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Voltammetric and atomic force microscopy characterization of chymotrypsin, trypsin and caspase activities of proteasome
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Voltammetric and atomic force microscopy characterization of chymotrypsin, trypsin and caspase activities of proteasome Catarina Sofia Henriques de Jesus a , Ana-Maria Chiorcea Paquim a , Victor Constantin Diculescu b,∗ a b
Laboratory of Electroanalysis and Corrosion, Instituto Pedro Nunes, 3030-199, Coimbra, Portugal National Institute of Material Physics, 077125, Magurele, Romania
a r t i c l e
i n f o
Article history: Received 29 September 2016 Received in revised form 12 December 2016 Accepted 3 January 2017 Available online xxx Keywords: Proteasome Proteolysis Voltammetry Atomic force microscopy
a b s t r a c t Proteasome is a multicatalytic enzyme complex responsible for proteolysis of damaged proteins and an important target for drug discovery in the pharmaceutical industry. Development of fast and economic strategies for detection of proteasome activity and inhibition is a topic of intensive research. The activity of the 20S proteasome was investigated by voltammetry and atomic force microscopy. The hydrolysis of peptide bonds was studied in incubated solutions of proteasome with oligopeptide sequences specific to each chymotrypsin, trypsin and caspase activity of proteasome, before and after inhibition with epoxomicin. The time-dependence of the proteolysis and the effect of substrate and inhibitor concentrations on the rate of enzymatic reaction were investigated. Different interaction mechanisms were characterized and enzyme kinetic parameters determined. The adsorption patterns of reaction mixture components were characterized by atomic force microscopy in order to understand the processes when saturation of enzyme catalytic centres occurs for high substrate concentrations. © 2017 Elsevier B.V. All rights reserved.
1. Introduction One of the most important function of biomolecules is to act as catalysts in order to increase the rate of chemical reactions within a living cell [1]. Although nucleic acids (ribozymes and deoxyribozymes) are capable of catalysing some reactions [2,3] most biological reactions are catalyzed by enzymes. Cells contain thousands of different enzymes, and their activities determine the multitude of chemical reactions that take place in order to maintain the proper functioning and sustain life. Structural and conformational damages to proteins frequently occur and this represents the main cause of abnormal functioning which consequently can lead to undesired medical anomalies [4]. Proteasome is a multicatalytic enzyme system responsible for keeping the balance between proper-functioning and damaged proteins [5,6]. Essentially, proteasome function is to degrade unneeded or damaged proteins by catalysing the hydrolysis of peptide bond in long polypeptide chains which are broken down into shorter oligopeptides or even amino acid residues [7]. Proper pro-
∗ Corresponding author at: National Institute of Material Physics, Atomistilor Str. No. 405A, PO Box MG 7, 077125, Magurele, Romania. E-mail address: victor.diculescu@infim.ro (V.C. Diculescu).
teasome activity is essential for normal cellular functioning and deregulated proteasome action was observed in many malignancies [8]. In this context, fast, economic and reliable methodologies or strategies for detection of proteasome and to assess its activity are necessary for understanding the mechanisms of action, the enzyme kinetic and for development of inhibitors with potential medical applications [9]. Commonly, the in vitro measurement of proteasome catalytic activities is based on the use of fluorogenic peptides composed typically from a short amino acids sequence with C-terminally attached fluorescent probe [10], such as 2-naphtylamine, 7amino-4-methylcoumarin and 4-methoxy-2-naphtylamine [11]. The proteasome cleaves these substrates between the last amino acid and the probe, resulting in the release of the fluorescent molecule [12]. Similarly, a bioluminescent assay which enables the measurement of proteasome activities directly from cultured cells was reported [13]. On the other hand, the assembly of the proteasome was investigated by atomic force microscopy [14] while surface plasmon resonance was employed for development of methods for quantification of proteasome [15] and X-rays crystallography was used to study proteasome-inhibitor complexes [16]. Electrochemical methods are advantageous due to their fast response and the possibility to detect compounds at concentrations
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below nanomole level [17,18]. Voltammetry allows discriminating compounds through their redox potential values and represent one of the most economic strategies for detection of biological interactions [19]. Electrochemical methods were used for detection and assessment of the activity of different enzymes [20] including some proteases such as thrombin [21], -secretase [22] or trypsin [23]. Referring strictly to proteasome, voltammetry was used for quantification of proteasome [24] or of adenosine triphosphate through self-assembly of proteasome particles [25], but there is no report on the use of voltammetry for assessing proteasome activity. In the present work voltammetry was used to assess proteasome activity. The kinetic of the proteasome catalyzed-proteolysis was investigated before and after inhibition of proteasome with epoxomicin [26] and using several substrate specific to each chymotrypsin, trypsin and caspase activity. The interaction mechanisms between substrates and proteasome were characterized by voltammetry and atomic force microscopy was also used in order to understand the adsorption processes at electrode surface. The results are important in the context of development of fast and economic methodologies/strategies for detection of proteasome, its activity and inhibitors.
2. Experimental 2.1. Materials and reagents
2.3. Atomic force microscopy Atomic force microscopy (AFM) was performed in the acoustic AC (AAC) mode, with a PicoScan controller and a CS AFM S scanner with a scan range of 6 m in x-y and 2 m in z, from Agilent Technologies, USA. AppNano type FORT of 225 m length, 3.0 N m−1 spring constants and 47–76 kHz resonant frequencies in air (Applied NanoStructures, Inc., USA) were used. All AFM images were topographical and were taken with 512 samples/line x 512 lines and scan rates of 0.8–2.5 lines s−1 . When necessary, the AFM images were processed by flattening in order to remove the background slope and the contrast and brightness were adjusted. Highly oriented pyrolytic graphite (HOPG), grade ZYB of 15 × 15 × 2 mm3 dimensions, from Advanced Ceramics Co., USA, and mica, from Agilent Technologies, USA, were used as substrates in the AFM study, because they are atomically flat. The GCE used for the voltammetric characterization is rough and therefore unsuitable for AFM surface characterization. Furthermore, the voltammetric experiments using HOPG and GCE showed similar electrochemical behaviours. The HOPG was freshly cleaved with adhesive tape prior to each experiment and imaged by AFM in order to establish its cleanliness. The activity of 20S proteasome on the Suc-LLVY-AMC substrate of chymotrypsin was studied onto HOPG by AAC mode AFM in air, using incubated solutions of 0.5 g mL−1 20S proteasome with 10 M Suc-LLVY-AMC in PAB, during periods of time up to 24 h. Control experiments with the Suc-LLVY-AMC substrate were also performed using 10 M Suc-LLVY-AMC in PAB. 50 L samples of the desired solution were placed onto the freshly cleaved HOPG for 3 min, allowing the free adsorption of the molecules. The excess of solution was removed with Millipore Milli-Q water, and the modified HOPG was dried in a sterile atmosphere.
