Intracellular peptides in SARS-CoV-2-infected patients

Summary Intracellular peptides (InPeps) generated by the orchestrated action of the proteasome and intracellular peptidases have biological and pharmacological significance. Here, human plasma relative concentration of specific InPeps was compared between 175 patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and 45 SARS-CoV-2 non-infected patients; 2,466 unique peptides were identified, of which 67% were InPeps. The results revealed differences of a specific group of peptides in human plasma comparing non-infected individuals to patients infected by SARS-CoV-2, following the results of the semi-quantitative analyses by isotope-labeled electrospray mass spectrometry. The protein-protein interactions networks enriched pathways, drawn by genes encoding the proteins from which the peptides originated, revealed the presence of the coronavirus disease/COVID-19 network solely in the group of patients fatally infected by SARS-CoV-2. Thus, modulation of the relative plasma levels of specific InPeps could be employed as a predictive tool for disease outcome.


INTRODUCTION
Circulating blood plasma connects the various compartments of the organism, promoting tissues/organs crosstalk, and warranting functional organization and accordance of adaptive responses. Human plasma peptide contents have been for decades associated with secreted, short-lived, neuronal and hormonal peptides, such as insulin, glucagon, angiotensin, and bradykinin. Recent advances in high-sensitive electrospray ionization mass spectrometry coupled with high performance nano liquid reversed-phase chromatography (ESI-nLC-MS/MS), allowed us to investigate proteome and peptidome from human fluids in far greater details. 1 Perturbation of the human plasma proteome was shown to persist for up to 6 weeks following the first confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, allowing to track symptoms of severity and antibody responses. 2 Human urine peptidome in SARS-CoV-2-infected patients suggested that urinary peptide profiling generates candidate biomarkers even at early disease stages. 3,4 It is noteworthy to mention that human urine and plasma have almost complete distinctive peptide profiles, suggesting that human plasma has a large pool of resident peptides, maintained by reabsorption in detriment of excretion during renal filtration. [4][5][6] Despite the well-known function of bradykinin and angiotensin 7,8 and fibrinogen-derived peptides, [9][10][11][12][13][14][15] the biological function of most plasma resident peptides remains poorly investigated.
Considering that peptides play key roles in physiology as well as in several diseases, and are frequently employed for medication [16][17][18][19][20] or diagnosis, 4,21 the present report was designed to semi-quantitatively investigate, by using formaldehyde-derived isotope labeling, the human plasma peptidome in SARS-CoV-2non-infected individuals and in SARS-CoV-2-infected patients. The results show variations in the relative levels of specific peptides according to the defined clinical status of the patients (i.e., non-infected vs. SARS-CoV-2-infected with moderate, severe, or fatal infection). Peptides identified in human plasma were 67% derived from intracellular, non-secreted proteins. Plasma peptides identified herein and elsewhere 6,22 were compared with the collection of previously reported urinary peptides. Plasma and urinary peptidomes were distinctive in several biochemical aspects (i.e., precursor proteins, molecular weight, net charge, isoelectric point, hydrophobicity, aliphatic index, and instability index). The present results indicate that human plasma presents a large pool of resident peptides, whose relative levels vary according Table 2 shows peptides with frequency R30% across mass spectrometry runs, and their semi-quantitatively evaluated ratios among groups MC, S/C or D/C. Peptides such as GIFTDQVLSVLKGEE, VESTSNSPSSS, IKERVPDSPSPAPSLEE, and GEGDFLAEGGGVR were identified in more than 50% of SARS-CoV-2-infected patients, and may thus present relevance for diagnosis; these more frequent peptides originated from nine proteins. Fibrinogen gave rise to four peptides, DACH1 yielded three peptides, and DACH2 generated two peptides ( Table 2). The remaining proteins gave rise to only one peptide each (Table 2).
When categorized by sex, fewer peptides seem differentially modulated in patients infected by SARS-CoV-2, as compared to non-infected individuals (Table S5). Shortly, an increase in the peptide GIFTDQVLSVLKGEE occurred in females from groups M/C and S/C, and in males D/C. The peptide VESTSNSPSSS increased only in females, in M/C, S/C, and D/C. Peptide KERVPDSPSPAPSLEE increased in both males and females from groups M/C and S/C, but not in group D/C. The related peptide IKERVPDSPSPAPSLEE increased in both male and female from group M/C, and in males from group S/C, while the shorter peptide VPDSPSPAPSLEE increased only in females from the M/C group. Peptide KERIPESPSPAPSLEE increased in females from group M/C, whereas the peptide DGSDVSK was reduced in males from groups M/C and D/C. Fibrinogen peptide ADSGEGDFLAEGGGVR was increased in females from group S/C, the peptide SGEGDFLAEGGGVR was increased in males from M/C group, while the shorter peptide GEGDFLAEGGGVR appeared increased in males and females from M/C group.
