Modulation of Alpha-Synuclein Aggregation by Cytochrome c Binding

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Modulation of Alpha-Synuclein Aggregation by Cytochrome c Binding and Hetero-di-Tyrosine Adduct Formation Sumanta Ghosh, Anindita Mahapatra, and Krishnananda Chattopadhyay ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00393 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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ACS Chemical Neuroscience

Modulation of Alpha-Synuclein Aggregation by Cytochrome c Binding and the Hetero-di-Tyrosine Adduct Formation Sumanta Ghosh, Anindita Mahapatra, Krishnananda Chattopadhyay*

Protein Folding and Dynamics Laboratory, Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology Keywords: Collapse, Free Radicals, Di-tyrosine, Nucleation Correspondence to: [email protected]

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ABSTRACT: The aggregation of α-synuclein (α-Syn) has been implicated strongly in Parkinson’s disease (PD). Intrinsically disordered nature of α-Syn makes this protein prone to self-association with itself, or hetero-association with another protein and/or lipid. While the role of conformational fluctuation and free radical chemistry have been shown to play important roles in its ability towards self and hetero association, any systematic understanding of their contributions is missing. Here, we report an in-vitro investigation of the interaction between α-Syn and cytochrome c in the oxidized (cyt c III) and reduced forms (cyt c II), in which cyt c III was found to induce a large compaction of α-Syn and inhibit the aggregation by favoring a hetero-dityrosine bond formation. In contrast, the presence of cyt c II did not result in any compaction and its presence was found to facilitate α-Syn aggregation. The variation in the charge distribution of the surface residues of cyt c III and cyt II is expected to play decisive roles in their interaction with α-Syn.


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INTRODUCTION A series of neurodegenerative disorders classified as Synucleinopathies, such as Parkinson’s disease (PD), Dementia with Lewy bodies (DLB), Lewy body variant of Alzheimer’s disease, multiple system atrophy (MSA) etc., are characterized by the deposition of proteinaceous aggregates called Lewy bodies (LB) and Lewy neurites (LN)1 in the substantia nigra of the patients’ brain. The intrinsically disordered protein α-Syn has been found to be a major component of LB and LN. Despite extensive investigations, the exact relationship between αSyn accumulation and neurodegeneration has not yet been completely understood. The aggregation of α-Syn has been found to depend on several factors, and an important one is the presence of conformational fluctuations at the rapid microsecond time scale2. α-Syn is unstructured in aqueous buffer, but when bound to membranes3 or at low pH4 the protein is believed to adopt a compact state. Controversies exist regarding its structure inside the cellular environment, and the existence of both a compact tetramer and an extended monomer has been reported5. Using NMR, EPR spectroscopy and MD simulations, α-Syn has been shown to possess some nascent secondary structure along with some short and long range contacts6, which make this protein extremely dynamic with the presence of transient conformers of varying compactness in equilibrium, in which the population of an individual species would vary depending on the protein environment, binding partners and other factors7. Previous study from our group has shown that a compaction at the early stages of aggregation leads to overall inhibition of aggregation, whereas formation of early oligomers can speed up the same8. On the other hand compaction in presence of surfactants like SDS leads to acceleration in aggregation process9. Thus, we hypothesize that the tuning of the conformational landscape (towards more of compact or extended conformers) can help tune the aggregation landscape of the IDP in turn. Another crucial factor, which attenuates α-Syn aggregation, is the involvement of free radicals. The presence of four tyrosine residues (39, 125, 127, and 133) in α-Syn contribute to the oxidative stress mediated aggregation of the protein10. A key event in such processes is the abstraction of one electron from the tyrosine residue (tyrosinate) to form a tyrosyl radical, which could be mediated by hydroxyl radical, carbonate radical, oxo-metal centre or NO2 radical, which come from peroxynitrite or heme peroxidase/H2O2 /nitrite, under conditions of oxidative stress in the cell. At lower oxidant levels, or at higher substrate levels, two tyrosyl radicals encountering each other may readily form a di-tyrosine adduct due to high reactivity of free radical species, thereby resulting in α-Syn crosslinking via di-tyrosine formation11. Although it would be interesting to find a binding partner or small molecule, which would influence both these two components affecting the overall aggregation of α-Syn, no systematic investigation has been reported till date. The α-Syn/cyt c system has important implications in the pathology of PD. α-Syn is believed to bind with around 1% of the proteins inside the cellular environment that are implicated in different signaling cascades. The interaction of α-Syn with key binding partners and its physiological roles may depend on its monomeric/oligomeric nature12. This protein has been 2 ACS Paragon Plus Environment


