Epigallocatechin Gallate Remodels Fibrils of Lattice Corneal

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Epigallocatechin gallate remodels fibrils of Lattice Corneal Dystrophy protein, facilitating proteolytic degradation and preventing formation of membrane-permeabilizing species Marcel Stenvang, Gunna Christiansen, and Daniel Erik Otzen Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00063 • Publication Date (Web): 04 Apr 2016 Downloaded from http://pubs.acs.org on April 9, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biochemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Epigallocatechin gallate remodels fibrils of Lattice Corneal Dystrophy protein, facilitating proteolytic degradation and preventing formation of membranepermeabilizing species Marcel Stenvanga,b, Gunna Christiansenc, Daniel Otzena* a

Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and

Genetics, Center for insoluble Protein Structures (inSPIN), Aarhus University, Denmark; b

Sino-Danish Centre for Education and Research;

c

Department of Biomedicine, Aarhus University, Denmark;

ABSTRACT:

Lattice Corneal Dystrophy is associated with painful recurrent corneal erosions and amyloid corneal opacities induced by transforming growth factor β induced protein (TGFBIp) that impairs vision. The exact mechanism of amyloid fibril formation in Corneal Dystrophy is unknown but has been associated with destabilizing mutations in the fourth fasciclin 1 (Fas1-4) domain of TGFBIp. The green tea compound Epigallo-catechin gallate (EGCG) has been found to inhibit fibril formation of various amyloidogenic proteins in vitro. In this study we

ACS Paragon Plus Environment

1

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 56

investigated the effect of EGCG as a potential treatment in Lattice Corneal Dystrophy (LCD) using Fas1-4 with the naturally occurring LCD-inducing A546T mutation. A few molar excess of EGCG were found to inhibit fibril formation in vitro by directing Fas1-4 A546T into stable EGCG-bound protein oligomers. Incubation with two molar equivalent EGCG led to a 4-fold reduction in the aggregates’ membrane disruptive potential, potentially indicative of significantly lower cytotoxicity with regards to corneal erosions. EGCG did not induce oligomer formation by wildtype Fas1-4, indicating that treatment with EGCG would not interfere with the native function of the wild-type protein. Addition of EGCG to 10-day old fibrils reduced fibril content in a dose-dependent manner. Proteinase K was found to reduce the light scattering of non-treated fibrils by 31%, but reduced that of fibrils treated with eight molar equivalents of EGCG by 85%. This suggests that EGCG remodeling of fibril structure can facilitate aggregate removal by endogenous proteases and thus alleviate the protein deposits’ light scattering symptoms.

Introduction Transforming Growth Factor β Induced protein (TGFBIp) is secreted as a 68 kDa protein after cleavage of a signal peptide and is, as its name implies, upregulated by transforming growth factor β (1). The protein is found in several human tissues (2-9); of these, the cornea has a particularly high abundance of TGFBIp, ranging from 1% to 37% (depending on the corneal layer) relative to total protein abundance (10). TGFBIp contains an N-terminal EMI domain, four consecutive and homologous fasciclin1 (Fas1) domains and a C-terminal RGD sequence. More than 30 mutations in TGFBIp are associated with Corneal Dystrophy (CD). Deposits form within the corneal layers and cause visual impairment through light scattering. TGFBIp-


2

Page 3 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

associated CD mutations are inherited in an autosomally dominant fashion and are phenotypically heterogeneous. The phenotypes can generally be divided into Granular Corneal Dystrophies (GCD) and Lattice Corneal Dystrophies (LCD). LCD stains positive for amyloid fibrils with Congo red and are mutations mainly found in the Fas1-4 domain whereas GCD does not stain for amyloid and are mutations mainly found in the Fas1-1 domain (11-14). Amyloid fibrils are highly ordered inter-protein fiber structures with a common cross-β structure that are proposed to constitute a generic protein structure (15, 16). They have mainly been associated with neurodegenerative diseases such as Parkinson’s, Alzheimer’s and Creutzfeldt-Jakob’s disease as well as protein deposition diseases including LCDs. An ab initio small angle x-ray scattering structure model of TGFBIp is consistent with a linear ‘beads on a string’ arrangement where each Fas1 domain folds independently, consistent with the linear arrangement of Drosophila Fas1 domain 3 and 4 (17, 18). Nevertheless, the stability of the entire TGFBIp appears to be limited by the stability of the fourth Fas1 domain (Fas1-4), since mutations in this domain affect the stability of TGFBIp and Fas1-4 to the same extent (19). These observations indicate that Fas1-4 alone is a good model for the entire TGFBIp in studying Fas1-4 mutations. In this study we employ the naturally occurring Ala→Thr substitution at position 546 (A546T). The phenotype is classified as a LCD type IIIA with onset in the fourth decade of life, recurrent corneal erosions, and the patients present with thick rope-like lattice lines and amyloid deposits throughout the corneal stroma (20). Amyloid formation by Fas1-4 A546T in vitro is highly polymorphic with both fibrillation kinetics and fibril structure affected by protein and salt concentration as well as the presence of the glycosaminoglycan (GAG) heparin (21)


3

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 56

Epigallocatechin gallate (EGCG) is the major polyphenolic compound in Camillia Sinensis, the plant from which traditional tea is brewed (22). EGCG has been shown to be an antibacterial and antiviral compound with beneficial effects against cancers and inflammation (22-24). In vitro EGCG inhibits the amyloid formation of several fibrillation-prone proteins, including Huntingtin (25), Transthyretin (26), Lysozyme (27), Sup35 prion (28), ApoAI (29), α-synuclein and β-amyloid (30, 31). EGCG is not only able to inhibit the formation of amyloid fibrils but can also remodel already formed fibrils (32). In vivo studies have been limited but amyloid Transthyretin deposits in a mouse model were reduced by 50% after oral administration of EGCG (33). It remains unknown which mechanisms are involved in protein deposit clearance after EGCG remodeling. Here we show that EGCG can inhibit the formation of Fas1-4 A546T fibrils by rapid formation of oligomers which become SDS-stable over time. These EGCG-induced oligomers show significantly less membrane disruption potential and are thereby potentially less cytotoxic than non-treated protein oligomers. EGCG only appears to target unstable and/or fibrillation-prone Fas1-4 variants, since the more stable and less fibrillation-prone wild-type (WT) Fas1-4 protein resists EGCG-induced oligomer formation. Furthermore, we find that EGCG can remodel mature amyloid fibrils and the subsequent protein species can be proteolytically degraded. Based on our data, we propose a mechanism by which EGCG-treated fibrils can be cleared by proteinase activity and alleviate light scattering, with potential in vivo relevance.

Experimental procedures Chemicals. All chemicals were from Sigma (St. Louis, OH) except nitroblue tetrazolium (Biomol, Hamburg, Germany), 1,2-dioleoyl-sn-[phosphor-rac-(1—glycerol)] (DOPG) (Avanti


4

Page 5 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

polar lipids, Alabaster, AL) and boric acid (J.T. Baker, Deventer, Holland). Heparin was from porcine intestinal mucosa (#H4784) and Proteinase K was from Tritirachium album (#P2308). EGCG

was

used

at

a

purity

of

>95%

(#E4143).