From all 2,466 unique peptides identified herein, 67.3% were considered InPeps as they originated from proteins whose predominant subcellular localization was either cytosolic, nuclear or mitochondrial (Figure 2A, sA); similar results were obtained considering only the most frequent peptides shown on Table 2. A smaller proportion of peptides identified in the human plasma were from proteins preferentially  Figure 2A, sA), or devoted to the secretory pathway (9.91%; Figure 2A, sA).
Considering that one protein can originate more than one peptide, the percentage and the respective subcellular localization of the precursor proteins giving rise to unique peptides were then investigated. Secreted proteins were observed to give rise to the greatest number of plasma peptides per protein (13.60 peptides per precursor protein), while intracellular proteins generated 3.58 peptides per precursor protein, and membrane proteins generate 2.49 peptides per precursor protein.
Comparisons between the present study and previous plasma peptidomics studies 6,22 revealed both similarities and discrepancies ( Figure 2B). From 2,466 peptides presently identified, 6 peptides intersect with Magalhães et al., 6 76 peptide with Parker et al., 22 while 42 peptides were common to all these three mentioned studies ( Figure 2B; Table S6).
The entire pool of plasma peptides identified in this study and previously, 6,22 was compared to the collection of previously reported urinary peptides. 4,6 These data suggested that plasma peptides, compared to urinary peptides, were: (1) significantly lighter ( Figures Figure 3G). Thus, these data suggest the existence of a distinctive large pool of plasma peptides, whose biological significance have not yet been investigated.
Protein-protein interactions (PPI) networks (PIN) were constructed using genes encoding the proteins that originated the differentially expressed peptides identified from groups M/C, S/C, and D/C. These analyses sheds light on the biological significance of differentially regulated peptides, revealing that their enriched terms were commonly associated with SARS-CoV-2 infection (Figure 4). A higher fold enrichment in D/C group compared to M/C or S/C groups was identified for term related to complement and coagulation cascades, and neutrophil extracellular trap formation. Parkinson's disease term was enriched exclusively in S/C group (Figure 4), which could indicate propensity of these patients to develop post-COVID-19 neurological symptoms. The coronavirus disease/COVID-19 term was enriched only in the D/C group ( Figure 4).
Ligand short linear motifs (SLiMs) are functional modules that participate in PPI without requiring stable tertiary structure to accomplish their function. 26 Thus, over-representation analysis (ORA) using SLiMs in differentially regulated peptides evince their possible role in interfering with PPI, and impacting the severity of SARS-CoV-2 infection ( Figure 5). The prediction tool from ELM, a dedicated database and exploratory server for over 300 SLiMs classes, with experimental evidence manually curated from over 3,800 scientific publications, 26 was employed for these analyses.
The ELM tool predicted an average of 5.31 SLiMs per peptide, among 108 (94%) out of 114 (100%) distinct differentially regulated peptides from M/C, S/C, or D/C groups. After filtering the SLiMs to remove duplicates and to keep only those with annotated instances of Homo sapiens from the classes docking or ligand, a significant overrepresentation of the MAPK, NF-kB (NF-kB), phospholipase D, and Ras signaling pathways were identified in all group comparisons. However, T cell receptor and tumor necrosis factor (TNF) Using SLiMs, as a framework to conduct ORA can offer insights into the processes taking place in the body. Indeed, integrins possess the capability to bind to a vast array of ligands harboring SLiMs such as an RGD, 27 LDS or LDI 28 ; the presence of those cells' attachment motifs in spike (S) protein of SARS-CoV-2 (S-SARS-CoV-2) indicates its usage by the virus's cell entry system for pathogenic hijacking. 27 RGD and LDS, but not LDI, peptides were identified multiple times in this study ( Figure 6A); similarly, peptides containing the RGD and LDS motifs were also identified in previous plasma peptidome studies. 6,22 The ratio of peptides containing either RGD or LDS motif was not differentially regulated within any group (data not shown). However, considered as a class of peptides, those containing the RGD motif were increased in S/C and D/C groups, but not in the M/C group ( Figure 6B). Peptides containing the LDS motif were increased in M/C and S/C groups, but not in D/C group ( Figure 6C).

