The inducible Hsp70 as a marker of tumor immunogenicity

Growing evidence indicates that the stress response in general and heat shock proteins (Hsps) in particular have a profound impact on tumor immunogenicity. In this study, we show that tumor cells subjected to a nonlethal heat shock stress are unable to form tumors in syngenic mice, whereas they do so in athymic nude mice. Moreover, heat-shocked MethA immunity is tumor specific. Enhancement of T-cell-mediated immunogenicity correlates with the expression of the inducible Hsp70 but not the constitutive Hsc70. These observations have a bearing on the proposed functional role of Hsp-peptide association in antigen processing and presentation by major histocompatibility complex I molecules under normal and stressful conditions.     

Alterations in the mitochondrial proteome of neuroblastoma cells in response to complex 1 inhibition

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing 1D blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDAC1). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDAC1. Subcytotoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDAC1). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDAC1, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD.     

Quantitative mRNA expression profiling of heat-shock protein families in rainbow trout cells

We isolated multiple HSPs from rainbow trout Oncorhynchus mykiss RTG-2 cells and quantitatively compared their mRNA levels between unstressed and heat-shocked cells using real-time RT-PCR analysis. Consequently, we isolated nine cDNAs encoding HSPs from heat-shocked RTG-2 cells, namely, Hsp90betaa, Hsp90betab, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. Quantitative RT-PCR analyses, in which Hsp70b isolated previously was included, showed that the mRNA accumulation levels of Hsp70a, Hsp70b, Hsc70a, Hsc70b, and Hsp47 were significantly increased after heat shock, and the increased levels of two Hsp70s, Hsp70a, and Hsp70b, were most conspicuous. In the case of Hsc70s, the increased level of Hsc70b was more remarkable than that of Hsc70a. These results demonstrate the importance of a comprehensive expression analysis of HSPs for better understanding of the cellular stress response in fish, especially in tetraploid species such as rainbow trout.     

Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences

Hsp70s function as molecular chaperones. The protective chaperone activities of hsp70 help to confer tolerance to heat, glucose deprivation, and drought. Overexpression of hsp70s in many organisms correlates with enhanced thermotolerance, altered growth, and development. To better understand the roles of hsp70 proteins in Arabidopsis, the molecular and physiological consequences of altered expression of the major heat shock cognate, Hsc70-1, were analyzed. Extensive efforts to achieve underexpression of Hsc70-1 mRNA using a full-length antisense cDNA resulted in no viable transgenic plants, suggesting that reduced expression is lethal. Constitutive overexpression of Hsc70-1 also appeared to be deleterious to viability, growth, and development because fewer transformants were recovered, and most were dwarfed with altered root systems. Despite being dwarfed, the overexpression plants progressed normally through four selected developmental stages. Heat treatment revealed that Hsc70-1 overexpression plants were more tolerant to heat shock (44 degrees C for 10 min). The elevated basal levels of HSC70-1 in transgenic plants led to delayed heat shock response of several heat shock genes. The data in this study suggest that tight regulation of Hsc70-1 expression is critical for the viability of Arabidopsis and that the functions of HSC70-1 contribute to optimum growth, development, thermotolerance, and regulation of the heat shock response.     

BAG-1 modulates the chaperone activity of Hsp70/Hsc70.

The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG-1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG-1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy-terminal peptide-binding domain, and can be co-immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG-1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG-1 inhibits Hsp/Hsc70-mediated in vitro refolding of an unfolded protein substrate, whereas BAG-1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG-1 to one of its known cellular targets, Bcl-2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG-1 also protected certain cell lines from heat shock-induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG-1 may explain the diverse interactions observed between BAG-1 and several other proteins, including Raf-1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG-1 on Hsp/Hsc70 chaperone activity suggest that BAG-1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.     