Proteasome 20S human, proteasome substrates: Suc-LLVY-AMC of chymotrypsin, Ac-RLR-AMC and Boc-LRR-AMC of trypsine, and Z-LLE-AMC of caspase activity, epoxomicin inhibitor from Enzo Life Sciences and 4-amino 7-methylcoumarin (AMC) from SigmaAldrich, were used without further purification. Stock solutions of substrates and inhibitor in DMSO and of 20S proteasome in proteasome assay buffer were prepared and kept at +4 ◦ C until further utilisation. Solutions of different concentrations of enzyme, substrates or inhibitor were obtained by dilution of the appropriate volume in the proteasome assay buffer. The proteasome assay buffer (PAB) pH = 7.5 containing 50 mM Tris/HCl, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2 and 100 M SDS was prepared using analytical grade reagents and purified water from a Millipore Milli-Q system (conductivity ≤ 0.1 S cm−1 ). The pH measurements were carried out with a Crison 2001 pHmeter with an Ingold combined glass electrode.
The AMC calibration curves were constructed by increasing the AMC concentration from 0 to 20 M in the presence of decreasing concentrations of the desired substrate from 100 to 80 M simulating the proteasome activity. Between measurements the GCE surface was always cleaned. DP voltammograms were recorded in the potential range of +0.20 V and +1.00 V. Three measurements were performed for each sample (n = 3).
2.2. Voltammetric parameters and electrochemical cells
2.5. Incubation procedure; measurement of 20S proteasome activity
Voltammetric experiments were carried out using a CompactStat potentiostat running IviumSoft 2.471 from Ivium Technologies, The Netherlands. The measurements were performed using a threeelectrode system in a one compartment V-shape electrochemical cell of 3 mL maximum capacity. The conical bottom of the electrochemical cell allowed measurements in 50 L solution. A glassy carbon (GCE, d = 1.0 mm), a Pt wire, and a Ag/AgCl (3 M KCl) were used as working, auxiliary and reference electrodes, respectively. The experimental conditions for differential pulse (DP) voltammetry were: pulse amplitude of 50 mV, pulse width of 100 ms, interval time 1 s and scan rate of 5 mV s−1 . Before each measurement the GCE was cleaned by surface polishing using diamond spray, particle size 3 M (Kemet, UK), and conditioned in buffer by recording several cyclic voltammograms until a reproducible baseline was obtained. After mechanical or chemical cleaning the GCE was rinsed thoroughly with Milli-Q water.
Samples of 20S proteasome and the desired substrate were allowed to equilibrate for 10 min at the reaction temperature under continuous stirring at 750 rpm in an Eppendorf ThermoMixer C. The reactions were carried out in Eppendorf test tubes and started by the addition of substrate to the 20S proteasome solution. 50 L of the reaction mixture were collected after different time intervals and placed into the electrochemical cell where DP voltammograms were recorded. AMC released from the substrate by the specific proteasome activity were measured up to 60 min by recording the current of the AMC oxidation peak at Epa ∼ +0.82 V on the DP voltammogram. Between measurements the GCE surface was cleaned. In order to study the inhibition of 20S proteasome by epoxomicin, similar reaction mixtures were prepared and incubated in the presence of the inhibitor. For control experiments, similar reaction mixtures were prepared in the absence of proteasome.
2.4. Calibration curve
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Table 1 Linear parameters obtained for each calibration curve of AMC in the presence of different 20S proteasome substrates. For more details please see Section 2.4. substrate
slope (A cm−2 M−1 )
intercept (A cm−2 )
chymotrypsin
Suc-LLVY-AMC
0.26
−0.32
trypsin
Ac-RLR-AMC Boc-LRR-AMC
0.31 0.29
−0.06 −0.12
caspase
Z-LLE-AMC
0.21
−0.08
activity
2.6. Acquisition and presentation of data All DP voltammograms were smoothed and baseline-corrected using an automatic function included in the IviumSoft version 2.471. This mathematical treatment improves the visualisation and identification of the peaks over the baseline without introducing any artefact. All experiments have been carried out in triplicate. Marvin Sketch implemented into Marvin Beans software from Chem Axon was used for the presentation of all chemical structures. Origin Pro 9.0 SR2 from OriginLab Corporation was used for the presentation of all the experimental data reported in this work. 3. Results and discussion 3.1. Characterization of 20S proteasome activity 3.1.1. Voltammetric analysis The proteasome is responsible for three different proteolytic activities. The chymotrypsin activity is defined as the cleavage of peptide bonds where the carboxyl side of the amide bond is in general a hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). Similarly, trypsin activity is related to the hydrolysis at the site of lysine or arginine, while the caspase activity at aspartic or glutamic acids residues. In this study several peptide substrates specific to each proteolytic activity of the 20S proteasome were used, Table 1. All substrates present at the C-terminal end a small molecule, the 4-amino 7-methylcoumarin (AMC), which is released from the peptide chain upon the action of proteasome as described in Eq (1) for the specific case of a chymotrypsin-like activity: 20S proteasome
Suc-LLVY-AMC
→
Suc-LLVY + AMC
(1)
The free AMC is electroactive and the DP voltammogram in a solution of AMC showed the oxidation peak at +0.75 V, Fig. 1. Contrary, on the DP voltammograms recorded in individual solutions of each substrate, in which the AMC molecule is bound to the peptide chain, no oxidation peak appeared in this potential range, Fig. 1. As an exception, the Suc-LLVY-AMC peptide also presents an additional peak at +0.62 V due to the electroactive tyrosine (Y/Tyr) residue [27]. This experiment shows that the release of the AMC moiety from the peptide structure upon the action of 20S proteasome can be followed by voltammetry. In order to investigate the enzyme kinetics, calibration curves that relate the AMC oxidation peak current with the AMC concentration were recorded as described in Section 2.4. DP voltammetry was used since this technique allows lower detection limits when compared with other voltammetric methods. The calibration curves were constructed by increasing the AMC concentration in the presence of decreasing concentrations of the desired substrate from simulating the proteasome activity. Linearity was observed in all cases and for the whole range of concentrations used. The line parameters are listed in Table 1. The time-dependence of the 20S proteasome activity was investigated by incubating different substrates with the 20S proteasome,
Fig. 1. DP voltammograms recorded in solutions of 10 M AMC (red curve) and 100 M 20S proteasome substrates (black curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
as described in Section 2.5. For each specific substrate, DP voltammograms were sequentially recorded up to 60 min incubation time, Fig. 2. The GCE surface was always renewed between voltammograms in order to avoid possible interferences with the adsorption of any of the reaction mixture components and/or their oxidation products at the electrode surface. For all substrates the AMC oxidation peak increased linearly up to 60 min of incubation time, in agreement with the increase of AMC concentration upon action of the 20S proteasome, Fig. 2. Control experiments were also performed. DP voltammograms were recorded in individual solutions of substrates after different incubation times in PAB in the absence of proteasome. No AMC oxidation peak was observed showing that substrate degradation does not occur in these conditions. Also, the 20S proteasome activity was investigated for different concentrations of substrate (not shown). For each substrate concentration, the DP voltammograms were recorded after 60 min incubation time always with a clean GCE surface. The AMC oxidation peak increased with substrate concentration (not shown) in agreement with a higher reaction rate. In all cases, the IAMC at 60 min incubation time was transformed into concentration values by using the AMC calibration curves in Table 1. This mathematical treatment allows calculating the initial rates of enzymatic reaction (V0 ) as CAMC after 60 min incubation time, Fig. 3. Also, the enzyme specific activity for Suc-LLVY-AMC was calculated for 50 and 100 M substrate to be 8.66 and 16.50 nmol min−1 mg−1 , respectively. For comparison, the enzyme specific activity reported by fluorescence spectroscopy for 75 M Suc-LLVY-AMC substrate in the absence and presence of 1 mM SDS was 9.4 and 53.2 pmol min−1 mg−1 , respectively [28]. The graphs of the variation of initial rate V0 vs. substrate concentration are shown in Fig. 3.The plots present typical shapes in which the reaction rates reach constant values for high substrate concentrations due to the saturation of enzyme catalytic centre with substrate molecules. For each curve non-linear regression was applied to the experimental data points using Hill equation (2) V0 =
V max × [S]n KM n + [S]n
(2)
where n is the Hill coefficient and K0.5 the substrate concentration at which V0 = Vmax /2. As a particular case, for n = 1 it resembles the Michaelis-Menten equation. The fitting results are summarized in Table 2.