Y571–R588

peptide

(571-

YHIGDEILVSGGIGALVR-588) was generously provided by Prof. Jan Enghild (Aarhus University) and synthesized at EZBiolab Inc (Carmel, IN, USA) as in (34). Purification of Fas1-4. Plasmids containing Fas1-4-SUMO fusion protein gene were generously provided by Prof. Jan Enghild (Aarhus University) (21). The plasmid contains a kanamycin resistance gene and all growth media contained 50 mg/L Kanamycin. All materials used were sterilized by autoclave or filter. E. Coli BL21(DE3) cells were plasmid transfected by electroporation, spread on LB-agar and grown at 37º C overnight. A colony was picked and transferred to 1L LB-media and grown overnight at 37º C 120 RMP. To six shake flasks, each containing 2L LB-media, 50 mL overnight culture was each added and grown to an optical density at 600 nm (OD600nm) of 0.6-1 at 37º C 120 RPM before induction with 1mM Isopropyl βD-1-thiogalactopyranoside. Cells were further incubated at 37º C for 2 hr, left at 20º C overnight, harvested by centrifugation at 4650g for 20 min and resuspended in 50mM Tris-HCl, 60 mM KCl, 10% Glycerol, 5mM β-mercaptoethanol, 1 mM phenylmethylsulfonylfluoride and 1 vol% propanol pH 7.4. Resuspended cells were frozen in liquid nitrogen, thawed and lysed by sonication on ice using a Q500 sonicator (Qsonica, Newtown, CT) with a 6 mm tip at 40% power and 30 cycles (5 sec sonication/5 sec pause). Cell debris was pelleted by centrifugation at 33,000g for 30 min and supernatant was incubated with Ni Sepharose 6 Fast Flow beads (GE Healthcare) overnight at 4º C. Beads were washed with two times 25mL wash buffer (50mM Tris-HCl, 7mM MgCl2, 0.5 M NaCl, 5 mM β-mercptoethanol and 10% glycerol pH 7.4) by pelleting beads for 2 min at 200g, packed on a column and washed with 50 mL wash buffer with


5

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 56

20 mM Imidazole followed by 50mL wash buffer with 35 mM Imidazole. Protein was eluted from beads with wash buffer with 200 mM Imidazole. Protein sample was dialyzed against 5L 50 mM Tris-HCl, 95 mM NaCl pH 7.4 at 4º C overnight, supplemented with NaCl to 150mM, cleaved by 125U SUMO protease (Life technologies) at 4º C overnight to cleave off the SUMO domain. Sample was dialyzed into 5L sample buffer (20mM phosphate, 15 mM NaCl pH 7.4) overnight at 4º C, and the SUMO domain and SUMO protease were removed by binding to Ni Sepharose 6 Fast Flow beads. Pure Fas1-4 protein was collected in the flow-through. Protein was refolded by dialyzing against 1L 6M GdmCl 20mM phosphate, 15 mM NaCl pH 7.4 at room temperature and GdmCl was removed by dialyzing three times 5L sample buffer at 4º C for minimum 6 hr per dialysis. Protein concentration was determined by A280 with a molecular weight of 17,000 g/mol and extinction coefficient 2980 M-1 cm-1. Protein purity was estimated by SDS-PAGE. Secondary structure was determined by Circular Dichroism (J-810, Jasco, Easton, MD) by protein dilution to 0.25 mg/mL in a 1 mm cuvette. Detection of oligomeric species was carried out by eluting over a Superdex 200 increase 10/300 GL column (GE Healthcare Life Sciences) equilibrated with buffer and coupled to an ÄKTA basic system (Amersham Biosciences, Buckinghamshire, England) by 100 μL protein sample injection and measuring A215nm elution profile. Fibrillation kinetics followed by Thioflavin T fluorescence. Samples containing 500 μL of 0.5 mg/mL (29.4 μM) Fas1-4 with 40 μM Thioflavin T (ThT) (from 20mM stock in Ethanol), 1% dimethyl sulfoxide (DMSO) and different amounts of EGCG were mixed per sample. When used, heparin was included at 100 μg/mL. To a 96-well clear bottom plate (#265301, Thermo Fischer Nunc) 150 μL was added in triplicate and covered with clear sealing tape (#232701, Nunc). The plate was run in a Genius Pro plate reader (Tecan, Männedorf, Switzerland) with the


6

Page 7 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

settings: 37º C, excitation 448nm, emission 485nm, gain 50 and 40 μs integration time. For each measurement linear orbital shaking for 3 min at 2.2 mm amplitude was applied and then 10 single reads were averaged for each well. Measurements were done every 5 min. Mature fibrils are made by incubation in this assay for 10 days without DMSO. Mature fibrils were highly aggregated and to obtain a homogeneous suspension, samples were sonicated in a Sonorex Digitec ultrasonic bath (Bandelin, Berlin, Germany) for 10 min. To 495 μL of the mature fibrils, 5 μL EGCG in DMSO was added and incubated in triplicates as above to measure fibril degradation. The F571-R588 peptide was dissolved in DMSO at a concentration of 4.3 mM. This was diluted to 43 μM (0.08 mg/mL) in buffer containing EGCG, ThT, and DMSO (final concentration 2%) followed by incubation as described for Fas1-4. Mature fibrils were made by incubation in this assay for 4 days with 1% DMSO followed by sonication and addition of EGCG as described for Fas1-4 fibrils. 8-Anilinonaphthalene-1-sulfonic acid (ANS) assay. Samples were prepared and incubated as in the ThT assay with two modifications: Instead of ThT, 29 µM ANS and an excitation of 360 nm was used. Transmission Electron Microscopy (TEM). Samples were taken from the fibrillation plate and imaged as described (35). For the proteinase K assay, insoluble protein was pelleted by centrifugation at 25,000g for 30 min, washed with 3 kDa filtered buffer, pelleted again and resuspended. Sample was sonicated by Sonorex Digitec ultrasonic bath (Bandelin, Berlin, Germany) for 10 min to re-suspend sample and allowed to equilibrate for 1 hr at room temperature. Sample was removed and frozen in liquid nitrogen before imaging. Proteinase K (1


7

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 56

μL 0.5 mg/mL) was added to 100 μL protein sample and incubated at 37º C for 2 hr. Sample was frozen in liquid nitrogen and stored at -20º C until imaging. Protein secondary structure measurement by Fourier Transformed Infrared (FTIR) spectroscopy. FTIR spectra were recorded on a Tensor 27 (Bruker, Billerica, MA) by Attenuated Total Reflectance (ATR). On the ATR crystal 2-4 μL sample was dried by nitrogen gas and 64 spectra were acquired at 2 cm-1 resolution and averaged. Data was atmospherically compensated, baseline corrected, normalized and the 2nd derivative was calculated with 17 smoothing points in instrument software (Opus v5.5, Bruker). Note that the FTIR spectra recorded for A546T fibrils in this study are very similar to our previous study (21), with the exception that previous FTIR spectra showed the same peak position but a more dominant fibril peak. We attribute this difference to the fact that FTIR was only measured on the insoluble material in (21) whereas we measure on the entire sample. This is because the EGCG incubated samples cannot be pelleted at normal centrifugation speeds, so to allow comparison, both EGCGincubated and non-EGCG samples were recorded without pelleting. However, pelleting of our non-EGCG A546T sample greatly increases the amyloid peak (data not shown). Protein oligomer size measurement by Dynamic Light Scattering (DLS). DLS was performed on a Zetasizer Nano ZS (Malvern instruments, Malvern, UK) using a ZEN2112 cuvette with 50 μL sample. Samples were measured at 25º C, backscatter (173º), 4.20 mm position and automatic attenuator. A data point was collected by averaging 10 measurements of 30 sec each. For each sample, 5 data points were collected and averaged. The cuvette was cleaned by washing three times with 100 μL 3kDa filtered (Amicon Ultra-15 Centrifugal Filter, Millipore, Darmstadt, Germany) buffer. Cuvette cleanliness was checked by DLS between measurements; the cuvette was considered clean if the DLS instrument was unable to start (0.99) with a mean


17

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 56

value of 29.2±10.8nm in line with the measurements on TEM. To better visualize this development, we divided the data into large and small protein species (Fig. 4B). This made it clear that increasing EGCG concentration reduces the amount of large particles, by volume percentage, going from ~87% (no EGCG) to ~13% (8 molar equivalent EGCG).