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found to be immune-colocalized with the protein cyt c13 in Lewy bodies of patients with PD and other Synucleinopathies, suggesting direct interactions between these two proteins inside the cells. In addition, a study of aggregation involving α-Syn and cyt c would address both the above factors (the effect of compaction and that of free radical) as discussed below. Cyt c being a globular protein with opposite charge to that of α-Syn would be expected to interact electrostatically with it and such interaction may lead to wrapping of the extended conformer of α-Syn around the globular partner leading to collapse of its extended structure and subsequent compaction. This would lead to an abundance of compact conformers in the folding landscape of α-Syn (the effect of compaction). However, cyt c exists in two forms i.e. the reduced and oxidized forms, containing the Fe center in different oxidation states. Along with a difference in the oxidation state of the heme iron, the oxidized and reduced cytochrome c show other minor yet significant changes at the surface14 that may allow them to exhibit different interactions towards α-Syn inducing binding and collapse of variable extents. Moreover, the Fe centre in the heme moiety is capable of undergoing redox processes by virtue of transfer of a single electron, to and from a neighbouring interaction partner if there may be. In this context, the reduced and oxidised forms of the cytochrome ought to interact oppositely with α-Syn in case a redox process is possible between the tyrosinate/tyrosyl system and Fe (II)/Fe (III) systems of the interacting proteins (the effect of free radical chemistry). Hence cyt c is one of the few protein systems (and supposedly a direct physiological partner of α-Syn because of their co-localization in Lewy bodies of patient’s brain) that can be selected as a model, in which the effect of compaction and free radical involvement on α-Syn aggregation can be conveniently investigated by changing the oxidation state of cyt c. An additional aspect of α-Syn/cyt c aggregation biology and its relevance towards the pathology of PD may be worth discussing. The studies on aggregation of α-Syn induced by cytochrome c/H2O2 complexes or with other free radical generators15, suggest the role of peroxidase activity of cytochrome c (cyt c III) in seeding the aggregation of α-Syn. Cyt c as a peroxidase can form radicals on proteins and/or phospholipid membranes in its proximity, using hydrogen peroxide as a substrate. This secondary function of cyt c causes oxidative damage and eventual cell death. α-Syn has been shown to divert cyt c from the initial stages of acute apoptosis by acting as a sacrificial scavenger, i.e. by acting as the substrate for the peroxidase activity of cyt c which causes α-Syn to aggregate and accumulate as oligomers16. This may be regarded as a beneficial role of α-Syn, as it delays cyt c induced apoptosis. Our study investigates the interactions of α-Syn with cyt c alone, when cyt c has been found to be attenuating the aggregation of the Parkinsonian protein, opposite of what occurs under oxidative stress. Thus the present investigation brings to light an interesting interplay between the two proteins α-Syn and cyt c in combating neurodegeneration in both the absence and presence of oxidative stress. It is known that in presence of oxidative stress, α-Syn reduces cyt c induced apoptosis by sacrificing itself17. Now, we show that under normal conditions, cyt c hetero-dimerises with α-Syn inhibiting fibrillation of the latter protein. Overall, our investigations revealed that the oxidized cytochrome was able to bind α-Syn and result in a significant compaction, and this early collapse inhibited aggregation of the latter. We also showed that the mechanism of inhibition of aggregation involved the formation of 3 ACS Paragon Plus Environment

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hetero-di-tyrosine bonds between α-Syn and cyt c III, which is in competition with the selfdimerization of α-Syn via homo-di-tyrosine adduct formation between two molecules of α-Syn. The interaction of α-Syn with reduced cyt c on the other hand, did not result in a collapse and accelerated the process of its aggregation, which could again be correlated to the involvement of tyrosine free radicals. This study provides a clear modulation of the folding and aggregation landscape of the IDP by tuning of the oxidation states of a single interacting partner protein.

