Showing papers on "Heat shock protein published in 2001"
TL;DR: It is shown here that complexes of peptides with heat shock proteins hsp90, calreticulin, and hsp70 are also taken up by macrophages and dendritic cells and re-presented by MHC class I molecules.
1,119 citations
TL;DR: Intracellular inclusions of de-natured proteins are also characteristic features of many neurological diseases, including Amyotrophic Lateral Sclerosis, Alzheimer's disease, Parkinson's disease and several hereditary diseases caused by expansions of polyglutamine tracts (e.g., Huntington's Disease or the spinocerebellar ataxias).
1,030 citations
TL;DR: The heat shock response, characterized by increased expression of heat shock proteins (Hsps), is induced by exposure of cells and tissues to extreme conditions that cause acute or chronic stress, thereby providing a finely tuned balance between survival and death.
Abstract: The heat shock response, characterized by increased expression of heat shock proteins (Hsps) is induced by exposure of cells and tissues to extreme conditions that cause acute or chronic stress. Hsps function as molecular chaperones in regulating cellular homeostasis and promoting survival. If the stress is too severe, a signal that leads to programmed cell death, apoptosis, is activated, thereby providing a finely tuned balance between survival and death. In addition to extracellular stimuli, several nonstressful conditions induce Hsps during normal cellular growth and development. The enhanced heat shock gene expression in response to various stimuli is regulated by heat shock transcription factors (HSFs). After the discovery of the family of HSFs (i.e., murine and human HSF1, 2, and 4 and a unique avian HSF3), the functional relevance of distinct HSFs is now emerging. HSF1, an HSF prototype, and HSF3 are responsible for heat-induced Hsp expression, whereas HSF2 is refractory to classical stressors. HSF4 is expressed in a tissue-specific manner; similar to HSF1 and HSF2, alternatively spliced isoforms add further complexity to its regulation. Recently developed powerful genetic models have provided evidence for both cooperative and specific functions of HSFs that expand beyond the heat shock response. Certain specialized functions of HSFs may even include regulation of novel target genes in response to distinct stimuli.
1,005 citations
TL;DR: It is shown that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis.
Abstract: To maintain quality control in cells, mechanisms distinguish among improperly folded peptides, mature and functional proteins, and proteins to be targeted for degradation. The molecular chaperones, including heat-shock protein Hsp90, have the ability to recognize misfolded proteins and assist in their conversion to a functional conformation. Disruption of Hsp90 heterocomplexes by the Hsp90 inhibitor geldanamycin leads to substrate degradation through the ubiquitin-proteasome pathway, implicating this system in protein triage decisions. We previously identified CHIP (carboxyl terminus of Hsc70-interacting protein) to be an interaction partner of Hsc70 (ref. 4). CHIP also interacts directly with a tetratricopeptide repeat acceptor site of Hsp90, incorporating into Hsp90 heterocomplexes and eliciting release of the regulatory cofactor p23. Here we show that CHIP abolishes the steroid-binding activity and transactivation potential of the glucocorticoid receptor, a well-characterized Hsp90 substrate, even though it has little effect on its synthesis. Instead, CHIP induces ubiquitylation of the glucocorticoid receptor and degradation through the proteasome. By remodelling Hsp90 heterocomplexes to favour substrate degradation, CHIP modulates protein triage decisions that regulate the balance between protein folding and degradation for chaperone substrates.
976 citations
TL;DR: A review of apoptosis-regulatory function of HSPs concludes that HSP27 and HSP70 may participate in carcinogenesis and should be considered as suspects in the development of cancer.
829 citations
TL;DR: To understand the proinflammatory nature of HSP, signaling induced by human and chlamydial HSP60 is analyzed and revealed that adjuvanticity of H SP60 operates similar to that of classical pathogen-derived ligands.
816 citations
TL;DR: The 2.7 Å structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its α-crystallin domain and flanking extensions assemble into a dodecameric double disk, and provides a model by which members of the sHSP protein family bind unfolded substrates.
Abstract: The 2.7 A structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its alpha-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the alpha-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.
710 citations
TL;DR: The antiapoptotic mechanism can be understood via expression of protective genes such as heat shock proteins, Bcl-2 as well as direct inhibition of the apoptotic caspase family proteases by S-nitrosylation of the cysteine thiol.
528 citations
TL;DR: It is found that high levels of HSP70 did indeed afford protection against neurodegeneration in invertebrate animal models of polyglutamine disorders, and enhancing chaperone activity could mitigate the phenotype in a mammalian model.
