The COVID-19 Cell Signalling Problem: Spike, RAGE, PKC, p38, NFκB & IL-6 Hyper-Expression and the Human Ezrin Peptide, VIP, PKA-CREB Solution

Round 1

Reviewer 1 Report

SARS-CoV-2 infection induces several cellular responses that are meditated  by various intracellular signaling cascades in different cell types and different time frames. Among other, the infection inhibits interferon expression, and elevates the expression of proinflammatory cytokines. In this review, Dr. Holms describes the intracellular signaling pathways that are involved in or dysregulated by SARS-CoV-2 infection, and eventually trigger  the chronic inflammation associated with COVID. Overall, it is a timely and interesting review that covers most of the known SARS-CoV-2-induced processes and the signaling pathways that mediate them. However, parts of the review are confusing, as the background given is not sufficient, the sequence of chapters and sections is not always logical, and  some of the signaling details are not accurate. These, as well as a few other points described below should be corrected in order to allow publication.

Comments.

1. The review lacks a proper background on the effect of SARS-CoV-2 infection, and the sequence of physiological/pathological events that it generates over time in different cell types. For example, the introduction jumps immediately into the description of therapeutic peptides, and the regulation of innate and adaptive immune responses. It is recommended to better describe the general effects of the virus on lung and other cells, explaining in detail what is the logic of the sequence of presentation of the different signaling pathways. This can make the review much clearer, particularly to the signaling experts that are not so familiar with viral infections. Finally, it is suggested to give more background details, including recent reviews, on the main signaling pathways described.
2. In several cases, the context, duration and function of the activated intracellular signaling is not clear. It is recommended to always mention the upstream activation (e.g. ACE2, TLR4) and the process regulated.
3. Names are not always accurate, and in some cases can be shortened. For example, in some cases (but not all), the MAP3Ks are named MAPK3. MAPKp38 that appears in some places should be changed to p38MAPK. MAPK3s>MKK3/6>MAPKp38 can be shortened to p38MAPK cascade, and Raf>MEK>ERK>p90RSK, can be shortened to ERK cascade.
4. according to the current literature, IKKs are not directly regulated by p38, but rather by TAK1 or other mechanisms. If the SARS-CoV-2 indeed induces a direct activation of the NFkB pathway by activated p38, the study that shows this particular pathway should be mentioned.
5. In Fig. 1 and the accompanied text, it is not clear whether all components shown are forming one big complex, or whether there are various distinct smaller complexes that each can induce a distinct signaling pathway. What regulates the production of these complexes?
6. In Fig. 2, it should be noted that TAK1 is a MAP3K by itself, so it is not clear why there are separate lines to MKK3/6. As mentioned above, MAPK3s should be MAP3Ks. TABs can activate p38 without the involvement of TAK1 and MKK3/6, is it the case also upon SARS-CoV-2 infection? The role of Ezrin and Actin in the bottom left side is not clear. It is also recommended to show the mechanism by which the distinct components translocate to the nucleus. Please note that the same comments apply for Fig. 3 as well.
7. In Fig. 4, it is recommended to show the phosphorylation sites and the kinases that mediate them, as mentioned in the text (page 12, 15 lines from bottom).
8. In Fig. 5, the role of the cAMP above the ERK cascade is not clear. Is it affecting the cascade via EPAC? What is VCAP1 and how is it involved in the infection? What induces cAMP in the bottom part of the figure close to the PKA? Please note that some of the comments for Fig. 2 apply here as well.

Author Response

Author Response to Reviewer 1

Thank you for taking the time to make a detailed analysis of my manuscript.

I have made changes to the draft manuscript, based on your recommendations.

Regarding your comments:

1. I have added a recent general review reference on the pathological effects of SARS-CoV-2 in different cell types and tissues, and a recent general review reference on inflammation and intra-cellular signalling in COVID.

Trougakos, I. P.; Stamatelopoulos, K.; Terpos, E.; Tsitsilonis, O.E.; Aivalioti, E.; Paraskevis, D.; EfsKastritis, E.; Pavlakis, G.N.; Meletios A. Dimopoulos, M.A. “Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments

that target COVID-19 clinical complications” J Biomed Sci (2021) 28:9

https://doi.org/10.1186/s12929-020-00703-5

Choudhary, S.; Sharma, K. ; Silakari O.; “The interplay between inflammatory pathways and COVID-19: A critical review on pathogenesis and therapeutic options”

Microbial Pathogenesis 150 (2021) 104673

2. Please assist me on the page /paragraph location where the upstream activation receptor is missing. Generally, I have been drawing attention to the fact that different receptors have related intracellular signalling pathways.

