The ATPase domain of hsp70 possesses a unique binding specificity for 3'-sulfogalactolipids

The region(s) of hsp70 critical for sulfogalactolipid (SGL) recognition has been defined through deletion analysis and site-directed mutagenesis. Truncated polymerase chain reaction products of hsp70 generated N-terminal fragments of 43, 35, 29, and 22 kDa. The C terminus substrate-binding domain (28 kDa) was also expressed. The N-terminal ATPase domain (rP43) shared the binding specificity of hsp70, because only sulfogalactosyl ceramide and sulfogalactosyl glycerolipid were recognized by both TLC overlay and RELISA. The C-terminal domain showed no binding. SGL binding of rP29 and rP22 was severely reduced. The loss of SGL binding for rP35 by RELISA but not TLC overlay was considered as a function of receptor presentation. The truncation of rP43 to rP35 demonstrates that residues 318-387 (the base of the ATP binding cleft) are critical for high affinity SGL binding. Mutagenesis showed that Arg(342) and Phe(198) are crucial for this process. SGL binding, mediated by these conserved residues within the ATPase domain of hsp70, implies that this binding specificity is evolutionarily conserved.     

The aglycone of sulfogalactolipids can alter the sulfate ester substitution position required for hsc70 recognition

3'-Sulfogalactolipids(SGLs), sulfogalactosyl ceramide (SGC), and sulfogalactoglycerolipid (SGG) bind to the N-terminal ATPase-containing domain of members of the heat shock protein 70 family. We have probed this binding specificity using a series of synthetic positional sulfated or phosphorylated glycolipid analogues, containing either a long-chain bisalkyl hydrocarbon-2-(tetradecyl)hexadecane (B30) or C(18) ceramide (SGC(18)) backbone. By TLC overlay and receptor ELISA, recombinant hsc70 bound ceramide-based glycoconjugates having 3'- or 4'-sulfogalactose glycone moieties and the 4'-sulfogalactose positional isomer conjugated to B30. Hsc70 binding was significantly decreased to the 3'-sulfogalactose conjugated to the long-chain branched alkane. 3'-Sulfoglucose conjugated to B30 was not bound, nor were similarly conjugated di-, tri-, and tetra-sulfated or phosphorylated galactolipids. These results highlight the importance of the position, rather than the number of sulfate esters within the galactose ring. This binding selectivity was shared by the sea urchin hsp70-related sperm receptor. A 3'-SGC-based soluble inhibitor, in which the acyl chain was replaced with an adamantyl group, inhibited binding of hsc70 to both 3'- and 4'-SGC species with an IC(50) of 50 and 75 microM, respectively, indicating a shared sulfogalactose binding site. These studies demonstrate the highly specific nature of hsc70/SGL binding and show, for the first time, that the lipid aglycone can alter the substitution position requirement for glycolipid recognition.     

3'Sulfogalactolipid binding specifically inhibits Hsp70 ATPase activity in vitro

A method for the generation of soluble glycosphingolipid derivatives that retain the receptor activity of the parent (BBRC 257:391-394, Carb Res 335:91-100) was used to investigate the consequence of 3'sulfogalactolipid (SGL) specific binding within the N-terminal ATPase-containing domain of Hsc70. Sulfogalactosyl ceramide (SGC) was deacylated, and the resulting sulfogalactosylsphingosine coupled to an alpha-adamantane or a norbornane rigid hydrophobic frame. The resulting conjugate preferentially partitioned into water, as opposed to organic solvent. In the range of 100-300 microM, these conjugates inhibited the specific binding of bovine brain Hsc70 to immobilized SGLs. A similar dose-related inhibition of bovine brain Hsc70 ATPase activity was seen between 100 and 300 microM adamantylSGC (adaSGC). Adamantyl conjugates of glycolipids not bound by Hsp70s had no effect. Kinetic analysis indicated that adaSGC was a noncompetitive inhibitor of Hsc70 ATPase activity, a special case of mixed inhibition since the K(m) values were not statistically different, 0.89 +/- 0.024 microM to 0.93 +/- 0.038 microM, but the V(max) decreased from 0.20 +/- 0.012 pmol min(-1) microg(-1) to 0.15 +/- 0.016 pmol min(-1) microg(-1). A reproducible 5 min lag was observed prior to ATPase inhibition that could be eliminated by preincubation of adaSGC with Hsc70 or by adding the cochaperone Hdj-1. The dependence of ATPase inhibition on the rate of hydrolysis indicates that adaSGC binding occurs at a specific stage of the ATPase cycle. These studies identify a new mechanism for the regulation of Hsp70 ATPase activity.     