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Fig. 2. DP voltammograms recorded after different incubation times in reaction mixtures containing 5 g mL−1 20S proteasome and: A) 100 M Suc-LLVY-AMC substrate of chymotrypsin, B) 100 M Ac-RLR-AMC and C) 200 M Boc-LRR-AMC of trypsin and D) 200 M Z-LLE-AMC of caspase-like activity. Table 2 K0.5 , Vmax and n values derived from non-linear regression of enzyme kinetic plots in Fig. 3 with Hill Eq. (2). activity
substrate
K0.5 (M)
vmax (M min−1 )
n
chymotrypsin
Suc-LLVY-AMC
127.9
0.113
1.1
trypsin
Ac-RLR-AMC Boc-LRR-AMC
158.8 201.5
0.059 0.055
1.4 3.9
caspase
Z-LLE-AMC
1070.0
0.052
1.0
For Suc-LLVY-AMC substrate of chymotrypsin-like activity, the data were alternatively fitted with Michaelis-Menten equation, Fig. 3A. Although the values of Vmax on one hand, and those of K0.5 and KM on the other hand were similar, the standard deviations were SD = 3.3 × 10−3 M min−1 and SD = 7.73 × 10−18 M min−1 for Michaelis-Menten and Hill equations, respectively. However, the n = 1.1, Table 2, shows that the enzyme follows a close Michaelis-Menten mechanism. A similar behaviour was observed in the case of Z-LLE-AMC of caspase activity and the enzyme followed a pure Michaelis-Menten kinetic with n = 1.0, Fig. 3D. Contrary, for substrates Ac-RLR-AMC and Boc-LRR-AMC of trypsin-like activity, Fig. 3B and C, the curves presented more pronounced sigmoidal aspects and the values of Hill coefficient n > 1.0 indicated positive cooperativity, Table 2. The analysis of the curves in Fig. 3 shows that, for increased substrates concentrations, the enzyme activity achieves constant maximal values in agreement with the saturation of all catalytic centres. In order to prove that the effects observed are due to pure
enzyme kinetic and not to electrode saturation effects atomic force microscopy imaging of the reaction mixture components at carbon electrodes was performed. 3.1.2. Atomic force microscopy The catalytic activity of 20S proteasome on the proteolysis of the Suc-LLVY-AMC substrate of chymotrypsin was investigated by AFM in air. For a correct evaluation of the degree of substrate degradation in the presence of 20S proteasome, small concentrations of 0.5 g mL−1 20S and 10 M Suc-LLVY-AMC in PAB were used. First, AFM was employed to study the free adsorption of SucLLVY-AMC peptide. AFM images showed the formation of a very smooth monolayer, of 0.46 ± 0.08 nm of height, Fig. 4A. Higher magnification images showed the predisposition of the Suc-LLVY-AMC peptides to adsorb close to each other, Fig. 4B, forming packed layers, oriented mainly along three directions dictated by the threefold symmetry of the graphite and by the interactions established with the surface. The Suc-LLVY-AMC peptides were not oriented at a fixed angle relative to the direction of the step edges of the HOPG, indicating that the orientation of the adsorbed layers was not induced by particular surface defects at HOPG step edges, but induced by epitaxial growth on the perfectly flat HOPG terraces. On the other hand, AFM images of free 20S proteasome are difficult to obtain, since the HOPG surface have the tendency to denaturate the protein. The activity of the 20S proteasome on Suc-LLVY-AMC substrate of chymotrypsine activity of proteasome was also investigated by AFM using incubated solutions of 0.5 g mL−1 20S with 10 M Suc-LLVY-AMC, Fig. 5. This procedure led to the co-adsorption of
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Fig. 3. Initial reaction rate (V0 ) function of substrate concentration for: A) Suc-LLVY-AMC substrate of chymotrypsin, B) Ac-RLR-AMC and C) Boc-LRR-AMC of trypsin and D) Z-LLE-AMC of caspase-like activity of the 20S proteasome; V0 values were determined using the calibration curves in Table 1 and the IAMC after 60 min incubation time. In A), the black dotted lines were obtained from non-linear least square fit of the rate data to the Hill Eq. (2) while the grey line with Michaelis-Menten equation. Fitting parameters are provided in Table 2.
Fig. 4. AFM images of 10 M Suc-LLVY-AMC substrate of chymotrypsin. The scale bars represent 100 nm.
20S proteasome, Suc-LLVY-AMC and the reaction products SucLLVY peptides and AMC. Also, the adsorption of 20S–Suc-LLVY-AMC complexes could not be neglected. After 0 h incubation, Fig. 5A and B, in freshly prepared solution, the AFM image shows the HOPG surface with the thin film of SucLLVY-AMC peptides and a number of 20S proteasome molecules (white arrows). Next to the 20S proteasomes, a number of small globular fragments were present, that were associated with aggregated AMC and/or Suc-LLVY molecules (red arrows). The measured
full width at half-maximum height (fwhm) of 20S proteasome was approximately 16–28 nm, which overestimates 20S diameter and length, due to the presence of the Suc-LLVY-AMC substrate and the convolution effect of the tip radius. Better representation of the 20S diameter is given by the height measurements, which are not limited by the tip radius. The free 20S proteasome molecules presented highs of 8.22 ± 0.26 nm, slightly smaller than the expected diameter of ∼11 nm, due to the molecules being dehydrated, and due to the vertical forces exercised by the AFM tip.
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Fig. 5. AFM images of 0.5 M 20S proteasome incubated with 10 M Suc-LLVY-AMC substrate of chymotrypsin, after A, B) 0 h, C), D) 4 h and E, F) 24 h incubation. A, C, E) Topographical and B, D, F) phase images. The scale bars represent 100 nm.
Increasing the incubation time to 4 h, a large number of small pores opened into the Suc-LLVY-AMC film, Fig. 5C and D, and the number of aggregates also increased. After 24 h incubation, the HOPG coverage by Suc-LLVY-AMC peptides decreased drastically and a large number of aggregates were formed, Fig. 5E and F. The AFM results are essential in understanding the adsorption processes in reaction mixtures containing 20S proteasome and its substrate. Although adsorption at the electrode surface was observed especially for substrate, the adsorption pattern drastically changes after proteolysis reaction and the presence of large pores into the adsorbed layer confirms that the effects observed in Fig. 3 are essentially due to enzyme saturation with substrate molecules.
