Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ

Chaperones of the Hsp70 family bind to unfolded or partially folded polypeptides to facilitate many cellular processes. ATP hydrolysis and substrate binding, the two key molecular activities of this chaperone, are modulated by the cochaperone DnaJ. By using both genetic and biochemical approaches, we provide evidence that DnaJ binds to at least two sites on the Escherichia coli Hsp70 family member DnaK: under the ATPase domain in a cleft between its two subdomains and at or near the pocket of substrate binding. The lower cleft of the ATPase domain is defined as a binding pocket for the J-domain because (i) a DnaK mutation located in this cleft (R167H) is an allele-specific suppressor of the binding defect of the DnaJ mutation, D35N and (ii) alanine substitution of two residues close to R167 in the crystal structure, N170A and T173A, significantly decrease DnaJ binding. A second binding determinant is likely to be in the substrate-binding domain because some DnaK mutations in the vicinity of the substrate-binding pocket are defective in either the affinity (G400D, G539D) or rate (D526N) of both peptide and DnaJ binding to DnaK. Binding of DnaJ may propagate conformational changes to the nearby ATPase catalytic center and substrate-binding sites as well as facilitate communication between these two domains to alter the molecular properties of Hsp70.     

Control of the DnaK chaperone cycle by substoichiometric concentrations of the co-chaperones DnaJ and GrpE

The polypeptide binding and release cycle of the molecular chaperone DnaK (Hsp70) of Escherichia coli is regulated by the two co-chaperones DnaJ and GrpE. Here, we show that the DnaJ-triggered conversion of DnaK.ATP (T state) to DnaK.ADP.Pi (R state), as monitored by intrinsic protein fluorescence, is monophasic and occurs simultaneously with ATP hydrolysis. This is in contrast with the T-->R conversion in the absence of DnaJ which is biphasic, the first phase occurring simultaneously with the hydrolysis of ATP (Theyssen, H., Schuster, H.-P., Packschies, L., Bukau, B., and Reinstein, J. (1996) J. Mol. Biol. 263, 657-670). Apparently, DnaJ not only stimulates ATP hydrolysis but also couples it with conformational changes of DnaK. In the absence of GrpE, DnaJ forms a tight ternary complex with peptide.DnaK.ADP.Pi (Kd = 0.14 microM). However, by monitoring complex formation between DnaK (1 microM) and a fluorophore-labeled peptide in the presence of ATP (1 mM), DnaJ (1 microM), and varying concentrations of the ADP/ATP exchange factor GrpE (0.1-3 microM), substoichiometric concentrations of GrpE were found to shift the equilibrium from the slowly binding and releasing, high-affinity R state of DnaK completely to the fast binding and releasing, low-affinity T state and thus to prevent the formation of a long lived ternary DnaJ. substrate.DnaK.ADP.Pi complex. Under in vivo conditions with an estimated chaperone ratio of DnaK:DnaJ:GrpE = 10:1:3, both DnaJ and GrpE appear to control the chaperone cycle by transient interactions with DnaK.     

Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action

Applying stopped-flow fluorescence spectroscopy for measuring conformational changes of the DnaK molecular chaperone (bacterial Hsp70 homologue) and its binding to target peptide, we found that after ATP hydrolysis, DnaK is converted to the DnaK*(ADP) conformation, which possesses limited affinity for peptide substrates and the GrpE cochaperone but efficiently binds the DnaJ chaperone. In the presence of DnaJ (bacterial Hsp40 homologue), the DnaK*(ADP) form is converted back to the DnaK conformation, and the resulting DnaJ-DnaK(ADP) complex binds to peptide substrates more tightly. Formation of the DnaJ(substrate-DnaK(ADP)) complex is a rate-limiting reaction. The presence of GrpE and ATP hydrolysis promotes the fast release of the peptide substrate from the chaperone complex and converts DnaK to the DnaK*(ADP) conformation. We conclude that in the presence of DnaJ and GrpE, the binding-release cycle of DnaK is stoichiometrically coupled to the adenosine triphosphatase activity of DnaK.     

Regulation and function of collagen-specific molecular chaperone, HSP47

Muscarinic cholinergic stimulation of submandibular acinar cells results in the activation of Ca(2+)-dependent ion-transport pathways responsible for the secretion of primary saliva. Decreased saliva production is common among elderly people and may compromise oral health with implications for systemic health, nutrition, and quality of life. The density and affinity of muscarinic receptors in the submandibular gland of rats and the Ca2+ responses to stimulation of these receptors in the acinar cells were examined. An increase in the number of receptors and increases in the affinities of the receptors were found as the rats age from 7 weeks to 11 months. However, the coupling of the receptors to the intracellular Ca2+ signals in acinar cell clusters was substantially reduced in the older animals.     

