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PUBLISHED VERSION: Understanding Selenium and Glutathione as Antiviral Factors in COVID-19: Does the Viral M pro Protease Target Host Selenoproteins and Glutathione Synthesis?

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Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally link selenium and glutathione, which both show correlations with clinical outcomes in COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact with an inactive C145A mutant of Mpro , the main cysteine protease of SARS-CoV-2, but not with catalytically active wild-type Mpro. This seemingly anomalous result is what might be expected if GPX1 is a substrate for the active protease, leading to its fragmentation. We show that the GPX1 active site sequence is substantially similar to a known Mpro cleavage site, and is identified as a potential cysteine protease site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent with previously documented effects of recombinant SARS-CoV Mpro in transfected cells, including increased reactive oxygen species and NF-κB activation. Because NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could contribute to increased inflammation and cytokine storms observed in COVID-19. Using web-based protease cleavage site prediction tools, we show that Mpro may be targeting not only GPX1, but several other selenoproteins including SELENOF and thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme for glutathione synthesis. This hypothesized proteolytic knockdown of components of both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby enhancing virion production. The resulting "collateral damage" of increased oxidative stress and inflammation would be exacerbated by dietary deficiencies of selenium and glutathione precursors.
Comparison of known and predicted Mpro cleavage sites. The most relevant positions P4-P1′ of the 11 known SARS coronavirus Mpro cleavage sites in nonstructural proteins are shown (for nsp4 through nsp16), aligned with predicted sites in several human selenoproteins (GPX1, SelenoF, SelenoP, and TXNRD1), as well as glutaredoxin-1 (GLRX-1) and the rate-limiting enzyme for glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC, with 2 predicted cleavage sites a and b). The known nsp cleavage sites shown are all from SARS-CoV-2, with the addition of the nsp5/6 site from SARS-CoV (marked *). The sites identified in human proteins are displayed next to the known Mpro cleavage site to which they are most similar, highlighted in matching color. The experimentally determined catalytic efficiencies of SARS-CoV Mpro at each of the known nsp cleavage sites are shown as relative kcat/Km ([10]). Three different methods for prediction of cysteine protease cleavage sites were used: PROSPER, with numbers in parenthesis showing the score for the predicted site, any score > 0.8 being significant; NetCorona, where scores > 0.5 are considered positive, and ProCleave, where every possible P4-P4′ octamer in a protein sequence is evaluated, scored and ranked. Some aligned non-identical residues highlighted in italics are nonetheless chemically and structually similar to residues found in the same position in the known cleavage sites, e.g., serine (S) vs. threonine (T), Leucine (L) or isoleucine (I) vs. valine (V), and glutamine (Q) vs. asparagine (N); all of these pairs differ only by a single carbon atom (CH3 or CH2 unit). The predicted Mpro cleavage sites in human proteins labeled A to G are discussed in the text.
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PERSPECTIVE
published: 02 September 2020
doi: 10.3389/fnut.2020.00143
Frontiers in Nutrition | www.frontiersin.org 1September 2020 | Volume 7 | Article 143
Edited by:
Christophe Matthys,
KU Leuven, Belgium
Reviewed by:
Alan Diamond,
University of Illinois at Chicago,
United States
Anna Kipp,
Friedrich Schiller University
Jena, Germany
*Correspondence:
Ethan Will Taylor
ewtaylor@uncg.edu
Specialty section:
This article was submitted to
Nutritional Immunology,
a section of the journal
Frontiers in Nutrition
Received: 26 June 2020
Accepted: 21 July 2020
Published: 02 September 2020
Citation:
Taylor EW and Radding W (2020)
Understanding Selenium and
Glutathione as Antiviral Factors in
COVID-19: Does the Viral Mpro
Protease Target Host Selenoproteins
and Glutathione Synthesis?
Front. Nutr. 7:143.
doi: 10.3389/fnut.2020.00143
Understanding Selenium and
Glutathione as Antiviral Factors in
COVID-19: Does the Viral Mpro
Protease Target Host Selenoproteins
and Glutathione Synthesis?
Ethan Will Taylor*and Wilson Radding
Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, NC, United States
Glutathione peroxidases (GPX), a family of antioxidant selenoenzymes, functionally
link selenium and glutathione, which both show correlations with clinical outcomes in
COVID-19. Thus, it is highly significant that cytosolic GPX1 has been shown to interact
with an inactive C145A mutant of Mpro, the main cysteine protease of SARS-CoV-2,
but not with catalytically active wild-type Mpro. This seemingly anomalous result is
what might be expected if GPX1 is a substrate for the active protease, leading to its
fragmentation. We show that the GPX1 active site sequence is substantially similar
to a known Mpro cleavage site, and is identified as a potential cysteine protease
site by the Procleave algorithm. Proteolytic knockdown of GPX1 is highly consistent
with previously documented effects of recombinant SARS-CoV Mpro in transfected
cells, including increased reactive oxygen species and NF-κB activation. Because
NF-κB in turn activates many pro-inflammatory cytokines, this mechanism could
contribute to increased inflammation and cytokine storms observed in COVID-19.
Using web-based protease cleavage site prediction tools, we show that Mpro may
be targeting not only GPX1, but several other selenoproteins including SELENOF and
thioredoxin reductase 1, as well as glutamate-cysteine ligase, the rate-limiting enzyme
for glutathione synthesis. This hypothesized proteolytic knockdown of components of
both the thioredoxin and glutaredoxin systems is consistent with a viral strategy to
inhibit DNA synthesis, to increase the pool of ribonucleotides for RNA synthesis, thereby
enhancing virion production. The resulting “collateral damage” of increased oxidative
stress and inflammation would be exacerbated by dietary deficiencies of selenium and
glutathione precursors.
Keywords: coronavirus, COVID-19, selenium, glutathione, glutathione peroxidase 1, protease, selenoprotein,
thioredoxin reductase
Taylor and Radding Selenoproteins as SARS-CoV-2 Protease Substrates
INTRODUCTION
Several independent studies have now established a significant
association between the outcome of COVID-19 and previously
documented regional selenium (Se) status in Chinese cities (1),
and a similar relationship between serum Se and mortality in a
European cohort of COVID-19 patients (2). These observations
invite questions about the mechanisms involved, particularly
because they fit into a consistent pattern of a role for Se that has
been reported over several decades for a variety of RNA viruses
(enteroviruses, hantaviruses, and influenza A) and viruses with
an RNA stage (HIV-1 and Hepatitis B virus), as reviewed by
various authors (35).
Most (but not all) of the biological roles of Se, both
as selenocysteine in selenoproteins, and as redox-active Se-
containing metabolites, involve interactions with cysteine thiols
and disulfides in proteins and peptides, and their various
oxidized forms. Like Se, the essential antioxidant and free radical
scavenger glutathione (GSH), a tripeptide thiol, has also proven
to be an important factor in various viral infections, particularly
in HIV/AIDS, as reviewed in section 2.1.2. of Taylor (6), and
most recently, in COVID-19 (7,8). Given their intertwined
biochemical roles, there are likely to be common factors and
mechanisms underlying the therapeutic importance of Se and
GSH in viral infections.
