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Proteasome activator PA28g-dependent degradation of coronavirus
disease (COVID-19) nucleocapsid protein
Haiyang Zhang 1, Jialu Tu 1, Chulei Cao, Ting Yang, Liangcai Gao*
Shanghai Key Laboratory of Regulatory Biology, Shanghai Key Laboratory of Brain Functional Genomics (Ministry of Education), Institute of Biomedical
Sciences, School of Life Sciences, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, China
a r t i c l e i n f o
Article history:
Received 2 June 2020
Accepted 12 June 2020
Available online 16 June 2020
Keywords:
PA28g
SARS-CoV-2
nCoV N
Protein degradation
COVID-19
a b s t r a c t
The nucleocapsid protein is signi!cant in the formation of viral RNA of the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2), accounting for the largest proportion of viral structural proteins.
Here, we report for the !rst time that the 11S proteasomal activator PA28g regulates the intracellular
abundance of the SARS-CoV-2 N protein (nCoV N). Furthermore, we have identi!ed proteasome activator
PA28g as a nCoV N binding protein by co-immunoprecipitation assay. As a result of their interaction,
nCoV N could be degraded by PA28g-20S in vitro degradation assay. This was also demonstrated by
blocking de novo protein synthesis with cycloheximide. The stability of nCoV N in PA28g-knockout cells
was greater than in PA28g-wildtype cells. Notably, immuno”uorescence staining revealed that knockout
of the PA28g gene in cells led to the transport of nCoV N from the nucleus to the cytoplasm. Overexpression
of PA28g enhanced proteolysis of nCoV N compared to that in PA28g-N151Y cells containing a
dominant-negative PA28g mutation, which reduced this process. These results suggest that PA28g
binding is important in regulating 20S proteasome activity, which in turn regulates levels of the critical
nCoV N nucleocapsid protein of SARS-CoV-2, furthering our understanding of the pathogenesis of COVID-
19.
© 2020 Elsevier Inc. All rights reserved.
1. Introduction
As of May 31, 2020, nearly 6 million cases of coronavirus disease
2019 (COVID-19) and over 350,000 deaths from the disease, have
been reported worldwide [1]. A novel coronavirus is the cause of
COVID-19. Taxonomically, SARS-CoV-2 forms a clade within the
subgenus sarbecovirus, orthocoronavirinae subfamily [2]. SARSCoV-
2 has a positive single-stranded RNA genome, approximately
29.8 kb, including a various number (from 6 to 11) of open reading
frames (ORFs) [3]. The !rst ORF, representing over 60% of the entire
genome, encodes 16 non-structural proteins, while the remaining
ORFs encode auxiliary proteins and four structural proteins [4]. The
four structural proteins are the small envelope protein (E), matrix
protein (M), spike surface glycoprotein (S), and nucleocapsid protein
(N) [5].
The SARS-CoV-2 nucleocapsid protein (hereafter, referred to as
nCoV N) accounts for the largest proportion of viral structure
proteins and is the most abundant protein in virus-infected cells. Its
primary function is to package the viral RNA genome into a ribonucleoprotein
complex, the capsid [6]. The nucleocapsid protein
encoded by SARS-CoV-2 can act as a viral inhibitory factor of RNA
interference in cells [7]. Furthermore, it has been shown that the N
protein of SARS-CoV can modulate the host cellular machinery and
it may serve in a regulatory role during the viral life cycle [8].
Therefore, the nucleocapsid protein is a crucial multifunctional
protein, involved in the process of virus infection, replication, and
packaging [9].
In general, viral nuclear proteins can enter the host nucleus and
interact with a variety of host proteins to interfere with the life
cycle of the host cell. It has been shown that the coronavirus N
protein is not only localized in the cytosol but also, to a certain
extent, translocated into the nucleus where it may interact with
various cellular proteins that modulate cellular functions [10]. This
process may depend on interaction of the N protein with the proteasome
activator PA28g, which is localized in the nucleus. PA28g
could be critical for degrading the SARS-CoV-19 nCoV N protein in
the nucleus as part of the 20S proteasome, which acts to degrade
proteins in a ubiquitin-independent manner, such as seen in the
* Corresponding author.
E-mail address: lcgao@bio.ecnu.edu.cn (L. Gao).
1 These authors contributed equally to this work.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
https://doi.org/10.1016/j.bbrc.2020.06.058
0006-291X/© 2020 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications 529 (2020) 251e256
hepatitis C virus (HCV) core protein [11].
The proteasome has an important role in the degradation of
unneeded or damaged proteins by proteolysis. Two distinct proteasomes
differentially target proteins for degradation. The 26S
proteasome, formed by association of the 20S catalytic core
(composed of a and b subunits) with the 19S regulator, is responsible
for degradation of the majority of proteins through a ubiquitin
(Ub)- and ATP-dependent pathway [12]. Additionally, the 20S
proteasome, which is required for the Ub- and ATP-independent
degradation of speci!c target substrates, is generated by a combination
of one 20S catalytic core and one proteasomal activator 28
(PA28) member [13]. Of the three PA28 family members, PA28g
(also called REGg, 11Sg, PSME3, or Ki antigen) is implicated in
tumorigenesis because it regulates cell proliferation and apoptosis,
and it predominantly exerts its function through nuclear proteolysis
[14]. PA28g has been known to target numerous intact proteins
directly through proteasomal degradation. This establishes the
function of PA28g in a variety of biological processes with physiological
and pathological relevance. In addition, PA28g can also
regulate some viruses such as the HCV core protein, hepatitis B
virus X protein, and human immunode!ciency virus type 1 Tat [15].