DISCUSSION
One of the major findings of the present report was to reveal in human plasma the presence of a large pool of peptides derived from intracellular protein precursors (i.e., InPeps). The relative levels of InPeps frequently identified in SARS-CoV-2-infected patients was increased, suggesting these to be interesting candidates for disrupting the PIN related to SARS-CoV-2 infection severity. The biological function of the InPeps identified herein remains elusive and deserves further investigation, as prior evidences indicates the potential for both biological [29][30][31][32] and medical 4,16-21,25 relevance of plasma-resident peptides. 6,22,33 InPeps IKERVPDSPSPAPSLEE and KERVPDSPSPAPSLEE, derived from the intracellular protein DACH1, were identified in patients with a frequency of 58% and 33%, respectively; these peptides were significantly increased in SARS-CoV-2-infected patients (i.e., M/C, S/C and D/C). A similar DACH1-derived peptide lacking three N-terminal residues, VPDSPSPAPSLEE, identified with a frequency of 66.67% among all patients, was also increased in SARS-CoV-2-infected patients with moderate or severe symptoms. These data imply that proteolytic processing (i.e., by aminopeptidases) differentially regulates the levels of plasma peptides, according to the severity of COVID-19. It is worth mentioning that InPeps functions correlates with those of the precursor proteins, [34][35][36][37] indicating that InPeps could be contributing to SARS-CoV-2 symptoms playing a role similar to those of their precursor proteins. In line with that, a recent report showed that the protein DACH1 is associated with SARS-Cov-2 infection 38 ; in addition, the expression of DACH1 is relatively increased in patients infected with influenza. 39 DACH1 represses the transcriptional level of matrix metalloprotease 9 (MMP9) by interacting with p65 and c-Jun at the NF-kB and AP-1 binding sites in the MMP9  iScience Article promoter, respectively. 40 MMP9 was also suggested as an early indicator of respiratory failure in patients infected by SARS-Cov-2. 41,42 The association of DACH1 and p65 promotes the recruitment of HDAC1 to the NF-kB binding at the MMP9 promoter, reducing p65 acetylation level and transcriptional activity, and inhibiting the metastasis of breast cancer cells downregulating the expression of MMP9, 40 and reducing the canonical NF-kB-driven inflammation. 43 Plasma peptides reported herein and elsewhere 6,22 have very little overlap to previously reported urinary peptides. 4,6 This fact may indicate that renal clearance selects specific peptides for excretion based on their biochemical characteristics; thereby, enabling the persistence of numerous peptides in the plasma (i.e., human plasma resident peptides). This observation suggests demonstrate a potential undiscovered pivotal role for plasma peptides in human physiology, as well as in disease states as SARS-CoV-2 infection. These distinguishable biochemical characteristics of plasma and urine peptides shown in the present study, provides a rationale for improving the pharmacokinetics of functional rationally design peptides. This could be of interest to improve the half-life of peptides in the plasma, reducing rapid urinary excretion.
The protein from which the peptide originates can provide valuable information about its biological role. [34][35][36][37] Supporting this notion, PIN enrichment analyses revealed coronavirus disease/COVID-19 enrichment in D/C group only. Furthermore, these analyses showed a higher fold enrichment of the PIN terms related to complement and coagulation cascades, and neutrophil extracellular trap formation in patients that died from SARS-CoV-2 infection. Additionally, considering the frequent association of neurological SLiMs are compelling candidates for deciphering the roles played by peptide sequences, suggesting that the biological functions of peptides can be predicted from their SLiMs. 47,48 Specifically, these analyses can reveal enriched terms commonly associated with SARS-CoV-2 infection, underscoring the potential usefulness of SLiMs as a means for comprehending the pathophysiology of this disease. Such functional motifs consist of short linear sequences, typically containing 3-15 residues, 49,50 and can serve as a proxy for peptides' biological activities. In fact, an in-depth exploration through ORA of differentially regulated peptides' SLiMs brought up a large number of enriched terms commonly linked to SARS-CoV-2 infection. 51,52 To spotlight the relationship between findings from SLiMs ORA and SARS-CoV-2 infection, it is noteworthy that in all infected groups, NF-kB, a central player in SARS-CoV-2 cytokine storm, 53 is enriched alongside the MAPK signaling pathway. In circulating immune cells, MAPK is activated during the active phase of SARS-CoV-2 infection, especially in severe cases. 54 Additionally, activation of this pathway could partially be responsible for the increase in platelet activation and aggregation observed with this infection. 55 Considering that Ras serves as the initial module in the MAPK cascade, it is unsurprising to observe its enrichment across all infected groups. Significance of Ras in COVID-19 becomes apparent through evidence showcasing that inhibition of its downstream axis impedes SARS-CoV-2 replication. 56 Another aspect of SLiMs ORA is what distinguishes a fatal from a non-fatal SARS-CoV-2 infection. Viral replication iScience Article can be potently inhibited through selective activation of the TLR3/TLR4-IRF3 pathway. 57 SARS-CoV-2 infected patients with an unfavorable outcome presented lower TLR3 expression and enhanced expression of TLR4, which could be related to the inflammatory response of patients with severe COVID-19. 58 Enrichment of T cell receptor and TNF signaling exclusively in patients who survived suggests that peptides, through their SLiMs, competitively inhibit protein interactions 59,60 that would otherwise trigger extensive T cell activation and TNF signaling, which are associated with poor clinical outcomes. 61,62 Moreover, exclusive enrichment of RNA degradation in individuals who succumbed to the infection can indicate pathogen's exploitation of cellular machinery to accelerate degradation of cytosolic cellular mRNAs, which facilitates viral takeover of the mRNA pool in infected cells. 63,64 The S-SARS-CoV-2, which plays a key role in the receptor recognition binding and cell membrane fusion process, is composed of subunits S1 and S2. The S1 subunit of S-SARS-CoV-2 contains a receptor-binding domain (RBD) that recognizes and binds to the host receptor angiotensin-converting enzyme 2 (ACE2); the S2 subunit of S-SARS-CoV-2 mediates viral cell membrane fusion. 65 The mechanisms surrounding SARS-CoV-2 infection have been widely attributed to ACE2-mediated pathways. 66,67 However, SARS-CoV-2 infection was observed in many extra-pulmonary tissues, such as brain tissue, cardiovascular tissue, and lymphoid tissue, where ACE2 expression was very low. 68 Integrins are a family of a/b heterodimeric cell surface adhesion receptors, which have also been suggested as a possible receptor candidate for SARS-CoV-2 cell infection. 28,65,69 The RBD of S-SARS-CoV-2 has three potential integrin-binding motifs: RGD (Arg403-Gly404-Asp405), LDS (Leu441-Asp442-Ser443) and LDI (Leu585-Asp586-Ile587). 28,65,69 The S-SARS-CoV-2 iScience Article was shown to depend on its integrin RGD motif to elicit vascular leakage events, which can be prevented by the RGD-cyclic peptide compound cilengitide. 70,71 Herein, the relative ratio of InPeps containing RGD or LDS motifs were reported to be increased in patients infected by SARS-CoV-2. Moreover, peptides presenting LDS motif were increased in M/C and S/C groups, but not in D/C group, which may help patients to survive the SARS-CoV-2 infection. On the other hand, binding of plasma RGD peptides to integrins may prevent the binding of S-SARS-CoV-2, thereby leaving it unhindered to interact with ACE2 facilitating a more robust viral cell entry, as previously suggested. 70 Plasma peptides containing RGD motifs, might also regulate the activation of transforming growth factor b, 72 which could lead to tissue fibrosis and augmented coagulation. 73 Therefore, the relative increase in peptides containing the RGD motif in patients from D/C groups may contribute to SARS-CoV-2 fatal effect. Nonetheless, these captivating possibilities warrant further experimental exploration to unveil additional clinical implications of plasma peptides/InPeps.

