The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson’s disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.


Introduction
Protein homeostasis, or proteostasis, refers to the cell's inherent biological pathways and networks which ensure that proteins acquire their native and functional form with their levels being maintained within a strict regime to ensure optimal cell functionality [1,2]. When proteostasis is compromised, proteins can partially unfold, misfold, and aggregate, leading to a plethora of deleterious consequences including cell death [3]. Proteostasis dysregulation underlies more than 55 human disorders including type-II diabetes, cataract, oxidized DA [3]. Taken together, since αBc and Hsp27 are present in the dopaminergic neurons that are primarily affected in PD pathophysiology, they are potential targets for modification by DA ox .
Herein, we investigated, structurally and functionally, the interaction of DA ox with αBc and Hsp27. Upon modification and cross-linking with DA ox at an equivalent molar level, the sHsps were capable, to a degree comparable to that of the unmodified protein, of preventing amorphous and amyloid fibrillar protein aggregation, thereby corroborating the chaperones' robust nature and their ability, in a cellular context, to mitigate the deleterious effects of protein unfolding and aggregation [5][6][7]42].

Oxidized Dopamine-Modified sHsps Prevent Amorphous and Amyloid Fibrillar Protein Aggregation
Conventional methods to monitor the ability of DA ox -modified sHsps (sHsps:DA ox ) to inhibit amyloid fibril and amorphous target protein aggregation could not be employed. For example, DA ox quenches fluorescence of thioflavin T, the dye that binds to the β-sheet region of amyloid fibrils and is therefore used routinely to monitor amyloid fibril formation [43]. Furthermore, sHsps:DA ox are large oligomeric species that scatter light, which skews turbidity measurements that are used to monitor amorphous protein aggregation.
Hence, a SDS-PAGE-based method was devised to monitor the soluble component of the aggregating protein over time, independent of sHsps:DA ox . The target protein was co-incubated with the sHsps:DA ox under conditions that promoted unfolding and aggregation of the former. At each time point, the co-incubated sample was centrifuged to separate soluble protein from aggregated protein. Then, an aliquot of the soluble component (supernatant) was removed and flash frozen. Over time, a decrease in the concentration of soluble protein occurred due to the target protein aggregating. After the final time point, the frozen soluble components were thawed and run on SDS-PAGE. Due to lower mass of the aggregating target protein compared to the sHsps:DA ox , the band intensity of the target protein could be quantified and plotted against time. As expected, a decrease in the band intensity of the aggregating protein occurred over time, which enabled target protein aggregation to be monitored, without the interference of sHsps:DA ox . The normalized band intensities of the target proteins at the end of the experiment provided a comparison of the relative chaperone effectiveness of the native and DA ox -modified sHsps.
The alanine to threonine point mutation at position 53 (A53T) in αSyn has been identified in patients with familial PD [44]. Under physiological conditions, A53T αSyn aggregates to form amyloid fibrils at a faster rate than the wild-type protein [40,41]. The aggregation of A53T αSyn was monitored over 6.5 days by SDS-PAGE, in the absence and presence of the native and DA ox -modified sHsps at a 10:1 molar ratio of A53T αSyn to the native sHsp or the sHsp modified with an equivalent or five-fold molar excess of DA ox (e.g., αBc:DA ox (1:5)). The complete gels for the time course of A53T αSyn aggregation in the presence of native and DA ox -modified sHsps are presented Figure 1A,B. As described above, in all cases, the band associated with soluble A53T αSyn (at~14 kDa in mass) decreased with time. The other bands arise from αBc at~20 kDa and Hsp27 at~27 kDa along with their oligomeric forms (dimer, trimer, tetramer, etc.) due to crosslinking associated with reaction with DA ox . Large aggregates were present at the top of the gel which most likely arise from fibrillar A53T αSyn species and aggregated sHsps (more below). None of the gels exhibited intermediate species associated with A53T αSyn aggregation. Consistent with this, during chaperone action, our studies have shown that sHsps interact with the monomeric form of the target protein to prevent its association to form high molecular weight (HMW) species (e.g., [41,45]). When complete inhibition of the aggregation of an amyloid fibril-forming target protein (e.g., αSyn) occurs during chaperone interaction with sHsps, there are no HMW species formed, including a complex between the two [46].
Quantification of the reduction in the gel band from monomeric αSyn in the presence and absence of native and DA ox -modified sHsps is also shown in Figure 1A,B. Upon incubation of αBc:DA ox with A53T αSyn, there was little difference (within the error of the experiment) between the chaperone ability of αBc modified with an equivalent and fivefold molar excess of DA ox (αBc:DA ox 1:1 and 1:5) compared to native αBc (αBc:DA ox 1:0) ( Figure 1A, Table 1). Similarly, the chaperone ability of unmodified Hsp27, or modified with DA ox at the two molar ratios, to inhibit A53T αSyn aggregation was comparable within the error of the experiment ( Figure 1B, Table 1). The chaperone ability of both sHsps modified with an equivalent and a five-fold molar excess of DA ox (Supplementary Figure S1) and αBc modified with much higher molar equivalents of DA ox (1:10, 1:20 and 1:30) (Supplementary Figure S2) was investigated over 24 h with another amyloid fibril-forming protein, reduced and carboxymethylated (RCM) α-lactalbumin (αLA). The highest ratio of αBc:DA ox almost completely abrogated the chaperone ability of αBc (Supplementary Figure S2). Thus, large-scale modification of sHsps with DA ox leads to impairment of sHsp chaperone ability against RCM αLA fibril formation whereas mild DA ox modification causes little, if any, reduction in chaperone functionality against the two fibril-forming target proteins investigated.
Apo αLA undergoes well-characterized amorphous aggregation over 20-odd hours at physiological pH and temperature upon reduction of its four disulfide bonds [45]. When αBc which had been modified with DA ox at an equimolar ratio was co-incubated with αLA, the aggregation of αLA was inhibited to an equivalent degree to that of native, unmodified αBc (i.e., αBc:DA (1:0)) ( Figure 1C, Table 1). αBc modified with a five-fold higher concentration of DA ox (αBc:DA ox (1:5)) was still capable of inhibiting the aggregation of αLA but to a reduced degree compared to αBc:DA ox (1:1), suggesting that greater modification of αBc by DA ox decreases its chaperone ability. Upon Hsp27 modification with DA ox at the same levels, a comparable trend was observed to αBc:DA ox in its ability to inhibit the amorphous aggregation of αLA ( Figure 1D, Table 1).  was allowed to aggregate in 50 mM phosphate, 150 mM NaCl, pH 7.4 at 37 °C with shaking at 1,500 rpm, in a total volume of 300 μL. Every ~12 h, the sHsp and αSyn co-incubation was centrifuged at 19,391 rcf for 15 min at room temperature. A volume of 15 μL of the supernatant was flash frozen and run on SDS-PAGE in a chronological order of time under standard conditions. The band at ~14 kDa in (A,B) reflects soluble monomeric αSyn and are data from one of the replicates. The band intensity was quantified using ImageJ software. The αSyn band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean. 100 μM Apo αLA with or without DAox-modified 4 μM αBc (C) or Hsp27 (D) was allowed to aggregate in 50 mM phosphate, 100 mM NaCl, 2.5 mM EDTA, pH 7.4 at 37 °C with shaking at 200 rpm. Every 45 min, the sHsp and αLA co-incubation was centrifuged at 19,391 rcf for 15 min at room temperature. A volume of 20 μL of the supernatant was flash frozen, and run on SDS-PAGE in a chronological order of time under standard conditions. The bands displayed in (C,D) reflect soluble monomeric αLA at ~14 kDa. The band intensity was then quantified using ImageJ software. The αLA band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean.

