ERC-BiP Functional Protein Pathway for Assessing Endoplasmic Reticulum Stress Induced by SARS-CoV-2 Replication after Cell Invasion

Background SARS-CoV-2 induces apoptosis and amplifies the immune response by continuously stressing the endoplasmic reticulum (ER) after invading cells. This study aimed to establish a protein-metabolic pathway associated with ER dysfunction based on the invasion mechanism of SARS-CoV-2. Methods This study included 17 healthy people and 46 COVID-19 patients, including 38 mild patients and 8 severe patients. Proteomics and metabolomics were measured in the patient plasma collected at admission and one week after admission. The patients were further divided into the aggravation and remission groups based on disease progression within one week of admission. Results Cross-sectional comparison showed that endoplasmic reticulum molecular chaperone-binding immunoglobulin protein (ERC-BiP), angiotensinogen (AGT), ceramide acid (Cer), and C-reactive protein (CRP) levels were significantly increased in COVID-19 patients, while the sphingomyelin (SM) level was significantly decreased (P  <  0.05). In addition, longitudinal comparative analysis found that the temporal fold changes of ERC-BiP, AGT, Cer, CRP, and SM were significantly different between the patients in the aggravation and remission groups (P  <  0.05). ERC-BiP, AGT, and Cer levels were significantly increased in aggravation patients, while SM was significantly decreased (P  <  0.05). Meanwhile, ERC-BiP was significantly correlated with AGT (r = 0.439; P  <  0.001). Conclusions ERC-BiP can be used as a core index to reflect the degree of ER stress in COVID-19 patients, which is of great value for evaluating the functional state of cells. A functional pathway for AGT/ERC-BiP/glycolysis can directly assess the activation of unfolded protein reactions. The ERC-BiP pathway is closer to the intracellular replication pathway of SARS-CoV-2 and may help in the development of predictive protocols for COVID-19 exacerbation.


Introduction
Te surface S protein of the SARS-CoV-2 virus invades the cytoplasm by binding to the host cell receptor angiotensinconverting enzyme 2 (ACE2) [1].Te virus then replicates in the endoplasmic reticulum (ER) [1].Te properties of rapid membrane fusion promote SARS-CoV-2 replication, propagation, and migration along the respiratory tract, inducing innate immune and infammatory responses [2,3].Respiratory symptoms are the main clinical manifestation of COVID-19 patients [2].Tere is pathological evidence of bilateral difuse alveolar injury, hyaline membrane formation, fbrin deposition, and exfoliation of pulmonary epithelial cells in COVID-19 patients [3].Mild COVID-19 patients may experience none or only respiratory symptoms, while severe patients may develop acute respiratory distress syndrome (ARDS) or multiple organ dysfunction syndrome (MODS) [4].As a result, the evaluation of COVID-19 exacerbation has attracted much interest.
Te increased risk of COVID-19 is closely related to the viral burden and infammatory storm.ER is one of the largest organelles in the cell where protein folding and secretion, membrane synthesis, and lipid synthesis occur.It is also one of the largest assembly regions for SARS-CoV-2, providing the membrane structure requisite for virus replication and release [5,6].ER controls lipid metabolism, calcium storage, and protein folding balance [7,8].ER membrane is in a dynamic equilibrium state.ER also acts as a regulator, detecting and repairing incorrectly translated proteins [9].Direct injury and indirect infammation induced by SARS-CoV-2 disrupt the stable protein synthesis environment of ER [9].Te accumulation of misfolded proteins triggers unfolded protein reaction (UPR), causing ER stress [9].A persistent stress signal triggers autophagy, leading to apoptosis and immune response amplifcation [6].
Terefore, the evaluation of ER functions in COVID-19 patients could provide insights into the mechanism of SARS-CoV-2-induced cellular dysfunction.Heat shock protein (HSP70, also named BiP) has a molecular weight of 70 kD.HSP70 is a ubiquitous molecular chaperone involved in a variety of cellular functions, protecting cells from the efects of various viral invasive stresses [10].However, it has also been suggested that Hsp70 family proteins interact directly with viral polymerases to enhance viral replication or that they may promote the formation of viral replication complexes and/or maintain the stability of complex proteins [11].As an important macromolecule assisting in protein folding and assembly, BiP plays diferent functions in different parts.In ER, BiP can regulate the activity and transport of proteins.For this kind of Hsp70 (BiP), the unifed name is ERC-BiP.Endoplasmic reticulum molecular chaperone-binding immunoglobulin protein (ERC-BiP), which regulates protein folding, plays a key role in ER homeostasis [12].Furthermore, ERC-BiP maintains the ER permeability barrier during protein translocation and retrograde translocation for misfolded proteins [13].However, proteasomes can degrade ERC-BiP, thus promoting ER calcium storage and UPR activation by sensing stress [13].Terefore, ERC-BiP can be used to evaluate the function of ER.Te formation of a protein network may aid in assessing homeostasis deviation caused by SARS-CoV-2.In this study, ERC-BiP was selected to evaluate the state of ER stress induced by SARS-CoV-2 and establish a systematic functional protein pathway, which may provide insights into the mechanism of COVID-19 disease progression and help in the development of predictive protocols for COVID-19 exacerbation.Clinical data and blood samples of patients were collected at admission and one week after admission [14].Te patients were divided into two groups based on their conditions at the two time points, except for 5 patients without longitudinal samples, as follows: the aggravation group (n � 29) (patients with signifcant respiratory symptom exacerbation, increased area of pulmonary imaging lesions, and decreased oxygenation index after admission) and the remission group (n � 12) (patients with reduced symptoms, decreased pulmonary lesions, or improved clinical indices).Te evaluation was conducted by three researchers, two laboratory physicians, and two clinicians based on the patients' examination results or symptoms.Moreover, 17 healthy controls were included.

