Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation

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Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ‑D Crystallin Liliana Quintanar,*,† José A. Domínguez-Calva,† Eugene Serebryany,‡ Lina Rivillas-Acevedo,§ Cameron Haase-Pettingell,‡ Carlos Amero,§ and Jonathan A. King*,‡ †

Departamento de Química, Centro de Investigación y de Estudios Avanzados (Cinvestav), 07360 Mexico City, México Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States § Centro de Investigaciones Químicas, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, 62209 Cuernavaca, México ‡

S Supporting Information *

ABSTRACT: Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

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proteins, and they function as molecular chaperones by recognizing exposed hydrophobic patches in partially folded β- and γ-crystallins and complexing them to prevent their aggregation.2,6,7 The β- and γ-crystallins are composed of duplicated domains that share double Greek key β-sheet folds. Although the β-crystallin family members form dimers and oligomers, the γ-crystallins are monomeric. Human γD (HγD) crystallin is one of the more abundant crystallins in the core of the lens, and its nonamyloid aggregation is associated with cataracts.5 HγD crystallin is a highly stable protein, resisting denaturation by heat (80 °C) and chemical agents (8 M urea or 2−3 M guanidinium chloride).8 However, when partially folded molecules of HγD crystallin are formed, these are prone to aggregation in the absence of chaperones.9,10 Aggregation of partially folded proteins into high-molecularweight aggregates has emerged as a major hallmark of degenerative diseases.11−14 Particular attention has been focused on cases in which the aggregated state is an amyloid

ataract is the leading cause of blindness in the world, and it is projected to affect 50 million people by 2050 in the U.S. alone.1 Cataracts are formed upon aggregation of lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity.2,3 The currently available treatment for cataract is eye surgery, which though effective, is costly and not risk-free. Developed countries like the United States spend billions of dollars per year in cataract surgery, while in developing countries, cataract has become the major cause of visual disability. The eye lens is responsible for transparency and focusing of light onto the retina, essential for normal vision. The lens is formed of elongated fiber cells with high protein content, depleted of nuclei and organelles; this unique differentiation occurs in the embryonic stage.4 Fully differentiated fiber cells have a very low metabolism, and they are void of protein synthesis and degradation machineries. Crystallins constitute the most abundant proteins in the lens, and their solubility and stability are essential to maintain its transparency throughout the lifetime of an individual.2,3 Crystallins are classified as α-, β-, and γ- crystallins, and they constitute more than 90% of the protein content in the human lens.5 α-crystallins belong to the family of small heat shock © 2015 American Chemical Society

Received: July 16, 2015 Accepted: November 18, 2015 Published: November 18, 2015 263

DOI: 10.1021/acschembio.5b00919 ACS Chem. Biol. 2016, 11, 263−272

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Figure 1. Effect of divalent metal ions in the aggregation of HγD crystallin. (A) Turbidity assays of HγD crystallin (50 μM) in the absence (black) or presence of 10 equiv of Mn(II) (purple), Ca(II) (pink), Fe(II) (orange), Ni(II) (gold), Zn(II) (red), and Cu(II) (blue). (B) Turbidity assays of HγD crystallin (50 μM) in the absence (gray) or presence of 1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces). (C) Turbidity assays of HγD crystallin (50 μM) in the absence (gray) or presence of 2, 4, 6, 8, and 10 equiv of Zn(II) (yellow to orange, to red traces). In all cases, temperature was 37 °C, and the absorbance at 405 nm is reported as a function of time after the addition of the metal ion.

Figure 2. Electron microscopy images of the metal-induced aggregates of HγD crystallin. Samples obtained at the end of turbidity assays of HγD crystallin with 2 equiv of Cu (A), 4 equiv of Cu (B), and 4 equiv of Zn (C) were analyzed by EM. Protein aggregates are indicated by arrowheads.

fibril, including type 2 diabetes, Alzheimer’s (AD), and Parkinson’s (PD) diseases. However, there are numerous other diseases in which the aggregated state is not amyloid, including α1-antitrypsin deficiency and cataract disease. The origin of the conformational change leading to aggregation has been subject of intense investigation. Metal ions have emerged as important players in protein aggregation. For example, copper ions specifically induce amyloid aggregation of αsynuclein,15 the main protein component of Lewi bodies in PD; both copper and zinc ions are known to affect the aggregation of the β-amyloid peptide present in extracellular plaques in AD.16,17 Metal-induced conformational changes and metalcatalyzed oxidative damage have been proposed as mechanisms by which metal ions influence protein aggregation. In the case of cataract, a variety of experimental and etiological studies implicate metals as a potential etiological agent. Cataract is well-documented among workers in metalworking industries.18 Copper and zinc concentrations in cataractous lenses are increased significantly, as compared to normal lenses, suggesting a potential role of these metals in cataract disease.19−21 A recent inductively coupled plasma mass spectrometry study reports an elegant mapping of copper, zinc, and iron concentrations across the healthy lens, reporting a range of concentrations from 0.4 to 10 μg of metal per gram of wet lens tissue.22 Metal ions are essential for the human body as cofactors of several metallo-enzymes; in spite of their low metabolism, the fiber cells are not an exception. Essential enzymes such as CuZn superoxide dismutase, as well as proteins that control metal ion homeostasis, such as metal-

