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Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that plays multiple roles in protein homeostasis. In these various tasks, the activity of Hsp70 is shaped by interactions with co-chaperones, such as Hsp40. The Hsp40 family of co-chaperones binds to Hsp70 through a conserved J-domain, and these factors stimulate ATPase and protein-folding activity. Using chemical screens, we identified a compound, 115-7c, which acts as an artificial co-chaperone for Hsp70. Specifically, the activities of 115-7c mirrored those of a Hsp40; the compound stimulated the ATPase and protein-folding activities of a prokaryotic Hsp70 (DnaK) and partially compensated for a Hsp40 loss-of-function mutation in yeast. Consistent with these observations, NMR and mutagenesis studies indicate that the binding site for 115-7c is adjacent to a region on DnaK that is required for J-domain-mediated stimulation. Interestingly, we found that 115-7c and the Hsp40 do not compete for binding but act in concert. Using this information, we introduced additional steric bulk to 115-7c and converted it into an inhibitor. Thus, these chemical probes either promote or inhibit chaperone functions by regulating Hsp70–Hsp40 complex assembly at a native protein–protein interface. This unexpected mechanism may provide new avenues for exploring how chaperones and co-chaperones cooperate to shape protein homeostasis.

A number of missense mutations in the two related small heat shock proteins HspB8 (Hsp22) and HspB1 (Hsp27) have been associated with the inherited motor neuron diseases (MND) distal hereditary motor neuropathy and Charcot-Marie-Tooth disease. HspB8 and HspB1 interact with each other, suggesting that these two etiologic factors may act through a common biochemical mechanism. However, their role in neuron biology and in MND is not understood. In a yeast two-hybrid screen, we identified the DEAD box protein Ddx20 (gemin3, DP103) as interacting partner of HspB8. Using co-immunoprecipitation, chemical cross-linking, and in vivo quantitative fluorescence resonance energy transfer, we confirmed this interaction. We also show that the two disease-associated mutant HspB8 forms have abnormally increased binding to Ddx20. Ddx20 itself binds to the survival-of-motor-neurons protein (SMN protein), and mutations in the SMN1 gene cause spinal muscular atrophy, another MND and one of the most prevalent genetic causes of infant mortality. Thus, these protein interaction data have linked the three etiologic factors HspB8, HspB1, and SMN protein, and mutations in any of their genes cause the various forms of MND. Ddx20 and SMN protein are involved in spliceosome assembly and pre-mRNA processing. RNase treatment affected the interaction of the mutant HspB8 with Ddx20 suggesting RNA involvement in this interaction and a potential role of HspB8 in ribonucleoprotein processing.

Neurodegenerative diseases caused by abnormal accumulation of the microtubule associated protein tau (MAPT, tau) are collectively called tauopathies. The most devastating tau related disorder is Alzheimer’s disease (AD). Molecular chaperones such as heat shock proteins (Hsp) have emerged as critical regulators of tau stability. Several studies from our group and others have shown that the chaperone network can be targeted for the development of therapeutic strategies for AD and other neurodegenerative diseases. Here we will discuss a recent paper and current work from our laboratory where we have manipulated the ATPase activity of the 70-kDa heat shock protein (Hsp70) to regulate tau turnover. A high-throughput screening assay revealed several compounds that activated or inhibited Hsp70’s ATPase activity. Inhibitors dramatically and rapidly reduced tau levels, whereas activators stabilized tau, both in cells and brain tissue. Moreover, increased levels of Hsp70 improved ATPase inhibitor efficacy, suggesting that therapies aimed at inducing Hsp70 levels followed by inhibition of its ATPase activity may be a very effective strategy to treat AD. These findings demonstrate that Hsp70 ATPase activity can be targeted to modify the pathologies of AD and other tauopathies.

HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.

Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.

Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

Saccharomyces cerevisiae has a single integral plasma membrane heat shock protein (Hsp). This Hsp30 is induced by several stresses, including heat shock, ethanol exposure, severe osmostress, weak organic acid exposure and glucose limitation, Plasma membrane H+-ATPase activities of heat shocked and weak acid-adapted, hsp30 mutant and wild-type cells, revealted that Hsp30 induction leads to a downregulation of the stress-stimulation of this H+-ATPase. Plasma membrane H+-ATPase activity consumes a substantial fraction of the ATP generated by the cell, a usage that will be increased by the H+-ATPase stimulation occurring with several Hsp30-inducing stresses. Hsp30 might therefore provide an energy conservation role, limiting excessive ATP consumption by plasma membrance H+-ATPase during prolonged stress exposure or glucose limitation, Consistent with the role of Hsp30 being energy conservation, Hsp30 null cultures give lower final biomass yields. They also have lower ATP levels, consistent with higher H+-ATPase activity, at the glucose exhaustion stage of batch fermentations (diauxic lag), when Hsp30 is normally induced. Loss of Hsp30 does not affect several strees tolerances but it extends the time needed for cells to adapt to growth under several stressful conditions where the maintenance of homeostasis will demand an unusually high usage of energy. hsp30 is the first yeast gene identified as both weak organic acid-inducible and assisting the adaptation to growth in the presence of these acids.

Abstract

The goal of this study was to identify whether heat-shock protein 90 (Hsp90) regulates the production of superoxide and other reactive oxygen species from the NADPH oxidases (Nox). We found that pharmacological and genetic inhibition of Hsp90 directly reduced Nox5-derived superoxide without secondarily modifying signaling events. Coimmunoprecipitation and bioluminescence resonance energy transfer studies suggest that the C-terminus of Nox5 binds to Hsp90. Long-term Hsp90 inhibition reduced Nox5 expression and provides further evidence that Nox5 is an Hsp90 client protein. Inhibitors of Hsp90 also reduced superoxide from Nox1, Nox2 (neutrophils), and Nox3. However, Nox4, which emits only hydrogen peroxide, was unaffected by Hsp90 inhibitors. Hydrogen peroxide production from the other Nox enzymes was not affected by short-term inhibition of Hsp90, but long-term inhibition reduced production of all reactive oxygen species coincident with loss of enzyme expression. Expression of chimeric Nox enzymes consisting of N-terminal Nox1 or Nox3 and C-terminal Nox4 resulted in only hydrogen peroxide formation that was insensitive to Hsp90 inhibitors. We conclude that Hsp90 binds to the C-terminus of Noxes1–3 and 5 and is necessary for enzyme stability and superoxide production. Hsp90 does not bind to the C-terminus of Nox4 and is not required for hydrogen peroxide formation. Antioxid. Redox Signal. 14, 2107–2119.

The heat shock protein 70 (Hsp70) family of chaperones play crucial roles in protein folding and have been linked to numerous diseases. We were interested in developing a generally applicable assay format for the Hsp70 family and have developed fluorescence polarization based assays for both the mammalian Hsp72 and its bacterial counterpart, DnaK. These assays are comparable in assay set-up, incubation conditions and buffer components. Both unfolded polypeptides and synthetic peptides can be utilized as tracers to detect binding although peptides meeting the minimum seven residue length for Hsp70 binders have weaken binding when modified with fluorescein presumably due to steric effects. Although we did not identify a suitable general substrate for all Hsp70 proteins, fluorescein tagged peptide substrates that gave high affinity binding were identified for both DnaK and hsp72. We would predict that these assays will be suitable for identifying both selective chemical probes of Hsp70 family members and “pan” Hsp70 inhibitors.