A CONFIRMED MOLECULAR
INTERACTION BETWEEN SARS-CoV-2
PROTEASE AND A HUMAN
SELENOPROTEIN
Correlations between COVID-19 clinical outcomes and both
host Se and GSH status provide important context to a related
observation emerging from a proteomics-based study of possible
cellular targets of SARS-CoV-2 (SCoV2) proteins. Using affinity-
purification mass spectrometry, Gordon et al. identified high-
confidence protein-protein interactions between 26 of the SCoV2
proteins and human proteins (9). As bait for interacting human
proteins, one of the proteins they used was the SCoV2 main viral
protease Mpro, a cysteine protease also known as nonstructural
protein 5 (nsp5). The study also included a catalytically inactive
C145A Mpro mutant (lacking the active site cysteine), which
was also used as a bait protein, in order to discriminate false
positives that might bind non-specifically to the Mpro active site
cysteine via disulfide bond formation. One of the interactions
they identified involved the cytosolic form of the selenoprotein
glutathione peroxidase, GPX1, which bound strongly to the
inactive C145A Mpro mutant. However, this interaction with
GPX1 was not observed with the wild type Mpro .This is precisely
what one might expect to see if GPX1 is a protease substrate,
because its cleavage would produce two fragments that would
necessarily have reduced affinity to the enzyme relative to GPX1.
Significantly, as shown in Figure 6 in their Extended Data (9),
Gordon et al. identified one other host protein (TRMT1) that
bound only to the inactive Mpro C145A mutant, and concluded
it was a likely Mpro substrate, because they were able to identify
a putative Mpro cleavage site in the TRMT1 protein sequence
(PRLQ/ANFT), where the slash (/) represents the cleavage site.
Thus, the only piece of evidence lacking to draw a similar
conclusion for GPX1 is a candidate Mpro cleavage site, which most
algorithms will fail to find, e.g., if they are highly stringent about
requiring a Q in the P1 position (Figure 1).
As shown in Figure 1A for the 2003 SARS coronavirus (SARS-
CoV, SCoV) and in Figure 1B for the 2019 SCoV2, the consensus
logo patterns of 11 Mpro cleavage sites in the viral 1ab polyprotein
are essentially identical for the 2 viruses. Specifically, 8 of the
11 sites are fully identical over the 10 residues spanning the
P5 to P5positions, and only two of the 11 cleavage sites have
more than a single amino acid difference within the 10 residue
span (Figure S1). This means that (as discussed below) functional
studies of the 2003 SCoV protease are highly relevant to that of
the 2019 virus. From Figures 1A,B, the most important residue
positions for Mpro recognition are, in descending order, P1, P2,
P1, P4, and P3, with the first 3 being the major determinants,
with the minimal P2-P1consensus being LQ/(S,A,N,G). The
downstream sequence past P1is highly variable.
The GPx1 active site has the sequence LUG, where U is
the catalytic selenocysteine; this matches the observed Mpro
consensus core target sequence combination LQ/G in 2 of 3
positions. Furthermore, as shown in Figure 1C, the important
upstream side of the known Mpro cleavage site at the nsp13/14
junction, NVATLQ/A, is remarkably similar to that of the active
site sequence of GPx1, NVASLU/G, where the selenocysteine
(U) lines up with a glutamine (Q) in the viral sequence. These
two amino acids (U and Q) are not highly similar, but are both
midrange in size, and polar in nature, because the selenol residue
is predominantly ionized at physiological pH. The other two
“mismatches” in the important positions P3 and P1, i.e., S vs.
T and G vs. A, both differ only slightly, by the size of a methyl
group, and in any case, having a glycine (G) in the P1position is
a permitted residue (Figure 1A), as observed in the SCoV Mpro
cleavage site at the nsp5/6 junction (indicated by in Figure 2).
Significantly, the Procleave protease cleavage prediction server
(procleave.erc.monash.edu.au) (13) identified the GPX1 active
site octameric sequence ASLU/GTTV as a highly ranked possible
cleavage site for a cathepsin S-like cysteine protease (Figure 2G).
For several reaction intermediates in the GPX1 mechanism, Se
is attached to either oxygen, or to nitrogen, as a selenenylamide.
The latter is quite stable, and may accumulate during GSH
depletion, because GSH is needed to regenerate the selenolate
(14). Selenenylamide is more similar than selenocysteine to
glutamine, so that form might be preferentially targeted by Mpro.
Because the mutation rate in cellular genes is thousands of
times slower than that for an RNA virus, if there is a physical
interaction between these two proteins as Gordon et al. observed
using the inactive mutant Mpro, it is almost certainly because
the virus has evolved to target the host for some reason, rather
than the converse. The most parsimonious hypothesis is simply
that this is a case of a viral protease targeting a cellular gene for
cleavage, for which there are abundant precedents, including the
proposed Mpro targeting of TRMT1 and HDAC2 (9), and HIV-
1 protease, which can be toxic to cells, via its action at various
cellular targets (15).
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Taylor and Radding Selenoproteins as SARS-CoV-2 Protease Substrates
FIGURE 1 | Coronavirus Mpro cleavage site consensus sequence logo plots and comparisons. Logo plots from multiple alignments of the known Mpro cleavage sites
are shown for the 2003 SCoV (A) and 2019 SCoV2 (B). The height of a letter at each position reflects its probability in the alignment; each of the logos shown
represents the consensus of 11 Mpro cleavage sites from a single virus. Plots were generated using WebLogo (weblogo.berkeley.edu/logo.cgi), from the alignments in
Figure S1.(C) Comparison of the GPX1 active site sequence containing selenocysteine (U) to the known SCoV2 Mpro cleavage site at the nsp13/14 junction; this site
is identical in the 2003 and 2019 coronaviruses. (D) Comparison of a predicted Mpro cleavage site in human selenoprotein F to a known SCoV2 Mpro cleavage site at
the nsp12/13 junction.
FUNCTIONAL EFFECTS OF MPRO IN
TRANSFECTED CELLS ARE CONSISTENT
WITH GPX1 KNOCKDOWN
As detailed above, the original 2003 SCoV Mpro has essentially
identical substrate sequence specificity as that of SCoV2. More
specifically, the 10 residue sequence of the cleavage site at
the nsp13/14 junction shown in Figure 2A, with the greatest
overall similarity to the GPX1 active site sequence, is 100%
identical in the 2003 and 2019 SARS coronaviruses (Figure S1).
Thus, the results of a 2006 study on the effects of transfecting
cells with a SCoV Mpro expression vector, in order to study
the effects of Mpro alone in the absence of intact virus
(16), are highly relevant for COVID-19. In transfected human
cells, the results were exactly what one would expect from
knockdown of GPX1: increased oxidative stress via production
of reactive oxygen species and activation of transcription factor
NF-κB (16). Both of these responses can be produced by
exposing cells to hydrogen peroxide, and inhibited by increasing
GPX1 activity, either by overexpression or via the addition of
sodium selenite to cell culture media (17,18). Cells expressing
the SCoV Mpro also became apoptotic, which is a known
consequence of NF-κB activation, especially in combination
with a high burden of ROS (19), which is even more likely
to occur if other antioxidant proteins are also degraded, as
discussed below.