lothienin, are known to exist in the lens. However, recent studies suggest that loss of metal ion homeostasis may occur with aging.23−26 The interaction of copper and zinc with lens proteins has been relatively unexplored. Perhaps the most relevant finding is that the two α-crystallin chaperones present in the lens, human αA- and αB-crystallins (HαA and HαB, respectively), are able to bind Cu(II) and Zn(II) ions.27−33 A hydrophobic region termed mini-crystallin is highly conserved in this family and has been associated with their chaperone activity.34,35 This region has been identified as the putative copper binding site.27,31 Because of this overlap, a recent study has suggested that, as the chaperone forms complexes with HγD crystallin, its ability to prevent copper-induced oxidative damage decreases.36 Interestingly, copper treatment of lens epithelial cells leads to an upregulation of both HαA and HαB crystallins.37 In this study, we show that Cu(II) and Zn(II) exert distinct and specific effects in the nonamyloid aggregation of HγD crystallin. Surprisingly, the interaction of these metal ions with HγD crystallin impacts the folding of this very stable protein. Particularly, copper ions induce a large conformational change in HγD crystallin, leading to high-molecular-weight lightscattering aggregates. Our work provides insights into the mechanisms of metal-induced aggregation of one of the most stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease. 264


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Figure 3. SDS-PAGE analysis of the metal-induced aggregates of HγD crystallin. Aggregates obtained at the end of turbidity assays of HγD crystallin with increasing amounts of Cu(II) (0,1, 1.5, 2, 2.5, 3, 3.5, 4, 6, 8, and 10 equiv) were analyzed by SDS-PAGE after denaturation by boiling samples in SDS solutions with β-mercaptoethanol (A) or in the absence of reducing agents (C). Aggregates obtained at the end of turbidity assays of HγD crystallin with increasing amounts of Zn(II) (2, 4, 6, 8, and 10 equiv) were analyzed by SDS-PAGE after denaturation by boiling samples in SDS solutions with β- mercaptoethanol (B) or in the absence of reducing agents (D).

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RESULTS AND DISCUSSION Cu(II) and Zn(II) Specifically Induce Aggregation of HγD Crystallin. The effect of several divalent metal ions in the aggregation of HγD crystallin was evaluated by turbidity assays. In a turbidity assay, an increase in absorbance is indicative of the formation of large protein aggregates that scatter light. HγD crystallin was incubated at 37 °C with 10 equiv of each of the following metal ions: Mn(II), Ca(II), Fe(II), Ni(II), Zn(II), and Cu(II). Absorbance at 405 nm was monitored as a function of time after the addition of the metal ion (Figure 1A). In the absence of metal ions, purified HγD crystallin remains soluble and it exhibited no tendency to aggregate at the concentration used in these assays 50 μM (∼1 mg mL−1). Figure 1A shows that Zn(II) and Cu(II) specifically induced the aggregation of HγD crystallin, while all other divalent metal ions showed no effect. Copper-induced aggregation of HγD crystallin was concentration-dependent (Figure 1B). Although the addition of the first two equiv of Cu(II) ions caused a mild and steady increase in turbidity over time, a drastic increase in turbidity was observed immediately after adding >3 equiv equivalents of copper, followed by a further gradual increase in scattering over time in a sigmoidal pattern. Consistently, small protein aggregates were observed at the end of the turbidity assay with 2 equiv of Cu(II) (Figure 2A), while much larger

aggregates were formed with 4 equiv of copper (Figure 2B). At low Cu:protein ratios (≤3 equiv), the mechanism of Cuinduced aggregation was temperature sensitive (Figure S1A), suggesting the involvement of temperature-dependent species, such as partially folded intermediates. In contrast, at high Cu:protein ratios (≥4 equiv), the rapid initial turbidity, which is associated with the formation of protein aggregates (Figure S2), was not affected by temperature (Figure S1B), suggesting the involvement of temperature-independent metal-bridged aggregates. Thus, different competing mechanisms may be involved in copper-induced aggregation of HγD crystallin, and the activation of each mechanism is highly dependent on the relative copper to protein ratio. In contrast, zinc-induced aggregation of HγD crystallin seems to follow a simpler mechanism, involving only temperaturedependent species. While the aggregation increased with Zn(II) concentration (Figure 1C), it was also temperature sensitive (Figure S1C) and showed a mild and steady growth of aggregates for all the Zn:protein ratios studied. The effect of Zn(II) ions was much less pronounced than that of Cu(II) ions, and zinc-induced aggregates were smaller (Figure 2D) as compared to the copper-induced aggregates (Figure 2C). Overall, these results suggest that the nature of the metalprotein interactions and the mechanism of metal-induced aggregation are very different for copper and zinc, suggesting 265


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Figure 4. SDS-PAGE analysis of the copper-induced aggregates of HγD crystallin. Aggregates obtained at the end of turbidity assays of HγD crystallin with increasing amounts of Cu(II) (0, 1, 2, 3, 4, 6, and 8 equiv) were centrifuged to separate the supernatant from the aggregates. Both fractions were analyzed by SDS-PAGE after denaturation by boiling samples in SDS with β-mercaptoethanol (A and B) or in the absence of reducing agents (C and D).