Figure 4. EGCG inhibits the formation of large protein structure. (A) DLS size distribution fitting by particle volume of samples collected at the end of the fibrillation time course shown in Fig. 2A. Color and arrow indicate increasing EGCG molar ratio: 0:1 (black), 1:1 (green), 2:1 (blue), 4:1 (orange), 8:1 (red) EGCG:protein monomer. For clarity, each EGCG concentration is offset by 10%. Error bars depicts standard deviation of triplicates from fibrillation assay. (B) Data from panel A are divided into small and large protein particles (below or above 100 nm in diameter) and their relative populations plotted over time. To better understand how these oligomers are formed, the protein monomer was incubated at 37º C and samples were removed at different times. The WT protein is also included to estimate whether the mutation has any effect on the EGCG-protein interaction. Changes in the monomer population over time could give valuable insights into the interaction with EGCG. The


18

Page 19 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

backscatter intensity in DLS depends strongly on size in a complex manner. For our purposes, the most noticeable effect is that very small particles (such as protein monomers) will be eclipsed by even small amounts of oligomers, making DLS inappropriate for this analysis. Instead we employed FPLC-SEC on a Superose 6 column with a separation range of 5 kDa to 5 MDa (Fig. 5A). For ease of interpretation, the spectra were divided into three different species: monomer, small oligomer and large oligomer (Fig. 5A, summarized in Fig. 5B). Without EGCG, we observed no difference in WT protein species after 24 hr incubation (data not shown), demonstrating that the WT protein was stable as a monomer during our measurement. However, EGCG-free A546T slowly formed larger oligomers during the first 8 hr, after which the monomer population had been reduced to 77% of the initial value. After 24 hr, the total signal intensity had declined to 58%, likely due to precipitation and inability to enter the column material. With EGCG we observed very different elution profiles. The total signal increases from 0 to 2 hr with a slight further increase at later time points. The simplest explanation for this signal increase was that EGCG binds to A546T and contributes to the signal at 215 nm. As the signal was not immediately increased, EGCG must either bind either slowly, bind to species which are not present at time t = 0 or bind weakly to species present at time t = 0. Since we do not know the exact binding of EGCG to different oligomeric species, signal intensities should be compared with caution. However, given that EGCG binding will increase signal intensity, the reduction in monomer intensity in the presence of EGCG must mean that monomer was lost more rapidly with EGCG than without. With 10 molar equivalents of EGCG, no monomer was detected after 2 hr (data not shown). Two possible scenarios can be proposed: either EGCG binds monomer and induces the formation of oligomers, or EGCG stabilizes oligomers. These two scenarios are difficult to distinguish. The oligomers only increase slightly in size from 2 to 8 hr,


19

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 56

indicating that EGCG inhibits the formation of very large species as we observed with DLS. Very little difference was observed in the first 8 hr of WT incubated with EGCG. Only after 24 hr did we observe a large difference with a total signal increase. In the presence of EGCG, WT forms some small oligomers and very limited amount of large species. Interestingly, an increase in monomer signal is observed, indicating that EGCG binds the monomer Fas1-4 WT after prolonged incubation.


20

Page 21 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Figure 5. EGCG induces oligomers in the destabilized A546T mutant but has limited effect on WT. (A) FPLC-SEC fractionation elution profile by absorbance at 215nm normalized to monomer peak at 0 hr of A546T (top), 2:1 EGCG:A546T molar ratio (middle), and 2:1


21

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 56

EGCG:WT molar ratio (bottom). Time series depicted for time points 0 (black), 2 (green), 8 (blue) and 24 (red) hr. Separation of monomer (17.7 mL to 20 mL), small oligomer (13.5 mL to 17.7 mL) and large oligomer (5 to 13.5 mL) indicated with black vertical lines. (B) Summary of A data by integration to determine signal from monomer (dark color), small oligomer (medium color) and large oligomer (light color). The three columns correspond to A546T (black), A546T with EGCG (red), and WT with EGCG (green). Data is normalized to the total signal at 0 hr.

Figure 6. EGCG forms SDS-stable oligomers rapidly with A546T but more slowly with WT. (A) SDS-PAGE gel of A546T (left) and WT (right) oligomer samples from Fig. 5 as well as samples with 10:1 molar ratio EGCG:protein. Protein ladder molar weight shown in kDa. Contrast adjusted to better visualize faint bands. (B) SDS-PAGE gel of 2:1 and 10:1 molar ratio


22

Page 23 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

EGCG:protein samples electroblotted onto PVDF membrane and stained with NBT to visualize the presence of EGCG. Next, we wanted to establish whether the oligomers formed by EGCG were SDS-stable. We approached this using protein gel separation by SDS-PAGE. Without incubation, a single band in SDS-PAGE at 17 kDa for both WT and AT was observed, corresponding to monomeric protein (Fig. 6A). After incubation, bands emerge with molecular weights consistent with dimer, trimer and up; after 24 hr, some oligomers are too large to enter the gel. According to FLPC-SEC, only small changes in oligomer size were observed, indicating that SDS-stable oligomers form independent of oligomer size in solution. No oligomers were observed with WT together with 2 molar equivalent EGCG, consistent with FPLC-SEC data. An increase in EGCG concentration to 10 molar equivalent does induce stable SDS oligomers, although to a lesser extent than A546T. We used the dye nitroblue tetrazolium (NBT) to detect EGCG in complex with Fas1-4. NBT can be reduced through redox cycling at alkaline pH in the presence of quinones (found in EGCG) to produce formazan that can be visually detected (56). Although SDS-PAGE Coomassie stain and SEC-FPLC suggests that WT protein was largely unaffected by the presence of two molar equivalent EGCG, weak bands of formazan stain were found for both WT protein monomer and larger species (Fig. 6B). Except for t = 0 hr, A546T monomer and oligomers bind EGCG with a greater intensity than WT at both low and high EGCG concentrations. The fact that EGCG stays bound even when boiled in SDS and exposed to a strong electric field during gel separation suggests that EGCG may be covalently bound to the protein, although tight non-covalent binding cannot be ruled out. The formation of the SDSstable oligomers could then be a product of EGCG crosslinking.


23

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 56

Taken together, our data suggest that Fas1-4 A546T fibrillation inhibition occurs by directing monomer into non-fibrillating oligomers that are cross-linked by EGCG over time. This effect is much less pronounced with WT, indicating that chemical or structural changes caused by the mutation allow for EGCG structural modification. EGCG inhibits protein induced leakage from artificial vesicles. We previously observed that A546T oligomers are able to cause leakage from artificial vesicles, providing a potential protein-aggregation-dependent explanation for cellular toxicity and corneal erosions (21). Small molecule leakage from vesicles can be probed by forming DOPG vesicles under high selfquenching concentrations of the fluorophore calcein. Purification of these vesicles allows for detection of membrane lysis, pore formation or membrane disruption as calcein is released and thereby diluted, resulting in increased fluorescence (57). Hence, we investigated the membrane disruptive properties of A546T protein species, and the effect of EGCG on this reaction, by mixing calcein-loaded vesicles with different amounts of samples of A546T preincubated for 24 hr with or without EGCG (Fig. 7). The EGCG-free A546T had a strong membrane disruptive potential and was able to cause 50% leakage at ~3000 lipid molecules pr. protein monomer. Incubation with 2 molar equivalent EGCG caused a ~4-fold reduction of the membrane disruptive potential at 50% leakage to ~700 lipid molecules pr. protein monomer. Since 2 molar equivalent had a limited effect on amyloid inhibition (Fig. 2A) and only reduced large oligomers from 87% to 39% (Fig. 4B), we only expected a reduction, and not a complete elimination, of membrane perturbation. The 10 molar equivalent EGCG had a much stronger inhibitory effect on A546T aggregation (Fig. 2A), and indeed we saw strong inhibition of the membrane disruption potential at this EGCG:A546T molar ratio (Fig. 7). At the highest protein concentration used, 40


24

Page 25 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

lipid molecules pr. protein monomer, only 31% membrane leakage was observed, and above 321 lipid molecules pr. protein monomer ratio, no membrane leakage was detected.


25

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 56

Figure 7. EGCG inhibits protein induced membrane leakage. (A) Normalized leakage of calcein from DOPG vesicles after 1 hr incubation with different amounts of Fas1-4 A546T incubated with 0 (black), 2 (green) and 10 (red) molar equivalents of EGCG for 24 hr. Error bars denote standard deviation (n=3). Points are connected with lines to guide the eye. No effect of EGCG on vesicles or calcein fluorescence was observed (data not shown). EGCG is also effective against the amyloidogenic Fas1-4 fragment F571-Y588. In protein plaques isolated from CD patients carrying the A546D or V624M TGFBIp mutations, the TGFBIp F571-Y588 tryptic peptide was detected (58, 59). Furthermore this peptide segment was found to constitute the fibril core structure of in vitro fibrillated Fas1-4 A546T (34). Two mechanisms can be suggested in vivo: either the amyloidogenic fragment is released due to proteolytic digestion of the monomer that then forms the amyloid structure, or fibrillation occurs and the resulting amyloid is proteolytically trimmed but leaves the fibril core structure intact. To investigate the effect of EGCG on the first proposed mechanism, the F571-Y588 peptide was incubated with EGCG and fibrillation was followed with ThT fluorescence (Fig 8A, top).