Darwinian adaptation, population genetics and the streetcar theory of evolution

This paper investigates the problem of how to conceive a robust theory of phenotypic adaptation in non-trivial models of evolutionary biology. A particular effort is made to develop a foundation of this theory in the context of n-locus population genetics. Therefore, the evolution of phenotypic traits is considered that are coded for by more than one gene. The potential for epistatic gene interactions is not a priori excluded. Furthermore, emphasis is laid on the intricacies of frequency-dependent selection. It is first discussed how strongly the scope for phenotypic adaptation is restricted by the complex nature of 'reproduction mechanics' in sexually reproducing diploid populations. This discussion shows that one can easily lose the traces of Darwinism in n-locus models of population genetics. In order to retrieve these traces, the outline of a new theory is given that I call 'streetcar theory of evolution'. This theory is based on the same models that geneticists have used in order to demonstrate substantial problems with the 'adaptationist programme'. However, these models are now analyzed differently by including thoughts about the evolutionary removal of genetic constraints. This requires consideration of a sufficiently wide range of potential mutant alleles and careful examination of what to consider as a stable state of the evolutionary process. A particular notion of stability is introduced in order to describe population states that are phenotypically stable against the effects of all mutant alleles that are to be expected in the long-run. Surprisingly, a long-term stable state can be characterized at the phenotypic level as a fitness maximum, a Nash equilibrium or an ESS. The paper presents these mathematical results and discusses - at unusual length for a mathematical journal - their fundamental role in our current understanding of evolution.     

Structure, function, and evolution of the oviducts of squamate reptiles, with special reference to viviparity and placentation

In lizards and snakes, the oviducts function in fertilization, sperm storage, egg transport, eggshell deposition, maintenance of the early embryo, and expulsion of the egg or fetus. In viviparous forms they also contribute to placentae responsible for gas exchange and nutrient provision to the fetus. Dissections of species of 30 genera coupled with data from the literature indicate that squamate oviducts vary interspecifically in seven macroscopic features, including the extent and nature of regional differentiation, vascular supply, topographic asymmetry, number of oviducts, vaginal pouches, and relationship to the cloaca. The uterus, infundibulum, and vagina differ histologically in their epithelia, glands, and myometrial layers. Season cyclicity occurs in all three oviductal regions, most prominently in the uterus, and is under endocrinological control. Regional and cytological specializations reflect the diverse functions performed by the oviduct. Definitive evidence for oviductal albumen production and egg resorption is lacking. In viviparous squamates, three uterine specializations may facilitate maternal-fetal gas exchange: an attenuated epithelium, reduced uterine glands (and a reduced shell membrane), and increased vascularization. Contrary to previous reports, pregnant uteri show no epithelial erosion or capillary exposure. Specializations for nutrient provision to the fetus include mucosal hypertrophy, enlarged glandular epithelia, and multicellular glands whose secretions are absorbed by the chorioallantois. Comparisons with other amniotes indicate that squamates inherited the oviduct as an organ with capabilities for egg uptake and transport, fertilization, eggshell deposition, and oviposition. Other features have evolved convergently among squamates: infundibular sperm receptacles, unilateral oviduct loss, uterine gestation, placentation, and specializations for placentotrophy. Cladistic analysis indicates that oviductal features associated with deposition of tertiary egg investments in reptiles reflect evolutionary convergence as well as secondary simplification, rather than a unidirectional trend towards increased specialization.     

The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review

The 90-kDa molecular chaperone family (which comprises, among other proteins, the 90-kDa heat-shock protein, hsp90 and the 94-kDa glucose-regulated protein, grp94, major molecular chaperones of the cytosol and of the endoplasmic reticulum, respectively) has become an increasingly active subject of research in the past couple of years. These ubiquitous, well-conserved proteins account for 1-2% of all cellular proteins in most cells. However, their precise function is still far from being elucidated. Their involvement in the aetiology of several autoimmune diseases, in various infections, in recognition of malignant cells, and in antigen-presentation already demonstrates the essential role they likely will play in clinical practice of the next decade. The present review summarizes our current knowledge about the cellular functions, expression, and clinical implications of the 90-kDa molecular chaperone family and some approaches for future research.     

Heat shock protein 70 family: multiple sequence comparisons, function, and evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus "matches" 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins.     

Cellular adaptation to drug exposure: evolution of the drug-resistant phenotype

The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.     

Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity

In this work we show that the GroEL (Hsp60 equivalent) chaperone protein can protected purified Escherichia coli RNA polymerase (RNAP) holoenzyme from heat inactivation better than the DnaK (Hsp70 equivalent) chaperone can. In this protection reaction, the GroES protein is not essential, but its presence reduces the amount of GroEL required. GroEL and GroES can also reactivate heat-inactivated RNAP in the presence of ATP. The mutant GroEL673 protein, with or without GroES, is incapable of reactivating heat-inactivated RNAP. GroEL673 can only protect RNAP, and this protecting ability is not stimulated by GroES. The mechanism by which the DnaJ and GrpE heat shock proteins contribute to DnaK's ability to reactivate heat-inactivated RNAP GroEL673 has also been investigated. We found that the DnaJ protein substantially reduces the levels of DnaK protein needed in this reactivation assay. However, the observed lag in reactivation is diminished only in the additional presence of the GrpE protein. Hence, DnaJ and GrpE are involved in both steps of this reactivation reaction (recognition of substrate and release of chaperone from the substrate-chaperone complex) while, in the case of the GroEL-dependent reaction, GroES is involved only during the release of chaperone from the substrate-chaperone complex.     

