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Amyloid formation by intrinsically disordered trans-activation domain of cMyb
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Amyloid formation by intrinsically disordered trans-activation domain of cMyb Kundlik Gadhave, Rajanish Giri* Indian Institute of Technology Mandi, School of Basic Sciences, VPO Kamand, Himachal Pradesh, 175005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 December 2019 Accepted 20 January 2020 Available online xxx
The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291e315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the diseaseassociated amyloid-forming proteins such as Ab, Tau, a-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in bsheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6e16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions. © 2020 Elsevier Inc. All rights reserved.
Keywords: Protein aggregation cMyb Trans-activation domain Intrinsically disordered proteins Transcriptional regulation Amyloid
1. Introduction Intrinsically disordered proteins (IDPs) do not form stable structures when they are free in solution, though they still show biological activity [1,2]. These are strongly associated with cellular regulation (such as cell cycle, transcription, and translation) and are widespread from prokaryotes to eukaryotes [2e4]. IDPs have made a new paradigm called Disorder-Function-Paradigm [5] and mutations or changes in the cellular content of IDPs are involved in several human disease pathways include neurodegenerative diseases, cancer, diabetes, and cardiovascular diseases [2,6]. The selfassembly of proteins into pathogenic insoluble aggregates causes
Abbreviations: Ab, Amyloid beta; AD, Alzheimer’s disease; Bis-ANS, 4,40 -bis 1anilinonaphthalene 8-sulfonate; CBP, CREB-binding protein; CD, circular dichroism; DMEM, dulbecco’s modified eagle’s medium; HR-TEM, high-resolution transmission electron microscopy; IDPs, intrinsically disordered proteins; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PD, Parkinson’s disease; TAD, transcriptional activation domain; ThT, thioflavin t. * Corresponding author. School of Basic Sciences, Indian Institute of Technology Mandi, Himachal Pradesh, 175005, India. E-mail address: [email protected] (R. Giri).
amyloid diseases [7]. Amyloid diseases, also known as “conformational diseases”, occur due to misfolding of proteins/peptides into intermolecular b-sheet aggregated structures. These structures get stabilized by intermolecular interactions and further lead to the formation of oligomers, proto-fibrils, and fibrils [8]. Many factors trigger the onset of protein aggregation such as mutations, environmental changes or chemical modifications, reducing the conformational stability of the protein [8,9]. Importantly, several human diseases are allied with the amyloid formation of IDPs, such as Ab in Alzheimer’s disease (AD) [10], Tau in tauopathy and AD [11], a-synuclein in Parkinson’s disease (PD) [12], and amylin in type 2 diabetes mellitus (T2DM) [13]. Aggregation of IDPs may occur due to their unique structural features enabling them to fold into multiple different conformations [14]. Therefore, in recent years, disordered proteins have been the attention of intensive research. In general, very little information on the aggregation of the trans-activation domain (TAD) is available, example includes TAD domain of tumor suppressor protein p53, which forms amyloid aggregates in vitro at the acidic condition which are cytotoxic to human SH-SY5Y neuroblastoma cells [15]. The high existence of disordered regions in the TAD domains
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Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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and their participation in diverse human diseases increases the importance of generating information on the amyloid nature of TAD domains. As an essential step in that direction, we have studied the amyloid fibrilization of the transactivation domain of cMyb (cMyb TAD). The proto-oncogene cMyb is highly conserved among the vertebrates that encode nuclear transcription factor and functions in proliferation and differentiation [16,17]. cMyb is expressed in tissues and cells of hematopoietic origin, which is essential for normal hematopoiesis in vertebrates [16,17]. Importantly, the mutation in full-length cMyb has been reported to cause defects at the stages of hematopoiesis [18]. cMyb consists of the N-terminal DNAbinding domain (DBD) comprises of three imperfect repeats R1, R2 and R3, a central hydrophilic and slightly acidic transcriptional activation domain (TAD) and a C-terminal negative regulatory region [16,17] which are highly interdependent for the regulation of transcription [17]. Thus, change in the function (aberrant modification) in one domain of cMyb may affect the function of the whole protein and further transcriptional process. The knowledge of co-regulators of the transcription factors is crucial for a complete understanding of its mechanism of action. In response to that, extensive research has identified numerous cMyb TAD interaction partners (Fig. 1A) to recognize how cMyb transcriptional activity is regulated. These studies were performed extensively to study the folding and binding mechanisms between cMyb TAD and KIX. However, what would be the effect of change in buffer conditions or stress environment on the aggregation behaviour of these domains has not been studied yet. Accordingly, using the standard approaches, in our study, we have extensively studied the aggregation behaviour of the KIX binding domain of cMyb TAD, and this is the first report of the same. Aggregates formed by cMyb TAD are rich in b-sheet structure and are cytotoxic
