Julia Aguirre

Roles of heat-shock protein 90 in maintaining and facilitating the neurodegenerative phenotype in tauopathies

Neurodegeneration, a result of multiple dysregulatory events, is a lengthy multistep process manifested by accrual of mutant variants and abnormal expression, posttranslational modification, and processing of certain proteins. Accumulation of these dysregulated processes requires a mechanism that maintains their functional stability and allows the evolution of the neurodegenerative phenotype. In malignant cells, the capacity to buffer transformation has been attributed to heat-shock protein 90 (Hsp90). Although normal proteins seem to require limited assistance from the chaperone, their aberrant counterparts seem to be highly dependent on Hsp90. Whereas enhanced Hsp90 affinity for mutated or functionally deregulated client proteins has been observed for several oncoproteins, it is unknown whether Hsp90 plays a similar role for neuronal proteins and thus maintains and facilitates the transformed phenotype in neurodegenerative diseases. Tauopathies are neurodegenerative diseases characterized by aberrant phosphorylation and/or expression of Tau protein, leading to a time-dependent accumulation of Tau aggregates and subsequent neuronal death. Here, we show that the stability of p35, a neuronal protein that activates cyclin-dependent protein kinase 5 through complex formation leading to aberrant Tau phosphorylation, and that of mutant but not WT Tau protein is maintained in tauopathies by Hsp90. Inhibition of Hsp90 in cellular and mouse models of tauopathies leads to a reduction of the pathogenic activity of these proteins and results in elimination of aggregated Tau. The results identify important roles played by Hsp90 in maintaining and facilitating the degenerative phenotype in these diseases and provide a common principle governing cancer and neurodegenerative diseases.

The yeast Hsp110, Sse1p, exhibits high-affinity peptide binding.

Hsp110s are divergent relatives of Hsp70 chaperones that hydrolyze ATP. Hsp110s serve as Hsp70 nucleotide exchange factors and act directly to maintain polypeptide solubility. To date, the impact of peptide binding on Hsp110 ATPase activity is unknown and an Hsp110/peptide affinity has not been measured. We now report on a peptide that binds to the yeast Hsp110, Sse1p, with a K D of ∼2 nM. Surprisingly, the binding of this peptide fails to stimulate Sse1p ATP hydrolysis. Moreover, an Hsp70‐binding peptide is unable to associate with Sse1p, suggesting that Hsp70s and Hsp110s possess partially distinct peptide recognition motifs.

High-Throughput Screening Fluorescence Polarization Assay for Tumor-Specific Hsp90

Heat shock protein 90 (Hsp90) is a molecular chaperone that has emerged as an important target in cancer and several other diseases, such as neurodegenerative diseases, nerve injuries, inflammation, and infection. Discovery of novel agents that inhibit Hsp90 and have druglike properties is therefore a major focus in several academic and industrial laboratories. In this study, the authors describe the development and optimization in a 384-well format of a novel assay for the identification of Hsp90 inhibitors using fluorescence polarization, which measures competitive binding of red-shifted fluorescently labeled geldanamycin (GM-cy3B) to Hsp90 found in the NCI-N417 small-cell lung carcinoma cells. The authors demonstrate that GMcy3B binds with high affinity and specificity to cellular Hsp90. The assay results in excellent signal-to-noise ratios (>10) and Z′ values (>0.75) at tracer concentrations greater than 4 nM and 1 µg/well of total NCI-N417 protein, indicating a robust assay. It also equilibrates after 5 h of incubation at room temperature and remains stable for up to 24 h. Furthermore, it is a simple mix-and-read format that is cost-effective and uses only low amounts of fluorophore and cell lysates. A study using more than 15,000 compounds from the National Institutes of Health Molecular Libraries Screening Center Network was performed to validate its performance in a high-throughput screening format. (Journal of Biomolecular Screening 2007:915-924)

Design of a fluorescence polarization assay platform for the study of human Hsp70

The 70kDa heat shock proteins (Hsp70) are molecular chaperones that assist in folding of newly synthesized polypeptides, refolding or denaturation of misfolded proteins, and translocation of proteins across biological membranes. In addition, Hsp70 play regulatory roles in signal transduction, cell cycle, and apoptosis. Here, we present a novel assay platform based on fluorescence polarization that is suitable for investigating the yet elusive molecular mechanics of human Hsp70 allosteric regulation.

Design of a Flexible Cell-Based Assay for the Evaluation of Heat Shock Protein 70 Expression Modulators

Heat shock protein 70 (Hsp70) is a chaperone protein that helps protect against cellular stress, a function that may be co-opted to fight human diseases. In particular, the upregulation of Hsp70 can suppress the neurotoxicity of misfolded proteins, suggesting possible therapeutic strategies in neurodegenerative diseases. Alternatively, in cancer cells where high levels of Hsp70 inhibit both intrinsic and extrinsic apoptotic pathways, a reduction in Hsp70 levels may induce apoptosis. To evaluate and identify, in a single assay format, small molecules that induce or inhibit endogenous Hsp70, we have designed and optimized a microtiter assay that relies on whole-cell immunodetection of Hsp70. The assay utilizes a minimal number of neuronal or cancer cells, yet is sufficiently sensitive and reproducible to permit quantitative determinations. We further validated the assay using a panel of Hsp70 modulators. In conclusion, we have developed an assay that is fast, robust, and cost efficient. As such, it can be implemented in most research laboratories. The assay should greatly improve the speed at which novel Hsp70 inducers and inhibitors of expression can be identified and evaluated.