Free messenger ribonucleoprotein complexes of chicken primary muscle cells following modification of protein synthesis by heat-shock treatment

When primary cultures of chicken myoblasts were subjected to incubation at a temperature higher than their normal growing temperature of 36-37 degrees C, the pattern of protein synthesis was altered. This condition of heat shock induced a vigorous production of a number of proteins collectively known as 'heat-shock proteins'. The synthesis of heat-shock proteins was achieved without a significant decrease in the production of a broad spectrum of proteins by muscle cells. The synthesis of three major heat-shock polypeptides with Mr values of 81 000, 65 000 and 25 000 was observed in both mononucleated dividing myoblast cells and terminally differentiated myotubes. Two-dimensional electrophoretic separation of the heat-induced polypeptides synthesized by myogenetic cultures further established that same set of polypeptides with Mr of 65 000 (pI 6.0 and 5.5), 81 000 (pI 6.2) and 25 000 (pI 5.6 and 5.3) were produced in myoblasts and myotubes. The effect of the changes in pattern of protein synthesis on the mRNA and protein moieties of non-polysomal cytoplasmic mRNA-protein complexes (free mRNP) was examined. Free mRNP complexes sedimenting at 20-35 S were isolated from the post-ribosomal supernatant of both normal and heat-shocked myotube cultures by centrifugation in a sucrose gradient. A 10-20S RNA fraction isolated from these complexes stimulated protein synthesis in a cell-free system. The RNA fraction obtained from heat-shocked cells appeared to direct the synthesis of all three major heat-shock proteins. In contrast, synthesis of these polypeptides was not detected when RNA from free mRNP complexes of normal cells was used for translation. The free mRNP complexes of both normal and heat-shocked cells showed a buoyant density of 1.195 g/cm3 in metrizamide gradients. A large number of polypeptides of Mr = 35 000-105 000 were present in the highly purified free mRNP complexes isolated from the metrizamide gradient. Similar sets of polypeptides were found in these complexes from both normal and heat-shocked myotube culture. However, the relative proportion of a 65 000-Mr polypeptide was dramatically increased in the free mRNP complexes of heat-shocked cells. Two-dimensional gel electrophoretic analysis revealed that this polypeptide and the 65 000-Mr heat-shock polypeptide exhibit similar electrophoretic migration properties. These observations suggest that, following heat-shock treatment of chicken myotube cultures, the changes in the pattern of protein synthesis is accompanied by alteration of the mRNA and protein composition of free mRNP complexes.     

Over-expression of highly conserved mitochondrial 70-kDa heat-shock protein in the sea anemone Anemonia viridis

Organisms react to cellular stress by inducing the synthesis of heat-shock proteins (Hsp). One such protein is the mitochondrial 70-kDa Hsp (mHsp70). The expression of mHsp70 in organisms that undergo stress in their natural habitat is unknown. We used a biochemical approach enabling to identify an mHsp70 from the sea anemone Anemonia viridis, which is abundant in highly fluctuating marine habitats. Antibodies raised specifically against yeast mHsp70 recognized a 70-kDa protein from A. viridis. We found that A. viridis mHsp70 is constitutively expressed at 22–23 °C, but over-expressed upon exposure to heat shock (31 °C) or to temperature fluctuations, suggesting that mHsp70 plays a significant role in adaptation of sessile marine invertebrates to highly fluctuating environmental conditions. Using an affinity column we were able to obtain a partially purified fraction of this protein. Partial amino acid sequences proved that the purified Hsp70 functions in the mitochondria.     

Characterization of the thermotolerant cell. II. Effects on the intracellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes

Here we further characterize a number of properties inherent to the thermotolerant cell. In the preceding paper, we showed that the acquisition of the thermotolerant state (by a prior induction of the heat-shock proteins) renders cells translationally tolerant to a subsequent severe heat-shock treatment and thereby results in faster kinetics of both the synthesis and subsequent repression of the stress proteins. Because of the apparent integral role of the 70-kD stress proteins in the acquisition of tolerance, we compared the intracellular distribution of these proteins in both tolerant and nontolerant cells before and after a severe 45 degrees C/30-min shock. In both HeLa and rat embryo fibroblasts, the synthesis and migration of the major stress-induced 72-kD protein into the nucleolus and its subsequent exit was markedly faster in the tolerant cells as compared with the nontolerant cells. Migration of preexisting 72-kD into the nucleolus was shown to be dependent upon heat-shock treatment and independent of active heat-shock protein synthesis. Using both microinjection and immunological techniques, we observed that the constitutive and abundant 73-kD stress protein similarly showed a redistribution from the cytoplasm and nucleus into the nucleolus as a function of heat-shock treatment. We show also that other lesions that occur in cells after heat shock can be prevented or at least minimized if the cells are first made tolerant. Specifically, the heat-induced collapse of the intermediate filament cytoskeleton did not occur in cells rendered thermotolerant. Similarly, the disruption of intranuclear staining patterns of the small nuclear ribonucleoprotein complexes after heat-shock treatment was less apparent in tolerant cells exposed to a subsequent heat-shock treatment.     

Adaptive cellular response to hyperthermia: 31P-NMR studies

Dynamic intracellular ATP and Pi levels were measured non-invasively for Chinese hamster V79 cells by 31P-NMR under conditions of thermotolerance and heat-shock protein induction. High densities of cells were embedded in agarose strands, placed within a standard NMR sample tube, and perfused with medium maintained either at 37 or 43 degrees C at pH 7.35. Cell survival and heat-shock protein synthesis were assessed either from parallel monolayer cultures or cells dislodged from the agarose strands post-treatment. Thermotolerance (heat resistance) and heat-shock protein synthesis was induced by a 1 h exposure to 43 degrees C followed by incubation for 5 h at 37 degrees C. After the 5 h incubation at 37 degrees C, marked thermal resistance was observed in regard to survival with concomitant synthesis of two major heat-shock proteins at 70 and 103 kDa. Studies were also conducted where tolerance and heat-shock protein synthesis were partially inhibited by depletion of cellular glutathione (GSH) prior to and during heat treatment. Dynamic measurement of intracellular ATP of cells heated with or without GSH depletion revealed no change in steady-state levels immediately after heating or during the 5 h post-heating incubation at 37 degrees C where thermotolerance and heat-shock proteins develop. These data are consistent with other reported data for mammalian cells and indicate that the steady-state ATP levels in mammalian cells remain unchanged during and after the acquisition of the thermotolerant state.     

The small heat shock protein Hsp22 of Drosophila melanogaster is a mitochondrial protein displaying oligomeric organization

Drosophila melanogaster has four main small heat shock proteins (Hsps), D. melanogaster Hsp22 (DmHsp22), Hsp23 (DmHsp23), Hsp26 (DmHsp26), and Hsp27 (DmHsp27). These proteins, although they have high sequence homology, show distinct developmental expression patterns. The function(s) of each small heat shock protein is unknown. DmHsp22 is shown to localize in mitochondria both in D. melanogaster S2 cells and after heterologous expression in mammalian cells. Fractionation of mitochondria indicates that DmHsp22 resides in the mitochondrial matrix, where it is found in oligomeric complexes, as shown by sedimentation and gel filtration analysis and by cross-linking experiments. Deletion analysis using a DmHsp22-EGFP construct reveals that residues 1-17 and an unknown number of residues between 17-28 are necessary for import. Site-directed mutagenesis within a putative mitochondrial motif (WRMAEE) at positions 8-13 shows that the first four residues are necessary for mitochondrial localization. Immunoprecipitation results indicate that there is no interaction between DmHsp22 and the other small heat shock proteins. The mitochondrial localization of this small Hsp22 of Drosophila and its high level of expression in aging suggests a role for this small heat shock protein in protection against oxidative stress.     

Evidence for a novel set of small heat-shock proteins that associates with the mitochondria of murine PC12 cells and protects NADH:ubiquinone oxidoreductase from heat and oxidative stress.

Several previously unreported small heat-shock proteins (sHsps) were detected in mitochondria from heat-stressed rat PC12 cells, but not in unstressed controls. Functional inactivation of the mitochondrial sHsps with murine Hsp25 antibody indicated that these sHsps protect NADH:ubiquinone oxidoreductase and NADH dehydrogenase activity (i.e., complex I) in submitochondrial vesicles during heat and oxidative stress. These results (i) confirm the existence of multiple sHsps in mammals and indicate that several of these sHsps associate with the mitochondria, (ii) indicate a conserved function between plant and mammalian mitochondrial sHsps in protecting electron transport during stress, and (iii) suggest that these sHsps may play an important role in diseases whose etiology is based upon oxidative damage of complex I.     