The nuclear retention and stability of PA28g are regulated via a
PA28g-dependent pathway through which HCV pathogenesis may
be exerted [16]. Moreover, the HCV core protein can decrease 20S
proteasome activity in the presence of PA28g [17].
It has been proposed that the ubiquitin-proteasome system
plays a critical role during various stages of the coronavirus infection
cycle [18]. In turn, the proteasomal inhibitor MG132 strongly
inhibits SARS-CoV replication by interfering with early steps of the
viral life cycle [19]. However, the potential role of the Ub- and ATPindependent
degradation pathway in the !eld of coronavirus
research is unknown. A previous study found that the SARS-CoV N
protein can interact with the host cell proteasome subunit p42, a
subunit of the 26S proteasome [20]. The two activators, PA28 and
26S, can bind to the Ub- and ATP-independent 20S proteasome
simultaneously [21]. Thus, there may be a connection between the
SARS-CoV N protein and PA28. Owing to over 90% amino acid
sequence similarity between SARS-CoV N protein and the SARSCoV-
2 N protein [22], the latter nCoV N protein may be presumed
to play the same role in this process. The precise role of PA28g in
the degradation of coronavirus proteins is still unclear. In the present
study, we found that the N protein of SARS-CoV-2 could be
degraded by PA28g in vitro. This may indicate that PA28g is a
regulator for SARS-CoV-2 N protein degradation. We also investigated
the interaction between SARS-CoV-2 N protein and the
PA28g-20S proteasome system through a co-immunoprecipitation
assay. This new !nding provides a clue for understanding the
previously unresolved physiological roles of the proteasomedependent
degradation of the SARS-CoV-2 N protein during pathogenesis
of COVID-19.
2. Materials and methods
2.1. Plasmids and reagents
Plasmid Flag-PA28g, FRT-PA28g, and FRT-PA28gN151Y were
previously generated [23]. HA-2019-nCoV-N was generated by
polymerase chain reaction using the primers Forward: 50-
CCGCTCGAGATGAGCGATAACGGTCCGC-30 and Reverse: 50-
CGCGGATCCTTACGCTTGGGTGCTATCCGC-30 and was inserted into
the pSG5 vector. The template gene, pET-32a-N-protein, was kindly
provided by Dr. Tao from Shanghai Center for Systems Biomedicine,
Key Laboratory of Systems Biomedicine (Ministry of Education),
Shanghai Jiao Tong University. The clone was constructed following
standard molecular cloning technology and con!rmed by DNA
sequence analysis.
The sources of the following antibodies and reagents were as
follows: rabbit anti-HA (HUABIO, Hangzhou, China), anti- mouse
Flag (MBL, Beijing, China), mouse anti-b-actin (MBL), and PA28g
(Abcam, Cambridge, UK); Flag-M2 beads, HA-beads, and cycloheximide
(Sigma-Aldrich, St. Louis, MO).
2.2. Cell culture and transfection
The 293T cells were purchased from ATCC; the 293T (PA28gknockout)
cell was constructed by TALENT. Cells were cultured in
Dulbecco’s modi!ed Eagle’s medium. Fetal bovine serum 10% and
antibiotics were added to the media.
The 293T cells (PA28g WT and PA28g knockout) were transiently
transfected with the plasmid HA-pSG5-nCoV-N, and Lipofectamine
2000 transfection reagent (Invitrogen) was used
depending on the manufacturer’s instructions.
2.3. Protein degradation in vitro assay
The PA28g and 20S proteasome proteins were puri!ed well [24].
The SARS-CoV-2 N proteinwas created by in vitro translation using
the TNT kit (Promega).
2.4. Immuno!uorescence (IF) staining
The day before the experiment, the cells were inserted into 60-
mmdishes covered with sterile slides and maintained at 37 !C in 5%
CO2 until they were attach to the plate about 24 h later. The cells
were then !xed using 4% paraformaldehyde for 10 min at room
temperature. After washing with PBS. Again, cells were blocked
using 4% serum for 1 h. Next, anti-HAwas added and the cells were
incubated at 4 !C overnight. Subsequently, cells were incubated
with “uorescent secondary antibody for 1 h at room temperature.
Cell images were visualized using a “uorescence microscope.
2.5. Co-immunoprecipitation (Co-IP)
Cells were collected and lysed on ice using the following lysis
buffer. The 10% cell lysate was collected to be the input of Co-IP
assay.
3. Results
3.1. PA28g interacts with nCoV N
The previous studies have demonstrated that PA28g can bind to
the core protein of HCV and promote its degradation [11]. Thus, to
investigate whether exogenous PA28g can interact with nCoV N, we
carried out a co-IP assay. The data support an interaction between
PA28g and nCoV N. Both plasmids Flag-PA28g and HA-nCoV-N
were transfected into 293T cells. PA28g was immunoprecipitated
with an anti-Flag antibody (Fig. 1A). nCoV N could easily be
observed in the immunoprecipitate by Western blot analysis.
