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Review 2: "Protein arginylation is regulated during SARS-CoV-2 infection"

This preprint looks at how SARS-CoV-2 infection modulates arginylation, a modification that tags proteins for degradation, and finds a specific arginylation signature in some cell types. The reviewers found the claims potentially informative.

Published onJan 26, 2022
Review 2: "Protein arginylation is regulated during SARS-CoV-2 infection"
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Protein arginylation is regulated during SARS-CoV-2 infection
Description

ABSTRACTIn 2019, the world witnessed the onset of an unprecedented pandemic. In September 2021, the infection by SARS-CoV-2 had already been responsible for the death of more than 4 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER-stress and activation of unfolded protein response (UPR) pathway. The degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-end rule pathway. Here we present, for the first time, data on the role of arginylation during SARS-CoV-2 infection. We studied the modulation of protein arginylation in Vero CCL-81 and Calu-3 cells infected after 2h, 6h, 12h, 24h, and 48h. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure the levels of ATE1 and arginylated proteins. This regulation is seen specifically during infections by coronaviruses. We demonstrate that during SARS-CoV-2 infection there is an increase in the expression of the ATE1 enzyme associated with regulated levels of specific arginylated proteins. On the other hand, infected macrophages showed no ATE1 regulation. An important finding revealed that modulation of the N-end rule pathway differs between different types of infected cells. We also confirmed the potential of tannic acid to reduce viral load, and furthermore, to modulate ATE1 levels during infection. In addition, the arginylation inhibitor merbromin (MER) is also capable of both reducing viral load and reducing ATE1 levels. Taken together, these data show the importance of arginylation during the progression of SARS-CoV-2 infection and open the door for future studies that may unravel the role of ATE1 and its inhibitors in pathogen infection.

RR:C19 Evidence Scale rating by reviewer:

  • Potentially informative. The main claims made are not strongly justified by the methods and data, but may yield some insight. The results and conclusions of the study may resemble those from the hypothetical ideal study, but there is substantial room for doubt. Decision-makers should consider this evidence only with a thorough understanding of its weaknesses, alongside other evidence and theory. Decision-makers should not consider this actionable, unless the weaknesses are clearly understood and there is other theory and evidence to further support it.

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Review:

Arginylation is an important but underappreciated post-translational modification (PTM). Catalyzed by the eukaryotic enzyme ATE1, the PTM arginylation is beginning to emerge as an essential regulator of intracellular homeostasis—not only through its degradative properties in tagging proteins for ubiquitination and proteasomal degradation via the N-degron pathway— but also through its non-degradative control of protein stability and oligomerization. Coming fresh off the heels of recent revelations that arginylation is important in maintaining the stability of Lentiviruses, such as the human immunodeficiency virus (HIV), it is perhaps unsurprising to find that the level of arginylation may be regulated by infection of Coronaviruses like SARS-CoV-2.

This preprint by Macedo-da-Silva et. al demonstrates the effects of SARS-CoV-2 infection on eukaryotic intracellular arginylation. Using several model cell lines, such as kidney epithelial cells (Vero E6) and lung adenocarcinoma (Calu-3 cells), cellular protein arginylation was studied over two days post-SARS-CoV-2 infection. Basal levels of enzymes involved in the N-degron pathway and their levels post-infection were assessed in human and monkey cell lines. Unfortunately, despite the large size and amount of the data collected, very little data are actually presented in the preprint; rather, some broad conclusions are presented (e.g., Figs. 2, 3, 5). In general, it appears that infection of SARS-CoV-2: a) does increase ATE1 expression (except in macrophages); b) displays a corollary between increases in ATE1 expression and several endoplasmic-reticulum centered events; c) shows a change in arginylation-related proteins associated with the cytoskeleton, cytoplasm, and nucleus; and d) does not increase ATE1 in patient-derived macrophages but does increase BiP and hspA5 gene expression. These effects seem to be limited to the Coronaviridae family; however, actual data to support this statement appear to be lacking in Fig. S5. Indeed, the lack of available data effectively requires the reader to put trust in the interpretation of the authors where they should otherwise convince the reader that these statements are true.

One large weakness of the work lies in the exploration of the inhibitors of ATE1-catalyzed arginylation such as tannic acid and merbromin. While the authors show limited data that the viral load in Calu-3 cells treated with tannic acid and merbromin is diminished, it is unclear (and not presented) as to whether these inhibitors actually prevent virus entry into the cell or instead affect further downstream processes. Unfortunately, the current understanding of how tannic acid and/or merbromin inhibit ATE1 is completely unknown at the atomic level. It is also unclear whether tannic acid is capable of entering the cell and having a direct or (rather) indirect effect on ATE1 function. Moreover, while merbromin is a known antiseptic, the presence of the mercury moiety may have off-target effects on other Cys-rich intracellular proteins. The authors claim that the cellular viability was not affected by either inhibitor (although the data are not shown), but what may occur after long-term exposure to either of these inhibitors is unknown and untested. Thus, readers should be extremely cautious as these compounds are neither approved nor explored clinically as SARS-CoV-2 infection inhibitors, and thus more work is absolutely necessary to validate this aspect of the preprint.

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