GroES in the asymmetric GroEL14-GroES7 complex exchanges via an associative mechanism.
(9/1959)
The interaction of the chaperonin GroEL14 with its cochaperonin GroES7 is dynamic, involving stable, asymmetric 1:1 complexes (GroES7.GroEL7-GroEL7) and transient, metastable symmetric 2:1 complexes [GroES7.GroEL7-GroEL7.GroES7]. The transient formation of a 2:1 complex permits exchange of free GroES7 for GroES7 bound in the stable 1:1 complex. Electrophoresis in the presence of ADP was used to resolve free GroEL14 from the GroES7-GroEL14 complex. Titration of GroEL14 with radiolabeled GroES7 to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex. No stable 2:1 complex was detected. Preincubation of the asymmetric GroES7.GroEL7-GroEL7 complex with excess unlabeled GroES7 in the presence of ADP demonstrated GroES7 exchange. The rates of GroES7 exchange were proportional to the concentration of unlabeled free GroES7. This concentration dependence points to an associative mechanism in which exchange of GroES7 occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL14. Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES7. In agreement with recent structural studies, this indicates that conformational changes in GroEL14 following the dissociation of GroES7 must precede ADP release. These results explain how the GroEL14 cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes. (+info)
Opposite behavior of two isozymes when refolding in the presence of non-ionic detergents.
(10/1959)
GroEL has a greater affinity for the mitochondrial isozyme (mAAT) of aspartate aminotransferase than for its cytosolic counterpart (cAAT) (Mattingly JR Jr, Iriarte A, Martinez-Carrion M, 1995, J Biol Chem 270:1138-1148), two proteins that share a high degree of sequence similarity and an almost identical spatial structure. The effect of detergents on the refolding of these large, dimeric isozymes parallels this difference in behavior. The presence of non-ionic detergents such as Triton X-100 or lubrol at concentrations above their critical micelle concentration (CMC) interferes with reactivation of mAAT unfolded in guanidinium chloride but increases the yield of cAAT refolding at low temperatures. The inhibitory effect of detergents on the reactivation of mAAT decreases progressively as the addition of detergents is delayed after starting the refolding reaction. The rate of disappearance of the species with affinity for binding detergents coincides with the slowest of the two rate-limiting steps detected in the refolding pathway of mAAT. Limited proteolysis studies indicate that the overall structure of the detergent-bound mAAT resembles that of the protein in a complex with GroEL. The mAAT folding intermediates trapped in the presence of detergents can resume reactivation either upon dilution of the detergent below its CMC or by adding beta-cyclodextrin. Thus, isolation of otherwise transient productive folding intermediates for further characterization is possible through the use of detergents. (+info)
A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus.
(11/1959)
Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A approximately 63-kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that tomato yellow leaf curl virus (TYLCV) particles displayed a specific affinity for the B. tabaci 63-kDa GroEL homologue. Feeding whiteflies anti-Buchnera GroEL antiserum before the acquisition of virions reduced TYLCV transmission to tomato test plants by >80%. In the haemolymph of these whiteflies, TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph. (+info)
Human 60-kDa heat-shock protein: a danger signal to the innate immune system.
(12/1959)
Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-alpha; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-gamma in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation. (+info)
Endothelial cytotoxicity mediated by serum antibodies to heat shock proteins of Escherichia coli and Chlamydia pneumoniae: immune reactions to heat shock proteins as a possible link between infection and atherosclerosis.
(13/1959)
BACKGROUND: Growing evidence suggests that an immunological reaction against heat shock proteins (HSPs) may be involved in atherogenesis. Because HSPs show a high degree of amino acid sequence homology between different species, from prokaryotes to humans, we investigated the possibility of "antigenic mimicry" caused by an immunological cross-reaction between microorganisms and autoantigens. METHODS AND RESULTS: Serum antibodies against the Escherichia coli HSP (GroEL) and the 60-kDa chlamydial HSP (cHSP60) from subjects with atherosclerosis were purified by use of affinity chromatography. Western blot analyses and competitive ELISAs confirmed the cross-reaction of the eluted antibodies with human HSP60 and the bacterial counterparts. The cytotoxicity of anti-GroEL and anti-cHSP60 antibodies was determined on human endothelial cells labeled with 51Cr. A significant difference (40% versus 8%) was observed in the specific 51Cr release of heat-treated (42 degrees C for 30 minutes) and untreated cells, respectively, in the presence of these anti-HSP antibodies and complement. This effect was blocked by addition of 100 microg/mL recombinant GroEL. In addition, seropositivity against specific non-HSP60 Chlamydia pneumoniae antigens is more prominent among high-anti-HSP titer sera than low-titer sera. CONCLUSIONS: Serum antibodies against HSP65/60 cross-react with human HSP60, cHSP60, and GroEL; correlate with the presence of antibodies to C pneumoniae and endotoxin; and mediate endothelial cytotoxicity. These findings suggest that humoral immune reactions to bacterial HSPs, such as cHSP60 and GroEL, may play an important role in the process of vascular endothelial injury, which is believed to be a key event in the pathogenesis of atherosclerosis. (+info)
The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells.
(14/1959)
The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system. (+info)
GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase. Obligatory interaction of chaperonins with an alpha beta dimeric intermediate.
(15/1959)
The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1. Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers. (+info)
Mechanisms for GroEL/GroES-mediated folding of a large 86-kDa fusion polypeptide in vitro.
(16/1959)
Our understanding of mechanisms for GroEL/GroES-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated chaperonin-dependent folding of a large 86-kDa fusion polypeptide, in which the mature maltose-binding protein (MBP) sequence was linked to the N terminus of the alpha subunit of the decarboxylase (E1) component of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. The fusion polypeptide, MBP-alpha, when co-expressed with the beta subunit of E1, produced a chimeric protein MBP-E1 with an (MBP-alpha)2beta2 structure, similar to the alpha2 beta2 structure in native E1. Reactivation of MBP-E1 denatured in 8 M urea was absolutely dependent on GroEL/GroES and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged E1. Chaperonin-mediated refolding of the MBP-alpha fusion polypeptide showed that the folding of the MBP moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an MBP-alpha. GroEL binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same GroEL scaffold. Moreover, we show that when the alpha.GroEL complex (molar ratio 1:1) was incubated with GroES, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the MBP-alpha.GroEL (1:1) complex was capped by GroES exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the GroEL cavity that is not sequestered by GroES. (+info)