Intracellular distribution of heat-induced stress glycoproteins

Cellular heat stress results in elevated heat-shock protein (HSP) synthesis and in thermotolerance development. Recently, we demonstrated that protein glycosylation is also an integral part of the stress response with the identification of two major stress glycoproteins, GP50, associated with thermotolerance, and P-SG67, the “prompt” stress glycoprotein induced immediately during acute heat stress. In the present study, we characterized the subcellular location and redistribution of these proteins during the cellular injury and recovery phase. In unheated and heated CHO cells, both stress glycoproteins were present in each subcellular fraction isolated by differential centrifugation. However, the subcellular redistribution in the course of cellular recovery after heat stress was specific for each stress glycoprotein. GP50 was present in all subcellular fractions before heat stress, but showed relatively little redistribution after heat stress. By 24 h of recovery following stress, GP50 showed partial depletion from lysosomes and microsomes, and was mainly present in the mitochondria. Glycosylated P-SG67 was redistributed in a more complex fashion. It was seen predominantly in the lysosomes and microsomes immediately following heat-stress, but after 6 h of recovery following heat stress, it largely disappeared from the microsomes and was present mainly in the cytosol. By 24 h of recovery following heat stress, it was found predominantly in the nucleus-rich fraction and mitochondria. The localization of GP50 and P-SG67 by subcellular fractionation is consistent with immunolocalization studies and contrasts with the translocation of HSP70 after heat stress from cytosol to nuclei and nucleoli. These results reflect a characteristic distribution for each stress glycoprotein; their presence in virtually all subcellular fractions suggests multifunctional roles for the various stress glycoproteins in the cellular heat stress response. J. Cell. Biochem. 66:98–111, 1997. © 1997 Wiley-Liss, Inc.     

The molecular chaperone Hsp78 confers compartment-specific thermotolerance to mitochondria

Hsp78, a member of the family of Clp/Hsp100 proteins, exerts chaperone functions in mitochondria of S. cerevisiae which overlap with those of mitochondrial Hsp70. In the present study, the role of Hsp78 under extreme stress was analyzed. Whereas deletion of HSP78 does not affect cell growth at temperatures up to 39 decrees C and cellular thermotolerance at 50 degrees C, Hsp78 is crucial for maintenance of respiratory competence and for mitochondrial genome integrity under severe temperature stress (mitochondrial thermotolerance). Mitochondrial protein synthesis is identified as a thermosensitive process. Reactivation of mitochondrial protein synthesis after heat stress depends on the presence of Hsp78, though Hsp78 does not confer protection against heat-inactivation to this process. Hsp78 appears to act in concert with other mitochondrial chaperone proteins since a conditioning pretreatment of the cells to induce the cellular heat shock response is required to maintain mitochondrial functions under severe temperature stress. When expressed in the cytosol, Hsp78 can substitute for the homologous heat shock protein Hsp104 in mediating cellular thermotolerance, suggesting a conserved mode of action of the two proteins. Thus, proteins of the Clp/Hsp100-family located in the cytosol and within mitochondria confer compartment-specific protection against heat damage to the cell.     

A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.

The synthesis of 40 polypeptides in mitochondria was found to be stimulated after transient exposure of human endothelial cells to sublethal levels of hydroperoxides, such as H(2)O(2), using comparative two-dimensional polyacrylamide gel electrophoresis. Eleven proteins were identified; these include 60 kDa heat shock protein (HSP60), a mitochondrial type of 70 kDa HSP (mtHSP70), manganese-dependent superoxide dismutase (MnSOD), three metabolic enzymes in citric acid cycle, two components for respiratory chain complexes, a ribosomal protein for translation in mitochondria (RM12), and an unnamed protein. These proteins are involved in reduction-oxidation and protein biogenesis, suggesting that their synthesis, which is triggered under oxidative stress conditions, is aimed at playing a defensive role in mitochondria. Moreover, mtHSP70, HSP60, MnSOD, and RM12 were revealed as their respective precursor proteins with mitochondrial targeting sequences. The preproteins of HSP60 and mtHSP70 were transiently accumulated in mitochondria after the removal of H(2)O(2) in a processing competent state, while the accumulated preprotein of MnSOD localized inside mitochondria and remained unchanged. Membrane potential of mitochondria and cellular ATP levels were unchanged under these conditions. Taken together, these results suggest that hydroperoxide stress leads to preprotein accumulation, possibly due to the impairment of the protein-processing system in mitochondria, independent of membrane potential dissipation and ATP depletion.     