disulfide-bridged dimer at ∼40 kDa was evident in the supernatant at low Cu:protein ratios (1−2 equiv, Figure 4A,C), when the Cu-induced aggregation was very mild. At higher Cu:protein ratios (>3 equiv), most of the protein was aggregated and SDS-PAGE analysis under nonreducing conditions indicated that the aggregate is composed of not only disulfide-bridged dimers and higher molecular species but also a significant amount of monomeric protein (Figure 4D). Overall, these results indicate that, although copper ions promote the formation of disulfide-bridged dimers, this is not the determining event for the drastic Cu-induced aggregation observed at high Cu:protein ratios, which yields aggregates composed of noncovalently linked monomers and disulfidebridged species. Copper-Induced Aggregation Does Not Require Oxygen. Copper is a redox active transition metal ion that is capable of activating oxygen. Metal-catalyzed oxidation of γcrystallins has been proposed to play a role in crystallin aggregation and cataract disease. Hyperbaric oxygen causes oxidation of lens crystallins, and this effect is inhibited by copper chelators,39 suggesting that copper is involved in oxygen activation and protein oxidative damage. We therefore evaluated the effect of oxygen in the copper-induced aggregation of HγD crystallin. Very similar turbidity assay results were obtained by adding 4 equiv of Cu(II) to HγD crystallin under anaerobic conditions (Figure S4A), whereas SDS-PAGE analysis showed no evidence for the formation of the dimeric species in the absence of oxygen (Figure S4B).

site-specific interactions. Still, in both cases, increased turbidity correlated with the formation of larger aggregates and the loss of soluble monomeric protein. Finally, the metal-induced aggregates were not amyloid in nature, as confirmed by ThT assay (Figure S3) and electron microscopy (Figure 2). SDS-PAGE Analysis of Metal-Induced Aggregates of HγD Crystallin. The protein solutions at the end points of the turbidity assays shown in Figure 1B,C were denatured under reducing conditions (with β-mercaptoethanol) and analyzed by SDS-PAGE. In all cases, the denatured aggregates led to a very intense band at ∼20 kDa, which corresponds to the molecular weight of the monomeric protein (Figures 3A,B). In the case of the Cu-induced aggregates, a very small amount of a second species at ∼40 kDa was detected at higher Cu:protein ratios (>3 equiv, Figure 3A). This species became more evident when the Cu-induced aggregates were denatured in the absence of reducing agents (without β-mercaptoethanol), while other higher molecular weight species were observed (Figure 3C), suggesting the presence of disulfide-bridged dimers in the aggregate. Indeed, an early study observed similar dimeric-like species in the aggregates of HγB and HγS crystallins formed in the presence of 7 to 10 equiv of Cu(II), concluding that copper-induced oxidation of crystallin proteins was implicated in the mechanism of aggregation.38 In contrast, no evidence for disulfide-bridged species was observed in the Zn-induced aggregates (Figure 3D). The Cu-induced aggregates were further analyzed after separation from the supernatant (Figure 4). The presence of a 266


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Figure 5. Effect of Cu(II) and Zn(II) ions in the folding (A and B) and the thermal denaturation (C and D) of HγD crystallin. Titrations of HγD crystallin (2 μM, gray spectrum) with 1, 2, 3, 4, 5, 6, 8, and 10 equiv of Cu(II) (light to dark blue traces in (A) or Zn(II) (yellow to orange, to red traces in (B) were followed by CD. Representative titration data at 37 °C are shown in panels A and B, while the relative CD intensity at 218 nm is shown in the insets as a function of number of equivalents of metal ion at 25, 37, and 42 °C. Thermal denaturation of HγD crystallin (2 μM) was followed by CD (C and D) in the presence of 0, 1, 2, 3, 4, or 5 equiv of Cu(II) (C) or Zn(II) (D). The fraction of unfolded protein is plotted as a function of temperature, and the associated Tm values are listed in Table S1.

leading to only a 10% decrease in the CD intensity at 218 nm upon addition of 10 equiv of Zn(II) (inset in Figure 5B). Thermal denaturation experiments are often used to assess protein stability. Here, we determined the midpoint of thermal denaturation curves for HγD crystallin in the absence and presence of Cu(II) and Zn(II) ions, using circular dichroism (Figure 5C,D) and fluorescence (Figure S7) spectroscopies. HγD crystallin is a highly stable protein; it can be heated up to 75 °C without losing secondary structure, and thus, its CD and Trp fluorescence spectra showed no changes at all in the temperature range of 20 to 75 °C. As the temperature was further increased, the negative CD signal at 218 nm in the spectrum of HγD crystallin decreased significantly, indicative of loss of secondary structure and denaturation of the protein. Figure 5C shows the thermal denaturation curve for HγD crystallin (black dots), expressed as the fraction of the protein that has unfolded as a function of temperature. Consistent with previous reports,40 this curve has a midpoint of 82.91 ± 0.30 °C, the temperature at which approximately half of the protein retains its native conformation (Tm). When the thermal denaturation experiment was performed with 1 equiv of Cu(II), the Tm decreased to 78.06 ± 0.28 °C (Figure 5C and Table S1), indicating that the interaction of Cu(II) with HγD crystallin significantly decreases the stability of the protein. The addition of more Cu(II) ions caused a further decrease of the Tm by ∼3.5 °C (Figure 5C and Table S1). In contrast, the addition of Zn(II) ions had no significant effect on the thermal stability of HγD crystallin (Figure 5D and Table S1). Overall, these results show that the interaction of Cu(II) ions with HγD crystallin causes significant changes in the conformation of HγD crystallin, and it decreases its thermal stability. Thus, copper-induced aggregation of HγD crystallin