Similarly, PA28g was readily detected using an anti-HA antibody
(Fig. 1B). Our results demonstrate that exogenous PA28g can
physically interact with nCoV N.
3.2. nCoV N regulation in PA28g-WT and PA28g-KO cell lines
Next we determined whether endogenous PA28g plays a role in
the degradation of nCoV N. The 293T cells (PA28g-WT and PA28g-
KO) were transfected with the plasmid HA-pSG5-nCoV N and then
treated with cycloheximide (CHX) for the indicated times. We
found that after 12 h, the expression of nCoV N was higher in
252 H. Zhang et al. / Biochemical and Biophysical Research Communications 529 (2020) 251e256
PA28g-KO cells than inWT cells (Fig.1C), indicating that the protein
was more stable in the absence of PA28g in the cells. Moreover, the
stability of nCoV N protein in PA28g-WT was lower than that in the
PA28g-KO cell line over time (Fig. 1D). Taken together, the data
show that endogenous PA28g may have an effect on the degradation
of nCoV N protein through interaction.
3.3. PA28g promotes the degradation of nCoV N
PA28g has been known to direct proteasomal degradation of a
number of complete proteins. And the 20S proteasome is responsible
for Ub- and ATP-independent degradation of speci!c target
substrates, in combination with PA28g. Therefore, to further validate
the role of PA28g in the degradation of nCoV N, we performed
an in vitro proteolytic analysis of nCoV N. We found that nCoV N
exhibits remarkable down-regulation in the presence of PA28g and
the 20S proteasome, while PA28g or 20S proteasome alone serves
no function in the degradation of nCoV N (Fig. 1E and F). These data
indicate that PA28g can promote the degradation of nCoV N in a 20S
proteasome- dependent manner.
3.4. PA28g-mediated degradation of nCoV N
To verify the effect of PA28g on the degradation of nCoV N, we
co-transfected PA28g and HA-nCoV N into 293T-PA28g KO cell
lines. EV-pSG5þPA28gWT, EV-pSG5þPA28g, N151Y, and HAnCoV
þ EV-FRT were transfected into cells as negative controls for
comparison. As expected, the level of nCoV N was signi!cantly
reduced when exogenous PA28g was normally expressed in the
cell. In addition, when the inactive mutant N151Y PA28g was
transfected into the cells with HA-nCoV, we found that the level of
nCoV N remained relatively equal to that of the control (Fig. 2B, C
and D). This con!rmed that the degradation of nCoV N could be
mediated by PA28g. We further investigated the effectiveness of
the expression of PA28g on the degradation of nCoV N by adding an
equal amount of EGFP expression plasmid to detect the expression
ef!ciency. We found that the increased expression level of PA28g
resulted in a marked reduction in nCoV N protein in a dose
dependent manner (Fig. 2A), con!rming that it was the PA28g that
promotes the degradation of nCoV N and its effectiveness is related
to dosage.
3.5. Location and expression of nCoV N in 293T cells
To more intuitively re”ect the promoting effect of PA28g on
nCoV N degradation, immuno”uorescence staining assay was used.
The same amount of HA-nCoV-N was transfected into 293T cells
lines (PA28g-WT and PA28g-KO). The expression level of nCoV N
Fig. 1. PA28g can regulate nCoV N by binding and degradation. (A) The 293T cell lines were co-transfected with HA-nCoV-N and Flag-PA28g plasmids and then subjected to
immunoprecipitation with conjugated anti-Flag beads. (B) The 293T cell lines were co-transfected with the indicated plasmids and then subjected to immunoprecipitation with
conjugated anti-HA beads. All samples were analyzed by western blotting using the indicated antibodies. (C) Degradation dynamics of nCoV N following a time-course treatment
with cycloheximide (CHX, protein synthesis inhibitor, 100 mg/ml) in 293T PA28g wild-type and 293T PA28g knockout cell lines. HA-nCoV-N means the expression of nCoV N after
the transfection of the plasmid HA-pSG5-nCoV N, PA28g means the endogenous PA28g expression in 293T cell lines (it does not express in KO cell line), actin is used as a reference.
(D) According to Western blot bands intensity, the expression of nCoV N in 293T WT and KO could be calculated. Normalized with actin, the quanti!cation of nCoV N degradation
was demonstrated. WT, wild-type PA28g; KO, knockout PA28g. (E) In vitro proteolytic analysis of PA28g-mediated degradation of nCoV N. Puri!ed PA28g, 20S proteasome, and
in vitro-translated nCoV N were incubated as indicated and described in materials and methods. PA28g means the PA28g protein expression, HA-nCoV-N means the nCoV N
translated protein expression and GAPDH means the reference. (F) Quanti!cation of nCoV N relative expressionwas normalized with the vitro-translated nCoV N. Results presented
as the means ± SEM. *p < 0.05; **p < 0.01 versus control, n ¼ 3.
H. Zhang et al. / Biochemical and Biophysical Research Communications 529 (2020) 251e256 253
protein (red) in the PA28g-KO cell lines was observed to be
prominently higher than that in the PA28g-WT cell line (Fig. 3C and
D), con!rming that PA28g de!ciency promotes nCoV N in the cell
line.
