Small Heat Shock Protein Activity Is Regulated by Variable Oligomeric Substructure

Abstract

The α-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified α-crystallins, without altering the protein sequence, by refolding the α-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the α-crystallins in protein deposition diseases. The α-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone to adjuvant of aggregation. We replicated the oligomeric rearrangements observed for post-translationally modified α-crystallins, without altering the protein sequence, by refolding the α-crystallins in vitro. This refolding resulted in a loss of dimeric substructure concomitant with an augmentation of substrate affinity. We show that packaging of small heat shock proteins into dimeric units is used to control the level of chaperone function by regulating the exposure of hydrophobic surfaces. We propose that a bias toward monomeric substructure is responsible for the aberrant chaperone behavior associated with the α-crystallins in protein deposition diseases. The small heat shock proteins (sHSPs) 4The abbreviations used are: sHSP, small heat shock protein; MS, mass spectrometry; ANS, 1-anilino-8-naphthalenesulfonic acid; PBS, phosphate-buffered saline; MS/MS, tandem MS; αBR, recombinant αB. 4The abbreviations used are: sHSP, small heat shock protein; MS, mass spectrometry; ANS, 1-anilino-8-naphthalenesulfonic acid; PBS, phosphate-buffered saline; MS/MS, tandem MS; αBR, recombinant αB. αA- and αB-crystallin are most prevalent in the vertebrate eye lens, where they are found co-assembled and maintain lens transparency by preventing other proteins from forming light-scattering aggregates (1Brady J.P. Garland D. Duglas-Tabor Y. Robison Jr., W.G. Groome A. Wawrousek E.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 884-889Crossref PubMed Scopus (298) Google Scholar), but are also found in other tissues (2Kappe G. Franck E. Verschuure P. Boelens W.C. Leunissen J.A. de Jong W.W. Cell Stress Chaperones. 2003; 8: 53-61Crossref PubMed Scopus (375) Google Scholar). It is clear that these proteins have the ability to sequester substrates that have become partially unfolded under conditions of stress to preserve them in a state competent for refolding (3Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (669) Google Scholar, 4Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (571) Google Scholar, 5Narberhaus F. Microbiol. Mol. Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (467) Google Scholar, 6van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (379) Google Scholar). However, molecular mechanisms of their function and regulation remain incompletely understood, particularly regarding aberrant behavior in the transitions that occur to bring about disease states (7Sun Y. MacRae T.H. FEBS J. 2005; 272: 2613-2627Crossref PubMed Scopus (285) Google Scholar). Structurally, the sHSPs are characterized by their low monomeric molecular mass, their assembly into oligomers, and the presence of a well conserved “α-crystallin” domain (3Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (669) Google Scholar, 4Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (571) Google Scholar, 5Narberhaus F. Microbiol. Mol. Biol. Rev. 2002; 66: 64-93Crossref PubMed Scopus (467) Google Scholar, 6van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (379) Google Scholar). Although crystal structures exist for some of the monodisperse members of the family (3Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (669) Google Scholar), the extreme polydispersity of many of the mammalian sHSPs, including the α-crystallins, has hampered crystallographic analysis (4Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (571) Google Scholar). Common to the sHSPs for which high resolution structures have been published is the existence of dimers as “building blocks” of the larger oligomer (3Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (669) Google Scholar, 6van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (379) Google Scholar). We have reported that recombinant αA and αB form a range of oligomers with a notable preference for an even number of subunits (9Aquilina J.A. Benesch J.L.P. Ding L.L. Yaron O. Horwitz J. Robinson C.V. J. Biol. Chem. 2004; 279: 28675-28680Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 10Aquilina J.A. Benesch J.L.P. Ding L.L. Yaron O. Horwitz J. Robinson C.V. J. Biol. Chem. 2005; 280: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). This suggests that dimeric substructure is a key characteristic of the sHSPs, and indeed, dimers have been proposed as the active “chaperoning unit” of the sHSPs (6van Montfort R. Slingsby C. Vierling E. Adv. Protein Chem. 2002; 59: 105-156Crossref Scopus (379) Google Scholar). In our previous study on αB isolated from the lens under denaturing conditions, we observed a difference in the proportion of oligomers containing an even or odd number of subunits (11Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (208) Google Scholar). This observation, along with recent work showing structural changes in the α-crystallins upon renaturation from urea (12Saha S. Das K.P. Protein J. 