POTENTIAL MPRO SITES IN OTHER
SELENOPROTEINS AND
GLUTATHIONE-RELATED PROTEINS
The question then arises, could targeting of host selenoproteins
for proteolysis be part of a more extensive viral strategy
contributing to the correlations between Se and GSH status
and COVID-19 outcome? To investigate this possibility, we
undertook a systematic search for potential Mpro cleavage sites
in the human selenoproteome and in several GSH-related
proteins including glutaredoxin, a small thioredoxin-like CXXC
protein that is recycled by GSH, rather than directly by a
reductase enzyme. For this analysis, we used three online
resources: (1) NetCorona (cbs.dtu.dk/services/NetCorona) (12),
the only resource specific for predictions of Mpro cleavage sites
in coronaviruses (although not limited to just the SARS type
coronaviruses), (2) PROSPER, the Protease Specificity Prediction
Server (prosper.erc.monash.edu.au) (11), which was rated in a
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Taylor and Radding Selenoproteins as SARS-CoV-2 Protease Substrates
FIGURE 2 | Comparison of known and predicted Mpro cleavage sites. The most relevant positions P4-P1of the 11 known SARS coronavirus Mpro cleavage sites in
non-structural proteins are shown (for nsp4 through nsp16), aligned with predicted sites in several human selenoproteins (GPX1, SelenoF, SelenoP, and TXNRD1), as
well as glutaredoxin-1 (GLRX-1) and the rate-limiting enzyme for glutathione synthesis, glutamate-cysteine ligase catalytic subunit (GCLC, with 2 predicted cleavage
sites A,B). The known nsp cleavage sites shown are all from SARS-CoV-2, with the addition of the nsp5/6 site from SARS-CoV (marked*). The sites identified in
human proteins are displayed next to the known Mpro cleavage site to which they are most similar, highlighted in matching color. The experimentally determined
catalytic efficiencies of SARS-CoV Mpro at each of the known nsp cleavage sites are shown as relative kcat/Km(10). Three different methods for prediction of cysteine
protease cleavage sites were used: PROSPER (11), with numbers in parenthesis showing the score for the predicted site, any score >0.8 being significant; NetCorona
(12), where scores >0.5 are considered positive, and Procleave (13), where every possible P4-P4octamer in a protein sequence is evaluated, scored and ranked.
Some aligned non-identical residues highlighted in italics are nonetheless chemically and structurally similar to residues found in the same position in the known
cleavage sites, e.g., serine (S) vs. threonine (T), Leucine (L) or isoleucine (I) vs. valine (V), and glutamine (Q) vs. asparagine (N); all of these pairs differ only by a single
carbon atom (CH3or CH2unit). The predicted Mpro cleavage sites in human proteins labeled (A–G) are discussed in the text. The computational results cited are
collated in Figure S2.
recent study as one of the most accurate resources for prediction
of HIV-1 protease sites (20), and (3) Procleave (cited above),
which was only used if either of the first 2 methods failed to
identify a site. Both Procleave and PROSPER do not specifically
predict coronavirus cysteine protease sites, but make predictions
for more generic cysteine proteases (e.g., cathepsins B, K, L,
or S). Thus, for both PROSPER and Procleave, the results had
to be screened manually for matches to the Mpro cleavage
site consensus as shown in Figures 1A,B. This search is also
predicated on the assumption that the approach of Gordon
et al. (9) might fail to identify some protease targets, because
their high affinity interactions are mostly limited to an 8–
10 residue sequence, and in some cases may be unable to
withstand the conditions needed to be isolable and observable by
mass spectrometry.
Figure 2 presents the most notable and highly ranked
potential Mpro sites identified computationally (the order A-G
is not significant, as placement in the listing was determined by
similarity to the known nsp sites). The most remarkable instance
is in selenoprotein F (SELENOF), an ER protein involved
in glycoprotein folding quality control. Over the 8 residues
spanning P4 to P4, it has 7 of 8 identical to the Mpro site at
the nsp12/nsp13 junction, with the only mismatch at the highly
variable P3position (Figure 1D), giving a near-perfect match to
the Mpro consensus. This was the highest scored NetCorona hit,
also highly scored by PROSPER, as shown in Figure 2F, and is
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Taylor and Radding Selenoproteins as SARS-CoV-2 Protease Substrates
significant because coronavirus assembly begins in the ER with
spike protein accumulation on the ER membrane surface.
Equally important is the hit on the selenoprotein thioredoxin
reductase 1 (TXNRD1), which also was highly scored by both
NetCorona and PROSPER (Figure 2E). Note that in a larger set
of 77 human coronaviruses, a serine (S) in P4 is very common
(12), and in addition the sequence SI in the TXNRD1 site is
isosteric to TV, the aligned residues in the nsp12/13 site, because
transfer of a methyl group from threonine to valine would result
in serine and isoleucine. Thus, SI should occupy about the
same volume as TV in the Mpro active site. Functionally, this
predicted cleavage site is only 5 residues from the C terminal of
TXNRD1, and would result in removal of the C-terminal redox
center of TXNRD1 (AGCUG), making the enzyme incapable of
regenerating reduced thioredoxin.
A predicted site in selenoprotein P (SELENOP, Figure 2C)
is at position 56, 3 residues prior to the first selenocysteine,
such that cleavage here would likely interfere with the N-
terminal redox activity, possibly without affecting the Se-
transport function of the rest of the protein.
Two possible sites were identified in the catalytic subunit of
glutamate-cysteine ligase (GCLC), the rate limiting enzyme for
GSH synthesis. The first of these, GCLC-a (Figure 2B), would
cleave after position 217, and is an exact match to P4-P1 of
nsp5/6, but with an A in P1, a highly favorable residue for that
position, although SCoV has a G there, and SCoV2 has an S.
Site GCLC-b (Figure 2A), at position 443, is an exact match to
P4-P1 of nsp4/5, but with a G in P1. The two coronavirus sites
with exact P4-P1 matches to GCLC are those at either end of
nsp5, i.e., Mpro itself, which have the highest catalytic efficiency
for cleavage of any of the viral Mpro targets (100 and 41%). This
suggests that Mpro might be particularly efficient at disrupting
GSH synthesis, as compared to action at some of the other target
sites predicted here, thereby contributing to the clinical findings
in COVID-19 (7).
Finally (Figure 2D), we include a non-canonical hit that
would cleave at position 90 of glutaredoxin-1 (GLRX-1). This site
was very highly scored by PROSPER, as well as Procleave, despite
the glutamine (Q) at P1, which has an additional carbon atom
relative to the asparagine (N) found at P1in the nsp8/9 site, to
which it is otherwise most similar.
WHY WOULD SARS-CoV-2 TARGET
COMPONENTS OF THE THIOREDOXIN
AND GLUTAREDOXIN SYSTEMS?
Along with GSH reductase, GSH and glutaredoxin comprise the
glutaredoxin system, one of two cellular redox systems involved
in maintaining reduced thiol states, protein folding and repair,
and providing electrons to ribonucleotide reductase (RNR). The
other redox system with similar roles is the thioredoxin system,
comprised of thioredoxin and thioredoxin reductase (21).
Because DNA is an “add-on” to RNA biosynthesis,
deoxyribonucleotides can only be synthesized via the reduction
of ribonucleotides by RNR, a process which is unsustainable
without the participation of one or both of the thioredoxin
and glutaredoxin systems. As a consequence of this basic fact
of biochemistry, one should expect that RNA viruses might
exploit various mechanisms to interfere with components of the
thioredoxin and glutaredoxin systems, in order to minimize the
diversion of ribonucleotides into DNA synthesis. This is simply
the inverse of a strategy used by some large DNA viruses to
maximize DNA production, by encoding their own thioredoxin-
like proteins, glutaredoxins and even entire RNR genes (22).
Consistent with this hypothesis, the results presented here
suggest coronaviral targeting of TXNRD1, glutaredoxin-1, and
GCLC, a key enzyme for GSH synthesis, for proteolytic cleavage.
This would lead to knockdown of both of the essential redox
systems required to sustain DNA synthesis, and thereby conserve
ribonucleotides to enhance RNA synthesis for virus production.
