Salicylic acid-mediated potentiation of Hsp70 induction correlates with reduced apoptosis in tobacco protoplasts.

BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.     

Tissue-specific variations in the induction of Hsp70 and Hsp64 by heat shock in insects

The patterns of heat-induced synthesis (37 degrees C to 45 degrees C) of heat shock proteins (Hsps) in different tissues of grasshoppers and cockroaches from natural populations and in laboratory-reared gram-pest (Heliothis armigera) were examined by 35S-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis fluorography. Whereas 45 degrees C was lethal in most cases, optimal induction of Hsp synthesis was seen between 37 degrees C and 42 degrees C. The ongoing protein synthesis was not much affected at these temperatures, except in the tissues of adult H. armigera exposed to 42 degrees C. The profiles of the Hsps induced in the tissues of the insects, however, were different. From the relative abundance of the synthesis of 70-kDa (Hsp70) and 64-kDa (Hsp64) polypeptides, three categories of heat shock response were identified: (1) induction of abundant Hsp70 but little Hsp64 (malpighian tubules, male accessory glands, and ovaries of adult grasshoppers), (2) abundant Hsp64 but little Hsp70 (testes of adult grasshoppers, testes and malpighian tubules of adult cockroaches, and testes, malpighian tubules, and fat bodies of H. armigera larvae), and (3) induction of both Hsp70 and Hsp64 in more or less equal abundance (ovaries of adult cockroaches, salivary glands of H. armigera larvae, and malpighian tubules, male accessory glands, testes, and ovaries of adult H. armigera). Cockroaches collected from storerooms showed detectable synthesis of Hsp64 and/or Hsp70 only after heat shock, but those collected from drains showed detectable synthesis of both Hsp70 and Hsp64 in different tissues without heat stress. Western blotting showed that the 64-kDa polypeptide in these insects is a member of the Hsp60 family. Grasshopper testes, which synthesized negligible Hsp70 but abundant Hsp64 after heat shock, developed thermotolerance. Thus, heat shock response is modulated by developmental and environmental factors in different tissues of insects.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70-specific antibodies

Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade, it became evident that apart from their intracellular localization, members of the heat shock protein 90 (Hsp90; HSPC) and Hsp70 (HSPA) family are also found on the cell surface. In this review, we will focus on Hsp70 (HSPA1A), the major stress-inducible member of the human Hsp70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells, Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells, Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein, or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.     

Hsp70 accelerates the recovery of nucleolar morphology after heat shock.

The major heat-shock protein, hsp70, is synthesized by cells of many organisms in response to stress. In the present study, Drosophila hsp70 was expressed from cloned genes in mouse L cells and monkey COS cells and detected by immunofluorescence using monoclonal antibodies. Hsp70 is found mostly but not exclusively in the nucleus of unstressed cells. For several hours after a short heat shock, however, it is strongly concentrated in nucleoli. Nucleoli are transiently damaged by such a heat shock: their morphology changes and assembly and export of ribosomes is blocked for several hours. This block can be visualized by addition of actinomycin D: under normal conditions pre-ribosomes are chased out of nucleoli, and the latter shrink dramatically, but no such shrinking is seen in heat-shocked cells. High levels of hsp70 can be produced in unstressed COS cells by transfecting them with an appropriate expression plasmid. Such cells show a more rapid recovery of nucleolar morphology following a heat shock than do untransfected cells. Furthermore, heat shock does not prevent shrinkage of their nucleoli in the presence of actinomycin, which indicates that ribosome export also recovers rapidly when pre-synthesized hsp70 is present. I suggest that an important function of hsp70 is to catalyze reassembly of damaged pre-ribosomes and other RNPs after heat shock.     

Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70

Heat shock proteins play a major role in the process of protein folding, and they have been termed molecular chaperones. Two members of the Hsp70 family, Hsc70 and Hsp70, have a high degree of sequence homology. But they differ in their expression pattern. Hsc70 is constitutively expressed, whereas Hsp70 is stress inducible. These 2 proteins are localized in the cytosol and the nucleus. In addition, they have also been observed in close proximity to cellular membranes. We have recently reported that Hsc70 is capable of interacting with a lipid bilayer forming ion-conductance channels. In the present study, we found that both Hsc70 and Hsp70 interact with lipids and can be differentiated by their characteristic induction of liposome aggregation. These proteins promote the aggregation of phosphatidylserine liposomes in a time- and protein concentration-dependent manner. Although both proteins are active in this process, the level and kinetics of aggregation are different between them. Calcium ions enhance Hsc70 and Hsp70 liposome aggregation, but the effect is more dramatic for Hsc70 than for Hsp70. Addition of adenosine triphosphate blocks liposome aggregation induced by both proteins. Adenosine diphosphate (ADP) also blocks Hsp70-mediated liposome aggregation. Micromolar concentrations of ADP enhance Hsc70-induced liposome aggregation, whereas at millimolar concentrations the nucleotide has an inhibitory effect. These results confirm those of previous studies indicating that the Hsp70 family can interact with lipids directly. It is possible that the interaction of Hsp70s with lipids may play a role in the folding of membrane proteins and the translocation of polypeptides across membranes.     

Neutral analogs of the heat shock protein 70 (Hsp70) inhibitor,JG-98

The heat shock protein 70 (Hsp70) family of molecular chaperones are highly expressed in tumors. Inhibitors containing a pyridinium-modified benzothiazole, such as JG-98, bind to a conserved, allosteric site in Hsp70, showing promising anti-proliferative activity in cancer cells. When bound to Hsp70, the charged pyridinium makes favorable contacts; however, this moiety also increases the inhibitor’s fluorescence, giving rise to undesirable interference in biochemical and cell-based assays. Here, we explore whether the pyridinium can be replaced with a neutral pyridine. We report that pyridine-modified benzothiazoles, such as compound 17h (JG2-38), have reduced fluorescence, yet retain promising anti-proliferative activity (EC₅₀ values ~0.1 to 0.07 µM) in breast and prostate cancer cell lines. These chemical probes are expected to be useful in exploring the roles of Hsp70s in tumorigenesis and cell survival.     

Lamina-associated polypeptide 2alpha forms complexes with heat shock proteins Hsp70 and Hsc70 in vivo

Lamina-associated polypeptide 2α (LAP2α), one of the alternatively spliced isoforms of the LAP2 gene, is a nucleoplasmic protein which forms oligomers and presumably associates to chromosomes via the LEM- and LEM-like regions. To characterize components of the LAP2α-containing complexes, we have expressed the α-specific C-terminal domain of LAP2α in HeLa cells and, after immunopurification, found that the heat shock proteins Hsp70 and Hsc70 reproducibly co-purified with this domain. Association between endogenous LAP2α and Hsp70 in non-transfected cells was confirmed by co-immunoprecipitation. The association was not mediated by the retinoblastoma protein.     

Hsp70 displaces small heat shock proteins from aggregates to initiate protein refolding

Small heat shock proteins (sHsps) are an evolutionary conserved class of ATP-independent chaperones that protect cells against proteotoxic stress. sHsps form assemblies with aggregation-prone misfolded proteins, which facilitates subsequent substrate solubilization and refolding by ATP-dependent Hsp70 and Hsp100 chaperones. Substrate solubilization requires disruption of sHsp association with trapped misfolded proteins. Here, we unravel a specific interplay between Hsp70 and sHsps at the initial step of the solubilization process. We show that Hsp70 displaces surface-bound sHsps from sHsp–substrate assemblies. This Hsp70 activity is unique among chaperones and highly sensitive to alterations in Hsp70 concentrations. The Hsp70 activity is reflected in the organization of sHsp–substrate assemblies, including an outer dynamic sHsp shell that is removed by Hsp70 and a stable core comprised mainly of aggregated substrates. Binding of Hsp70 to the sHsp/substrate core protects the core from aggregation and directs sequestered substrates towards refolding pathway. The sHsp/Hsp70 interplay has major impact on protein homeostasis as it sensitizes substrate release towards cellular Hsp70 availability ensuring efficient refolding of damaged proteins under favourable folding conditions.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels

Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membrane-bound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37 degrees C), and following non-lethal heat shock at 41.8 degrees C. A concentration of 1 microM and an incubation period of 2 h were determined as non-lethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heat-shocked cells under these conditions. Our results show that tubulin-interacting agents, including vincristine and paclitaxel, but not DNA-interacting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semi-quantitative Western blot analysis. Mechanistically, a vincristine- and paclitaxel-induced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.     