26

Page 27 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

The fibrillation kinetics of the peptide fragment, in the absence of EGCG, shows significant variation among the three samples tested, but an increase in ThT fluorescence was observed in all cases. However, EGCG fibril inhibition was very effective, and even at a 1:4 EGCG:peptides ratio no increase in ThT fluorescence was detected. Some soluble peptides were detected in the presence of EGCG but not in its absence (Fig 8A, top insert). Formation of SDS-stable higher oligomers were not detected (data not shown), in contrast to Fas1-4 (Fig. 6), likely due to the absence of lysine residues in the peptide (60). The resulting structure was visualized with TEM, revealing straight, broad and twisted fibrils in the absence of EGCG similar to previously observed (34) (Fig. 8B, top left). Very thin fibers were also observed which suggest protofilaments (not shown). In the presence of EGCG, very little material was detected; most consists of tiny dots (Fig. 8, top right).


27

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 56

Figure 8. EGCG alters fibrillation and fibril structure of the amyloidogenic Fas1-4 peptide fragment F571-Y588. (A) ThT fibrillation assay of 45 μM peptide incubated with increasing concentrations of EGCG denoted by color. 0 (black), 1:4 (green), 1:2 (blue) or 1:1 (red) EGCG:peptide ratios are shown (higher EGCG concentrations were similar to 1:1 ratio). EGCG incubated samples are shown as average of triplicates but due to high sample variation, data collected in the absence of EGCG (black) are shown as individual fibrillation samples. Top is monomer and bottom is 4-day old fibrils incubated with EGCG respectively. Insert in top: Silver stained Tris-Tricine SDS-PAGE gel with the indicated EGCG:peptide molar ratio and a freshly dissolved peptide solution as control “C”. (B) TEM images of samples from A. Left column is peptide structures formed in the absence of EGCG and right column is with EGCG presence (1:4 EGCG:peptide ratio). Top row: monomer samples from A top. Bottom row: fibril samples from A bottom. Scale bar is 200 nm. In the second proposed mechanism for accumulation of fibrils of the F571-Y588 fragment (full length fibrillation followed by proteolytic trimming), EGCG inhibition of fibril formation from


28

Page 29 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

the Fas1-4 A546T protein would likely block peptide formation. Nevertheless, to investigate if EGCG can affect the peptide fibril structure after fibrillation has occurred, 4-day old fibrils were incubated with EGCG (Fig. 8A bottom). Addition of EGCG to the preformed fibrils leads to a rapid reduction in ThT fluorescence which we attribute to ThT displacement by EGCG. ThT has been shown to inhibit EGCG binding to human islet amyloid polypeptide (hIAPP) and the reverse can thus be expected to occur in the presence of excess EGCG (61). The initial decrease in ThT fluorescence upon EGCG binding to Aβ40 amyloid fibrils could be restored if the EGCG was removed by washing. However, the ThT fluorescence was unchanged after washing fibrils which had been treated with EGCG for 24 hr (60). It is thus important to confirm fibril remodeling by other techniques. The peptide was not returned to its soluble state since no peptide was detected in the supernatant on SDS-PAGE (data not shown) but structural changes were observed by TEM. As expected, the fibril structure in the absence of EGCG was very similar to those found for incubated monomer peptide (Fig. 8B, bottom left). After addition of EGCG, fibril structure was not seen; rather, we observed amorphous aggregates of variable structure (Fig. 8B, bottom right). These results indicate that EGCG could be effective against the proposed peptide fragment mechanisms of LCDs. Incubation of both monomer and fibril with low concentrations of EGCG results in a protein structure different from fibrils. EGCG protects against peptide aggregation, whereas EGCG treated fibrils remain insoluble. EGCG can remodel mature amyloid Fas1-4 A546T fibrils to protease-sensitive species. Although the F571-Y588 peptide fragment was enriched in protein plaques, larger fragments spanning the entire Fas1-4 domain were also detected (59). Hence, fibril deposits of the entire Fas1-4 domain may still play a significant role in vivo. To be an effective therapeutic treatment


29

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 56

against amyloid plaques in vivo, EGCG must not only prevent fibrillation but also remove existing amyloid fibrils. Accordingly, 10-day old amyloid fibrils of full-length Fas1-4 were incubated with EGCG at various concentrations together with ThT (Fig. 9A). Addition of EGCG reduces ThT fluorescence intensity in a dose dependent manner, indicative of fibril structure loss. TEM images of the untreated fibril sample showed the expected combination of small aggregates, worm-like fibrils and longer, straight, and highly bundled fibrils (Fig. 9B, top). The addition of EGCG to fibrils reveals different structures from EGCG incubated with monomer (Fig. 9B, bottom). The sample consists of small spherical particles and worm-like fibrils, but no straight long fibrils were observed. It is difficult to establish whether the worm-like fibril population showed the same structure with and without EGCG. FTIR spectra of EGCG-treated and non-treated fibrils only showed minor differences in secondary structure (data not shown). Similar results were found for EGCG remodeling of heparin induced fibrils (data not shown).

Figure 9. EGCG remodels Fas1-4 A546T fibril structure. (A) 10-day old Fas1-4 A546T fibrils to which EGCG is added and incubated in a ThT fibrillation assay. Color and arrow indicate increasing EGCG concentration: 0:1 (black), 1:1 (green), 2:1 (blue), 4:1 (orange), 8:1 (red) EGCG:protein monomer. Error bars depicts standard deviation of triplicates every 5 hr. (B)


30

Page 31 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

TEM images of 0:1 (top) and 8:1 (bottom) EGCG:protein samples from A after the fibrillation assay. Scale bar is 200 nm. Isolating the insoluble fraction by centrifugation and pellet re-solubilizing by sonication reveals larger differences between EGCG treated and non-treated fibrils (Fig. 10A, left). Nontreated sample contains mostly short straight fibrils with some other undefined structures, whereas the EGCG-treated sample was highly polydisperse with a majority of spherical aggregates of various size and some short fibrils. These differences were also revealed in DLS with the major species size of 38 and 220nm in diameter of treated and non-treated sample respectively (data not shown). As insoluble protein was still present after EGCG treatment, it is clear that EGCG did not solubilize fibrils. Although a decrease in aggregate size was observed after sonication, we were unable to achieve a stable DLS signal due to protein precipitation in both the non-treated and EGCG treated samples without sonication. As both the EGCG treated and non-treated samples were precipitation prone, it is unlikely that EGCG treatment alone will facilitate any major changes to light scattering in vivo. However, remodeling might facilitate subsequent clearance by proteolysis. In two lattice corneal dystrophy TGFBIp mutations, V624M and A546D, the High-temperature requirement A1 (HtrA1) serine protease was found co-localized with protein plaques (58, 59). The co-localization indicates that HtrA1 is unsuccessfully recruited to facilitate the breakdown of the protein aggregates. As HtrA1 is not commercially available, we substitute with another serine protease, Proteinase K. Large differences were observed in the addition of Proteinase K to the insoluble fraction of non-treated and treated fibrils (Fig. 10A). While many fibril structures were still present in the non-treated sample, very little material was observed with treated fibrils. The structures remaining in the treated sample resemble short fibrils. These results indicate that intact fibril structures are at best


31

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 56

slowly degraded by Proteinase K, whereas EGCG remodeled structures are much more susceptible to degradation.