THE POTENTIAL MPRO TARGETS TXNRD1
AND GCLC ARE STRONGLY
UPREGULATED BY VITAMIN D3
In light of accruing evidence that vitamin D3 status is inversely
correlated with severity of COVID-19 (2326), it is significant
that, via its hormone-like actions on gene expression, D3 has
been shown to be a potent activator of both TXNRD1 and
GCLC. In a microarray study, 6 h after exposure to 1,25-
dihydroxyvitamin D3, TXNRD1 mRNA was upregulated 7.1X,
and GCLC was upregulated 2.5X (27). These effects of vitamin
D3, as well as a resulting increase in GSH levels, have been
functionally documented in cell culture studies (28,29) and in
human subjects (30). Vitamin D3-mediated activation of both
TXNRD1 and GSH biosynthesis could substantially counteract the
proteolytic knockdown of TXNRD1 and GCLC predicted by our
hypothesis, which, if validated, would thus represent an important
mechanism contributing to a role for vitamin D3 in moderating
the severity of COVID-19. However, the therapeutic potential of
D3 in COVID-19 is the subject of ongoing debate (31). Perhaps
inconsistencies in the findings of various studies of this question
may be explained in part by a 20-year old observation, that the
effective upregulation of TXNRD1 by vitamin D3 requires the
presence of an adequate level of Se (28). So the full potential of
vitamin D3 vs. COVID-19 may only be seen in combination with
optimal Se intake, and possibly, vice-versa.
DISCUSSION AND CONCLUSIONS
Altogether, given the protein interaction and functional data
(9,16), it is a very strong hypothesis that GPX1 is an Mpro
substrate. Of the other sites we propose, some are more
likely than others, but those in TXNRD1 and SELENOF are
particularly convincing, and all of the predicted sites map to
surface accessible regions of the targeted proteins (Figure S3).
If validated, these results offer new insights into COVID-19
pathogenesis. If SCoV2 is targeting GSH biosynthesis as well as
TXNRD1 and GPx1 for proteolytic knockdown, in infected cells,
the resulting decreases in these critical antioxidant molecules
would contribute to increased oxidative stress, NF-κB activation
and pro-apoptotic signaling (17,18). Because NF-κB is an
Frontiers in Nutrition | www.frontiersin.org 5September 2020 | Volume 7 | Article 143
Taylor and Radding Selenoproteins as SARS-CoV-2 Protease Substrates
activator of many pro-inflammatory cytokines, including IL-6
(32), this could contribute to increased inflammation and the
cytokine storms observed in COVID-19, and be a significant
basis of pathogenic effects associated with SCoV2 infection of
various tissues, including the lung, gastrointestinal tract and
cardiovascular system. Importantly, these consequences of virus-
mediated proteolysis would be taking place in everyone who is
actively infected, regardless of their Se status, and would thus
be consistent with results suggesting that Se intakes up to
several times the minimal dietary requirement are associated
with an increase in cure rate from COVID-19 (1). But people
with suboptimal nutritional status could be particularly at
risk, because their GSH and selenoprotein levels might be
low to begin with, making them more vulnerable to the
detrimental effects of virus-induced proteolysis. Still, an infection
resulting from low dose exposure to virus and limited by a
strong immune response in a person with excellent dietary
status still might have minimal impact on cellular and patient
health, because proteolytic knockdown of host proteins is
likely to be incomplete, due to low catalytic efficiencies at
some target sites (Figure 2) and the stochastic nature of such
molecular interactions.
These results are not unprecedented—our lab has shown
that some RNA viruses target host mRNAs encoding isoforms
of thioredoxin reductase via RNA:RNA antisense interactions,
which, like proteolysis, would likely also result in host
selenoprotein knockdown, but by a different mechanism (22,
33). If our hypothesis is confirmed (i.e., some of these host
cleavage sites prove to be functional), that leaves us with an
interesting question—what evolutionary advantages are driving
some viruses to go so far as to degrade or block the synthesis of
GSH and specific host selenoproteins?
Understanding why RNA viruses may have developed such
strategies presents an interesting challenge for future research.
DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the
article/Supplementary Material.
AUTHOR CONTRIBUTIONS
This study was conceived and designed by ET, who wrote the
initial draft. ET and WR worked on the data analysis and
revisions of the manuscript. All authors contributed to the article
and approved the submitted version.
FUNDING
This work has been supported by a recurring unrestricted gift
from the Dr. Arthur and Bonnie Ennis Foundation, Decatur,
IL, USA.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnut.2020.
00143/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Copyright © 2020 Taylor and Radding. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms.
Frontiers in Nutrition | www.frontiersin.org 7September 2020 | Volume 7 | Article 143
Supplementary Materials
for
Taylor & Radding, 2020
Mpro site 2003 SARS-CoV 2019 SARS-CoV-2 # Non-identical
nsp4/5 SAVLQ/SGFRK SAVLQ/SGFRK 0
nsp5/6 GVTFQ/GKFKK GVTFQ/SAVKR 4
nsp6/7 VATVQ/SKMSD VATVQ/SKMSD 0
nsp7/8 RATLQ/AIASE RATLQ/AIASE 0
nsp8/9 AVKLQ/NNELS AVKLQ/NNELS 0
nsp9/10 TVRLQ/AGNAT TVRLQ/AGNAT 0
nsp10/11 EPLMQ/SADAS EPMLQ/SADAQ 3
nsp12/13 HTVLQ/AVGAC HTVLQ/AVGAC 0
nsp13/14 VATLQ/AENVT VATLQ/AENVT 0
nsp14/15 FTRLQ/SLENV FTRLQ/SLENV 0
nsp15/16 YPKLQ/ASQAW YPKLQ/SSQAW 1
Figure S1. The 11 known Mpro cleavage sites in the 2003 and 2019 SARS coronaviruses with a
comparison of the 10-residue protein sequences spanning the cleavages sites in the two viruses.
The alignments underneath each of the virus names (2003 SARS-CoV and 2019 SARS-CoV-2)
were used to generate the logos shown in Fig. 1A and 1B. The cleavage sites and sequences for
the SARS-CoV (Genbank NC_004718.3) are given in reference (10), and the equivalent sequences
in SARS-CoV-2 were taken from its Genbank Reference Sequence, NC_045512.
6:HEVHUYHUSURWHDVHFOHDYDJHVLWHSUHGLFWLRQUHVXOWVFLWHGLQ)LJXUH
*HQEDQNVRXUFHILOHVIRUWKHSURWHLQVHTXHQFHV*&/&13B6(/(12313B
*/5;13B7;15'13B6(/(12)13B*3;13B
61HW&RURQDUHVXOWVIURPFEVGWXGNVHUYLFHV1HW&RURQD
1HW&RURQD6HUYHUSUHGLFWLRQUHVXOWV
7HFKQLFDO8QLYHUVLW\RI'HQPDUN
165 SelenoF
MVAMAAGPSGCLVPAFGLRLLLATVLQAVSAFGAEFSSEACRELGFSSNLLCSSCDLLGQFNLLQLDPDCRGCCQEEAQF 80
ETKKLYAGAILEVCGCKLGRFPQVQAFVRSDKPKLFRGLQIKYVRGSDPVLKLLDDNGNIAEELSILKWNTDSVEEFLSE 160
KLERI Selenocysteine (U) at #96 changed to C for search as NetCorona ignores U
..........................C..................................................... 80
................................................................................ 160
.....