We then investigated the cellular localization and expression of
nCoV N in 293T cells under the regulation of PA28g by immuno-
“uorescence staining. The same amount of nCoV N was transfected
into 293T cell lines (PA28g-WT and PA28g-KO). In the PA28g-WT
cell lines, the nCoV N protein (red) was prominently expressed in
the cytoplasm, while in the PA28g-KO cell lines it existed both in
the nucleus and the cytoplasm (Fig. 3A). In addition, the total
expression of nCoV N was quanti!ed and, as expected, PA28g-WT
cell lines exhibited a decrease in nCoV N (Fig. 3B). PA28g has been
known to express in the nucleus [11]. Therefore, our results indicate
that the nCoV N in the nucleus was degraded under the regulation
of PA28g, while that in the cytoplasm was not.
3.6. Cartoon depicting the nCoV N protein degradation process
through the PA28g-20S system
A schematic diagram of the degradation process shows that
PA28g !rst binds with nCoV N and transports it to the 20S proteasome
subunit where internal degradation occurs (Fig. 4). Thus,
the time of existence of nCoV N in the nucleus is much shorter
compared to that in the cytoplasm.
4. Discussion
An increasing number of studies have shown that the proteasome
is associated with viral infection, and there is evidence that
HCV core proteins can be degraded through the PA28g-20S system.
That is, the nuclear retention and stability of HCV core proteins are
regulated by PA28g-dependent pathways such that the
pathogenicity of HCV may be achieved through this pathway [16].
In addition, the human immunode!ciency virus-1 (HIV-1) Tat
protein can inhibit the peptidase activity of the 20S proteasome by
competing with the 11S/PA28 regulator (REG) for binding at the
REG/Tat-proteasome-binding (RTP) site and interfering with antigen
processing [25]. It has been shown that the hepatitis B virus X
protein-derived polypeptide, harboring the a4 proteasome subunit
binding motif, impairs the activation of 20S proteasomes by PA28
[26]. The coxsackievirus infection can be enhanced by proteasome
activator PA28g promoting p53 degradation [27], similar to the
mechanism of degradation observed in the HBx virus [28].
Furthermore, protein p30 interactions with PA28g may also affect
ATM functions and increase cell survival [29]. On the other hand,
PA28g acts as a co-repressor of HTLV-1 p30 to suppress virus
replication and is required for the maintenance of control viral latency
[33]. Therefore, previous studies have shown that PA28g is
closely related to the HTLV-1 virus and plays an indelible role in the
formation and spread of the virus. Additionally, the ability of PA28g
to promote viral protein degradation suggests its involvement in
viral pathogenesis.
Studies also have demonstrated that the novel coronavirus
nucleocapsid protein (N) shares nearly 90% amino acid sequence
homology with SARS coronavirus. SARS coronavirus N protein antibodies
can cross-react with novel coronavirus, but cannot provide
cross-immunity. Similar to SARS-CoV, nCoV N proteins can inhibit
RNA interference (RNAi) to overcome the host defense [32]. Early
research revealed the involvement of the ubiquitin-proteasome
system (UPS) in multiple steps of the coronavirus infection cycle
and identi!ed UPS as a potential drug target to modulate the signi
!cance of coronavirus infection [18].
In the present study, we found that a novel ubiquitinationindependent
pathway could regulate the protein levels of nCoV
N, and PA28g could control the abundance of nCoV N by regulating
Fig. 2. PA28g-mediated degradation of nCoV N in 293T cells. (A) The 293T cells were co-transfected HA-nCoV-N with increasing amounts of PA28g. The nCoV N protein levels were
determined by western blotting. A constant amount of an enhanced green “uorescent protein (EGFP) expression plasmid was included to monitor transfection ef!ciency. (B) The
293T PA28g KO cells were transfected the same amount of only HA-nCoV-N, HA-nCoV-N with FRT-PA28g wt and HA-nCoV-N with FRT-PA28gN151Y. (C) Cells were transient
transfected with the same amount of HA-nCoV-N, FRT-PA28g wild-type, FRT-PA28g N151Y, HA-pSG5 vector, and FRT vector using Lipo2000 transfection reagent for 48 h. Protein
expression was detected using anti-PA28g, anti-HA, and anti-actin antibodies. Non-transfected 293T cells were used as a control. (D) Quanti!cation of nCoV N relative expression.
Data are presented as means ± SEM. *p < 0.05; **p < 0.01 versus control, n ¼ 3.
254 H. Zhang et al. / Biochemical and Biophysical Research Communications 529 (2020) 251e256
Fig. 3. Location and expression of nCoV N in 293T PA28gWT and PA28gKO cells. (A) Cells were transiently transfected with HA-nCoV-N, !xed, and immunostained with anti-HA
(red color). Cell nuclei were stained with DAPI (blue color). Scale bar: 50 mm. (B) Quanti!cation analysis of nCoV N expression as a percentage per cell of three independent experiments
(n ¼ 100; values represent the mean ± SEM; *p < 0.05; **p < 0.01 versus control). (C) Cells were transiently transfected with the same amount of HA-nCoV-N plasmid,
!xed, immune-stained with anti-HA (red color), and stained with DAPI (blue color). Scale bar: 100 mm. (D) Quanti!cation analysis of nCoV N expression as a percentage in the same
cell area. Each data set represents three independent experiments (n ¼ 300 . values represent the mean ± SEM; *p < 0.05; **p < 0.01 versus control). (For interpretation of the
references to color in this !gure legend, the reader is referred to the Web version of this article.)
