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Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 μM isopropyl-β- d -thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2–2.3-fold by coexpression of GroEL/ES, Trx, or DsbB–DsbD (DsbBD), while it was decreased by coexpression of DnaK–DnaJ–GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli.

Expression of 150 kda oxygen-regulated protein, ORP150, was examined in the atheromatous lesions on aortic valves in high-fat diet fed mice. Immunohistochemical staining revealed that ORP150 was expressed on the surface of plaque and was co-localized with phagocytes bearing Mac-3, a mouse macrophage differentiation antigen. These findings suggest that ORP150 is involved in the development of the atheromatous plaque. Titer of autoantibody against ORP150 was gradually elevated in parallel with the length of period of high-fat diet feeding. These results suggest that the deposition of immunocomplex toward ORP150 antigen is involved in atheromatous plaque progression.

金沢大学医薬保健研究域医学系, A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.

RpoH ( Escherichia coli ς 32 and its homologs) is the central regulator of the heat shock response in gram-negative proteobacteria. Here we studied salient regulatory features of RpoH in Agrobacterium tumefaciens by examining its synthesis, stability, and activity while increasing the temperature from 25 to 37°C. Heat induction of RpoH synthesis occurred at the level of transcription from an RpoH-dependent promoter, coordinately with that of DnaK, and followed by an increase in the RpoH level. Essentially normal induction of heat shock proteins was observed even with a strain that was unable to increase the RpoH level upon heat shock. Moreover, heat-induced accumulation of dnaK mRNA occurred without protein synthesis, showing that preexisting RpoH was sufficient for induction of the heat shock response. These results suggested that controlling the activity, rather than the amount, of RpoH plays a major role in regulation of the heat shock response. In addition, increasing or decreasing the DnaK-DnaJ chaperones specifically reduced or enhanced the RpoH activity, respectively. On the other hand, the RpoH protein was normally stable and remained stable during the induction phase but was destabilized transiently during the adaptation phase. We propose that the DnaK-mediated control of RpoH activity plays a primary role in the induction of heat shock response in A. tumefaciens , in contrast to what has been found in E. coli .

The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of alpha, delta and zeta-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the alpha and delta subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower alpha and delta subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the alpha and delta subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of alpha and delta subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells.

Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.

Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.

The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We show here that degradation of CCT in mammalian cells is inhibited by a proteasome-specific inhibitor, lactacystin. When CCT synthesis was inhibited by growth arrest of cells, the decrease in CCT levels was much slower in the presence of lactacystin than in its absence. Pulse-chase experiments indicated that degradation of CCT is inhibited 2- to 2.5-fold by addition of lactacystin. In addition, CCT degradation rate in ts85 cells that produce thermolabile ubiquitin-activating enzyme E1 was reduced 3-fold at the nonpermissive temperature compared to the degradation at the permissive temperature. These results indicate that the ubiquitin-proteasome system is involved in CCT degradation.

150 kDa oxygen-regulated protein (ORP150) is one of the endoplasmic reticulum (ER)-resident stress proteins. We have cloned and sequenced the entire human ORP150 gene covering over 15-kb. Analyses of transcription initiation sites and transcriptional regulatory sequences revealed that at least three distinct mRNA species were produced by alternative promoters: two of them starting from alternative exon 1 (1A or 1B), and the third one starting from exon 2, six nucleotides upstream of the first AUG initiation codon. Among them, the transcript that begins with exon 1B was preferentially induced by hypoxia or tunicamycin treatment. A cis-acting segment involved in the stress-dependent induction was found at the 5'-end of exon 1A, which could account for the selective induction of the transcription from exon 1B. Furthermore, in vitro analyses of translation of the third mRNA suggested the constitutive expression of the cytosolic ORP150 due to the lack of the signal peptide resulting from differential translation initiation.

To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

To examine the effects of overexpression of trigger factor (TF) on recombinant proteins produced in Escherichia coli , we constructed plasmids that permitted controlled expression of TF alone or together with the GroEL-GroES chaperones. The following three proteins that are prone to aggregation were tested as targets: mouse endostatin, human oxygen-regulated protein ORP150, and human lysozyme. The results revealed that TF overexpression had marked effects on the production of these proteins in soluble forms, presumably through facilitating correct folding. Whereas overexpression of TF alone was sufficient to prevent aggregation of endostatin, overexpression of TF together with GroEL-GroES was more effective for ORP150 and lysozyme, suggesting that TF and GroEL-GroES play synergistic roles in vivo. Although coexpression of the DnaK-DnaJ-GrpE chaperones was also effective for endostatin and ORP150, coexpression of TF and GroEL-GroES was more effective for lysozyme. These results attest to the usefulness of the present expression plasmids for improving protein production in E. coli .

The chaperonin containing TCP-1 (CCT) is a molecular chaperone consisting of eight subunit species and assists in the folding of actin, tubulin and some other cytosolic proteins. We examined the stress response of CCT subunit proteins in mammalian cultured cells using chemical stressors that cause accumulation of unfolded proteins. Levels of CCT subunit proteins in HeLa cells were coordinately and transiently upregulated under continuous chemical stress with sodium arsenite. CCT subunit levels in several mammalian cell lines were also upregulated during recovery from chemical stress caused by sodium arsenite or a proline analogue, L-azetidine-2-carboxylic acid. Several unidentified proteins that were newly synthesized and associated with CCT were found to increase concomitantly with CCT subunits themselves and known substrates during recovery from the stress. These results suggest that CCT plays important roles in the recovery of cells from protein damage by assisting in the folding of proteins that are actively synthesized and/or renatured during this period.