Studies on the binding of bacteria to glycolipids. Two species of Propionibacterium apparently recognize separate epitopes on lactose of lactosylceramide

Two species of Propionibacterium were analysed regarding their binding to glycosphingolipids. Bacteria were labeled with 125I and selective interaction with glycolipids on thin-layer chromatograms was revealed by autoradiography. The carbohydrate site in common for active molecular species appeared to be lactose. The two bacteria differed, however, in the overall binding pattern on the chromatogram, probably due to recognition of separate epitopes on lactose. P. freudenreichii bound only to lactosylceramide while P. granulosum also recognized substituted lactosylceramide: Gal alpha 1----3Gal beta 1----4Glc beta Cer, GlcNAc beta 1----3Gal beta 1----4Glc beta Cer and Gal beta 1----3GlcNAc beta 1----3Gal beta 1----4Glc beta Cer were active, but Gal-alpha 1----4Gal beta 1----4Glc beta Cer was inactive. Also, there was an interesting dependence on ceramide structure in the case of lactosylceramide. P. freudenreichii bound to lactosylceramide with sphingosine and non-hydroxy fatty acids but not to species with sphingosine and 2-hydroxy fatty acids, phytosphingosine and non-hydroxy fatty acids or phytosphingosine and 2-hydroxy fatty acids. For P. granulosum the situation was reversed. This may be explained by an influence of ceramide structure on the presentation of the two lactose epitopes at the assay surface. These results were supported by curves from the binding of labeled bacteria to glycolipids coated in microtiter wells and in part by binding to glycolipid-coated chicken erythrocytes.     

Ceramide composition of the psoriatic scale

This paper investigates the ceramide composition of the psoriatic scale compared with that of normal human SC. A method was optimalized, based on TLC separation followed by densitometry, allowing the provision of good resolution and quantification of ceramide fractions from both normal and pathological specimens. Seven ceramide fractions were isolated and submitted to compositional analysis. The obtained results suggested a revisitation of previous ceramide designation. Therefore a simple classification is suggested, based on grouping ceramides carrying structural similarities under common codes. According to these rules, ceramides were grouped into five classes designated as: (1) Cer[EOS], which contains ester-linked fatty acids, ω-OH fatty acids and sphingosines; (2) Cer[NS], which contains non-OH fatty acids and sphingosines; (3) Cer[NP], which contains non-OH fatty acids and phytosphingosines; (4) Cer[AS], which contains α-OH fatty acids and sphingosines; (5) Cer[AP], which contains α-OH fatty acids and phytosphingosines. Analysis of ceramides from the psoriatic scale, compared to those from normal human SC, resulted in an impairment of the Cer[EOS] content as well as of the ceramides containing phytosphingosine, with concurrent increase in ceramides containing sphingosine, being the total amount maintained identical. Since one of the suggested pathways for phytosphingosine biosynthesis involves the water addition to the corresponding sphingosine double bond, we can speculate that the observed alterarion is due to a deranged water bioavailability, associated with psoriaris.     