ATPase activity and molecular chaperone function of the stress70 proteins

The codons for the amino acid residues making up the proposed ATP-binding sites of the maize (Zea mays L.) endoplasmic reticulum and tomato (Lycopersicon esculentum) cytoplasmic Stress70 proteins were deleted from their respective cDNAs. The deletions had little effect on the predicted secondary structure characteristics of the encoded proteins. Both wild-type and mutant proteins were expressed in Escherichia coli and purified to electrophoretic homogeneity. The mutant recombinant proteins did not bind to immobilized ATP columns, had no detectable ATPase activity, and were unable to function in vitro as molecular chaperones. Additionally, the inability to bind ATP was associated with changes in the oligomerization state of the Stress70 proteins.     

Molecular phylogeography and the evolution and conservation of Amazonian mammals

The phylogeographic structure of 15 genera of Amazonian marsupials and rodents is summarized based on comparative sequence of the mitochondrial cytochrome b gene. The data are limited in geographical coverage, with samples widely scattered throughout Amazonia from the base of the Andes in Peru to the Guianan coast and eastern Brazil. We use this approach to define species boundaries, based minimally on the principle of reciprocal monophyly, in conjunction with morphological or other genetic discontinuities. The taxa so defined are older than previously appreciated, with many lineages dating from 1 to more than 3 Myr, and thus apparently predating the early Pleistocene. We relate patterns of concordant geographical shifts with underlying tectonic history and to current positions of major rivers. Finally, we provide comments on the utility of these data and patterns to conservation, articulating a need to incorporate phylogeographic information as part of the rationale in establishing conservation priorities at the organismal and geographical area levels.     

Structure, function and metabolism of sialic acids

Sialic acids represent a family of sugar molecules with an unusual and highly variable chemical structure that are found mostly in the terminal position of oligosaccharide chains on the surface of cells and molecules. These special features enable them to fulfil several important and even diametrical biological functions. Because of the great importance of sialic acids, it is also worth having a look at their metabolism in order to get an idea of the intimate connection between structure and function of these fascinating molecules and the often serious consequences that results from disturbances in the balance of metabolic reactions. The latter can be due to genetic disorders that result in the absence of certain enzyme activity, leading to severe illness or even to death.     

Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution

Virulence genes of pathogenic bacteria, which code for toxins, adhesins, invasins or other virulence factors, may be located on transmissible genetic elements such as transposons, plasmids or bacteriophages. In addition, such genes may be part of particular regions on the bacterial chromosomes, termed 'pathogenicity islands' (Pais). Pathogenicity islands are found in Gram-negative as well as in Gram-positive bacteria. They are present in the genome of pathogenic strains of a given species but absent or only rarely present in those of non-pathogenic variants of the same or related species. They comprise large DNA regions (up to 200 kb of DNA) and often carry more than one virulence gene, the G + C contents of which often differ from those of the remaining bacterial genome. In most cases, Pais are flanked by specific DNA sequences, such as direct repeats or insertion sequence (IS) elements. In addition, Pais of certain bacteria (e,g. uropathogenic Escherichia coli, Yersinia spp., Helicobacter pylori) have the tendency to delete with high frequencies or may undergo duplications and amplifications. Pais are often associated with tRNA loci, which may represent target sites for the chromosomal integration of these elements. Bacteriophage attachment sites and cryptic genes on Pais, which are homologous to phage integrase genes, plasmid origins of replication of IS elements, indicate that these particular genetic elements were previously able to spread among bacterial populations by horizontal gene transfer, a process known to contribute to microbial evolution.     

The evolution of gene expression, structure and function of transthyretin

Thyroxine, the most abundant thyroid hormone in blood, partitions into lipid membranes. In a network-like system, thyroxine-binding plasma proteins counteract this partitioning and establish intravascular, protein-bound thyroxine pools. These are far larger than the free thyroxine pools. In larger eutherians, proteins specifically binding thyroxine are albumin, transthyretin, and thyroxine-binding globulin. Some binding of thyroxine can also occur to lipoproteins. During evolution, transthyretin synthesis first appeared in the choroid plexus of the stem reptiles, about 300 million years ago. Transthretin synthesis in the liver evolved much later, independently, in birds, eutherians and some marsupial species. Analysis of 57 human transthyretin variants suggests that most mutations in transthyretin are not compatible with its normal metabolism and lead to its deposition as amyloid. Analysis of transthyretin or its gene in 20 different species shows that evolutionary changes of transthyretin predominantly occurred near the N-termini. A change in RNA splicing between exon 1 and exon 2 led to a decrease in hydrophobicity and length of the N-termini. It is proposed that the selection pressure producing these changes was the need for a more effective prevention of thyroxine partitioning into lipids. Lipid pools increased during evolution with the increases in relative sizes of brains and internal organs and changes in lipid composition of membranes in ectothermic and endothermic species.