Coexpression of two classes of folding accessory proteins, molecular chaperones and foldases, can be expected to improve the productivity of soluble and active recombinant proteins. In this study, horseradish peroxidase (HRP), which has four disulfide bonds, was selected as a model enzyme and overexpressed in Escherichia coli. The effects of coexpression of a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active HRP in E. coli were examined. Active HRP was produced by very mild induction with 1 microM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C, whereas the amount of active HRP produced by the induction with 1 mM IPTG was negligibly small. Active HRP production was increased significantly by coexpression of DsbA-DsbB (DsbAB) or DsbC-DsbD (DsbCD), while coexpression of molecular chaperones did not improve active HRP production. The growth of E. coli cells was inhibited significantly by the induction with 1 mM IPTG in a HRP single expression system. In contrast, when HRP was coexpressed with DsbCD, the growth inhibition of E. coli was not observed. Therefore, coexpression of Dsb proteins improves both the cell growth and the productivity of HRP.

The 150-kDa oxygen-regulated protein (ORP150) is a member of glucose-regulated proteins (GRPs), which are induced by stressful conditions such as oxygen or glucose deprivation. Here we investigated the highly abundant expression of ORP150 in mouse pancreas and its relationship with insulin secretion. Immunohistochemical analysis revealed that ORP150 expression was restricted to islets, especially to beta cells. The beta cell-specific expression was also observed in a mouse insulinoma cell line, MIN6, which secretes insulin in response to increased glucose concentration. Furthermore, ORP150 in islets dramatically diminished by fasting, concomitant with reduction of the serum insulin level. These results strongly suggest the role for ORP150 in insulin secretion.

In the paradigm of bacterial heat shock response studied with Escherichia coli, ς32 (encoded by rpoH) plays a key role in controlling the transcription of all major heat shock proteins (HSPs) in the cytoplasm, including GroE and DnaK chaperone machineries and ATP-dependent proteases, such as Clp and Lon (10, 42). Upon heat shock, the cellular level of ς32 increases rapidly both by enhanced translation of rpoH mRNA and by transient stabilization of ς32, and this increase in turn activates the transcription of heat shock genes. Homologs of rpoH have been identified from some 20 species of eubacteria that belong to the alpha, beta, and gamma subgroups of proteobacteria (1, 13, 15, 20, 21, 24, 30, 31); earlier work cited in reference (21). The rpoH homologs from many gamma proteobacteria share common structural features with E. coli rpoH, a downstream box, mRNA secondary structure, and highly conserved amino acid sequence of region C, that are important for thermoregulation of rpoH translation and ς32 stability and activity in E. coli (21). In these bacteria, the RpoH homologs indeed exhibit translational induction and stabilization upon heat shock very similar to that found with E. coli (22). In contrast, alpha and beta proteobacteria have diverged from the gamma subgroup in their modes of regulation of rpoH expression. Their rpoH genes do not contain the downstream box or mRNA secondary structure that are conserved among gamma proteobacteria. Instead, some rpoH genes from alpha proteobacteria contain an RpoH-dependent promoter that can be induced upon heat shock (24, 26, 40). Another important feature found in alpha but not in gamma proteobacteria is the presence of the CIRCE (for controlling inverted repeat of chaperone expression)-HrcA regulatory system. Recent studies of wide groups of eubacteria revealed a variety of heat shock regulatory mechanisms, either positive regulation by alternative ς factors or negative regulation by specific repressor-operator systems (see reviews in references 23 and 28). The most widely distributed system is the CIRCE-HrcA system, extensively characterized in Bacillus subtilis as the regulatory mechanism specific for the groE and dnaK operons (18, 19, 41, 43). CIRCE, with a consensus of TTAGCACTC-N9-GAGTGCTAA, represents a site for binding the HrcA repressor. HrcA could act as a stress sensor whose activity is modulated by the GroEL chaperone. This system has been found in gram-positive bacteria and proteobacteria and implicated in cyanobacteria and several other groups of eubacteria (5, 8, 9, 38, 39). Within proteobacteria, CIRCE and/or hrcA were shown to regulate groE operons of some alpha proteobacteria, including Agrobacterium tumefaciens (3, 27, 37). However a CIRCE-like sequence has not been detected in gamma proteobacteria, except for the groE operon of Chromatium vinosum (7). It seemed likely that the CIRCE-HrcA system is operative in the alpha and beta proteobacteria but not in most gamma proteobacteria. Thus, alpha and beta proteobacteria appeared to be unique in that they carry both the RpoH system, with regulatory features different from those of gamma proteobacteria, and the CIRCE-HrcA system for regulation of the heat shock response. In this study, we used A. tumefaciens as a model to study the differential roles of the RpoH and CIRCE-HrcA systems in the heat shock response in alpha proteobacteria. The groE and dnaK operons of A. tumefaciens were previously isolated and characterized (34–37), and the CIRCE element was found in the groE, but not in the dnaK, promoter region. Although the rpoH gene was available from our previous study (21), no information on hrcA was at hand. Successful isolation of hrcA and analysis of the deletion mutants lacking rpoH and/or hrcA revealed that RpoH plays a major and global role in the induction of HSPs, whereas the role of HrcA is restricted primarily to repressing the groE operon under nonstress conditions.

The chaperonin containing t-complex polypeptide 1 (CCT) is a heterooligomeric molecular chaperone assisting in the folding of actin, tubulin, and other cytosolic proteins. The expression levels of CCT subunits varied among seven mouse cell lines tested but showed a close correlation with growth rate. Both the CCT protein and mRNA levels in the human promyelolytic cell HL60 decreased concomitant with growth arrest during differentiation. More rapid decrease in CCT level occurred when the mouse interleukin (IL)-3-dependent myeloid DA3 cells were starved for IL-3. Readdition of IL-3 caused rapid resumption of CCT synthesis during synchronous growth: the maximum CCT protein and mRNA levels were observed at G1/S transition through early S phase. The turnover rate of CCT was nearly constant regardless of growth. Gel filtration and immunoprecipitation analyses indicated that CCT in vivo is associated with tubulin at early S phase, but not at G0/G1 phase. These results demonstrated that CCT expression is strongly up-regulated during cell growth especially from G1/S transition to early S phase and is primarily controlled at the mRNA level. CCT appears to play important roles for cell growth by assisting in the folding of tubulin and other proteins.