Abstract: Many neurodegenerative diseases are caused by gain-of-function mechanisms in which the disease-causing protein is altered, becomes toxic to the cell, and aggregates. Among these 'proteinopathies' are Alzheimer's and Parkinson's disease, prion disorders and polyglutamine diseases. Members of this latter group, also known as triplet repeat diseases, are caused by the expansion of unstable CAG repeats coding for glutamine within the respective proteins. Spinocerebellar ataxia type 1 (SCA1) is one such disease, characterized by loss of motor coordination due to the degeneration of cerebellar Purkinje cells and brain stem neurons. In SCA1 and several other polyglutamine diseases, the expanded protein aggregates into nuclear inclusions (NIs). Because these NIs accumulate molecular chaperones, ubiquitin and proteasomal subunits--all components of the cellular protein re-folding and degradation machinery--we hypothesized that protein misfolding and impaired protein clearance might underlie the pathogenesis of polyglutamine diseases. Over-expressing specific chaperones reduces protein aggregation in transfected cells and suppresses neurodegeneration in invertebrate animal models of polyglutamine disorders. To determine whether enhancing chaperone activity could mitigate the phenotype in a mammalian model, we crossbred SCA1 mice with mice over-expressing a molecular chaperone (inducible HSP70 or iHSP70). We found that high levels of HSP70 did indeed afford protection against neurodegeneration.
505 citations
TL;DR: Developmentally regulated expression of the heat-inducible Hsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family.
Abstract: We isolated cDNA clones for two nuclear-encoded, organellar members of the Arabidopsis hsp70 gene family, mtHsc70-2 (AF217458) and cpHsc70-2 (AF217459). Together with the completion of the genome sequence, the hsp70 family in Arabidopsis consists of 14 members unequally distributed among the five chromosomes. To establish detailed expression data of this gene family, a comprehensive reverse transcriptase-polymerase chain reaction analysis for 11 hsp70s was conducted including analysis of organ-specific and developmental expression and expression in response to temperature extremes. All hsp70s showed 2- to 20-fold induction by heat shock treatment except cpHsc70-1 and mtHsc70-1, which were unchanged or repressed. The expression profiles in response to low temperature treatment were more diverse than those evoked by heat shock treatment. Both mitochondrial and all cytosolic members of the family except Hsp70b were strongly induced by low temperature, whereas endoplasmic reticulum and chloroplast members were not induced or were slightly repressed. Developmentally regulated expression of the heat-inducible Hsp70 in mature dry seed and roots in the absence of temperature stress suggests prominent roles in seed maturation and root growth for this member of the hsp70 family. This reverse transcriptase-polymerase chain reaction analysis establishes the complex differential expression pattern for the hsp70s in Arabidopsis that portends specialized functions even among members localized to the same subcellular compartment.
463 citations
TL;DR: The diversity of the sHsp family indicates that care must be taken in generalizing biochemical properties and activities across different family members, and this chapter has a firmer structural foundation on which to design future experiments to build a biochemical mechanism of action.
Abstract: Publisher Summary The goal of this chapter is to clarify the diversity within the heat shock proteins (sHsps) family and to describe evidence indicating that sHsps have many different substrates and affect a wide range of cellular functions. The diversity of sHsp structure and expression patterns is immense, and their activities in vivo may involve multiple mechanisms. The sHsps and the structurally related vertebrate eye lens α-crystallins are the poor cousins in the family of molecular chaperones, and remain the least understood both structurally and functionally. The chaperone model for sHsp function provides a basic framework to explain the many proposed sHsp/protein interactions and potential functions. The diversity of the sHsp family, however, indicates that care must be taken in generalizing biochemical properties and activities across different family members. Nonetheless, the chapter has a firmer structural foundation on which to design future experiments to build a biochemical mechanism of action.
TL;DR: It is shown by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner.
Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder with no effective treatment. Geldanamycin is a benzoquinone ansamycin that binds to the heat shock protein Hsp90 and activates a heat shock response in mammalian cells. In this study, we show by using a filter retardation assay and immunofluorescence microscopy that treatment of mammalian cells with geldanamycin at nanomolar concentrations induces the expression of Hsp40, Hsp70 and Hsp90 and inhibits HD exon 1 protein aggregation in a dose-dependent manner. Similar results were obtained by overexpression of Hsp70 and Hsp40 in a separate cell culture model of HD. This is the first demonstration that huntingtin protein aggregation in cells can be suppressed by chemical compounds activating a specific heat shock response. These findings may provide the basis for the development of a novel pharmacotherapy for HD and related glutamine repeat disorders.