Pos Score Cleavage
___________________________
27 0.846 ATVLQ^AVSAF SelenoF
60 0.067 none SelenoF
65 0.073 none SelenoF
75 0.066 none SelenoF
79 0.064 none SelenoF
103 0.064 none SelenoF
105 0.219 none SelenoF
120 0.136 none SelenoF
___________________________
499 TXNRD1
MNGPEDLPKSYDYDLIIIGGGSGGLAAAKEAAQYGKKVMVLDFVTPTPLGTRWGLGGTCVNVGCIPKKLMHQAALLGQAL 80
QDSRNYGWKVEETVKHDWDRMIEAVQNHIGSLNWGYRVALREKKVVYENAYGQFIGPHRIKATNNKGKEKIYSAERFLIA 160
TGERPRYLGIPGDKEYCISSDDLFSLPYCPGKTLVVGASYVALECAGFLAGIGLDVTVMVRSILLRGFDQDMANKIGEHM 240
EEHGIKFIRQFVPIKVEQIEAGTPGRLRVVAQSTNSEEIIEGEYNTVMLAIGRDACTRKIGLETVGVKINEKTGKIPVTD 320
EEQTNVPYIYAIGDILEDKVELTPVAIQAGRLLAQRLYAGSTVKCDYENVPTTVFTPLEYGACGLSEEKAVEKFGEENIE 400
VYHSYFWPLEWTIPSRDNNKCYAKIICNTKDNERVVGFHVLGPNAGEVTQGFAAALKCGLTKKQLDSTIGIHPVCAEVFT 480
TLSVTKRSGASILQAGCCG (Penultimate U changed to C as NetCorona ignores U)
................................................................................ 80
................................................................................ 160
................................................................................ 240
...............................C................................................ 320
................................................................................ 400
................................................................................ 480
.............C.....
Pos Score Cleavage
___________________________
33 0.068 none TXNRD1
72 0.104 none TXNRD1
78 0.071 none TXNRD1
81 0.123 none TXNRD1
106 0.073 none TXNRD1
133 0.071 none TXNRD1
230 0.075 none TXNRD1
250 0.070 none TXNRD1
258 0.065 none TXNRD1
272 0.505 RVVAQ^STNSE TXNRD1
323 0.061 none TXNRD1
348 0.191 none TXNRD1
355 0.064 none TXNRD1
450 0.107 none TXNRD1
464 0.074 none TXNRD1
494 0.640 ASILQ^AGCCG TXNRD1
___________________________
635263(5WKH3URWHDVH6SHFLILFLW\3UHGLFWLRQ6HUYHUUHVXOWVIURPSURVSHUHUFPRQDVKHGXDX
6%
B. GCLC-a VTFQ/A WZK^WZ;ϭ͘ϬϬͿ
nsp5/6* VTFQ/G
6'
D. GLRX-1 VSLQ/Q WZK^WZ;ϭ͘ϯϬͿ
nsp8/9 VKLQ/N


 
6(
E. TXNRD1
SILQ/A WZK^WZ;ϭ͘ϭϰͿ
nsp12/13 TVLQ/A
 
6)
F. SelenoF TVLQ/A WZK^WZ;ϭ͘ϬϮͿ
nsp12/13 TVLQ/A
63URFOHDYHUHVXOWVIURPSURFOHDYHHUFPRQDVKHGXDX
6$
A. GCLC-b AVLQ/G WƌŽůĞĂǀĞηϳͬϱϵϮĨŽƌĂƚŚĞƉƐŝŶ^
nsp4/5 AVLQ/S
 
6%
B. GCLC-a VTFQ/A WƌŽůĞĂǀĞηϭϯŽĨϱϵϮĨŽƌĂƚŚĞƉƐŝŶ>
nsp5/6* VTFQ/G
6&
C. SelenoP
ALLQ/A WƌŽůĞĂǀĞηϯͬϯϳϰĨŽƌĂƚŚĞƉƐŝŶ>
nsp7/8 ATLQ/A
6'
D. GLRX-1 VSLQ/Q WƌŽůĞĂǀĞηϭͬϵϵĨŽƌĂƚŚĞƉƐŝŶ^
nsp8/9 VKLQ/N
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G. GPX1 ASLU/G WƌŽůĞĂǀĞηϮͬϭϵϲĨŽƌĂƚŚĞƉƐŝŶ^
nsp13/14 ATLQ/A
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... So, boosting the intracellular glutathione redox status and 25(OH)D level may constitute a novel therapeutic alternative for overcoming impaired immunity and inflammation in COVID-19 subjects. 38 By employing web-based predictive strategies that are relative to molecular interactions and focused on protease cleavage sites, it has been proved that the viral main protease targets glutamate−cysteine ligase involved in the rate-limiting step of glutathione synthesis, glutathione peroxidase (GPX1), and other selenoproteins like selenoprotein F (SELENOF) or thioredoxin reductase 1. 57 These observations showed consistency with the viral ability to interfere with DNA synthesis and to promote RNA synthesis via increase in the ribonucleotide profile by that enhancing multiplication. Moreover, these findings are confirmatory for the correlation between oxidative stress, associated inflammatory response, and the impairment in glutathione precursors and dietary selenium. ...
... Moreover, these findings are confirmatory for the correlation between oxidative stress, associated inflammatory response, and the impairment in glutathione precursors and dietary selenium. 43,57 Endogenous glutathione impairment can enhance the oxidative damage of the lungs induced by SARS-CoV-2, leading to acute respiratory distress syndrome, organ failure, and even death. With respect to the antiviral activity of glutathione, individuals with low glutathione levels seem to present a greater susceptibility for uncontrolled replication of the virus and, therefore, increased viral load. ...
Article
Viral pathologies encompass activation of pro-oxidative pathways and inflammatory burst. Alleviating overproduction of reactive oxygen species and cytokine storm in COVID-19 is essential to counteract the immunogenic damage in endothelium and alveolar membranes. Antioxidants alleviate oxidative stress, cytokine storm, hyperinflammation, and diminish the risk of organ failure. Direct antiviral roles imply: impact on viral spike protein, interference with the ACE2 receptor, inhibition of dipeptidyl peptidase 4, transmembrane protease serine 2 or furin, and impact on of helicase, papain-like protease, 3-chyomotrypsin like protease, and RNA-dependent RNA polymerase. Prooxidative environment favors conformational changes in the receptor binding domain, promoting the affinity of the spike protein for the host receptor. Viral pathologies imply a vicious cycle, oxidative stress promoting inflammatory responses, and vice versa. The same was noticed with respect to the relationship antioxidant impairment-viral replication. Timing, dosage, pro-oxidative activities, mutual influences, and interference with other antioxidants should be carefully regarded. Deficiency is linked to illness severity.
... Although GSH adduct formation might lower the efficacy of both wifA and win, it should be mentioned here that the host protein involved in the biosynthesis of GSH, glutamate-cysteine ligase, is a proposed substrate of M pro . 42 Hence, the attenuated levels of GSH in the infected cells would allow wifA and win to inhibit M pro , while the normal level of GSH in noninfected cells would play a protective role against any toxic effect of wifA and win. Additionally, depletion of GSH in SARS-CoV-2-infected cells may also occur due to excessive ROS production due to acute inflammation. ...
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The current COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a global health crisis. The ability of vaccines to protect immunocompromised individuals and from emerging new strains are major concerns. Hence antiviral drugs against SARS-CoV-2 are essential. The SARS-CoV-2 main protease Mpro is vital for replication and an important target for antivirals. Using CMap analysis and docking studies, withaferin A (wifA) and withanone (win), two natural products from the medicinal herb Withania somnifera (ashwagandha), were identified as promising candidates that can covalently inhibit the viral protease Mpro. Cell culture, enzymatic, LC-MS/MS, computational, and equilibrium dialysis based assays were performed. DFT calculations indicated that wifA and win can form stable adducts with thiols. The cytotoxicity of Mpro was significantly reduced by wifA and win. Both wifA and win were found to irreversibly inhibit 0.5 μM Mpro with IC50 values of 0.54 and 1.8 μM, respectively. LC-MS/MS analysis revealed covalent adduct formation with wifA at cysteines 145 and 300 of Mpro. The natural products wifA and win can irreversibly inhibit the SARS-CoV-2 main protease Mpro. Based on the work presented here we propose that both wifA and win have the potential to be safely used as preventative and therapeutic interventions for COVID-19.