Figure 10. Proteinase K can digest EGCG-remodeled Fas1-4 A546T fibrils. (A) TEM images of insoluble protein aggregates in 0:1 (top) and 8:1 (bottom) EGCG:Fas1-4 ratio samples from Fig. 9 (10 day old fibrils incubated for 2 days with 0:1 or 8:1 EGCG:Fas1-4 ratio). Insoluble material isolated by centrifugation, resolubilized in buffer and sonicated (left). Sample digested by addition of 5 μg/mL Proteinase K at 37º C for 2 hr (right). Scale bar is 200 nm. (B) Scattering intensity of 600 nm light measured at 90º of 0:1 (black) and 8:1 (red) EGCG:Fas4 A546T ratio samples from Fig. 9 at room temperature. At 60 min, 50 μg/mL proteinase K was added. Control sample with EGCG only (green). Ultimately these changes are only of interest if they reduce the visually impairing light scattering caused by amyloid deposits. To model the degradation of Fas1-4 fibrils and its effect on light scattering, we employ static light scattering (SLS) which measures the intensity of scattered light. Prior to SLS measurement, it was necessary to sonicate the sample to obtain a stable suspension during sample measurement. While proteinase K reduced scattering of both samples (Fig. 10B), the effect was clearly larger on EGCG-treated fibrils (85±1%), while


32

Page 33 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

scattering intensity from non-treated fibrils are only reduced by 31±8%. Combining this knowledge with the TEM images, we conclude that small aggregates in the non-treated are degraded while fibrils are left largely intact. Treatment with EGCG restructures most of the long straight fibrils and the resulting protein aggregates can be degraded by Proteinase K. Discussion We provide evidence that EGCG can inhibit the formation of fibrils by Fas1-4 A546T in a dose dependent manner through the rapid formation of ~30 nm diameter non-amyloid oligomers which are slowly cross-linked by EGCG. The EGCG induced A546T oligomers dramatically reduced the membrane disruptive potential compared to oligomers formed in the absence of EGCG. Fas1-4 WT does not fibrillate or oligomerize to any significant extent and is left largely unaffected by EGCG. We observed no long and straight fibrils of A546T after treatment of mature fibrils with EGCG, and treated fibrils are much more susceptible toward proteinase treatment than non-treated fibrils. Furthermore we found EGCG to be effective against fibrils formed in the presence of GAG as well as by the F571-Y588 peptide fragment. We summarize these observations in Fig. 11, which shows the effect of EGCG on different aggregation steps seen for A546T.


33

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 56

FIGURE 11. Illustration of proposed mechanism for EGCG effects on fibril structure in LCD. In the absence of EGCG Fas1-4 forms both oligomers, worm-like fibrils, and straight fibrils. Addition of EGCG before formation of fibrils leads to stable protein oligomers that are cross-linked over time. In contrast to EGCG-free oligomers, the EGCG induced oligomers are unable to cause leakage from membranes, and may protect against corneal erosions. Straight fibrils cannot be completely proteolytically degraded and may accumulate in the cornea. However, upon EGCG treatment, the straight fibrils disappear and proteolytic digestion can completely remove protein deposits and alleviate protein aggregation induced light scattering. Illustration courtesy of Mathias Vinther (iNANO, Aarhus University). EGCG inhibition of Fas1-4 aggregation is likely to be representative of other TGFBIpassociated LCD cases. More than 30 mutations are known to cause TGFBIp associated corneal


34

Page 35 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

dystrophies (12). Mutations are mainly found on R124 in the Fas1-1 domain and throughout the Fas1-4 domain. The thermodynamic stability of the entire TGFBIp was linked to the stability of Fas1-4 domain where TGFBIp and Fas1-4 stability were correlated with specific Fas1-4 mutations (19). This leads to a proposed coupling between disease and mutation-specific protein stability. TGFBIp has no known post-translational modification in the cornea (62), but Nterminal proteolytic processing in the cornea leaves a mostly intact Fas1-4 domain (63), and Fas1-4 fragments have been found in the protein plaques of LCD patients (58, 59). Together these observations make the recombinant Fas1-4 domain a highly relevant model for the effects of TGFBIp mutations in the Fas1-4 domain. The formation of oligomers by EGCG has been observed for other amyloidogenic proteins (26-31). The fact that WT Fas1-4 – but not A546T - is highly resistant toward EGCG-induced oligomerization nicely follows this trend, since the WT is not amyloidogenic, unlike A546T. Fas1-4 is unusual in that most amyloidogenic proteins tested with EGCG are natively unfolded proteins where the entire protein is solvent exposed, whereas Fas1-4 has a well-defined structure. The closest comparison is with the natively folded Transthyretin (TTR) but here EGCG binds WT just as well as the V30M mutation (26). Further, TTR’s proposed amyloid mechanism is different from Fas1-4. The amyloid potential of TTR mutations is linked to a decrease in tetramer stability, whereas Fas1-4 mutations are linked to monomer stability (19, 64). As TGFBIp associated corneal dystrophies are rare and autosomally dominantly inherited, most patients will be heterozygotes. The EGCG-resistance of the WT Fas1-4 implies that potential treatment with EGCG would not affect the normal function of the native WT TGFBIp. More than 20 mutations in Fas1-4 have been correlated with LCD (12). A question remains whether our results is representative of all mutations. Evidence of a common mechanism has


35

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 56

been found using laser microdissection and mass spectroscopy of corneas from patients with either A546D or V624M TGFBIp mutations and the phenotype of polymorphic corneal amyloidosis and atypical LCD respectively (12, 58, 59). Both mutations had similar spectral counts in mass spectroscopy of tryptic digested TGFBIp peptides with a high abundance of the Y571-R588 fragment, indicating similar structure in the aggregates of the two mutations. This fragment was also identified as the fibril core in in vitro fibrillated Fas1-4 A546T (34). Taken together, these results indicate a similar mechanism for Fas1-4 mutation LCDs and – by implication - a similar effect of EGCG. The identification of the Y571-R588 peptide in LCD corneas also suggests that only fragments of the protein are found in LCD aggregates in vivo. It remains unknown whether the fragment occurs as an endogenous attempt to remove TGFBIp fibrils by proteolysis or happens through the release of the amyloidogenic peptide by proteolytic digestion of TGFBIp induced by mutation destabilization. Nevertheless, our results indicate that EGCG could be effective in either scenario. In vivo potential use of EGCG. A general issue with EGCG in vivo is the low bioavailability (65). Oral administration of up to 1600 mg EGCG is well tolerated, but this only leads to a peak free plasma concentration of 6.4 μM (66), considerably lower than the concentrations used in our in vitro assay (29.4 to 412 μM). Furthermore it is not known how the plasma free concentration correlates with concentrations in the cornea. Tolerance and plasma concentrations have large individual variations (65), high doses cause hepatoxicity in mice (67), and green tea has been associated with hepatotoxicity in humans in some cases with doses that are normally considered safe (68). However, Corneal Dystrophies have a potential administration route that is not available by other amyloidosis diseases – direct application to the eye, potentially yielding local high concentrations with low systemic concentration. The efficiency of EGCG diffusion into the


36

Page 37 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

eye and eye tolerance is currently unknown. In two studies on mouse eyes, EGCG protects against dry eye disease and ultraviolet radiation damage (69, 70). These studies do not report on any tolerance issues of topical application of a 0.1% (2.2 mM) EGCG solution and the results indirectly indicate that EGCG is able to diffuse into the eye. EGCG has no effect on the viability of rat primary epithelial cells up to a concentration of 100 μM (the highest concentration tested) (71). However, very high concentrations should be avoided as they have been shown to cause irritation in rabbit eyes (application of 0.1g EGCG) that are reversible by stopping treatment (72). These promising results need support from additional in vivo experiments to assess the toxicity of EGCG eye application. EGCG has the potential to alleviate corneal erosions and visual impairment. Recurrent corneal erosions are a commonly observed in CD (14). In agreement with many other amyloid forming proteins, the oligomers, formed during fibrillation of A546T, are able to cause leakage from vesicles (21, 73). Due to the major differences in composition of artificial vesicles and cellular membranes, vesicle permeability does not necessarily equal cytotoxicity. However, cytotoxicity has been correlated with vesicle permeability and is hence an indicator of potential cytotoxicity (74). Consequently, prefibrillar oligomers could be involved in TGFBIp associated CD recurrent corneal erosions (21). Fas1-4 A546T preincubation with EGCG greatly reduced the membrane disruptive potential, indicating that EGCG could help alleviate recurrent corneal erosion symptoms in vivo. The characteristic symptom of Corneal Dystrophies is light scattering, and thereby vision impairment, by the formed protein plaques. Due to the non-flexible fibril cross-β structure, proteases do not degrade fibrils effectively, and this may explain the failure to clear amyloid in human diseases (34, 75-77). The HtrA1 protease is found co-localized with the protein