Plasma
Collection.Blood samples were collected from subjects early in the morning.Te samples were centrifuged at 3000 rpm and room temperature for 10 min within two hours.Te samples were then stored in isolation at −80 °C.Each sample was aspirated with 50 μl of plasma dispensed for further analysis [14].

Target Proteomics Analysis.
Target proteomics analyses were conducted using ultraperformance liquid chromatography (UPLC, ExionLC AD, Shanghai, China.https:// sciex.com.cn/) and tandem mass spectrometry (MS/MS.QTRAP ® , https://sciex.com.cn/).Trough the selective detection of specifc peptide sequences or target peptide segments, such as those undergoing post-translational modifcations, it is possible to achieve targeted relative quantifcation of ERC-BiP.Tis approach makes use of the selective detection capability of a quadrupole mass analyzer in the frst-stage mass spectrometry (Q1) to accurately identify the precursor ion information of the target peptide segment.Subsequently, the peptide is fragmented in the collision-induced dissociation (CID) cell, and the resulting For the detection and quantifcation of specifc metabolites, we utilized multiple reaction monitoring (MRM) in the mass spectrometer.MRM enables the precise selection of parent and daughter ion pairs corresponding to the metabolites of interest.Tis targeted detection strategy signifcantly enhances the sensitivity and specifcity of metabolite analysis, enabling the accurate quantifcation of low-abundance metabolites in complex biological samples.Te study primarily focused on lipid metabolites and energy cycle-related metabolites.

Standardization of Omics Data.
During the preprocessing of serum samples, 5 μl of internal standards, HETE-d8 and Phe-2, were added to each sample.Te peak area of each metabolite (Area i) was then divided by the peak area of the corresponding internal standard (area internal) in the same sample, resulting in a relative abundance value.Since an equal amount of internal standard was added to each sample, this calibration helps to normalize the metabolite levels to the time of internal standard addition.In PRM experiments based on mass spectrometry, the fragmentary signal entering the detector in the actual sample is collected by mass spectrometry and then the relative signal strength of the target protein is compared between diferent groups.
2.6.Statistical Analysis.Continuous variables were expressed as the median (interquartile range (IQR)), while categorical variables were expressed as the frequency.Te Mann-Whitney-Wilcoxon rank-sum test (two groups) or the Kruskal-Wallis test (three or more groups) was used to assess diferences in continuous variables.Te Chi-square test (two groups) or Fisher's exact test (three or more groups) was used to compare the categorical data.Spearman's correlation coefcient was calculated to explore the correlation between diferent metabolites and proteins.Metabolite and protein levels were separately measured at the two time points.Te change in metabolite levels was determined by fnding the diference between the values of the second measurement and the frst measurement.Partial least squares-discriminant analysis (PLS-DA) was used to screen metabolites.P < 0.05 was considered statistically signifcant.R software version 4.0.0 (R Core Team) was used for all statistical analyses.

