Vladimir F. Lazarev

Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease

The key feature of polyglutamine aggregates accumulating in the course of Huntington disease (HD) is their resistance to protein denaturants, and to date only chaperones are proved to prevent mutant protein aggregation. It was suggested that expanded polyglutamine chains (polyQ) of mutant huntingtin are cross-linked to other proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Here we clarify the roles of GAPDH and molecular chaperone Hsp70 in the formation of sodium dodecyl sulfate (SDS)-insoluble polyQ aggregates. First, the addition of pure GAPDH was found to enhance the aggregation of polyQ in a cell-free model of HD. Secondly, the immunodepletion of GAPDH dose-dependently decreased polyQ aggregation. Finally, siRNA-mediated inhibition of GAPDH protein in SK-N-SH neuroblastoma cells has also reduced the aggregation of cellular polyQ. Regulated over-expression of Hsp70 decreased the amount of GAPDH associated with SDS-insoluble polyQ aggregates. Physical association of Hsp70 and GAPDH in SK-N-SH cells was shown by reciprocal immunoprecipitation and confocal microscopy. Pure Hsp70 dose-dependently inhibited the formation of polyQ aggregates in cell-free model of HD by sequestering both GAPDH and polyQ. We demonstrated that Hsp70 binds to polyQ in adenosine triphosphate-dependent manner, which suggests that Hsp70 exerts a chaperoning activity in the course of this interaction. Binding of Hsp70 to GAPDH was nicotinamide adenine dinucleotide-dependent suggesting another type of association. Based on our findings, we conclude that Hsp70 protects cells in HD by removing/sequestering two intrinsic components of protein aggregates: the polyQ itself and GAPDH. We propose that GAPDH might be an important target for pharmacological treatment of HD and other polyglutamine expansion-related diseases.

Pharmacological protein targets in polyglutamine diseases: Mutant polypeptides and their interactors

Polyglutamine diseases are a group of pathologies affecting different parts of the brain and causing dysfunction and atrophy of certain neural cell populations. These diseases stem from mutations in various cellular genes that result in the synthesis of proteins with extended polyglutamine tracts. In particular, this concerns huntingtin, ataxins, and androgen receptor. These mutant proteins can form oligomers, aggregates, and, finally, aggresomes with distinct functions and different degrees of cytotoxicity. In this review, we analyze the effects of different forms of polyQ proteins on other proteins and their functions, which are considered as targets for therapeutic intervention.

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

Proteins 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.

Kinetics of chaperone activity of proteins Hsp70 and Hdj1 in human leukemia u-937 cells after preconditioning with thermal shock or compound u-133.

Kinetics of the chaperone activity of proteins Hsp70 and Hdj1 were analyzed in human U-937 promonocytes during their response to heat shock or to treatment with the echinochrome triacetyl glucoside derivative U-133. To measure the chaperone activity of both proteins, a special test was developed for their recognition and binding of a denatured protein. Using this test, the chaperone activity could be concurrently estimated in large numbers of cellular or tissue extracts. We also estimated the contents of both chaperones in cells by immunoblotting. The values for contents of Hsp70 and Hdj1 obtained by two independent test systems coincided, and this suggested that the substrate-binding activity could change proportionally to the chaperone content in the protein mixture. Therefore, the test developed by us can be employed for high throughput screening of drugs activating cellular chaperones. The analysis of quantity and activity of two cellular chaperones during the cell response to heat stress or to the drug-like substance U-133 showed that both factors caused the accumulation of chaperones with similar kinetics. We conclude that the efficiency of drug preconditioning could be close to the efficiency of hyperthermia and that the high activity of chaperones could be retained in human cells for no less than 1.5 days.

Hsp70 chaperone rescues C6 rat glioblastoma cells from oxidative stress by sequestration of aggregating GAPDH

The Hsp70 chaperone is known to elicit cytoprotective activity and this protection has a negative impact in anti-tumor therapy. In cancer cells subjected to oxidative stress Hsp70 may bind damaged polypeptides and proteins involved in apoptosis signaling. Since one of the important targets of oxidative stress is glyceraldehyde-3-phospate dehydrogenase (GAPDH) we suggested that Hsp70 might elicit its protective effect by binding GAPDH. Microscopy data show that in C6 rat glioma cells subjected to hydrogen peroxide treatment a considerable proportion of the GAPDH molecules are denatured and according to dot ultrafiltration data they form SDS-insoluble aggregates. Using two newly developed assays we show that Hsp70 can bind oxidized GAPDH in an ATP-dependent manner. Pharmacological up- or down-regulation of Hsp70 with the aid of U133 echinochrome or triptolide, respectively, reduced or increased the number of C6 glioma cells containing GAPDH aggregates and dying due to treatment with hydrogen peroxide. Using immunoprecipitation we found that Hsp70 is able to sequester aggregation-prone GAPDH and this may explain the anti-oxidative power of the chaperone. The results of this study led us to conclude that in cancer cells constantly exposed to conditions of oxidative stress, the protective power of Hsp70 should be abolished by specific inhibitors of Hsp70 expression.