RESULTS Oxidized cyt c binds to α-Syn inducing a collapse while reduced cyt c does not. We used Fluorescence Correlation Spectroscopy (FCS) to study the binding between α-Syn and cyt c in its oxidized (cyt c III) and reduced (cyt c II) forms. For this purpose, an Alexa-488 tagged G132C mutant of α-Syn (Alexa488Syn) was used. FCS monitors the fluorescence fluctuations of fluorescently labeled proteins inside a small volume element. Fluorescence fluctuations can result from the diffusion and/or conformational dynamics of the protein18. Figure 1A shows the correlation functions obtained from the FCS experiments (using 100nM labeled protein in the presence of ~650nM unlabeled protein) in the absence and presence of different concentrations of cyt c III. The correlation functions were fit to a model containing a single diffusion component with an additional exponential term19. The diffusion component is needed to account for the fluctuations of the labeled protein as a result of molecular diffusion (with diffusion time of D) in and out of the observation volume. The exponential component accounts for a possible rapid conformational fluctuation (with time constant R), where R

……………… (1)

< 𝐼(𝑡) > 2

In this expression, is the average of fluorescence signal over time, and δI(t) is the signal fluctuation at time t minus the average: ……………. (2)

< 𝛿𝐼(𝑡) >= 𝐼(𝑡) ―< 𝐼(𝑡) >

Correlation for a simple solution containing a single diffusing species represented as: 1

𝐺(𝜏) = 1 + 𝑁.

1 (1 +

.

𝜏 𝜏𝐷)

1

……………… (3)

𝜏 1/2

(1 + 𝑆2𝜏 ) 𝐷

Where τD is the characteristic diffusion time, N is the average number of particles in the observation volume, and S is the structural parameter. For a more complex system containing multiple diffusing species (excluding the triplet state contributions), the correlation function is: 1

𝐺(𝜏) = 1 + 𝑁.Ʃ𝑖

𝐴𝑖

1+

.

1

( ) (1 + 𝑆 ( )) 𝑡

𝜏𝐷

𝑖

2

𝑡 𝜏𝐷

1/2

…………… (4)

𝑖

Where τDi is the diffusion time of the ith diffusing species present in the solution and Ai is its relative amplitude. It is important to note that ……………. (5)

∑𝑖𝐴𝑖 = 1

Where τDi is the diffusion time of the ith component and Ai is its amplitude in the correlation function. The value of τD obtained from the diffusion coefficient (D) by the following equation:


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𝜔2

……………. (6)

𝜏𝐷 = 4𝐷

Size of the observation volume () was calculated using FCS measurements with Rhodamine 6G, whose value of D has been well established32. The value of the hydrodynamic radius (rH) can be obtained from D using the Stokes’ Einstein formalism (Equation11). 𝑘𝑇

……………... (7)

𝐷 = 6𝜋𝜂𝑟𝐻

Where η is the viscosity and k is the Boltzmann constant. It may be noted that Equation X assumes that the molecules under investigation are spherical. In order to study conformational change of α-Syn in presence of cyt c, we used different concentration of alexa488 labeled α-Syn (25, 75 and 100 nM) as in the binding experiments. Cyt c dynamics in presence of α-Syn was also where we used 200 nM of Tetra-methyl rhodamine (TMR) labeled cyt c with increasing concentrations of α-Syn up to 1600 nM. Conformation switch from dark sate to bright state corresponding to heme geometry of cyt c was also measured from these experiments. We minimized the effect of refractive index and viscosity on the correlation functions using previously published method. We optimized the FCS data by suitably changing the collar settings and the height of the objective and then, the protein data were normalized using the values of τD obtained with the free dye (Alexa488) measured under identical conditions. ThT binding assay. 500 µl of 200 μM α-Syn with different concentration of cyt c at different condition was used to study aggregation kinetics of α-Syn by ThT fluorescence assay, which binds with amyloids of the protein. Samples were agitated at 180 rpm at 37°C for 2-3 days and fluorescence intensity was measured at different time point of incubation. pH 7.4 was maintained before starting of each experiments. Slits for this experiment were 5 nm for both excitation and emission, while the integration time was 0.3 s. Typical excitation and emission wavelengths were 450 nm and 485 nm respectively. The path length of the quartz cuvette was 1 cm. Aggregation kinetics data were fit to the following equation: 𝐴