3.2. Inhibition of the 20S proteasome by epoxomicin Epoxomicin is a potent and selective proteasome inhibitor originally isolated from Actinomycetes strain based on its potent in vivo antitumor activity. It inhibits the chymotrypsin-like activity of the proteasome but also blocks trypsin and caspase-like activities [26]. The inhibitory potential of epoxomicin on chymotrypsin, trypsin and caspase-like activities of 20S proteasome was investigated in incubated solutions of 5 g mL−1 20S proteasome with 250 M substrate in the presence of 1 nM and 5 M epoxomicin, Fig. 6. DP voltammograms were recorded in reaction mixtures in the presence of epoxomicin after different incubation times. The AMC oxidation peak increased linearly in time but at a lower rate than
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Acknowledgments Financial support from Romanian Ministry of Education and National Authority for Scientific Research and Innovation (ANCSI) through Project POC P-37-689-NANOBIOSURF and Programme PN16-480101-Fenomene si procese fizico-chimice in sisteme nanometrice complexe, suprafete si interfete, and from Fundac¸ão para a Ciência e Tecnologia (FCT) – Portugal through PTDC/DTPFTO/0191/2012 and SFRH/BPD/92726/2013 Post-doctoral Grant (A.-M. Chiorcea-Paquim) is gratefully acknowledged. References
Fig. 6. 20S proteasome chymotrypsin, trypsin and caspase-like residual activities upon inhibition with different concentrations of epoxomicin.
in the absence of the inhibitor. The remaining enzyme activity was calculated following Eq. (3): activity =
I × 100% I0
(3)
where I is the peak current in the presence and I0 in the absence of epoxomicin. The results in Fig. 6 confirm epoxomicin as an inhibitor of each proteasome function. However, in the case of trypsin and caspaselike activities high concentrations of epoxomicin were needed. Nevertheless, the chymotrypsin-like activity underwent the highest inhibition even for low concentrations of epoxomicin. 4. Conclusion Proteasome catalyzed-hydrolysis of oligopeptide sequences was investigated by voltammetry and atomic force microscopy. The activity of the proteasome was detected through the release of an electroactive probe molecule from the peptide strands. Several substrates specific to each chymotrypsin, trypsin and caspase activity were investigated before and after inhibition of proteasome with epoxomicin. The time-dependence of the proteolysis and the effect of substrate concentrations were studied and interaction mechanisms ranging from pure Michaelis-Menten to positive cooperativity were characterized allowing determination of enzyme kinetic parameters. Saturation effects were observed for high substrate concentrations. Atomic force microscopy was also used in order to evaluate the adsorption processes in reaction mixtures of proteasome and substrate. Different adsorption patters with large pores especially for long incubation times were characterized. The results respond to the necessities for the development of fast and economic methodologies/strategies for detection of proteasome activity and inhibition.
[1] T. Johannes, M.R. Simurdiak, H. Zhao, Biocatalysis, in: S. Lee (Ed.), Encyclopedia of Chemical Processes, CRC Press, 2005, 2017, pp. 101–110. [2] M.J. Fedor, J.R. Williamson, Nat. Rev. Mol. Cell Biol. 6 (2005) 399–412. [3] M. Hollenstein, Molecules 20 (2015) 20777–20804. [4] M. Gardes-Albert, D. Bonnefont-Rousselot, Z. Abedinzadeh, D. Jore, Actual. Chim. 11–12 (2003) 91–96. [5] A.J. Marques, R. Palanimurugan, A.C. Matias, P.C. Ramos, R.J. Dohmen, Chem. Rev. 109 (2009) 1509–1536. [6] F. Cesari, Nat. Rev. Mol. Cell Biol. 9 (2008), 498-498. [7] A. Varshavsky, Trends Biochem. Sci. 30 (2005) 283–286. [8] A. Ciechanover, A.L. Schwartz, Hepatology 35 (2002) 3–6. [9] X. Huang, V.M. Dixit, Cell Res. 26 (2016) 484–498. [10] A. Liggett, L.J. Crawford, B. Walker, T.C.M. Morris, A.E. Irvine, Leukemia Res. 34 (2010) 1403–1409. [11] A.F. Kisselev, A.L. Goldberg, Monitoring activity and inhibition of 26S proteasome with fluorogenic peptide substrates, in: R.J. Deshaies (Ed.), Methods in Enzymology: Ubiquitin and Protein Degradation. Part A, vol. 398, Gulf Professional Publishing, 2005, 2017, pp. 364–378. [12] C.R. Berkers, H. Ovaa, Biochem. Soc. Trans. 38 (2010) 14–20. [13] R.A. Moravec, M.A. O’Brien, W.J. Daily, M.A. Scurria, L. Bernad, T.L. Riss, Anal. Biochem. 387 (2009) 294–302. [14] M. Gaczynska, P.A. Osmulski, Atomic force microscopy as a tool to study the proteasome assemblies, in: J. Bhanu (Ed.), Methods in Cell Biology, vol. 90, Elsevier, 2008, 2017, pp. 39–60. [15] E. Gorodkiewicz, H. Ostrowska, A. Sankiewicz, Microchim. Acta 175 (2011) 177–184. [16] N. Gallastegui, M. Groll, Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies, in: R.J. Dohmen, M. Scheffner (Eds.), Ubiquitin Family Modifiers and the Proteasome: Reviews and Protocols, vol. 832, Springer Science+Business Media, 2012, pp. 373–389, Methods in Molecular Biology. [17] I.A. Rebelo, J.A.P. Piedade, A.M. Oliveira-Brett, Talanta 63 (2004) 323–331. [18] V.C. Diculescu, M. Vivan, A.M. Oliveira Brett, Electroanalysis 18 (2006) 1808–1814. [19] O.M. Popa, V.C. Diculescu, J. Electroanal. Chem. 742 (2015) 54–61. [20] V.C. Diculescu, T.A. Enache, Anal. Chim. Acta 845 (2014) 23–29. [21] J. Ji, J. Gan, J. Kong, P. Yang, B. Liu, C. Ji, Electrochem. Commun. 16 (2012) 53–56. [22] N. Xia, P. Peng, S. Wang, J. Du, G. Zhu, W. Du, L. Liu, Sens. Actuators B 232 (2016) 557–563. [23] E. Gonzalez-Fernandez, N. Avlonitis, A.F. Murray, A.R. Mount, M. Bradley, Biosens. Bioelectron. 84 (2016) 82–88. [24] T.-Y. Lin, K.-Y. Lee, T.-L. Chang, C.C. Chang, Y.Z. Lin, Sens. Actuators B 160 (2011) 412–417. [25] Y.Z. Lin, T.L. Chang, C.C. Chang, Sens. Actuators B 190 (2014) 486–493. [26] L. Meng, R. Mohan, B.H.B. Kwok, M. Elofsson, N. Sin, C. Crews, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 10403–10408. [27] T.A. Enache, A.M. Oliveira-Brett, J. Electroanal. Chem. 655 (2011) 9–16. [28] http://www.enzolifesciences.com/fileadmin/files/manual/BML-AK740 insert. pdf.