to SH-SY5Y neuroblastoma cells. This amyloidogenic nature of the cMyb TAD domain under the stress condition could affect the function of whole cMyb protein as well as its binding with KIX. These processes may further affect the transcriptional regulation of target proteins. Therefore, in the future, understanding the complete mechanism of cMyb TAD and full-length cMyb protein fibrillization and its effect in vivo animal models is crucial to recognize its role in amyloid diseases. 2. Materials and methods 2.1. Amyloid aggregation of cMyb (291e315) TAD peptide cMyb (291e315) TAD peptide was dissolved in 50 mM phosphate buffer solution, pH 3. Samples for a peptide with the final concentration of 2.5 mg/mL were prepared and incubated at 4 C for 261 h under constant stirring at 1200 rpm. 2.2. Thioflavin T binding assay and ThT kinetics The kinetics of cMyb TAD aggregation samples were monitored at different time intervals in TECAN infinite M200 PRO plate reader in black 96-well plates. The excitation and emission wavelength was set at 450 nm and 490 nm respectively. At different incubation times, 25 mM samples from aliquots of the incubated samples of cMyb TAD peptide were taken and mixed with 20 mM ThT for fluorescence measurements. All measurements were done in triplicate, and the final value represents the average of three measurements. To obtain t50 value, the kinetic curve of cMyb aggregation were fitted to the following sigmoidal Boltzmann equation:
Fig. 1. cMyb TAD interacting partners and TANGO aggregation predictions. (A) cMyb TAD interaction with other proteins for transcription regulation. (a) cMyb: KIX complex (PDB ID: 1SB0). (b) Ternary cMyb: MLL: KIX complex (PDB ID: 2AGH). (c) Ternary HBZ77: KIX: cMyb complex (PDB ID: 6DNQ). (d) Ternary HBZ56: KIX: cMyb complex (PDB ID: 6DMX). (e) KIX: MLL/cMyb chimera (PDB ID: 5SVH). N and C represent N-terminal and C-terminal respectively. cMyb, KIX, MLL, HBZ77, and HBZ56 are showed in red, sea green, olive, magenta, and tan color respectively. (B) TANGO prediction for cMyb (291e315) TAD at different pH and temperature. (a) The b-sheet aggregation propensity at temperature 4 C, (b) 25 C, and (c) 37 C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
K. Gadhave, R. Giri / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
y ¼ A2 þ (A1-A2)/(1 þ exp((x-x0)/dx))
(1)
Where A1 is the initial fluorescence value, A2 represents final fluorescence value, x0 is the t50 value (time at which ThT fluorescence reached 50% of maximum intensity), and dx is time constant. 2.3. Bis-ANS binding assay Bis-ANS binding experiments were performed using 25 mM of cMyb TAD aggregated sample mixed with 15 mM of bis-ANS, a dye which is widely used to measure the exposed hydrophobic surfaces of proteins and their aggregates [19]. The excitation wavelength was set at 380 nm, and the emission spectra were recorded from 400 to 650 nm (data presented 414e650 nm) in black 96-well plates using TECAN infinite M200 PRO plate reader.
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treated with different concentrations (0, 1, 3.125, 12.5, and 50 mM) of cMyb TAD aggregates in 100 ml fresh culture medium and incubated at 37 C for 48 h. MTT was added to the culture medium with a final concentration of 0.5 mg/mL and incubated for 3 h in a CO2 incubator. Then 100 ml of DMSO solution was added to dissolve formazan crystals, incubated for 10 min and the absorbance for each well was recorded at 590 nm with a reference wavelength of 630 nm using TECAN infinite M200 PRO plate reader. The data are reported as the percentage of the MTT reduced by the cells, and cell viability was shown relative to control cells which were not treated with the cMyb TAD aggregates. 3. Results and discussion 3.1. cMyb (291e315) TAD has a strong intrinsic propensity to aggregate at acidic condition
2.4. Nanoparticle tracking analysis (NTA) The nanoparticle tracking analysis (NTA) allows particle-byparticle measurement of number concentration and size distribution of nanoparticles/aggregates in solution. NTA measurements were performed at room temperature according to the procedure reported by Lu et al., [20]. 5 ml of cMyb TAD peptide from 2.5 mg/mL solution was mixed with 995 ml of filtered phosphate buffer of pH 3 and injected into the sample chamber of Nanosight LM10 (Nanosight, Amesbury, UK) equipped with a 405 nm laser. 2.5. Circular dichroism (CD) spectroscopy Far-UV CD spectra for 25 mM cMyb TAD peptide was recorded in Jasco J-1500 Circular Dichroism Spectrophotometer using a 1 mm path-length quartz cuvette. All the measurements were done at 25 C temperature, 50 nm scanning speed, 1 nm bandwidth, and 0.5 nm data interval. The sample spectra were recorded after subtracting from buffer spectra. Each spectrum was the average of three scans. The secondary structure contents (a-helix, b-sheet, random coil, and turn) of the samples were assessed using the CONTIN algorithm with a reference set 7 which is accessible from the DICROWEB website [21]. 2.6. High-resolution transmission electron microscopy (HR-TEM) The morphology of cMyb TAD fibrils was observed with a highresolution transmission electron microscope (HR-TEM) (FP 5022/ 22-Tecnai G2 20 S-TWIN, FEI). 7 ml of twenty-fold diluted aggregated samples were mounted on 200 mesh carbon-coated copper grids (Ted Pella, Inc, USA). The negative staining was done by using a 3% ammonium molybdate solution, then grids were air-dried overnight, and images were captured in HR-TEM with accelerating voltage at 200 kV. Further, the diameter of fibrils was measured using the open-source image processing program ImageJ [22]. 2.7. Cell viability assay (MTT assay) The cytotoxicity of aggregated cMyb TAD samples was evaluated in SH-SY5Y human neuroblastoma cells which were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM F-12) supplemented with 10% fetal bovine serum (FBS). The aggregated peptide sample was centrifuged at 15000 rpm for 30 min. The pellets were resuspended in PBS and again centrifuged at 15000 rpm for 30 min. Finally, pellets were resuspended in the DMEM medium to obtain the required concentration. The cells were seeded in 96 well-plate with a cell density of 3000 cells/well in 150 ml culture medium and incubated for 24 h. Further, cells were
