhancing latent oscillation’s manifestations not present in the resting EEG. However, for adequate quantitative evaluation of the photic driving, these changes should be assessed on a statistical basis. With this aim, the sampling distribution of spectral F test was investigated. On this basis, confidence limits of the SFT-estimate could be obtained for different practical situations, in which the signal-to-noise ratio and the number of epochs used in the estimation may vary. The technique was applied to the EEG of 10 normal subjects during IPS, and allowed detecting responses not only at the fundamental IPS frequency but also at higher harmonics. It also permitted to assess the strength of the photic driving responses and to compare them in different derivations and in different subjects.<br>A fotoestimulação intermitente (FEI) é um importante teste functional, que pode induzir o fotorecrutamento no eletroencefalograma (EEG), sendo capaz de realçar manifestações latentes de oscilações não observadas no EEG de repouso. Entretanto, para uma análise quantitativa adequada do fotorecrutamento, tais alterações devem ser avaliadas com base estatística. Assim, a distribuição de probabilidade do teste-F espectral (TFE) foi investigada. Neste sentido, limites de confiança para a estimativa do TFE puderam ser obtidos para diferentes situações práticas, nas quais a razão sinal-ruído e o número de épocas usadas na estimação podem variar. A técnica foi aplicada ao EEG de 10 sujeitos normais durante FEI, e permitiu a detecção de respostas não somente na freqüência fundamental da FEI como também em seus harmônicos. Além disso, permitiu avaliar o grau de fotorecrutamento entre derivações distintas e entre diferentes sujeitos