The dramatically increased chaperone activity of small heat-shock protein IbpB is retained for an extended period of time after the stress condition is removed.

sHSP (small heat-shock protein) IbpB (inclusion-body-binding protein B) from Escherichia coli is known as an ATP-independent holding chaperone which prevents the insolubilization of aggregation-prone proteins by forming stable complexes with them. It was found that the chaperone function of IbpB is greatly modulated by the ambient temperature, i.e. when the temperature increases from normal to heat-shock, the chaperone activity of IbpB is dramatically elevated to a level that allows it to effectively bind the aggregation-prone client proteins. Although it is generally believed that the release and refolding of the client protein from the sHSPs depends on the aid of the ATP-dependent chaperones such as Hsp (heat-shock protein) 70 and Hsp100 when the ambient temperature recovers from heat-shock to normal, the behaviour of the sHSPs during this recovery stage has not yet been investigated. In the present study, we examined the behaviour and properties of IbpB upon temperature decrease from heat-shock to normal. We found that IbpB, which becomes functional only under heat-shock conditions, retains the chaperone activity for an extended period of time after the heat-shock stress condition is removed. A detail comparison demonstrates that such preconditioned IbpB is distinguished from the non-preconditioned IbpB by a remarkable conformational transformation, including a significant increase in the flexibility of the N- and C-terminal regions, as well as enhanced dynamic subunit dissociation/reassociation. Intriguingly, the preconditioned IbpB displayed a dramatic decrease in its surface hydrophobicity, suggesting that the exposure of hydrophobic sites might not be the sole determinant for IbpB to exhibit chaperone activity. We propose that the maintenance of the chaperone activity for such 'holdases' as sHSPs would be important for cells to recover from heat-shock stress.

Stepwise disassembly and apparent nonstepwise reassembly for the oligomeric RbsD protein.

Many cellular proteins exist as homo-oligomers. The mechanism of the assembly process of such proteins is still poorly understood. We have previously observed that Hsp16.3, a protein exhibiting chaperone-like activity, undergoes stepwise disassembly and nonstepwise reassembly. Here, the disassembly and reassembly of a nonchaperone protein RbsD, from Escherichia coli, was studied in vitro. The protein was found to mainly exist as decamers with a small portion of apparently larger oligomeric forms, both of which are able to refold/reassemble effectively in a spontaneous way after being completely unfolded. Disassembly RbsD intermediates including pentamers, tetramers, trimers, dimers, and monomers were detected by using urea-containing pore gradient polyacrylamide gel electrophoresis, while only pentamers were detected for its reassembly. The observation of stepwise disassembly and apparent nonstepwise reassembly for both a chaperone protein (Hsp16.3) and a nonchaperone protein (RbsD) strongly suggests that such a feature is most likely general for homo-oligomeric proteins.

The essential role of the flexible termini in the temperature-responsiveness of the oligomeric state and chaperone-like activity for the polydisperse small heat shock protein IbpB from Escherichia coli.

Small heat shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones that are believed to prevent irreversible aggregation of other cellular proteins under stress conditions. One of the most prominent features of sHSPs is that they exist as homo-oligomers. Examples of both monodisperse and polydisperse oligomers are found within this family. The small heat shock inclusion-body binding protein B (IbpB) of Escherichia coli, originally discovered as a component of inclusion bodies, exhibits a pronounced polydispersity in its oligomeric state. This research was performed to elucidate the temperature effect on the oligomeric state and chaperone-like activity of the polydisperse IbpB oligomers, as well as the structural basis for such a temperature effect. The data presented here demonstrate that the large oligomers of IbpB progressively dissociate into smaller ones at increasing heat-shock temperatures, accompanied by a notable enhancement of chaperone-like activities. The secondary structure, enriched mainly by beta-strands, is slightly changed with such temperature increases. The dimeric building blocks, which seem to be highly stable, act as the functional unit of IbpB. Limited proteolysis was used to identify the susceptible sites in IbpB that may compose the subunit interfaces, which indicated that the 11 residues at both the N and the C terminus are highly flexible and the removal of each will lead to the formation of dimers, as well as the disappearance of chaperone-like activities. Truncation of 11 residues from either end, using recombinant DNA technology, also led to the formation of dimeric mutant IbpB proteins lacking chaperone-like activities. Taken together, the flexible termini appear to be essential for small heat shock protein IbpB to generate various temperature-responsive oligomers, which exhibit various levels of chaperone-like activities, by interlinking or separating the dimer building blocks.

Chaperone-like activity of beta-casein.

The caseins are major components of milk for most mammals and are secreted as large colloidal aggregates termed micelles. They have less ordered secondary and tertiary structures in comparison with typical globular proteins. In this work, beta-casein, a member of the casein family, has been demonstrated to exhibit chaperone-like activity, being able to suppress the thermal and chemical aggregation of such substrate proteins as insulin, lysozyme, alcohol dehydrogenase, and catalase by forming stable complexes with the denaturing substrate proteins. Meanwhile, beta-casein was found to not only prevent aggregation of the substrate proteins, but also solubilize the protein aggregates already formed. Data also show that beta-casein exhibits a higher chaperone-like activity than alpha-casein, likely due to the difference in the number of proline residues present and/or in the extent of exposed hydrophobic surfaces. The implications for their in vivo functions of the caseins, based on their exhibiting such in vitro chaperone-like activities, are discussed.