... Superoxide dismutase (SOD) [131], heme oxygenase-1 (HO-1) [90], catalase (CAT) [154], glutathione peroxidase (GPX) [243], glutathione S-transferase (GST) [210], peroxiredoxin (PrxI) [112], and nuclear factor erythroid 2-related factor 2 (Nrf2) [55] are enzymes that mitigate oxidative stress and are implicated in the pathogenesis of COVID-19. The correlation between decreased expression of SOD3 in lungs and severity of COVID-19 has been reported in elderly patients [131]. ...
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The Coronavirus Disease-2019 (COVID-19) pandemic urges researching possibilities for prevention and management of the effects of the virus. Carotenoids are natural phytochemicals of anti-oxidant, anti-inflammatory and immunomodulatory properties and may exert potential in aiding in combatting the pandemic. This review presents the direct and indirect evidence of the health benefits of carotenoids and derivatives based on in vitro and in vivo studies, human clinical trials and epidemiological studies and proposes possible mechanisms of action via which carotenoids may have the capacity to protect against COVID-19 effects. The current evidence provides a rationale for considering carotenoids as natural supportive nutrients via antioxidant activities, including scavenging lipid-soluble radicals, reducing hypoxia-associated superoxide by activating antioxidant enzymes, or suppressing enzymes that produce reactive oxygen species (ROS). Carotenoids may regulate COVID-19 induced over-production of pro-inflammatory cytokines, chemokines, pro-inflammatory enzymes and adhesion molecules by nuclear factor kappa B (NF-κB), renin-angiotensin-aldosterone system (RAS) and interleukins-6- Janus kinase-signal transducer and activator of transcription (IL-6-JAK/STAT) pathways and suppress the polarization of pro-inflammatory M1 macrophage. Moreover, carotenoids may modulate the peroxisome proliferator-activated receptors γ by acting as agonists to alleviate COVID-19 symptoms. They also may potentially block the cellular receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), human angiotensin-converting enzyme 2 (ACE2). These activities may reduce the severity of COVID-19 and flu-like diseases. Thus, carotenoid supplementation may aid in combatting the pandemic, as well as seasonal flu. However, further in vitro, in vivo and in particular long-term clinical trials in COVID-19 patients are needed to evaluate this hypothesis.
... GSH interacts and, in certain conditions, decreases the main SARS-CoV-2 protease activity [100]. Bioinformatic tools predicted that SARS-CoV-2 main protease targets GPX1, an enzyme involved in neutralizing lipid peroxides and hydrogen peroxide in organisms [101], as well as a glutamate-cysteine ligase, an enzyme involved in GSH synthesis [102]. In a study on 60 hospitalized COVID-19 patients, GSH deficiency was observed compared to healthy controls. ...
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The coronavirus disease (COVID-19) pandemic is a leading global health and economic challenge. What defines the disease’s progression is not entirely understood, but there are strong indications that oxidative stress and the defense against reactive oxygen species are crucial players. A big influx of immune cells to the site of infection is marked by the increase in reactive oxygen and nitrogen species. Our article aims to highlight the critical role of oxidative stress in the emergence and severity of COVID-19 and, more importantly, to shed light on the underlying molecular and genetic mechanisms. We have reviewed the available literature and clinical trials to extract the relevant genetic variants within the oxidative stress pathway associated with COVID-19 and the anti-oxidative therapies currently evaluated in the clinical trials for COVID-19 treatment, in particular clinical trials on glutathione and N-acetylcysteine.
... Note that high doses of oral NAC could result in intolerable gastrointestinal adverse effects such as nausea, vomiting, and diarrhoea [159]. Other micronutrients, such as zinc [194], selenium [106,195,196], magnesium, vitamins A, C, and D [113,116,197], should form part of a successful integrative protocol [110], while sulfur-donors such as MSM, allicin [157], or marine-derived sulfated PSs can also be considered. MSM is entirely safe and effective, taken at daily dosages of up to 4 g to prevent infection and modulate the immune response [136]. ...
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The airway epithelial glycocalyx plays an important role in preventing severe acute respiratory syndrome coronavirus 2 entry into the epithelial cells, while the endothelial glycocalyx contributes to vascular permeability and tone, as well as modulating immune, inflammatory, and coagulation responses. With ample evidence in the scientific literature that coronavirus disease 2019 (COVID19) is related to epithelial and endothelial dysfunction, preserving the glycocalyx should be the main focus of any COVID-19 treatment protocol. The most studied functional unit of the glycocalyx is the glycosaminoglycan heparan sulfate, where the degree and position of the sulfate groups determine the biological activity. N-acetylcysteine (NAC) and other sulfur donors contribute to the inorganic sulfate pool, the rate-limiting molecule in sulfation. NAC is not only a precursor to glutathione but also converts to hydrogen sulfide, inorganic sulfate, taurine, Coenzyme A, and albumin. By optimising inorganic sulfate availability, and therefore sulfation, it is proposed that COVID-19 can be prevented or at least most of the symptoms attenuated. A comprehensive COVID19 treatment protocol is needed to preserve the glycocalyx in both the prevention and treatment of COVID-19. The use of NAC at a dosage of 600 mg bid for the prevention of COVID-19 is proposed, but a higher dosage of NAC (1200 mg bid) should be administered upon the first onset of symptoms. In the severe to critically ill, it is advised that IV NAC should be administered immediately upon hospital admission, and in the late stage of the disease, IV sodium thiosulfate should be considered. Doxycycline as a protease inhibitor will prevent shedding and further degradation of the glycocalyx.
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The immune-inflammatory response during the acute phase of COVID-19, as assessed using peak body temperature (PBT) and peripheral oxygen saturation (SpO2), predicts the severity of chronic fatigue, depression and anxiety symptoms three to four months later. The present study was performed to examine the effects of SpO2 and PBT during acute infection on immune, oxidative and nitrosative stress (IO&NS) pathways and neuropsychiatric symptoms of Long COVID. This study assayed SpO2 and PBT during acute COVID-19, and C-reactive protein (CRP), malondialdehyde (MDA), protein carbonyls (PCs), myeloperoxidase (MPO), nitric oxide (NO), zinc, and glutathione peroxidase (Gpx) in 120 Long COVID individuals and 36 controls. Cluster analysis showed that 31.7% of the Long COVID patients had severe abnormalities in SpO2, body temperature, increased oxidative toxicity (OSTOX) and lowered antioxidant defenses (ANTIOX), and increased total Hamilton Depression (HAMD) and Anxiety (HAMA) and Fibromylagia-Fatigue (FF) scores. Around 60% of the variance in the neuropsychiatric symptoms of Long COVID (a factor extracted from HAMD, HAMA and FF scores) was explained by OSTOX/ANTIOX ratio, PBT and SpO2. Increased PBT predicted increased CRP and lowered ANTIOX and zinc levels, while lowered SpO2 predicted lowered Gpx and increased NO production. Lowered SpO2 strongly predicts OSTOX/ANTIOX during Long COVID. In conclusion, the impact of acute COVID-19 on the symptoms of Long COVID is partly mediated by OSTOX/ANTIOX, especially lowered Gpx and zinc, increased MPO and NO production and lipid peroxidation-associated aldehyde formation. The results suggest that post-viral somatic and mental symptoms have a neuroimmune and neuro-oxidative origin.