Roles of ADAM13-regulated Wnt activity in early Xenopus eye development

Pericellular proteolysis by ADAM family metalloproteinases has been widely implicated in cell signaling and development. We recently found that Xenopus ADAM13, an ADAM metalloproteinase, is required for activation of canonical Wnt signaling during cranial neural crest (CNC) induction by regulating a novel crosstalk between Wnt and ephrin B (EfnB) signaling pathways (Wei et al., 2010b). In the present study we show that the metalloproteinase activity of ADAM13 also plays important roles in eye development in Xenopus tropicalis. Knockdown of ADAM13 results in reduced expression of eye field markers pax6 and rx1, as well as that of the pan-neural marker sox2. Activation of canonical Wnt signaling or inhibition of forward EfnB signaling rescues the eye defects caused by loss of ADAM13, suggesting that ADAM13 functions through regulation of the EfnB-Wnt pathway interaction. Downstream of Wnt, the head inducer Cerberus was identified as an effector that mediates ADAM13 function in early eye field formation. Furthermore, ectopic expression of the Wnt target gene snail2 restores cerberus expression and rescues the eye defects caused by ADAM13 knockdown. Together these data suggest an important role of ADAM13-regulated Wnt activity in eye development in Xenopus.     

Immunoelectron microscope localization of Mr 90000 heat shock protein and Mr 70000 heat shock cognate protein in the salivary glands of Chironomus thummi

The Mr 90000 heat shock protein (hsp 90) and one of the Mr 70000 heat shock cognate proteins (hsc 70) were localized by immunoelectron microscopy in salavary gland cells of normal and heat-shocked larvae of Chironomus thummi using polyclonal antibodies raised against Drosophila proteins. Immunoblotting after separation of proteins by gel electrophoresis shows that these antibodies cross-react with the corresponding proteins of Chironomus. Hsp 90 was localized both in the cytoplasm and in the nucleus, where it is associated with intrachromosomal and extrachromosomal ribonucleoprotein (RNP) fibrils, as well as with the peripheral region of compact chromatin. After heat shock the concentration of hsp 90 increases in the nucleus. This increase is prevented by actinomycin D administration during the heat shock. Hsp 90 is associated with the chromatin of puffs repressed by heat shock and with the RNP fibrils of actively transcribing heat shock puffs. Hsc 70 is mainly found in RNP fibrils and in the periphery of compact chromatin. During heat shock the concentration of hsc 70 decreases in the cytoplasm while it becomes more abundant in association with chromatin and intrachromosomal and extrachromosomal RNP fibrils. These results suggest a translocation of the existing protein from the cytoplasm toward the nucleus. They are supported by observations of the effect of heat shock carried out in the presence of actinomycin D.by D.P. Bazett-Jones     

Impact of aging on heat shock protein expression in the substantia nigra and striatum of the female rat

Many heat shock proteins are chaperones that help refold or degrade misfolded proteins and battle apoptosis. Because of their capacity to protect against protein misfolding, they may help keep diseases of aging at bay. A few reports have examined heat shock proteins (eg. Hsp25, Hsp60, Hsp70, and heat shock cognate 70 or Hsc70) as a function of age in the striatum and nigra. In the present study, we examined the impact of aging on Hsp25, heme oxygenase 1 (HO1 or Hsp32), Hsp40, Hsp60, Hsc70, Hsc/Hsp70 interacting protein (Hip), 78 kDa glucose-regulated protein (GRP78), Hsp90, and ubiquitinated proteins in the nigra and striatum of the female rat by infrared immunoblotting. Female animals are not typically examined in aging studies, adding further to the novelty of our study. Striatal HO1 and Hsp40 were both higher in middle-aged females than in the oldest group. Hsp60 levels were also highest in middle age in the nigra, but were highest in the oldest animals in the striatum. Striatal levels of Hsc70 and the co-chaperone Hip were lower in the oldest group relative to the youngest animals. In contrast, Hsp25 rose with advancing age in both regions. Hsp25 was also colocalized with tyrosine hydroxylase in nigral neurons. Ubiquitinated proteins exhibited a trend to rise in the oldest animals in both regions, and K48 linkage-specific ubiquitin rose significantly from 4–6 to 16–19 months in the striatum. Our study reveals a complex array of age-related changes in heat shock proteins. Furthermore, the age-related rises in some proteins, such as Hsp25, may reflect endogenous adaptations to cellular stress.     

