key: cord-0797000-fnkdvtwo authors: Süt, Burcu Biterge title: Molecular profiling of immune cell-enriched Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) interacting protein USP13 date: 2020-07-29 journal: Life Sci DOI: 10.1016/j.lfs.2020.118170 sha: 6a8c91573a113b20ba6b5b8589588884ceb80966 doc_id: 797000 cord_uid: fnkdvtwo AIMS: Coronavirus disease 2019 (COVID-19), which is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is a major health concern worldwide. Due to the lack of specific medication and vaccination, drug-repurposing attempts has emerged as a promising approach and identified several human proteins interacting with the virus. This study aims to provide a comprehensive molecular profiling of the immune cell-enriched SARS-CoV-2 interacting protein USP13. MATERIALS AND METHODS: The list of immune cell-enriched proteins interacting with SARS-CoV-2 was retrieved from The Human Protein Atlas. Genomic alterations were identified using cBioPortal. Survival analysis was performed via Kaplan-Meier Plotter. Analyses of protein expression and tumor infiltration levels were carried out by TIMER. KEY FINDINGS: 14 human proteins that interact with SARS-CoV-2 were enriched in immune cells. Among these proteins, USP13 had the highest frequency of genomic alterations. Higher USP13 levels were correlated with improved survival in breast and lung cancers, while resulting in poor prognosis in ovarian and gastric cancers. Furthermore, copy number variations of USP13 significantly affected the infiltration levels of distinct subtypes of immune cells in head & neck, lung, ovarian and stomach cancers. Although our results suggested a tumor suppressor role for USP13 in lung cancer, in other cancers, its role seemed to be context-dependent. SIGNIFICANCE: It is critical to identify and characterize human proteins that interact with SARS-CoV-2 in order to have a better understanding of the disease and to develop better therapies/vaccines. Here, we provided a comprehensive molecular profiling the immune cell-enriched SARS-CoV-2 interacting protein USP13, which will be useful for future studies. The recent pandemic of the coronavirus disease 2019 (COVID- 19) , which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become one of the major health concerns of the 21 st century [1] . The disease has spread to more than 8 million people worldwide, killing over 450.000 individuals [2] . In addition to the shortcomings of public health systems in providing sufficient healthcare to such massive number of patients, another great challenge in fighting the disease has been the lack of specific medication and vaccination. Therefore, drug-repurposing attempts aimed at identification of efficient therapeutic agents for the treatment of COVID-19 has emerged as a promising approach to compensate for this urgent need. In line with this, a recent study has identified 332 human proteins as potential targets for drug repurposing that SARS-CoV-2 interact upon entry into the host cell [3] . As for any other viral infection, a well-coordinated immune response is key to battling the SARS-CoV-2 infection effectively. The innate and the adaptive immune systems of the host cooperate to fight the virus off by inhibiting viral replication and its further spread, through secretion of proinflammatory cytokines, induction of inflammation and activation of cytotoxic (CD8+) and helper (CD4+) T-cells [4, 5] . Furthermore, host cells that are infected with the virus are targeted and cleared by the natural killer (NK) cells, which comprise a subset of innate immune cells [6] . Dendritic cells (DC) are also critical for the elimination of these infections via their antigenpresenting mechanisms [7] . The significance of immune system and immune cells in resolving SARS-CoV-2 infection is further emphasized by studies that suggest immune dysregulation and reduced levels of T-lymphocytes in patients infected with SARS-CoV-2 [8] . Likewise, a significant correlation between the host's immune system response and the severity of the disease has been proposed [9, 10]. Thus, it is of utmost importance to identify and characterize proteins that might play a role in the proper functioning of the immune system during SARS-CoV-2 infection. The list of immune cell-enriched proteins interacting with SARS-CoV-2 identified by Gordon et al. is retrieved from The Human Protein Atlas (https://www.proteinatlas.org) [3, 17] . The Human Protein Atlas offers categorization of proteins according to their mRNA expression levels in specific cell types or compartments, based on RNA-seq data. The shortlist of immune cellenriched SARS-CoV-2 interacting proteins were identified by narrowing down all SARS-CoV-2 interacting proteins by enrichment in immune cells. Mutations and copy number alterations within the genes encoding immune cell-enriched SARS-CoV-2 interacting proteins across different cancer types were