Differential proteomic analysis of highly purified placental cytotrophoblasts in pre-eclampsia demonstrates a state of increased oxidative stress and reduced cytotrophoblast antioxidant defense

Proteomics were performed using highly (99.99%) purified cytotrophoblasts from six normal and six pre-eclamptic placentas. Eleven proteins were found which decreased in pre-eclampsia (actin, glutathione S-transferase, peroxiredoxin 6, aldose reductase, heat shock protein 60 (Hsp60), two molecular forms of heat shock protein 70 (Hsp70) β-tubulin, subunit proteasome, ezrin, protein disulfide isomerase, and phosphoglycerate mutase 1). Only one protein, α-2-HS-glycoprotein (fetuin), was found to increase its expression. Western blots of actin, Hsp70, ezrin, and glutatione S-transferase confirmed decrease in protein expression. Many of the proteins that decreased are consistent with a state of oxidative stress in the pre-eclamptic placenta and a decreased cytotrophoblast defense against and response to oxidative stress.     

Comparative genomic analysis of the Hsp70s from five diverse photosynthetic eukaryotes

We have identified 24 members of the DnaK subfamily of heat shock 70 proteins (Hsp70s) in the complete genomes of 5 diverse photosynthetic eukaryotes. The Hsp70s are a ubiquitous protein family that is highly conserved across all domains of life. Eukaryotic Hsp70s are found in a number of subcellular compartments in the cell: cytoplasm, mitochondrion (MT), chloroplast (CP), and endoplasmic reticulum (ER). Although the Hsp70s have been the subject of intense study in model organisms, very little is known of the Hsp70s from early diverging photosynthetic lineages. The sequencing of the complete genomes of Thalassiosira pseudonana (a diatom), Cyanidioschyzon merolae (a red alga), and 3 green algae (Chlamydomonas reinhardtii, Ostreococcus lucimarinus, Ostreococcus tauri) allow us to conduct comparative genomics of the Hsp70s present in these diverse photosynthetic eukaryotes. We have found that the distinct lineages of Hsp70s (MT, CP, ER, and cytoplasmic) each have different evolutionary histories. In general, evolutionary patterns of the mitochondrial and endoplasmic reticulum Hsp70s are relatively stable even among very distantly related organisms. This is not true of the chloroplast Hsp70s and we discuss the distinct evolutionary patterns between "green" and "red" plastids. Finally, we find that, in contrast to the angiosperms Arabidopsis thaliana and Oryza sativa that have numerous cytoplasmic Hsp70, the 5 algal species have only 1 cytoplasmic Hsp70 each. The evolutionary and functional implications of these differences are discussed.     

Hsp70 promotes epithelial sodium channel functional expression by increasing its association with coat complex II and its exit from endoplasmic reticulum

The epithelial sodium channel (ENaC) plays an important role in the homeostasis of blood pressure and of the airway surface liquid, and inappropriate regulation of ENaC results in refractory hypertension (in Liddle syndrome) and impaired mucociliary clearance (in cystic fibrosis). The regulation of ENaC by molecular chaperones, such as the 70-kDa heat shock protein Hsp70, is not completely understood. Building on the previous suggestion by our group that Hsp70 promotes ENaC functional and surface expression in Xenopus oocytes, we investigated the mechanism by which Hsp70 acts upon ENaC in epithelial cells. In Madin-Darby canine kidney cells stably expressing epitope-tagged αβγ-ENaC and with tetracycline-inducible overexpression of Hsp70, treatment with 1 or 2 μg/ml doxycycline increased total Hsp70 expression ~2-fold and ENaC functional expression ~1.4-fold. This increase in ENaC functional expression corresponded to an increase in ENaC expression at the apical surface of the cells and was not present when an ATPase-deficient Hsp70 was similarly overexpressed. The increase in functional expression was not due to a change in the rate at which ENaC was retrieved from the apical membrane. Instead, Hsp70 overexpression increased the association of ENaC with the Sec24D cargo recognition component of coat complex II, which carries protein cargo from the endoplasmic reticulum to the Golgi. These data support the hypothesis that Hsp70 promotes ENaC biogenesis and trafficking to the apical surface of epithelial cells.     