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Voltammetric and atomic force microscopy characterization of chymotrypsin, trypsin and caspase activities of proteasome Catarina Sofia Henriques de Jesus a , Ana-Maria Chiorcea Paquim a , Victor Constantin Diculescu b,∗ a b
Laboratory of Electroanalysis and Corrosion, Instituto Pedro Nunes, 3030-199, Coimbra, Portugal National Institute of Material Physics, 077125, Magurele, Romania
a r t i c l e
i n f o
Article history: Received 29 September 2016 Received in revised form 12 December 2016 Accepted 3 January 2017 Available online xxx Keywords: Proteasome Proteolysis Voltammetry Atomic force microscopy
a b s t r a c t Proteasome is a multicatalytic enzyme complex responsible for proteolysis of damaged proteins and an important target for drug discovery in the pharmaceutical industry. Development of fast and economic strategies for detection of proteasome activity and inhibition is a topic of intensive research. The activity of the 20S proteasome was investigated by voltammetry and atomic force microscopy. The hydrolysis of peptide bonds was studied in incubated solutions of proteasome with oligopeptide sequences specific to each chymotrypsin, trypsin and caspase activity of proteasome, before and after inhibition with epoxomicin. The time-dependence of the proteolysis and the effect of substrate and inhibitor concentrations on the rate of enzymatic reaction were investigated. Different interaction mechanisms were characterized and enzyme kinetic parameters determined. The adsorption patterns of reaction mixture components were characterized by atomic force microscopy in order to understand the processes when saturation of enzyme catalytic centres occurs for high substrate concentrations. © 2017 Elsevier B.V. All rights reserved.
1. Introduction One of the most important function of biomolecules is to act as catalysts in order to increase the rate of chemical reactions within a living cell [1]. Although nucleic acids (ribozymes and deoxyribozymes) are capable of catalysing some reactions [2,3] most biological reactions are catalyzed by enzymes. Cells contain thousands of different enzymes, and their activities determine the multitude of chemical reactions that take place in order to maintain the proper functioning and sustain life. Structural and conformational damages to proteins frequently occur and this represents the main cause of abnormal functioning which consequently can lead to undesired medical anomalies [4]. Proteasome is a multicatalytic enzyme system responsible for keeping the balance between proper-functioning and damaged proteins [5,6]. Essentially, proteasome function is to degrade unneeded or damaged proteins by catalysing the hydrolysis of peptide bond in long polypeptide chains which are broken down into shorter oligopeptides or even amino acid residues [7]. Proper pro-
∗ Corresponding author at: National Institute of Material Physics, Atomistilor Str. No. 405A, PO Box MG 7, 077125, Magurele, Romania. E-mail address: victor.diculescu@infim.ro (V.C. Diculescu).
teasome activity is essential for normal cellular functioning and deregulated proteasome action was observed in many malignancies [8]. In this context, fast, economic and reliable methodologies or strategies for detection of proteasome and to assess its activity are necessary for understanding the mechanisms of action, the enzyme kinetic and for development of inhibitors with potential medical applications [9]. Commonly, the in vitro measurement of proteasome catalytic activities is based on the use of fluorogenic peptides composed typically from a short amino acids sequence with C-terminally attached fluorescent probe [10], such as 2-naphtylamine, 7amino-4-methylcoumarin and 4-methoxy-2-naphtylamine [11]. The proteasome cleaves these substrates between the last amino acid and the probe, resulting in the release of the fluorescent molecule [12]. Similarly, a bioluminescent assay which enables the measurement of proteasome activities directly from cultured cells was reported [13]. On the other hand, the assembly of the proteasome was investigated by atomic force microscopy [14] while surface plasmon resonance was employed for development of methods for quantification of proteasome [15] and X-rays crystallography was used to study proteasome-inhibitor complexes [16]. Electrochemical methods are advantageous due to their fast response and the possibility to detect compounds at concentrations
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below nanomole level [17,18]. Voltammetry allows discriminating compounds through their redox potential values and represent one of the most economic strategies for detection of biological interactions [19]. Electrochemical methods were used for detection and assessment of the activity of different enzymes [20] including some proteases such as thrombin [21], -secretase [22] or trypsin [23]. Referring strictly to proteasome, voltammetry was used for quantification of proteasome [24] or of adenosine triphosphate through self-assembly of proteasome particles [25], but there is no report on the use of voltammetry for assessing proteasome activity. In the present work voltammetry was used to assess proteasome activity. The kinetic of the proteasome catalyzed-proteolysis was investigated before and after inhibition of proteasome with epoxomicin [26] and using several substrate specific to each chymotrypsin, trypsin and caspase activity. The interaction mechanisms between substrates and proteasome were characterized by voltammetry and atomic force microscopy was also used in order to understand the adsorption processes at electrode surface. The results are important in the context of development of fast and economic methodologies/strategies for detection of proteasome, its activity and inhibitors.
2. Experimental 2.1. Materials and reagents
2.3. Atomic force microscopy Atomic force microscopy (AFM) was performed in the acoustic AC (AAC) mode, with a PicoScan controller and a CS AFM S scanner with a scan range of 6 m in x-y and 2 m in z, from Agilent Technologies, USA. AppNano type FORT of 225 m length, 3.0 N m−1 spring constants and 47–76 kHz resonant frequencies in air (Applied NanoStructures, Inc., USA) were used. All AFM images were topographical and were taken with 512 samples/line x 512 lines and scan rates of 0.8–2.5 lines s−1 . When necessary, the AFM images were processed by flattening in order to remove the background slope and the contrast and brightness were adjusted. Highly oriented pyrolytic graphite (HOPG), grade ZYB of 15 × 15 × 2 mm3 dimensions, from Advanced Ceramics Co., USA, and mica, from Agilent Technologies, USA, were used as substrates in the AFM study, because they are atomically flat. The GCE used for the voltammetric characterization is rough and therefore unsuitable for AFM surface characterization. Furthermore, the voltammetric experiments using HOPG and GCE showed similar electrochemical behaviours. The HOPG was freshly cleaved with adhesive tape prior to each experiment and imaged by AFM in order to establish its cleanliness. The activity of 20S proteasome on the Suc-LLVY-AMC substrate of chymotrypsin was studied onto HOPG by AAC mode AFM in air, using incubated solutions of 0.5 g mL−1 20S proteasome with 10 M Suc-LLVY-AMC in PAB, during periods of time up to 24 h. Control experiments with the Suc-LLVY-AMC substrate were also performed using 10 M Suc-LLVY-AMC in PAB. 50 L samples of the desired solution were placed onto the freshly cleaved HOPG for 3 min, allowing the free adsorption of the molecules. The excess of solution was removed with Millipore Milli-Q water, and the modified HOPG was dried in a sterile atmosphere.
Proteasome 20S human, proteasome substrates: Suc-LLVY-AMC of chymotrypsin, Ac-RLR-AMC and Boc-LRR-AMC of trypsine, and Z-LLE-AMC of caspase activity, epoxomicin inhibitor from Enzo Life Sciences and 4-amino 7-methylcoumarin (AMC) from SigmaAldrich, were used without further purification. Stock solutions of substrates and inhibitor in DMSO and of 20S proteasome in proteasome assay buffer were prepared and kept at +4 ◦ C until further utilisation. Solutions of different concentrations of enzyme, substrates or inhibitor were obtained by dilution of the appropriate volume in the proteasome assay buffer. The proteasome assay buffer (PAB) pH = 7.5 containing 50 mM Tris/HCl, 25 mM KCl, 10 mM NaCl, 1 mM MgCl2 and 100 M SDS was prepared using analytical grade reagents and purified water from a Millipore Milli-Q system (conductivity ≤ 0.1 S cm−1 ). The pH measurements were carried out with a Crison 2001 pHmeter with an Ingold combined glass electrode.