3. MAP3Ks (not MAPK3) corrected and p38MAPK (not MAPKp38) corrected.

Generally I want to show the full activation cascade using > to denote each phosphorylation / activate step. In contrast I use + to show protein+protein association / binding.

4. MAPKp38 activation of IKK

Reference

Rian Craig, Andrea Larkin, Amy M. Mingo, Donna J. Thuerauf, Catherine Andrews,

Patrick M. McDonough, and Christopher C. Glembotski: “p38 MAPK and NF-kB Collaborate to Induce Interleukin-6 Gene Expression and Release”, THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 31, Issue of August 4, pp. 23814–23824, 2000

See diagram on p23822: Tak1/Tab>MKK6>p38>IKKbeta>IkB/NFkB>NFkB>IL-6

5. Multi-protein-complexes.

Specific protein+protein associations at the membrane are extensively described in the literature and in the case of ezrin, multiple distinct binding zones have been established for cell surface receptors, adhesion molecules, ion channels and kinases. These complexes form spontaneously depending on the topology and electrostatic fields associated with proteins and lipids. However, if different proteins are attracted to the same binding zone, clearly there will be competition. There is growing evidence that the array of: ACE2+CFTR+NHERF+NHE+EZRIN+PKC+PI3K+PKA+F-ACTIN

is physically possible, based on the binding zones and relative size of proteins involved (note in Figure 1, the protein images have been scaled based on the dimensions of alpha helix size common to all the proteins). However, the FERM domain of ezrin may be too small to bind CD44, CD43, ICAM-1, EGFR, SOS Grb2 and Ras etc, simultaneously.

6. In Fig 1 and Fig 2, I have amended the diagrams to reflect that TAK1 is one of the MAP3Ks. Generally, I have tried to keep the diagrams simple to focus on the central role of p38MAPK and NFkB in inflammatory signalling in COVID.

7. The main phosphorylation sites on ezrin are: tyrosine Y146, tyrosine Y354, Serine S366, tyrosine Y478, Serine S535 and Tyrosine Y567 (phosphorylated by ROCK2 and PKC/PRKCI), but I would prefer to keep this ezrin conformation diagram simple: the main idea is the folded inactive state of ezrin in the cyptoplasm, the elongated active conformation on the submembrane surface, and a potential transition conformation where ezrin has its Alpha domain exposed on the cell surface (which can detected by anti-ezrin alpha-domain monoclonal antibodies). I have added reference to the main phosphorylation sites and binding sites of ezrin:

 UniProtKB - P15311 (EZRI_HUMAN) https://www.uniprot.org/uniprot/P15311

8. I have amended the diagram in Fig 5 to avoid the confusion that cAMP is effecting the ERK pathway ( and copied across other corrections relating to MAP3Ks and p38).

There are two potential CREB activation routes from PKA+ezrin activation.

1. VIP binds VACP1 and stimulates Adenylyl Cyclase (AC), resulting in increasing cAMP intracellular concentration of cAMP, activation of ezrin-bound PKA. Activated PKA migrates then phosphorylates and activates CREB. Ezrin peptides may also cooperate in this activation of PKA.
2. Ezrin peptides may also induce an allosteric change in ezrin, stimulating Ras+SOS+FERM (ezrin) to activate the ERK cascade, which may also result in CREB activation.

Ezrin, an actin binding protein, and EPAC1 a cAMP-sensor, cooperate together to

activate cell spreading and other functions, in response to elevations in intracellular cAMP.

Parnell, E. Koschinski, A. Zaccolo M., Cameronc R, Baillie, G, Baillie, G. Porter, A. McElroy, S., Yarwood, S. “Phosphorylation of ezrin on Thr567 is required for the synergistic activation of cell spreading by EPAC1 and protein kinase A in HEK293T cells”. Biochimica et Biophysica Acta 1853 (2015) 1749–1758

Best wishes

Dr Rupert Holms

CEO, NewalR&D

Reviewer 2 Report

The paper revealed intimate mechanism of COVID Cell Signalling. The paper distinguishes as comprehensive, thorough and argumentatetive text. The article is consistent within itself. The references are relevant and recent. The cited sources are referenced correctly. Appropriate and key studies are included. The flow is logical and the data is presented critically.