Typically, proteins/peptides comprise some sequence stretches that possess inherent aggregation propensity. According to the ProtParam server [23], the cMyb TAD sequence (291-KEKRIKELELLLMSTENELKGQQVL-315) has a large number of hydrophilic residues (15 polar amino acids), among them, 5 residues are positively charged, and 5 residues are negatively charged. Similar features of cMyb TAD were observed in the case of disordered p53 (residues 1e63) TAD domain [15], that favor the aggregation of proteins. We performed in silico amyloidogenic propensity prediction by using TANGO [24] aggregation predictor at different pH conditions for the identification of amyloid-forming residues and calculated amyloidogenic tendency of cMyb TAD peptide. Interestingly, we found that the region ‘299-ELLLMSTE-306’ is showing aggregation propensity in all pH (pH 3e10) in the case of 4 C and 25 C temperature (Fig. 1a and b). However, at 37 C, pH 3 and 4 show a low aggregation score (Fig. 1c) and all other pHs (pH 5e10) display no aggregation propensity. Particularly at pH 3 and 4 C temperature, above-mentioned region displays a significant aggregation propensity as compared with other pH (pH 4e10) conditions (Fig. 1a). These observations revealed that cMyb TAD has a high aggregation propensity in acidic pH as compared with neutral or basic pH. In general, the rate of amyloid fibril formation is pHdependent [25]. Moreover, the studies have been revealed that the affinity of cMyb for KIX is pH-dependent. In acidic pH (pH 5.5), cMyb:KiX affinity is six-fold higher as compared with physiological pH (pH 7.0) [26]. Thus, we selected pH (pH 3) and temperature (4 C) for in vitro aggregation studies and further experiments are performed to demonstrate its amyloid nature. 3.2. cMyb (291e315) TAD aggregates are ThT and ANS-Positive We analyzed cMyb TAD binding to ThT dye by observing the increase of fluorescence at a different time. An increase in ThT fluorescence is an indication of the formation of ordered aggregates of amyloid types [15]. We found that, cMyb TAD aggregates displays near eight-fold increase in ThT fluorescence intensity as compared with ThT only with buffer (Fig. 2A). Further, aggregation kinetics of cMyb TAD monitored by ThT assay (Fig. 3A). Each point in Fig. 3A indicates the ThT fluorescence intensity in the presence of a fixed amount of the incubated sample (25 mM) taken at several time points of incubation. The fibril formation kinetics of cMyb TAD represents a typical phase of aggregation. Generally, the steps involved include first a lag phase (nucleation), second an elongation phase (growth), and third a saturation phase (steady-state) [27]. From ThT kinetics, we observed that the length of the lag phase was approximately 85 h which is significantly longer. Following the lag phase, cMyb TAD entered a growth phase. From 100 h the exponential phase was faster. Here, we determine the half
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 2. Dye binding assay and particle size measurement for cMyb (291e315) TAD aggregates. (A) ThT fluorescence spectra. The black and red curve indicates the ThT fluorescence spectra of ThT control and aggregated cMyb TAD respectively. (B) Bis-ANS fluorescence spectra. The black and red line shows ANS fluorescence spectra of Bis-ANS control and aggregated cMyb TAD respectively. (C) Particle hydrodynamic size and number concentration measurement using NTA. The black and red line indicates the cMyb TAD monomers and aggregates respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
time (t50), that denote the midpoint of the exponential phase of the reaction (time where fibril formation is 50% complete). A greater t50 value indicates a longer time required to form amyloid fibrils. The kinetic trace of cMyb TAD fibril formation was sigmoidal and represents t50 value of 124 ± 2.07 h that represent cMyb TAD takes a longer time for aggregation. Fibril formation by cMyb TAD was considered complete when ThT fluorescence reached a steadystate. After 175 h fibril growth slowed down/remains the same and plateu phase appeared. The kinetic species within each of the three phases of cMyb TAD fibrillization needs to be characterized to know which species are more toxic to the human cells. Maybe we or some other group can take up this task. It is evident that the expose of a hydrophobic amino acid residue to an external environment leads to the formation of b-sheet rich structured amyloid fibrils [28]. Bis-ANS is a hydrophobic dye that binds with hydrophobic surfaces (characteristic of protein aggregates and partially structured folding intermediates) and shows an
increase in fluorescence and characteristic blue shift [15]. Results obtained in this work agree with such an observation. Bis-ANS showed insignificant fluorescence with the highest peak at 547 nm without cMyb TAD aggregates but an increase in bis-ANS fluorescence intensity and a blue shift from 547 nm to 515 nm were observed in the presence of cMyb TAD aggregates, indicates cMyb TAD peptide aggregates may expose hydrophobic residues that result into enhanced fluorescence along with blue shift (Fig. 2B). Furthermore, to measure the hydrodynamic size (and change in size) and the number of particles in cMyb TAD we used NTA. The freshly dissolved cMyb TAD peptide shows the presence of very few particles of ~38 nm diameter and the number concentration of particles was 1.4 106 particles/mL. Upon 261 h, aggregates reached a large hydrodynamic diameter and the number concentration increased from 1.4 106 to 1.3 107 particles/mL (Fig. 2C), indicates the formation of the large-sized aggregates of cMyb TAD.