Retroviral integrase enzymes have a nonspecific endonuclease activity that is stimulated by certain compounds, suggesting that integrase could be manipulated to damage viral DNA. To identify integrase stimulator (IS) compounds as potential antiviral agents, we have developed a nonradioactive assay that is suitable for high-throughput screening. The assay uses a 49-mer oligonucleotide that is 5'-labeled with a fluorophore, 3'-tagged with a quencher, and designed to form a hairpin that mimics radioactive double-stranded substrates in gel-based nicking assays. Reactions in 384-well plates are analyzed on a real-time PCR machine after a single heat denaturation and subsequent cooling to a point between the melting temperatures of unnicked substrate and nicked products (no cycling is required). Under these conditions, unnicked DNA reforms the hairpin and quenches fluorescence, whereas completely nicked DNA yields a large signal. The assay was linear with time, stimulator concentration, and amount of integrase, and 20% concentrations of the solvent used for many chemical libraries did not interfere with the assay. The assay had an excellent Z'-factor, and it reliably detected known IS compounds. This assay, which is adaptable to other nonspecific nucleases, will be useful for identifying additional IS compounds to develop the novel antiviral strategy of stimulating integrase to destroy retroviral DNA.

Abstract

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex.

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a Vmax of 32.5 nmol Pi/min and a KM of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg2+ and K+ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.

Background

A newly identified mechanism of smooth muscle relaxation is the interaction between the small heat shock protein 20 (HSP20) and 14-3-3 proteins. Focusing upon this class of interactions, we describe here a novel drug target screening approach for treating airflow obstruction in asthma.

Methods

Using a high-throughput fluorescence polarization (FP) assay, we screened a library of compounds that could act as small molecule modulators of HSP20 signals. We then applied two quantitative, cell-based biophysical methods to assess the functional efficacy of these molecules and rank-ordered their abilities to relax isolated human airway smooth muscle (ASM). Scaling up to the level of an intact tissue, we confirmed in a concentration-responsive manner the potency of the cell-based hit compounds.

Results

Among 58,019 compound tested, 268 compounds caused 20% or more reduction of the polarized emission in the FP assay. A small subset of these primary screen hits, belonging to two scaffolds, caused relaxation of isolated ASM cell in vitro and attenuated active force development of intact tissue ex vivo.

Conclusions

This staged biophysical screening paradigm provides proof-of-principle for high-throughput and cost-effective discovery of new small molecule therapeutic agents for obstructive lung diseases.

Saccharomyces cerevisiae contain a multigene family related to the Drosophila heat shock gene hsp70. Two members of this family, YG100 and YG101, have been previously characterized (Ingolia et al., Mol. Cell. Biol. 2:1388-1398, 1982), and only YG100 was found to have elevated levels of transcription after heat shock. The yeast hsp70 genes contained on YG100 and YG101 were truncated and fused to the Escherichia coli lacZ gene contained on pMC1587 (Casadaban et al., Methods Enzymol. 100:283-308, 1983). The resulting plasmids directed synthesis of the beta-galactosidase gene as measured by in vitro enzyme assays and by colorimetric assays on plates. The expression level from the YG101 gene was constant under all the conditions tested, whereas expression driven by the YG100 gene could be induced over 50-fold. Other stimuli besides heat, including recovery from anoxia and high cell density, were found to strongly induce YG100 gene expression. Most physical and chemical stimuli tested, including UV irradiation, zymolyase treatment, and ethanol, did not stimulate expression of this heat shock gene.

The green fluorescent protein (GFP) gene offers many advantages as a viability reporter for high-throughput antimicrobial drug screening. However, screening for antituberculosis compounds by using GFP driven by the heat shock promoter, hsp60, has been of limited utility due to the low signal-to-noise ratio. Therefore, an alternative promoter was evaluated for its enhanced fluorescence during microplate-based culture and its response to 18 established antimicrobial agents by using a green fluorescent protein microplate assay (GFPMA). Mycobacterium tuberculosis strains H37Rv, H37Ra, and Erdman were transformed with pFPCA1, which contains a red-shifted gfp gene driven by the acetamidase promoter of M. smegmatis mc2155. The pFPCA1 transformants achieved higher levels of GFP-mediated fluorescence than those carrying the hsp60 construct, with signal-to-noise ratios of 20.6 to 27.8 and 3.8 to 4.5, respectively. The MICs of 18 established antimicrobial agents for all strains carrying pFPCA1 in the GFPMA were within 1 to 2 twofold dilutions of those determined by either the fluorometric or the visual microplate Alamar Blue assay (MABA). No significant differences in MICs were observed between wild-type and pFPCA1 transformants by MABA. The optimized GFPMA is sufficiently simple, robust, and inexpensive (no reagent costs) to be used for routine high-throughput screening for antituberculosis compounds.