Molecular evolution of the HSP70 multigene family

Eukaryotic genomes encode multiple 70-kDa heat-shock proteins (HSP70s). The Saccharomyces cerevisiae HSP70 family is comprised of eight members. Here we present the nucleotide sequence of the SSA3 and SSB2 genes, completing the nucleotide sequence data for the yeast HSP70 family. We have analyzed these yeast sequences as well as 29 HSP70s from 24 additional eukaryotic and prokaryotic species. Comparison of the sequences demonstrates the extreme conservation of HSP70s; proteins from the most distantly related species share at least 45% identity and more than one-sixth of the amino acids are identical in the aligned region (567 amino acids) among all proteins analyzed. Phylogenetic trees constructed by two independent methods indicate that ancient molecular and cellular events have given rise to at least four monophyletic groups of eukaryotic HSP70 proteins. Each group of evolutionarily similar HSP70s shares a common intracellular localization and is presumed to be comprised of functional homologues; these include heat-shock proteins of the cytoplasm, endoplasmic reticulum, mitochondria, and chloroplasts. HSP70s localized in mitochondria and plastids are most similar to the DnaK HSP70 homologues in purple bacteria and cyanobacteria, respectively, which is consistent with the proposed prokaryotic origin of these organelles. The analyses indicate that the major eukaryotic HSP70 groups arose prior to the divergence of the earliest eukaryotes, roughly 2 billion years ago. In some cases, as exemplified by the SSA genes encoding the cytoplasmic HSP70s of S. cerevisiae, more recent duplication events have given rise to subfamilies within the major groups. The S. cerevisiae SSB proteins comprise a unique subfamily not identified in other species to date. This subfamily appears to have resulted from an ancient gene duplication that occurred at approximately the same time as the origin of the major eukaryotic HSP70 groups. Correspondence to: E.A. Craig     

A novel hsp70-like protein (P70) is present in mouse spermatogenic cells.

Mouse spermatogenic cells contain relatively large amounts of a 70-kilodalton protein (P70) that is closely related to hsp70, the major inducible heat shock protein. When hsp70 from spermatogenic cells is heat induced, it migrates to the same location as does P70 on two-dimensional polyacrylamide gels, indicating that it has an apparently identical mass and isoelectric point. P70 reacts strongly and specifically with an anti-Drosophila hsp70 monoclonal antibody that is specific for products of the hsp70 gene family. Both P70 and hsp70 are also ATP-binding proteins and are purified by using ATP-affinity chromatography. However, P70 and hsp70 are unique proteins on the basis of peptide map analysis and are regulated differently in germ cells. P70 appears to be a novel heat shock protein of spermatogenic cells which is synthesized in association with germ cell differentiation.     

Sphingolipid Content of Human Adipose Tissue: Relationship to Adiponectin and Insulin Resistance

Ceramides (Cer) are implicated in obesity‐associated skeletal muscle and perhaps adipocyte insulin resistance. We examined whether the sphingolipid content of human subcutaneous adipose tissue and plasma varies by obesity and sex as well as the relationship between ceramide content and metabolic indices. Abdominal subcutaneous adipose biopsies were performed on 12 lean adults (males = 6), 12 obese adults (males = 6) for measurement of sphingolipid content and activity of the main ceramide metabolism enzymes. Blood was sampled for glucose, insulin (to calculate homeostasis model assessment‐estimated insulin resistance (HOMA_IR)) adiponectin, and interleukin‐6 (IL‐6) concentrations. Compared to lean controls, total ceramide content (pg/adipocyte) was increased by 31% (P < 0.05) and 34% (P < 0.05) in obese females and males, respectively. In adipocytes from obese adults sphingosine, sphinganine, sphingosine‐1‐phosphate, C14‐Cer, C16‐Cer, and C24‐Cer were all increased. C18:1‐Cer was increased in obese males and C24:1‐Cer in obese females. For women only, there was a negative correlation between C16‐Cer ceramide and plasma adiponectin (r = ?0.77, P = 0.003) and a positive correlation between total ceramide content and HOMA_IR (r = 0.74, P = 0.006). For men only there were significant (at least P < 0.05), positive correlations between adipocyte Cer‐containing saturated fatty acid and plasma IL‐6 concentration. We conclude that the sexual dimorphism in adipose tissue behavior in humans extends to adipose tissue sphingolipid content its association with adiponectin, IL‐6 and insulin resistance.     

Hsp 70 expression and function during gametogenesis

The dramatic transformations in nuclear content and cellular organization that occur during gametogenesis require unique regulation and execution of the mitotic and meiotic cell cycle, apoptotic cell death, DNA recombination and repair, and cellular differentiation. These processes are accompained by the constitutive and developmentally regulated expression of a number of hsp70 genes encoding 70 kDa heat shock proteins (Hsp70), including several hsp70s whose expression is unique to male germ cells. Examining the expression and function of Hsp70s in germ cells has provided significant insights into mechanisms of hsp70 gene regulation and Hsp70 protein function, as well as the developmental processes of gametogenesis.     