Constitutive expression of heat shock proteins Hsp90, Hsc70, Hsp70 and Hsp60 in neural and non-neural tissues of the rat during postnatal development

Heat shock proteins (Hsps) are a group of highly conserved proteins, that are constitutively expressed in most cells under normal physiological conditions. Previous work from our laboratory has shown that neurons in the adult brain exhibit high levels of Hsp90 and Hsc70 mRNA and protein, as well as basal levels of Hsp70 mRNA. We have now investigated the expression of Hsp90, Hsc70, Hsp60 and Hsp70 in neural and non-neural tissues of the rat during postnatal development, a time of extensive cell differentiation. Western blot analysis revealed constitutive expression of these Hsps early in postnatal development. Developmental profiles of these Hsps suggest that they are differentially regulated during postnatal development of the rat. For example, while levels of Hsp90 decrease somewhat in certain developing brain regions, levels of Hsp60 show a developmental increase, and Hsc70 protein is abundant throughout postnatal neural development. Low basal levels of Hsp70 are also observed in the developing and adult brain. A pronounced decrease in Hsp90 and Hsc70 was observed during postnatal development of the kidney while levels of Hsp60 increased. In addition, tissue-specific differences in the relative levels of these Hsps between brain and non-brain regions were found. Immunocytochemical studies demonstrated a neuronal localization of Hsp90, Hsc70 and Hsp60 at all stages of postnatal development examined as well as in the adult, suggesting a role for Hsps in both the developing and fully differentiated neuron. The developmental expression of subunit IV of cytochrome oxidase was similar to that of Hsp60, a protein localized predominantly to mitochondria.     

Valproic acid-mediated Hsp70 induction and anti-apoptotic neuroprotection in SH-SY5Y cells

Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, has been reported to exert neuroprotection against a variety of insults. We now show that VPA attenuates rotenone (a potent complex I inhibitor)-induced apoptosis through the induction of heat shock protein 70, which may interact with apoptotic-protease-activating factor 1. Activation of p-Akt, p-Bcl-2, as well as p-Erk1/2 by VPA may be co-contributors to the protection.     

Myocardial accumulation and localization of the inducible 70-kDa heat shock protein, Hsp70, following exercise

Exercise increases the 70-kDa heat shock protein (Hsp70) in the myocardium, and this exercise-induced increase is associated with significantly improved cardiac recovery following insult. However, while heat shock has been shown to elevate Hsp70 primarily in the cardiac vasculature of the myocardium, the localization following exercise is unknown. Male Sprague-Dawley rats performed continuous treadmill running at 30 m/min for 60 min (2% incline) on either 1 or 5 consecutive days. At 30 min and 24 h following exercise, hearts were extirpated, and the left ventricle was isolated, OCT-cork mounted, and sectioned for immunofluorescent analysis. Whereas immunofluorescent analysis revealed little to no Hsp70 in control hearts and 30 min postexercise, the accumulation of Hsp70 24 h after a single exercise bout or 5 days of training was predominantly located in large blood vessels and, in particular, colocalized with a marker of smooth muscle. Furthermore, higher core temperatures attained during exercise led to more abundant accumulation in smaller vessels and the endothelium. It is concluded that the accumulation of myocardial Hsp70 following acute exercise predominantly occurs in a cell type-specific manner, such that changes in the cardiac vasculature account for much of the increase. This accumulation appears first in the smooth muscle of larger vessels and then increases in smaller vessels and the endothelium, as core temperature attained during exercise increases. This finding supports the observations after heat shock and further suggests that the vasculature is a primary target in exercise-induced cardioprotection.