COVID-19 Research: Study Reveals That That SARS-CoV-2 Targets Proteins Involved In Endocytosis And Autophagy, Hijacking These Cellular Processes

COVID-19 Research: A new study by researchers from the European Molecular Biology Laboratory (EMBL)-Germany, Heidelberg University-Germany,  Fundación Instituto Leloir-Argentina, University College Dublin-Ireland, Merck KGaA-Germany and Universidad Nacional de San Martín-Argentina has identified sequences in human proteins that might be used by SARS-CoV-2 to infect cells.


 
The study team discovered that the SARS-CoV-2 virus targets proteins Involved In endocytosis and autophagy and also hijacks these cellular processes.
 
The SARS-CoV-2 coronavirus enters cells through endocytosis upon binding to the cell surface receptor ACE2 and potentially others, including integrins. Utilizing bioinformatics, the study team predicted the presence of short amino acid sequences, called short linear motifs (SLiMs), in the cytoplasmic tails of ACE2 and various integrins that may engage the endocytic and autophagic machinery. Using affinity binding assays,I t was not only confirmed that many of these predicted SLiMs interacted with target peptides in various components of the endocytosis and autophagy machinery but it was also found that these interactions were regulated by the phosphorylation of SLiM-adjacent amino acids. Together, these findings have identified a potential link between autophagy and integrin signaling and could lead to new ways to prevent viral infection.
 
The study findings were published in the peer reviewed journal: Science Signaling.
https://stke.sciencemag.org/content/14/665/eabd0334
 
During the early days of the COVID-19 pandemic, it was established that SARS-CoV-2 infects cells by binding to the human protein ACE2, which plays a role in regulating blood pressure. But ACE2 is almost absent in human lung cells, so how can the lungs be one of the most affected organs in COVID-19? This gave researchers a hint that ACE2 might be more than just a blood pressure regulator, and might not be the only player in the SARS-CoV-2 infection mechanism.
 
Interestingly the viral spike protein also has an RGD motif, suggesting that cell surface integrins may be co-receptors. The study team examined the sequences of ACE2 and integrins with the Eukaryotic Linear Motif (ELM) resource and identified candidate short linear motifs (SLiMs) in their short, unstructured, cytosolic tails with potential roles in endocytosis, membrane dynamics, autophagy, cytoskeleton, and cell signaling.
 
The team focused on short strings of amino acids called short linear motifs (SLiMs), which are involved in transmitting information between the inside and outside of cells. Quick identification and comparison of SLiMs was possible thanks to the Eukaryotic Linear Motif (ELM) resource, the largest curated SLiMs database, which the team and collaborators have been developing for 20 years.
 
These SLiM candidates are highly conserved in vertebrates and may interact with the μ2 subunit of the endocytosis-associated AP2 adaptor complex, as well as with various protein domains (namely, I-BAR, LC3, PDZ, PTB, and SH2) found in human signaling and regulatory proteins. Several motifs overlap in the tail sequences, suggesting that th ey may act as molecular switches, such as in response to tyrosine phosphorylation status. Candidate LC3-interacting region (LIR) motifs are present in the tails of integrin β3 and ACE2, suggesting that these proteins could directly recruit autophagy components.
 
The study findings identified several molecular links and testable hypotheses that could uncover mechanisms of SARS-CoV-2 attachment, entry, and replication against which it may be possible to develop host-directed therapies that dampen viral infection and disease progression. Several of these SLiMs have now been validated to mediate the predicted peptide interactions.
 
The study team saw that ACE2 and several integrins contain SLiMs that are probably involved in endocytosis and autophagy ie cellular processes of uptake and disposal of substances, respectively. This result suggests previously unknown roles of ACE2 and integrins in cell physiology.
 
First author of the study, Dr Bálint Mészáros from the Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL) told Thailand Medical News, “If SARS-CoV-2 targets proteins involved in endocytosis and autophagy, it means these processes might be hijacked by the virus during infection.”
 
