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Nine neurodegenerative disorders are caused by the abnormal expansion of polyglutamine (polyQ) regions within distinct proteins. Genetic and biochemical evidence has documented that the molecular chaperone, heat shock protein 70 (Hsp70), modulates polyQ toxicity and aggregation, yet it remains unclear how Hsp70 might be used as a potential therapeutic target in polyQ-related diseases. We have utilized a pair of membrane-permeable compounds that tune the activity of Hsp70 by either stimulating or by inhibiting its ATPase functions. Using these two pharmacological agents in both yeast and PC12 cell models of polyQ aggregation and toxicity, we were surprised to find that stimulating Hsp70 solubilized polyQ conformers and simultaneously exacerbated polyQ-mediated toxicity. By contrast, inhibiting Hsp70 ATPase activity protected against polyQ toxicity and promoted aggregation. These findings clarify the role of Hsp70 as a possible drug target in polyQ disorders and suggest that Hsp70 uses ATP hydrolysis to help partition polyQ proteins into structures with varying levels of proteotoxicity. Our results thus support an emerging concept in which certain kinds of polyQ aggregates may be protective, while more soluble polyQ species are toxic.

Molecular chaperones, such as heat shock protein 70 (Hsp70) and its bacterial ortholog DnaK, play numerous important roles in protein folding. In vitro, this activity can be observed by incubating purified chaperones with denatured substrates and measuring the recovery of properly folded protein. In an effort to rapidly identify small molecules that modify this folding activity, we modified an existing method for use in 96-well plates. In this assay, denatured firefly luciferase was treated with a mixture of DnaK and prospective chemical modulators. The luminescence of refolded luciferase was used to follow the reaction progress, and counterscreens excluded compounds that target luciferase; thus, hits from these screens modify protein folding via their effects on the function of the chaperone machine. Using this platform, we screened a pilot chemical library and found five new inhibitors of DnaK and one compound that promoted folding. These chemical probes may be useful in studies aimed at understanding the many varied roles of chaperones in cellular protein folding. Moreover, this assay provides the opportunity to rapidly screen for additional compounds that might regulate the folding activity of Hsp70.

Alzheimer disease is a neurological disorder that is characterized by the presence of fibrils and oligomers composed of the amyloid beta (Abeta) peptide. In models of Alzheimer disease, overexpression of molecular chaperones, specifically heat shock protein 70 (Hsp70), suppresses phenotypes related to Abeta aggregation. These observations led to the hypothesis that chaperones might interact with Abeta and block self-association. However, although biochemical evidence to support this model has been collected in other neurodegenerative systems, the interaction between chaperones and Abeta has not been similarly explored. Here, we examine the effects of Hsp70/40 and Hsp90 on Abeta aggregation in vitro. We found that recombinant Hsp70/40 and Hsp90 block Abeta self-assembly and that these chaperones are effective at substoichiometric concentrations (approximately 1:50). The anti-aggregation activity of Hsp70 can be inhibited by a nonhydrolyzable nucleotide analog and encouraged by pharmacological stimulation of its ATPase activity. Finally, we were interested in discerning what type of amyloid structures can be acted upon by these chaperones. To address this question, we added Hsp70/40 and Hsp90 to pre-formed oligomers and fibrils. Based on thioflavin T reactivity, the combination of Hsp70/40 and Hsp90 caused structural changes in oligomers but had little effect on fibrils. These results suggest that if these chaperones are present in the same cellular compartment in which Abeta is produced, Hsp70/40 and Hsp90 may suppress the early stages of self-assembly. Thus, these results are consistent with a model in which pharmacological activation of chaperones might have a favorable therapeutic effect on Alzheimer disease.

NtcA is a transcription factor found in a wide variety of cyanobacteria. It is a key component in the control of the nitrogen metabolism, and regulates genes involved in ammonia assimilation, heterocyst differentiation and nitrogen fixation. NtcA expression is subject to nitrogen control, but there is also evidence that the binding of NtcA to DNA can be regulated by a redox mechanism involving the two cysteine residues in the NtcA protein from Anabaena PCC 7120. In order to investigate this further, the two cysteine residues in NtcA were mutated into alanine to give four variants of the protein: wild-type NtcA, the point-mutated variants Cys157Ala and Cys164Ala, as well as the double mutant Cys157Ala/Cys164Ala. The binding of a DNA probe containing a palindromic NtcA-binding motif was investigated by gel mobility shift analysis under non-reducing and reducing conditions. The experiments show that the DNA binding in vitro is stronger in the presence of the reducing agent DTT than in its absence. However, this effect is not due to breaking of a disulfide bond between the cysteine residues, since the double mutant containing no cysteines was also affected by DTT. A molecular model of a monomer of NtcA, based on the homologous cAMP receptor protein structure, was created in order to locate the positions of the cysteine residues. The NtcA model suggested that the positions of the sulfur atoms are not compatible with formation of a bond between them.

A recursive in vitro selection among random DNA sequences was used for analysis of the cyanobacterial transcription factor NtcA-binding motifs. An eight-base palindromic sequence, TGTA-(N-8)-TACA, ...