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.     

Interaction of heat and salt shock in cultured tobacco cells

Cultured tobacco cells (Nicotiana tabacum L. var Wisconsin-38) developed tolerance to otherwise nonpermissive 54°C treatment when heat-shocked at 38°C (2 h) but not at 42°C. Heat-shocked cells (38°C) exhibited little normal growth when the 54°C stress came immediately after heat shock and normal growth when 54°C stress was administered 8 hours after heat shock. Heat shock extended the length of time that the cells tolerated 54°C. Tobacco cells developed tolerance to otherwise lethal 2% NaCl treatment when salt-shocked (1.2% NaCl for 3 hours). The time course for salt tolerance development was similar to that of thermotolerance. Heat-shocked cells (38°C) developed tolerance of nonpermissive salt stress 8 hours after heat shock. Alternatively, cells heat-shocked at 42°C exhibited immediate tolerance to lethal salt stress followed by a decline over 8 hours. Radioactive methionine incorporation studies demonstrated synthesis of heat shock proteins at 38°C. The apparent molecular weights range from 15 to 115 kilodaltons with a protein complex in the 15 to 20 kilodalton range. Synthesis of heat shock proteins appeared to persist at 42°C but with large decreases in incorporation into selected heat shock protein. During salt shock, the synthesis of normal control proteins was reduced and a group of salt shock proteins appeared 3 to 6 h after shock. Similarities between the physiology and salt shock proteins/heat shock proteins suggest that both forms of stress may share common elements.     

Cytoskeletal stability and heat shock-mediated thermoprotection of central pattern generation in Locusta migratoria

Prior exposure to extreme temperatures can induce thermoprotection in migratory locusts, which is important for survival in their natural environment. An important motor activity that needs to be protected is ventilation. The mechanism underlying heat shock is not fully understood, and our goal was to test the idea that cytoskeletal stability is critical for such thermoprotection. Cytoskeletal stabilizers (concanavalin A) and destabilizers (colchicine) were bath-applied in semi-intact locust preparations in both control (C) and pre-treated heat-shocked (3 h, 45 degrees C) animals. We measured parameters of the ventilatory motor pattern during maintained high temperature (43 degrees C) and recorded the times taken for motor pattern generation to fail and then recover on returning to room temperature. We found that concanavalin A mimicked the effects of a prior heat stress in control animals by increasing time to failure and decreasing time to recovery of motor pattern generation. However, colchicine destroyed protection in heat-shocked animals by decreasing time to failure and increasing time to recovery. Our findings confirm that the cytoskeleton has a mechanistic role in preserving neural function at high temperatures, possibly through stabilizing ion channels and other integral membrane proteins (e.g. Na(+)/K(+) ATPase) and their interactions with heat shock proteins.     

Tissue-specific Expression of Dominant Negative Mutant Drosophila HSC70 Causes Developmental Defects and Lethality

The Drosophila melanogaster HSC3 and HSC4 genes encode Hsc70 proteins homologous to the mammalian endoplasmic reticulum (ER) protein BiP and the cytoplasmic clathrin uncoating ATPase, respectively. These proteins possess ATP binding/hydrolysis activities that mediate their ability to aid in protein folding by coordinating the sequential binding and release of misfolded proteins. To investigate the roles of HSC3 (Hsc3p) and HSC4 (Hsc4p) proteins during development, GAL4-targeted gene expression was used to analyze the effects of producing dominant negatively acting Hsc3p (D231S, K97S) and Hsc4p (D206S, K71S) proteins, containing single amino acid substitutions in their ATP-binding domains, in specific tissues of Drosophila throughout development. We show that the production of each mutant protein results in lethality over a range of developmental stages, depending on the levels of protein produced and which tissues are targeted. We demonstrate that the functions of both Hsc3p and Hsc4p are required for proper tissue establishment and maintenance. Production of mutant Hsc4p, but not Hsc3p, results in induction of the stress-inducible Hsp70 at normal temperatures. Evidence is presented that lethality is caused by tissue-specific defects that result from a global accumulation of misfolded protein caused by lack of functional Hsc70. We show that both mutant Hsc3ps are defective in ATP-induced substrate release, although Hsc3p(D231S) does undergo an ATP-induced conformational change. We believe that the amino acid substitutions in Hsc3p interfere with the structural coupling of ATP binding to substrate release, and this defect is the basis for the mutant proteins' dominant negative effects in vivo.     