Soluble and membrane-associated heat shock proteins in soybean root

Summary Localization of heat shock proteins (Hsp) in endomembranes and determination of whether they are integral or peripheral membrane proteins will aid in understanding the physiological function of the heat shock response. Radiolabeled endomembranes (endoplasmic reticulum, Golgi, and plasma membrane), obtained by sucrose gradient centrifugation of heat-shocked soybean (Glycine max L.) root tissue were solubilized and the polypeptides separated by two-dimensional IEF-SDS-PAGE. Autoradiography revealed three groups of Hsp. A diverse group fo 25 low mol wt Hsp (18 to 24 kDa) with isoelectric point (pI) between 5 and 7; an intermediate mol wt group (30 to 47 kDa) with pI of 5.5 to 6.0; and a group of two high mol wt Hsp (75 to 80 kDa) with pI 4.8 to 5.2. The plasma membrane fraction lacked the Hsp pair of 47 kDa detected in the endoplasmic reticulum and Golgi fractions but possessed a unique Hsp of 30 kDa, pI 5.5.Comparison of soluble and microsome fractions revealed a difference in the pattern of the low mol wt Hsp class. The soluble fraction contained Hsp of 16–20 kDa with pI between 5 and 7.8 while the microsome fraction was characterized by Hsp of 18–24 kDa with pI between 5.8 and 6.5.The microsomal Hsp were not released by 1 M KCl. Treatment of the microsome fraction with Triton X-100 selectively released several Hsp, and Na₂CO₃ treatment removed additional Hsp from the membrane fraction.Abbreviations Hsp heat-shock protein(s) - GA Golgi apparatus - PM plasma membrane - 2 D two-dimensional     

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.     

Meta-analysis of heat- and chemically upregulated chaperone genes in plant and human cells

Molecular chaperones are central to cellular protein homeostasis. In mammals, protein misfolding diseases and aging cause inflammation and progressive tissue loss, in correlation with the accumulation of toxic protein aggregates and the defective expression of chaperone genes. Bacteria and non-diseased, non-aged eukaryotic cells effectively respond to heat shock by inducing the accumulation of heat-shock proteins (HSPs), many of which molecular chaperones involved in protein homeostasis, in reducing stress damages and promoting cellular recovery and thermotolerance. We performed a meta-analysis of published microarray data and compared expression profiles of HSP genes from mammalian and plant cells in response to heat or isothermal treatments with drugs. The differences and overlaps between HSP and chaperone genes were analyzed, and expression patterns were clustered and organized in a network. HSPs and chaperones only partly overlapped. Heat-shock induced a subset of chaperones primarily targeted to the cytoplasm and organelles but not to the endoplasmic reticulum, which organized into a network with a central core of Hsp90s, Hsp70s, and sHSPs. Heat was best mimicked by isothermal treatments with Hsp90 inhibitors, whereas less toxic drugs, some of which non-steroidal anti-inflammatory drugs, weakly expressed different subsets of Hsp chaperones. This type of analysis may uncover new HSP-inducing drugs to improve protein homeostasis in misfolding and aging diseases.     

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.     

Causal links between protein folding in the ER and events along the secretory pathway

The 70-kDa heat shock protein (Hsp70) family comprises the most abundant and important group of molecular chaperones. Hsp70s cooperate with a number of cofactors, which define their functions. We recently reported that a yeast protein, Rot1, is a putative cofactor of BiP, an endoplasmic reticulum (ER)-localized Hsp70. Rot1 is an essential ER membrane protein and may be involved in protein folding. Mutation of the ROT1 gene caused defects in cell wall synthesis and lysis of autophagic bodies. We suggest that Rot1 is required for folding of proteins engaged in these cellular processes.     