Fig. 4. Schematic diagram of the progress of PA28g-20S degradation of nCoV N. nCoV N enters the nucleus, binds to PA28g, and is then degraded internally under the action of the
20 S proteasome. nCoV N is internally after 20S proteasome binding to PA28g.
H. Zhang et al. / Biochemical and Biophysical Research Communications 529 (2020) 251e256 255
its stability. In addition, we showed that PA28g interacts with nCoV
N and promotes its intracellular degradation. SARS coronavirus
nucleocapsid protein is a very important viral structural protein
and an important indicator in early diagnosis due to its abundance
and high conservation in cells. Studying nucleocapsid protein
structural function, how it participates in transcription and translation,
the molecular mechanism in virus-infected cells, and the
regulation of gene expression will help to thoroughly understand
SARS coronaviruses and !nd effective methods for prevention and
treatment of related diseases [6]. When SARS-CoV-2 invades the
human body, it makes contact with human immune cells, thus
causing the immune cells to produce massive IFN-g, and IFN-g can
induce PA28g [32]. This in turn stimulates proteasome activity
resulting in degradation of the coronavirus N protein. As a result,
virus production is blocked and proliferation and diffusion are
greatly amelioraten.
In conclusion, our study substantiates that PA28g could mediate
the degradation of nCoV N. These results suggest that the PA28g
interaction has an important role in regulating 20S proteasome
activity and furthers our understanding of the pathogenesis of
2019-nCoV. However, this interaction correlates to the of truncated
nCoV N, further work is necessary to locate the accurate region of
nCoV N interact with PA28g. In addition, some mechanisms may be
found. Understanding the precise function of PA28g may give us
new insight into virus-cell interactions and lead to a greater understanding
of the pathogenicity of 2019-nCoV infection.
Declaration of competing interest
The authors declare that they have no known competing
!nancial interests or personal relationships that could have
appeared to in”uence the work reported in this paper.
Acknowledgments
This workwas supported by Shanghai Committee of Science and
Technology. (no. 16DZ2348900).
References
[1] WHO. Coronavirus Disease 2019 (COVID-19): Situation Reportd132, May 31,
2020.
[2] N. Zhu, D.Y. Zhang, W.L. Wang, X.W. Li, B. Yang, J.D. Song, X. Zhao, B.Y. Huang,
W.F. Shi, R.J. Lu, P.H. Niu, F.X. Zhan, X.J. Ma, D.Y. Wang, W.B. Xu, G.Z. Wu,
G.G.F. Gao, W.J. Tan, C.N. Coronavirus, A novel coronavirus from patients with
pneumonia in China, 2019, N. Engl. J. Med. 382 (2020) 727e733.
[3] Z.Q. Song, Y.F. Xu, L.L. Bao, L. Zhang, P. Yu, Y.J. Qu, H. Zhu, W.J. Zhao, Y.L. Han,
C. Qin, From SARS to MERS, thrusting coronaviruses into the spotlight, Viruses-
Basel vol. 11 (2019).
[4] J. Cui, F. Li, Z.L. Shi, Origin and evolution of pathogenic coronaviruses, Nat. Rev.
Microbiol. 17 (2019) 181e192.
[5] A.P. Wu, Y.S. Peng, B.Y. Huang, X. Ding, X.Y. Wang, P.H. Niu, J. Meng, Z.Z. Zhu,
Z. Zhang, J.Y. Wang, J. Sheng, L.J. Quan, Z.X. Xia, W.J. Tan, G.H. Cheng, T.J. Jiang,
Genome composition and divergence of the novel coronavirus (2019-nCoV)
originating in China, Cell Host Microbe 27 (2020) 325e328.
[6] C.K. Chang, M.H. Hou, C.F. Chang, C.D. Hsiao, T.H. Huang, The SARS coronavirus
nucleocapsid protein – forms and functions, Antivir. Res. 103 (2014) 39e50.
[7] J. Mu, J. Xu, L. Zhang, T. Shu, D. Wu, M. Huang, Y. Ren, X. Li, Q. Geng, Y. Xu,
Y. Qiu, X. Zhou, SARS-CoV-2-encoded nucleocapsid protein acts as a viral
suppressor of RNA interference in cells, Sci. China Life Sci. (2020).
[8] M. Surjit, B.P. Liu, V.T.K. Chow, S.K. Lal, The nucleocapsid protein of severe
acute respiratory syndrome-coronavirus inhibits the activity of cyclin-cyclindependent
kinase complex and blocks S phase progression in mammalian
cells, J. Biol. Chem. 281 (2006) 10669e10681.
[9] J. Fu, R. Chen, J. Hu, H. Qu, Y. Zhao, S. Cao, X. Wen, Y. Wen, R. Wu, Q. Zhao,
X. Ma, X. Huang, Identi!cation of a novel linear B-cell epitope on the nucleocapsid
protein of porcine deltacoronavirus, Int. J. Mol. Sci. 21 (2020).
[10] K.A. Timani, Q.J. Liao, L.B. Ye, Y.C. Zeng, J. Liu, Y. Zheng, L. Ye, X.J. Yang,
L.B. Kong, J.R. Gao, Y. Zhu, Nuclear/nucleolar localization properties of C-terminal
nucleocapsid protein of SARS coronavirus, Virus Res. 114 (2005) 23e34.
