A sensor for the enzymatic activity and inhibition of the 20S proteasome was developed by immobilizing the synthetic peptide ABZ-VVSYAMG-(O2Oc)2-OH at Au electrodes. The detection principle is based on the electroactivity of ABZ, part of the ABZ-VVSY-OH moiety released from the peptide upon 20S proteasome chymotrypsin action. The peptide was immobilized on a para-amino thiophenol (PATP) self-assembled monolayer on Au electrode by cross-linking its amino group to the -(O2Oc)2-OH moiety of the peptide (Au/PATP/peptide). The immobilization of the peptide and its interaction with 20S proteasome was investigated by SEM, QCM, SPR, ATR-FTIR and electrochemistry. The activity of 20S proteasome was assessed electrochemically by cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) after the immersion Au/PATP/peptide in 20S proteasome solution. CV study showed a decrease in both capacitive and faradaic currents corresponding to the ABZ-VVSY-OH removal, allowing the quantification of the 20S proteasome activity. The EIS study revealed that the resistance corresponding to charge transfer reactions at the peptide/solution interface correlated to the ABZ redox reaction, decreased linearly with increasing the incubation time in 20S proteasome solution. The perfected assay was applied for the investigation of the inhibitory effect of one synthetic, bortezomib, and two naturally occurring, epoxomicin, and lactacystin inhibitors.
Understanding intracellular protein degradation is a topic of huge interest for researchers because of the need to monitor, control and develop new pathways against several mortal diseases. Proteases are often used as biomarkers since they are known to play a significant role in many pathologies, such as cardiovascular diseases, HIV, Alzheimer's disease, thrombosis, diabetes and cancer, including microbial contamination and infection [1].
Among the proteases, 20S proteasome is a multicatalytic ubiquitin proteinase system and represents the core part of the 26S proteasome nonlysomal proteolytic structure [2]. The proteasome has been first localized in the nucleus and in the cytoplasm of all eukaryotic cell [3], cell membrane[4] and in human alveolar space [5]. However, the recently discovered 20S proteasome secreted outside the cell, circulating proteasome [6], [7], has the most significant diagnostic value, its presence and high enzymatic activity being observed in several types of cancer malignancies[8], where it has been associated with abnormally fast division of cancer cell [7]. This connection between the level and high enzymatic activity of proteasomes and certain types of cancer malignancies has made its detection and monitoring of enzymatic activity important for clinical trials. As a result, highly specific proteasome inhibitors have been approved for the treatment of several types of cancer, especially hematological cancer [9]. The need to understand the inhibitor therapeutic action as well as the importance in developing new inhibitors for cancer treatment has made the detection of proteasome activity extremely important and of high demand. Proteasome activity has been investigated using fluorescently tagged substrates specific for the three main components of proteasome activity, being based on the release of the fluorescent molecule [10] (most commonly, 7-amino-4-methylcoumarin (AMC)) following proteasomal cleavage of the substrate. Taking advantage on the AMC electroactivity, fast and reliable electrochemical methods have been also recently developed [11], [12], [13], [14]. Recently a dual strategy for the electrochemical detection of 20S proteasome was developed, based on a capture monoclonal antibody (Abβ): (i) through the cleavage of an AMC-marked substrate by the bounded 20S proteasome and (ii) through the enzymatic activity of alkaline phosphatase (AlkP) by using an AlkP labelled detection antibody [14]. All the above-mentioned electrochemical assays have applicability of 20S proteasome inhibitor detection.
In this context, there is considerable interest in developing sensitive and selective assays and sensors that monitor protease activity, which at this point can be classified into two main groups: i ) homogeneous assays, using colorimetry, mass spectrometry and fluorescence) and ii) heterogeneous systems which are the electrochemical assays, surface-enhanced Raman scattering and surface plasmon resonance [15]. All the above methods require specially designed peptides marked with a detectable tag that enable protease activity monitoring, There are several commercially available peptides marked with fluorogenic reporter group, with rather low sensitivity and specificity [16] and new strategies to improve their use are being reported [17]. Other approaches involve immobilizing magnetic nanoparticles to the sensor surface using peptides [18], or nanoparticles detectable using mass spectrometry [19]. Amongst the above mentioned methods used for protease sensing, the electrochemical peptide-based biosensors are particularly of interest, especially those based on redox tagged peptides that are immobilized onto an electrode surface, since they can offer high sensitivity, fast response times, they can be integrated in multiplexing system with use of relatively cheap instrumentation, and nevertheless ease of miniaturization for point-of-care (PoC) applications [20], [21], [22] Using microelectrodes and self-assembled monolayers of redox-tagged short chain proteins, a biosensor system was successfully used for the protease activity monitoring [23]. Synthetic peptides were also currently designed to access the proteolytic activity of proteasome, with the possibility to target with high accuracy and specificity just one proteolytic active subunit of the proteasome [24], [25]. and have applicability mostly in proteasome inhibition studies [26]. Since the chymotrypsin-like sites are the most important in protein degradation and most proteasome inhibitors are specific to it [27], the majority of synthetic peptides are targeting these active site and are used to investigate inhibitor’s mechanism of action and to further identify new possible compounds with inhibitor effect, for anticancer treatment development.