Oxidation of glycosphingolipids under basic conditions: synthesis of glycosyl "serine acids" as opposed to "ceramide acids". Precursors for neoglycoconjugates with increased ligand binding affinity.

Two types of oxidative cleavage of the double bond of glycosphingolipids (GSLs) are described. Oxidation of peracetylated GSL precursors with stoichiometric proportions of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system, gave nearly quantitative yields of the glycosyl ceramide acid, 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)oxybutyric acid [Mylvaganam, M., and Lingwood, C. A. (1999) J. Biol. Chem. 274, 20725-20732]. However, if the reaction medium was made alkaline, the hydroxyallylic function of the sphingolipid, as a whole, was oxidized and the glycosyl serine acid, 2-(N-acyl)-3-(O-glycosyl)oxypropionic acid, was obtained in good yield. This represents a new type of oxidation reaction. Optimized conditions gave glycosyl ceramide or serine acids with greater than 90% selectivity and in good yields (90%). Oxidation of dGSLs gave serine and ceramide oligosaccharides, devoid of hydrocarbon chains. An intriguing glycosyl species containing 5-hydroxy-4-oxo-3-hydroxy-2-(N-acyl)sphingosine (hydroxy-acyl intermediate) was identified via ESMS analyses. We propose that further oxidation of this intermediate is pH-dependent and will be oxidized to either serine or ceramide acids. On the basis of MS-MS analysis of specific homologues of serine and ceramide acids, two types of collision-induced dissociation (CID) patterns have been established. These CID patterns were then used in the identification of serine and ceramide acids synthesized from natural GSL samples. Also, on a qualitative basis, this oxidation protocol, in conjunction with ESMS, provides a novel method for characterizing the aglycone composition (acyl chain length, unsaturation position, dihydrosphingosine content, etc.) of natural GSLs. A novel class of neohydrocarbon conjugates were synthesized by coupling the acids to rigid hydrocarbon frames such as 2-aminoadamantane. Preliminary studies with conjugates derived from globotriaosyl ceramide (Gb3C), lactosyl ceramide (LC), and galactosyl ceramide (GalC) bound verotoxin with the expected specificity but with affinities much greater than that of the natural glycolipid. Also, the ceramide acid-based conjugates were better ligands than serine acid conjugates.     

Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2

In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(1), an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.     

Essential roles of 70kDa heat inducible proteins

The 70 kDa heat inducible proteins (hsp70s) are a highly conserved family of proteins found in every organism examined. Some hsp70 proteins are essential for cell viability. Recent work has revealed that these proteins are involved in the movement of proteins into and through various compartments of the eukaryotic cell.     

A human gene family with sequence homology to Drosophila melanogaster Hsp70 heat shock genes

A growing literature describes the structure and regulation of prokaryotic and eukaryotic heat shock genes. We here report the isolation of several members of a human heat shock protein 70 (hsp 70) multigene family which contains at least 10 different genes and/or pseudogenes exhibiting sequence homology to the hsp70 gene of Drosophila melanogaster. Eight nonoverlapping recombinant lambda phages from a lambda-Charon4A human genomic library were studied by restriction mapping. One lambda clone was sequenced and characterized as a hsp70 pseudogene inserted into a rearranged human HindIII 1.9-kilobase repeated DNA sequence. This pseudogene is probably located on the X chromosome. Its predicted amino acid sequence shows extensive homology to those of Drosophila hsp70, trout hsp70, Xenopus hsp70, yeast hsp70, and some homology to the heat-inducible dnaK gene product of Escherichia coli. Amino acid homology is clustered, suggesting evolutionary conservation of domains critical to the function of this protein in both prokaryotic and eukaryotic cells.     

Glycosphingolipids in cestodes. Chemical structures of ceramide monosaccharide, disaccharide, trisaccharide and tetrasaccharide from metacestodes of the fox tapeworm, Taenia crassiceps (Cestoda: Cyclophyllidea).