The AMC calibration curves were constructed by increasing the AMC concentration from 0 to 20 M in the presence of decreasing concentrations of the desired substrate from 100 to 80 M simulating the proteasome activity. Between measurements the GCE surface was always cleaned. DP voltammograms were recorded in the potential range of +0.20 V and +1.00 V. Three measurements were performed for each sample (n = 3).
2.2. Voltammetric parameters and electrochemical cells
2.5. Incubation procedure; measurement of 20S proteasome activity
Voltammetric experiments were carried out using a CompactStat potentiostat running IviumSoft 2.471 from Ivium Technologies, The Netherlands. The measurements were performed using a threeelectrode system in a one compartment V-shape electrochemical cell of 3 mL maximum capacity. The conical bottom of the electrochemical cell allowed measurements in 50 L solution. A glassy carbon (GCE, d = 1.0 mm), a Pt wire, and a Ag/AgCl (3 M KCl) were used as working, auxiliary and reference electrodes, respectively. The experimental conditions for differential pulse (DP) voltammetry were: pulse amplitude of 50 mV, pulse width of 100 ms, interval time 1 s and scan rate of 5 mV s−1 . Before each measurement the GCE was cleaned by surface polishing using diamond spray, particle size 3 M (Kemet, UK), and conditioned in buffer by recording several cyclic voltammograms until a reproducible baseline was obtained. After mechanical or chemical cleaning the GCE was rinsed thoroughly with Milli-Q water.
Samples of 20S proteasome and the desired substrate were allowed to equilibrate for 10 min at the reaction temperature under continuous stirring at 750 rpm in an Eppendorf ThermoMixer C. The reactions were carried out in Eppendorf test tubes and started by the addition of substrate to the 20S proteasome solution. 50 L of the reaction mixture were collected after different time intervals and placed into the electrochemical cell where DP voltammograms were recorded. AMC released from the substrate by the specific proteasome activity were measured up to 60 min by recording the current of the AMC oxidation peak at Epa ∼ +0.82 V on the DP voltammogram. Between measurements the GCE surface was cleaned. In order to study the inhibition of 20S proteasome by epoxomicin, similar reaction mixtures were prepared and incubated in the presence of the inhibitor. For control experiments, similar reaction mixtures were prepared in the absence of proteasome.
2.4. Calibration curve
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Table 1 Linear parameters obtained for each calibration curve of AMC in the presence of different 20S proteasome substrates. For more details please see Section 2.4. substrate
slope (A cm−2 M−1 )
intercept (A cm−2 )
chymotrypsin
Suc-LLVY-AMC
0.26
−0.32
trypsin
Ac-RLR-AMC Boc-LRR-AMC
0.31 0.29
−0.06 −0.12
caspase
Z-LLE-AMC
0.21
−0.08
activity
2.6. Acquisition and presentation of data All DP voltammograms were smoothed and baseline-corrected using an automatic function included in the IviumSoft version 2.471. This mathematical treatment improves the visualisation and identification of the peaks over the baseline without introducing any artefact. All experiments have been carried out in triplicate. Marvin Sketch implemented into Marvin Beans software from Chem Axon was used for the presentation of all chemical structures. Origin Pro 9.0 SR2 from OriginLab Corporation was used for the presentation of all the experimental data reported in this work. 3. Results and discussion 3.1. Characterization of 20S proteasome activity 3.1.1. Voltammetric analysis The proteasome is responsible for three different proteolytic activities. The chymotrypsin activity is defined as the cleavage of peptide bonds where the carboxyl side of the amide bond is in general a hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). Similarly, trypsin activity is related to the hydrolysis at the site of lysine or arginine, while the caspase activity at aspartic or glutamic acids residues. In this study several peptide substrates specific to each proteolytic activity of the 20S proteasome were used, Table 1. All substrates present at the C-terminal end a small molecule, the 4-amino 7-methylcoumarin (AMC), which is released from the peptide chain upon the action of proteasome as described in Eq (1) for the specific case of a chymotrypsin-like activity: 20S proteasome
Suc-LLVY-AMC
→
Suc-LLVY + AMC
(1)
The free AMC is electroactive and the DP voltammogram in a solution of AMC showed the oxidation peak at +0.75 V, Fig. 1. Contrary, on the DP voltammograms recorded in individual solutions of each substrate, in which the AMC molecule is bound to the peptide chain, no oxidation peak appeared in this potential range, Fig. 1. As an exception, the Suc-LLVY-AMC peptide also presents an additional peak at +0.62 V due to the electroactive tyrosine (Y/Tyr) residue [27]. This experiment shows that the release of the AMC moiety from the peptide structure upon the action of 20S proteasome can be followed by voltammetry. In order to investigate the enzyme kinetics, calibration curves that relate the AMC oxidation peak current with the AMC concentration were recorded as described in Section 2.4. DP voltammetry was used since this technique allows lower detection limits when compared with other voltammetric methods. The calibration curves were constructed by increasing the AMC concentration in the presence of decreasing concentrations of the desired substrate from simulating the proteasome activity. Linearity was observed in all cases and for the whole range of concentrations used. The line parameters are listed in Table 1. The time-dependence of the 20S proteasome activity was investigated by incubating different substrates with the 20S proteasome,
Fig. 1. DP voltammograms recorded in solutions of 10 M AMC (red curve) and 100 M 20S proteasome substrates (black curves). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
as described in Section 2.5. For each specific substrate, DP voltammograms were sequentially recorded up to 60 min incubation time, Fig. 2. The GCE surface was always renewed between voltammograms in order to avoid possible interferences with the adsorption of any of the reaction mixture components and/or their oxidation products at the electrode surface. For all substrates the AMC oxidation peak increased linearly up to 60 min of incubation time, in agreement with the increase of AMC concentration upon action of the 20S proteasome, Fig. 2. Control experiments were also performed. DP voltammograms were recorded in individual solutions of substrates after different incubation times in PAB in the absence of proteasome. No AMC oxidation peak was observed showing that substrate degradation does not occur in these conditions. Also, the 20S proteasome activity was investigated for different concentrations of substrate (not shown). For each substrate concentration, the DP voltammograms were recorded after 60 min incubation time always with a clean GCE surface. The AMC oxidation peak increased with substrate concentration (not shown) in agreement with a higher reaction rate. In all cases, the IAMC at 60 min incubation time was transformed into concentration values by using the AMC calibration curves in Table 1. This mathematical treatment allows calculating the initial rates of enzymatic reaction (V0 ) as CAMC after 60 min incubation time, Fig. 3. Also, the enzyme specific activity for Suc-LLVY-AMC was calculated for 50 and 100 M substrate to be 8.66 and 16.50 nmol min−1 mg−1 , respectively. For comparison, the enzyme specific activity reported by fluorescence spectroscopy for 75 M Suc-LLVY-AMC substrate in the absence and presence of 1 mM SDS was 9.4 and 53.2 pmol min−1 mg−1 , respectively [28]. The graphs of the variation of initial rate V0 vs. substrate concentration are shown in Fig. 3.The plots present typical shapes in which the reaction rates reach constant values for high substrate concentrations due to the saturation of enzyme catalytic centre with substrate molecules. For each curve non-linear regression was applied to the experimental data points using Hill equation (2) V0 =
V max × [S]n KM n + [S]n
(2)
where n is the Hill coefficient and K0.5 the substrate concentration at which V0 = Vmax /2. As a particular case, for n = 1 it resembles the Michaelis-Menten equation. The fitting results are summarized in Table 2.