Overall, these results indicate that the copper-induced aggregation of HγD crystallin does not require oxygen. While Cu(II) ions might be promoting the formation of disulfide bridges (vide supra), the major mechanism of copper-induced aggregation of HγD crystallin does not involve the formation of reactive oxygen species, and it does not involve oxidative protein modifications caused by copper-activated oxygen species. Copper Ions Induce Partial Unfolding of HγD Crystallin and Decrease Its Thermal Stability. We next evaluated the effect of Cu(II) and Zn(II) ions in the folding and thermal stability of HγD crystallin. The protein was titrated with increasing amounts of these metal ions at 37 °C, and circular dichroism (CD) data were collected in the UV region (Figure 5A,B). HγD crystallin contains four Greek key motifs, and consistently, its CD spectrum displays an intense negative transition at 218 nm and a positive band at 195 nm (Figure S5), indicative of β-sheet folding. Upon addition of Cu(II) ions, the intensity of the 218 nm signal decreased significantly as a function of Cu added, indicating an important loss of β-sheet structure. At lower protein concentrations, it was possible to observe a decrease in the 195 nm signal upon addition of Cu (Figure S5), confirming loss of β-sheet structure. After adding 10 equiv of Cu(II), 26% of the CD intensity at 218 nm was lost at 37 °C, while the effect was even more pronounced at higher temperatures (Figure 5A, inset). It should be noted that no aggregation was observed during the course of these experiments and that the loss of CD intensity is not due to loss of soluble protein, as determined by absorption spectroscopy and by the CD detector voltage (Figure S6). In contrast to the case of copper, the addition of Zn(II) ions caused very small changes in the CD spectrum of HγD crystallin (Figure 5B), 267


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Figure 6. Cu(II) and Zn(II) binding to HγD crystallin as detected by NMR. Overlaid 1H- 15N HSQC spectra of HγD crystallin (100 μM) with 0.2 (black), 0.6 (red), 1 (blue), and 1.5 (turquoise blue) equivalents of Cu(II) (A) or Zn(II) (D). The full 1H−15N HSQC spectra are shown in Figure S8. Overlaid weighted intensities changes (I/Io profiles) for the HSQC signals of each residue at different concentrations of metal ion: 0.2 (black bars), 0.6 (red bars), 1.0 (blue bars), and 1.5 (turquoise blue bars) equiv of Cu(II) (B) or Zn(II) (E). The metal-induced signal intensity changes have been mapped into the crystal structure of HγD crystallin (1HK0) and color-coded based on the I/Io profiles with a linear gradient ramp from cyan (no changes or small changes) to magenta (largest changes), for Cu(II) (C) and Zn(II) (F). Unassigned residues are shown in gray.

signals. This became evident in the I/Io profiles for each amide signal (Figure 6B). Because Cu(II) is a paramagnetic ion, its binding to the protein causes signal broadening in the vicinity of the binding site, due to the paramagnetic relaxation enhancement effect.42 The most affected residues with the addition of 0.2 and 0.6 equiv of Cu(II) were those in the region linking the N- and C- terminal domains: His83, Ser84, Ser86, His87, which are in proximity to the C-terminal residues 168− 173 (Figure 6B,C). Residues around Cys18 in the N-terminal domain were also affected significantly at substoichiometric ratios of Cu to protein. At higher Cu:protein ratios (1 to 1.5 equiv), residues 136−138 at the C-terminal domain and residues around Cys41 at the N-terminal region were affected. Upon addition of Zn(II) ions, some signals in the 1H−15N HSQC spectrum of HγD crystallin lost intensity and shifted significantly leading to new signals (Figure 6D and Figure S8).

must involve a direct interaction of the metal ion with the protein that promotes the formation of partially folded intermediates that are prone to nonamyloid aggregation. Zn(II) ions have a smaller effect in the folding of HγD crystallin, supporting the notion that these two metal ions have distinct and site-specific interactions with the protein. Site-Specific Interactions of Cu(II) and Zn(II) Ions with HγD Crystallin. Nuclear magnetic resonance (NMR) spectroscopy was used to probe the interactions of Cu(II) and Zn(II) ions with HγD crystallin. 94% of the N−H amide resonances (not counting prolines) were assigned on the basis of previously reported assignments for P23T HγD crystallin.41 The 1 H− 15 N heteronuclear single quantum coherence (HSQC) spectrum of HγD crystallin was affected when the protein was titrated with Cu(II) (Figure 6A and Figure S8), showing broadening and loss of intensity of several N−H amide 268


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Figure 7. Effect of the chaperone HαB and the mini-crystallin KFVIFLDVKHFSPEDLTVK in the copper-induced aggregation of HγD crystallin. Turbidity assays of HγD crystallin (50 μM) with 0, 2, 3, 4, and 10 equiv of Cu(II) in the absence (light to dark blue traces) or presence (purple traces with markers) of 4 equiv of HαB (A). Turbidity assays of HγD crystallin (50 μM) with 0, 2, 3, 4, and 10 equiv of Cu(II) in the absence (light to dark blue traces) or presence (purple traces with markers) of 4 equiv of mini-crystallin (B). In both cases, temperature was 37 °C, and the absorbance at 405 nm is reported as a function of time after the addition of the metal ion.