The dnaK operon of Streptomyces coelicolor contains four genes (5'-dnaK-grpE-dnaJ-hspR). The fourth gene encodes a novel heat shock protein, HspR, which appears so far to be unique to the high-G+C actinomycete group of bacteria. HspR binds with high specificity to three inverted repeat sequences in the promoter region of the S. coelicolor dnaK operon, strongly suggesting a direct role for HspR in heat shock gene regulation. Here we present genetic and biochemical evidence that HspR is the repressor of the dnaK operon. Disruption of hspR leads to high-level constitutive transcription of the dnaK operon. Parallel transcriptional analyses of groESL1 and groEL2 expression demonstrated that heat shock regulation of the groE genes was essentially unaffected in an hspR null mutant, although the basal (uninduced) level of groEL2 transcription was slightly elevated compared with the wild type. The results of HspR titration experiments, where the dnaK operon promoter region was cloned at ca. 50 copies per chromosome, were consistent with the prediction that HspR functions as a negative autoregulator. His-tagged HspR, overproduced and purified from Escherichia coli, was shown to repress transcription from the dnaK operon promoter in vitro, providing additional evidence for the proposal that HspR directly regulates transcription of the dnaK operon. These studies indicate that there are at least two transcriptional mechanisms for controlling heat shock genes in S. coelicolor--one controlling the dnaK operon and another controlling the groE genes.

Malaria infects 500 million people annually, a number that is likely to rise as drug resistance to currently used antimalarials increases. During its intraerythrocytic stage, the causative parasite, Plasmodium falciparum, metabolizes hemoglobin and releases toxic heme, which is neutralized by a parasite-specific crystallization mechanism to form hemozoin. Evidence suggests that chloroquine, the most successful antimalarial agent in history, acts by disrupting the formation of hemozoin. Here we describe the development of a 384-well microtiter plate screen to detect small molecules that can also disrupt heme crystallization. This assay, which is based on a colorimetric assay developed by Ncokazi and Egan (K. K. Ncokazi and T. J. Egan, Anal. Biochem. 338:306-319, 2005), requires no parasites or parasite-derived reagents and no radioactive materials and is suitable for a high-throughput screening platform. The assay's reproducibility and large dynamic range are reflected by a Z factor of 0.74. A pilot screen of 16,000 small molecules belonging to diverse structural classes was conducted. The results of the target-based assay were compared with a whole-parasite viability assay of the same small molecules to identify small molecules active in both assays.

The induction of heat shock genes (HSPs) is thought to be primarily regulated by heat shock transcription factors (HSFs), which bind target sequences on HSP promoters, called heat shock elements (HSEs). In this study, we investigated the 5′ untranslated regions of the Tetrahymena thermophila HSP70-1 gene, and we found, in addition to the canonical and divergent HSEs, multiple sets of GATA elements that have not been reported previously in protozoa. By means of in vivo analysis of a green fluorescent protein reporter transgene driven by the HSP70-1 promoter, we demonstrate that HSEs do not represent the minimal regulatory elements for heat shock induction, since the HSP70-1 is tightly regulated by both HSE and GATA elements. Electrophoretic mobility shift assay also showed that HSFs are constitutively bound to the HSEs, whereas GATA elements are engaged only after heat shock. This is the first demonstration by in vivo analysis of functional HSE and GATA elements in protozoa. Furthermore, we provide evidence of a functional link between HSE and GATA elements in the activation of the heat shock response.

Background & Aims

Heat shock proteins (HSPs) are highly conserved and serve a multitude of functions that mediate cell survival. HSP70, the only inducible form of the 70-kilodalton subfamily of HSPs, is overexpressed in pancreatic cancer cells and has been shown to inhibit caspase-dependent apoptosis. We aimed to elucidate the mechanism by which HSP70 inhibits apoptosis in cancer cells.