Nuclear-mitochondrial cross-talk during heat shock in Arabidopsis cell culture

Apart from energy generation, mitochondria perform a signalling function determining the life and death of a cell under stress exposure. In the present study we have explored patterns of heat-induced synthesis of Hsp101, Hsp70, Hsp17.6 (class I), Hsp17.6 (class II) and Hsp60, and the development of induced thermotolerance in Arabidopsis thaliana cell culture under conditions of mitochondrial dysfunction. It was shown that treatment by mitochondrial inhibitors and uncouplers at the time of mild heat shock downregulates HSP synthesis, which is important for induced thermotolerance in plants. The exposure to elevated temperature induced an increase in cell oxygen consumption and hyperpolarization of the inner mitochondrial membrane. Taken together, these facts suggest that mitochondrial functions are essential for heat-induced HSP synthesis and development of induced thermotolerance in A. thaliana cell culture, suggesting that mitochondrial-nuclear cross-talk is activated under stress conditions. Treatment of Arabidopsis cell culture at 50 degrees C initiates a programmed cell death determined by the time course of viability decrease, DNA fragmentation and cytochrome c release from mitochondria. As treatment at 37 degrees C protected Arabidopsis cells from heat-induced cell death, it may be suggested that Hsp101, Hsp70 and small heat-shock proteins, the synthesis of which is induced under these conditions, are playing an anti-apoptotic role in the plant cell. On the other hand, drastic heat shock upregulated mitochondrial Hsp60 synthesis and induced its release from mitochondria to the cytosol, indicating a pro-apoptotic role of plant Hsp60.     

The impact of oxidative stress on Arabidopsis mitochondria

Treatment of Arabidopsis cell culture for 16 h with H2O2, menadione or antimycin A induced an oxidative stress decreasing growth rate and increasing DCF fluorescence and lipid peroxidation products. Treated cells remained viable and maintained significant respiratory rates. Mitochondrial integrity was maintained, but accumulation of alternative oxidase and decreased abundance of lipoic acid-containing components during several of the treatments indicated oxidative stress. Analysis of the treatments was undertaken by IEF/SDS-PAGE, comparison of protein spot abundances and tandem mass spectrometry. A set of 25 protein spots increased >3-fold in H2O2/menadione treatments, a subset of these increased in antimycin A-treated samples. A set of 10 protein spots decreased significantly during stress treatments. A specific set of mitochondrial proteins were degraded by stress treatments. These damaged components included subunits of ATP synthase, complex I, succinyl CoA ligase, aconitase, and pyruvate and 2-oxoglutarate dehydrogenase complexes. Nine increased proteins represented products of different genes not found in control mitochondria. One is directly involved in antioxidant defense, a mitochondrial thioredoxin-dependent peroxidase, while another, a thioredoxin reductase-dependent protein disulphide isomerase, is required for protein disulfide redox homeostasis. Several others are generally considered to be extramitochondrial but are clearly present in a highly purified mitochondrial fraction used in this study and are known to play roles in stress response. Using H2O2 as a model stress, further work revealed that this treatment induced a protease activity in isolated mitochondria, putatively responsible for the degradation of oxidatively damaged mitochondrial proteins and that O2 consumption by mitochondria was significantly decreased by H2O2 treatment.     

Heat-Stress Response of Maize Mitochondria

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.     