Horizontal Comparison of Protein and Metabolite Levels in COVID-19
Patients.Te patients were divided into the aggravation group (n � 29) and the remission group (n � 12) based on the changes in their condition after one week of admission to assess the disease progression.Te temporal fold change was defned as the fold change between the second measurement of metabolites and proteins and the frst measurement, i.e., the second measurement value/the frst measurement value.Te distributions of the temporal fold changes are shown using heatmap in Figure 1(a).PLS-DA analysis showed signifcant separation of the temporal diferences among the aggravation and remission groups in Figure 1(c).Te detailed values of the temporal fold changes in each group are shown in Table 3. Te temporal fold changes of ERC-BiP, CRP, AGT, Cer, and SM were signifcantly diferent between aggravation group and remission group (P < 0.05).However, the temporal fold Canadian Journal of Infectious Diseases and Medical Microbiology changes of melatonin were not signifcantly diferent among these groups.
Te longitudinal temporal changes in metabolites and proteins in each group are expressed in histograms in Figure 2. ERC-BiP, AGT, and Cer levels were signifcantly increased in the aggravation group after one week, while SM was signifcantly decreased (P < 0.05).Moreover, CRP was signifcantly decreased in the remission group (P < 0.05), while SM was slightly elevated.ERC-BiP, AGT, and Cer levels were also slightly elevated in the remission group (P > 0.05).Tere were no signifcantly diferences of melatonin in either aggravation group or remission group.
Te correlations between clinical indicators and metabolites/proteins are calculated in Figure 3. Te detailed values of correlation coefcients and P values are shown in Supplementary Table 2. Te correlation analyst showed that the ERC-BiP was not signifcantly correlated with the immune cell count (WBC, NEU, LYM, and MONO) and infammatory markers (SAA and PCT).However, ERC-BiP was signifcantly positively correlated with AGT (r � 0.439; P < 0.001).ERC-BiP was also signifcantly correlated with Cer and lactate acid (r � 0.534 and P < 0.001; r � 0.3528 and P � 0.005).

Discussion
In this study, lymphocytes of COVID-19 patients were signifcantly decreased.Moreover, ERC-BiP, which refects the ER function, was signifcantly diferent between patients and healthy subjects and was signifcantly increased in severe patients.AGT, which mediates viral transmembrane action, was also associated with disease exacerbation.In COVID-19 patients, the equilibrium between anaerobic and aerobic circulation, the major pathway for protein folding in ER, was signifcantly altered.Furthermore, the activity of the TCA cycle (standardized population) and lactic acid accumulation was decreased in COVID-19 patients.As a result, signifcant infammatory factor release syndrome occurred in severe patients, as shown by the signifcantly increased IL-6, CRP, and SAA levels.

ERC-BiP Refects ER Stress and Cell Pyroptosis.
Te ERC-BiP levels in COVID-19 patients were signifcantly higher than those in healthy controls, while those in the severe group were signifcantly higher than those in the mild group.Moreover, longitudinal comparative analysis found that the temporal diferences of ERC-BiP were signifcantly diferent between the patients in the aggravation and remission groups, and the ERC-BiP levels were signifcantly elevated in aggravation patients.
Proteins misfold during translation under normal conditions.Terefore, abnormal proteins should be timely removed to maintain intracellular homeostasis through ERC-BiP [12].SARS-CoV-2 afects the normal function of ER, resulting in the emergence of several abnormally folded   proteins, which then activate the protective stress state of the ER.Stress is mainly characterized by UPR [9].UPR can promote apoptosis, angiogenesis, autophagy, innate immunity, and proinfammatory efects [6].Terefore, rapid protein folding is crucial for the survival of ER-stressed cells [15].UPR can inhibit protein translation, thus reducing the ER load.However, the upregulation of misfolded protein clearance factors restores ER function [16].Terefore, the SARS-CoV-2-induced stress signal activates the massive release of ERC-BiP to recognize incomplete folding of glycosylated proteins in response to ER stress [9,13], thus preventing the transformation of stress efect from protection to injury.Terefore, ERC-BiP can inhibit the intracellular abnormalities induced by SARS-CoV-2 replication after invasion, thereby delaying the risk of systemic infammation caused by scoria following decompensated accumulation of abnormal proteins.