Synthesis of heat shock proteins in Drosophila melanogaster embryos

The pattern of polypeptides synthesized in a cell-free protein synthesizing system containing polysomes isolated from heat-shocked (37 C) Drosophila embryos showed significant differences when compared with the pattern obtained when polysomes from normal embryos were used. The synthesis of normal embryonal proteins was reduced and the heat shock proteins were the major products of elongation. After short, 10 min, heat treatment mainly quantitative changes were observed suggesting that normal mRNAs were still present on polysomes, and their products could be completed in vitro in the heterologous cell-free system. The mRNAs coding for normal embryonal proteins were present in almost unchanged amounts in heat-shocked embryos as could be judged from the pattern of proteins synthesized in heterologous cell-free system supplemented with cytoplasmic RNA from normal and heat-shocked embryos. Thus the change in protein synthesis in heat-shocked embryos is not associated with degradation of normal embryonal mRNAs but with their inaccessibility for translation.     

Chronic morphine alters the presynaptic protein profile: identification of novel molecular targets using proteomics and network analysis

Opiates produce significant and persistent changes in synaptic transmission; knowledge of the proteins involved in these changes may help to understand the molecular mechanisms underlying opiate dependence. Using an integrated quantitative proteomics and systems biology approach, we explored changes in the presynaptic protein profile following a paradigm of chronic morphine administration that leads to the development of dependence. For this, we isolated presynaptic fractions from the striata of rats treated with saline or escalating doses of morphine, and analyzed the proteins in these fractions using differential isotopic labeling. We identified 30 proteins that were significantly altered by morphine and integrated them into a protein-protein interaction (PPI) network representing potential morphine-regulated protein complexes. Graph theory-based analysis of this network revealed clusters of densely connected and functionally related morphine-regulated clusters of proteins. One of the clusters contained molecular chaperones thought to be involved in regulation of neurotransmission. Within this cluster, cysteine-string protein (CSP) and the heat shock protein Hsc70 were downregulated by morphine. Interestingly, Hsp90, a heat shock protein that normally interacts with CSP and Hsc70, was upregulated by morphine. Moreover, treatment with the selective Hsp90 inhibitor, geldanamycin, decreased the somatic signs of naloxone-precipitated morphine withdrawal, suggesting that Hsp90 upregulation at the presynapse plays a role in the expression of morphine dependence. Thus, integration of proteomics, network analysis, and behavioral studies has provided a greater understanding of morphine-induced alterations in synaptic composition, and identified a potential novel therapeutic target for opiate dependence.     

Exposure to heat shock affects thermosensitivity of the locust flight system

The natural habitat of the migratory locust, Locusta migratoria, is likely to result in locusts being heat stressed during their normal adult life. It is known that locusts exhibit a heat-shock response: exposure to 45°C for 3 h induces thermotolerance and the expression of heat-shock proteins. We investigated the effects of exposure to heat-shock conditions on the thermosensitivity of flight rhythm generation in tethered, intact animals and in deafferented preparations. Heat shock had no effect on wingbeat frequency measured at the start of flight sequences, nor did it affect the postimaginal maturation of this parameter. During sustained flight, heat shock slowed the characteristic asymptotic reduction of wingbeat frequency. Wingbeat frequency of heat-shocked animals was less sensitive to temperature in the range 24° to 47°C than that of control animals, and the upper temperature limit, above which flight rhythms could not be produced, was 6° to 7°C higher in heat-shocked animals. These results were mirrored in the response of deafferented preparations, indicating that modifications in the properties of the flight neuromuscular system were involved in mediating the response of the intact animal. We propose that exposure to heat shock had the adaptive consequences of reducing thermosensitivity of the neural circuits in the flight system and allowing them to operate at higher temperatures. © 1996 John Wiley & Sons, Inc.