TL;DR: Functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.
Abstract: Bag (Bcl2-associated athanogene) domains occur in a class of cofactors of the eukaryotic chaperone 70-kilodalton heat shock protein (Hsp70) family. Binding of the Bag domain to the Hsp70 adenosine triphosphatase (ATPase) domain promotes adenosine 5'-triphosphate-dependent release of substrate from Hsp70 in vitro. In a 1.9 angstrom crystal structure of a complex with the ATPase of the 70-kilodalton heat shock cognate protein (Hsc70), the Bag domain forms a three-helix bundle, inducing a conformational switch in the ATPase that is incompatible with nucleotide binding. The same switch is observed in the bacterial Hsp70 homolog DnaK upon binding of the structurally unrelated nucleotide exchange factor GrpE. Thus, functional convergence has allowed proteins with different architectures to trigger a conserved conformational shift in Hsp70 that leads to nucleotide exchange.
TL;DR: Evidence is presented that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.
Abstract: Bacterial lipopolysaccharide (LPS), the major structural component of the outer wall of Gram-negative bacteria, is a potent initiator of an inflammatory response and serves as an indicator of bacterial infection. Although CD14 has been identified as the main LPS receptor, accumulating evidence has suggested the possible existence of other functional receptor(s). In this study, using affinity chromatography, peptide mass fingerprinting and fluorescence resonance energy transfer, we have identified four new proteins that form an activation cluster after LPS ligation and are involved in LPS signal transduction. Here we present evidence that implicates heat shock proteins 70 and 90, chemokine receptor 4 and growth differentiation factor 5 as the main mediators of activation by bacterial lipopolysaccharide.
TL;DR: The functional interactions between the stress response and the apoptotic cell death pathway are discussed and how these might impact on determining cellular survival are discussed.
TL;DR: This work characterized a sigE mutant of M. tuberculosis H37Rv that was more sensitive than the wild‐type strain to heat shock, SDS and various oxidative stresses, and showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock.
Abstract: In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.
TL;DR: It is shown that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress, and cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.
TL;DR: The physiological role of heat shock proteins is described and their protective potential against various stress agents in the cardiovascular system is discussed.
Abstract: In the eukaryotic cell an intrinsic mechanism is present providing the ability to defend itself against external stressors from various sources. This defense mechanism probably evolved from the presence of a group of chaperones, playing a crucial role in governing proper protein assembly, folding, and transport. Upregulation of the synthesis of a number of these proteins upon environmental stress establishes a unique defense system to maintain cellular protein homeostasis and to ensure survival of the cell. In the cardiovascular system this enhanced protein synthesis leads to a transient but powerful increase in tolerance to such endangering situations as ischemia, hypoxia, oxidative injury, and endotoxemia. These so-called heat shock proteins interfere with several physiological processes within several cell organelles and, for proper functioning, are translocated to different compartments following stress-induced synthesis. In this review we describe the physiological role of heat shock proteins and discuss their protective potential against various stress agents in the cardiovascular system.
TL;DR: It is demonstrated that αB-crystallin is a novel negative regulator of apoptosis that acts distally in the conserved cell death machinery by inhibiting the autocatalytic maturation of caspase-3.
TL;DR: Findings indicate the potential importance of developing procedures for elevating hsp expression in a safe and efficient manner in human individuals using either pharmacological or gene therapy procedures.
Abstract: The heat shock proteins (hsps) are expressed in normal cells but their expression is enhanced by a number of different stresses including heat and ischaemia. They play important roles in chaperoning the folding of other proteins and in protein degradation. In the heart a number of studies have shown that prior induction of the hsps by a mild stress has a protective effect against a more severe stress. Moreover, over-expression of an individual hsp in cardiac cells in culture or in the intact heart of either transgenic animals or using virus vectors, also produces a protective effect, directly demonstrating the ability of the hsps to produce protection. These findings indicate the potential importance of developing procedures for elevating hsp expression in a safe and efficient manner in human individuals using either pharmacological or gene therapy procedures.
TL;DR: The results indicate that NaCl stress damaged Complex I via oxidative stress and suggests that sHsps may protect Complex I as antioxidants, but NaCl damaged Complex II directly.