Article
Objective Glutathione peroxidase 1 (GPX1) is a major selenoprotein in most animal tissues, primarily expressed in the cytoplasm and mitochondria of cells and peroxidase structures of certain cells. GPX1 expression is highly correlated with carcinogenesis and disease progression. The goal of the study was to determine the association between GPX1 expression and tumor therapy, and to identify GPX1 prognostic value in various malignancies. Methods The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Human Protein Atlas (HPA) databases were used to detect the levels of GPX1 expression in human tumor tissues and normal tissues. Indeed, correlations between GPX1 and tumor purity, tumor mutation burden (TMB), microsatellite instability (MSI), and DNA mismatch repair genes (MMRs) were explored using the TCGA cohort. Functional and enrichment analyses were performed by the GeneMANIA database and Gene Set Enrichment Analysis (GSEA), respectively. Cox regression models and Kaplan – Meier curves were used to screen for independent risk factors and estimate brain lower-grade glioma (LGG) survival probability. The Chinese Glioma Genome Atlas (CGGA) database was used to determine whether GPX1 had a race-specific effect on overall survival (OS) in LGG. The cross-interaction between GPX1 and chemoradiotherapy on LGG OS was determined by Kaplan – Meier curves. Logistic regression models of multiplicative interactions were constructed. Furthermore, the relationship between GPX1 and LGG treatment regimens was also explored through the Genomics of Drug Sensitivity in Cancer (GDSC) database. Results GPX1 was highly expressed in various tumors, GPX1 overexpression was significantly correlated with the poor prognosis of LGG. GPX1 was found to be an independent predictive factor for LGG in both univariate and multivariate Cox models. The nomogram showed a high predictive accuracy (C-index: 0.804, 95% CI: 0.74-0.86). In addition, GPX1 was significantly associated with TMB, MSI, and MMRs in diverse cancers. GPX1 was involved in IL6/JAK/STAT3, inflammatory response, and apoptosis signaling pathways. Besides, non-radiotherapy, chemotherapy, and low GPX1 expression were important factors affecting the better prognosis of LGG. GPX1 acted as a tumor promoter, which has taken the worst effect on LGG survival, but a multiplicative interaction of GPX1*chemoradiotherapy may improve the poor clinical outcome. GPX1 was negatively correlated with the half inhibition concentration (IC50) of temozolomide (TMZ) (Spearman = -0.44, P = 4.52×10⁻²⁶). Conclusion In LGG patients, high GPX1 expression was linked to a shorter OS. The interaction between GPX1 and chemoradiotherapy exhibits a beneficial clinical effect and chemotherapy was recommended for LGG patients, especially for those with high GPX1 expression. Besides, high GPX1 expression can predict TMZ sensitivity in LGG, providing potential evidence for chemotherapy. On the whole, this study presents a wealth of biological as well as clinical significance for the roles of GPX1 in human tumors, particularly in LGG.
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Corona virus induced disease-19 caused by SARS-CoV-2 has presented unprecedented health and economic crisis worldwide. Substantial progress has been made in last two years to develop vaccines against SARS-CoV-2. Along...
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In this review, the relevance of selenium (Se) to viral disease will be discussed paying particular attention to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease (COVID-19). Se, the active centre in selenoproteins has an ongoing history of reducing the incidence and severity of viral infections. Host Se deficiency increased the virulence of RNA viruses such as influenza A and coxsackievirus B3, the latter of which is implicated in the development of Keshan disease in north-east China. Significant clinical benefits of Se supplementation have been demonstrated in HIV-1, in liver cancer linked to hepatitis B, and in Chinese patients with hantavirus that was successfully treated with oral sodium selenite. China is of particular interest because it has populations that have both the lowest and the highest Se status in the world. We found a significant association between COVID-19 cure rate and background Se status in Chinese cities; the cure rate continued to rise beyond the Se intake required to optimise selenoproteins, suggesting an additional mechanism. Se status was significantly higher in serum samples from surviving than non-surviving COVID-19 patients. As regards mechanism, SARS-CoV-2 may interfere with the human selenoprotein system; selenoproteins are important in scavenging reactive oxygen species, controlling immunity, reducing inflammation, ferroptosis and endoplasmic reticulum (ER) stress. We found that SARS-CoV-2 significantly suppressed mRNA expression of GPX4 , of the ER selenoproteins, SELENOF , SELENOM , SELENOK and SELENOS and down-regulated TXNRD3 . Based on the available data, both selenoproteins and redox-active Se species (mimicking ebselen, an inhibitor of the main SARS-CoV-2 protease that enables viral maturation within the host) could employ their separate mechanisms to attenuate virus-triggered oxidative stress, excessive inflammatory responses and immune-system dysfunction, thus improving the outcome of SARS-CoV-2 infection.
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SARS-CoV-2 is an RNA virus responsible for the COVID-19 pandemic that already claimed more than 340,000 lives worldwide as of May 23, 2020, the majority of which are elderly. Selenium (Se), a natural trace element, has a key and complex role in the immune system. It is well-documented that Se deficiency is associated with higher susceptibility to RNA viral infections and more severe disease outcome. In this article, we firstly present evidence on how Se deficiency promotes mutations, replication and virulence of RNA viruses. Next, we review how Se might be beneficial via restoration of host antioxidant capacity, reduction of apoptosis and endothelial cell damages as well as platelet aggregation. It also appears that low Se status is a common finding in conditions considered at risk of severe COVID-19, especially in the elderly. Finally, we present a rationale for Se use at different stages of COVID-19. Se has been overlooked but may have a significant place in COVID-19 spectrum management, particularly in vulnerable elderly, and might represent a game changer in the global response to COVID-19.
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Background SARS‐CoV‐2 coronavirus infection ranges from asymptomatic through to fatal COVID‐19 characterised by a “cytokine storm” and lung failure. Vitamin D deficiency has been postulated as a determinant of severity. Objectives To review the evidence relevant to vitamin D and COVID‐19 Methods Narrative review Results Regression modelling shows that more northerly countries in the Northern Hemisphere are currently (May 2020) showing relatively high COVID‐19 mortality, with an estimated 4.4% increase in mortality for each 1 degree latitude north of 28 degrees North (P=0.031) after adjustment for age of population. This supports a role for ultraviolet B acting via vitamin D synthesis. Factors associated with worse COVID‐19 prognosis include old age, ethnicity, male sex, obesity, diabetes and hypertension and these also associate with deficiency of vitamin D or its response. Vitamin D deficiency is also linked to severity of childhood respiratory illness. Experimentally, vitamin D increases the ratio of angiotensin converting enzyme 2 (ACE2) to ACE, thus increasing angiotensin II hydrolysis and reducing subsequent inflammatory cytokine response to pathogens and lung injury. Conclusions Substantial evidence supports a link between vitamin D deficiency and COVID‐19 severity but it is all indirect. Community‐based placebo‐controlled trials of vitamin D supplementation may be difficult. Further evidence could come from study of COVID‐19 outcomes in large cohorts with information on prescribing data for vitamin D supplementation or assay of serum unbound 25(OH) vitamin D levels. Meanwhile vitamin D supplementation should be strongly advised for people likely to be deficient.