DAox Promotes Cross-Linking of sHsps
The structural alterations to the sHsps as a result of DAox modification were investigated by a variety of biophysical methods. Via SDS-PAGE, DAox modification of αSyn leads to the formation of a well-defined, characteristic cross-link fingerprint of monomeric, dimeric, trimeric and larger oligomeric species [50]. The oligomers are not broken down into the monomer by SDS and boiling, consistent with covalent cross-links being responsible for oligomerization. Similarly, as determined by SDS-PAGE, both αBc:DAox and Hsp27:DAox formed higher molecular weight (HMW) species with a well-defined oligomer fingerprint of dimer, trimer, tetramer, etc. (Figure 2A, Table 2), a pattern that was not affected by the presence of an excess of the reducing agent, dithiothreitol (DTT) (Supplementary Figure S4). At a five-fold molar excess of DAox relative to the sHsps compared The band at~14 kDa in (A,B) reflects soluble monomeric αSyn and are data from one of the replicates. The band intensity was quantified using ImageJ software. The αSyn band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean. 100 µM Apo αLA with or without DA ox -modified 4 µM αBc (C) or Hsp27 (D) was allowed to aggregate in 50 mM phosphate, 100 mM NaCl, 2.5 mM EDTA, pH 7.4 at 37 • C with shaking at 200 rpm. Every 45 min, the sHsp and αLA co-incubation was centrifuged at 19,391 rcf for 15 min at room temperature. A volume of 20 µL of the supernatant was flash frozen, and run on SDS-PAGE in a chronological order of time under standard conditions. The bands displayed in (C,D) reflect soluble monomeric αLA at~14 kDa. The band intensity was then quantified using ImageJ software. The αLA band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean.
For both sHsps interacting with amyloid fibril-forming αSyn ( Figure 1A,B) and RCM αLA (Supplementary Figures S1 and S2) and amorphously aggregating apo αLA ( Figure 1C,D), a reduction in monomeric sHsps occurred with time, as monitored by densitometry (Supplementary Figure S3). For the DA ox -modified sHsps, some of this decrease in intensity arises from oligomerization of the sHsps due to cross-linking. Under the relatively rapid stirring conditions at pH 7 and 37 • C used for all these experiments (apart from those with RCM αLA), native αBc forms amyloid fibrils over 28 h (Kumar et al., unpublished results). Aggregation arises from shear effects that cause partial protein unfolding and amyloid fibril formation [47]. Consistent with this, mammalian sHsps, including αBc and Hsp27, have a high amyloid fibril-forming propensity, particularly within their ACD [48]. The day-long timeframe for sHsp fibril formation provides an explanation for the loss with time of native and DA ox -modified sHsps in the experiments summarized in Supplementary Figure S3. Our previous studies showed that in its amyloid fibrillar state, αBc retains, and in some cases increases, chaperone activity in preventing the amorphous and fibrillar aggregation of target proteins [49]. Thus, even though both sHsps form amyloid fibrils themselves during the time-course of the chaperone assays, their alteration in chaperone ability arises from DA ox modification.