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 3. Aggregation kinetics and CD spectra for cMyb (291e315) TAD amyloid formation. (A) The time-dependent ThT kinetics of cMyb TAD in the presence of the incubated samples. ( ) represents experimental data and ( ) represents sigmoidal fitting. (B) Far-UV CD spectra of freshly dissolved cMyb TAD peptide sample (black) and the sample incubated for 261 h (red). Black and red bar represents the percentage of b-sheet secondary structure content for freshly dissolved and aggregated cMyb TAD peptide respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Fibrils formed by cMyb (291e315) TAD are rich in b-sheet secondary structure The conversion of the secondary structure of protein/peptides to a cross b-sheet structure is the representative feature of amyloid fibril formation [29]. Our results demonstrate that freshly dissolved cMyb TAD shows a negative peak at 202 nm which is the characteristic of a secondary structure composed mostly of the random coil, suggesting that the cMyb TAD monomers are disordered. This is in line with the published report [30]. After 261 h incubation, random coil structure reduced, and new minima at 218 nm with relatively reduced ellipticity were observed, which is characteristic of a b-sheet rich structure (Fig. 3B), suggesting that the cMyb TAD amyloid fibrils were occupied mainly in the aggregation medium with higher-order b-sheet structure (Fig. 3B). Further, the structural features of unaggregated and aggregated cMyb peptide were analyzed using the software DichroWeb [21] (Table 1). In the monomeric unaggregated state the disordered content was significantly high. The conformations changed at 261 h from random coil to a fibrillar state, the contents of unstructured secondary structures reduced significantly to 34.05%, and the b-sheet content increased to ~40.3%. These results denote, the cMyb TAD undergoes a conformational change from their disordered to the b-sheet rich structure that renders them to oligomerize and form fibrils. 3.4. cMyb (291e315) TAD displays typical amyloid fibrils in HRTEM and cytotoxicity to SH-SY5Y neuroblastoma cells In light of the fibrillar nature of cMyb TAD aggregated samples indicated by the ThT assay, bis-ANS assay, and NTA we performed an HR-TEM analysis to gain more insights into the morphological features of these fibrils. Incubation of cMyb TAD peptide for a
longer period (261 h) produced a long threadlike amyloid fibril. Our electron microscopic analysis displays typical amyloid morphology, characterized by long, straight and branched fibrils with a diameter of 6e16 nm (Fig. 4A). These fibrils are exhibited either in branching that connects long fibrils or in the bunch (black arrow) or either as single fibrils (white arrow). It is widely believed that aggregated/fibrillar amyloid proteins are toxic to human neurons and neuron-like cells [31]. Among all the reported amyloid-forming protein and natively unstructured protein aggregates play a more crucial role to produce cytotoxic species [32]. We were thus interested in determining whether disordered cMyb TAD fibrils are cytotoxic to human cells. Cytotoxicity of the aggregates formed by cMyb TAD was assessed on SHSY5Y neuroblastoma cells using MTT assay (Fig. 4B). We observed that the viability of cells gradually reduces at higher cMyb TAD amyloid concentrations (the ability of cells to reduce MTT was less at high concentration), suggesting that cMyb TAD has cytotoxic activities. 4. Conclusions Binding of the KIX domain to cMyb TAD is crucial for cMybdependent gene activation and repression, which is indeed vital for definitive hematopoiesis. Any changes in the cMyb TAD domain due to stress conditions such as a change in pH and temperature may affect the binding between them. Our results demonstrated that the incubation in an acidic stress condition resulted in the folding of cMyb TAD in higher-order b-sheet secondary structure with long fibrillar morphologies of 6e16 nm diameter. CD spectroscopy results revealed that the fibril formation accompanied by the conformational change (random coil to b-sheet structure) in monomeric cMyb TAD. These fibrils reduce cell viability, however,
Table 1 Estimates of the secondary structural contents of cMyb (291e315) TAD during aggregation as obtained from CD spectral analyses. Incubation time (hours) Secondary structure content (%)
a-helix b-sheet Turn Disordered
Freshly dissolved (0 h) cMyb (291e315) TAD
Aggregated (261 h) cMyb (291e315) TAD
10.35% 19.9% 13.3% 56.4%
2.95% 40.3% 22.65% 34.05%
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 4. Morphologies and cell viability assay for cMyb (291e315) TAD amyloid fibrils. (A) HR-TEM images were recorded at 200 kV. Scale bar represents 0.5 mm, and 100 nm respectively. Black and white arrows indicate a bunch of fibrils and single fibril respectively. The diameter of fibrils ranging from 6 to 16 nm is labeled on fibrils. (B) The cell viability assay on SH-SY5Y neuroblastoma cells at different concentration cMyb TAD aggregates. Error bar represents standard error mean (SEM).
exactly how is the effect of these fibrils being still must be studied. As many TAD domains have sequences and overall architectures analogous to cMyb TAD, the amyloid-like structures for a wider range of TAD domains need to be described in detail. In conclusion, this study provides valuable information on the amyloidogenic nature of cMyb TAD that will help in the future to elucidate whether aggregates generated by cMyb TAD are involved in disease pathologies. Author contributions RG: Conception, design, data analysis, and writing of the manuscript; KG: acquisition of data, analysis, and writing of the manuscript. Declaration of competing interest Authors declare that there is no conflict of interest. Acknowledgments Authors would like to thank IIT Mandi for all the facilities. RG is grateful to the Department of Biotechnology (DBT), Government of India for funding (Project: BT/PR15453/BRB/10/1460/2015). References [1] H.J. Dyson, Expanding the proteome: disordered and alternatively folded proteins, Q. Rev. Biophys. 44 (2011) 467e518, https://doi.org/10.1017/ S0033583511000060. [2] P.E. Wright, H.J. Dyson, Intrinsically disordered proteins in cellular signalling and regulation, Nat. Rev. Mol. Cell Biol. 16 (2015) 18e29, https://doi.org/ 10.1038/nrm3920. [3] V.N. Uversky, A decade and a half of protein intrinsic disorder: biology still waits for physics, Protein Sci. 22 (2013) 693e724, https://doi.org/10.1002/
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Amyloid formation by intrinsically disordered trans-activation domain of cMyb Kundlik Gadhave, Rajanish Giri* Indian Institute of Technology Mandi, School of Basic Sciences, VPO Kamand, Himachal Pradesh, 175005, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 December 2019 Accepted 20 January 2020 Available online xxx
The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291e315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the diseaseassociated amyloid-forming proteins such as Ab, Tau, a-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in bsheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6e16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions. © 2020 Elsevier Inc. All rights reserved.
