In vivo chaperone activity of heat shock protein 70 and thermotolerance.
Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells. (+info)
Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.
The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. cholerae is ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed from toxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR and htpG was comparable to that by the parental strain. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease in htpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro. (+info)
Cloning and expression of the dnaK gene of Campylobacter jejuni and antigenicity of heat shock protein 70.
Campylobacter jejuni is a leading cause of infectious diarrhea throughout the world. In addition, there is growing evidence that Guillain-Barre syndrome, an inflammatory demyelinating disease of the peripheral nervous system, is frequently preceded by C. jejuni infection. In the present study, the hrcA-grpE-dnaK gene cluster of C. jejuni was cloned and sequenced. The dnaK gene consists of an open reading frame of 1,869 bp and encodes a protein with a high degree of homology to other bacterial 70-kDa heat shock proteins (HSPs). The overall percentages of identity to the HSP70 proteins of Helicobacter pylori, Borrelia burgdorferi, Chlamydia trachomatis, and Bacillus subtilis were calculated to be 78.1, 60.5, 57.2, and 53. 8%, respectively. Regions similar to the Escherichia coli sigma70 promoter consensus sequence and to a cis-acting regulatory element (CIRCE) are located upstream of the hrcA gene. Following heat shock, a rapid increase of dnaK mRNA was detectable, which reached its maximum after 20 to 30 min. A 6-His-tagged recombinant DnaK protein (rCjDnaK-His) was generated in E. coli, after cloning of the dnaK coding region into pET-22b(+), and purified by affinity and gel filtration chromatography. Antibody responses to rCjDnaK-His were significantly elevated, compared to those of healthy individuals, in about one-third of the serum specimens obtained from C. jejuni enteritis patients. (+info)
Heat shock protein 70 (Hsp70) protects postimplantation murine embryos from the embryolethal effects of hyperthermia.
Previous work has shown that there is a positive correlation between the induction of Hsp70 and its transient nuclear localization and the acquisition and loss of induced thermotolerance in postimplantation rat embryos. To determine whether Hsp70 is sufficient to induce thermotolerance in postimplantation mammalian embryos, we used a transgenic mouse in which the normally strictly inducible Hsp70 is constitutively expressed in the embryo under the control of a beta-actin promoter. Day 8.0 mouse embryos heterozygous for the Hsp70 transgene were not protected from the embryotoxic effects of hyperthermia (43 degrees C); however, homozygous embryos, expressing approximately twice as much Hsp70 as heterozygous embryos, were partially protected (increased embryo viability) from the embryolethal effects of hyperthermia. Although the viability of transgenic embryos was significantly increased compared with that of nontransgenic embryos, this protection did not extend to embryo growth and development. To determine whether the failure to achieve a more robust protection was related to the expression of insufficient Hsp70 in transgenic embryos, we undertook experiments to determine whether the level of Hsp70 correlated with the level of thermotolerance induced by various lengths of a 41 degrees C heat shock. A 41 degrees C, 5-minute heat shock failed to induce Hsp70 or thermotolerance, a 41 degrees C, 15-minute heat shock induced Hsp70 and a significant level of thermotolerance, while a 41 degrees C, 60-minute heat shock induced an even higher level of Hsp70 as well as a higher level of thermotolerance. Quantitation of Hsp70 levels indicated that thermotolerance was associated with levels of Hsp70 of 820 pg/microg embryo protein or greater. Subsequent quantitation of the amount of Hsp70 expressed in homozygous transgenic embryos indicated a level of 577 pg/microg embryo protein, that is, a level below that associated with induced thermotolerance. Overall, results presented indicate that Hsp70 does play a direct role in the induction of thermotolerance in postimplantation mouse embryos; however, the level of thermotolerance is dependent on the level of Hsp70 expressed. (+info)
Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.
Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution. (+info)
Gene expression and chromatin organization during mouse oocyte growth.
Mouse oocytes can be classified according to their chromatin organization and the presence [surrounded nucleolus (SN) oocytes] or absence [nonsurrounded nucleolus (NSN) oocytes] of a ring of Hoechst-positive chromatin around the nucleolus. Following fertilization only SN oocytes are able to develop beyond the two-cell stage. These studies indicate a correlation between SN and NSN chromatin organization and the developmental competence of the female gamete, which may depend on gene expression. In the present study, we have used the HSP70.1Luc transgene (murine HSP70.1 promoter + reporter gene firefly luciferase) to analyze gene expression in oocytes isolated from ovaries of 2-day- to 13-week-old females. Luciferase was assayed on oocytes after classification as SN or NSN type. Our data show that SN oocytes always exhibit a higher level of luciferase activity, demonstrating a higher gene expression in this category. Only after meiotic resumption, metaphase II oocytes derived from NSN or SN oocytes acquire the same level of transgene expression. We suggest that the limited availability of transcripts and corresponding proteins, excluded from the cytoplasm until GVBD in NSN oocytes, could explain why these oocytes have a lower ability to sustain embryonic development beyond the two-cell stage at which major zygotic transcription occurs. With this study we have furthered our knowledge of epigenetic regulation of gene expression in oogenesis. (+info)
Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.
The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves. (+info)
Role of heat shock protein HSP70-2 in spermatogenesis.
The HSP70 heat-shock proteins are molecular chaperones that assist other proteins in their folding, transport and assembly into complexes. Most of these proteins are either constitutively expressed or their expression is induced by heat shock and other stresses. However, two members of the Hsp70 family (HSP70-2 and HSC70T in mice) are regulated developmentally and expressed specifically in spermatogenic cells. The HSP70-2 protein is synthesized during the meiotic phase of spermatogenesis and is abundant in pachytene spermatocytes. The knockout approach was used to determine whether HSP70-2 is a chaperone for proteins involved in meiosis. Male mice lacking HSP70-2 were infertile while females lacking HSP70-2 were fertile. Spermatogenic cell development was arrested in prophase of meiosis I at the G2-M-phase transition and late pachytene spermatocytes were eliminated by apoptosis, resulting in an absence of spermatids. HSP70-2 is required for Cdc2 to form a heterodimer with cyclin B1, suggesting that it is a chaperone necessary for the progression of meiosis in the germ cells of male mice. HSP70-2 is also associated with the synaptonemal complex and desynapsis is disrupted in male mice lacking this protein. Homologues of HSP70-2 are present in the testes of many animals, suggesting that the role of this spermatogenic cell chaperone is conserved across phyla. (+info)