Limitations of the study
The present study has some important limitations. This was a single-center study. Blood samples from SARS-CoV-2-infected patients from groups M, S, and D were collected early during the first wave of infection. Blood from SARS-CoV-2 non-infected individuals was collected later during the course of the COVID-19 pandemic, with unusual limited clinical information about the patients. There were no available data iScience Article about the vaccinal status or previous SARS-CoV-2 infections from control individuals non-infected by SARS-CoV-2 at the moment of the blood test. Therefore, differences in the relative ratio of human plasma peptides presented herein should be taken as the differences between individuals that: (1) tested positive for SARS-CoV-2 infection by RT-PCR test in nasopharyngeal and throat swabs, and by typical chest computed tomography (CT)-scan findings at the moment of their blood collection; and (2) compared to individuals that tested negative for SARS-CoV-2 infection by RT-PCR test in nasopharyngeal and throat swabs at the moment of their blood collection. Therefore, SARS-CoV-2 derived peptides were identified in blood of individuals that tested either negative or positive for SARS-CoV-2 infection at the moment of blood collection (data not shown). Differences in peptidome caused by distinctive SARS-CoV-2 variants or those caused by vaccination status were beyond the scope of this study. The statistical analyses presented herein have not been further correlated with additional clinical conditions of the patients/individuals, including obesity, diabetes, and/or hypertension, hospitalization period (days), need for mechanical ventilation, or time under mechanical ventilation if needed (days). The limited number of differentially modulated peptides iScience Article according to sex suggests the need for a larger patient cohort to further investigate the modulation of plasma peptidome. Urine and plasma were not simultaneously collected from patients investigated in the present study, which prevented the parallel analysis of plasma and urine peptidome under similar experimental protocols.