DA ox Promotes Cross-Linking of sHsps
The structural alterations to the sHsps as a result of DA ox modification were investigated by a variety of biophysical methods. Via SDS-PAGE, DA ox modification of αSyn leads to the formation of a well-defined, characteristic cross-link fingerprint of monomeric, dimeric, trimeric and larger oligomeric species [50]. The oligomers are not broken down into the monomer by SDS and boiling, consistent with covalent cross-links being responsible for oligomerization. Similarly, as determined by SDS-PAGE, both αBc:DA ox and Hsp27:DA ox formed higher molecular weight (HMW) species with a well-defined oligomer fingerprint of dimer, trimer, tetramer, etc. (Figure 2A, Table 2), a pattern that was not affected by the presence of an excess of the reducing agent, dithiothreitol (DTT) (Supplementary Figure S4). At a five-fold molar excess of DA ox relative to the sHsps compared to an equivalent concentration of DA ox , SDS-PAGE showed that the monomeric band decreased in intensity concomitantly with an increase in intensity of the HMW bands. Thus, a higher concentration of DA ox led to greater cross-linked oligomerization of the sHsps.
The oligomeric state of sHsps:DA ox in solution, in the absence of the denaturant SDS, was investigated by size-exclusion chromatography. Using a buffer mimicking physiological conditions, sHsps:DA ox , particularly Hsp27, eluted from the size-exclusion column earlier than the native sHsps, consistent with them being larger in mass than the native sHsps ( Figure 2B,C). Both sHsps:DA ox exhibited an increase in intensity of their size-exclusion peak compared to their unmodified counterparts, which is attributed to the aromatic ring of DA ox also absorbing at 280 nm [26], and being bound to and incorporated into the sHsps. Negative-stained transmission electron microscopy (TEM) revealed that there was an increase in the overall mean diameter of the αBc:DA ox and Hsp27:DA ox spherical oligomers in comparison to native αBc and Hsp27 ( Figure 2D,E). For αBc, this increase in diameter was from 11.9 ± 2.7 nm to 14.5 ± 5.4 nm, and for Hsp27, the increase was from 12.5 ± 2.6 nm to 17.3 ± 3.3 nm upon modification with DA ox . The spherical morphology of the oligomeric sHsps was not altered upon modification with DA ox . to an equivalent concentration of DAox, SDS-PAGE showed that the monomeric band decreased in intensity concomitantly with an increase in intensity of the HMW bands. Thus, a higher concentration of DAox led to greater cross-linked oligomerization of the sHsps.  The oligomeric state of sHsps:DAox in solution, in the absence of the denaturant SDS, was investigated by size-exclusion chromatography. Using a buffer mimicking physiological conditions, sHsps:DAox, particularly Hsp27, eluted from the size-exclusion column earlier than the native sHsps, consistent with them being larger in mass than the native sHsps ( Figure 2B,C). Both sHsps:DAox exhibited an increase in intensity of their size-exclusion peak compared to their unmodified counterparts, which is attributed to the aromatic ring of DAox also absorbing at 280 nm [26], and being bound to and incorporated into the sHsps. Negative-stained transmission electron microscopy (TEM) revealed that

sHsps:DA ox Retain Their β-Sheet Secondary Structure in the ACD
Characterization of sHsps:DA ox using fluorescence spectroscopy, both intrinsic (i.e., fluorescence of the two tryptophan residues in the NTR of αBc) and extrinsic (i.e., 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence to monitor exposed hydrophobicity), was not possible due to interference from the fluorescence of the incorporated DA ox cross-links in the sHsps (Supplementary Figure S5). However, far-UV circular dichroism (CD) spectroscopy was used to monitor the effects of DA ox on the overall secondary structure of the sHsps. The β-sheet-rich ACD of αBc interacts with amyloid-fibril forming proteins to prevent their aggregation [12]. Native sHsps have a CD spectrum indicative of the presence of significant anti-parallel β-sheet within the ACD, i.e., a positive peak at 198 nm and a broad negative minimum at~218 nm [51,52]. When both sHsps were modi-fied with DA ox , there was no substantial change in the overall CD spectrum ( Figure 3A,B), consistent with a retention of the β-sheet secondary structure in the ACD of αBc and Hsp27. links in the sHsps (Supplementary Figure S5). However, far-UV circular dichroism (CD) spectroscopy was used to monitor the effects of DAox on the overall secondary structure of the sHsps. The β-sheet-rich ACD of αBc interacts with amyloid-fibril forming proteins to prevent their aggregation [12]. Native sHsps have a CD spectrum indicative of the presence of significant anti-parallel β-sheet within the ACD, i.e., a positive peak at ~198 nm and a broad negative minimum at ~218 nm [51,52]. When both sHsps were modified with DAox, there was no substantial change in the overall CD spectrum ( Figure 3A,B), consistent with a retention of the β-sheet secondary structure in the ACD of αBc and Hsp27.