ERC-BiP Refects ER Stress and Cell Pyroptosis.
Angiotensinogen (AGT) and melatonin are key proteins afecting ER stress.In this study, AGT levels were signifcantly increased in COVID-19 patients and were signifcantly correlated with ERC-BiP.Melatonin levels were signifcantly increased in COVID-19 patients.However, melatonin levels were not signifcantly diferent between mild and severe patients.AGT plays a crucial role in SARS-CoV-2 binding to cell surface sites.Te ACE2 transmembrane protein receptor is the binding site of S-spike protein on the SARS-CoV-2 coat [15,16].Free ACE2 (sACE2) levels are elevated in COVID-19 patients, indicating its mediating function in SARS-CoV-2 invasion progression [17].Terefore, COVID-19 infection is closely related to the reninangiotensin system (RAS).AGT is the rate-limiting substrate of RAS.Cafero et al. showed that AGT enhances SARS-CoV-2 binding to cells, tissues, and organs [18].AGT is upstream of ERC-BiP and acts as a "valve" regulating ER function.ACE2 degrades AGT.Te expression of ACE2 on the membrane surface decreases after SARS-CoV-2 infection [19].Terefore, the decreased AGT conversion rate may increase AGT levels.Te AGT-SACE2 system is associated with susceptibility to COVID-19 [20].Elevated AGT levels may result in overactivation of the downstream Ang II/ AT1R axis [21], thus increasing the risk of MODs and ARDS.However, it is unknown whether the increased AGT level is benefcial or harmful.Wang et al. [17], Hatmal et al. [22], and Cui et al. [23] have extensively studied the RAS pathway.In this study, the expression level of ACE2 was associated with COVID-19 progression.AGT can also control the replication process of the virus in ER.Melatonin acts as another "valve" controlling stress intensity.Melatonin has antioxidant and anti-infammatory efects.It can afect the UPR pathway and reduce ER stress intensity [2,9].In this study, melatonin levels were signifcantly higher in COVID-19 patients than in healthy people.However, the longitudinal time diference analysis showed that melatonin secretion did not have a linear trend, indicating that melatonin release is not associated with the aggravated ER stress state [24][25][26].Te infection process requires cooperation between the virus and the host cell [27].ERC-BiP does not independently play a protective role since substances upstream and downstream also interact with each other.ERC-BiP has both promotive and inhibitory efects, and the overall function of pathways is often diversifed.Although melatonin was not correlated with AGT, external complementary intervention can inhibit ER stress and reduce the adverse efects of abnormal protein accumulation.Many studies have also shown that melatonin supplementation has a clinical value in COVID-19 treatment [28,29].

Functional Evaluation of ER Synthesis and Protein Release.
Cer-SM (ceramide-sphingomyelin), a metabolic pathway produced in ER and closely associated with cell pyrosis, was used to further explore the abnormalities of ER function in COVID-19 patients and the exact role of ERC-BiP.Te twoprotein pathway can be used to evaluate the impact of COVID-19 on ER function and the infammatory state of the body.Clarke et al. [30] indicated that sphingolipids can afect the physical properties of membranes.ER induces membrane rearrangement and self-fusion into two-membrane-vesicle-structured autophagosomes under normal conditions to remove cell debris or pathogens.Te balance of Cer-SM sphingolipid metabolism can maintain the dynamic equilibrium of the ER membrane.However, SARS-CoV-2 inhibits autophagosome and lysosome fusion and the cell clearance mechanism [24].In this study, the Cer-SM pathway analysis showed that increased Cer and SM hydrolysis could lead to the stagnation of the cell cycle.Moreover, the amplifcation of stress signal can lead to pyroptosis [31].Although Cer is widely distributed in the body, omics technology cannot accurately detect the Cer level in ER.However, the increased Cer level negatively afected the alleviating efect of UPR on abnormal protein folding.Terefore, ER can maintain its internal homeostasis by enhancing ERC-BiP even after the invasion of SARS-CoV-2 since ERC-BiP is signifcantly correlated with Cer.4.4.Energy Supply.Te overall level of the TCA cycle decreases in COVID-19 patients while the lactic acid level increases.Protein folding and repair are energyconsuming processes whether ER is in the infected or defense states.SARS-CoV-2 invades the airway epithelium and triggers respiratory symptoms.Tis leads to hypoxia which inhibits the TCA activity, leading to the Warburg efect [32].Pyruvate converts to lactic acid and accumulates in the cell.Lactic acidosis synergistically activates UPR and infammatory response [33], thus affecting the ER membrane activity and protein folding and repair.Furthermore, the correlation between lactic acid and ERC-BiP indicates that energy supply disturbance afects the ER function.
In this study, a functional protein network was established to assess the ER stress state (Figure 4).ERC-BiP was selected as the key pathway protein to evaluate the ER function.Upstream AGT is the key protein essential for the entry of SARS-CoV-2, while downstream melatonin is the ER stress-inhibiting protein.Te two act "valves" regulating pathway activation, thus controlling UPR intensity.Te imbalance of Cer-SM, which is closely associated with membrane function, further indicates that ER dysfunction occurs in COVID-19 patients.TCA circulation is inhibited in COVID-19 patients due to hypoxia and mitochondrial dysfunction.Te accumulation of lactic acid further destroys the ER protein synthesis system and activates the pyroptosis efect.Both virus and infammation eventually result in cytokine release syndrome (CRS), which is the leading cause of multiple organ damage and death in severe COVID-19 patients [34].Over-release of cytokines, injury of respiratory epithelial cells, and accumulation of immune cells lead to a vicious cycle of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) [6].