[11] R. Suzuki, K. Moriishi, K. Fukuda, M. Shirakura, K. Ishii, I. Shoji, T. Wakita,
T. Miyamura, Y. Matsuura, T. Suzuki, Proteasomal turnover of hepatitis C virus
core protein is regulated by two distinct mechanisms: a ubiquitin-dependent
mechanism and a ubiquitin-independent but PA28 gamma-dependent
mechanism, J. Virol. 83 (2009) 2389e2392.
[12] O. Coux, K. Tanaka, A.L. Goldberg, Structure and functions of the 20S and 26S
proteasomes, Annu. Rev. Biochem. 65 (1996) 801e847.
[13] C.P. Ma, C.A. Slaughter, G.N. DeMartino, Identi!cation, puri!cation, and
characterization of a protein activator (PA28) of the 20 S proteasome (macropain),
J. Biol. Chem. 267 (1992) 10515e10523.
[14] I. Mao, J. Liu, X. Li, H. Luo, REGgamma, a proteasome activator and beyond?
Cell. Mol. Life Sci. 65 (2008) 3971e3980.
[15] H. Sivakumaran, A. van der Horst, A.J. Fulcher, A. Apolloni, M.H. Lin, D.A. Jans,
D. Harrich, Arginine methylation increases the stability of human immunode
!ciency virus type 1 Tat, J. Virol. 83 (2009) 11694e11703.
[16] K. Moriishi, T. Okabayashi, K. Nakai, K. Moriya, K. Koike, S. Murata, T. Chiba,
K. Tanaka, R. Suzuki, T. Suzuki, T. Miyamura, Y. Matsuura, Proteasome activator
PA28gamma-dependent nuclear retention and degradation of hepatitis
C virus core protein, J. Virol. 77 (2003) 10237e10249.
[17] Y. Zheng, S. Shimamoto, T. Maruno, Y. Kobayashi, Y. Matsuura, K. Kawahara,
T. Yoshida, T. Ohkubo, N-terminal HCV core protein fragment decreases 20S
proteasome activity in the presence of PA28gamma, Biochem. Biophys. Res.
Commun. 509 (2019) 590e595.
[18] M. Raaben, C.C. Posthuma, M.H. Verheije, E.G. te Lintelo, M. Kikkert,
J.W. Drijfhout, E.J. Snijder, P.J.M. Rottier, C.A.M. de Haan, The ubiquitinproteasome
system plays an important role during various stages of the
coronavirus infection cycle, J. Virol. 84 (2010) 7869e7879.
[19] M. Schneider, K. Ackermann, M. Stuart, C. Wex, U. Protzer, H.M. Schatzl,
S. Gilch, Severe acute respiratory syndrome coronavirus replication is severely
impaired by MG132 due to proteasome-independent inhibition of M-calpain,
J. Virol. 86 (2012) 10112e10122.
[20] Q. Wang, C.A. Li, Q.F. Zhang, T. Wang, J.D. Li, W.X. Guan, J.S. Yu, M.F. Liang,
D.X. Li, Interactions of SARS Coronavirus Nucleocapsid Protein with the host
cell proteasome subunit p42, Virol. J. 7 (2010).
[21] K.B. Hendil, S. Khan, K. Tanaka, Simultaneous binding of PA28 and PA700
activators to 20 S proteasomes, Biochem. J. 332 (Pt 3) (1998) 749e754.
[22] S. Kannan, P.S.S. Ali, A. Sheeza, K. Hemalatha, COVID-19 (Novel Coronavirus
2019) – recent trends, Eur. Rev. Med. Pharmacol. Sci. 24 (2020) 2006e2011.
[23] Q. Zuo, S. Cheng, W. Huang, M.Z. Bhatti, Y. Xue, Y. Zhang, B. Zhang, L. Li, L. Wu,
J. Fu, J. Chen, X. Li, REGgamma contributes to regulation of hemoglobin and
hemoglobin delta subunit, Oxid. Med. Cell. Longev. 2017 (2017) 7295319.
[24] X. Gao, J. Li, G. Pratt, S. Wilk, M. Rechsteiner, Puri!cation procedures determine
the proteasome activation properties of REG gamma (PA28 gamma),
Arch. Biochem. Biophys. 425 (2004) 158e164.
[25] X.H. Huang, U. Seifert, U. Salzmann, P. Henklein, R. Preissner, W. Henke,
A.J. Sijts, P.M. Kloetzel, W. Dubiel, The RTP site shared by the HIV-1 Tat protein
and the 11 S regulator subunit alpha is crucial for their effects on proteasome
function including antigen processing, J. Mol. Biol. 323 (2002) 771e782.
[26] R. Stohwasser, H.G. Holzhutter, U. Lehmann, P. Henklein, P.M. Kloetzel, Hepatitis
B virus HBx peptide 116-138 and proteasome activator PA28 compete
for binding to the proteasome alpha4/MC6 subunit, Biol. Chem. 384 (2003)
39e49.
[27] G. Gao, J. Wong, J. Zhang, I. Mao, J. Shravah, Y. Wu, A. Xiao, X. Li, H. Luo,
Proteasome activator REGgamma enhances coxsackieviral infection by facilitating
p53 degradation, J. Virol. 84 (2010) 11056e11066.