Lysine Residues Are Involved in the Formation of DAox-Induced HMW Species of sHsps
As there was little change in β-sheet ACD secondary structure upon modification of the sHsps with DAox, residues in the mostly unstructured NTR and CTR could be a target for DAox. The highly solvent exposed, unstructured, flexible and short C-terminal extension in mammalian sHsps [17,18,53] is a prime candidate for modification by DAox. As mentioned above, DA modifies a range of amino acids resulting in cross-linking of peptides and proteins [24]. In particular, the ε-amino group of the lysine side chain reacts with the catechol ring of a DAox intermediate [24,54], for example the DA o-quinone, to form a Schiff base linkage [55]. The C-terminal extension in both αBc and Hsp27 is well served with lysine residues, for example at their extreme C-terminus (Lys174 and Lys175 in αBc and Lys205 in Hsp27).
The lysine residues of αBc and Hsp27 were selectively dimethylated. The dimethylation of all lysine residues (and the amino terminus) of both sHsps was confirmed by mass spectrometry (Supplementary Table S1). The methylated sHsps were reacted with DAox, as described above, to determine whether the sHsp lysine residues were involved in DAoxmediated cross-linking ( Figure 4). To determine the extent of cross-linking and hence

Lysine Residues Are Involved in the Formation of DA ox -Induced HMW Species of sHsps
As there was little change in β-sheet ACD secondary structure upon modification of the sHsps with DA ox , residues in the mostly unstructured NTR and CTR could be a target for DA ox . The highly solvent exposed, unstructured, flexible and short C-terminal extension in mammalian sHsps [17,18,53] is a prime candidate for modification by DA ox . As mentioned above, DA modifies a range of amino acids resulting in cross-linking of peptides and proteins [24]. In particular, the ε-amino group of the lysine side chain reacts with the catechol ring of a DA ox intermediate [24,54], for example the DA o-quinone, to form a Schiff base linkage [55]. The C-terminal extension in both αBc and Hsp27 is well served with lysine residues, for example at their extreme C-terminus (Lys174 and Lys175 in αBc and Lys205 in Hsp27).
The lysine residues of αBc and Hsp27 were selectively dimethylated. The dimethylation of all lysine residues (and the amino terminus) of both sHsps was confirmed by mass spectrometry (Supplementary Table S1). The methylated sHsps were reacted with DA ox , as described above, to determine whether the sHsp lysine residues were involved in DA ox -mediated cross-linking ( Figure 4). To determine the extent of cross-linking and hence HMW oligomerization induced by DA ox , the monomeric band intensity in SDS-PAGE was quantified. The intensity of the monomeric band retained upon modification was compared with that of the monomeric band intensity of the unmodified sHsps. For both methylated and non-methylated (native) sHsps, a gradual loss of the monomeric band intensity (and hence an increase in HMW species) was observed as the concentration of DA ox increased. Methylated αBc retained comparable monomeric band intensities to its non-methylated counterpart up to a 10-fold molar excess of DA ox . At a 20-fold molar excess of DA ox , substantially more monomer was retained for methylated αBc compared to its non-methylated counterpart ( Figure 4A,B). Methylated Hsp27 retained comparable monomeric band intensities up to a molar equivalent of DA ox . However, at a five-fold molar excess of DA ox , substantially more monomer was retained for methylated Hsp27 ( Figure 4C,D). Similar to methylated αBc, significantly more of the monomeric band was retained when the lysine residues were dimethylated in comparison to non-methylated Hsp27 upon reaction with a 20-fold molar excess of DA ox . Overall, methylated Hsp27 was less susceptible to modification by DA ox than methylated αBc. Thus, lysine methylation of the sHsps leads to less cross-linking due to reaction with DA ox , particularly at higher ratios of sHsp:DA ox , more so for Hsp27 than αBc. The implication is that lysine residues (via their ε-amino groups) are involved in reacting with DA ox to form covalent cross-links resulting in HMW sHsp species. excess of DAox, substantially more monomer was retained for methylated Hsp27 ( Figure  4C,D). Similar to methylated αBc, significantly more of the monomeric band was retained when the lysine residues were dimethylated in comparison to non-methylated Hsp27 upon reaction with a 20-fold molar excess of DAox. Overall, methylated Hsp27 was less susceptible to modification by DAox than methylated αBc. Thus, lysine methylation of the sHsps leads to less cross-linking due to reaction with DAox, particularly at higher ratios of sHsp:DAox, more so for Hsp27 than αBc. The implication is that lysine residues (via their ε-amino groups) are involved in reacting with DAox to form covalent cross-links resulting in HMW sHsp species.

Acidic pH and the Antioxidant, Glutathione, Rescue sHsps from DA ox -Induced Oligomerization
The protective mechanisms in the brain that prevent the oxidation of DA were mimicked in vitro to determine if they discouraged DA-induced cross-linking of sHsps. DA readily oxidizes at physiological pH. Hence, in vivo, DA is sequestered into acidic vesicles, thereby maintaining its protonated form [21,22]. The sHsps were reacted with DA in buffer at pH values of 2.5, 3.6, 4.8, 5.9, 7.1, 8.3 and 10.5, followed by examination via SDS-PAGE. At acidic pH (e.g., pH 2.5), no HMW sHsp species formed, i.e., only the monomeric form was observed ( Figure 5A,B). With increasing pH, a greater number and intensity of HMW bands were observed for both sHsps. The HMW species were most prominent around physiological pH, i.e., at pH 7.1 and 8.3.

