Heat shock and other proteotoxic stresses cause accumulation of nonnative proteins that trigger activation of heat shock protein (Hsp) genes. A chaperone/Hsp functioning as repressor of heat shock transcription factor (HSF) could make activation of hsp genes dependent on protein unfolding. In a novel in vitro system, in which human HSF1 can be activated by nonnative protein, heat, and geldanamycin, addition of Hsp90 inhibits activation. Reduction of the level of Hsp90 but not of Hsp/c70, Hop, Hip, p23, CyP40, or Hsp40 dramatically activates HSF1. In vivo, geldanamycin activates HSF1 under conditions in which it is an Hsp90-specific reagent. Hsp90-containing HSF1 complex is present in the unstressed cell and dissociates during stress. We conclude that Hsp90, by itself and/or associated with multichaperone complexes, is a major repressor of HSF1.

Molecular chaperones, ubiquitin ligases and proteasome impairment have been implicated in several neurodegenerative diseases, including Alzheimer's and Parkinson's disease, which are characterized by accumulation of abnormal protein aggregates (e.g. tau and alpha-synuclein respectively). Here we report that CHIP, an ubiquitin ligase that interacts directly with Hsp70/90, induces ubiquitination of the microtubule associated protein, tau. CHIP also increases tau aggregation. Consistent with this observation, diverse of tau lesions in human postmortem tissue were found to be immunopositive for CHIP. Conversely, induction of Hsp70 through treatment with either geldanamycin or heat shock factor 1 leads to a decrease in tau steady-state levels and a selective reduction in detergent insoluble tau. Furthermore, 30-month-old mice overexpressing inducible Hsp70 show a significant reduction in tau levels. Together these data demonstrate that the Hsp70/CHIP chaperone system plays an important role in the regulation of tau turnover and the selective elimination of abnormal tau species. Hsp70/CHIP may therefore play an important role in the pathogenesis of tauopathies and also represents a potential therapeutic target.

Transcriptional activity of heat shock (hsp) genes is controlled by a heat-activated, group-specific transcription factor(s) recognizing arrays of inverted repeats of the element NGAAN. To date genes for two human factors, HSF1 and HSF2, have been isolated. To define their properties as well as the changes they undergo during heat stress activation, we prepared polyclonal antibodies to these factors. Using these tools, we have shown that human HeLa cells constitutively synthesize HSF1, but we were unable to detect HSF2. In unstressed cells HSF1 is present mainly in complexes with an apparent molecular mass of about 200 kDa, unable to bind to DNA. Heat treatment induces a shift in the apparent molecular mass of HSF1 to about 700 kDa, concomitant with the acquisition of DNA-binding ability. Cross-linking activation of transcription of hsp genes is indeed mediated by denatured proteins (4), suggesting protein denaturation as the common denominator of many of the disparate treatments inducing the response. Earlier studies have also suggested that hsp gene expression may be subject to autoregulation (15). In concordance with findings that members of the hsp7O family of proteins are capable of binding to denatured proteins and peptides (18,36,38), as well as of associating with nascent polypeptides (8), several recent studies showing down-regulation of the transcriptional stress response following overexpression of hsp7O (52) and the propensity of hsp7O to directly bind HSF (2, 7) have implicated hsp70 as the autoregulatory factor. After the cloning of HSF genes from a variety of organisms (13, 41, 43-45, 51, 63), support for a negative mode of regulation of HSF activity has come from observations of constitutive transcriptional activity of mutated Saccharomyces cerevisiae HSF (9, 35) and of constitutive DNA-binding activity of wild-type Drosophila and human HSF expressed in bacteria (13,41,45). Further evidence for such a regulatory mechanism has been provided by experiments showing that derepression of DNA-binding activity of human HSF can be reproduced in vitro by treatment with heat (33; see reference 13 for analogous experiments with Drosophila cells) and with agents affecting protein conformation (34). Surprisingly, considering the divergence of transcriptional mechanisms, quite analogous findings concerning the role of denatured proteins in triggering the stress response (21) and its autoregulation by hsp7O (DnaK) and other hsps (20,(53)(54)(55)(56) were made with bacteria.The second question with which this study is mainly concerned is directed toward the biochemical analysis of molecular events occurring when HSF is converted from an

Heat shock protein (hsp) genes, a group of ubiquitous genes, are activated by various metabolic stresses. The suggestion that denaturation of intracellular proteins may be produced by the metabolic stresses and then signal the activation of the hsp genes was examined by co-injection of purified proteins and hsp genes into frog oocytes. Activation of hsp genes was observed if the proteins were denatured prior to injection but not if they were introduced in their native form. Furthermore, the activation of hsp genes by abnormal proteins and by heat shock appears to occur by a common mechanism. A model for the transcriptional regulation of the genes is based on competition for degradation between abnormal intracellular proteins and a labile regulatory factor.

The promoters of heat shock protein genes are among the best-studied inducible eucaryotic promoters. Regions responsible for heat regulation have been identified previously by deletion experiments with several different heat shock genes. In this paper the critical importance of two novel features of heat shock regulatory elements was investigated. First, the elements were modular and, as a consequence, displayed a characteristic 5-nucleotide periodicity produced by multiple GAA blocks that were arranged in alternating orientations and at 2-nucleotide intervals. Functional heat shock regulatory elements appeared to include three or more of these blocks. Second, the nucleotides at the two positions immediately upstream from GAA segments played an important role in defining the competence of regulatory elements.Heat shock protein (hsp) genes occur in all cell types examined so far and are typically silent at the temperature of normal growth but are expressed at exceedingly high levels at elevated temperatures or in cells suffering from other types of stress (36). Drosophila melanogaster hsp70 genes encoding a major hsp of 70 kilodaltons (kDa) were the first hsp genes to be introduced into a variety of different cell types, such an NIH 3T3 cells (9), monkey COS cells (24, 30), Xenopus oocytes (3, 43), Drosophila cells (6,7,10,11,19,20), and others (see reference 27 for review). In most cell types, the genes were expressed in a heat-regulated fashion. A region located about 45 to 65 nucleotides upstream from the transcription start site of hsp70 genes was found to be essential for heat regulation in monkey cells and Xenopus oocytes (3,24,30). A second region containing related sequences, between about -65 and -90, was required in addition to the above region for high activity in Drosophila cells (1,11,39).Comparison of the -45 to -65 sequence of Drosophila hsp70 genes and of analogous sequences in other hsp genes led to the establishment of a heat shock consensus sequence, CNNGAANNTTCNNG, (31), where N is any nucleotide.Factors binding to this type of sequence have been identified (17,28,37,45,47,48), and two groups have purified such factors from Drosophila nuclear extracts and have shown that they are specifically involved in transcriptional activation of hsp genes (28,45,48).Since the sequences described above are the only elements specifically required for heat regulation and are also binding sites for hsp gene-specific transcription factors, they are of central importance for the understanding of heat shock regulation. Earlier studies in this laboratory (2) as well as by another group (40) had indicated that the heat shock consensus sequence that had been accepted for many years as the prototype heat shock regulatory sequence did not by itself function as a regulatory element. We have attempted here to define experimentally the nature of heat shock regulatory elements.MATERIALS AND METHODS Plasmid constructions. Plasmids ( Fig. 1 and 2) were derived from constructs D88 and D50 (2). In the latter constructs, D. m...