[28] S. Yeom, H. Jeong, S.S. Kim, K.L. Jang, Hepatitis B virus X protein activates
proteasomal activator 28 gamma expression via upregulation of p53 levels to
stimulate virus replication, J. Gen. Virol. 99 (2018) 655e666.
[29] R. Anupam, A. Datta, M. Kesic, K. Green-Church, N. Shkriabai, M. Kvaratskhelia,
M.D. Lairmore, Human T-lymphotropic virus type 1 p30 interacts with
REGgamma and modulates ATM (ataxia telangiectasia mutated) to promote
cell survival, J. Biol. Chem. 286 (2011) 7661e7668.
[32] K. Schwarz, M. van Den Broek, S. Kostka, R. Kraft, A. Soza, G. Schmidtke,
P.M. Kloetzel, M. Groettrup, Overexpression of the proteasome subunits LMP2,
LMP7, and MECL-1, but not PA28 alpha/beta, enhances the presentation of an
immunodominant lymphocytic choriomeningitis virus T cell epitope,
J. Immunol. 165 (2000) 768e778.
[33] N.L. Ko, Taylor J.M. Bellon, M. Bai, X.T. Shevtsov, S.P. Dundr, M. Nicot C., Blood
121 (2013), https://doi.org/10.1182/blood-2012-03-420414.
256 H. Zhang et al. / Biochemical and Biophysical Research Communications 529 (2020) 251e256

This article presents a critical review of the paper “Purification and characterization of a novel extracellular, alkaline, thermoactive, and detergent-compatible lipase from Aeromonas caviae LipT51 for application in detergent industry” by Sumeyra Gurkok and Murat Ozdal [1]. According to the paper the application of enzymes in Industries has been a beneficial action at many points in the past few decades, such as increased washing quality at detergent industries, eco-friendly and economically. The purpose of this experiment was to isolate the best bacterial source from extreme environments to introduce and identify a lipase (triglycerides hydrolysis enzyme) well-adjusted to washing detergents’ environment and ideally works on oil-stain removal. The authors collected samples and isolated their experiment participants from hot springs and oil-contaminated soil, particularly to be matched with the extreme washing environment, then screened their lipase activity based on hydrolysis zone formation on the Tributyrin Agar (TBA) plates to find the best of lipase production. They continued by cultivating the isolate contains the largest hydrolysis zone to inoculate with the Tributyrin broth (TBB) media toward production of lipase. They analyzed lipase activity by spectrophotometery; p-nitrophenol palmitate (pNPP) used as substrate. On the other hand the lipase enzyme identified as A. caviae LipT51 by its 16S rRNA gene sequences deposited in the NCBI database and conventional identification methods. Furthermore they used centrifugation along with ammonium sulfate precipitation and anion exchange chromatography to purify the enzyme. To characterize the purified lipase enzyme, they tried to define kinetic constants by SDS-PAGE and obtain the optimum pH and temperature for lipase activity through enzyme assay over variable pH range with constant temperature and vice versa while enzyme and substrate concentration both were constant all the time. In Continue with characterizing, the enzyme activity was measured individually at the addition of metal ions, organic solvents, surfactants, and detergents then compared with the control in order to demonstrate their effects on the enzyme activity. Their final work was to evaluate the enzyme washing performance by using a washing solution containing essentials plus the enzyme, as it represents the usual washing environments. The focus of this paper will be on some aspects appeared to strongly have approved the whole idea of the experiment and evaluating the figures content and their consistency to the trend of the experiment. Furthermore, this paper will spot some points of the experiment which need to expand if it is necessary to assure its readers about what it claims to have been achieved.

What has done well in the experiment was setting up a well-specified process to claim the benefits of collaborating lipase enzyme with detergents for commercial use.  Reasonably collected isolates, Set of controls, and relative activity measuring through the whole experiment, have helped it to be convincible. Also, they did an accurate job identifying the effects of metal ions, organic solvents, and surfactants on lipase activity, even though it recommends investigating the lipase activity under the influence of their combinations besides individually.

In Figure 1 the hydrolysis zone of A. caviae LipT51 on TBA plate has shown but there is no image of the other participant plates. The text claims that the highest lipase activity belonged to A. caviae LipT51 among all of the isolated colonies. If authors have made a comparison, it is necessary to give pieces of evidence for the readers to see themselves such as images in comparison.

To investigate the molecular weight of the lipase, SDS page has run and reported approximately 31.6 kDa. As a lipase previously reported from A. caviae AU04 having slightly higher weight of 39 kDa [2], it would be more persuader if the authors had ran a repeat line 3 and had shown it at Figure.2  to assure lipase introduced in the paper firmly have 31.6 kDa weight as its newly reporting.

Table 1 instance for the washing solutions and the control contents prepared to evaluate the washing performance. It located at the result part 3.2 which refers to the purification of lipase from A. caviae LipT51. the Table basically relates to both result 3.4, and the method and materials 2.8, in this case, it should have been located at result 3.4 or should have been explained in the text of the 2.8 method and materials according to its contents. It is not common to reference figures and tables at the method and materials; if the Table form is a better exhibition of the process, it would be better to had brought at the result section of its own.