DAox-Modified sHsps Are Not Toxic to Cells
Finally, the effect of sHsps:DAox on cell viability was investigated. When HEK293T cells, which possess neuronal-like characteristics due to the presence of neurofilaments, vimentin and other proteins characteristic of a cell line with neuronal lineage [58], were treated with 1, 5, 10 and 20 μM of αBc:DAox ( Figure 6A) or Hsp27:DAox ( Figure 6B), there was no significant difference between cell viability in the presence of unmodified αBc or a phosphate-buffered saline (PBS) blank. The percentage values of cell viability were normalized to those of cells without sHsps or PBS added. The values in Figure 6 greater than 100% arise because the presence of sHsps increased cell viability relative to the controls, i.e., sHsps had a slight protective effect on the cells which is consistent with their ability as molecular chaperones to stabilize proteins. It is concluded that modification of sHsps with DAox did not induce cell toxicity over the experimental timeframe.  GSH is a reducing agent that is present at mM levels in the brain and other organs to protect from oxidative stressors such as DA ox . The concentration of antioxidants such as GSH declines with age, which contributes to the pathogenesis of diseases associated with oxidative stress including PD [23,56]. Accordingly, varying in vitro concentrations of GSH were co-incubated with the sHsps and DA at pH 7.4 prior to the formation of sHsps:DA ox ( Figure 5C). For both αBc:DA ox and Hsp27:DA ox , substantially fewer HMW species were visible on SDS-PAGE in the presence of elevated levels of GSH. Thus, when 2 mM GSH was co-incubated with Hsp27 and DA ox , no species greater in mass than the dimer were observed. At the same concentration of GSH in the presence of αBc and DA ox , HMW species greater in mass than the dimer were visible via SDS-PAGE, but to a lesser extent in comparison to when the αBc:DA ox co-incubation lacked GSH. Thus, for both sHsps, GSH at mM levels provided protection against DA ox modification and cross-linking. GSH inhibits the oxidation of DA and hence its modification of the sHsps to form covalent cross-links. If formed, GSH does not dissociate the sHsp:DA ox oligomers which are very stable as exemplified in Figure 2A where, even after treatment with a reducing agent (DTT), SDS and boiling, they do not dissociate. Similarly, Supplementary Figure S4 shows that the addition of DTT, at a 10-fold higher concentration in comparison to normal SDS-PAGE protocols, after the formation of the sHsp oligomers, did not lead to their dissociation.
The effect of GSH on αBc and Hsp27 should also be considered. In their native state, sHsps are highly dynamic, oligomeric species that are undergoing continuous subunit exchange, either via their monomeric or dimeric forms [5][6][7][8][9][10]. The presence of GSH in the surrounding medium is unlikely to affect the dissociation of sHsps. Most likely because of subunit exchange, DA ox reacts with the dissociated sHsp species. GSH is unlikely to react with αBc as it does not contain any free sulfhydrl groups (it has no cysteine residues). Hsp27 has a single cysteine (Cys137) that could be glutathionylated. However, this is unlikely to have much effect on the protein as studies of modification at this site (e.g., mutation, S-thiolation and reductive methylation) did not reveal conformational change, let alone large alteration in the oligomeric state of Hsp27 [57].

DA ox -Modified sHsps Are Not Toxic to Cells
Finally, the effect of sHsps:DA ox on cell viability was investigated. When HEK293T cells, which possess neuronal-like characteristics due to the presence of neurofilaments, vimentin and other proteins characteristic of a cell line with neuronal lineage [58], were treated with 1, 5, 10 and 20 µM of αBc:DA ox ( Figure 6A) or Hsp27:DA ox ( Figure 6B), there was no significant difference between cell viability in the presence of unmodified αBc or a phosphate-buffered saline (PBS) blank. The percentage values of cell viability were normalized to those of cells without sHsps or PBS added. The values in Figure 6 greater than 100% arise because the presence of sHsps increased cell viability relative to the controls, i.e., sHsps had a slight protective effect on the cells which is consistent with their ability as molecular chaperones to stabilize proteins. It is concluded that modification of sHsps with DA ox did not induce cell toxicity over the experimental timeframe.

DAox-Modified sHsps Are Not Toxic to Cells
Finally, the effect of sHsps:DAox on cell viability was investigated. When HEK293T cells, which possess neuronal-like characteristics due to the presence of neurofilaments, vimentin and other proteins characteristic of a cell line with neuronal lineage [58], were treated with 1, 5, 10 and 20 μM of αBc:DAox ( Figure 6A) or Hsp27:DAox ( Figure 6B), there was no significant difference between cell viability in the presence of unmodified αBc or a phosphate-buffered saline (PBS) blank. The percentage values of cell viability were normalized to those of cells without sHsps or PBS added. The values in Figure 6 greater than 100% arise because the presence of sHsps increased cell viability relative to the controls, i.e., sHsps had a slight protective effect on the cells which is consistent with their ability as molecular chaperones to stabilize proteins. It is concluded that modification of sHsps with DAox did not induce cell toxicity over the experimental timeframe.