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The biosynthesis of DNA inherently competes with RNA synthesis because it depends on the reduction of ribonucleotides (RNA precursors) to 2’-deoxyribonucleotides by ribonucleotide reductase (RNR). Hence, RNA viruses can increase viral RNA production in cells by partially blocking the synthesis of DNA, e.g. by downregulating the mammalian selenoprotein thioredoxin reductase (TR), which normally acts to sustain DNA synthesis by regenerating reduced thioredoxin, a hydrogen donor for RNR. Computational and preliminary experimental evidence supports the hypothesis that a number of pathogenic RNA viruses, including HIV-1, Ebola, Zika, some flu viruses, and SARS-CoV-2, target TR isoforms by antisense. TR knockdown would create a host antioxidant defect that could be partially rectified by increased selenium intake, or be exacerbated by selenium deficiency, contributing to viral pathogenesis. There are several non-selenium-dependent means that viruses might also exploit to slow DNA synthesis, such as targeting RNR itself, or components of the glutaredoxin system, which serves as a backup redox system for RNR. HIV-1 substantially downregulates glutathione synthesis, so it interferes with both the thioredoxin and glutaredoxin systems. Computational results suggest that, like Ebola, SARS-CoV-2 targets TR3 by antisense. TR3 is the only TR isoform that includes an N-terminal glutaredoxin domain, so antisense knockdown of TR3 may also affect both redox systems, favoring RNA synthesis. In contrast, some DNA viruses encode their own glutaredoxins, thioredoxin-like proteins and even RNR homologues – so they are doing just the opposite, favoring DNA synthesis. This is clear evidence that viruses can benefit from shifting the RNA:DNA balance to their advantage.
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Higher rates of serious illness and death from coronavirus SARS-CoV-2 (COVID-19) infection among older people and those who have comorbidities suggest that age- and disease-related biological processes make such individuals more sensitive to environmental stress factors including infectious agents like coronavirus SARS-CoV-2. Specifically, impaired redox homeostasis and associated oxidative stress appear to be important biological processes that may account for increased individual susceptibility to diverse environmental insults. The aim of this Viewpoint is to justify (1) the crucial roles of glutathione in determining individual responsiveness to COVID-19 infection and disease pathogenesis and (2) the feasibility of using glutathione as a means for the treatment and prevention of COVID-19 illness. The hypothesis that glutathione deficiency is the most plausible explanation for serious manifestation and death in COVID-19 patients was proposed on the basis of an exhaustive literature analysis and observations. The hypothesis unravels the mysteries of epidemiological data on the risk factors determining serious manifestations of COVID-19 infection and the high risk of death and opens real opportunities for effective treatment and prevention of the disease.
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The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 2.3 million people, killed over 160,000, and caused worldwide social and economic disruption1,2. There are currently no antiviral drugs with proven clinical efficacy, nor are there vaccines for its prevention, and these efforts are hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To address this, we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), identifying 332 high-confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (29 FDA-approved drugs, 12 drugs in clinical trials, and 28 preclinical compounds). Screening a subset of these in multiple viral assays identified two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the Sigma1 and Sigma2 receptors. Further studies of these host factor targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.
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Selenium status is an established factor in the incidence, outcome or virulence of various RNA viral infections. Because China has geographic regions that range from extremely high to extremely low selenium intakes, we hypothesized that selenium status might influence the outcome of COVID-19 in these areas. For our analysis, we used reported cumulative case and outcome data for a snapshot of the COVID-19 outbreak, as of 2-18-2020. We found that in the city of Enshi, which is renown for having the highest selenium intakes in China, the cure rate was 3 times as high as that for all the other cities in Hubei Province, where Wuhan is located (p < 0.0001). In contrast, in Heilongjiang Province, where Keshan is located and extreme selenium deficiency is endemic, the death rate was almost 5 times as high as that for all the other Provinces and Municipalities outside of Hubei (p < 0.0001). Finally, for a set of 17 cities outside of Hubei, using published city data on average levels of selenium in human hair (a reliable measure of dietary intake), a significant linear correlation with the cure rate for COVID-19 was observed (R squared = 0.72, P < 0.0001).
Article
SARS-CoV-2 infections underlie the current coronavirus disease (COVID-19) pandemic and are causative for a high death toll particularly among elderly subjects and those with comorbidities. Selenium (Se) is an essential trace element of high importance for human health and particularly for a well-balanced immune response. The mortality risk from a severe disease like sepsis or polytrauma is inversely related to Se status. We hypothesized that this relation also applies to COVID-19. Serum samples (n = 166) from COVID-19 patients (n = 33) were collected consecutively and analyzed for total Se by X-ray fluorescence and selenoprotein P (SELENOP) by a validated ELISA. Both biomarkers showed the expected strong correlation (r = 0.7758, p < 0.001), pointing to an insufficient Se availability for optimal selenoprotein expression. In comparison with reference data from a European cross-sectional analysis (EPIC, n = 1915), the patients showed a pronounced deficit in total serum Se (mean ± SD, 50.8 ± 15.7 vs. 84.4 ± 23.4 µg/L) and SELENOP (3.0 ± 1.4 vs. 4.3 ± 1.0 mg/L) concentrations. A Se status below the 2.5th percentile of the reference population, i.e., [Se] < 45.7 µg/L and [SELENOP] < 2.56 mg/L, was present in 43.4% and 39.2% of COVID samples, respectively. The Se status was significantly higher in samples from surviving COVID patients as compared with non-survivors (Se; 53.3 ± 16.2 vs. 40.8 ± 8.1 µg/L, SELENOP; 3.3 ± 1.3 vs. 2.1 ± 0.9 mg/L), recovering with time in survivors while remaining low or even declining in non-survivors. We conclude that Se status analysis in COVID patients provides diagnostic information. However, causality remains unknown due to the observational nature of this study. Nevertheless, the findings strengthen the notion of a relevant role of Se for COVID convalescence and support the discussion on adjuvant Se supplementation in severely diseased and Se-deficient patients.
Preprint
Background: COVID-19 is a major pandemic that has killed more than 196,000 people. The COVID-19 disease course is strikingly divergent. Approximately 80-85% of patients experience mild or no symptoms, while the remainder develop severe disease. The mechanisms underlying these divergent outcomes are unclear. Emerging health disparities data regarding African American and homeless populations suggest that vitamin D insufficiency (VDI) may be an underlying driver of COVID-19 severity. To better define the VDI-COVID-19 link, we determined the prevalence of VDI among our COVID-19 intensive care unit (ICU) patients. Methods: In an Institutional Review Board approved study performed at a single, tertiary care academic medical center, the medical records of COVID-19 patients were retrospectively reviewed. Subjects were included for whom serum 25-hydroxycholecalcifoerol (25OHD) levels were determined. COVID-19-relevant data were compiled and analyzed. We determined the frequency of VDI among COVID-19 patients to evaluate the likelihood of a VDI-COVID-19 relationship. Results: Twenty COVID-19 patients with serum 25OHD levels were identified; 65.0% required ICU admission.The VDI prevalence in ICU patients was 84.6%, vs. 57.1% in floor patients. Strikingly, 100% of ICU patients less than 75 years old had VDI. Coagulopathy was present in 62.5% of ICU COVID-19 patients, and 92.3% were lymphocytopenic. Conclusions: VDI is highly prevalent in severe COVID-19 patients. VDI and severe COVID-19 share numerous associations including hypertension, obesity, male sex, advanced age, concentration in northern climates, coagulopathy, and immune dysfunction. Thus, we suggest that prospective, randomized controlled studies of VDI in COVID-19 patients are warranted.