Tau Protein Aggregation Associated With SARS-CoV-2 Main Protease
Raphael Josef Eberle, Mônika Aparecida Coronado, Ian Gering, Simon Sommerhage, Karolina Korostov, Anja Stefanski, Kai Stühler, Victoria Kraemer-Schulien, Lara Blömeke, Oliver Bannach, Dieter Willbold
Abstract
The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-β, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of tau aggregation by the SARS-CoV-2 3CL protease (3CLpro) possibly relevant in neuropathology. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.
Introduction
Viral pathogens encode their protease(s) or use host proteases for their replication cycle. In the case of acute respiratory syndrome coronavirus 2 (SARS-CoV-2), proteolytic cleavage of the two virus polyproteins generates the various viral proteins needed to form a replication complex required for transcription and replication of the viral genome and subgenomic mRNAs. The key viral enzymes responsible are the papain-like (PLP, nsp3) and 3-chymotrypsin-like proteases (3CLpro) [1–3]. In addition, host cell protein cleavage is a critical component of viral pathogenicity [4], including diverting cellular processes to viral replication, defeating antiviral responses and immune response modulation. Many large-scale analyses of the SARS-CoV-2 infected-cell transcriptome, proteome, phosphoproteome and interactomes are described [5–7]. Regarding the 3CLpro human substrate repertoire, also known as the degradome [8], Pablos et al., 2021 identified over 100 substrates and 58 additional high confidence candidate substrates out of SARS-CoV-2 infected human lung and kidney cells [9].
Material and methods
Preparation of alpha-synuclein, TDP-43 and 2N4R tau
Alpha-synuclein was cloned, expressed and purified, as described previously [35]. TDP-43 sample was kindly provided by Dr Jeanine Kutzsche (IBI-7, Forschungszentrum Jülich). The gene for human tau (2N4R isoform, uniprot ID: P10636-8) encodes a protein of 441 amino acids. The respective gene was commercially synthesised and cloned into the pET28A(+) vector (Genentech, San Francisco, USA), without His-tag. Protein expression was performed as described previously [36]. Protein extraction began by dissolving the cell pellet of 1 L expression in 30 ml buffer 1 (50 mM HEPES pH 7.5, 500 mM KCL, 5 mM β-ME and 1 mM EDTA). The dissolved cell pellets were heated for 30 min at 85°C followed by 10 min on ice, and samples were sonicated 3 x 40 seconds at a power setting of 5 in an ultrasonic cell disruptor Modell 250 (Branson Ultrasonic, Brookfield, USA). Bacterial debris was pelleted for 50 min at 10,000 x g. Soluble tau protein was precipitated from the supernatant by adding 40 ml of a saturated ammonium sulfate solution and incubated for 30 minutes at room temperature.
Results
Purification of 2N4R tau with a precipitation approach and characterisation of the protein
2N4R tau was expressed in BL21 (DE3) (T1) E. coli and purified by a precipitation approach. 2N4R tau consists of 441 amino acids with an approximated molecular weight of 46 kDa. The purity was assessed by SDS PAGE (S1 Fig). However, the protein presented a single band on a denaturing SDS-PAGE gel with an apparent molecular mass of around 67 kDa, this behavior was described previously [42, 43]. A western blot with the specific antibody (Tau13, Biolegend) confirmed the target protein (S2A Fig). Following successful purification, 2N4R tau was characterised to compare the properties to those previously reported [44–47]. It is well known that tau, in the monomeric state, is inherently unfolded, with predominantly random-coil conformation. Our CD analysis confirmed this observation for the purified protein, with minimum peaks around 200 nm (S2B Fig). Tau aggregation was investigated using ThT assay and heparin as an inducer [48]. The results of the ThT assay indicated that heparin promoted the induction and acceleration of tau aggregation within 24h (S2C Fig). The structural changes of tau in the presence of heparin were followed by CD spectroscopy, demonstrating a shift of the absorbance spectrum from 202 (random-coil conformation) to 213 (beta-sheet conformation) nm (S2B Fig). As a control, the CD spectrum of 2.5 μM heparin was measured, the spectra showed no specific minimum at 213 nm (S3B Fig).
Discussion
Surface-based fluorescence intensity distribution analysis
Surface-based fluorescence intensity distribution analysis (sFIDA) was performed to quantify the tau oligomers and aggregates after treatment with 3CLpro. The technique employs a similar biochemical setup as ELISA-like techniques. However, sFIDA uses the same epitope to capture and detect antibodies and features single-particle sensitivity through a microscopy-based readout (Herrmann et al., 2017). Recently, sFIDA was applied to quantify tau aggregates in cerebrospinal fluid (CSF) and demonstrated its applicability in clinical settings [37]. Initial sFIDA experiments include analysis of tau monomers, tau aggregates and tau SiNaPs (Fig 5A–5C). To quantify tau aggregates formed by 3CLpro proteolysis, two approaches were tested: tau plus active 3CLpro and tau plus inactivated 3CLpro (with the addition of disulfiram). As shown in Fig 5D, tau samples containing active 3CLpro yielded a similar aggregate-specific readout compared with tau samples in the presence of inactivated protease. Compared to the tau aggregate control, however, only a small fraction of the employed tau substrate was converted into aggregates.
Conclusion
The proportion of older adults in the population is increasing in almost all countries. Worldwide, around 55 million people have dementia, which is expected to increase to 78 million in 2030 and 139 million in 2050 [67]. Different dementias show a conformationally altered tau, the protein detaches from microtubules and aggregates into oligomers and neurofibrillary tangles, which can be secreted from neurons, and spread through the brain during disease progression. The COVID-19 pandemic has increasingly moved virus infections into the scientific spotlight and has shown that this infection can damage the brain in many ways. The molecular underpinnings of neurodegenerative processes need to be investigated to develop appropriate therapies. Proteolysis of tau protein may be a crucial factor in forming toxic aggregates. Our results demonstrated that the SARS-CoV-2 3CLpro can cleave 2N4R tau into fragments and thus induce protein aggregation in vitro.
Acknowledgments
The authors gratefully acknowledge the electron microscopy training, imaging and access time granted by the life science EM facility of the Ernst-Ruska Centre at Forschungszentrum Jülich. We want to thank the support of the Institute of Biological Information Processing (IBI-7) Forschungszentrum Jülich, Germany.
Citation: Eberle RJ, Coronado MA, Gering I, Sommerhage S, Korostov K, Stefanski A, et al. (2023) Tau protein aggregation associated with SARS-CoV-2 main protease. PLoS ONE 18(8): e0288138. https://doi.org/10.1371/journal.pone.0288138
Editor: Maria Gasset, Consejo Superior de Investigaciones Cientificas, SPAIN
Received: March 16, 2023; Accepted: June 20, 2023; Published: August 21, 2023
Copyright: © 2023 Eberle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: D.W. is supported by Deutsche Forschungsgemeinschaft (DFG, German Research Foundation), Project-ID 267205415, SFB 1208. sFIDA was supported by the programs “Biomarkers Across Neurodegenerative Diseases I + II” of The Alzheimer’s Association, Alzheimer’s Research UK and the Weston Brain Institute (11084 and BAND-19-614337). We are also grateful for support from The Michael J. Fox Foundation for Parkinson’s Research (14977, 009889), from the ALS Association and from the Packard Center (19-SI-476). We received further funding from the Deutsche Forschungsgemeinschaft (INST 208/616-1 FUGG, INST 208/794-1 FUGG) and the Helmholtz Association (HVF0079).
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0288138#abstract0