domain.40 On the other hand, zinc affected several residues at the interface between the N- and C-terminal domains, such as Phe56, Leu57, Gln54, and Gln142, some of which are known to be important for the folding of the protein.45 The N-terminal domain of HγD crystallin has a decreased stability as compared to the C-terminal domain, the latter serving as a template to stabilize the folding of the N-terminal domain.3,40 Both, direct perturbation of the β-sheets in the N-terminal domain, or any alteration of the interdomain interface could lead to partial unfolding of the protein, and cause aggregation. Chaperone HαB Crystallin Protects HγD Crystallin from Metal-Induced Aggregation. HαB crystallin is one of the lens chaperones and it binds one Cu(II) ion in the minicrystallin region (KFVIFLDVKHFSPEDLTVK). Here we evaluated the effect of HαB and the mini-crystallin in the metal-induced aggregation of HγD crystallin. Figure 7A shows that HαB crystallin could prevent copper-induced aggregation of HγD crystallin, regardless of the concentration of Cu(II) ions. A similar result was observed in the case of zinc-induced aggregation (data not shown). The binding affinity of HγD crystallin for Cu(II) is not known; however, HαB crystallin binds only one Cu(II) ion with picomolar affinity.27,29,32,33 Even if the affinity of HαB crystallin for copper were much greater than that of HγD crystallin, its ability to sequester metal ions in this assay is limited by the stoichiometry. For example, with 10 equiv of Cu, assuming saturation of the binding site in HαB crystallin, there would still be 6 equiv of Cu available to bind HγD crystallin and cause aggregation. The fact that no turbidity was observed under these conditions indicates that HαB is acting as a chaperone and not as a chelating agent. In contrast, the mini-crystallin only slightly prevented the aggregation induced by Cu(II) ions (Figure 7B); its effect is likely due to its ability to chelate Cu(II) ions, buffering the copper-induced aggregation only to the extent that the peptide can compete for Cu(II) ions with HγD crystallin. Our results indicate that HαB crystallin can still function as a chaperone, even in the presence of high Cu concentrations, suggesting that its affinity for the substrate is higher than that for copper. Still, in the aged lens, α-crystallins are gradually depleted,2,46 and a disturbance of metal homeostasis could have an important impact in the stability of lens proteins, as more chaperone

The Zn(II)-protein interactions fall into the slow exchange regime, and it was not possible to unambiguously assign all new signals. Therefore, the analysis of the effect of Zn(II) was based on the loss of intensity of the original peaks, as shown in Figure 6E,F. The most affected residues were 39−41 and 57−62 at the N-terminal domain, and residues 79−87 in the interdomain loop. The C-terminal domain residues 125−130 and 140−142 were also affected. In contrast to the cases of Cu(II) and Zn(II), the addition of 1.0 equiv of Mn(II) or Ca(II) did not cause any significant changes in the 1H−15N HSQC spectrum of HγD crystallin (Figure S9), indicating that these ions do not interact with the protein. These results are consistent with the fact that Mn(II) and Ca(II) did not cause the aggregation of the protein, as observed by the turbidity assays (Figure 1A). Our NMR results reveal site-specific interactions of Cu(II) and Zn(II) ions with HγD crystallin, involving several His and Cys residues, which are amenable ligands to coordinate Cu(II) and Zn(II) ions. Although the specific residues involved in the mechanisms of metal-induced aggregation remain to be discovered through site-directed mutagenesis studies, our preliminary NMR data provides some mechanistic insights. Notably, both ions interact with the region linking the N- and C-terminal domains, including His83 and His87 and the proximal Cys41, which is part of an inner β-sheet that includes the Trp42 hydrophobic core. Mutations at Trp42 for hydrophilic residues cause unfolding of the N-terminal domain and subsequent aggregation of HγD crystallin.43,44 If the metal ion is strong enough to pull Cys41 into its coordination sphere, it is likely to cause unfolding of this β-sheet, exposing Trp42 and other hydrophobic residues, causing aggregation. In such a model, metal binding at this site and the effects of substitutions or oxidations in the hydrophobic core may have a synergistic impact to induce the aggregation of HγD crystallin. Other regions of the protein were specifically affected by only one metal ion and not the other, supporting the notion that Cu(II) and Zn(II) ions display distinct and specific interactions with HγD crystallin that lead to different mechanisms of metalinduced aggregation. Copper perturbed the region around Cys18, which is part of the first β-hairpin of the N-terminal domain, where several cataractogenic mutations have been identified; it is also in the vicinity of Tyr16, whose aromatic pairing with Tyr28 is known to contribute to the stability of this 269


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acrylamide gels, and electrophoresis was run for 1 h at 170 V. Gels were stained with Coomassie blue and digitalized. SDS-PAGE analysis was performed at least in duplicates, and representative images are reported. Transmission Electron Microscopy. Protein aggregates from the end point of turbidity assays were diluted by 5-fold with water, incubated onto glow-discharged carbon coated Cu-Formvar grids (Ted Pella) for 5 min, blotted with filter paper, stained with uranyl acetate (1% w/v) for 45 s, and dried. Grids were imaged in a Jeol-1200 transmission electron microscope. CD Spectroscopy. Protein folding of HγD crystallin was monitored by circular dichroism in the absence and presence of metal ions. A 2 μM solution of protein in 5 mM potassium phosphate buffer pH 7.0 with 5 mM NaCl was titrated by adding small volumes of aqueous solutions of CuSO4 or ZnSO4 salts. CD spectra in the UV region (200−250 nm) were recorded at each titration point in a Jasco J-815 CD spectropolarimeter, equipped with a Peltier system to control temperature, and using a 1 cm path length Suprasil quartz cell. Triplicate experiments were performed for each condition and each temperature. Thermal Stability Measurements by CD and Fluorescence Spectroscopies. The thermal stability of HγD crystallin was determined by circular dichroism and by fluorescence, in the absence and presence of metal ions. Independent solutions of 2 μM protein in 5 mM potassium phosphate buffer pH 7.0 with 5 mM NaCl with the indicated amount of metal ion were prepared. The temperature was increased from 20 to 95 °C at a rate of 3 °C/min using a Peltier system, while the CD intensity at 218 nm was monitored in a Jasco J815 CD spectropolarimeter. For fluorescence measurements, an excitation wavelength of 295 nm was used, and the emission intensity at 325 and 350 nm was monitored, using a Cary Eclipse spectrofluorimeter. Triplicate experiments were performed for each condition. The fraction of unfolded protein at each temperature was calculated as described in SI. NMR Spectroscopy. For NMR measurements, solutions of 0.5 mM HγD crystallin in 10 mM ammonium acetate pH 7.0 and 50 mM NaCl were prepared. All NMR spectra were recorded on a Varian 700 MHz VNMR-S spectrometer equipped with a cryogenically cooled triple resonance pulsed field gradient probe. Backbone resonance assignments were obtained using HNCACB, HNCA, and HNCO triple resonance spectra at 20 °C. All spectra were processed with NMRPipe and analyzed using CARA. Interactions between HγD and Cu(II)/Zn(II) were examined using a lower concentration of protein (100 μM) at 20 °C to prevent metal-induced aggregation, and HSQC spectra were collected in 1 h. Nevertheless, in some cases the samples presented turbidity. Residues with low signal or heavily overlapped signals were ignored.