This work comes as a response to the need of having highly sensitive, low cost, performant tools to determine the specific catalytic activity and inhibition of 20S proteasome using synthetic peptides with high specificity and sensitivity. The peptide sequence used in the present work is ABZ-VVSYAMG-(O2Oc)2-OH (the VVSYAMG is the amino acid sequence consisting in valine-valine-serine-tyrosine-alanine-methionine-glycine, and O2Oc is 8-amino-3,6-dioxooctanoic acid, which acts as a linker), which targets the chymotrypsin-like activity of the 20S proteasome releasing the ABZ-VVSY-OH fragment, and where the electroactive ABZ (2-aminobezoic acid) is electroactive. The peptide is immobilized at a para-amino-thiophenol (PATP) modified Au electrode surface by cross-linking the -(O2Oc)2-OH terminal group with carbodiimide EDC in the presence of N-hydroxysuccinimide (NHS). The peptide modified electrode is schematically represented in Scheme 1, with the proteolysis action of proteasome upon its incubation in proteasome solutions highlighted.
The stepwise modification of the Au electrode with the peptide was monitored via electrochemical methods, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) together with surface analysis techniques, scanning electron microscopy (SEM), surface plasmon resonance (SPR) spectroscopy, Fourier transformed infrared spectroscopy (FT-IR). Surface analysis techniques enabled also to determine the effect of 20S proteasome on the peptide upon Au/PATP/peptide incubation in 20S proteasome solution. The activity of proteasome is investigated electrochemically by recording CV and an EIS spectra after pre-defined immersion times of the peptide modified electrode in the 20S solution. After the proteasome activity assay perfected, inhibition studies were also performed. Proteasome inhibitors with therapeutic application usually act on its chymotrypsin-like sites, therefore the inhibitors used in the present study inhibit the chymotrypsin-type proteolysis and were bortezomib, a synthetic inhibitor, and two natural occurring inhibitors: epoxomicin, and lactacystin [28], [29].
Proteasome 20S (human) was from Enzo Life Sciences (USA). Peptide substrates of the 20S proteasome were synthesized in agreement with Section 2.2. Bortezomib, epoxomicin and lactacystin proteasome inhibitors were from Selleck Chemicals (USA). 4-Aminothiophenol (PATP), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), N-N-dimethylformamide (DMF), EDC, NHS were from Sigma-Aldrich. The reagents were of analytical grade and were used without further purification. The proteasome assay buffer
The objective of this investigation is the development of a sensor for the detection of enzymatic activity and inhibition of the 20S proteasome. The proteolytic activity of the 20S proteasome was investigated in synergy with the peptide sequence ABZ-VVSYAMG-(O2Oc)2-OH, which upon the chymotrypsin action of 20S proteasome releases ABZ-VVSY-OH fragment, where the ABZ (2-aminobenzoic acid) moiety is electroactive. The design of the sensor consists in the immobilization of the peptide at a PATP
An electrochemical sensor for the detection of the 20S proteasome enzymatic activity and inhibition was developed using a synthetic peptide, ABZ-VVSYAMG-(O2Oc)2-OH, with ABZ as an electroactive moiety, immobilized through cross-linking at the surface of a para-amino thiophenol (PATP) modified Au electrode. The oriented immobilization of the peptide and the proteolytic action of the 20S proteasome were evidenced by scanning electron microscopy, gravimetric analysis and surface plasmon resonance
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Financial support from the Romanian Ministry of Research and Innovation through Operational Programme Competitiveness 2014-2020, Project: NANOBIOSURF-SMIS 103528; and Romanian Ministry of Research, Innovation and Digitalization, Project PN19-03 (contract no. 21N/08.02.2019). The authors thank G. Stan for the FTIR measurements.