The heat shock response inEscherichia coli depends on a transient increase in the intracellular level of ς32 that results from both increased synthesis and transient stabilization of normally unstable ς32. Although the membrane-bound ATP-dependent protease FtsH (HflB) plays an important role in degradation of ς32, our previous results suggested that several cytosolic ATP-dependent proteases including HslVU (ClpQY) are also involved in ς32 degradation (Kanemori, M., Nishihara, K., Yanagi, H., and Yura, T. (1997) J. Bacteriol.179, 7219–7225). We now report on the ATP-dependent proteolysis of ς32 by purified HslVU protease and its unusual dependence on high temperature: ς32 was rapidly degraded at 44 °C, but with much slower rates (∼15-fold) at 35 °C. FtsH-dependent degradation of ς32 also gave similar results. In agreement with these results in vitro, the turnover of ς32 in normally growing cells at high temperature (42 °C) was much faster than at low temperature (30 °C). Taken together with other evidence, these results suggest that the ς32 level during normal growth is primarily determined by the stability (susceptibility to proteases) and synthesis rate of ς32 set by ambient temperature, whereas fine adjustment such as transient stabilization of ς32 observed upon heat shock is brought about through monitoring changes in the cellular state of protein folding.

The chaperonin-containing TCP-1 (CCT) is a hetero-oligomeric molecular chaperone that mediates protein folding in the cytosol of eukaryotes. Eight (or nine in testis) subunit species are assembled in the CCT hexadecamer complex. We have cloned seven CCT subunit genes, Cctb, Cctd, Ccte, Cctz-1, Cctz-2 (testis specific), Ccth and Cctq, from mouse genomic DNA libraries, in addition to the Ccta and Cctg genes reported previously, and the entire nucleotide sequences of these DNA clones were determined. These genes are approximately 15-20 kb in length except for Cctz-2 which is longer than 35 kb, and all the Cct genes consist of 11-16 exons. Primer extension analyses of testis RNA indicate one to several potential transcription start sites 50-150 bp upstream from the translation start codon of each Cct gene. There are several possible Sp1-binding sequences, but no obvious TATA box was observed around the potential start sites. From 5'-flanking regions to the first introns, the Cct genes are rich in CpG dinucleotides. In reporter gene assays using these regions, five of eight Cct genes showed strong transcriptional activity comparable with the combination of SV40 promoter and enhancer in HeLa cells. We also show, by Western and Northern blot analyses, that CCT expression levels vary widely among different tissues but the expression patterns are very similar among the eight subunit species. It is likely that expression levels of the eight different subunits are tightly co-regulated to maintain a constant ratio of these subunits which constitute the CCT hexadecamer complex with a fixed subunit arrangement.

Induction of heat shock proteins in Escherichia coli is primarily caused by increased cellular levels of the heat shock sigma-factor sigma32 encoded by the rpoH gene. Increased sigma32 levels result from both enhanced synthesis and stabilization. Previous work indicated that sigma32 synthesis is induced at the translational level and is mediated by the mRNA secondary structure formed within the 5'-coding sequence of rpoH, including the translation initiation region. To understand the mechanism of heat induction of sigma32 synthesis further, we analyzed expression of rpoH-lacZ gene fusions with altered stability of mRNA structure before and after heat shock. A clear correlation was found between the stability and expression or the extent of heat induction. Temperature-melting profiles of mRNAs with or without mutations correlated well with the expression patterns of fusion genes carrying the corresponding mutations in vivo. Furthermore, temperature dependence of mRNA-30S ribosome-tRNAfMet complex formation with wild-type or mutant mRNAs in vitro agreed well with that of the expression of gene fusions in vivo. Our results support a novel mechanism in which partial melting of mRNA secondary structure at high temperature enhances ribosome entry and translational initiation without involvement of other cellular components, that is, intrinsic mRNA stability controls synthesis of a transcriptional regulator.

The heat shock response in Escherichia coli depends primarily on the increased synthesis and stabilization of otherwise scarce and unstable ς 32 ( rpoH gene product), which is required for the transcription of heat shock genes. The heat-induced synthesis of ς 32 occurs at the level of translation, and genetic evidence has suggested the involvement of a secondary structure at the 5′ portion (nucleotides −19 to +247) of rpoH mRNA in regulation. We now present evidence for the mRNA secondary structure model by means of structure probing of RNA with chemical and enzymatic probes. A similar analysis of several mutant RNAs with a mutation predicted to alter a base pairing or with two compensatory mutations revealed altered secondary structures consistent with the expression and heat inducibility of the corresponding fusion constructs observed in vivo. These findings led us to assess the possible roles of each of the stem-loop structures by analyzing an additional set of deletions and base substitutions. The results indicated not only the primary importance of base pairings between the translation initiation region of ca. 20 nucleotides (the AUG initiation codon plus the “downstream box”) and the internal region of rpoH mRNA but also the requirement of appropriate stability of mRNA secondary structures for characteristic thermoregulation, i.e., repression at a low temperature and induction upon a temperature upshift.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), transcription of glucose-regulated proteins (GRPs) representing ER-resident molecular chaperones is markedly induced via the unfolded protein response (UPR) pathway. In contrast to recent progress in the analysis of yeast UPR, bothcis-acting elements and transactivators responsible for mammalian UPR have remained obscure. Here, we analyzed the promoter regions of human GRP78, GRP94, and calreticulin genes and identified a novel element designated the ER stress response element (ERSE). ERSE, with a consensus of CCAATN9CCACG, was shown to be necessary and sufficient for induction of these GRPs. Using yeast one-hybrid screening, we isolated a human cDNA encoding a basic leucine zipper (bZIP) protein, ATF6, as a putative ERSE-binding protein. When overexpressed in HeLa cells, ATF6 enhanced transcription of GRP genes in an ERSE-dependent manner, whereas CREB-RP, another bZIP protein closely related to ATF6, specifically inhibited GRP induction. Endogenous ATF6 constitutively expressed as a 90-kDa protein was converted to a 50-kDa protein in ER-stressed cells, which appeared to be important for the cellular response to ER stress. These results suggest that, as in yeast, bZIP proteins are involved in mammalian UPR, acting through newly defined ERSE.