The presence of glycosphingolipids in the metacestodes of the fox tapeworm, Taenia crassiceps, has been established. The normal-phase TLC pattern of the neutral-fraction glycolipids revealed groups of bands corresponding to homologous components of increasing sugar chain length. The three simplest glycolipid components have been isolated and their chemical constitution determined as being of the neogala series: Gal beta 1Cer, Gal beta 6Gal beta 1Cer and Gal beta 6Gal beta 6Gal beta 1Cer. The ceramide tetrasaccharide fraction has been found to consist of a mixture of neogalatetraosylceramide, as an elongation of the neogala series, Gal beta 6Gal beta 6Gal beta 6Gal beta 1Cer and the component Gal alpha 4Gal beta 6Gal beta 6Gal beta 1Cer (both occurring in approximately equimolar proportions). The long-chain bases of the ceramide monogalactoside, digalactoside, trigalactoside and tetragalactosides contain, as well as small amounts of sphingosine, predominantly dihydrosphingosine/phytosphingosine in the approximate ratios 1.7:1, 1.4:1, 1:1 and 2.3:1, respectively. The major ceramide fatty acids have particularly long chains, with hexacosanoic and octacosanoic acids predominating. Upon reverse-phase TCL, the glycolipid components ceramide monogalactoside, digalactoside and trigalactoside were each separable into five component bands. Parent glycolipid components therefore show component band distributions comparable to one another in being governed by similar ceramide constitutions.     

Mitochondrial GrpE is present in a complex with hsp70 and preproteins in transit across membranes.

We characterized a 24-kDa protein associated with matrix hsp70 (mt-hsp70) of Neurospora crassa and Saccharomyces cerevisiae mitochondria. By using specific antibodies, the protein was identified as MGE, a mitochondrial homolog of the prokaryotic heat shock protein GrpE. MGE extracted from mitochondria was quantitatively bound to hsp70. It was efficiently released from hsp70 by the addition of Mg-ATP but not by nonhydrolyzable ATP analogs or high salt. A mutant mt-hsp70, which was impaired in release of bound precursor proteins, released MGE in an ATP-dependent manner, indicating that precursor proteins and MGE bind to different sites of hsp70. A preprotein accumulated in transit across the mitochondrial membranes was specifically coprecipitated by either antibodies directed against MGE or antibodies directed against mt-hsp70. The preprotein accumulated at the outer membrane was not coprecipitated by either antibody preparation. After being imported into the matrix, the preprotein could be coprecipitated only by antibodies against mt-hsp70. We propose that mt-hsp70 and MGE cooperate in membrane translocation of preproteins.     

Cer1p Functions as a Molecular Chaperone in the Endoplasmic Reticulum of Saccharomyces cerevisiae

Cer1p/Lhs1p/Ssi1p is a novel Hsp70-related protein that is important for the translocation of a subset of proteins into the yeast Saccharomyces cerevisiae endoplasmic reticulum. Cer1p has very limited amino acid identity to the hsp70 chaperone family in the N-terminal ATPase domain but lacks homology to the highly conserved hsp70 peptide binding domain. The role of Cer1p in protein folding and translocation was assessed. Deletion of CER1 slowed the folding of reduced pro-carboxypeptidase Y (pro-CPY) approximately twofold in yeast. In wild-type yeast under reducing conditions, pro-CPY can be found in a complex with Cer1p, while partially purified Cer1p is able to bind directly to peptides. Together, this suggests that Cer1p has a chaperoning activity required for proper refolding of denatured pro-CPY which is mediated by direct interaction with the unfolded polypeptide. Cer1p peptide binding and oligomerization could be disrupted by addition of ATP, confirming that Cer1p possesses a functional ATP binding site, much like Kar2p and other members of the hsp70 family. Interestingly, replacing the signal sequence of a CER1-dependent protein with that of a CER1-independent protein did not relieve the requirement of CER1 for import. This result suggests that an interaction with the mature portion of the protein also is important for the translocation role of Cer1p. The CER1 RNA levels increase at lower temperatures. In addition, the effects of deletion on folding and translocation are more severe at lower temperatures. Therefore, these results suggest that Cer1p provides an additional chaperoning activity in processes known to require Kar2p. However, there appears to be a greater requirement for Cer1p chaperone activity at lower temperatures.     