molecules would be employed to prevent metal-induced aggregation.

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CONCLUSIONS Our study shows that copper and zinc ions specifically induce the nonamyloid aggregation of HγD crystallin, one of the more abundant crystallins in the core of the lens involved in cataract disease. Metal-induced aggregation depended strongly on temperature and was prevented by the human lens chaperone HαB crystallin, implicating partially folded intermediates in the aggregation process and underscoring the role that the lens chaperones would play under conditions of environmental metal exposure or loss of metal ion homeostasis in the aged lens. Zinc- and copper-induced aggregation of HγD crystallin involve distinct site-specific interactions with the protein, and also distinct mechanisms, as Zn(II) is a nonredox active metal, while Cu(II) ions promote the formation of disulfide-bridged dimers. Although oxidative damage to proteins has been proposed as a mechanism for copper-induced aggregation, we have shown that Cu(II) ions cause significant loss of protein stability, and even in the absence of oxygen, promote the formation of high-molecular-weight light-scattering aggregates. Thus, we conclude that the major mechanism of copperinduced aggregation of HγD crystallin does not involve oxidative protein modifications caused by copper-activated oxygen species. Our work provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

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METHODS

Protein Expression and Purification. Recombinant HγD crystallin was expressed using a pET16b plasmid in BL21-RIL E. coli, and purified by ammonium sulfate precipitation followed by sizeexclusion chromatography (SEC). For a more detailed description of the procedure, please refer to Supporting Information. Eluted HγD crystallin fractions were analyzed by SDS-PAGE to verify purity, and fractions were pooled and stored at 4 °C. Protein concentration was determined by electronic absorption spectroscopy, using the extinction coefficient ε = 41 cm−1 mM−1 for HγD crystallin wild type. Peptide Synthesis and Purification. The mini-crystallin peptide, with sequence KFVIFLDVKHFSPEDLTVK, was synthesized by solidphase synthesis and Fmoc strategy. The peptide was acetylated at the amino terminus, and the carboxylic terminal was amidated. The crude peptide was purified to >95% by reversed-phase HPLC and characterized by electrospray ionization mass spectrometry (ESI-MS). Turbidity Assays. HγD crystallin (50 μM) was incubated in 10 mM ammonium sulfate buffer pH 7 with 50 mM NaCl, in the absence or presence of metal ions, at 37 °C (unless otherwise stated), in a total volume of 200 μL. Samples were incubated and analyzed in a FluoStar Optima 96-well plate reader. Absorbance at 405 nm was monitored every 60 s, shaking 5 s before each measurement. CuSO4 and ZnSO4 salts were used as source of Cu(II) and Zn(II) ions, respectively; however, it should be noted that no effect of the counterions was observed, as CuCl2 and zinc acetate salts yielded identical results to those obtained using the corresponding sulfate salts. No turbidity was detected in the control experiments with sample buffer only, or sample buffer with 10 equiv of metal ions in the absence of protein. For each experiment, kinetic traces were baseline corrected using the control trace of sample buffer only. Turbidity assays were run at least four times for each condition. SDS-PAGE Analysis. Protein aggregates from the end point of turbidity assays were denatured by incubation with 2% SDS, 60 mM Tris, pH 6.8, with or without 5% β-mercaptoethanol, in a bath of boiling water, for 5 min. Samples were then loaded into 14%

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00919. HγD crystallin expression and purification methods. Calculation of the fraction of unfolded protein from CD and fluorescence data. Effect of temperature in the metalinduced aggregation of HγD crystallin. Characterization of initial burst in turbidity at 10 equiv of Cu(II). Thioflavin T assay for the HγD crystallin aggregates. Effect of oxygen in the copper-induced aggregation of HγD crystallin. Effect of Cu(II) ions in the folding of HγD crystallin by CD. Control experiments to show that no aggregation occurs in the course of the CD folding experiment. Thermal denaturation curves for HγD crystallin in the presence of Cu(II) and Zn(II), followed by Trp fluorescence. Table with Tm values for HγD crystallin in the presence of different concentrations of 270


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Cu(II) or Zn(II) ions. 1H 15N-HSQC spectra of HγD crystallin in the presence of Cu(II), Zn(II), Mn(II) or Ca(II) ions (PDF)