37

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 56

aggregates in vivo and is a candidate for proteolytic processing of aggregated TGFBIp (59). Here we show that, in the case of Fas1-4, EGCG treatment allows for proteinase digestion of the Fas14 aggregates in vitro, leading to a substantial reduction in light scattering. Thus we propose a potential mechanism where EGCG remodels TGFBIp amyloid into proteinase digestible aggregates, thereby relieving protein plaques by HtrA1 digestion and regain of vision. This may open up for new therapeutic strategies against corneal dystrophies.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * To whom correspondence should be addressed: Tel.: 45-87155441; Fax:45-89123178; E-mail: [email protected]. iNANO, Aarhus University, DK-8000 Aarhus C Funding Sources M.S. is funded by the Sino-Danish Centre for Education and Research. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Prof. Jan Enghild (Aarhus University) for generously providing plasmids and the peptide fragment F571-Y588, Prof. Jan Skov Pedersen (iNANO, Aarhus University) for clarifying and illuminating discussions on light scattering, Prof. Jørgen Kjems (iNANO, Aarhus


38

Page 39 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

University) for the use of the Bio-Rad imager and Malvern Zetaziser Nano ZS and Mathias Vinther (iNANO, Aarhus University) for figure illustration. ABBREVIATIONS TGFBIp, Transforming Growth Factor β Induced protein; Fas1-4, fourth domain of TGFBIp; ECM, extracellular matrix; CD, Corneal Dystrophy; GCD, Granular Corneal Dystrophies; LCD, Lattice Corneal Dystrophies; EGCG, Epigallocatechin gallate; ThT, Thioflavin T, NBT, nitroblue tetrazolium; GAG, glycosaminoglycan; DMSO, dimethyl sulfoxide; DOPG, 1,2dioleoyl-sn-[phosphor-rac-(1—glycerol)]; transmission electron microscopy (TEM); FPLC-SEC, Fast protein liquid chromatography size exclusion chromatography; DLS, dynamic light scattering; SLS, static light scattering; WT, Fas1-4 wild-type; A546T, Fas1-4 A546T; HtrA1, High-temperature requirement A1

Table of Contents Graphic

REFERENCES 1.

2.

Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G. D., and Purchio, A. F. (1992) cDNA cloning and sequence analysis of beta ig-h3, a novel gene induced in a human adenocarcinoma cell line after treatment with transforming growth factor-beta, DNA Cell Biol. 11, 511-522. LeBaron, R. G., Bezverkov, K. I., Zimber, M. P., Pavelec, R., Skonier, J., and Purchio, A. F. (1995) Beta IG-H3, a novel secretory protein inducible by transforming growth factor-


39

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.

4.

5.

6.

7.

8.

9. 10.

11.

12. 13. 14. 15. 16.

17.

Page 40 of 56

beta, is present in normal skin and promotes the adhesion and spreading of dermal fibroblasts in vitro, J Invest Dermatol 104, 844-849. Kitahama, S., Gibson, M. A., Hatzinikolas, G., Hay, S., Kuliwaba, J. L., Evdokiou, A., Atkins, G. J., and Findlay, D. M. (2000) Expression of fibrillins and other microfibrilassociated proteins in human bone and osteoblast-like cells, Bone 27, 61-67. Lee, S. H., Bae, J. S., Park, S. H., Lee, B. H., Park, R. W., Choi, J. Y., Park, J. Y., Ha, S. W., Kim, Y. L., Kwon, T. H., and Kim, I. S. (2003) Expression of TGF-beta-induced matrix protein betaig-h3 is up-regulated in the diabetic rat kidney and human proximal tubular epithelial cells treated with high glucose, Kidney Int 64, 1012-1021. Norris, R. A., Kern, C. B., Wessels, A., Wirrig, E. E., Markwald, R. R., and Mjaatvedt, C. H. (2005) Detection of betaig-H3, a TGFbeta induced gene, during cardiac development and its complementary pattern with periostin, Anat Embryol (Berl) 210, 1323. Sciandra, F., Morlacchi, S., Allamand, V., De Benedetti, G., Macchia, G., Petrucci, T. C., Bozzi, M., and Brancaccio, A. (2008) First molecular characterization and immunolocalization of keratoepithelin in adult human skeletal muscle, Matrix Biol. 27, 360-370. Ferguson, J. W., Thoma, B. S., Mikesh, M. F., Kramer, R. H., Bennett, K. L., Purchio, A., Bellard, B. J., and LeBaron, R. G. (2003) The extracellular matrix protein betaIG-H3 is expressed at myotendinous junctions and supports muscle cell adhesion, Cell Tissue Res. 313, 93-105. Carson, D. D., Lagow, E., Thathiah, A., Al-Shami, R., Farach-Carson, M. C., Vernon, M., Yuan, L., Fritz, M. A., and Lessey, B. (2002) Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by highdensity microarray screening, Mol Hum Reprod 8, 871-879. Klintworth, G. K., Enghild, J., and Valnickova, Z. (1994) Discovery of a Novel Protein (Beta-Ig-H3) in Normal Human Cornea, Invest Ophthalmol Vis Sci 35, 1938-1938. Dyrlund, T. F., Poulsen, E. T., Scavenius, C., Nikolajsen, C. L., Thogersen, I. B., Vorum, H., and Enghild, J. J. (2012) Human cornea proteome: identification and quantitation of the proteins of the three main layers including epithelium, stroma, and endothelium, J Proteome Res 11, 4231-4239. Stenvang, M., Andreasen, M., Enghild, J. J., and Otzen, D. E. (2014) Chapter 16 - The Molecular Basis For TGFBIp-Related Corneal Dystrophies, In Bio-nanoimaging (Lyubchenko, V. N. U. L., Ed.), pp 179-188, Academic Press, Boston. Kannabiran, C., and Klintworth, G. K. (2006) TGFBI gene mutations in corneal dystrophies, Hum. Mutat. 27, 615-625. Runager, K., Enghild, J. J., and Klintworth, G. K. (2008) Focus on molecules: Transforming growth factor beta induced protein (TGFBIp), Exp Eye Res 87, 298-299. Klintworth, G. K. (2009) Corneal dystrophies, Orphanet J Rare Dis 4, 7. Dobson, C. M. (1999) Protein misfolding, evolution and disease, Trends Biochem. Sci. 24, 329-332. Jahn, T. R., Makin, O. S., Morris, K. L., Marshall, K. E., Tian, P., Sikorski, P., and Serpell, L. C. (2010) The common architecture of cross-beta amyloid, J. Mol. Biol. 395, 717-727. Basaiawmoit, R. V., Oliveira, C. L., Runager, K., Sorensen, C. S., Behrens, M. A., Jonsson, B. H., Kristensen, T., Klintworth, G. K., Enghild, J. J., Pedersen, J. S., and


40

Page 41 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

18.

19.

20.

21.

22.

23.

24. 25.

26.

27.

28.

29.