Methods

HSP70 expression was down-regulated in cultured pancreatic cancer cells by exposure to quercetin, triptolide, or short interfering RNAs. Intracellular Ca2+, cytosolic cathepsin B activity, caspase-3 activity, cell viability, and lysosome integrity were measured using colorimetric assays. Immunofluorescence assays were used to localize cathepsin B and Lamp2. BAPTA-AM was used to chelate intracellular Ca2+.

Results

Inhibition of HSP70 increased intracellular Ca2+ levels in pancreatic and colon cancer cell lines and led to loss of lysosome integrity in pancreatic cancer cells. The release of intracellular Ca2+ and lysosomal enzymes activated caspase-dependent apoptosis independently and simultaneously.

Conclusions

HSP70 inhibits apoptosis in cancer cells by 2 mechanisms: attenuation of cytosolic calcium and stabilization of lysosomes. HSP70-mediated cell survival might occur in other types of cancer cells.

Caulobacter crescentus cells respond to a sudden increase in temperature by transiently inducing the synthesis of several polypeptides. Two of the proteins induced, Hsp62 and Hsp70, were shown to be analogous to the heat shock proteins of Escherichia coli, GroEL and DnaK, respectively, by immunological cross-reactivity with antibodies raised against the E. coli proteins. Two-dimensional gel electrophoretic resolution of extracts of cells labeled with [35S]methionine during heat shock led to the identification of 20 distinct Hsps in C. crescentus which are coordinately expressed, in response to heat, at the various stages of the cell division cycle. Thus, a developmental control does not seem to be superimposed on the transient activation of the heat shock genes. Nonetheless, under normal temperature conditions, four Hsps (Hsp70, Hsp62, Hsp24b, and Hsp23a) were shown to be synthesized, and their synthesis was cell cycle regulated.

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Mitochondria contain a protein, hsp60, that is induced by heat shock and has been shown to function as a chaperonin in the assembly of mitochondrial enzyme complexes composed of proteins encoded by nuclear genes and imported from the cytosol. To determine whether products of mitochondrial genes are also assembled through an interaction with hsp60, we looked for association between hsp60 and proteins synthesized by isolated mitochondria. We have determined by electrophoretic, centrifugal, and immunological assays that at least two of those proteins become physically associated with hsp60. In mitochondrial matrix extracts, this association could be disrupted by the addition of Mg-ATP. One of the proteins that formed a stable association with hsp60 was the alpha subunit of the multicomponent complex F1-ATPase. We have not identified the other protein. These results indicate that hsp60 can function in the folding and assembly of mitochondrial proteins encoded by both mitochondrial and nuclear genes.

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The effects of both H-2 and non-H-2 genes on antibody responses to two Chlamydia trachomatis heat shock proteins (hsp60 and hsp70) were investigated. These chlamydial proteins are homologs of Escherichia coli GroEL (hsp60) and DnaK (hsp70) and are highly sequence conserved between bacterial and mammalian sources. Antibody responses among 17 different strains of mice immunized with C. trachomatis serovar B and serovar C elementary bodies were evaluated by immunoblot, radioimmunoprecipitation and enzyme-linked immunosorbent assay. Antibody responses to the two proteins displayed host genetic restriction. Of six distinctive H-2 haplotypes, only H-2d generated high antibody responses to hsp70. Five of the six H-2 haplotypes, i.e., H-2a, H-2d, H-2k, H-2q, and H-2s, produced high antibody responses to hsp60. Only the H-2b-bearing strain had low antibody responses to hsp60. By using congenic and H-2 recombinant strains, the genes responsible for regulating antibody responses to hsp70 and hsp60 were mapped to the K-IA region of the H-2 locus. In F1 hybrid crosses between high and low responders, high responses to hsp60 and hsp70 were dominant traits. Other genes outside the H-2 locus also influenced antibody responses to hsp60 and hsp70, since inbred strains of identical H-2 but different background genes displayed variable antibody responses to the proteins. The genetic control of murine immune responses to C. trachomatis hsp60, a putative chlamydial immunopathologic antigen, suggests that a similar genetic mechanism may also exist in humans, and this observation may help to explain the observed variability in the spectrum of chlamydial diseases seen in humans.