Keywords: Protein aggregation cMyb Trans-activation domain Intrinsically disordered proteins Transcriptional regulation Amyloid
1. Introduction Intrinsically disordered proteins (IDPs) do not form stable structures when they are free in solution, though they still show biological activity [1,2]. These are strongly associated with cellular regulation (such as cell cycle, transcription, and translation) and are widespread from prokaryotes to eukaryotes [2e4]. IDPs have made a new paradigm called Disorder-Function-Paradigm [5] and mutations or changes in the cellular content of IDPs are involved in several human disease pathways include neurodegenerative diseases, cancer, diabetes, and cardiovascular diseases [2,6]. The selfassembly of proteins into pathogenic insoluble aggregates causes
Abbreviations: Ab, Amyloid beta; AD, Alzheimer’s disease; Bis-ANS, 4,40 -bis 1anilinonaphthalene 8-sulfonate; CBP, CREB-binding protein; CD, circular dichroism; DMEM, dulbecco’s modified eagle’s medium; HR-TEM, high-resolution transmission electron microscopy; IDPs, intrinsically disordered proteins; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PD, Parkinson’s disease; TAD, transcriptional activation domain; ThT, thioflavin t. * Corresponding author. School of Basic Sciences, Indian Institute of Technology Mandi, Himachal Pradesh, 175005, India. E-mail address: [email protected] (R. Giri).
amyloid diseases [7]. Amyloid diseases, also known as “conformational diseases”, occur due to misfolding of proteins/peptides into intermolecular b-sheet aggregated structures. These structures get stabilized by intermolecular interactions and further lead to the formation of oligomers, proto-fibrils, and fibrils [8]. Many factors trigger the onset of protein aggregation such as mutations, environmental changes or chemical modifications, reducing the conformational stability of the protein [8,9]. Importantly, several human diseases are allied with the amyloid formation of IDPs, such as Ab in Alzheimer’s disease (AD) [10], Tau in tauopathy and AD [11], a-synuclein in Parkinson’s disease (PD) [12], and amylin in type 2 diabetes mellitus (T2DM) [13]. Aggregation of IDPs may occur due to their unique structural features enabling them to fold into multiple different conformations [14]. Therefore, in recent years, disordered proteins have been the attention of intensive research. In general, very little information on the aggregation of the trans-activation domain (TAD) is available, example includes TAD domain of tumor suppressor protein p53, which forms amyloid aggregates in vitro at the acidic condition which are cytotoxic to human SH-SY5Y neuroblastoma cells [15]. The high existence of disordered regions in the TAD domains
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Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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and their participation in diverse human diseases increases the importance of generating information on the amyloid nature of TAD domains. As an essential step in that direction, we have studied the amyloid fibrilization of the transactivation domain of cMyb (cMyb TAD). The proto-oncogene cMyb is highly conserved among the vertebrates that encode nuclear transcription factor and functions in proliferation and differentiation [16,17]. cMyb is expressed in tissues and cells of hematopoietic origin, which is essential for normal hematopoiesis in vertebrates [16,17]. Importantly, the mutation in full-length cMyb has been reported to cause defects at the stages of hematopoiesis [18]. cMyb consists of the N-terminal DNAbinding domain (DBD) comprises of three imperfect repeats R1, R2 and R3, a central hydrophilic and slightly acidic transcriptional activation domain (TAD) and a C-terminal negative regulatory region [16,17] which are highly interdependent for the regulation of transcription [17]. Thus, change in the function (aberrant modification) in one domain of cMyb may affect the function of the whole protein and further transcriptional process. The knowledge of co-regulators of the transcription factors is crucial for a complete understanding of its mechanism of action. In response to that, extensive research has identified numerous cMyb TAD interaction partners (Fig. 1A) to recognize how cMyb transcriptional activity is regulated. These studies were performed extensively to study the folding and binding mechanisms between cMyb TAD and KIX. However, what would be the effect of change in buffer conditions or stress environment on the aggregation behaviour of these domains has not been studied yet. Accordingly, using the standard approaches, in our study, we have extensively studied the aggregation behaviour of the KIX binding domain of cMyb TAD, and this is the first report of the same. Aggregates formed by cMyb TAD are rich in b-sheet structure and are cytotoxic
