Functional Analysis of Hsp70 Inhibitors

The molecular chaperones of the Hsp70 family have been recognized as targets for anti-cancer therapy. Since several paralogs of Hsp70 proteins exist in cytosol, endoplasmic reticulum and mitochondria, we investigated which isoform needs to be down-regulated for reducing viability of cancer cells. For two recently identified small molecule inhibitors, VER-155008 and 2-phenylethynesulfonamide (PES), which are proposed to target different sites in Hsp70s, we analyzed the molecular mode of action in vitro. We found that for significant reduction of viability of cancer cells simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8) is necessary. The compound VER-155008, which binds to the nucleotide binding site of Hsp70, arrests the nucleotide binding domain (NBD) in a half-open conformation and thereby acts as ATP-competitive inhibitor that prevents allosteric control between NBD and substrate binding domain (SBD). Compound PES interacts with the SBD of Hsp70 in an unspecific, detergent-like fashion, under the conditions tested. None of the two inhibitors investigated was isoform-specific.     

A Mutant of Arabidopsis thaliana Which Lacks Activation of RuBP Carboxylase In Vivo

A mutant of Arabidopsis thaliana has been isolated in which ribulose-1,5-bisphosphate carboxylase is present in a nonactivatable form in vivo. The mutation appears to affect carboxylase activation specifically, and not any other enzyme of the photosynthesis or photorespiratory cycles. The effect of the mutation on carboxylase activation is indirect, inasmuch as the properties of ribulose-1,5-bisphosphate carboxylase purified from the mutant are not distinguishable from those of the wild type enzyme. The mutant requires high levels of atmospheric CO₂ for growth because photosynthesis is severely impaired in atmospheres containing normal levels of CO₂, irrespective of the atmospheric O₂ concentration. In this respect, the mutant is distinguished from previously described high-CO₂ requiring mutants of Arabidopsis which have defects in photorespiratory carbon or nitrogen metabolism.     

Activation of phospholipase A2 by Hsp70 in vitro

We recently suggested a novel mechanism for the activation of phospholipase A2 (PLA2), with a (catalytically) highly active oligomeric state, which subsequently becomes inactivated by conversion into amyloid. This process can be activated by lysophosphatidylcholine which promotes both oligomerization and amyloid activation/inactivation. The heat shock protein 70 (Hsp70), has been demonstrated to be able to revert the conversion of α-synuclein and Alzheimer β-peptide to amyloid fibrils in vitro. Accordingly, we would expect Hsp70 to sustain the lifetime of the active state of the enzyme oligomer by attenuating the conversion of the enzyme oligomers into inactive amyloid. Here we show that Hsp70 activates PLA2 in vitro, in a manner requiring ATP and Mg(2+).     

In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH₂-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH₂-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis–dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.     

Blue Light-Induced In Vivo Absorbance Changes and In Vitro Activation of Nitrate Reductase in a Nitrate-Starved Chlorella Mutant

Illuminating a colorless mutant of Chlorella vulgaris 11h (M125)with blue light caused a reversible photoreduction of b-typecytochrome, i.e., absorbance increases at 423, 525 and 557 nm.This light-induced reduction of cytochrome b was most pronouncedin nitrate-starved cells, which showed some blue light responsesin carbon metabolism, including enhancement of respiration byblue light as reported previously. Prolonged illumination withblue light caused a decrease in the rate of the reduction. The photoactivation of nitrate reductase in the mutant cellswas studied in both cell-free crude extract and purified enzyme.The absorption spectrum of purified enzyme showed three peaksat 423, 525 and 557 nm after the addition of a reductant, indicatingthat the spectrum is that of cytochrome b associated with nitratereductase. Nitrate reductase activity was easily enhanced byblue light illumination after 1 min; red light had no effecton it. The blue light activation of nitrate reductase was notsignificant in growing cells, which showed its high activity. The relationship between the blue light-induced reduction ofcytochrome b and carbon metabolism is discussed. (Received September 30, 1987; Accepted February 9, 1988)     

An Intramolecular Signaling Element that Modulates Dynamin Function In Vitro and In Vivo