The heat shock response inEscherichia coli and related bacteria is primarily mediated byσ32 or its homologue (RpoH protein) specifically required for transcription of heat shock genes encoding molecular chaperones and proteases. Extensive work inE. coli revealed some of the mechanisms controlling the cellular level and activity ofσ32 during the heat shock response. Recent isolation of a number of RpoH homologues from γ, β and α proteobacteria provided an opportunity to examine evolutionary conservation and diversity of regulatory mechanisms in these bacteria. We here summarize the present status of this aspect of the stress response not only by comparative sequence analysis but by examining the response of representative RpoH homologues of the γ subgroup to heat shock stress. Current evidence indicates that the basic strategy of enhancing RpoH level as a primary response to heat shock stress is well conserved, but the detailed mechanisms for enhancement of the heat shock σ factor level vary among different species that may reflect diverse ecological niches.

Transcriptional induction of thehsp70 gene is mediated by heat shock factor 1 (HSF1) rapidly activated upon heat and other stresses. HSF2 has been thought to be responsible for accumulation of HSP70 during hemin-induced differentiation of human K562 erythroleukemia cells because of accompanying acquisition of HSF2 DNA binding activity. However, there has not been any direct evidence for such a functional role of HSF2. The purpose of this study is to clarify the roles of HSF1 and HSF2 in HSP70 induction in hemin-treated K562 cells. We show here that a chimeric polypeptide of HSF2 and GAL4 DNA binding domain (GAL4-BD–HSF2) was unable to induce a GAL4 binding site-containing luciferase reporter gene in response to hemin and that exogenously overproduced HSF2 also failed to increase expression of a heat shock element-containing reporter. On the contrary, expression of a GAL4-BD–HSF1 chimeric protein responded to hemin treatment as well as to heat shock, and transiently overexpressed HSF1 caused hemin-responsive induction of the reporter gene in a dose-dependent manner. These results indicate that HSF1, rather than HSF2, primarily mediates the hemin-induced transcription of the hsp70 gene.

Although heat shock factor 2 (HSF2) binds to heat shock element (HSE) constitutively during differentiation, development and spermatogenesis, little is known about the nature and mechanism of transcriptional control of heat shock genes by HSF2. We screened a human testis cDNA library for proteins that can associate with HSF2 by the yeast two-hybrid system, and isolated clones encoding a novel protein, designated HSF2 binding protein (HSF2BP), that associates with HSF2 in vitro and in vivo and is specifically expressed in testis. The interaction seemed to occur between the trimerization domain of HSF2 and the amino terminal hydrophilic region of HSF2BP that comprises two leucine zipper motifs. HSF2BP may therefore be involved in modulating HSF2 activation in testis.

DNA binding of heat shock factor 2 (HSF2) is induced during hemin-induced differentiation of human erythroleukemia cell line K562. To identify the transcriptional activation and the regulatory domains of HSF2, we constructed a series of deletion derivatives fused to the yeast GAL4 DNA binding domain and analyzed their transactivation activity. A minimal transactivation domain of HSF2 was localized to the C-terminus (residues 472-536), as in HSF1, although amino acid sequence similarity for these regions was rather limited and the potential transactivation ability was about 25% that of HSF1. The transactivation mediated by this region of HSF2 was found to be negatively regulated by the adjacent 18 amino acid segment (residues 428-445) under normal conditions. Furthermore, the latter segment, when fused to the GAL4 activation domain, markedly inhibited GAL4 activity. Extract containing most derivatives of HSF2 retaining this segment exhibited doublet or triplet bands in gel mobility shift assays with heat shock element-containing DNA, suggesting possible involvement of some factors interacting with that segment in the negative regulation. Another putative transactivation domain and two negative regulatory regions were also localized within the internal region.