to SH-SY5Y neuroblastoma cells. This amyloidogenic nature of the cMyb TAD domain under the stress condition could affect the function of whole cMyb protein as well as its binding with KIX. These processes may further affect the transcriptional regulation of target proteins. Therefore, in the future, understanding the complete mechanism of cMyb TAD and full-length cMyb protein fibrillization and its effect in vivo animal models is crucial to recognize its role in amyloid diseases. 2. Materials and methods 2.1. Amyloid aggregation of cMyb (291e315) TAD peptide cMyb (291e315) TAD peptide was dissolved in 50 mM phosphate buffer solution, pH 3. Samples for a peptide with the final concentration of 2.5 mg/mL were prepared and incubated at 4 C for 261 h under constant stirring at 1200 rpm. 2.2. Thioflavin T binding assay and ThT kinetics The kinetics of cMyb TAD aggregation samples were monitored at different time intervals in TECAN infinite M200 PRO plate reader in black 96-well plates. The excitation and emission wavelength was set at 450 nm and 490 nm respectively. At different incubation times, 25 mM samples from aliquots of the incubated samples of cMyb TAD peptide were taken and mixed with 20 mM ThT for fluorescence measurements. All measurements were done in triplicate, and the final value represents the average of three measurements. To obtain t50 value, the kinetic curve of cMyb aggregation were fitted to the following sigmoidal Boltzmann equation:
Fig. 1. cMyb TAD interacting partners and TANGO aggregation predictions. (A) cMyb TAD interaction with other proteins for transcription regulation. (a) cMyb: KIX complex (PDB ID: 1SB0). (b) Ternary cMyb: MLL: KIX complex (PDB ID: 2AGH). (c) Ternary HBZ77: KIX: cMyb complex (PDB ID: 6DNQ). (d) Ternary HBZ56: KIX: cMyb complex (PDB ID: 6DMX). (e) KIX: MLL/cMyb chimera (PDB ID: 5SVH). N and C represent N-terminal and C-terminal respectively. cMyb, KIX, MLL, HBZ77, and HBZ56 are showed in red, sea green, olive, magenta, and tan color respectively. (B) TANGO prediction for cMyb (291e315) TAD at different pH and temperature. (a) The b-sheet aggregation propensity at temperature 4 C, (b) 25 C, and (c) 37 C. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
K. Gadhave, R. Giri / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
y ¼ A2 þ (A1-A2)/(1 þ exp((x-x0)/dx))
(1)
Where A1 is the initial fluorescence value, A2 represents final fluorescence value, x0 is the t50 value (time at which ThT fluorescence reached 50% of maximum intensity), and dx is time constant. 2.3. Bis-ANS binding assay Bis-ANS binding experiments were performed using 25 mM of cMyb TAD aggregated sample mixed with 15 mM of bis-ANS, a dye which is widely used to measure the exposed hydrophobic surfaces of proteins and their aggregates [19]. The excitation wavelength was set at 380 nm, and the emission spectra were recorded from 400 to 650 nm (data presented 414e650 nm) in black 96-well plates using TECAN infinite M200 PRO plate reader.
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treated with different concentrations (0, 1, 3.125, 12.5, and 50 mM) of cMyb TAD aggregates in 100 ml fresh culture medium and incubated at 37 C for 48 h. MTT was added to the culture medium with a final concentration of 0.5 mg/mL and incubated for 3 h in a CO2 incubator. Then 100 ml of DMSO solution was added to dissolve formazan crystals, incubated for 10 min and the absorbance for each well was recorded at 590 nm with a reference wavelength of 630 nm using TECAN infinite M200 PRO plate reader. The data are reported as the percentage of the MTT reduced by the cells, and cell viability was shown relative to control cells which were not treated with the cMyb TAD aggregates. 3. Results and discussion 3.1. cMyb (291e315) TAD has a strong intrinsic propensity to aggregate at acidic condition
2.4. Nanoparticle tracking analysis (NTA) The nanoparticle tracking analysis (NTA) allows particle-byparticle measurement of number concentration and size distribution of nanoparticles/aggregates in solution. NTA measurements were performed at room temperature according to the procedure reported by Lu et al., [20]. 5 ml of cMyb TAD peptide from 2.5 mg/mL solution was mixed with 995 ml of filtered phosphate buffer of pH 3 and injected into the sample chamber of Nanosight LM10 (Nanosight, Amesbury, UK) equipped with a 405 nm laser. 2.5. Circular dichroism (CD) spectroscopy Far-UV CD spectra for 25 mM cMyb TAD peptide was recorded in Jasco J-1500 Circular Dichroism Spectrophotometer using a 1 mm path-length quartz cuvette. All the measurements were done at 25 C temperature, 50 nm scanning speed, 1 nm bandwidth, and 0.5 nm data interval. The sample spectra were recorded after subtracting from buffer spectra. Each spectrum was the average of three scans. The secondary structure contents (a-helix, b-sheet, random coil, and turn) of the samples were assessed using the CONTIN algorithm with a reference set 7 which is accessible from the DICROWEB website [21]. 2.6. High-resolution transmission electron microscopy (HR-TEM) The morphology of cMyb TAD fibrils was observed with a highresolution transmission electron microscope (HR-TEM) (FP 5022/ 22-Tecnai G2 20 S-TWIN, FEI). 7 ml of twenty-fold diluted aggregated samples were mounted on 200 mesh carbon-coated copper grids (Ted Pella, Inc, USA). The negative staining was done by using a 3% ammonium molybdate solution, then grids were air-dried overnight, and images were captured in HR-TEM with accelerating voltage at 200 kV. Further, the diameter of fibrils was measured using the open-source image processing program ImageJ [22]. 2.7. Cell viability assay (MTT assay) The cytotoxicity of aggregated cMyb TAD samples was evaluated in SH-SY5Y human neuroblastoma cells which were cultured in Dulbecco’s modified Eagle’s medium/Nutrient Mixture F-12 Ham (DMEM F-12) supplemented with 10% fetal bovine serum (FBS). The aggregated peptide sample was centrifuged at 15000 rpm for 30 min. The pellets were resuspended in PBS and again centrifuged at 15000 rpm for 30 min. Finally, pellets were resuspended in the DMEM medium to obtain the required concentration. The cells were seeded in 96 well-plate with a cell density of 3000 cells/well in 150 ml culture medium and incubated for 24 h. Further, cells were