Dynamin exhibits a high basal rate of GTP hydrolysis that is enhanced by self-assembly on a lipid template. Dynamin''s GTPase effector domain (GED) is required for this stimulation, though its mechanism of action is poorly understood. Recent structural work has suggested that GED may physically dock with the GTPase domain to exert its stimulatory effects. To examine how these interactions activate dynamin, we engineered a minimal GTPase-GED fusion protein (GG) that reconstitutes dynamin''s basal GTPase activity and utilized it to define the structural framework that mediates GED''s association with the GTPase domain. Chemical cross-linking of GG and mutagenesis of full-length dynamin establishes that the GTPase-GED interface is comprised of the N- and C-terminal helices of the GTPase domain and the C-terminus of GED. We further show that this interface is essential for structural stability in full-length dynamin. Finally, we identify mutations in this interface that disrupt assembly-stimulated GTP hydrolysis and dynamin-catalyzed membrane fission in vitro and impair the late stages of clathrin-mediated endocytosis in vivo. These data suggest that the components of the GTPase-GED interface act as an intramolecular signaling module, which we term the bundle signaling element, that can modulate dynamin function in vitro and in vivo.     

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP^C) converts into a misfolded isoform (PrP^Sc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP^C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP^Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP^Sc-specific conformational epitopes. In contrast to PrP^Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP^Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP^Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP^Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.     

Endopeptidase-24.15 Is the Primary Enzyme that Degrades Luteinizing Hormone Releasing Hormone Both In Vitro and In Vivo

The concentration of luteinizing hormone releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), which reaches the anterior pituitary via the hypothalamo-hypophyseal portal system, appears to be controlled in part by the rate of LHRH degradation within the hypothalamus and/or pituitary. Specific, active site-directed endopeptidase inhibitors synthesized in our laboratory were used to identify the enzyme(s) involved in LHRH degradation by hypothalamic and pituitary membrane preparations, and by an intact anterior pituitary tumor cell line (AtT20). Incubation of LHRH with pituitary and hypothalamic membrane preparations led to the formation of pGlu-His-Trp (LHRH1-3) as the main reaction product. Under the same conditions, addition to the incubation mixtures of captopril, an inhibitor of the angiotensin converting enzyme, led to accumulation of pGlu-His-Trp-Ser-Tyr (LHRH1-5) and, to a lesser extent, pGlu-His-Trp-Ser-Tyr (LHRH1-6). The degradation of LHRH and the formation of the N-terminal tri- and pentapeptides was blocked by N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific, active site directed inhibitor of endopeptidase-24.15. Some inhibition of LHRH degradation and formation of the N-terminal hexapeptide was also obtained in the presence of N-[1-carboxy-2-phenylethyl]-Phe-p-aminobenzoate (cFE-F-pAB), an inhibitor of endopeptidase-24.11. Similar results were obtained with AtT20 cell membranes and with intact AtT20 cells in monolayer culture. Following cleavage by endopeptidases the C-terminal part of LHRH was rapidly degraded by aminopeptidases. Superactive analogs of LHRH in which Gly6 was replaced by a D-amino acid are resistant to degradation by both endopeptidase-24.11 and -24.15. In vivo, when LHRH was injected directly into the third ventricle of rats, the presence of cFP-AAF-pAB inhibited LHRH degradation. It is concluded that LHRH degradation is primarily initiated by the membrane-bound form of endopeptidase-24.15 to yield pGlu-His-Trp-Ser-Tyr and to a lesser extent by endopeptidase-24.11 to yield pGlu-His-Trp-Ser-Tyr-Gly.     

Mechanisms for regulation of Hsp70 function by Hsp40

The Hsp70 family members play an essential role in cellular protein metabolism by acting as polypeptide-binding and release factors that interact with nonnative regions of proteins at different stages of their life cycles. Hsp40 cochaperone proteins regulate complex formation between Hsp70 and client proteins. Herein, literature is reviewed that describes the mechanisms by which Hsp40 proteins interact with Hsp70 to specify its cellular functions.     