Otzen, D. E. (2011) SAXS models of TGFBIp reveal a trimeric structure and show that the overall shape is not affected by the Arg124His mutation, J. Mol. Biol. 408, 503-513. Clout, N. J., Tisi, D., and Hohenester, E. (2003) Novel fold revealed by the structure of a FAS1 domain pair from the insect cell adhesion molecule fasciclin I, Structure 11, 197203. Runager, K., Basaiawmoit, R. V., Deva, T., Andreasen, M., Valnickova, Z., Sorensen, C. S., Karring, H., Thogersen, I. B., Christiansen, G., Underhaug, J., Kristensen, T., Nielsen, N. C., Klintworth, G. K., Otzen, D. E., and Enghild, J. J. (2011) Human phenotypically distinct TGFBI corneal dystrophies are linked to the stability of the fourth FAS1 domain of TGFBIp, J. Biol. Chem. 286, 4951-4958. Dighiero, P., Drunat, S., Ellies, P., D'Hermies, F., Savoldelli, M., Legeais, J. M., Renard, G., Delpech, M., Grateau, G., and Valleix, S. (2000) A new mutation (A546T) of the betaig-h3 gene responsible for a French lattice corneal dystrophy type IIIA, Am. J. Ophthalmol. 129, 248-251. Andreasen, M., Nielsen, S. B., Runager, K., Christiansen, G., Nielsen, N. C., Enghild, J. J., and Otzen, D. E. (2012) Polymorphic fibrillation of the destabilized 4th fasciclin-1 domain mutant A546T of the transforming growth factor-beta induced protein (TGFBIp) occurs through multiple pathways with different oligomeric intermediates, J. Biol. Chem. 287, 34730-34742. Steinmann, J., Buer, J., Pietschmann, T., and Steinmann, E. (2013) Anti-infective properties of epigallocatechin-3-gallate (EGCG), a component of green tea, Br. J. Pharmacol. 168, 1059-1073. Riegsecker, S., Wiczynski, D., Kaplan, M. J., and Ahmed, S. (2013) Potential benefits of green tea polyphenol EGCG in the prevention and treatment of vascular inflammation in rheumatoid arthritis, Life Sci. 93, 307-312. Ahmad, N., and Mukhtar, H. (1999) Green tea polyphenols and cancer: biologic mechanisms and practical implications, Nutr. Rev. 57, 78-83. Ehrnhoefer, D. E., Duennwald, M., Markovic, P., Wacker, J. L., Engemann, S., Roark, M., Legleiter, J., Marsh, J. L., Thompson, L. M., Lindquist, S., Muchowski, P. J., and Wanker, E. E. (2006) Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models, Hum Mol Genet 15, 2743-2751. Ferreira, N., Cardoso, I., Domingues, M. R., Vitorino, R., Bastos, M., Bai, G., Saraiva, M. J., and Almeida, M. R. (2009) Binding of epigallocatechin-3-gallate to transthyretin modulates its amyloidogenicity, FEBS Lett. 583, 3569-3576. He, J., Xing, Y. F., Huang, B., Zhang, Y. Z., and Zeng, C. M. (2009) Tea catechins induce the conversion of preformed lysozyme amyloid fibrils to amorphous aggregates, J. Agric. Food Chem. 57, 11391-11396. Roberts, B. E., Duennwald, M. L., Wang, H., Chung, C., Lopreiato, N. P., Sweeny, E. A., Knight, M. N., and Shorter, J. (2009) A synergistic small-molecule combination directly eradicates diverse prion strain structures, Nat. Chem. Biol. 5, 936-946. Nakajima, H., Nishitsuji, K., Kawashima, H., Kuwabara, K., Mikawa, S., Uchimura, K., Akaji, K., Kashiwada, Y., Kobayashi, N., Saito, H., and Sakashita, N. (2016) The polyphenol (-)-epigallocatechin-3-gallate prevents apoA-IIowa amyloidosis in vitro and protects human embryonic kidney 293 cells against amyloid cytotoxicity, Amyloid 23, 17-25.


41

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30.

31.

32. 33. 34.

35.

36. 37.

38.

39. 40.

41.

42.

43. 44.

Page 42 of 56

Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst, M., Masino, L., Lurz, R., Engemann, S., Pastore, A., and Wanker, E. E. (2008) EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers, Nat. Struct. Mol. Biol. 15, 558566. Bieschke, J., Russ, J., Friedrich, R. P., Ehrnhoefer, D. E., Wobst, H., Neugebauer, K., and Wanker, E. E. (2010) EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity, Proc Natl Acad Sci U S A 107, 7710-7715. Bieschke, J. (2013) Natural compounds may open new routes to treatment of amyloid diseases, Neurotherapeutics 10, 429-439. Almeida, M. R., and Saraiva, M. J. (2012) Clearance of extracellular misfolded proteins in systemic amyloidosis: experience with transthyretin, FEBS Lett. 586, 2891-2896. Sorensen, C. S., Runager, K., Scavenius, C., Jensen, M. M., Nielsen, N. S., Christiansen, G., Petersen, S. V., Karring, H., Sanggaard, K. W., and Enghild, J. J. (2015) Fibril Core of Transforming Growth Factor Beta-Induced Protein (TGFBIp) Facilitates Aggregation of Corneal TGFBIp, Biochemistry 54, 2943-2956. Seviour, T., Hansen, S. H., Yang, L., Yau, Y. H., Wang, V. B., Stenvang, M. R., Christiansen, G., Marsili, E., Givskov, M., Chen, Y., Otzen, D. E., Nielsen, P. H., Geifman-Shochat, S., Kjelleberg, S., and Dueholm, M. S. (2015) Functional Amyloids Keep Quorum-sensing Molecules in Check, J. Biol. Chem. 290, 6457-6469. Schagger, H. (2006) Tricine-SDS-PAGE, Nat. Protocols 1, 16-22. Hyldgaard, M., Mygind, T., Vad, B. S., Stenvang, M., Otzen, D. E., and Meyer, R. L. (2014) The antimicrobial mechanism of action of epsilon-poly-l-lysine, Appl. Environ. Microbiol. 80, 7758-7770. Lorenzen, N., Nielsen, S. B., Yoshimura, Y., Andersen, C. B., Betzer, C., Vad, B. S., Kaspersen, J. D., Christiansen, G., Pedersen, J. S., Jensen, P. H., Mulder, F. A., and Otzen, D. E. (2014) How epigallogatechin gallate can inhibit α-synuclein oligomer toxicity in vitro, J. Biol. Chem. 289, 21299-21310. Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to study proteins by circular dichroism, Biochimica Et Biophysica Acta-Proteins and Proteomics 1751, 119-139. Micsonai, A., Wien, F., Kernya, L., Lee, Y. H., Goto, Y., Refregiers, M., and Kardos, J. (2015) Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy, Proc Natl Acad Sci U S A 112, 3095-3103. Underhaug, J., Koldso, H., Runager, K., Nielsen, J. T., Sorensen, C. S., Kristensen, T., Otzen, D. E., Karring, H., Malmendal, A., Schiott, B., Enghild, J. J., and Nielsen, N. C. (2013) Mutation in transforming growth factor beta induced protein associated with granular corneal dystrophy type 1 reduces the proteolytic susceptibility through local structural stabilization, Biochim. Biophys. Acta 1834, 2812-2822. Lindgren, M., Sorgjerd, K., and Hammarstrom, P. (2005) Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy, Biophys. J. 88, 4200-4212. Krebs, M. R., Bromley, E. H., and Donald, A. M. (2005) The binding of thioflavin-T to amyloid fibrils: localisation and implications, Journal of structural biology 149, 30-37. Groenning, M. (2010) Binding mode of Thioflavin T and other molecular probes in the context of amyloid fibrils-current status, Journal of chemical biology 3, 1-18.


42

Page 43 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

45.

46.

47. 48.

49.

50.

51.

52.

53. 54.

55.

56. 57. 58.

59.