The whole idea was a delicate attitude to prevention of washing wastage contamination and utilizing fewer chemicals by increasing washing quality due to lipase application in detergent commercials, we need such scientific approaches to the Industries inveterate least concerns about environmental pollution, therefore they set a well-organized experiment with reasonable controls and well-assumed participants even though there were some advancements to apply on figures and data confirmation to accomplish its perfect level.

References:

1. Gurkok, S., & Ozdal, M. (2021). Purification and characterization of a novel extracellular , alkaline , thermoactive , and detergent-compatible lipase from Aeromonas caviae LipT51 for application in detergent industry. Protein Expression and Purification, 180(January), 105819. https://doi.org/10.1016/j.pep.2021.105819
2. Yasuda, M., & Ishikawa, H. (2000). Purification and Characterization of O rganic Solvent-Stable Lipase from O rganic Solvent-Tolerant Pseudomonas aeruginosa LST-03, 89(5), 451–457.

1
Guidelines to writing critical reviews
Read through the Introduction section of the paper assigned and try to identify its
main objectives. What did the researchers set out to investigate? Why is it important?
What is known about the proteins/processes the authors are interested in from previous
research? Summarize this information in a short paragraph and come back to it as you
read through the rest of the paper.
Next, read through the Materials and Methods section and sketch out a diagram or
a flow-chart of the experiments performed. Try to imagine yourself doing these
experiments in the lab. Will you be able to repeat them without any outside help? Is
something unclear? If so, try to use the Internet to find answers. If you are unable to
make sense of the paper􀂶s experimental methods, you are unlikely to make sense of the
paper in general. So, find answers yourself or ask for help from your instructor or W TA.
Once you understand all of the techniques described in the paper, read the Materials and
Methods section again and revise your diagram or flow-chart. Consult it as you read
through the Results section of the paper.
Turn your attention now to the Results section. Remember that the main focus of
your review should rest with this section. So, read it carefully more than once, making
notes as you go along. Read the Figure legends closely. Make sure you understand what
results are presented where. Are there negative or positive controls? Are the images
presented in Figures uncluttered and clear? Do you find that the text in the Results
section is justified by what is being presented in the Figures and Tables? If something is
unclear, why is it so? This is the critical aspect of your review. Don􀂶t worry about
making mistakes just now. Ask questions of the paper􀂶s authors as you read through and
write them down.
To get a good mark on this assignment, you should have at least THREE critical
points. It may help to think of these points as questions you may ask the authors. These
do not all have to be negative. If you find, for example, that the authors provided
convincing experimental evidence that protein A interacts with protein B, say so. If, on
the other hand, they failed, in your opinion, to provide good experimental evidence for
their claim(s), or if you find that there may be other possibilities accounting for the
observed results, you should explore them.
Finally, read the Discussion section and review it in the context of the obtained
results. Are the conclusions the authors draw from their results seem well argued,
reasoned and supported by the experimental data? Would you have come to similar
conclusions on your own? If not, why do you think you might have come to different
conclusions?
Now, go back to the beginning, look through all of the notes you have taken and
write the opening paragraph of your critical review, stating in the last sentence of this
paragraph what the focus of your paper is going to be. In the subsequent paragraphs of
2
your paper, advance your critical points, one at a time, and justify each of them. Your
last, concluding paragraph should come back to the beginning, so close your paper in a
meaningful way.
Style
Avoid reviews of the Materials and Methods and/or Results sections without a
true critical focus. Your job is to critically evaluate the experiments and results described
in the paper and to comment on them, not repeat in your own words what experiments
were done.
THE PAGE LIMIT for CRITICAL REVIEW 1 IS 3 DOUBLE-SPACED
PAGES WITH LETTERS IN 12 POINT ARIAL FONT AND STANDARD
MARGINS (EXCLUDING REFERENCES. You shouldn􀂶t ha􀁙e man􀁜 references
because your focus should rest with the paper under review. But you will have to
cite the paper you are reviewing, so at least one reference is necessary.)
THE PAGE LIMIT for CRITICAL REVIEW 2 IS 2 DOUBLE-SPACED
PAGES WITH LETTERS IN 12 POINT ARIAL FONT AND STANDARD
MARGINS (EXCLUDING REFERENCES)
Be specific: if something is unclear, write out why it is unclear to you. If you
think of a different or follow-up experiment, suggest it in specific terms, not just 􀂳more
experiments needed to be done.􀂴 Remember that you don􀂶t have to be negative: if you
think that experiments were well designed and executed, you should say so.
Avoid long-winded introductions and extensive review of literature at all costs.
You are reviewing THIS PARTICULAR paper, so stick to it. Look at the experimental
design, evaluate the results, and carefully consider the discussion. Many students get
caught up in reviewing the literature and going on tangents about previous work. Two or
three (at most) sentences should suffice to summarize the goals of the paper. Don􀂶t get
caught up summarizing the materials and methods described in the paper or regurgitate
the experimental data obtained.
Finally, it􀂶s better to write about what you know than to venture into speculation.
In our labs we immune-precipitated beta-tubulin (IP), cast and ran SDS-PAGE, did
Western blotting, and examined microscopy slides for immunofluorescence (IF). You are
expected to comment on these experiments in the paper under your review. You may
skip sections of the paper you are not familiar with.
Final note
Express your thoughts clearly and simply. Read your sentences our loud.

A”few”notes”about”cri0cal”
thinking”
Or,”sugges0ons”for”wri0ng”good”
cri0cal”reviews