Abstract: High soil sodium (Na) is a common stress in natural and agricultural systems. Roots are usually the first tissues exposed to Na stress and Na stress-related impairment of mitochondrial function is likely to be particularly important in roots. However, neither the effects of NaCl on mitochondrial function, nor its protection by several potential adaptive mechanisms, have been well studied. This study investigated the effects of NaCl stress on maize (Zea mays) mitochondrial electron transport and its relative protection by osmoprotectants (proline, betaine, and sucrose), antioxidants (ascorbate, glutathione, and α-tocopherol), antioxidant enzymes (catalase and Cu/Zn-superoxide dismutase), and mitochondrial small heat shock proteins (sHsps). We demonstrate that Complex I electron transport is protected by antioxidants and sHsps, but not osmoprotectants, whereas Complex II is protected only by low concentrations of proline and betaine. These results indicate that NaCl stress damaged Complex I via oxidative stress and suggests that sHsps may protect Complex I as antioxidants, but NaCl damaged Complex II directly. This is the first study to demonstrate that NaCl stress differentially affects Complex I and II in plants and that protection of Complex I and II during NaCl stress is achieved by different mechanisms.
TL;DR: It is suggested that induction of HO‐1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.
Abstract: This is the first report on the protective effect of heme oxygenase-1 (HO-1) overexpression against oxidative stress-mediated neuronal cell death and demonstration of a decreased production of oxygen free radicals when HO-1 levels are increased. HO-1 is the heat shock/stress cognate of the heat shock protein 32 family of proteins. A known function of these proteins is alpha-meso bridge-specific cleavage of the heme molecule. For the present study, we used cerebellar granular neurons (CGNs) isolated from homozygous transgenic (Tg) mice that overexpress HO-1 under neuron-specific enolase control and nontransgenic (Ntg) littermates. The Tg mouse CGNs were characterized by increased levels of HO-1 mRNA and protein, a lower resting intracellular calcium concentration, and a reduced HO-1 transcriptional response to glutamate-mediated oxidative stress. Compared with the Ntg neurons, when exposed to glutamate (30 microM or 3 mM), the magnitude of cell viability was increased and the number of cells exhibiting membrane permeability and chromatin condensation were significantly decreased in the Tg CGN cultures. The population of neurons surviving glutamate toxicity decreased when HO-1 activity was inhibited by a peptide inhibitor. The neuroprotective effect by HO-1 was extended to H(2)O(2)-induced cell death. The mechanism of protection may involve in part a reduced production of reactive oxygen species upon exposure to glutamate. We suggest that induction of HO-1 by pharmacological means may be a novel approach to amelioration of oxidative insults to neurons.
TL;DR: The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.
Abstract: The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle. A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members. The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively. The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD. All of the AtHsp90 sequences include a conserved adenosine triphosphate–binding domain, whereas only the cytoplasmic and endoplasmic reticulum–resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences. The occurrence of multiple AtHsp90 proteins in the cyto...
TL;DR: Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heatshock and concomitantly abolished the suppressive effect of mild heat shock on UV‐induced JNK activation and apoptosis.
Abstract: Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43°C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.
TL;DR: Data show that Hsp 90 is essential for eNOS-dependent ·NO production and that inhibition of ATP-dependent conformational changes in Hsp90 uncouples eN OS activity and increases eNos-dependent O⨪2production.
TL;DR: The results suggest that extracellular Hsp70 may provide a means by which glia can affect neuronal function, perhaps enhancing neuronal stress tolerance.
TL;DR: The array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.
Abstract: Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43°C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.
TL;DR: The gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana was characterized and its expression was induced by heat and osmotic stress, as well as during seed development, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression.
Abstract: Owing to their sessile lifestyle, it is crucial for plants to acquire stress tolerance. The function of heat-shock proteins, including small heat-shock proteins (smHSPs), in stress tolerance is not fully explored. To gain further knowledge about the smHSPs, the gene that encoded the cytosolic class II smHSP in Arabidopsis thaliana (At-HSP17.6A) was characterized. The At-HSP17.6A expression was induced by heat and osmotic stress, as well as during seed development. Accumulation of At-HSP17.6A proteins could be detected with heat and at a late stage of seed development, but not with osmotic stress, suggesting stress-induced post-transcriptional regulation of At-HSP17.6A expression. Overproduction of At-HSP17.6A could increase salt and drought tolerance in Arabidopsis. The chaperone activity of At-HSP17.6A was demonstrated in vitro.