Heat-shock response and its contribution to thermotolerance of the nitrogen-fixing cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain L-31

Compared to Escherichia coli, the nitrogen-fixing soil cyanobacterium Anabaena sp. strain L-31 exhibited significantly superior abilities to survive prolonged and continuous heat stress and recover therefrom. Temperature upshift induced the synthesis of heat-shock proteins of similar molecular mass in the two microbes. However, in Anabaena sp. strain L-31 the heat-shock proteins (particularly the GroEL proteins) were synthesised throughout the stress period, were much more stable and accumulated during heat stress. In contrast, in E. coli the heat-shock proteins were transiently synthesised, quickly turned over and did not accumulate. Nitrogenase activity of Anabaena cells of sp. strain L-31 continuously exposed to heat stress for 7 days rapidly recovered from thermal injury, although growth recovery was delayed. Exposure of E. coli cells to >4.5 h of heat stress resulted in a complete loss of viability and the ability to recover. Marked differences in the synthesis, stability and accumulation of heat-shock proteins appear to distinguish these bacteria in their thermotolerance and recovery from heat stress.     

A heat shock protein is encoded within mitochondria of higher plants

A temperature shift from 25 to 41 degrees C initiates the synthesis of a specific set of proteins in maize, including a peptide of 60 kilodaltons. Using an in vitro mitochondrial protein synthesizing system, we provide evidence that this 60-kDa heat shock protein is encoded within the organelle. Further support for this heat-induced protein being encoded within mitochondria is that its synthesis is inhibited in whole seedlings by chloramphenicol. This 60-kDa heat shock protein is induced in all lines of maize we examined. Additionally, a heat-induced peptide of similar size (62 kDa) can be detected in isolated mitochondria of a dicot plant, Brassica campestris. The function of the heat shock protein encoded within the mitochondria remains unknown.     

Mutational analysis of G-protein coupled receptor--FFA2

As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.     

Archaebacterial heat-shock proteins

The response to heat shock was examined in seven archaebacterial strains from the genus Halobacterium. Upon heat shock each strain preferentially synthesized a limited number of proteins which fell into three narrow mol. wt. ranges. Further examination of the heat-shock response in H. volcanii revealed that heat-shock protein (hsp) synthesis was greatest at 60°C. Synthesis of hsps at this induction temperature was both rapid and transient. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. H. volcanii cells also responded with a `heat shock-like' response to salt dilution, a natural environmental stress for these organisms. These results indicate that the heat shock or stress response which is charactertistic of eukaryotic and eubacterial cells is also present among members of the archaebacterial genus Halobacterium.     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

Mitochondrial alterations in human gastric carcinoma cell line

We compared mitochondrial function, morphology, and proteome in the rat normal gastric cell line RGM-1 and the human gastric cancer cell line AGS. Total numbers and cross-sectional sizes of mitochondria were smaller in AGS cells. Mitochondria in AGS cells were deformed and consumed less oxygen. Confocal microscopy indicated that the mitochondrial inner membrane potential was hyperpolarized and the mitochondrial Ca²⁺ concentration was elevated in AGS cells. Interestingly, two-dimensional electrophoresis proteomics on the mitochondria-enriched fraction revealed high expression of four mitochondrial proteins in AGS cells: ubiquinol-cytochrome c reductase, mitochondrial short-chain enoyl-coenzyme A hydratase-1, heat shock protein 60, and mitochondria elongation factor Tu. The results provide clues as to the mechanism of the mitochondrial changes in cancer at the protein level and may serve as potential cancer biomarkers in mitochondria. two-dimensional gel electrophoresis proteomics; biomarker; cancer     

Stress Protein and Crystallin Synthesis During Heat Shock and Transdifferentiation of Embryonic Chick Neural Retina Cells

Embryonic chick neural retina responds to heat shock by the synthesis of "stress" polypeptides with molecular weights of 85 and 70 kd. Both stress proteins are synthesised from newly-transcribed messenger RNA. Sodium arsenite induces an additional stress protein of MW 25 kd. The heat shock response does not change during culture and subsequent transdifferentiation, and crystallin synthesis is not coinducible with the heat-shock proteins. We have also examined the pattern of protein synthesis at various stages of culture in both monolayer and aggregate systems; although changes in the protein synthetic profine are evident, there is no stress protein induction above basal levels at any time. Whilst mammalian α crystallin (B₂ chain) exhibits considerable homology to four small Drosophila heat-shock proteins, no significant antigenic similarity is apparent between δ crystallin and the major avian heat shock proteins. Thus during transdifferentiation, (a) the crystallin proteins do not behave in a manner analogous to stress proteins; moreover (b) crystallin production is not mediated by stress proteins resulting from a culture-induced stress response.