The transcription factor NtcA from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli. In order to optimize the expression of NtcA, random silent mutations were introduced at the 5' end of the DNA encoding the protein. To get as high a yield of pure protein as possible, different strategies of expression as well as purification conditions were used. Under optimal expression conditions, a high-level expression clone of NtcA was coexpressed with GroEL-ES at 37 degrees C. A hexahistidine tag was added to the N-terminus of the protein in order to allow purification on an IMAC affinity column. Expression followed by one purification step using IMAC affinity chromatography gave a yield of 30-40 mg pure NtcA protein per liter of bacterial culture. Gel-shift experiments showed that the recombinant NtcA was active in binding a DNA sequence containing an NtcA-specific site.

The very evolutionarily conserved human carbonic anhydrase-related polypeptide (CA-RP VIII) lacks the carbon-dioxide hydration-activity, characteristic of the enzymatically active carbonic anhydrases. We have expressed HCA-RP VIII as a glutathione-S-transferase fusion protein (GST-HCA-RP VIII). The purified HCA-RP VIII showed a substantially higher apparent molecular weight by gel-filtration compared to the molecular weight calculated from the amino acid sequence, indicating a larger than expected Stoke's radius. Like other studied CA's, the protein unfolds through two transitions at increasing concentrations of guanidine hydrochloride. The far-UV CD spectra of HCA-RP VIII indicates a secondary structure similar to that of the catalytically active HCA II. The very high sequence identity between human and mouse CA-RP VIII (98%), might indicate that the function of the protein involves binding of another protein. However, an attempt to use the GST-HCA-RP VIII fusion protein to affinity purify a ligand was unsuccessful.

Proteins with expanded polyglutamine (polyQ) segments cause a number of fatal neurodegenerative disorders, including Huntington's disease (HD). Previous high-throughput screens in cellular and biochemical models of HD have revealed compounds that mitigate polyQ aggregation and proteotoxicity, providing insight into the mechanisms of disease and leads for potential therapeutics. However, the structural diversity of natural products has not yet been fully mobilized toward these goals. Here, we have screened a collection of ~11 000 natural product extracts for the ability to recover the slow growth of ΔProQ103-expressing yeast cells in 384-well plates (Z' ~ 0.7, CV ~ 8%). This screen identified actinomycin D as a strong inhibitor of polyQ aggregation and proteotoxicity at nanomolar concentrations (~50-500 ng/mL). We found that a low dose of actinomycin D increased the levels of the heat-shock proteins Hsp104, Hsp70 and Hsp26 and enhanced binding of Hsp70 to the polyQ in yeast. Actinomycin also suppressed aggregation of polyQ in mammalian cells, suggesting a conserved mechanism. These results establish natural products as a rich source of compounds with interesting mechanisms of action against polyQ disorders.

In Saccharomyces cerevisae, expanded polyglutamine (polyQ) fragments are assembled into discrete cytosolic aggregates in a process regulated by the molecular chaperones Hsp26, Hsp70, Hsp90, and Hsp104. To better understand how the different chaperones might cooperate during polyQ aggregation, we used sequential immunoprecipitations and mass spectrometry to identify proteins associated with either soluble (Q25) or aggregation-prone (Q103) fragments at both early and later times after induction of their expression. We found that Hsp26, Hsp70, Hsp90, and other chaperones interact with Q103, but not Q25, within the first 2 h. Further, Hsp70 and Hsp90 appear to be partially released from Q103 prior to the maturation of the aggregates and before the recruitment of Hsp104. To test the importance of this seemingly ordered process, we used a chemical probe to artificially enhance Hsp70 binding to Q103. This treatment retained both Hsp70 and Hsp90 on the polyQ fragment and, interestingly, limited subsequent exchange for Hsp26 and Hsp104, resulting in incomplete aggregation. Together, these results suggest that partial release of Hsp70 may be an essential step in the continued processing of expanded polyQ fragments in yeast.

Molecular chaperones, such as Hsp70 and Hsp90, are responsible for a variety of protective, anti-apoptotic functions. While inhibitors of Hsp90, such as geldanamycin and its derivative 17-AAG, are well known and important anti-cancer leads, Hsp70 has received less attention. Interesting lead candidates for Hsp70 share a dihydropyrimidine core; however, the preferred display of pendant functionality is still not clear. Here, we take advantage of the versatility of peptides to explore the requirements for activity. An exploratory compound collection was assembled by performing a Biginelli cyclocondensation at the terminus of a resin-bound beta-peptide. Liberation from solid support yielded peptide-modified dihydropyrimidines and, within this series, we uncovered compounds that alter the ATPase activity of Hsp70 and its bacterial ortholog, DnaK. Moreover, we identified important contributions made by aromatic, hydrophobic groups. These chemical probes could be used to study the roles of this molecular chaperone in disease.

DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.

NtcA is a transcription factor that acts as a global nitrogen regulator in cyanobacteria. Cyanobacteria are photosynthetic prokaryotic organisms, some genera of which can fix nitrogen under conditions of nitrogen deprivation. NtcA from Anabaena PCC 7120 is a dimeric protein that consists of 223 amino acids with a molecular weight of 25 kDa per subunit. It belongs to the cAMP receptor-protein (CAP) family and is involved in the regulation of several of the genes acting in the nitrogen-fixation process. Here, the crystallization and preliminary X-ray data of NtcA are described. The crystallization was made possible by an improved purification method, which provides a stable NtcA protein at concentrations suitable for crystallization. The protein was crystallized using the hanging-drop method. Data were collected to 2.5 A resolution using synchrotron radiation and the crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 69.23, b = 69.23, c = 162.15 A, alpha = beta = gamma = 90 degrees. The phases necessary to solve the structure of NtcA could not be obtained by molecular replacement based on the CAP structure using various models.