Amdursky, N., Erez, Y., and Huppert, D. (2012) Molecular rotors: what lies behind the high sensitivity of the thioflavin-T fluorescent marker, Accounts of chemical research 45, 1548-1557. Runager, K., Basaiawmoit, R. V., Deva, T., Andreasen, M., Valnickova, Z., Sørensen, C. S., Karring, H., Thøgersen, I. B., Christiansen, G., Underhaug, J., Kristensen, T., Nielsen, N. C., Klintworth, G. K., Otzen, D. E., and Enghild, J. J. (2011) Human phenotypically distinct TGFI corneal dystrophies are linked to the stability of the fourth Fas1 domain of TGFBIp, J. Biol. Chem. 286, 4951-4958. Krimm, S., and Bandekar, J. (1986) Vibrational Spectroscopy and Conformation of Peptides, Polypeptides, and Proteins, Adv. Protein Chem. 38, 181-364. Zandomeneghi, G., Krebs, M. R. H., McCammon, M. G., and Fändrich, M. (2004) FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils, Protein Sci. 13, 3314-3321. Uversky, V. N., Winter, S., and Löber, G. (1998) Self-association of 8-anilino-1naphthalene-sulfonate molecules: spectroscopic characterization and application to the investigation of protein folding, Biochim. Biophys. Acta 1388, 133-142. Andreasen, M., Nielsen, S. B., Runager, K., Christiansen, G., Nielsen, N. C., Enghild, J. J., and Otzen, D. E. (2012) Polymorphic fibrillation of the destabilized fourth fasciclin-1 domain mutant A546T of the transforming growth factor--induced protein (TGFBIp) occurs through different pathways with different oligomeric intermediates, J. Biol. Chem. 287, 34730-34742. Ariga, T., Miyatake, T., and Yu, R. K. (2010) Role of proteoglycans and glycosaminoglycans in the pathogenesis of Alzheimer's disease and related disorders: amyloidogenesis and therapeutic strategies--a review, J. Neurosci. Res. 88, 2303-2315. Nielsen, S. B., Yde, P., Giehm, L., Sundbye, S., Christiansen, G., Mathiesen, J., Jensen, M. H., Jensen, P. H., and Otzen, D. E. (2012) Multiple roles of heparin in the aggregation of p25alpha, J. Mol. Biol. 421, 601-615. Suk, J. Y., Zhang, F., Balch, W. E., Linhardt, R. J., and Kelly, J. W. (2006) Heparin accelerates gelsolin amyloidogenesis, Biochemistry 45, 2234-2242. Maji, S. K., Perrin, M. H., Sawaya, M. R., Jessberger, S., Vadodaria, K., Rissman, R. A., Singru, P. S., Nilsson, K. P., Simon, R., Schubert, D., Eisenberg, D., Rivier, J., Sawchenko, P., Vale, W., and Riek, R. (2009) Functional amyloids as natural storage of peptide hormones in pituitary secretory granules, Science 325, 328-332. Pacella, E., Pacella, F., De Paolis, G., Parisella, F. R., Turchetti, P., Anello, G., and Cavallotti, C. (2015) Glycosaminoglycans in the human cornea: age-related changes, Ophthalmol Eye Dis 7, 1-5. Paz, M. A., Fluckiger, R., Boak, A., Kagan, H. M., and Gallop, P. M. (1991) Specific detection of quinoproteins by redox-cycling staining, J. Biol. Chem. 266, 689-692. Allen, T. M., and Cleland, L. G. (1980) Serum-Induced Leakage of Liposome Contents, Biochim. Biophys. Acta 597, 418-426. Karring, H., Runager, K., Thogersen, I. B., Klintworth, G. K., Hojrup, P., and Enghild, J. J. (2012) Composition and proteolytic processing of corneal deposits associated with mutations in the TGFBI gene, Exp Eye Res 96, 163-170. Karring, H., Poulsen, E. T., Runager, K., Thogersen, I. B., Klintworth, G. K., Hojrup, P., and Enghild, J. J. (2013) Serine protease HtrA1 accumulates in corneal transforming growth factor beta induced protein (TGFBIp) amyloid deposits, Mol Vis 19, 861-876.


43

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60.

61.

62.

63.

64.

65. 66.

67.

68.

69.

70.

71.

72.

73.

74.

Page 44 of 56

Palhano, F. L., Lee, J., Grimster, N. P., and Kelly, J. W. (2013) Toward the molecular mechanism(s) by which EGCG treatment remodels mature amyloid fibrils, J. Am. Chem. Soc. 135, 7503-7510. Suzuki, Y., Brender, J. R., Hartman, K., Ramamoorthy, A., and Marsh, E. N. (2012) Alternative Pathways of Human Islet Amyloid Polypeptide Aggregation Distinguished by (19)F Nuclear Magnetic Resonance-Detected Kinetics of Monomer Consumption, Biochemistry. Andersen, R. B., Karring, H., Moller-Pedersen, T., Valnickova, Z., Thogersen, I. B., Hedegaard, C. J., Kristensen, T., Klintworth, G. K., and Enghild, J. J. (2004) Purification and structural characterization of transforming growth factor beta induced protein (TGFBIp) from porcine and human corneas, Biochemistry 43, 16374-16384. Karring, H., Runager, K., Valnickova, Z., Thogersen, I. B., Moller-Pedersen, T., Klintworth, G. K., and Enghild, J. J. (2010) Differential expression and processing of transforming growth factor beta induced protein (TGFBIp) in the normal human cornea during postnatal development and aging, Exp Eye Res 90, 57-62. Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) The amyloidogenic potential of transthyretin variants correlates with their tendency to aggregate in solution, FEBS Lett. 418, 297-300. Mereles, D., and Hunstein, W. (2011) Epigallocatechin-3-gallate (EGCG) for clinical trials: more pitfalls than promises?, Int J Mol Sci 12, 5592-5603. Ullmann, U., Haller, J., Decourt, J. P., Girault, N., Girault, J., Richard-Caudron, A. S., Pineau, B., and Weber, P. (2003) A single ascending dose study of epigallocatechin gallate in healthy volunteers, J Int Med Res 31, 88-101. Lambert, J. D., Kennett, M. J., Sang, S., Reuhl, K. R., Ju, J., and Yang, C. S. (2010) Hepatotoxicity of high oral dose (-)-epigallocatechin-3-gallate in mice, Food Chem. Toxicol. 48, 409-416. Mazzanti, G., Menniti-Ippolito, F., Moro, P. A., Cassetti, F., Raschetti, R., Santuccio, C., and Mastrangelo, S. (2009) Hepatotoxicity from green tea: a review of the literature and two unpublished cases, Eur. J. Clin. Pharmacol. 65, 331-341. Lee, H. S., Chauhan, S. K., Okanobo, A., Nallasamy, N., and Dana, R. (2011) Therapeutic efficacy of topical epigallocatechin gallate in murine dry eye, Cornea 30, 1465-1472. Chen, M. H., Tsai, C. F., Hsu, Y. W., and Lu, F. J. (2014) Epigallocatechin gallate eye drops protect against ultraviolet B–induced corneal oxidative damage in mice, Mol Vis 20, 153-162. Cia, D., Vergnaud-Gauduchon, J., Jacquemot, N., and Doly, M. (2014) Epigallocatechin gallate (EGCG) prevents H2O2-induced oxidative stress in primary rat retinal pigment epithelial cells, Curr Eye Res 39, 944-952. Isbrucker, R. A., Edwards, J. A., Wolz, E., Davidovich, A., and Bausch, J. (2006) Safety studies on epigallocatechin gallate (EGCG) preparations. Part 2: dermal, acute and shortterm toxicity studies, Food Chem. Toxicol. 44, 636-650. Andreasen, M., Lorenzen, N., and Otzen, D. (2015) Interactions between misfolded protein oligomers and membranes: A central topic in neurodegenerative diseases?, Biochim. Biophys. Acta 1848, 1897-1907. Lorenzen, N., Nielsen, S. B., Yoshimura, Y., Vad, B. S., Andersen, C. B., Betzer, C., Kaspersen, J. D., Christiansen, G., Pedersen, J. S., Jensen, P. H., Mulder, F. A., and


44

Page 45 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

75.

76.

77.

Otzen, D. E. (2014) How epigallocatechin gallate can inhibit alpha-synuclein oligomer toxicity in vitro, J. Biol. Chem. 289, 21299-21310. Frare, E., Mossuto, M. F., de Laureto, P. P., Dumoulin, M., Dobson, C. M., and Fontana, A. (2006) Identification of the core structure of lysozyme amyloid fibrils by proteolysis, J. Mol. Biol. 361, 551-561. Miake, H., Mizusawa, H., Iwatsubo, T., and Hasegawa, M. (2002) Biochemical characterization of the core structure of alpha-synuclein filaments, J. Biol. Chem. 277, 19213-19219. Teppo, A. M., and Maury, C. P. J. (1983) Do Serine Proteases Degrade Amyloid-a Fibrils, Scand. J. Immunol. 18, 363-366.


45

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 1 61x28mm (300 x 300 DPI)


Page 46 of 56

Page 47 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig 2 141x162mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 3 63x36mm (300 x 300 DPI)


Page 48 of 56

Page 49 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig 4 77x33mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 5 180x668mm (300 x 300 DPI)


Page 50 of 56

Page 51 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig 6 119x115mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 8 88x44mm (300 x 300 DPI)


Page 52 of 56

Page 53 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig 9 62x33mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 10 75x30mm (300 x 300 DPI)


Page 54 of 56

Page 55 of 56

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

Fig 10 75x30mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig 11 127x89mm (300 x 300 DPI)


Page 56 of 56

Epigallocatechin Gallate Remodels Fibrils of Lattice Corneal

Recommend Documents