Ceramide metabolism is affected by obesity and diabetes in human adipose tissue

Ceramide is involved in development of insulin resistance. However, there are no data on ceramide metabolism in human adipose tissue. The aim of our study was to examine sphingolipid metabolism in fat tissue from obese nondiabetic (n = 11), obese diabetic (n = 11), and lean nondiabetic (n = 8) subjects. The content of ceramide (Cer), dihydroceramide (dhCer), sphingosine (SPH), sphinganine (SPA), sphingosine‐1‐phosphate (S1P; pmol/mg of protein), the expression (mRNA) and activity of key enzymes responsible for Cer metabolism: serine palmitoyltransferase (SPT), neutral and acidic sphingomyelinase (nSMase and aSMase, respectively), and neutral and acidic ceramidase (nCDase and aCDase, respectively) were examined in human adipose tissue. The contents of SPA and Cer were significantly lower whereas the content of dhCer was higher in both obese groups than the respective values in the lean subjects. The expression of examined enzymes was elevated in both obese groups. The SPT and CDases activity increased whereas aSMase activity deceased in both obese groups. We have found correlation between adipose tissue Cer content and plasma adiponectin concentration (r = 0.69, P < 0.001) and negative correlation between total Cer content and HOMA‐IR index (homeostasis model of insulin resistance) (r = ?0.67, P < 0.001). We have found that both obesity and diabetes affected pathways of sphingolipid metabolism in the adipose tissue. J. Cell. Physiol. 227: 550–557, 2012. © 2011 Wiley Periodicals, Inc.     

Evidence that heat shock protein-70 associated with progesterone receptors is not involved in receptor-DNA binding.

In the absence of hormone, human progesterone receptors (PR) are recovered in the cytosolic fraction of cell lysates as a multimeric complex containing the steroid-binding polypeptide, heat shock protein-90 (hsp90), and heat shock protein-70 (hsp70). Activated forms of human PR that acquire the ability to bind to DNA are dissociated from hsp90, but retain association with hsp70. The present study has examined whether associated hsp70 has a function in receptor-DNA binding. When activated PR was bound to specific target DNA in a gel shift assay, no hsp70 was detectable in the PR-DNA complex, as evidenced by the failure of several antibodies to hsp70 to affect the mobility or the amount of complexes. To determine whether hsp70 might indirectly influence DNA-binding activity, we have examined the effect of hsp70 dissociation on PR-DNA-binding activity. Dissociation was achieved either by treatment of immunoaffinity-purified immobilized PR complexes with ATP or by the binding of PR complexes to ATP-agarose, followed by elution with high salt. Under both conditions, dissociation from hsp70 neither enhanced nor impaired the ability of PR to bind to specific DNA. These results suggest that hsp70 is not involved in PR binding to DNA, either directly by participating in DNA binding or indirectly by modulating PR-DNA-binding activity. This implies that hsp70 functions at an earlier stage in the receptor activation pathway. Consistent with the known involvement of hsp70 in stabilizing unfolded states of other target proteins, we propose that hsp70 may assist in nuclear transport of PR or in assembly-disassembly of the 8-10S multimeric complex.     

嗜热四膜虫五个hsp70基因的表达分析

鉴定得到嗜热四膜虫13个含有完整保守结构域的hsp70基因,对其中5个高度相似且无内含子的hsp70基因进行表达分析。在37、39和41℃热激条件下,实时荧光定量PCR结果表明,hsp70-2基因对热激响应最敏感。在四膜虫生长、饥饿和接合生殖这3种生理或发育状态下,Microarray结果显示,hsp70-4基因恒定且高表达;在热激条件下,hsp70-4基因的表达水平随着温度的升高而略微增加,证实hsp70-4基因为热休克相关蛋白hsc70基因;克隆的hsp70-4基因全长2208bp,开放阅读框长1959bp,编码653个氨基酸。Microarray结果提示,hsp70-3可能参与四膜虫饥饿早期(0～12h)的耐受和接合生殖后期(6～10h)的新大小核形成,老大核凋亡等事件;hsp70-5可能参与四膜虫饥饿晚期(12～15h)的耐受和接合生殖早期(0～6h)的小核减数分裂、小核交换和原核(pronuclear)融合事件。Blast2GO分析表明,与hsp70-3和hsp70-5共表达的基因分别参与不同的生物学过程,进一步反映了hsp70-3和hsp70-5这两个基因在功能上是存在差异的。     

hsp70 is not required for high affinity binding of purified calf uterine estrogen receptor to estrogen response element DNA in Vitro

Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg²⁺, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [³H]estradiol (E₂) or [³H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.