Conclusions
SARS-CoV-2 infection perturbs the plasma peptidome. PIN related to SARS-CoV-2 infection was deciphered through peptidome signature. The presence of resident InPeps in human plasma may have implications for health and diseases, and differs from the urinary profile indicating selective renal filtration.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:    80 This step was important because, despite all the care taken to work with samples with the highest possible quality, some contaminants, such as drug vehicles, were still identified in mass spectrometry: when the peak of one of these contaminants coincides with the expected mass time for a labeled peptide, the Mascot algorithm will consider it as the peptide; consequently, later quantitation of the ratio between the labeled peptides will be influenced.
After removing contaminants, data analyses processes continued with the search for peptides using the Mascot search engine. No cleavage site was specified and a fragment ion mass tolerance of G0.5 Da was applied to the MS and MS/MS ions. The search parameters were no enzyme specificity; precursor mass tolerance set to G0.5 Da; no modifications included. The identified peptides were filtered to keep only those in which the ratio between the number of peptide-matched ions and the number of peptide residues minus one were greater than 0.4 (Table S2).

Peptide relative levels and statistical analyses
In isotope-labeled mass spectrometry, the peptide ratio value among groups is indicative of its increase or decrease. We assessed whether the peptide ratios in patient groups (M/C, S/C, and D/C) were significantly different from the null effect of 1 using the One-Sample Wilcoxon Signed Rank Test. All analyses were performed using R (v.4.1). p-values %0.05 were significantly different.

PIN enrichment analysis and SLiMs
Active subnetworks identification in a PIN and the subsequent enrichment analyses were performed using the R library pathfindR (v.1.6.4) 81 with the genes coding the proteins that were precursors of differentially regulated peptides as input. Biogrid was selected as the reference PIN, and KEGG as the reference gene set. Only pathways with at least two genes involved were kept. SLiMs from differentially regulated peptides were identified using the eukaryotic linear motif (ELM) prediction tool. 26 Only SLiMs with Homo sapiens annotated instances from docking or ligand classes were included for further analyses. Enrichment of SLiMs selected through this process was evaluated by calculating their enrichment ORA per KEGG term, using Fisher's hypergeometric test. 82 The reference set for these analyses was the ELM 26 database of SLiMs for Homo sapiens KEGG terms. A p value %0.05 were considered significant. Odds ratio was assumed as fold enrichment.

Multiple sequences alignments
Alignments of peptides containing arginine-glycine-aspartate (RGD) and leucine-aspartic acid-seryne (LDS) SLiMs were performed using the msa interface for ClustalW algorithm. 83 RGD and LDS peptide ratios were assessed using the One-Sample Wilcoxon Signed Rank Test. All analyses were performed using R (v.4.1). p-values %0.05 were significantly different.

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