Relationship between ERC-BiP and HSP70
. BiP is a molecular chaperone of HSP70 located within the endoplasmic reticulum (ER) cavity, which binds to newly synthesized proteins when they are translocated into the ER and maintains them in a state suitable for subsequent folding and oligomerization.BiP is also an important part of the translocation mechanism and plays a role in retrograde transport of abnormal proteins through the ER (which is eventually degraded by the proteasome).BiP is an abundant protein under all growth conditions, but its synthesis is signifcantly induced under conditions that lead to the accumulation of unfolded peptides in ER [35].As an ER molecular chaperone, BiP is also required to introduce peptides into the ER cavity or ER membrane in an ATPdependent manner.ATPase mutants of BiP have been found to block translocation of many proteins (sucrase, carboxypeptidase Y, and factor a) into the endoplasmic network cavity [36][37][38].However, there are no studies to explore the relationship between HSP70 and ERC-BiP in the plasma of COVID-19 patients.Although this study accurately detected the value of ERC-BiP, demonstrating a linear correlation between ERC-BiP and disease progression of COVID-19, the detection of HSP70 may provide another new diagnostic marker for the disease, which needs further research in the future.Canadian Journal of Infectious Diseases and Medical Microbiology

Conclusion
Te ER plays an important role in the activation of infammation and innate immunity since it is the main organelle for SARS-CoV-2 assembly.Terefore, the ER functional correlation pathway analysis is crucial for evaluating the mechanism of COVID-19 progression.In this study, ERC-BiP was used as the central protein of the pathway to evaluate the ER function.ERC-BiP was linearly correlated with disease progression.A functional pathway of AGT/ERC-BiP/glycolysis combined with the operation mode of intracellular metabolic pathways was established to evaluate the stress state of intracellular ER after SARS-CoV-2 invasion.Terefore, this study may provide insights into the mechanism of COVID-19 disease progression.

Figure 1 :
Figure 1: (a) Heatmap showing the levels of diferent proteins/metabolites at admission (left) and the diference between the second measurement and the frst measurement.(b) Te PLS-DA scores plot showing the metabolites and proteins in the healthy control, mild patients, and severe patients (R 2 � 0.779, Q 2 � 0.706, and P < 0.05).(c) Te PLS-DA scores plot showing the diferences of metabolites and proteins in the aggravation group, constant group, and remission group (R 2 � 0.993, Q 2 � 0.978, and P < 0.05).SM: sphingomyelin; CRP: C-reactive protein; AGT: angiotensinogen; ERC-BiP: endoplasmic reticulum companion-BiP; Cer: ceramide acid.

8
2.1.Inclusion Criteria.A total of 46 COVID-19 patients were recruited from the Eighth Hospital of Guangzhou Medical University.Te patients were divided into the mild group (n = 38) (patients with mild clinical symptoms and mild pneumonia on imaging) and the severe group (n = 8) (respiratory rate ≥30 breaths/min, SaO 2 ≤93%, and PaO 2 /FiO 2 ≤300 mmHg at the resting state, imaging indicating a lung exudate lesion area progressing by more than 50% within 24-48 hours, and the necessity for mechanical ventilation, etc.) based on the ninth edition of the COVID-19 guidelines of the National Health Commission of China.

Table 1 :
Basic information of healthy controls and COVID-19 patients.

Table 2 :
Comparative analysis of protein and metabolites between healthy controls and COVID-19 patients.

Table 3 :
Comparative analysis of the temporal fold changes of protein and metabolite between patients in aggravation group and remission group.