Induction of heat shock proteins represents a ubiquitous and homeostatic cellular response to cope with the accumulation of unfolded and misfolded proteins following exposure to heat or other stresses. In Escherichia coli, the induction occurs primarily by activating the transcription of heat shock genes by a transient increase in the cellular level of ς32 (10), encoded by the rpoH gene (8, 36). The level of ς32 during steady-state growth is very low (ca. 10 to 30 molecules per cell at 30°C [4]) due to the extreme instability (half life, 1 min) and restricted translation of rpoH mRNA (8, 37). Upon exposure to higher temperatures (e.g., 42°C), both rapid stabilization of ς32 and translational activation of rpoH mRNA result in a marked and transient increase in the ς32 level, which in turn activates the transcription of heat shock genes such as those encoding molecular chaperones (e.g., DnaK and GroEL) and ATP-dependent proteases (e.g., Lon and ClpP) (7, 8). Whereas stabilization of ς32 is thought to occur by accumulation of partially unfolded proteins that can titrate chaperones away from ς32 (2, 4), translational activation probably occurs by resolving extensive mRNA secondary structure formed under nonstress conditions (17, 37, 38). Previous work on heat-induced synthesis of ς32–β-galactosidase fusion protein in E. coli revealed that two proximal coding regions (A and B) of rpoH mRNA are involved in translational control of ς32 synthesis (17, 37). Region A is similar to the “downstream box,” which is complementary to part of 16S rRNA (27), and acts as a positive element by enhancing translation, whereas region B is a negative element which forms an extensive secondary structure with region A. In addition, a segment of ς32 (region C; around residues 122 to 144 [18]) that corresponds to an area further downstream of rpoH was shown to be important for the DnaK/DnaJ-mediated negative feedback control of the heat shock response (28, 30, 31). Region C affects both the synthesis and stability of the fusion protein (18, 37), presumably by providing sites for interaction with DnaK (14). Recent work in this and other laboratories led to the isolation and characterization of rpoH genes encoding ς32 (RpoH) homologs from a number of gram-negative bacteria (1, 5, 6, 16, 19–23, 25, 34). Some of them were isolated by directly selecting for clones that can complement the defects of an E. coli ΔrpoH mutant which grows only at or below 20°C (1, 19, 21). Comparison of nucleotide and amino acid sequences among these homologs revealed several conserved sequences that seemed to be of regulatory importance for the heat shock response. Specifically, both downstream box and predicted mRNA secondary structures similar to those seen in E. coli were found among the homologs from most members of the gamma but not alpha proteobacteria (1, 19, 34, 36). Besides, a stretch of 9 amino acid residues, highly conserved among all ς32 homologs but absent in other ς factors (designated the RpoH box [19, 36]), was detected within region C, implicated in the chaperone-mediated feedback control (18; see above). These interesting developments prompted us to examine the regulatory roles of ς32 homologs in the heat shock response of some of the representative bacteria. In this report, we analyzed the regulatory and functional properties of ς32 homologs (RpoH) both in E. coli and in cognate bacteria. Several members of the gamma proteobacteria (E. coli, Serratia marcescens, Proteus mirabilis, and Pseudomonas aeruginosa) and one of the alpha proteobacteria (Agrobacterium tumefaciens) were chosen for this study. The ability of these rpoH homologs to complement, at least partially, the growth defect of the E. coli ΔrpoH mutant had suggested that they can be expressed to appreciable extents in E. coli and can functionally replace ς32 to varying degrees. Such expectations were fully supported by the present results. Most significantly, they revealed substantial conservation and some divergence in the regulatory strategy of ς32 homologs, particularly among the gamma proteobacteria.

When unfolded proteins are accumulated in the endoplasmic reticulum (ER), an intracellular signaling pathway termed the unfolded protein response (UPR) is activated to induce transcription of ER-localized molecular chaperones and folding enzymes in the nucleus. In Saccharomyces cerevisiae, at least six lumenal proteins including essential Kar2p and Pdi1p are known to be regulated by the UPR. We and others recently demonstrated that the basic-leucine zipper protein Hac1p/Ern4p functions as a trans-acting factor responsible for the UPR. Hac1p binds directly to the cis-acting unfolded protein response element (UPRE) responsible for Kar2p induction. Moreover, we showed that the KAR2 UPRE contains an E box-like palindrome separated by one nucleotide (CAGCGTG) that is essential for its function. We report here that the promoter regions of each of five target proteins (Kar2p, Pdi1p, Eug1p, Fkb2p, and Lhs1p) contain a single UPRE sequence that is necessary and sufficient for induction and that binds specifically to Hac1p in vitro. All of the five functional UPRE sequences identified contain a palindromic sequence that has, in four cases, a spacer of one C nucleotide. This unique characteristic of UPRE explains why only a specific set of proteins are induced in the UPR to cope with ER stress.

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates an intracellular signaling pathway from the ER to the nucleus, termed the unfolded protein response. We and others recently identified transcription factor Hac1p/Ern4p responsible for the response in Saccharomyces cerevisiaeand found that Hac1p expression is controlled by the regulated splicing of HAC1 mRNA. Walter and co-workers (Sidrauski, C., Cox, J. S., and Walter, P. (1996) Cell, 87, 405–413) further showed that the splicing requires tRNA ligase but not spliceosome. In this report, we carried out mutational analysis ofHAC1 mRNA and revealed several unique features of the splicing. First, a mutation or deletion of the branchpoint-like sequence present in HAC1 intron did not affect the splicing. Second, cleavage of the splice sites was sequence-specific and thus completely blocked by some point mutations introduced at the 5′ or 3′ splice site. Third, cleavage of the 5′ and 3′ splice sites could occur independently as judged by the nature of splicing intermediates accumulated. Fourth, swapping the nucleotide sequences of the 5′ and 3′ splice sites inhibited the ligation but not the cleavage step. We conclude that signaling from the ER activates putative endonucleases that can carry out sequence-specific cleavage of the splice sites in a random order.

Heat shock factor 2 (HSF2) acquires DNA binding activity during hemin-induced differentiation of human K562 erythroleukemia cells. To investigate the mechanisms responsible for the regulation of HSF2 activity, we searched for proteins that can associate with HSF2 by the yeast two-hybrid system. Nucleoporin p62, a major component of the nuclear pore complex, was cloned from cDNA libraries of K562 cells. We demonstrated physical interaction between HSF2 and p62 both by a glutathione S-transferase (GST) pull-down assay in vitro and by a two-hybrid assay in K562 cells. HSF1 is also able to interact with p62 on a GST pull-down assay, but not on a mammalian two-hybrid system. Furthermore, it was shown that this interaction occurred between the trimerization domain of HSF2 and the C-terminal α-helical coiled-coil domain of p62. These data suggest the possibility that p62 is involved in the activation or regulation of HSF2.

As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons.