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Fig. 2. DP voltammograms recorded after different incubation times in reaction mixtures containing 5 g mL−1 20S proteasome and: A) 100 M Suc-LLVY-AMC substrate of chymotrypsin, B) 100 M Ac-RLR-AMC and C) 200 M Boc-LRR-AMC of trypsin and D) 200 M Z-LLE-AMC of caspase-like activity. Table 2 K0.5 , Vmax and n values derived from non-linear regression of enzyme kinetic plots in Fig. 3 with Hill Eq. (2). activity
substrate
K0.5 (M)
vmax (M min−1 )
n
chymotrypsin
Suc-LLVY-AMC
127.9
0.113
1.1
trypsin
Ac-RLR-AMC Boc-LRR-AMC
158.8 201.5
0.059 0.055
1.4 3.9
caspase
Z-LLE-AMC
1070.0
0.052
1.0
For Suc-LLVY-AMC substrate of chymotrypsin-like activity, the data were alternatively fitted with Michaelis-Menten equation, Fig. 3A. Although the values of Vmax on one hand, and those of K0.5 and KM on the other hand were similar, the standard deviations were SD = 3.3 × 10−3 M min−1 and SD = 7.73 × 10−18 M min−1 for Michaelis-Menten and Hill equations, respectively. However, the n = 1.1, Table 2, shows that the enzyme follows a close Michaelis-Menten mechanism. A similar behaviour was observed in the case of Z-LLE-AMC of caspase activity and the enzyme followed a pure Michaelis-Menten kinetic with n = 1.0, Fig. 3D. Contrary, for substrates Ac-RLR-AMC and Boc-LRR-AMC of trypsin-like activity, Fig. 3B and C, the curves presented more pronounced sigmoidal aspects and the values of Hill coefficient n > 1.0 indicated positive cooperativity, Table 2. The analysis of the curves in Fig. 3 shows that, for increased substrates concentrations, the enzyme activity achieves constant maximal values in agreement with the saturation of all catalytic centres. In order to prove that the effects observed are due to pure
enzyme kinetic and not to electrode saturation effects atomic force microscopy imaging of the reaction mixture components at carbon electrodes was performed. 3.1.2. Atomic force microscopy The catalytic activity of 20S proteasome on the proteolysis of the Suc-LLVY-AMC substrate of chymotrypsin was investigated by AFM in air. For a correct evaluation of the degree of substrate degradation in the presence of 20S proteasome, small concentrations of 0.5 g mL−1 20S and 10 M Suc-LLVY-AMC in PAB were used. First, AFM was employed to study the free adsorption of SucLLVY-AMC peptide. AFM images showed the formation of a very smooth monolayer, of 0.46 ± 0.08 nm of height, Fig. 4A. Higher magnification images showed the predisposition of the Suc-LLVY-AMC peptides to adsorb close to each other, Fig. 4B, forming packed layers, oriented mainly along three directions dictated by the threefold symmetry of the graphite and by the interactions established with the surface. The Suc-LLVY-AMC peptides were not oriented at a fixed angle relative to the direction of the step edges of the HOPG, indicating that the orientation of the adsorbed layers was not induced by particular surface defects at HOPG step edges, but induced by epitaxial growth on the perfectly flat HOPG terraces. On the other hand, AFM images of free 20S proteasome are difficult to obtain, since the HOPG surface have the tendency to denaturate the protein. The activity of the 20S proteasome on Suc-LLVY-AMC substrate of chymotrypsine activity of proteasome was also investigated by AFM using incubated solutions of 0.5 g mL−1 20S with 10 M Suc-LLVY-AMC, Fig. 5. This procedure led to the co-adsorption of
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Fig. 3. Initial reaction rate (V0 ) function of substrate concentration for: A) Suc-LLVY-AMC substrate of chymotrypsin, B) Ac-RLR-AMC and C) Boc-LRR-AMC of trypsin and D) Z-LLE-AMC of caspase-like activity of the 20S proteasome; V0 values were determined using the calibration curves in Table 1 and the IAMC after 60 min incubation time. In A), the black dotted lines were obtained from non-linear least square fit of the rate data to the Hill Eq. (2) while the grey line with Michaelis-Menten equation. Fitting parameters are provided in Table 2.
Fig. 4. AFM images of 10 M Suc-LLVY-AMC substrate of chymotrypsin. The scale bars represent 100 nm.
20S proteasome, Suc-LLVY-AMC and the reaction products SucLLVY peptides and AMC. Also, the adsorption of 20S–Suc-LLVY-AMC complexes could not be neglected. After 0 h incubation, Fig. 5A and B, in freshly prepared solution, the AFM image shows the HOPG surface with the thin film of SucLLVY-AMC peptides and a number of 20S proteasome molecules (white arrows). Next to the 20S proteasomes, a number of small globular fragments were present, that were associated with aggregated AMC and/or Suc-LLVY molecules (red arrows). The measured
full width at half-maximum height (fwhm) of 20S proteasome was approximately 16–28 nm, which overestimates 20S diameter and length, due to the presence of the Suc-LLVY-AMC substrate and the convolution effect of the tip radius. Better representation of the 20S diameter is given by the height measurements, which are not limited by the tip radius. The free 20S proteasome molecules presented highs of 8.22 ± 0.26 nm, slightly smaller than the expected diameter of ∼11 nm, due to the molecules being dehydrated, and due to the vertical forces exercised by the AFM tip.
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Fig. 5. AFM images of 0.5 M 20S proteasome incubated with 10 M Suc-LLVY-AMC substrate of chymotrypsin, after A, B) 0 h, C), D) 4 h and E, F) 24 h incubation. A, C, E) Topographical and B, D, F) phase images. The scale bars represent 100 nm.
Increasing the incubation time to 4 h, a large number of small pores opened into the Suc-LLVY-AMC film, Fig. 5C and D, and the number of aggregates also increased. After 24 h incubation, the HOPG coverage by Suc-LLVY-AMC peptides decreased drastically and a large number of aggregates were formed, Fig. 5E and F. The AFM results are essential in understanding the adsorption processes in reaction mixtures containing 20S proteasome and its substrate. Although adsorption at the electrode surface was observed especially for substrate, the adsorption pattern drastically changes after proteolysis reaction and the presence of large pores into the adsorbed layer confirms that the effects observed in Fig. 3 are essentially due to enzyme saturation with substrate molecules.