There are no major flaws associated with this paper.

Author Response

Author Response to Reviewer 2

Thank you for your very positive summary of my manuscript. I shall amend the manuscript based on all Reviewer comments.

Best wishes

Dr Rupert Holms

CEO, NewalR&D

Reviewer 3 Report

The author explains the signaling cascade induced in mild, medium, and severe cases of SARS-Cov2 patients. The author starts with the mechanism involved with VIP and Ezrin related to CREB and TIRAP inhibition. Next, the manuscript describes how interferons act as a defense mechanism; that is disabled in SARS-Cov2 due to the activation of NFkB. Activation of NFkB scavenges all the CBP and P300, which are essential for the initiation of interferons. Following this, the author elucidates the signaling cascade between the different disease progression and the importance of cell surface m-RAGE. m-RAGE binds to damage-associated molecular patterns and pathogen-associated molecular patterns, triggering inflammatory pathways. Further, the manuscript discusses the role of S100 protein, which was elevated in severe COVID patients and related to the disease progression. m-RAGE phosphorylates MAPKp38 and activates its downstream targets ERK1/2 and MEK1/2, creating a cytokine storm of proinflammatory cytokines. Finally, the review discusses two therapeutic approaches: Ezrin, a sub-membrane adaptor protein containing specific binding sites for ROA and RAS, and thus regulated m-RAGE+TIRAP+S100 protein complex causing suppression of inflammatory signaling. The second target discussed in the manuscript is the Vasoactive intestinal peptide. High levels of VIP correlated with the survival of COVID, and that administration of VIP causes activation of PKA and its downstream target CREB. This signaling suppresses the secretion of proinflammatory cytokines. VIP administration caused adverse side effects such as high blood pressure and diarrhea, making it unsuitable for a long-term therapeutic approach. Overall, this manuscript explains the signaling pathways involved in SARS-CoV2 and the potential therapeutic targets of Ezrin and VIP.

• Any thoughts on whether AMPK activators such as metformin would work better than Ezrin since they target the same pathway downstream?
• Many references are missing when you state an experiment such as A549, statements on Ezrin anchored protein as a cell-cell junction, m-RAGE KO mice study, siRNA study, and other patient studies that are explained.
• What are your thoughts on MNK1/2 inhibitor drugs? Would that block NFkB signaling and thus increase the chances of survival?
• Does Ezrin bind to any particular serine and threonine site? You may want to mention if there is one.
• References are not formatted correctly.

Author Response

Author Response to Reviewer 3

Thank you for your detailed summary of my manuscript and your comments on References.

I have focused on attaching references to specific statements, which got disconnected from sources during re-organisation of paragraphs (however, in some cases, this results in duplication of references).

Thank you for suggesting I consider Metformin, which decreases COVID severity and mortality, probably by inhibiting p65 phosphorylation and NFkB mediated pro-inflammatory cytokine expression (Metformin does not activate CREB). In contrast, cAMP-PKA antagonizes Metformin activity by phosphorylating and inhibiting AMPK alpha sub-unit. In addition, Metformin increases half-life and cell surface expression of ACE2, which could increase SARS-CoV-2 infection susceptibility.

Olexandr Kamyshnyi, Victoriya Matskevych, Tetyana Lenchuk, Olha Strilbytska,

Kenneth Storey, Oleh Lushchak. “Metformin to decrease COVID-19 severity and mortality: Molecular mechanisms and therapeutic potential.” Biomedicine & Pharmacotherapy 144 (2021) 112230

Atul Malhotra, Mark Hepokoski, Karen C. McCowen and John Y-J Shyy

“ACE2, Metformin, and COVID-19” iScience 23, 101425, September 25, 2020

Ling He, Evan Chang, Jinghua Peng, Hongying An, Sara M. McMillin, Sally Radovick, Constantine A. Stratakis and Fredric E. Wondisford. “Activation of the cAMP-PKA pathway Antagonizes Metformin Suppression of Hepatic Glucose Production” THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 20, pp. 10562–10570, May 13, 2016 DOI 10.1074/jbc.M116.719666

Best wishes

Dr Rupert Holms

CEO, NewalR&D

Round 2

Reviewer 1 Report

I have no more comments