BiP and PDI cooperate in the oxidative folding of antibodies in vitro

Immunoglobulin heavy chain binding protein (BiP), a member of the Hsp70 chaperone family, and the oxidoreductase protein-disulfide isomerase (PDI) play an important role in the folding and oxidation of proteins in the endoplasmic reticulum. However, it was not clear whether both cooperate in this process. We show here that BiP and PDI act synergistically in the in vitro folding of the denatured and reduced Fab fragment. Several ATP-dependent cycles of binding, release, and rebinding of the unfolded antibody chains by BiP are required for efficient reactivation. Our data suggest that in the absence of BiP unfolded antibody chains collapse rapidly upon refolding, rendering cysteine side chains inaccessible for PDI. BiP binds the unfolded polypeptide chains and keeps them in a conformation in which the cysteine residues are accessible for PDI. These findings support the idea of a network of folding helper proteins in the endoplasmic reticulum, which makes this organelle a dedicated protein-processing compartment.     

A complex of chaperones and disulfide isomerases occludes the cytosolic face of the translocation protein Sec61p and affects translocation of the prion protein

Secretion of newly synthesized proteins across the mammalian rough endoplasmic reticulum (translocation) is supported by the membrane proteins Sec61p and TRAM, but may also include accessory factors, depending on the particular translocation substrate. Studies designed to investigate the binding of anti-peptide antibodies to the carboxyl terminus of the alpha-subunit of Sec61 (Sec61palpha) lead us to the isolation of a complex of proteins that occlude the cytosolic face of Sec61palpha in microsomes that have been prepared by standard protocols used to study translocation in vitro [Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93]. This complex was shown by nanospray tandem mass spectrometry to be composed of protein disulfide isomerase (PDI), calcium binding protein 1 (CABP1/P5), 72 kDa endoplasmic reticulum protein (ERp72), and BiP (heat shock protein A5/HSPA5), and has been named TR-PDI for "translocon-resident protein disulfide isomerase complex". This constitutes a novel location for these proteins, which are known to be major constituents of the lumen of the rough endoplasmic reticulum. We have not established the function of TR-PDI at this location, but did observe that the absence of this complex results in a relative loss of correct topology of prion protein insertion across RER membranes, indicating the possibility of a functional role in vivo.     

A lysine-rich region within fungal BAG domain-containing proteins mediates a novel association with ribosomes

Heat shock protein 70 (Hsp70) is a highly conserved molecular chaperone that assists in the folding of nascent chains and the repair of unfolded proteins through iterative cycles of ATP binding, hydrolysis, and nucleotide exchange tightly coupled to polypeptide binding and release. Cochaperones, including nucleotide exchange factors (NEFs), modulate the rate of ADP/ATP exchange and serve to recruit Hsp70 to distinct processes or locations. Among three nonrelated cytosolic NEFs in Saccharomyces cerevisiae, the Bag-1 homolog SNL1 is unique in being tethered to the endoplasmic reticulum (ER) membrane. We demonstrate here a novel physical association between Snl1 and the intact ribosome. This interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. Additionally, we demonstrate a ribosome association with the Candida albicans Snl1 homolog and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. We therefore propose that the Snl1 family of NEFs serves a previously unknown role in fungal protein biogenesis based on the coincident recruitment of ribosomes and Hsp70 to the ER membrane.     

The heat shock protein 70 family: Highly homologous proteins with overlapping and distinct functions

The human heat shock protein 70 (Hsp70) family contains at least eight homologous chaperone proteins. Endoplasmatic reticulum and mitochondria have their specific Hsp70 proteins, whereas the remaining six family members reside mainly in the cytosol and nucleus. The requirement for multiple highly homologous although different Hsp70 proteins is still far from clear, but their individual and tissue-specific expression suggests that they are assigned distinct biological tasks. This concept is supported by the fact that mice knockout for different Hsp70 genes display remarkably discrete phenotypes. Moreover, emerging data suggest that individual Hsp70 proteins can bring about non-overlapping and chaperone-independent functions essential for growth and survival of cancer cells. This review summarizes our present knowledge of the individual members of human Hsp70 family and elaborate on the functional differences between the cytosolic/nuclear representatives.     

二硫键异构酶

天然二硫键的形成是许多蛋白正确折叠中的限速步骤,在稳定蛋白质构象和保持蛋白质活性方面起重要作用。讨论的二硫键异构酶是内质网中一种重要的蛋白折叠催化剂,它催化蛋白二硫键的形成和错误配对二硫键的重排,并有抑制错误折叠蛋白聚集的分子伴侣活性。PDI广泛应用于基因工程上提高外源蛋白表达水平。     

Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.