Independent regulation of Hsp70 and Hsp90 chaperones by Hsp70/Hsp90-organizing protein Sti1 (Hop1)

Hsp70 and Hsp90 protein chaperones cooperate in a protein-folding pathway required by many "client" proteins. The co-chaperone Sti1p coordinates functions of Hsp70 and Hsp90 in this pathway. Sti1p has three tetratricopeptide repeat (TPR) domains. TPR1 binds Hsp70, TPR2a binds Hsp90, and the ligand for TPR2b is unknown. Although Sti1p is thought to be dedicated to the client folding pathway, we earlier showed that Sti1p regulated Hsp70, independently of Hsp90, in a way that impairs yeast [PSI+] prion propagation. Using this prion system to monitor Sti1p regulation of Hsp70 and an Hsp90-inhibiting compound to monitor Hsp90 regulation, we identified Sti1p mutations that separately affect Hsp70 and Hsp90. TPR1 mutations impaired Sti1p regulation of Hsp70, but deletion of TPR2a and TPR2b did not. Conversely, TPR2a and TPR2b mutations impaired Sti1p regulation of Hsp90, but deletion of TPR1 did not. All Sti1p mutations variously impaired the client folding pathway, which requires both Hsp70 and Hsp90. Thus, Sti1p regulated Hsp70 and Hsp90 separately, Hsp90 is implicated as a TPR2b ligand, and mutations separately affecting regulation of either chaperone impair a pathway that is dependent upon both. We further demonstrate that client folding depended upon bridging of Hsp70 and Hsp90 by Sti1p and find conservation of the independent regulation of Hsp70 and Hsp90 by human Hop1.     

Proteasome Inhibitors Which Induce Neurite Outgrowth From PC12h Cells Cause Different Subcellular Accumulations of Multi-Ubiquitin Chains

The effects of two proteasome inhibitors on neurite outgrowth from PC12h cells were investigated in terms of the mean length of the neurites and the frequency of occurrence of cells with long neurites. Benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) and benzyloxycarbonyl-isoleucyl-t-butyl-glutamyl-leucinal (PSI) caused a significant elongation of PC12h cell neurites. Since ZLLLal is known to inhibit both calpain and proteasome activity, we examined the effects of benzyloxycarbonyl-leucyl-leucinal (ZLLal) which inhibits calpain activity to the same degree as ZLLLal, but which inhibits proteasome activity only weakly. ZLLal did not induce the significant elongation of neurites at any of the concentrations we studied. These results show that the inhibition of proteasome activity causes neurite elongation. We also quantified subcellular levels of multi-ubiquitin chains and free ubiquitin after treatments with PSI, ZLLLal and ZLLal. Treatment with ZLLal had no effects on levels of water- and urea-soluble multi-ubiquitin chains or of free ubiquitin either in the nucleus or in the cytoplasm. PSI and ZLLLal induced a large accumulation of water- and urea-soluble multi-ubiquitin chains and free ubiquitin in the nucleus. Similarly, PSI and ZLLLal increased cytoplasmic levels of urea-soluble multi-ubiquitin chains. On the contrary, PSI and ZLLLal had no effect on levels of water-soluble multi-ubiquitin chains or free ubiquitin in the cytoplasm. This is the first study to demonstrate subcellular differences in the accumulation of multi-ubiquitin chains and free ubiquitin during the neurite elongation induced by proteasome inhibitors.     

Novel compounds that increase expression of Hsp70 and its biological activity

The Hsp70 protein has chaperone activity, which is associated with the protective function that demonstrated in experiments using several cell lines and animals. Therefore, the search for the substances able to harmlessly elevate the chaperone concentration in the organism cells and tissues is particularly important. In our work, we screened more than 60 compounds and revealed two chemicals, i.e., derivatives of shikonin and echinochrome, that, at micromolar concentrations, were able to increase the chaperone level in various human cells. Under the effects of both of these substances, in human erythroleukemia K-562 cells, a significant increase in the absolute Hsp70 chaperone activity was also observed, which can indicate the mobilization of the entire cellular chaperone mechanism. The estimation of the biological activity of these two substances has shown that their action upon the treatment of cells with severe heat stress, hydrogen peroxide, and staurosporine leads to a decrease in cell mortality by 20–50% depending on the cytotoxic factor. These results show that, by effecting noncomplicated chemical modifications of the chosen substances, it is possible to obtain pharmacogenic agents of the wide profile of action.     

The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis

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Highlights? Yeast Hsp70 SSB binds cotranslationally to a defined set of nascent polypeptides ? SSB shows specificity for substrates highly challenged in cotranslational folding ? The heterodimeric cochaperone RAC modulates cotranslational SSB specificity ? Loss of SSB function leads to widespread misfolding and aggregation