Glycation-altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age-related disease (in nondiabetics).

Epidemiologic studies indicate that the risks for major age-related debilities including coronary heart disease, diabetes, and age-related macular degeneration (AMD) are diminished in people who consume lower glycemic index (GI) diets, but lack of a unifying physiobiochemical mechanism that explains the salutary effect is a barrier to implementing dietary practices that capture the benefits of consuming lower GI diets. We established a simple murine model of age-related retinal lesions that precede AMD (hereafter called AMD-like lesions). We found that consuming a higher GI diet promotes these AMD-like lesions. However, mice that consumed the lower vs. higher GI diet had significantly reduced frequency (P < 0.02) and severity (P < 0.05) of hallmark age-related retinal lesions such as basal deposits. Consuming higher GI diets was associated with > 3 fold higher accumulation of advanced glycation end products (AGEs) in retina, lens, liver, and brain in the age-matched mice, suggesting that higher GI diets induce systemic glycative stress that is etiologic for lesions. Data from live cell and cell-free systems show that the ubiquitin-proteasome system (UPS) and lysosome/autophagy pathway [lysosomal proteolytic system (LPS)] are involved in the degradation of AGEs. Glycatively modified substrates were degraded significantly slower than unmodified substrates by the UPS. Compounding the detriments of glycative stress, AGE modification of ubiquitin and ubiquitin-conjugating enzymes impaired UPS activities. Furthermore, ubiquitin conjugates and AGEs accumulate and are found in lysosomes when cells are glycatively stressed or the UPS or LPS/autophagy are inhibited, indicating that the UPS and LPS interact with one another to degrade AGEs. Together, these data explain why AGEs accumulate as glycative stress increases.

Disassembly intermediates of RbsD protein remain oligomeric despite the loss of an intact secondary structure.

Many proteins exist as homo-oligomers in living organisms wherein the change of oligomeric status apparently serves as an effective means for modulating their biological activities. We have previously reported that the homo-decameric RbsD from Escherichia coli undergoes stepwise disassembly and non-stepwise reassembly. Here the structural status of the urea-induced RbsD disassembly intermediates was examined, mainly using urea-containing polyacrylamide gel electrophoresis and chemical cross-linking. Such intermediates were found to remain oligomeric while losing their intact secondary structures. Such disassembly intermediates were able to effectively refold when the concentration of the urea denaturant was reduced to a lower level, or to refold/reassemble into the native decamers when urea was completely removed, as detected by non-denaturing polyacrylamide gel electrophoresis. These novel observations strongly suggest that the assembly of oligomeric proteins may occur before the completion of subunit folding.

Phylogenetic and biochemical studies reveal a potential evolutionary origin of small heat shock proteins of animals from bacterial class A.

Small heat shock proteins (sHSPs), as one subclass of molecular chaperones, are important for cells to protect proteins under stress conditions. Unlike the large HSPs (represented by Hsp60 and Hsp70), sHSPs are highly divergent in both primary sequences and oligomeric status, with their evolutionary relationships being unresolved. Here the phylogenetic analysis of a representative 51 sHSPs (covering the six subfamilies: bacterial class A, bacterial class B, archae, fungi, plant, and animal) reveals a close relationship between bacterial class A and animal sHSPs which form an outgroup. Accumulating data indicate that the oligomers from bacterial class A and animal sHSPs appear to exhibit polydispersity, while those from the rest exhibit monodispersity. Together, the close evolutionary relationship and the similarity in oligomeric polydispersity between bacterial class A and animal sHSPs not only suggest a potential evolutionary origin of the latter from the former, but also imply that their oligomeric polydispersity is somehow a property determined by their primary sequences.

The association of small heat shock protein Hsp16.3 with the plasma membrane of Mycobacterium tuberculosis: dissociation of oligomers is a prerequisite.

Hsp16.3, a small heat shock protein from Mycobacterium tuberculosis (MTB), was originally identified as an immuno-dominant antigen and later found to be a major membrane protein. In vitro studies show that Hsp16.3 exists as nonamers and undergoes dynamic dissociation/re-association equilibrium in solutions. Nevertheless, neither the details nor the physiological implications of the presence of Hsp16.3 in the plasma membrane have been studied. In this study, we demonstrated that the purified Hsp16.3 proteins were able to interact with the MTB plasma membrane in a specific and reversible manner, suggesting that there might be subunit exchange between membrane-bound Hsp16.3 and soluble Hsp16.3 oligomers. The dissociation of Hsp16.3 oligomers appears to be a prerequisite for its membrane binding, which is interesting in view that the dissociation of small heat shock protein oligomers was also found to be necessary for it to bind denaturing substrate proteins. Furthermore, the oligomeric structure of Hsp16.3 seems to be more dynamic and flexible when incubating with the mycobacterium lipids. The physiological implications of these observations for Hsp16.3, and small heat shock proteins in general, are discussed.