(15) Binolfi, A., Quintanar, L., Bertoncini, C. W., Griesinger, C., and Fernandez, C. O. (2012) Bioinorganic Chemistry of Copper Coordination to Alpha-Synuclein: Relevance to Parkinson’s Disease. Coord. Chem. Rev. 256, 2188−2201. (16) Bush, A. I. (2003) The metallobiology of Alzheimer’s disease. Trends Neurosci. 26, 207−214. (17) Faller, P., Hureau, C., and La Penna, G. (2014) Metal ions and intrinsically disordered proteins and peptides: From Cu/Zn amylodbeta to general principles. Acc. Chem. Res. 47, 2252−2259. (18) Donald, H. (1962) The Diseases of Occupations, 3rd ed., Little, Brown and Co., Boston, MA. (19) Dawczynski, J., Blum, M., Winnefeld, K., and Strobel, J. (2002) Increased content of zinc and iron in human cataractous lenses. Biol. Trace Elem. Res. 90, 15−23. (20) Srivastava, V. K., Varshney, N., and Pandey, D. C. (1992) Role of trace elements in senile cataract. Acta Ophthalmol. 70, 839−841. (21) Cekic, O. (1998) Effect of cigarette smoking on copper, lead, and cadmium accumulation in human lens. Br. J. Ophthalmol. 82, 186− 188. (22) Konz, I., Fernandez, B., Fernandez, M. L., Pereiro, R., GonzalezIglesias, H., Coca-Prados, M., and Sanz-Medel, A. (2014) Quantitative bioimaging of trace elements in the human lens by LA-ICP-MS. Anal. Bioanal. Chem. 406, 2343−2348. (23) Hancock, S. M., Finkelstein, D. I., and Adlard, P. A. (2014) Glia and zinc in ageing and Alzheimer’s disease: a mechanism for cognitive decline? Front. Aging Neurosci. 6, 137. (24) James, S. A., Roberts, B. R., Hare, D. J., de Jonge, M. D., Birchall, I. E., Jenkins, N. L., Cherny, R. A., Bush, A. I., and McColl, G. (2015) Direct in vivo imaging of ferrous iron dyshomeostasis in ageing Caenorhabditis elegans. Chem. Sci. 6, 2952−2962. (25) Opazo, C. M., Greenough, M. A., and Bush, A. I. (2014) Copper: from neurotransmission to neuroproteostasis. Front. Aging Neurosci. 6, 143. (26) Pushkar, Y., Robison, G., Sullivan, B., Fu, S. X., Kohne, M., Jiang, W., Rohr, S., Lai, B., Marcus, M. A., Zakharova, T., and Zheng, W. (2013) Aging results in copper accumulations in glial fibrillary acidic protein-positive cells in the subventricular zone. Aging Cell 12, 823− 832. (27) Ahmad, M. F., Singh, D., Taiyab, A., Ramakrishna, T., Raman, B., and Rao, C. M. (2008) Selective Cu2+ binding, redox silencing, and cytoprotective effects of the small heat shock proteins αA- and αBcrystallin. J. Mol. Biol. 382, 812−824. (28) Biswas, A., and Das, K. P. (2008) Zn2+ enhances the molecular chaperone function and stability of α-crystallin. Biochemistry 47, 804− 816. (29) Ganadu, M. L., Aru, M., Mura, G. M., Coi, A., Mlynarz, P., and Kozlowski, H. (2004) Effects of divalent metal ions on the αBcrystallin chaperone-like activity: spectroscopic evidence for a complex between copper(II) and protein. J. Inorg. Biochem. 98, 1103−1109. (30) Laganowsky, A., Benesch, J. L. P., Landau, M., Ding, L., Sawaya, M. R., Cascio, D., Huang, Q., Robinson, C. V., Horwitz, J., and Eisenberg, D. (2010) Crystal structures of truncated αA- and αBcrystallins reveal structural mechanisms of polydispersity important for eye lens function. Protein Sci. 19, 1031−1043. (31) Raju, M., Santhoshkumar, P., Henzl, T. M., and Sharma, K. (2011) Identification and characterization of a copper-binding site in αA-crystallin. Free Radical Biol. Med. 50, 1429−1436. (32) Singh, D., Tangirala, R., Bakthisaran, R., and Chintalagiri, M. R. (2009) Synergistic effects of metal ion and the pre-senile cataractcausing G98R αA-crystallin: Self-aggregation propensities and chaperone activity. Mol. Vis. 15, 2050−2060. (33) Mainz, A., Bardiaux, B., Kuppler, F., Multhaup, G., Felli, I. C., Pierattelli, R., and Reif, B. (2012) Structural and mechanistic implications of metal binding in the small heat-shock protein αBcrystallin. J. Biol. Chem. 287, 1128−1138. (34) Kumar, R. S., and Sharma, K. K. (2000) Chaperone-like activity of a synthetic peptide toward oxidized γ-crystallin. J. Pept. Res. 56, 157−164.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research has been supported by NIH grant EY015834 (to J.A.K.), MIT-Mexico Seed Funds grant (to J.A.K. and L.Q.), and Consejo Nacional de Ciencia y Tecnologiá (Conacyt) grant nos. 221134 (to L.Q.) and 151780 (to C. A.). J.A.D.C. thanks Conacyt for a PhD fellowship. C.A. is thankful for access to NMR facilities at Laboratorio Nacional de Estructuras de Macromoléculas (LANEM). L.Q. is thankful for the following fellowships: AMC-FUMEC “Estancias de Verano para Investigadores Jóvenes”, Conacyt “Estancias Sabáticas en el extranjero”, and Fulbright-Garcı ́a Robles fellowship.