We have purified recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) produced in human lymphoblastoid Namalwa cells. From the results of tunicamycin treatment and N-glycosidase F digestion, it was demonstrated that Namalwa-derived hGM-CSF was highly glycosylated at two potential N-glycosylation sites and several O-glycosylation sites as previously shown for naturally occurring hGM-CSF. We classified the hGM-CSF molecules into three groups according to the molecular weight corresponding to the degree of N-glycosylation: the molecules with two N-glycosylation sites occupied (designated 2N), the molecules with either site glycosylated (1N), and the molecules lacking N-glycosylation (0N). Despite such varied degrees of N-glycosylation, almost all molecules were O-glycosylated. To investigate the role of carbohydrate moieties of hGM-CSF, we isolated each form of hGM-CSF and examined its biological properties. The 2N-type showed 200-fold less in vitro specific activity compared with unglycosylated Escherichia coli-derived hGM-CSF, although the activity of the 0N-type was equivalent to that of the E. coli-derived material. The 1N-type showed an intermediate level of activity. However, in terms of clearance from blood circulation in the rat, the 2N-type showed a half-life five times longer than that of the 0N-type and E. coli-derived hGM-CSF. From these findings, we concluded that N-linked carbohydrate moieties of hGM-CSF play conflicting physiological roles in the efficacy of the protein in vivo but that O-linked carbohydrate moieties do not have such effects.

Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18 degrees C), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate, and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

The levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) are controlled by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element (ERSE), the consensus sequence of which is CCAAT-N(9)-CCACG. We recently proposed that ER stress response factor (ERSF) binding to ERSE is a heterologous protein complex consisting of the constitutive component NF-Y (CBF) binding to CCAAT and an inducible component binding to CCACG and identified the basic leucine zipper-type transcription factors ATF6alpha and ATF6beta as inducible components of ERSF. ATF6alpha and ATF6beta produced by ER stress-induced proteolysis bind to CCACG only when CCAAT is bound to NF-Y, a heterotrimer consisting of NF-YA, NF-YB, and NF-YC. Interestingly, the NF-Y and ATF6 binding sites must be separated by a spacer of 9 bp. We describe here the basis for this strict requirement by demonstrating that both ATF6alpha and ATF6beta physically interact with NF-Y trimer via direct binding to the NF-YC subunit. ATF6alpha and ATF6beta bind to the ERSE as a homo- or heterodimer. Furthermore, we showed that ERSF including NF-Y and ATF6alpha and/or beta and capable of binding to ERSE is indeed formed when the cellular UPR is activated. We concluded that ATF6 homo- or heterodimers recognize and bind directly to both the DNA and adjacent protein NF-Y and that this complex formation process is essential for transcriptional induction of ER chaperones.

Dsb proteins (DsbA, DsbB, DsbC, and DsbD) catalyze formation and isomerization of protein disulfide bonds in the periplasm of Escherichia coli . By using a set of Dsb coexpression plasmids constructed recently, we analyzed the effects of Dsb overexpression on production of horseradish peroxidase (HRP) isozyme C that contains complex disulfide bonds and tends to aggregate when produced in E. coli . When transported to the periplasm, HRP was unstable but was markedly stabilized upon simultaneous overexpression of the set of Dsb proteins (DsbABCD). Whereas total HRP production increased severalfold upon overexpression of at least disulfide-bonded isomerase DsbC, maximum transport of HRP to the periplasm seemed to require overexpression of all DsbABCD proteins, suggesting that excess Dsb proteins exert synergistic effects in assisting folding and transport of HRP. Periplasmic production of HRP also increased when calcium, thought to play an essential role in folding of nascent HRP polypeptide, was added to the medium with or without Dsb overexpression. These results suggest that Dsb proteins and calcium play distinct roles in periplasmic production of HRP, presumably through facilitating correct folding. The present Dsb expression plasmids should be useful in assessing and dissecting periplasmic production of proteins that contain multiple disulfide bonds in E. coli .

The chaperonin containing t-complex polypeptide 1 (CCT) is a molecular chaperone assisting in the folding of proteins in eukaryotic cytosol, and the Ccta (encoding the alpha subunit of CCT)/t-complex polypeptide 1 gene encodes the alpha subunit of CCT. We show here that transcription of the mouse Ccta gene is regulated by selenocysteine tRNA gene transcription activating factor (Staf) family zinc-finger transcription factors ZNF143 and ZNF76. Reporter gene assay using HeLa cells indicated that the Ccta gene promoter contains two 18-base pair-long cis-acting elements with similar sequences at -70 and -20 base pairs (designated CCT alpha subunit gene transcription activating element 1 (CAE1) and CAE2, respectively). By yeast one-hybrid screening of CAE1-binding factors, we isolated human ZNF143, which is known to activate transcription of selenocysteine tRNA and small nuclear RNA genes. DNA binding domains of ZNF143 and ZNF76 produced in E. coli recognized CAE1 and CAE2 elements in electrophoretic mobility shift assay. HeLa cell nuclear extract contained a protein that specifically binds to CAE1 and CAE2 and recognized by anti-ZNF143 antibody. Transcription from a minimal Ccta promoter containing CAE2 element in HeLa cells was enhanced by overexpression of full-length ZNF143 and ZNF76 but inhibited by that of their DNA binding domains alone. These results demonstrate that the Staf family proteins control transcription of at least one of the chaperone-encoding genes besides that of tRNA and small nuclear RNA genes. These RNA and chaperone genes are suggested to be coregulated to facilitate synthesis of mature proteins during active cell growth.