Small heat-shock proteins function in the insoluble protein complex.

Small heat-shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones. The current model proposes that sHSPs function to prevent irreversible aggregation of non-native proteins by forming soluble complex. The chaperone activity of sHSPs is usually determined by the capacity to suppress thermally or chemically induced protein aggregation. However, sHSPs were frequently found in the insoluble complex particularly in vivo. In this report, it is clearly revealed that the insoluble sHSP/substrate complex is formed when sHSP is overloaded with non-native substrates, which is the very case under in vivo conditions. The proposal that sHSPs function to prevent the protein aggregation seems misleading. sHSPs appear to promote the elimination of protein aggregates by incorporating into the insoluble protein complex.

A dual role for the N-terminal region of Mycobacterium tuberculosis Hsp16.3 in self-oligomerization and binding denaturing substrate proteins.

The N-terminal regions, which are highly variable in small heat-shock proteins, were found to be structurally disordered in all the 24 subunits of Methanococcus jannaschii Hsp16.5 oligomer and half of the 12 subunits of wheat Hsp16.9 oligomer. The structural and functional roles of the corresponding region (potentially disordered) in Mycobacterium tuberculosis Hsp16.3, existing as nonamers, were investigated in this work. The data demonstrate that the mutant Hsp16.3 protein with 35 N-terminal residues removed (DeltaN35) existed as trimers/dimers rather than as nonamers, failing to bind the hydrophobic probe (1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid) and exhibiting no chaperone-like activity. Nevertheless, another mutant protein with the C-terminal extension (of nine residues) removed, although existing predominantly as dimers, exhibited efficient chaperone-like activity even at room temperatures, indicating that pre-existence as nonamers is not a prerequisite for its chaperone-like activity. Meanwhile, the mutant protein with both the N- and C-terminal ends removed fully exists as a dimer lacking any chaperone-like activity. Furthermore, the N-terminal region alone, either as a synthesized peptide or in fusion protein with glutathione S-transferase, was capable of interacting with denaturing proteins. These observations strongly suggest that the N-terminal region of Hsp16.3 is not only involved in self-oligomerization but also contains the critical site for substrate binding. Such a dual role for the N-terminal region would provide an effective mechanism for the small heat-shock protein to modulate its chaperone-like activity through oligomeric dissociation/reassociation. In addition, this study demonstrated that the wild-type protein was able to form heterononamers with DeltaN35 via subunit exchange at a subunit ratio of 2:1. This implies that the 35 N-terminal residues in three of the nine subunits in the wild-type nonamer are not needed for the assembly of nonamers from trimers and are thus probably structurally disordered.

Periplasmic protein HdeA exhibits chaperone-like activity exclusively within stomach pH range by transforming into disordered conformation.

The extremely acidic environment of the mammalian stomach, with a pH range usually between 1 and 3, represents a stressful challenge for enteric pathogenic bacteria such as Escherichia coli before they enter into the intestine. The hdeA gene of E. coli was found to be acid inducible and was revealed by genetic studies to be important for the acid survival of the strain. This study was performed in an attempt to characterize the mechanism of the activity of the HdeA protein. Our data provided in this report strongly suggest that HdeA employs a novel strategy to modulate its chaperone activity: it possesses an ordered conformation that is unable to bind denatured substrate proteins under normal physiological conditions (i.e. at neutral pH) and transforms into a globally disordered conformation that is able to bind substrate proteins under stress conditions (i.e. at a pH below 3). Furthermore, our data indicate that HdeA exposes hydrophobic surfaces that appear to be involved in the binding of denatured substrate proteins at extremely low pH values. In light of our observations, models are proposed to explain the action of HdeA in both a physiological and a molecular context.

Inter-subunit cross-linking suppressed the dynamic oligomeric dissociation of Mycobacterium tuberculosis Hsp16.3 and reduced its chaperone activity.

Small heat shock proteins (sHsps) usually exist as dynamic oligomers and oligomeric dissociation was believed to be a prerequisite for their chaperone activities. The truth of this hypothesis was verified in our present study on Hsp16.3, one member of sHsps from Mycobacterium tuberculosis, mainly by utilizing chemical cross-linking. Analysis using size exclusion chromatography demonstrated that the heat-induced oligomeric dissociation of Hsp16.3 was severely blocked due to highly efficient inter-subunit cross-linkages generated by chemical cross-linking, as well as its chaperone activity being reduced. Further analysis by non-denaturing pore gradient polyacrylamide gel electrophoresis and fluorescence spectrometry revealed that the dynamic oligomeric dissociation/reassociation process of Hsp16.3 at room temperature was suppressed by inter-subunit cross-linkages, accompanied by significantly decreased exposure of hydrophobic surfaces that are usually hidden in oligomers. These findings supported the hypothesis that substrate-binding sites of sHsps are exposed presumably by dissociation of larger oligomers into smaller active oligomers, and therefore such a dissociation process could be adjusted to modulate chaperone activities.