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Unregulated cellular proliferation, caused by mutation or dysregulation of growth-promoting proteins, is an underlying cause of cancer. Many such growth-promoting proteins exhibit an increased dependence on the activity of the chaperone heat-shock protein 90 (Hsp90) for correct folding and maturation in the cell. One can therefore envision that inhibition of Hsp90 would be an effective and broadly applicable strategy for the development of anticancer agents. Hsp90 functions in multichaperone complexes driven by the binding and hydrolysis of ATP. Encouraging results have been obtained by inhibiting Hsp90 with 17-AAG, an active-site binding ATP analog. Here we present the results of a different approach to inhibiting Hsp90 by disrupting its interaction with a cochaperone named Hsp organizing protein (HOP). We have used an AlphaScreen technology based high-throughput in vitro screen to identify compounds that inhibit this interaction. In addition, we demonstrate that these compounds are active in vivo. Treatment of human breast cancer cell lines BT474 and SKBR3 with these compounds decreases the levels of the Hsp90-dependent client protein HER2, with associated cell death.

HspR is a repressor known to control expression of heat shock operons in a number of Eubacteria. In mycobacteria and in several other actinobacteria, this protein is synthesized from the dnaKJE-hspR operon. Previous investigations revealed that HspR binds to the operon promoter, thereby controlling its expression in an autoregulatory manner. DnaK, which is a product of the same operon, further aids this autoregulatory process by stimulating the operator binding activity of HspR. The molecular mechanism by which DnaK assists HspR in executing its function is not clearly understood. In this study, it has been shown that DnaK can augment DNA binding activity of HspR by two mechanisms: (i) DnaK can restore the activity of completely denatured HspR by forming a complex with it, and (ii) DnaK can prevent thermal instability of HspR renatured by other means. Unlike the first mechanism, the latter function does not involve complex formation. The C-terminal hydrophobic tail of HspR was found to play a significant role in determining its thermal stability and DnaK dependence properties. A deletion mutant in which this region is removed does not respond to thermal stress and functions independent of DnaK. The hydrophobic C-terminal tails of HspRs of Mycobacterium tuberculosis and related Actinomycetales therefore may have evolved to make these HspRs more sensitive to thermal stress and, at the same time, subject to regulation by DnaK.

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.

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OBJECTIVE: The relationship between pregnancy outcome and expression of the heat shock proteins (hsps) or hsp-antibody complexes of 60kD (hsp60), 70kD (hsp70), and 90kD (hsp90) in placental tissue and circulating antibodies to hsps was evaluated. METHOD: Expression of hsp60, hsp70, and hsp90 in placentae from 12 women with preterm birth, eight with intrauterine growth restriction (IUGR), and 10 with term birth, as well as the presence of the corresponding antibodies, was investigated by a new carbocyanine double fluorescence technique. Results were compared with microbiological findings and circulating antibodies to hsps in sera. RESULTS: In each placental specimen examined, hsp60, hsp70, and hsp90 were identified. However, hsp70-antibody complexes were detected in only four of the preterm labor cases. Similarly, hsp60-antibody complexes were detected in only five preterm labor patients and in one patient with IUGR. None of the placentae contained hsp90-antibody complexes. In the preterm birth group, all patients with hsp60-antibody complexes were also positive for circulating antibodies to hsp60. The presence of hsp70-antibody complexes also correlated with hsp70 antibody in sera. CONCLUSIONS: Formation of hsp60- and hsp70-antibody complexes in the placenta may contribute to the induction of preterm birth. Women sensitized to these antibodies may be at increased risk for adverse pregnancy outcome.