2007; 26: 315-326Crossref PubMed Scopus (16) Google Scholar), suggests that in vitro refolding provides a means of modulating the quaternary structure of these proteins. Here, we exploit this phenomenon to examine the structural and functional properties of α-crystallins with identical sequence yet differing quaternary structure. Using novel mass spectrometry (MS) approaches, we show that not only do these proteins exist as different sized oligomers, but also that these oligomers have variable building blocks. Moreover, we correlate the loss of dimeric substructure with an augmentation of substrate binding and suggest that it is these changes in local oligomeric organization that provide the molecular mechanism for sHSP regulation. Expression and Purification—The expression vector pET21b(+) containing the genes for either bovine αA-crystallin or human αB-crystallin was introduced into the BL21(DE3) strain of Escherichia coli by standard methods. Protein expression and purification were performed using methods described previously (13Horwitz J. Huang Q.L. Ding L. Bova M.P. Methods Enzymol. 1998; 290: 365-383Crossref PubMed Scopus (194) Google Scholar). 1-Anilino-8-naphthalenesulfonic Acid (ANS) Binding—αA and αB solutions of 13.5 mg/ml were used. To effect unfolding of the protein, 12 mg of urea was added to 20 μl of each solution, giving a final volume of 30 μl (6.67 m urea), followed by incubation at room temperature for 30 min. The proteins were refolded by dilution to 0.2 mg/ml with 1320 μl of phosphate-buffered saline (PBS; 100 mm sodium chloride and 50 mm phosphate, pH 7.2). Matching controls were prepared by adding the urea after dilution. ANS (5 μl of a 20 mm stock solution in methanol) was added to each sample (resulting in a methanol concentration of 0.37% (v/v) in the samples), and the fluorescence emission was measured between 400 and 600 nm at an excitation wavelength of 370 nm using a Hitachi F4500 fluorometer. The experiment was also performed, as described above, using 200 mm ammonium acetate instead of PBS as diluent. Kynurenine Binding—60 mg of urea was added to 100 μl of 4 mg/ml αB in PBS and left at room temperature for 30 min. The protein was refolded by dilution to 1 ml with PBS. Matching controls were prepared by adding the urea after dilution. Urea was removed from the diluted solutions by five consecutive concentration and dilution steps using Biomax concentrators (Millipore). 25 μl of kynurenine (5 mg/ml in PBS) and 2 μl of chloroform were added before being sealed and incubated at 37 °C for 14 days. No significant proteolysis was observed to occur by MS analysis. MS Analysis—Nanoelectrospray MS of the kynureninemodified αB was performed using a Q-ToF Ultima spectrometer (Waters). Samples were desalted and denatured using C18 ZipTips (Millipore) prior to analysis. Tandem MS (MS/MS) experiments were performed on a modified Q-ToF 2 instrument as described previously (11Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (208) Google Scholar). Prior to analysis, ∼2 m g of α-crystallin (30 mg/ml in PBS with or without 6.67 m urea) was loaded onto a Superdex 200 gel filtration column (GE Healthcare) and eluted at 0.3 ml/min with 200 mm ammonium acetate. Chromatography resulted in refolding and/or buffer exchange. Chaperone Assay—Apo-α-lactalbumin (0.5 mg/ml final concentration) was mixed with αB or recombinant αB(αBR; both at 0.24 mg/ml final concentration) in the presence of 20 mm dithiothreitol. The final sample volumes were 100 μl in 200 mm ammonium acetate. The mixtures and a control of apo-α-lactalbumin were transferred to identical cuvettes and placed in a heated (37 °C) multicell block, and the apparent absorption due to light scattering at 360 nm was monitored using a Cary 400 Scan spectrophotometer. In Vitro Refolding Enhances the Substrate Affinity of αB-crystallin via Increased Surface Exposure—To structurally reorganize recombinant αB, we subjected the protein to in vitro refolding from a high concentration of urea. We have termed this protein αBR. To investigate any surface changes in the oligomers associated with this refolding, we employed two molecular probes: ANS, which fluoresces in a hydrophobic environment, and the reactive small molecule kynurenine, which can covalently bind to exposed His residues in αB (14Aquilina J.A. Truscott R.J. Biochem. Biophys. Res. Commun. 2001; 285: 1107-1113Crossref PubMed Scopus (20) Google Scholar). Fig. 1A shows the ANS fluorescence for equimolar concentrations of αB and αBR in both PBS and ammonium acetate. In both cases, the fluorescence intensity of the ANS probe was greater for the refolded protein relative to the original, indicating that a small but significant increase in exposed hydrophobic surface area had occurred. This is in agreement with a recent study examining the biophysical properties of α-crystallin (12Saha S. Das K.P. Protein J. 2007; 26: 315-326Crossref PubMed Scopus (16) Google Scholar). Fig. 1B shows the stoichiometries of relative binding of kynurenine to the surface-exposed His residues of αB, with αBR demonstrating ∼70% more total binding. Furthermore, whereas only doubly modified chains were identified in the case of αB, a