Discussion
PD is characterized by an increase in DA ox [20] and protein aggregation, particularly of αSyn. Intracellularly, protein aggregation is mitigated by sHsps such as αBc and Hsp27 which form the first line of defense against proteostatic dysregulation. Of relevance to this study, sHsps are immunopositive in Lewy bodies and are overexpressed in disease states such as PD [37][38][39]. Despite the importance of sHsps in cellular proteostasis, how DA ox affects their structure or function has not been investigated to date.
DA is inherently unstable in the presence of oxygen at physiological pH whereby it undergoes a cascade of reactions commencing with autoxidation leading to deprotonation of its hydroxyl groups. A further one electron oxidation leads to the formation of a DA o-semiquinone radical, which immediately forms a DA o-quinone and then undergoes cyclisation into an aminochrome. The aminochrome further reacts to form 5,6-dihydroxyindole, and then indole-5,6-quinone which self-polymerizes [21,22]. In the human brain, the polymerized DA ox is the major component of neuromelanin, a dark pigment found within the cytoplasm of cells [21,59]. Neuromelanin also contains lipids and proteins related to protein degradation, autophagy, lysosomal and ubiquitin proteasome degradation systems including αBc and Hsp27, suggesting that neuromelanin is a storage site for these molecules which cannot be adequately degraded by the cell's existing mechanisms [60,61]. Although the accumulation of neuromelanin progressively increases with age, in PD, there is a direct correlation between the amount of neuromelanin present in the cell and its death [62,63]. Hence, the oxidation of DA plays a key role in the pathogenesis of PD.
The present study demonstrated that the sHsps retain, to a significant degree, their chaperone activity after modification with DA ox at equivalent or five-fold excess levels, against the amorphous aggregation of αLA and the amyloid fibrillar aggregation of A53T αSyn and RCM αLA. In general, chaperone ability was reduced at the higher level of DA ox modification, more so at very high levels of DA ox modification in preventing RCM αLA fibril formation. There is a plethora of evidence illustrating the reduction in protein function when modified with DA ox . For example, the E3 ligase activity of the protein parkin, a key enzyme in the proteosomal system, is inactivated upon modification with DA o-quinone [25]. In cells, upon the modification of actin and tubulin with aminochrome, the cytoskeleton of the cell was disrupted and microtubule polymerization was inhibited [27,64]. Most relevant to this study, αSyn forms unstructured adducts and oligomers upon the interaction with DA ox [32,33,[65][66][67]. Recently, there has been evidence suggesting that, in comparison to its unmodified counterpart, these αSyn oligomers are more effective at cross-seeding with Tau, a protein whose aggregation to form intracellular tangles is associated with Alzheimer's disease [68], a process which may further exacerbate the disease's pathogenesis. In a neuroblastoma cell line, PD-related proteins such as ubiquitin carboxy-terminal hydrolase L1 and DJ-1 were found to be conjugated with DA o-quinone [69]. In addition, a decrease in mitochondrial proteins such as MtCK and mitofilin occurred [69], which may cause a decrease in mitochondrial function to mitigate oxidative stress. Similarly, the structures formed during the oxidation of DA directly degenerate dopaminergic neurons [70] and induce neuroinflammation [71]. Hence, for αBc and Hsp27 to retain chaperone activity after cross-linking with DA ox implies that they are highly robust chaperone proteins. Previous studies are consistent with this characteristic of sHsps. For example, when αBc was covalently bound to a solid-phase support [72] or formed amyloid fibrils [49], it still retained chaperone activity. However, the decrease in chaperone effectiveness of both sHsps at high molar excess of DA ox implies that large-scale modification (cross-linking) of sHsps reduces their chaperone functionality.
When the sHsps:DA ox were analyzed by SDS-PAGE after heating and the addition of reducing agent, they demonstrated a well-ordered pattern of monomer, dimer, trimer, tetramer, pentamer, etc. (Figure 2A, Table 2), as observed for other DA ox -modified proteins such as αSyn [50] and parkin [25]. Even after the addition of a 10-fold molar excess of DTT, the SDS-PAGE pattern was not altered (Supplementary Figure S4), i.e., the cross-linking was not affected by reducing agent. Moreover, as the concentration of DA ox increased, the band intensity of the monomeric band for both sHsps decreased, and the higher molecular weight bands increased. The decrease in chaperone activity against αLA amorphous aggregation for the sHsps modified with a five-fold excess of DA ox may be due to the decrease in concentration of the sHsp monomer, as the monomer is proposed to be the most chaperone-active species, compared to the sHsp oligomer [9]. The doubling of the Hsp27 HMW bands in SDS-PAGE (Figures 2A and 4C) may be due the modification of Cys137 in the ACD of Hsp27. Cysteine residues are highly susceptible to modification by DA ox , in which a carbon atom of the DA o-quinone readily attacks the sulfhydrl group on the cysteine sidechain, forming a 5-S-cysteinyldopamine adduct [24][25][26]. In addition, αSyn, which does not contain a cysteine residue, was not as reactive towards DA o-quinone or other species in the DA oxidation cascade [26]. As αBc does not contain a cysteine residue, no doubling of its HMW bands was observed in SDS-PAGE (Figures 2A and 4A). Cys137 is responsible for regulating Hsp27 dimerization, and responding to cellular oxidative stress [73,74]. DA adduct formation to this key cysteine may affect the dissociation of the Hsp27 oligomer, with a concomitant effect on its chaperone function and its regulation of cellular stress. There was no substantial alteration in the far-UV CD spectra of both sHsps upon modification with DA ox (Figure 3), suggesting that the antiparallel β-sheet characteristics of the ACD remained intact, along with the structure of the mostly disordered NTR and CTR, which is consistent with the retention, predominantly, of chaperone activity upon mild DA ox modification.