3.2. Inhibition of the 20S proteasome by epoxomicin Epoxomicin is a potent and selective proteasome inhibitor originally isolated from Actinomycetes strain based on its potent in vivo antitumor activity. It inhibits the chymotrypsin-like activity of the proteasome but also blocks trypsin and caspase-like activities [26]. The inhibitory potential of epoxomicin on chymotrypsin, trypsin and caspase-like activities of 20S proteasome was investigated in incubated solutions of 5 g mL−1 20S proteasome with 250 M substrate in the presence of 1 nM and 5 M epoxomicin, Fig. 6. DP voltammograms were recorded in reaction mixtures in the presence of epoxomicin after different incubation times. The AMC oxidation peak increased linearly in time but at a lower rate than
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Acknowledgments Financial support from Romanian Ministry of Education and National Authority for Scientific Research and Innovation (ANCSI) through Project POC P-37-689-NANOBIOSURF and Programme PN16-480101-Fenomene si procese fizico-chimice in sisteme nanometrice complexe, suprafete si interfete, and from Fundac¸ão para a Ciência e Tecnologia (FCT) – Portugal through PTDC/DTPFTO/0191/2012 and SFRH/BPD/92726/2013 Post-doctoral Grant (A.-M. Chiorcea-Paquim) is gratefully acknowledged. References
Fig. 6. 20S proteasome chymotrypsin, trypsin and caspase-like residual activities upon inhibition with different concentrations of epoxomicin.
in the absence of the inhibitor. The remaining enzyme activity was calculated following Eq. (3): activity =
I × 100% I0
(3)
where I is the peak current in the presence and I0 in the absence of epoxomicin. The results in Fig. 6 confirm epoxomicin as an inhibitor of each proteasome function. However, in the case of trypsin and caspaselike activities high concentrations of epoxomicin were needed. Nevertheless, the chymotrypsin-like activity underwent the highest inhibition even for low concentrations of epoxomicin. 4. Conclusion Proteasome catalyzed-hydrolysis of oligopeptide sequences was investigated by voltammetry and atomic force microscopy. The activity of the proteasome was detected through the release of an electroactive probe molecule from the peptide strands. Several substrates specific to each chymotrypsin, trypsin and caspase activity were investigated before and after inhibition of proteasome with epoxomicin. The time-dependence of the proteolysis and the effect of substrate concentrations were studied and interaction mechanisms ranging from pure Michaelis-Menten to positive cooperativity were characterized allowing determination of enzyme kinetic parameters. Saturation effects were observed for high substrate concentrations. Atomic force microscopy was also used in order to evaluate the adsorption processes in reaction mixtures of proteasome and substrate. Different adsorption patters with large pores especially for long incubation times were characterized. The results respond to the necessities for the development of fast and economic methodologies/strategies for detection of proteasome activity and inhibition.
[1] T. Johannes, M.R. Simurdiak, H. Zhao, Biocatalysis, in: S. Lee (Ed.), Encyclopedia of Chemical Processes, CRC Press, 2005, 2017, pp. 101–110. [2] M.J. Fedor, J.R. Williamson, Nat. Rev. Mol. Cell Biol. 6 (2005) 399–412. [3] M. Hollenstein, Molecules 20 (2015) 20777–20804. [4] M. Gardes-Albert, D. Bonnefont-Rousselot, Z. Abedinzadeh, D. Jore, Actual. Chim. 11–12 (2003) 91–96. [5] A.J. Marques, R. Palanimurugan, A.C. Matias, P.C. Ramos, R.J. Dohmen, Chem. Rev. 109 (2009) 1509–1536. [6] F. Cesari, Nat. Rev. Mol. Cell Biol. 9 (2008), 498-498. [7] A. Varshavsky, Trends Biochem. Sci. 30 (2005) 283–286. [8] A. Ciechanover, A.L. Schwartz, Hepatology 35 (2002) 3–6. [9] X. Huang, V.M. Dixit, Cell Res. 26 (2016) 484–498. [10] A. Liggett, L.J. Crawford, B. Walker, T.C.M. Morris, A.E. Irvine, Leukemia Res. 34 (2010) 1403–1409. [11] A.F. Kisselev, A.L. Goldberg, Monitoring activity and inhibition of 26S proteasome with fluorogenic peptide substrates, in: R.J. Deshaies (Ed.), Methods in Enzymology: Ubiquitin and Protein Degradation. Part A, vol. 398, Gulf Professional Publishing, 2005, 2017, pp. 364–378. [12] C.R. Berkers, H. Ovaa, Biochem. Soc. Trans. 38 (2010) 14–20. [13] R.A. Moravec, M.A. O’Brien, W.J. Daily, M.A. Scurria, L. Bernad, T.L. Riss, Anal. Biochem. 387 (2009) 294–302. [14] M. Gaczynska, P.A. Osmulski, Atomic force microscopy as a tool to study the proteasome assemblies, in: J. Bhanu (Ed.), Methods in Cell Biology, vol. 90, Elsevier, 2008, 2017, pp. 39–60. [15] E. Gorodkiewicz, H. Ostrowska, A. Sankiewicz, Microchim. Acta 175 (2011) 177–184. [16] N. Gallastegui, M. Groll, Analysing properties of proteasome inhibitors using kinetic and X-ray crystallographic studies, in: R.J. Dohmen, M. Scheffner (Eds.), Ubiquitin Family Modifiers and the Proteasome: Reviews and Protocols, vol. 832, Springer Science+Business Media, 2012, pp. 373–389, Methods in Molecular Biology. [17] I.A. Rebelo, J.A.P. Piedade, A.M. Oliveira-Brett, Talanta 63 (2004) 323–331. [18] V.C. Diculescu, M. Vivan, A.M. Oliveira Brett, Electroanalysis 18 (2006) 1808–1814. [19] O.M. Popa, V.C. Diculescu, J. Electroanal. Chem. 742 (2015) 54–61. [20] V.C. Diculescu, T.A. Enache, Anal. Chim. Acta 845 (2014) 23–29. [21] J. Ji, J. Gan, J. Kong, P. Yang, B. Liu, C. Ji, Electrochem. Commun. 16 (2012) 53–56. [22] N. Xia, P. Peng, S. Wang, J. Du, G. Zhu, W. Du, L. Liu, Sens. Actuators B 232 (2016) 557–563. [23] E. Gonzalez-Fernandez, N. Avlonitis, A.F. Murray, A.R. Mount, M. Bradley, Biosens. Bioelectron. 84 (2016) 82–88. [24] T.-Y. Lin, K.-Y. Lee, T.-L. Chang, C.C. Chang, Y.Z. Lin, Sens. Actuators B 160 (2011) 412–417. [25] Y.Z. Lin, T.L. Chang, C.C. Chang, Sens. Actuators B 190 (2014) 486–493. [26] L. Meng, R. Mohan, B.H.B. Kwok, M. Elofsson, N. Sin, C. Crews, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 10403–10408. [27] T.A. Enache, A.M. Oliveira-Brett, J. Electroanal. Chem. 655 (2011) 9–16. [28] http://www.enzolifesciences.com/fileadmin/files/manual/BML-AK740 insert. pdf.
Please cite this article in press as: C.S. Henriques de Jesus, et al., Voltammetric and atomic force microscopy characterization of chymotrypsin, trypsin and caspase activities of proteasome, Catal. Today (2017), http://dx.doi.org/10.1016/j.cattod.2017.01.012