distinct proportion of αBR was triply modified. These two probing techniques demonstrate that the refolded proteins are sufficiently different in structure such that they are more susceptible to both ANS binding and kynurenine modification. To assess the effect of this structural reorganization on sHSP function, αB and αBR were assayed for their ability to prevent precipitation of the aggregation-prone substrate, reduced apo-α-lactalbumin (Fig. 1C). In the absence of chaperone, a rapid increase in apparent absorbance due to light scattering was observed, indicative of the aggregation of apo-α-lactalbumin. When αB was added, the onset of apo-α-lactalbumin aggregation was considerably delayed, demonstrating the protective ability of this protein. αBR was found to further reduce the level of aggregation, suggesting that αBR has a higher substrate affinity than αB. Therefore, taking our chaperone and surface probing experiments together, it appears that in vitro refolding results in an increase in substrate affinity or capacity via the exposure of new surfaces. MS/MS Characterizes the Polydisperse sHSP Assemblies—Over the last decade, MS analysis of intact protein assemblies has become a valuable addition to the structural biologists' tool kit, allowing the determination of both structural and dynamical parameters (15Robinson C.V. Sali A. Baumeister W. Nature. 2007; 450: 973-982Crossref PubMed Scopus (411) Google Scholar). To examine the quaternary arrangement of the α-crystallins, we used a MS/MS approach we previously developed whereby the range of oligomers that comprise the polydisperse ensemble may be identified (11Aquilina J.A. Benesch J.L.P. Bateman O.A. Slingsby C. Robinson C.V. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10611-10616Crossref PubMed Scopus (208) Google Scholar). Fig. 2A shows MS/MS spectra of αBR resulting from the selection and activation of ions in the peak at m/z 10,080 (supplemental Fig. S1). This peak corresponds to all oligomers, each carrying twice as many charges as subunits. At an accelerating voltage of 110 V, some undissociated ions remain, but two additional areas of signal are also observed, in the ranges of m/z 12,000–17,000 and 17,000–28,000 (Fig. 2A, lower panel). At 170 V, the same groups of peaks are observed; however, those at higher m/z dominate the spectrum (Fig. 2A, upper panel). These two ranges correspond to singly and doubly stripped oligomers, i.e. parent oligomers having lost one or two monomers, respectively. Monitoring the relative abundance of ions as a function of the accelerating voltage revealed that this dissociation is a sequential process and allowed us to determine the voltages at which the different dissociation steps occur (Fig. 2B). Fig. 2C shows an expansion of the area of the spectrum where doubly stripped oligomers carrying two charges less than subunits are found, i.e. [αBR(n-2)][n-4]+, where n is the number of subunits in the original parent oligomers. Doubly stripped species of 23–32 subunits, which correspond to original oligomers with 25–34 subunits, are clearly observed. In this way, not only can the different oligomeric species that comprise the heterogeneous assembly be identified, despite differing by <5% in mass, but also, from their intensities, their relative abundances can be quantified. In Vitro Refolding of αA and αB Results in the Loss of Dimeric Substructure—Fig. 3A shows the oligomeric distribution of αA and αB prior to and after in vitro refolding. These histograms were obtained from MS/MS experiments as described above. Intensities for peaks corresponding to the charge states of the different doubly stripped oligomers were quantified, and from these, the relative abundances of the original oligomers were obtained. αA and αB exhibit a distribution of oligomeric stoichiometries centered on a 26-mer and a 28-mer, respectively. Moreover, both proteins display a marked preference toward assembling into oligomers with an even number of subunits. αAR and αBR are centered on a 24-mer and 28-mer, respectively, and are therefore comparable with their wild-type equivalents in number of subunits. Notably, however, these proteins have lost their preference for forming even-numbered species. Thus, the dimeric substructure of the wild-type proteins, evidenced by an excess of oligomers with an even number of subunits, is lost upon refolding. Distinct Substructures within Protein Types Are Found by Gas-phase Dissociation—To further investigate the difference between the quaternary organization of these proteins, we employed a recently developed method of probing substructure by MS (16Benesch J.L.P. Aquilina J.A. Ruotolo B.T. Sobott F. Robinson C.V. Chem. Biol. 2006; 13: 597-605Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). This approach involves a detailed examination of the dissociation pathway of protein assemblies during MS/MS. Specifically, we examined the ease of the dissociation step in which an additional monomer is removed from the singly stripped oligomers, the rationale being that different local quaternary arrangements might effect differences in dissociation behavior. The percentage of species that are doubly stripped (relative