Both αBc:DA ox and Hsp27:DA ox eluted earlier from a size-exclusion column than their native counterparts, consistent with their larger size ( Figure 2B,C). In agreement with this, TEM revealed that sHsps:DA ox were larger in diameter than their native counterparts ( Figure 2D,E). Moreover, there was no significant alteration in the spherical shape of the oligomeric sHsps upon modification with DA ox compared to their native counterparts [75]. In contrast, the destabilized R120G mutant of αBc, which causes desmin-related myopa-thy, also has an increase in size as observed for αBc:DA ox but has significantly reduced chaperone ability [76].
The CTR of mammalian sHsps has a flexible, unstructured C-terminal extension at its extremity which is highly solvent exposed [17,18,53,77], making this region potentially susceptible to reaction with DA ox . For both αBc and Hsp27, there are more lysine residues in the CTR in comparison to the NTR and the ACD; 23% and 8.7% of residues are lysine in the CTR of αBc and Hsp27 compared to 7.2% and 5.0%, respectively, in the ACD. Moreover, lysine is the final amino acid of both proteins. sHsps with dimethylated lysine residues were less susceptible to DA ox modification ( Figure 4). As mentioned above, lysine readily reacts via its ε-amino group with the DA o-quinone to form a Schiff base linkage [24,55]. Similarly, upon reaction of α-crystallin proteins with galactose, lysine residues in the Cterminal extension were covalently modified via a Schiff base linkage between the aldehyde group of galactose and the lysine ε-amino group [78], a modification which is common in the diabetic eye lens. In vivo, lysine residues of sHsps could react with and thereby quench damaging oxidants, and in doing so, provide another function for these proteins in maintaining proteostasis. DA ox modification is not limited to lysine residues. Other amino acid residues are readily modified by DA o-quinone such as cysteine, methionine and histidine [24], with some being more reactive to one intermediate in the DA oxidation cascade than others [26]. Cys137 in the ACD of Hsp27 is a prime candidate for modification by DA ox . Thus, as discussed above, reaction of DA ox with Cys137 in Hsp27 could have a similar redox-regulating capability [9]. Furthermore, adduct formation via DA ox is not the only modification (e.g., hydrogen bonding) that could affect sHsps [24,33].
In vivo, cellular mechanisms which prevent DA oxidation potentially would discourage the formation of cross-linked, HMW species of sHsps:DA ox . Thus, DA secreted from the presynaptic terminals in neurons is immediately sequestered into strongly acidic, monoaminergic vesicles called VMAT2, ensuring that the hydroxyl groups of DA are fully protonated, and hence minimizing the possibility of oxidation [79]. Excess DA remaining in the cytosol, which is at neutral pH, is enzymatically degraded [21]. Consistent with this, in vitro, no cross-linked HMW sHsps formed at pH 2.5 in the presence of DA ox ( Figure 5A,B). Around pH 7.1 and 8.3, oligomer formation was most pronounced. GSH is present at mM levels in the brain to counteract the effects of oxidative stress. The presence of GSH at these levels decreased the in vitro formation of sHsps:DA ox oligomers ( Figure 5C), with Hsp27 exhibiting less susceptibility to modification by DA ox than αBc. The effect of GSH in protecting against DA-induced cytotoxicity is apparent from a variety of studies. αSyn oligomers formed with GSH-conjugated aminochrome are non-toxic [80] in comparison to those formed in the presence of DA ox [36]. In addition, GSH transferase, an enzyme which conjugates GSH to electrophilic compounds, protected a neuroblastoma cell line from aminochrome-induced neurotoxicity [81] and prevented degeneration in a mouse model of PD [82]. Conversely, DA o-quinone modifies and decreases the abundance of enzymes involved in GSH regulation [83], further exacerbating the susceptibility of the cell to oxidative stress. Consistent with these studies, sHsps:DA ox are not toxic to neuronal-like cells ( Figure 6).
In conclusion, the effect of DA ox on proteins and biological processes has been experimentally challenging to study due to the plethora of species produced upon DA oxidation, their variation in reactivity with different amino acids, and the absence of neuromelanin in mice, despite a recent model which mimicked the levels of neuromelanin-like structures [84]. This study has demonstrated that at relatively low levels of DA ox modification and cross-linking of αBc and Hsp27, chaperone activity was retained to a significant degree to prevent amyloid fibrillar and amorphous aggregation. Lysine residues are one of the sites of modification by DA ox . The sHsps, αBc and Hsp27, are key players in maintaining cellular proteostasis; they have resistance to oxidative stress, and their chaperone protective roles mitigate the deleterious effects of protein aggregation, including that of αSyn during PD pathogenesis.