The heat-shock response in Escherichia coli depends primarily on the transient increase in the cellular level of heat-shock sigma factor σ 32 encoded by the rpoH gene, which results from both enhanced synthesis and transient stabilization of normally unstable σ 32 . Heat-induced synthesis of σ 32 was previously shown to occur at the translation level by melting the mRNA secondary structure formed within the 5′ coding sequence of rpoH including the translation initiation region. The subsequent decrease in the σ 32 level during the adaptation phase has been thought to involve both shutoff of synthesis (translation) and destabilization of σ 32 -mediated by the DnaK–DnaJ chaperones, although direct evidence for translational repression was lacking. We now show that the heat-induced synthesis of σ 32 does not shut off at the translation level by using a reporter system involving translational coupling. Furthermore, the apparent shutoff was not observed when the synthesis rate was determined by a very short pulse labeling (15 s). Examination of σ 32 stability at 10 min after shift from 30 to 42°C revealed more extreme instability ( t 1/2 =20 s) than had previously been thought. Thus, the dynamic change in σ 32 stability during the heat-shock response largely accounts for the apparent shutoff of σ 32 synthesis observed with a longer pulse. These results suggest a mechanism for maintaining the intricate balance between the antagonistic pathways: the rpoH translation as determined primarily by ambient temperature and the turnover of σ 32 as modulated by the chaperone (and presumably protease)-mediated autogenous control.

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR) according to the needs within the ER. In Saccharomyces cerevisiae , expression of the UPR-specific transcription factor Hac1p is tightly regulated at the level of mRNA splicing that depends on an unconventional system. Thus, HAC1 precursor mRNA is constitutively expressed but not translated. A sensor molecule Ire1p/Ern1p-mediated signaling from the ER specifically removes an intron of 252 nucleotides from the precursor mRNA, and the resulting mature mRNA is translated to produce Hac1p. Because the 5′ splice site is located near the C-terminal end of the Hac1p-coding region, this splicing replaces the last 10 codons of the ORF with an exon encoding 18 aa without affecting the N-terminal 220-aa region which contains the DNA-binding domain. Here, we found that this C-terminal 18-aa segment functions as a potent activation domain. Therefore, the splicing event joins the HAC1 DNA-binding domain to its activation domain, allowing rapid posttranscriptional generation of a potent transcriptional activator (238-aa Hac1p) that activates the UPR efficiently. This suggests that the UPR is hardly activated by Hac1p produced without splicing (230-aa Hac1p) which may occur in the absence of Ire1p/Ern1p-mediated signaling from the ER. Based on these and other results, we propose that the control of expression and activity of Hac1p meets the requirements of the ER.

The chaperonin containing TCP-1 (CCT) is a eukaryotic molecular chaperone consisting of eight subunit species and assists in the folding of cytosolic proteins. We show here that all eight mouse CCT subunit genes contain sequences called heat shock elements for binding heat shock transcription factors (HSFs) by electrophoretic mobility shift assays and that these genes are transcriptionally activated by HSFs in reporter gene assays using HeLa cells transiently overexpressing HSFs. These results suggest that HSF1 and/or HSF2 play a role in Cct gene expression.

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

Escherichia coli mutants lacking activities of all known cytosolic ATP-dependent proteases (Lon, ClpAP, ClpXP, and HslVU), due to double deletions [Δ hslVU and Δ( clpPX-lon )], cannot grow at low (30°C) or very high (45°C) temperatures, unlike those carrying either of the deletions. Such growth defects were particularly marked when the deletions were introduced into strain MG1655 or W3110. To examine the functions of HslVU and other proteases further, revertants that can grow at 30°C were isolated from the multiple-protease mutant and characterized. The revertants were found to carry a suppressor affecting either ftsZ (encoding a key cell division protein) or sulA (encoding the SulA inhibitor, which binds and inhibits FtsZ). Whereas the ftsZ mutations were identical to a mutation known to produce a protein refractory to SulA inhibition, the sulA mutations affected the promoter-operator region, reducing synthesis of SulA. These results suggested that the growth defect of the parental double-deletion mutant at a low temperature was due to the accumulation of excess SulA without DNA-damaging treatment. Consistent with these results, SulA in the double-deletion mutant was much more stable than that in the Δ( clpPX-lon ) mutant, suggesting that SulA can be degraded by HslVU. As expected, purified HslVU protease degraded SulA (fused to the maltose-binding protein) efficiently in an ATP-dependent manner. These results suggest that HslVU as well as Lon participates in the in vivo turnover of SulA and that HslVU becomes essential for growth when the Lon (and Clp) protease level is reduced below a critical threshold.

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Plasmids that can be used for controlled expression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team were constructed in order to facilitate assessment of the effects of these chaperone teams on folding or assembly of recombinant proteins in Escherichia coli . A typical pACYC184-based plasmid which was obtained could express the major DnaK-DnaJ-GrpE and GroEL-GroES chaperone teams from separate promoters when l -arabinose and tetracycline, respectively, were added in a dose-dependent fashion. The model protein used to determine whether this system was useful was an allergen of Japanese cedar pollen, Cryj2, which was unstable when it was produced in E. coli K-12. The effects of chaperone coexpression on the folding, aggregation, and stability of Cryj2 were examined in the wild type and in several mutant bacteria. Coexpression of the DnaK-DnaJ-GrpE and/or GroEL-GroES chaperone team at appropriate levels resulted in marked stabilization and accumulation of Cryj2 without extensive aggregation. Experiments performed with mutants that lack each of the chaperone proteins (DnaK, DnaJ, GrpE, GroEL, and GroES) or heat shock transcription factor ς 32 revealed that both chaperone teams are critically involved in Cryj2 folding but that they are involved in distinct ways. In addition, it was observed that the two chaperone teams have synergistic roles in preventing aggregation of Cryj2 in the absence of ς 32 at certain temperatures.

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions (“ER stress”). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR inSaccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing ofHAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin–proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.