Typically, proteins/peptides comprise some sequence stretches that possess inherent aggregation propensity. According to the ProtParam server [23], the cMyb TAD sequence (291-KEKRIKELELLLMSTENELKGQQVL-315) has a large number of hydrophilic residues (15 polar amino acids), among them, 5 residues are positively charged, and 5 residues are negatively charged. Similar features of cMyb TAD were observed in the case of disordered p53 (residues 1e63) TAD domain [15], that favor the aggregation of proteins. We performed in silico amyloidogenic propensity prediction by using TANGO [24] aggregation predictor at different pH conditions for the identification of amyloid-forming residues and calculated amyloidogenic tendency of cMyb TAD peptide. Interestingly, we found that the region ‘299-ELLLMSTE-306’ is showing aggregation propensity in all pH (pH 3e10) in the case of 4 C and 25 C temperature (Fig. 1a and b). However, at 37 C, pH 3 and 4 show a low aggregation score (Fig. 1c) and all other pHs (pH 5e10) display no aggregation propensity. Particularly at pH 3 and 4 C temperature, above-mentioned region displays a significant aggregation propensity as compared with other pH (pH 4e10) conditions (Fig. 1a). These observations revealed that cMyb TAD has a high aggregation propensity in acidic pH as compared with neutral or basic pH. In general, the rate of amyloid fibril formation is pHdependent [25]. Moreover, the studies have been revealed that the affinity of cMyb for KIX is pH-dependent. In acidic pH (pH 5.5), cMyb:KiX affinity is six-fold higher as compared with physiological pH (pH 7.0) [26]. Thus, we selected pH (pH 3) and temperature (4 C) for in vitro aggregation studies and further experiments are performed to demonstrate its amyloid nature. 3.2. cMyb (291e315) TAD aggregates are ThT and ANS-Positive We analyzed cMyb TAD binding to ThT dye by observing the increase of fluorescence at a different time. An increase in ThT fluorescence is an indication of the formation of ordered aggregates of amyloid types [15]. We found that, cMyb TAD aggregates displays near eight-fold increase in ThT fluorescence intensity as compared with ThT only with buffer (Fig. 2A). Further, aggregation kinetics of cMyb TAD monitored by ThT assay (Fig. 3A). Each point in Fig. 3A indicates the ThT fluorescence intensity in the presence of a fixed amount of the incubated sample (25 mM) taken at several time points of incubation. The fibril formation kinetics of cMyb TAD represents a typical phase of aggregation. Generally, the steps involved include first a lag phase (nucleation), second an elongation phase (growth), and third a saturation phase (steady-state) [27]. From ThT kinetics, we observed that the length of the lag phase was approximately 85 h which is significantly longer. Following the lag phase, cMyb TAD entered a growth phase. From 100 h the exponential phase was faster. Here, we determine the half
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 2. Dye binding assay and particle size measurement for cMyb (291e315) TAD aggregates. (A) ThT fluorescence spectra. The black and red curve indicates the ThT fluorescence spectra of ThT control and aggregated cMyb TAD respectively. (B) Bis-ANS fluorescence spectra. The black and red line shows ANS fluorescence spectra of Bis-ANS control and aggregated cMyb TAD respectively. (C) Particle hydrodynamic size and number concentration measurement using NTA. The black and red line indicates the cMyb TAD monomers and aggregates respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
time (t50), that denote the midpoint of the exponential phase of the reaction (time where fibril formation is 50% complete). A greater t50 value indicates a longer time required to form amyloid fibrils. The kinetic trace of cMyb TAD fibril formation was sigmoidal and represents t50 value of 124 ± 2.07 h that represent cMyb TAD takes a longer time for aggregation. Fibril formation by cMyb TAD was considered complete when ThT fluorescence reached a steadystate. After 175 h fibril growth slowed down/remains the same and plateu phase appeared. The kinetic species within each of the three phases of cMyb TAD fibrillization needs to be characterized to know which species are more toxic to the human cells. Maybe we or some other group can take up this task. It is evident that the expose of a hydrophobic amino acid residue to an external environment leads to the formation of b-sheet rich structured amyloid fibrils [28]. Bis-ANS is a hydrophobic dye that binds with hydrophobic surfaces (characteristic of protein aggregates and partially structured folding intermediates) and shows an
increase in fluorescence and characteristic blue shift [15]. Results obtained in this work agree with such an observation. Bis-ANS showed insignificant fluorescence with the highest peak at 547 nm without cMyb TAD aggregates but an increase in bis-ANS fluorescence intensity and a blue shift from 547 nm to 515 nm were observed in the presence of cMyb TAD aggregates, indicates cMyb TAD peptide aggregates may expose hydrophobic residues that result into enhanced fluorescence along with blue shift (Fig. 2B). Furthermore, to measure the hydrodynamic size (and change in size) and the number of particles in cMyb TAD we used NTA. The freshly dissolved cMyb TAD peptide shows the presence of very few particles of ~38 nm diameter and the number concentration of particles was 1.4 106 particles/mL. Upon 261 h, aggregates reached a large hydrodynamic diameter and the number concentration increased from 1.4 106 to 1.3 107 particles/mL (Fig. 2C), indicates the formation of the large-sized aggregates of cMyb TAD.