Preparation of sHsps:DA ox
αBc and Hsp27 were incubated in 50 mM phosphate, 150 mM NaCl, pH 7.4 at 1:0, 1:0.5, 1:1, 1:2, 1:5, 1:10 and/or 1:20 molar ratios of DA HCl (Sigma) for 24 h at 37 • C under aerobic conditions to promote the oxidation of DA. The oxidation of DA was confirmed by the change in solution color from clear to brown-grey. The sHsp:DA mixture was then centrifuged at 19,391 rcf for 30 min at room temperature to remove any precipitates. The supernatant was transferred to a 0.5 mL 10 kDa mass cut off centrifugal filter to remove unreacted, excess DA. The filters were then spun at 14,000× g for 10 min. The eluate was discarded and the filters were filled with 50 mM phosphate, 150 mM NaCl, pH 7.4 and spun at 14,000× g for 10 min. This process was repeated five times before the filtrate was recovered. The filtrate was run on SDS-PAGE to confirm formation of the oligomeric, HMW sHsp species.

Chaperone Assays
Amorphous aggregation: 100 µM αLA (Sigma) with or without 4 µM αBc:DA ox or Hsp27:DA ox was allowed to aggregate in 50 mM phosphate, 100 mM NaCl, 2.5 mM EDTA, pH 7.4 at 37 • C with shaking at 200 rpm. Every 45 min, the sHsps and αLA co-incubation was centrifuged at 19,391 rcf for 15 min at room temperature. A volume of 20 µL of the supernatant was flash frozen, and run on SDS-PAGE in chronological order of time under standard conditions. The band intensity at~14 kDa, reflective of soluble monomeric αLA, was quantified using ImageJ software. The αLA band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean.
Amyloid fibrillar aggregation: 100 µM of monomeric A53T αSyn (previously 0.2 µm filtered and centrifuged to remove seeds) with or without 10 µM αBc:DA ox or Hsp27:DA ox was allowed to aggregate in 50 mM phosphate, 150 mM NaCl, pH 7.4 at 37 • C with shaking at 1500 rpm in a total volume of 300 µL. Every~12 h, the sHsp and αSyn co-incubation was centrifuged at 19,391 rcf for 15 min at room temperature. A volume of 15 µL of the supernatant was flash frozen and run on SDS-PAGE in chronological order of time under standard conditions. The band intensity at~14 kDa, reflective of soluble monomeric αSyn, was quantified using ImageJ software. The αSyn band intensity at each time point was normalized relative to the band intensity at 0 h. The data represent the average of three replicates. Error bars represent the standard error of the mean.
The amyloid fibrillar aggregation of RCM αLA in the absence and presence of αBc:DA ox or Hsp27:DA ox was undertaken as previously described [85].

SDS-PAGE
SDS-PAGE was performed using Bis-Tris 4-12% gradient gels (Invitrogen) in a MES running system. Each sample contained 6 x protein loading dye containing DTT and was boiled for 5 min at 95 • C. Precision Plus Protein TM Dual Color Standards (Biorad) or the Triple-color Protein Ladder One (Product No. 09547-74, Nacalai Tesque) were used as the molecular weight standards. The gel was run for 60 min at 140 V and stained with Coomassie blue (0.1% w/v Brilliant Blue R, Sigma), 40% methanol, 10% acetic acid in water and de-stained in 40% methanol, 10% acetic acid in water.

Mass Determination of sHsp:DA Oligomers from SDS-PAGE
ImageJ software (NIH) was used to determine the relative motility value (R f ) by dividing the y-axis value of the sHsp band by the y-axis value of the dye-front. A linear equation was fitted upon plotting the log 10 mass (x axis) against R f for the molecular weight standards from which the masses of the sHsps:DA ox bands were estimated .

Size-Exclusion Chromatography
1 mg/mL of sHsps:DA ox was loaded on a HiPrep 16/10 Sephacryl S-300 HR gel filtration column (GE Healthcare) at a flow rate of 1 mL/min in 50 mM phosphate buffer and 150 mM NaCl, pH 7.4, and eluted over one column volume (120 mL). Absorbance was monitored at a wavelength of 280 nm.

Circular Dichroism Spectroscopy
The far-UV CD spectra of 10 µM of sHsps:DA ox in 10 mM phosphate buffer, pH 7.4 at 37 • C were acquired from 190-260 nm, at a 0.5 nm interval (in a Spectrosil®Far-UV Quartz 21-Q-1 cuvette, Hellma). The total scan time was~9 min. All spectra were acquired on an Applied Photophysics Chirascan spectrophotometer.

Cell Viability Assays
Cell Counting Kit-8 (Sigma) was performed according to the manufacturer's guidelines. Briefly, cell viability was measured by the addition of WST-8, a compound which produces an orange formazan dye when reduced by cellular dehydrogenases. Production of the dye was assessed spectrophotometrically at a wavelength of 460 nm.

Selective Dimethylation of sHsp Lysine Residues via Reductive Alkylation
Selective dimethylation of sHsp lysine residues was performed according to the reductive alkylation kit protocol of Hampton Research. The protein concentration in the filtrate was quantified by measuring the absorbance at 280 nm. The extinction coefficients for αBc and Hsp27 were 19,000 [86] and 40,450 M −1 cm −1 , respectively, which were obtained from the bioinformatics tool, Expasy Protparam. Using the methylated sHsps, sHsps:DA ox were prepared as described above.

Mass Spectrometry
Mass spectra of the methylated and native (non-methylated) sHsps were acquired on an Orbitrap Elite Hybrid Ion Trap-Orbitrap mass spectrometer coupled with an UltiMate 3000 UHPLC (Thermo Scientific, USA). Samples were prepared in 50 mM phosphate buffer with 0.1% v/v formic acid.
Supplementary Materials: Supplementary Materials can be found at https://www.mdpi.com/ article/10.3390/ijms22073700/s1. Figure S1. The effect of modification of αBc and Hsp27 by DAox on their chaperone activity against reduced and carboxymethylated α-lactalbumin (RCM αLA); Figure S2. The effect of modification of αBc by a 10, 20 and 30 molar excess of DAox on its chaperone activity against reduced and carboxymethylated α-lactalbumin (RCM αLA). Figure S3. Densitometry of sHsp monomeric band intensity upon incubation with amorphous or amyloid fibrillar aggregating target proteins; Figure S4. HMW DAox-modified Bc species are not affected by the presence of excess DTT. Figure S5. ANS fluorescence of DAox-modified sHsps. Table S1. Masses determined by electrospray mass spectrometry of methylated sHsps.

Data Availability Statement:
The data presented in this study are available in this article and in its supplementary material.