TL;DR: The results illuminate an important mechanism for regulated protein turnover in which a unique targeting protein, whose own activity is regulated through specific signaling pathways, catalyzes the delivery of a specific substrate to a specific protease.
Abstract: All organisms use rapid degradation of specific proteins as one way to tightly control important regulatory factors and developmental switches. Regulation of biological functions by proteolysis requires accurate substrate recognition at a precise time. In eukaryotes, fidelity of protein degradation depends on ubiquitin-tagging, a process that occurs in multicomponent complexes involving regulatory factors and various ubiquitin pathway enzymes. The ubiquitinated proteins are then targeted to the proteasome for degradation (for review, see Voges et al. 1999). In Escherichia coli, an example of substrate tagging as a means for regulating proteolysis is the degradation of incomplete proteins made from truncated mRNAs. In this case, short peptides coded by the SsrA RNA are added cotranslationally to polypeptides that have become stalled on ribosomes, and the tagged proteins are then degraded (Keiler et al. 1996; Gottesman et al. 1998; Roche and Sauer 1999). In general, E. coli proteins regulated by degradation interact directly with the proteases themselves, with each of the five known E. coli ATP-dependent proteases degrading a different but somewhat overlapping set of substrates (Gottesman 1996; Wickner et al. 1999). For many of the highly unstable E. coli proteins, degradation is rapid under all conditions, and synthesis is tightly controlled.
However, the activity of some proteins in E. coli is controlled by regulated degradation. For example, σ32 (RpoH), the heat shock sigma factor, is rapidly degraded under normal growth conditions by the AAA protease, FtsH, in a reaction modulated by the DnaJ/DnaK/GrpE chaperone system. During heat shock, σ32 is transiently stabilized, and this stabilization results in the rapid increase in the synthesis of the heat shock proteins (Yura and Nakahigashi 1999).
The stationary phase sigma factor, σS (RpoS), is another example of a protein whose activity is controlled by regulated proteolysis. σS promotes expression of more than 50 genes involved in responses to many stresses, including starvation, osmotic stress, acid shock, cold shock, heat shock, and oxidative damage, as well as the transition to stationary phase (Loewen and Hengge-Aronis 1994; Hengge-Aronis 2000). Although σS is present at very low levels during exponential cell growth, owing largely to its rapid degradation (half-life of ∼2 min), its stability increases ∼10-fold following transition to stationary phase or other stress treatments. Regulated degradation plays a major role in determining the amount of σS in the cell, but σS accumulation is also regulated at the transcriptional and translational levels (Lange and Hengge-Aronis 1994). The protease responsible for σS turnover in exponentially growing cells is ClpXP (Schweder et al. 1996), an ATP-dependent protease consisting of a regulatory component, ClpX, and a proteolytic component, ClpP (Gottesman et al. 1993; Wojtkowiak et al. 1993).
Degradation of σS requires an additional protein, RssB (Regulator of Sigma S; also referred to as SprE in E. coli, MviA in Salmonella, and ExpM in Erwinia) that is homologous to response regulator proteins (Bearson et al. 1996; Muffler et al. 1996b; Pratt and Silhavy 1996; Andersson et al. 1999). Genetic evidence shows that RssB is required for σS degradation but not for another ClpXP substrate, λ O, which indicates that RssB specifically targets σS for degradation (Zhou and Gottesman 1998). One of the hallmarks of the response regulator component of two-component signal transduction systems in prokaryotes is the presence of a conserved aspartate that is phosphorylated by the cognate sensor component. The N-terminal domain of RssB contains this conserved aspartate, although a cognate sensor protein has not been identified. RssB, like many other response regulators, is phosphorylated by acetyl phosphate in vitro (Bouche et al. 1998). In addition, phosphorylated RssB forms a stable complex with σS in vitro (Becker et al. 1999).
In this report we investigate RssB-regulated degradation of σS by reconstituting the pathway of σS degradation in vitro with purified RssB and ClpXP. We discovered that RssB acts directly and catalytically in stimulating degradation of σS by ClpXP in a reaction requiring acetyl phosphate and ATP. We have isolated and characterized subassemblies of the degradation machinery including σS–RssB, σS–RssB–ClpX, and σS–RssB–ClpXP complexes, and suggest a probable pathway for the degradation of σS.
TL;DR: It is suggested that CD40 is critical in microbial HSP70 binding and stimulation of RANTES production and coimmunoprecipitation of Mtb HSP 70 with CD40 indicates a physical association between these molecules.