We have cloned a cDNA encoding the human 150 kDa oxygen-regulated protein (ORP150) from hypoxia-treated astrocytoma U373 cDNA library. The deduced amino acid sequence of 999 residues contains a signal peptide and an ER retention-like signal at the N- and C-termini, respectively. It has a striking sequence similarity (91% identity) with Chinese hamster 170 kDa glucose-regulated protein (GRP170). The N-terminal half of ORP150 exhibits significant similarity to the ATPase domain of HSP70 family proteins with well-conserved ATP binding motifs. Northern blot analysis revealed that induction of ORP150 in U373 cells was not limited to hypoxia but also observed by 2-deoxyglucose or tunicamycin treatment. Furthermore, tissue specificity of expression of ORP150 was quite similar to that of GRP78. These findings suggest that ORP150 participates in quality control of proteins in the ER in response to diverse environmental stresses.

The 150-kD oxygen-regulated protein (ORP150) was initially characterized based on its selective expression in astrocytes subjected to oxygen deprivation (Kuwabara, K., M. Matsumoto, J. Ikeda, O. Hori, S. Ogawa, Y. Maeda, K. Kitagawa, N. Imuta, K. Kinoshita, D.M. Stern, et al. 1996. J. Biol. Chem. 279:5025-5032). We have found that exposure of cultured human aortic smooth muscle cells and mononuclear phagocytes (MPs) to hypoxia (pO2 approximately 12-14 torr) induces ORP150 transcripts and production of the antigen, whereas incubation with either hydrogen peroxide, sodium arsenite, heat shock, or 2-deoxyglucose was without effect. Tissue extracts prepared from human atherosclerotic lesions demonstrated expression of ORP150 mRNA and antigen, vs lack of ORP150 in samples from nonatherosclerotic areas. In situ hybridization using ORP150 riboprobes showed the mRNA to be predominantly [correction of predominately] present in macrophages in in atherosclerotic plaques. Furthermore, autoantibody to ORP150 was demonstrated in the serum of patients with severe atherosclerosis, consistent with inducible in vivo expression of ORP150. Introduction of antisense oligonucleotide for ORP150 selectively diminished hypoxia-mediated induction of ORP150 antigen and reduced the viability of hypoxic MPs, especially in the presence of modified (oxidized/acetylated) LDL. In support of a role for ORP150 in the MPs' response to the microenvironment of an atheroma, the presence of oxidized LDL enhanced by approximately 10-fold ORP150 expression in hypoxic cultures. These data indicate that cells of the atherosclerotic vessel wall express ORP150 as part of a protective mechanism, potentially triggered by local hypoxia/hypoxemia and augmented by modified lipoproteins. The presence of antibody to ORP150 in sera of patients with severe atherosclerosis emphasizes the possibility that ORP150 may be a marker of vascular pathology.

Background: Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the transcriptional induction of molecular chaperones and folding enzymes localized in the ER. Thus, eukaryotic cells possess an intracellular signalling pathway from the ER to the nucleus, called the unfolded protein-response (UPR) pathway. In Saccharomyces cerevisiae, such induction is mediated by the cis-acting unfolded protein-response element (UPRE) which has been thought to be recognized by one or more transcription factor(s). Results: Extensive mutational analysis revealed that UPRE contains a partial palindrome with a spacer of one nucleotide (CAGCGTG) that is essential for its function. We then cloned the ERN4 (presumably identical with HAC1) gene using yeast one-hybrid screening, in which the GAL4-ERN4 fusion gene constitutively activates the UPR pathway. The ERN4 gene encodes a basic-leucine zipper protein (Ern4p) that specifically binds to UPRE in vitro and activates transcription in vivo. Cells lacking Ern4p are unable to induce transcription of any of the five target genes tested and exhibit sensitivity to ER stress and inositol requirement for growth. Conclusion: We concluded that Ern4p represents a major component of the putative transcription factor (UPRF) responsible for the UPR leading to the induction of ER-localized stress proteins.

We have achieved high-level expression of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in human lymphoblastoid Namalwa cells by introducing and subsequently amplifying an expression vector encoding human GM-CSF and mouse dihydrofolate reductase (DHFR). Transformants expressing elevated levels of both GM-CSF and DHFR were selected by a step-wise increase in the concentration of methotrexate (MTX). Several cell lines resistant in 800 nM MTX have been established that express GM-CSF at a rate of 10-20 micrograms/10(6) cells/day. The amplified genes are integrated and stably maintained in the chromosomes of these cell lines. Analysis of the GM-CSF produced in the amplified Namalwa cells revealed that the molecular-size distribution due to varied degrees of glycosylation was similar to that observed in the naturally-occurring molecules.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a hetero-oligomeric molecular chaperone that assists in the folding of actin, tubulin, and other cytosolic proteins. We recently reported that the expression level of CCT is closely correlated with growth rates of mammalian cultured cells. Here we examine the levels of CCT subunits and other molecular chaperones in tumor tissues of patients with hepatocelluar and colonic carcinoma, and compare them with nontumor tissues in the same patients. Expression levels of CCTbeta in tumor tissues was significantly higher than in nontumor tissues in all patients with hepatocellular carcinoma (n = 15) and 83% of patients with colonic carcinoma (n = 17). The increased level of CCT expression in colonic cancer cells was confirmed by immunohistochemistry with anti-CCTbeta antibody. The levels of CCTbeta were highly correlated (r = 0.606) with those of the proliferating cell nuclear antigen (PCNA), which was used as an indicator of cell growth. CCTalpha gave similar results, although the correlation with PCNA levels was weaker. Other cytosolic and endoplasmic reticulum chaperones also showed higher expression in significant numbers of tumor tissues but less frequently than that observed with CCT. These results suggest that CCT is up-regulated in rapidly proliferating tumor cells in vivo to effectively produce proteins required for growth, and may serve as a useful tumor marker because it is widely distributed in the cytosol.