…………….. (8)

𝑦 = 𝑦0 + 1 + exp (𝑘′(𝑡 ― 𝑡 )) 0.5

Where y0 was the signal base line at the lag phase; A was the total increase in fluorescence signal between the lag and stationary phase; k´ is the growth rate constant and t0.5 was its midpoint of the log phase. From this lag time is calculated by the following equation. ………………. (9)

tlag = t0.5 ―1/2k′

We calculated tlag of aggregation in the presence of different co-solvents, and this value was used as a measure of the aggregation propensity. tlag here is defined as the time point where amplitude of the transition is 10% of the whole transition. Regulation of redox states of cyt c analysed using Absorption spectroscopy. 1 µM cyt c III (ferric state) was titrated with increasing amounts of α-Syn such that final concentrations of α14 ACS Paragon Plus Environment


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Syn were 1, 5, 10, 15 and 20 µM respectively. A characteristic peak at 530 nm for cyt c III getting resolved into two peaks at 525 nm and 550 nm, is the indication of cyt c reduction. So, the absorbance at 550 nm was recorded in each case and plotted against increasing ratio of cyt c: α-Syn, to understand the effect of native (0 hr) α-Syn on cyt c III. To study this effect at later stages of aggregation, α-Syn (200 µM) was incubated with 2 and 20 µM of cyt c III under agitation at 180 rpm and 37 °C, and the redox profile of cyt c was recorded at different time points of agitation of the mixture. The absorbance at 550 nm at different time points, were plotted alongside the ThT emission values (Fig 5A and B) to measure the extent of reduction that cyt c underwent in the respective time points of aggregation. Again, to understand the effect of free radical quencher azide on this reduction profile, initially it was checked if azide by itself can reduce cyt c III under our experimental conditions. For this, 1 µM cy tc (ferric state) was titrated with increasing amounts of 10 mM sodium azide solution, such that final concentrations of azide ion in solution were 0.5 and 1 mM respectively. The absorbance at 550 nm was recorded in each case, and plotted against increasing ratio of cyt c : azide. Then two 100 µL samples, one containing 200 µM α-Syn and 20 µM cyt c in 20 mM sodium phosphate buffer (pH 7.5), and the other containing the same along with 1 mM sodium azide, were incubated under shaking condition at 37 degree Celsius, in 1.5 mL micro-centrifuge tubes. Aliquots of 10 µL were withdrawn from both samples at different time points of 0, 1, 2, 3, 28 and 69 hours and their absorbance values were recorded at 550 nm (OD550 nm). For each of the two samples, the OD550 nm was then plotted against time, for comparative monitoring of the amount of reduced cyt c produced with increasing time, as aggregation progressed. Circular Dichroism. A volume of 400 μL of 20 μM unlabeled α-Syn in 20 mM NaH2PO4 buffer at pH 7.4 was kept in cuvette with different concentration (2, 20 and 40 µM) of cyt c and the secondary structure change of each sample was monitored using a JASCO J 720 CD instrument. Other parameters included scan speed at 100 nm/ min, integration time of 1 s, and number of scans of 5. CD measurements were carried out between 200 and 250 nm using the permissible HT voltage to obtain optimum signal-to-noise ratio. A traditional CD cuvette of 0.1 cm path length was used for the far UV CD measurements. Change in secondary structure at the aggregated condition in the absence and presence of cyt c III and cyt c II was also measured using CD analyses. Monitoring and confirmation of hetero-oligomerization of cyt c and α-Syn during its aggregation by fluorescence spectroscopic analysis of di-tyrosine adducts. Four 50 µL samples, first one containing 200 µM α-Syn, second one containing 200 µM α-Syn with 1 mM sodium azide, third one containing 200 µM α-Syn with 20 µM cyt c, and fourth one containing 200 µM α-Syn with 20 µM cyt c as well as 1 mM sodium azide, all in 20 mM sodium phosphate buffer (pH 7.5), were incubated under shaking condition at 37 °C, in separate 1.5 mL microcentrifuge tubes, for different amounts of time i.e. 4, 24, 50 and 97 hours. A similar set of four samples were prepared but not incubated, to serve as the 0 hour control. The fluorescence spectroscopic analysis of di-tyrosine adducts for all samples was performed as per the protocol mentioned elsewhere with slight modification. Samples were diluted to 500 µL with buffer and were digested overnight with Proteinase K (final concentration being 40µg/ml) at 37 °C under 15 ACS Paragon Plus Environment