Glyceraldehyde 3‐phosphate dehydrogenase augments the intercellular transmission and toxicity of polyglutamine aggregates in a cell model of Huntington disease

The common feature of Huntington disease is the accumulation of oligomers or aggregates of mutant huntingtin protein (mHTT), which causes the death of a subset of striatal neuronal populations. The cytotoxic species can leave neurons and migrate to other groups of cells penetrating and damaging them in a prion‐like manner. We hypothesized that the glycolytic enzyme glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), previously shown to elevate the aggregation of mHTT, is associated with an increased efficiency of intercellular propagation of mHTT. GAPDH, on its own or together with polyglutamine species, was shown to be released into the extracellular milieu mainly from dying cells as assessed by a novel enzyme immunoassay, western blotting, and ultrafiltration. The conditioned medium of cells with growing GAPDH‐polyQ aggregates was toxic to naïve cells, whereas depletion of the aggregates from the medium lowered this cytotoxicity. The GAPDH component of the aggregates was found to increase their toxicity by two‐fold in comparison with polyQ alone. Furthermore, GAPDH‐polyQ complexes were shown to penetrate acceptor cells and to increase the capacity of polyQ to prionize its intracellular homolog containing a repeat of 25 glutamine residues. Finally, inhibitors of intracellular transport showed that polyQ‐GAPDH complexes, as well as GAPDH itself, penetrated cells using clathrin‐mediated endocytosis. This suggested a pivotal role of the enzyme in the intercellular transmission of Huntington disease pathogenicity. In conclusion, GAPDH occurring in complexes with polyglutamine strengthens the prion‐like activity and toxicity of the migrating aggregates.

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.

Knock-down of Hdj2/DNAJA1 co-chaperone results in an unexpected burst of tumorigenicity of C6 glioblastoma cells.

The chaperone system based on Hsp70 and proteins of the DnaJ family is known to protect tumor cells from a variety of cytotoxic factors, including anti-tumor therapy. To analyze whether this also functions in a highly malignant brain tumor, we knocked down the expression of Hsp70 (HSPA1A) and its two most abundant co-chaperones, Hdj1 (DNAJB1) and Hdj2 (DNAJA1) in a C6 rat glioblastoma cell line. As expected, tumor depletion of Hsp70 caused a substantial reduction in its growth rate and increased the survival of tumor-bearing animals, whereas the reduction of Hdj1 expression had no effect. Unexpectedly, a reduction in the expression of Hdj2 led to the enhanced aggressiveness of the C6 tumor, demonstrated by its rapid growth, metastasis formation and a 1.5-fold reduction in the lifespan of tumor-bearing animals. The in vitro reduction of Hdj2 expression reduced spheroid density and simultaneously enhanced the migration and invasion of C6 cells. At the molecular level, a knock-down of Hdj2 led to the relocation of N-cadherin and the enhanced activity of metalloproteinases 1, 2, 8 and 9, which are markers of highly malignant cancer cells. The changes in the actin cytoskeleton in Hdj2-depleted cells indicate that the protein is also important for prevention of the amoeboid-like transition of tumor cells. The results of this study uncover a completely new role for the Hdj2 co-chaperone in tumorigenicity and suggest that the protein is a potential drug target.

Properties of substances inhibiting aggregation of oxidized GAPDH: Data on the interaction with the enzyme and the impact on its intracellular content

This data is related to our paper “Small molecules preventing GAPDH aggregation are therapeutically applicable in cell and rat models of oxidative stress” (Lazarev et al. [1]) where we explore therapeutic properties of small molecules preventing GAPDH aggregation in cell and rat models of oxidative stress. The present article demonstrates a few of additional properties of the chemicals shown to block GAPDH aggregation such as calculated site for targeting the enzyme, effects on GAPDH glycolytic activity, influence on GAPDH intracellular level and anti-aggregate activity of pure polyglutamine exemplifying a denatured protein.

The development of modified human Hsp70 (HSPA1A) and its production in the milk of transgenic mice

The production of major human heat shock protein Hsp70 (HSPA1A) in a eukaryotic expression system is needed for testing and possible medical applications. In this study, transgenic mice were produced containing wild-type human Hsp70 allele in the vector providing expression in the milk. The results indicated that human Hsp70 was readily expressed in the transgenic animals but did not apparently preserve its intact structure and, hence, it was not possible to purify the protein using conventional isolation techniques. It was suggested that the protein underwent glycosylation in the process of expression, and this quite common modification for proteins expressed in the milk complicated its isolation. To check this possibility, we mutated all presumptive sites of glycosylation and tested the properties of the resulting modified Hsp70 expressed in E. coli. The investigation demonstrated that the modified protein exhibited all beneficial properties of the wild-type Hsp70 and was even superior to the latter for a few parameters. Based on these results, a transgenic mouse strain was obtained which expressed the modified Hsp70 in milk and which was easy to isolate using ATP columns. Therefore, the developed construct can be explored in various bioreactors for reliable manufacture of high quality, uniform, and reproducible human Hsp70 for possible medical applications including neurodegenerative diseases and cancer.