to singly stripped) for the four different proteins at a range of accelerating voltages, representing the ease of gas-phase removal of the second subunit, is in Fig. they have been to they an even or odd number of subunits. In all cases, the percentage of doubly stripped oligomers a the we obtained the in αAR and αBR, we not a significant difference between the dissociation pathway for and even-numbered oligomers, suggesting is difference in substructure between This with the from the which suggests that their organization is monomeric (Fig. In the case of αA and αB, however, is a significant difference between the and even-numbered oligomers, the from singly to doubly stripped oligomers at higher in the case of the even-numbered oligomers. These results suggest that is a difference in the substructure of and oligomers. Furthermore, the of the are for the oligomers of both αB and αBR and for αA and whereas they are different in the case of the even-numbered (supplemental S1). This suggests that the substructure of the different oligomers of αBR as well as αB with an odd of subunits is the but that a different substructure is for αB with an even number of subunits. The same properties were observed for αAR and this with the in Fig. that dimeric substructure is lost upon in vitro refolding, we suggest that wild-type αA and αB exist as a of the of which are oligomers with monomeric substructure and even-numbered oligomers with dimeric we have that the substructure of αA and αB can be by of αB (9Aquilina J.A. Benesch J.L.P. Ding L.L. Yaron O. Horwitz J. Robinson C.V. J. Biol. Chem. 2004; 279: 28675-28680Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, S. Horwitz J. Aquilina J.A. Benesch J.L.P. Robinson C.V. J.A. Biochem. J. 2007; PubMed Scopus Google and of αA J.A. Benesch J.L.P. Ding L.L. Yaron O. Horwitz J. Robinson C.V. J. Biol. Chem. 2005; 280: 14485-14491Abstract Full Text Full Text PDF PubMed Scopus (95) Google a and an increase in the of dimeric respectively. Here, we have this work to show that the proteins exist in an apparent between different with dimeric or monomeric building (Fig. This is in addition to both these proteins a polydisperse in an range of oligomeric (4Horwitz J. Exp. Eye Res. 2003; 76: 145-153Crossref PubMed Scopus (571) Google Scholar). we propose a new of these proteins, that they are heterogeneous in of both local and quaternary organization and that it is that this is to their in this without sequence we have observed that of the dimeric sHSP substructure to form oligomers of monomeric building results in an augmentation of chaperone Moreover, the chaperone function of these two states is the monomeric state has greater exposed hydrophobic surface area and is more active in protein small changes in differences in chaperone have been observed upon of the α-crystallins R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Sci. 2004; PubMed Scopus Google Scholar). Here, we have that it is not the changes in structure by but the in substructure that they that determine chaperone recent of sHSP chaperone function suggests that sHSP oligomers exist in two with a low affinity state and a high affinity state (3Haslbeck M. Franzmann T. Weinfurtner D. Buchner J. Nat. Struct. Mol. Biol. 2005; 12: 842-846Crossref PubMed Scopus (669) Google Scholar, P. J. Mol. Biol. PubMed Scopus Google Scholar). We propose in the case of the α-crystallins, these two correspond to oligomers with dimeric and monomeric (Fig. We suggest that it is the of these two that the of the chaperone or protein this by such that dimeric substructure is This to an increase in substrate affinity by the to unfolded substrate proteins. This ability to subunits into dimers for activation an mechanism of regulating the chaperone of these is a of in which αB has been reported to be in protein deposition (7Sun Y. MacRae T.H. FEBS J. 2005; 272: 2613-2627Crossref PubMed Scopus (285) Google Scholar), due to of the chaperone with substrate proteins in M.P. Yaron O. Huang Ding L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). for this that from the work is that or some other of may the dimeric substructure of αB such that a of binding is we have previously that of αB can in a loss of dimeric preference and behavior (9Aquilina J.A. Benesch J.L.P. Ding L.L. Yaron O. Horwitz J. Robinson C.V. J. Biol. Chem. 2004; 279: 28675-28680Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). an in αB has been to in protein deposition including and P. A. J.A. J. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, P. A. P. A. D. F. F. D. M. Nat. 1998; PubMed Scopus Google Scholar). In vitro of wild-type αB that a increase in substrate affinity is responsible for forming with the proteins M.P. Yaron O. Huang Ding L. Horwitz J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We that this chaperone associated with some and is the of a in the of dimeric to monomeric substructure of αB. we the level of αB function to be by on a of this aberrant chaperone behavior the is (Fig. Thus, the chaperone function of sHSPs might be as an that in the case of some such that the chaperone to the We Horwitz and Ding for the recombinant protein and of for of the with