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REFERENCES

(1) National Eye Institute. Projections for Cataract (2010−2030− 2050). nei.nih.gov/eyedata/cataract. (2) Moreau, K. L., and King, J. A. (2012) Protein misfolding and aggregation in cataract disease and prospects for prevention. Trends Mol. Med. 18, 273−282. (3) Serebryany, E., and King, J. A. (2014) The -crystallins: Native state stability and pathways to aggregation. Prog. Biophys. Mol. Biol. 115, 32−41. (4) Wride, M. A. (2011) Lens fibre cell differentiation and organelle loss: many paths lead to clarity. Philos. Trans. R. Soc., B 366, 1219− 1233. (5) Lampi, K. J., Ma, Z. X., Shih, M., Shearer, T. R., Smith, J. B., Smith, D. L., and David, L. L. (1997) Sequence analysis of beta A3, beta B3, and beta A4 crystallins completes the identification of the major proteins in young human lens. J. Biol. Chem. 272, 2268−2275. (6) Clark, A. R., Lubsen, N. H., and Slingsby, C. (2012) sHSP in the eye lens: Crystallin mutations, cataract and proteostasis. Int. J. Biochem. Cell Biol. 44, 1687−1697. (7) Acosta-Sampson, L., and King, J. (2010) Partially folded aggregation intermediates of human γD-, γC-, and γS-crystallin are recognized and bound by human αB-crystallin chaperone. J. Mol. Biol. 401, 134−152. (8) Kosinski-Collins, M. S., and King, J. (2003) In vitro unfolding, refolding, and polymerization of human γD crystallin, a protein involved in cataract formation. Protein Sci. 12, 480−490. (9) Das, P., King, J. A., and Zhou, R. H. (2011) Aggregation of γcrystallins associated with human cataracts via domain swapping at the C-terminal beta-strands. Proc. Natl. Acad. Sci. U. S. A. 108, 10514− 10519. (10) Moran, S. D., Zhang, T. Q. O., Decatur, S. M., and Zanni, M. T. (2013) Amyloid Fiber Formation in Human γD-Crystallin Induced by UV-B Photodamage. Biochemistry 52, 6169−6181. (11) Chiti, F., and Dobson, C. M. (2009) Amyloid formation by globular proteins under native conditions. Nat. Chem. Biol. 5, 15−22. (12) Horwich, A. (2002) Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions. J. Clin. Invest. 110, 1221−1232. (13) Dobson, C. M. (2003) Protein folding and misfolding. Nature 426, 884−890. (14) Bennett, M. J., Sawaya, M. R., and Eisenberg, D. (2006) Deposition diseases and 3D domain swapping. Structure 14, 811−824. 271


Articles

ACS Chemical Biology (35) Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) Synthesis and characterization of a peptide identified as a functional element in αA-crystallin. J. Biol. Chem. 275, 3767−3771. (36) Ghosh, K. S., Pande, A., and Pande, J. (2011) Binding of γcrystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone α-crystallin. Biochemistry 50, 3279−3281. (37) Hawse, J. R., Cumming, J. R., Oppermann, B., Sheets, N. L., Reddy, V. N., and Kantorow, M. (2003) Activation of metallothioneins and α-crystallin/sHSPs in in human lens epithelial cells by specific metals and the metal content of aging clear human lenses. Invest. Ophthalmol. Visual Sci. 44, 672−679. (38) Atalay, A., Ogus, A., Bateman, O., and Slingsby, C. (1998) Vitamin C induced oxidation of eye lens γ-crystallins. Biochimie 80, 283−288. (39) Padgaonkar, V. A., Leverenz, V. R., Fowler, K. E., Reddy, V. N., and Giblin, F. J. (2000) The effects of hyperbaric oxygen on the crystallins of cultured rabbit lenses: a possible catalytic role for copper. Exp. Eye Res. 71, 371−383. (40) Kong, F., and King, J. (2011) Contributions of aromatic pairs to the folding and stability of long-lived human γD-crystallin. Protein Sci. 20, 513−528. (41) Jung, J., Byeon, I.-J. L., Wang, Y., King, J., and Gronenborn, A. M. (2009) The Structure of the Cataract-Causing P23T Mutant of Human γD-Crystallin Exhibits Distinctive Local Conformational and Dynamic Changes. Biochemistry 48, 2597−2609. (42) Clore, G. M., and Iwahara, J. (2009) Theory, practice, and applications of paramagnetic relaxation enhancement for the characterization of transient low-populations states of biological macromolecules and their complexes. Chem. Rev. 109, 4108−4139. (43) Serebryany, E., and King, J. A. (2015) Wild-type human γDcrystallin promotes aggregation of its oxidation-mimicking, misfoldingprone W42Q mutant. J. Biol. Chem. 290, 11491−11503. (44) Wang, B., Yu, C., Xi, Y.-B., Cai, H.-C., Wang, J., Zhou, S., Zhou, S., Wu, Y., Yan, Y.-B., Ma, X., and Xie, L. (2011) A Novel CRYGD Mutation (p.Trp43Arg) Causing Autosomal Dominant Congenital Cataract in a Chinese Family. Hum. Mutat. 32, E1939−E1947. (45) Flaugh, S. L., Kosinski-Collins, M. S., and King, J. (2005) Interdomain side-chain interactions in human γD crystallin influencing folding and stability. Protein Sci. 14, 2030−2043. (46) Krishna-Sharma, K., and Santhoshkumar, P. (2009) Lens aging: effects of crystallins. Biochim. Biophys. Acta, Gen. Subj. 1790, 1095− 1108.

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Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation

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