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 3. Aggregation kinetics and CD spectra for cMyb (291e315) TAD amyloid formation. (A) The time-dependent ThT kinetics of cMyb TAD in the presence of the incubated samples. ( ) represents experimental data and ( ) represents sigmoidal fitting. (B) Far-UV CD spectra of freshly dissolved cMyb TAD peptide sample (black) and the sample incubated for 261 h (red). Black and red bar represents the percentage of b-sheet secondary structure content for freshly dissolved and aggregated cMyb TAD peptide respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
3.3. Fibrils formed by cMyb (291e315) TAD are rich in b-sheet secondary structure The conversion of the secondary structure of protein/peptides to a cross b-sheet structure is the representative feature of amyloid fibril formation [29]. Our results demonstrate that freshly dissolved cMyb TAD shows a negative peak at 202 nm which is the characteristic of a secondary structure composed mostly of the random coil, suggesting that the cMyb TAD monomers are disordered. This is in line with the published report [30]. After 261 h incubation, random coil structure reduced, and new minima at 218 nm with relatively reduced ellipticity were observed, which is characteristic of a b-sheet rich structure (Fig. 3B), suggesting that the cMyb TAD amyloid fibrils were occupied mainly in the aggregation medium with higher-order b-sheet structure (Fig. 3B). Further, the structural features of unaggregated and aggregated cMyb peptide were analyzed using the software DichroWeb [21] (Table 1). In the monomeric unaggregated state the disordered content was significantly high. The conformations changed at 261 h from random coil to a fibrillar state, the contents of unstructured secondary structures reduced significantly to 34.05%, and the b-sheet content increased to ~40.3%. These results denote, the cMyb TAD undergoes a conformational change from their disordered to the b-sheet rich structure that renders them to oligomerize and form fibrils. 3.4. cMyb (291e315) TAD displays typical amyloid fibrils in HRTEM and cytotoxicity to SH-SY5Y neuroblastoma cells In light of the fibrillar nature of cMyb TAD aggregated samples indicated by the ThT assay, bis-ANS assay, and NTA we performed an HR-TEM analysis to gain more insights into the morphological features of these fibrils. Incubation of cMyb TAD peptide for a
longer period (261 h) produced a long threadlike amyloid fibril. Our electron microscopic analysis displays typical amyloid morphology, characterized by long, straight and branched fibrils with a diameter of 6e16 nm (Fig. 4A). These fibrils are exhibited either in branching that connects long fibrils or in the bunch (black arrow) or either as single fibrils (white arrow). It is widely believed that aggregated/fibrillar amyloid proteins are toxic to human neurons and neuron-like cells [31]. Among all the reported amyloid-forming protein and natively unstructured protein aggregates play a more crucial role to produce cytotoxic species [32]. We were thus interested in determining whether disordered cMyb TAD fibrils are cytotoxic to human cells. Cytotoxicity of the aggregates formed by cMyb TAD was assessed on SHSY5Y neuroblastoma cells using MTT assay (Fig. 4B). We observed that the viability of cells gradually reduces at higher cMyb TAD amyloid concentrations (the ability of cells to reduce MTT was less at high concentration), suggesting that cMyb TAD has cytotoxic activities. 4. Conclusions Binding of the KIX domain to cMyb TAD is crucial for cMybdependent gene activation and repression, which is indeed vital for definitive hematopoiesis. Any changes in the cMyb TAD domain due to stress conditions such as a change in pH and temperature may affect the binding between them. Our results demonstrated that the incubation in an acidic stress condition resulted in the folding of cMyb TAD in higher-order b-sheet secondary structure with long fibrillar morphologies of 6e16 nm diameter. CD spectroscopy results revealed that the fibril formation accompanied by the conformational change (random coil to b-sheet structure) in monomeric cMyb TAD. These fibrils reduce cell viability, however,
Table 1 Estimates of the secondary structural contents of cMyb (291e315) TAD during aggregation as obtained from CD spectral analyses. Incubation time (hours) Secondary structure content (%)
a-helix b-sheet Turn Disordered
Freshly dissolved (0 h) cMyb (291e315) TAD
Aggregated (261 h) cMyb (291e315) TAD
10.35% 19.9% 13.3% 56.4%
2.95% 40.3% 22.65% 34.05%
Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110
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Fig. 4. Morphologies and cell viability assay for cMyb (291e315) TAD amyloid fibrils. (A) HR-TEM images were recorded at 200 kV. Scale bar represents 0.5 mm, and 100 nm respectively. Black and white arrows indicate a bunch of fibrils and single fibril respectively. The diameter of fibrils ranging from 6 to 16 nm is labeled on fibrils. (B) The cell viability assay on SH-SY5Y neuroblastoma cells at different concentration cMyb TAD aggregates. Error bar represents standard error mean (SEM).
exactly how is the effect of these fibrils being still must be studied. As many TAD domains have sequences and overall architectures analogous to cMyb TAD, the amyloid-like structures for a wider range of TAD domains need to be described in detail. In conclusion, this study provides valuable information on the amyloidogenic nature of cMyb TAD that will help in the future to elucidate whether aggregates generated by cMyb TAD are involved in disease pathologies. Author contributions RG: Conception, design, data analysis, and writing of the manuscript; KG: acquisition of data, analysis, and writing of the manuscript. Declaration of competing interest Authors declare that there is no conflict of interest. Acknowledgments Authors would like to thank IIT Mandi for all the facilities. RG is grateful to the Department of Biotechnology (DBT), Government of India for funding (Project: BT/PR15453/BRB/10/1460/2015). References [1] H.J. Dyson, Expanding the proteome: disordered and alternatively folded proteins, Q. Rev. Biophys. 44 (2011) 467e518, https://doi.org/10.1017/ S0033583511000060. [2] P.E. Wright, H.J. Dyson, Intrinsically disordered proteins in cellular signalling and regulation, Nat. Rev. Mol. Cell Biol. 16 (2015) 18e29, https://doi.org/ 10.1038/nrm3920. [3] V.N. Uversky, A decade and a half of protein intrinsic disorder: biology still waits for physics, Protein Sci. 22 (2013) 693e724, https://doi.org/10.1002/
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Please cite this article as: K. Gadhave, R. Giri, Amyloid formation by intrinsically disordered trans-activation domain of cMyb, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.110