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shaking condition. Digested samples were precipitated with cold perchloric acid (final concentration of acid being 0.53 N). Samples were allowed to stand for 10 min on an ice bath and centrifuged for 15 min at 3,000 g. After neutralization with 2 N KOH, the precipitate of potassium perchlorate crystals was discarded by centrifugation for 15 min at 3,000 g. Supernatants were collected and fluorescence was measured at 410 nm, for an excitation wavelength of 315 nm. The fold of increase in di-tyrosine fluorescence relative to that at 0 hour was calculated for each of the four different sets of samples and plotted against time. Di-tyrosine adduct formation for only 20 µM cyt c was also monitored in same way for the same time points, and the fold of increase in di-tyrosine fluorescence with time was compared for 200 µM α-Syn, 20 µM cyt c and a mixture of both. In another experiment, di-tyrosine adduct formation was monitored in presence of imidazole. For this, a 50 µL sample containing 20 µM cyt c, 200 µM α-Syn and 1 mM Imidazole in NaP buffer (pH 7.5), was incubated for 24 hours, followed by Proteinase K digestion and measurement of di-tyrosine fluorescence as already described. Another sample of same composition and volume was also digested and measured, without any prior incubation, to serve as the 0 hour control. The fold of increase in di-tyrosine fluorescence at 24 hour (relative to that at 0 hour), was calculated and plotted for this sample, alongside the same for α-Syn + cyt c and α-Syn + cyt c + azide, for comparison. Atomic Force Microscopy. AFM was performed using a Pico Plus 5500 AFM (Agilent Technologies, USA) with a piezoscanner having a maximum range of 9 μm. 10 μL of the Aggregated α-Syn incubated with cyt c in a ratio of 1:40 were deposited on a freshly cleaved muscovite ruby mica sheet (ASTM V1grade ruby mica from MICAFAB, Chennai). After 30 minutes the sample was dried with a vacuum drier. The cantilever resonance frequency was 150−300 kHz. The images (256 pixels × 256 pixels) were captured using a scan size of between 0.5 and 8 μm at a scan speed of 0.5 lines/s. The length, height, and width of protein fibrils were measured manually using PicoView1.10 software (Agilent Technologies, USA).

Associated Content: The Supporting Information is available free of charge on the ACS Publications website. The Supporting Information contains figures on the residual distribution analyses of FCS data (Figure S1), steady state fluorescence intensity variation of Alexa488Syn with different concentrations of cyt c III (Figure S2), differential CD spectra (Figure S3), residual distribution analyses of aggregation data at 5 hours (Figure S4), CD spectra of -syn after 96 hours in the absence and presence of cyt c III and cyt c II (Figure S5), AFM images in the absence and presence of cyt c III (Figure S6) and cyt c II (Figure S7), ThT fluorescence (Figure S8) and absorbance (Figure S9, Figure S10) data with cyt c, di-tyrosine fluorescence data (Figure S11) and a table with the percentage of secondary structure of -syn (Table S1).