Many of the findings were experimentally verified by Dr Ylva Ivarsson and her group at Uppsala University in Sweden. They confirmed the predicted protein interactions, and verified that these interactions are regulated by the naturally occurring addition of ions containing phosphorus.
 
EMBL team leader Dr Toby Gibson added, “Dr Ylva Ivarsson was the best person we knew of to test these predictions. We were delighted she agreed to join this project.”
 
Dr Ylva Ivarsson said, “Switching our work to SARS-CoV-2-related research helped us keep the spirit up in the lab during the pandemic.”
 
The study findings could lead to new therapeutic approaches for COVID-19.
 
Senior author Dr Lucía Chemes added, “SLiMs could ‘switch’ to turn viral entry signals on or off. This means that if we can find a way to reverse these switches using drugs, this might stop coronavirus from entering cells.”
 
Along with collaboration with pharmaceutical giant: Merck KGaA Darmstadt, the team gathered a list of existing drugs that interfere with endocytosis and autophagy. The list includes some surprising candidates, such as the antipsychotic chlorpromazine.
 
Dr Manjeet Kumar, a bioinformatics scientist in the Gibson team and a senior author in the study added, “If clinical trials prove some of these drugs to work against COVID-19, this could be a game changer.”
 
The study was initiated at the beginning of the first lockdown in Germany in spring 2020. The project was an opportunity to strengthen relations between scientists across continents.
 
Dr Chemes said, “Dr Toby and I have had a collaboration since 2012, when Argentina became an associate member of EMBL. Our previous experience enabled us now to work together on SARS-CoV-2.”
 
Conducting the research under lockdown conditions was not always easy. For example, one of the co-authors of the study, Dr Elizabeth Martínez Perez from Leloir Institute in Argentina, was unable to return from her secondment in the Gibson team at EMBL Heidelberg.
 
Also at the same time, Dr Manjeet Kumar had to adjust to working from home when his children were around. “I got kind support from our landlady to work in the attic of the building, though the internet signal wasn’t reaching there! Eventually, I bought a 35 meter internet cable and connected it to the attic. Once this was set, I got momentum in the project,” he recalls.
 
For the team working on the SARS-CoV-2 research, it  was an inspiring experience. “We wanted to contribute to combating COVID-19. This gave us a common aim,” says Dr Toby Gibson.
 
Dr Bálint Mészáros agreed, “It’s strange, thrilling, and a bit unsettling breaking new ground in the COVID-19 field. As researchers we’re enthusiastic about figuring out bits of the biology, but at the same time we’re thoroughly excited to work on such an important topic.”
 
The study team concluded, “In summary, we have presented evidence at the sequence level for SLiMs in ACE2 and β integrins with the potential to function in viral attachment, entry, and replication for SARS-CoV-2. We have identified several candidate molecular links and testable hypotheses that might help uncover the (still poorly understood) mechanisms of SARS-CoV-2 entry and replication. Because most of these motifs belong to host proteins acting as viral receptors, they are not revealed by virus-centered proteomic interaction assays. That they may well be functional, however, is indicated by sequence conservation, in some cases for hundreds of millions of years. In addition, the motifs are in appropriate cellular contexts to interact with their partner proteins. These putative motifs originally lacked direct experimental evidence, but the accompanying paper from Kliche et al. https://stke.sciencemag.org/content/14/665/eabf1117?intcmp=trendmd-stke shows that ACE2 YxxPhi, SH2, and PBM motifs and the integrin β₃ phospho-LIR bind to partner domains in vitro. Further experimental follow-up will yield insights into RME for the SARS-CoV-2 virus and, in addition, for the role of ACE2 in the normal cell, where it surely has much more functionality than its role as an ACE. Overall, the collection of candidate motifs in this system suggests that a range of HDTs might be explored including RGD inhibition, tyrosine kinase inhibition, endocytosis inhibition, and autophagy inhibition and/or activation.”
 
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