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Acknowledgements We are thankful to T. Muruganandan for his technical support in AFM. We thank Department of Science and Technology (EMR/2016/000310) for funding. SG and AM thank UGC and CSIR respectively for awarding fellowships. We thank the Director, CSIR-IICB for his encouragements. Author Contributions S.G. and A.M. carried out the experiments and analyzed the data. K.C. conceived the idea, coordinated the study and wrote the paper. All authors approved the final version of the manuscript.

Figure 1:


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Figure 1: [A] The variation in correlation functions obtained in the absence and presence of varying concentrations of cyt c. [B] Variation in diffusion time of Alexa488syn with varying concentration of cyt c, indicating collapse in this case. [C] CD profile of natively unfolded α-Syn in the absence and presence of cyt c III; inset shows CD profile of cyt c III. [D] Difference spectrum of α-Syn /cyt c III obtained by subtraction of only α-Syn and only cyt c III profiles from that of the mixture. [D] Contribution of dark state of alexa488syn (G7C and G132C) on addition of cyt c, indicating a C-terminal specific binding. Figure 2:

Figure 2: [A] Change of rH with varying concentrations of cyt c II, [B] Steady state fluorescence profile of Alexa488 labeled α-Syn in presence of cyt c II.



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Figure 3:

Figure 3: [A] Late stage ThT binding kinetics with 2, 20, 50, 200 and 400µM of cyt c III. [B] Late stage aggregation of α-Syn in presence of cyt c at the concentrations corresponding to mid-point of the transitions indicated subtle change in lag time. [C] Plot of lag time (tlag) versus concentration of cyt c III. [D] Early stage aggregation kinetics of α-Syn with 20 and 50µM of cyt c. [E] tlag value for early aggregation of α-Syn without and 19 ACS Paragon Plus Environment

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with cyt c (20 and 50 µM). [F] ThT-binding aggregation kinetics of 200 µM WT α-Syn (black) and the same with 2, 10 and 20µM reduced cyt c II.

Figure 4:

Figure 4: Comparison of both the reduction and aggregation profiles of 200 µM α-Syn in presence of [A] 2µM and [B] 20µM cyt c. [C] Absorbance profile of cyt c at 550 nm with α-Syn at different time point of agitation which indicated ~20-30% of reduced cyt c in solution, as measured from standard procedure. Inset shows complete absorption profile. [D] Change in diffusion time of TMR-labelled cyt c with increasing concentrations of αSyn.



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Figure 5:

Figure 5. [A] Titration of cyt c by α-Syn. [B] Titration of cyt c by azide. [C] Reduction of cyt c at different stages of α-Syn aggregation. [D] Fold of increase in di-tyrosine fluorescence (relative to 0 hour) after 0, 4, 24, 50 and 97 hours of incubation, for 200 µM α-Syn (black squares) and 200 µM α-Syn in presence of 20 µM cyt c (blue triangles). Red circles represent the values for 20 µM cyt c at same time points. [E] Fold of increase in di-tyrosine fluorescence (relative to 0 hour) after 4, 24, 50 and 97 hours of incubation, for 200 µM α-Syn (black), 200 µM α-Syn in presence of 1 mM azide (red), 200 µM α-Syn in presence of 20 µM cyt c (green), and 200 µM α-Syn in presence of 20 µM cyt c and 1 mM azide (blue). [F] Fold of increase in di-tyrosine fluorescence (relative to 0 hour) after 24 21 ACS Paragon Plus Environment

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hours of incubation, for 200 µM α-Syn in presence of 20 µM cyt c (red), 200 µM α-Syn in presence of 20 µM cyt c and 1 mM imidazole, and 200 µM α-Syn in presence of 20 µM cyt c and 1 mM azide. Figure 6:

Figure 6: Mechanistic outline of the aggregation and hetero-dityrosine formation of αSyn with cyt c III.



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Modulation of Alpha-Synuclein Aggregation by Cytochrome c Binding

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