identified using The cBio Cancer Genomics Portal (http://cbioportal.org) [18, 19] . The analyses were carried out on all TCGA PanCancer Atlas Studies available, which consisted of 32 studies and a total of 10967 samples. TCGA PanCancer Studies were selected as they provide the latest curated data regarding the dataset. Protein expression levels across all TCGA tumors were analyzed using the DiffExp module of TIMER: Tumor IMmune Estimation Resource (http://timer.cistrome.org/) [20] , which offers differential gene expression data (in log2 TPM-Transcript count Per Million) between tumor and adjacent normal tissues in the form of boxplots. Statistical significance between tumor/normal samples are automatically calculated by the tool using the Wilcoxon test. The effect of USP13 (Affymetrix ID: 226902_at) mRNA expression levels on overall survival was assessed via Kaplan-Meier Plotter (https://kmplot.com/analysis/) [21] . The analyses were run on 1764 breast cancer, 1144 lung cancer, 614 ovarian cancer and 631 gastric cancer patients using default settings. p-values below 0.05 (%5) were considered significant. The effect of somatic copy number alterations (SCNAs) in USP13 gene on tumor infiltration levels of B cells, CD4+ T cells, CD8+ T cells, macrophages, neutrophils and dendritic cells was analyzed using SCNA module of TIMER [20] . The algorithm defines SCNAs based on GISTIC 2.0 and the distributions of different immune cell types are depicted in relation to chromosomal deletions/amplifications. TIMER defines copy number variations on the basis of confidence levels between -2 to 2; as deep deletion (-2), arm-level deletion (-1), diploid/normal (0), arm-level gain (1) , and high amplification (2) . Statistical analysis uses a two-sided Wilcoxon rank-sum test. pvalues below 0.05 (%5) are considered significant. Among all SARS-CoV-2 interacting human proteins, 14 proteins namely F2RL1, FOXRED2, GGH, MARK3, NPTX1, PLD3, SCCPDH, SELENOS, SLC27A2, SMOC1, SPART, TLE3, USP13 and ZNF318 were identified as immune cell-enriched (Table 1) comprising E1, E2 and E3 ligases [23] ; while deubiquitinases (DUBs) are responsible for the removal of these marks [24] . Recently, it was reported that several viral proteins of SARS-CoV-2 interact with human proteins involved in the ubiquitin proteasome system, such as E3 ligases TRIM59 and MIB1, Cullin 2 (CUL2) RING E3 ligase complex, and USP13 [3] . E3 ubiquitin ligases are crucial mediators of activating the host's antiviral immune response (through NF-KB and IFN pathways) [25, 26] and are often taken over by the virus upon infection to facilitate its own replication and pathogenesis [27] . Similarly, USP13 has previously been implicated in downregulation of innate immune response against both DNA and RNA viruses through deubiquitination of a protein called STING [28, 29] . The data in the literature regarding the role of USP13 in carcinogenesis is controversial. Some studies propose that USP13 favors tumor progression and plays an oncogenic role in glioma [30] , melanoma [31] , ovarian cancer [32] and lung cancer [33] [39] . CD8+ T-cell infiltration was associated with better outcome in ovarian cancer, although it was limited only to high grade serous ovarian carcinomas [40, 41] . Head and neck cancer has different subtypes, which show different levels of lymphocyte infiltration; however, T-lymphocytes rather than B-lymphocytes, in particular CD8+ T-cells are linked with improved outcome [42] . Here we showed that copy number variations of USP13 significantly affected the infiltration levels of distinct subtypes of immune cells in HNSC, LUAD, LUSC, OV and STAD. These are also the cancer types, which had the highest levels of USP13 amplifications. Specifically, in LUSC, USP13 amplifications were correlated with higher infiltration of CD4+ T cells, CD8+ T cells, macrophages, neutrophils and dendritic cells, which is associated in the literature with better prognosis [39] . It is also noteworthy to point out that USP13 amplifications did not seem to increase B-cell infiltration in LUSC, which has the opposite effect on disease outcome [38] . Furthermore, the Kaplan-Meier survival analysis showed that higher levels of USP13 expression resulted in improved survival. Therefore, we can claim that immune cell-enriched SARS-CoV-2 interacting protein USP13 might have a tumor suppressor role in lung cancer, especially in LUSC subtype. On the other hand, although USP13 arm-level gains and high amplifications significantly increased lymphocyte infiltration in HNSC, OV and STAD, higher USP13 levels were correlated with shorter survival in ovarian and gastric cancers. One explanation for this unexpected result could be that different analyses might have been run on different datasets for the same cancer type. This is also why we were not able to perform survival analysis on HNSC. The other explanation is that the role of USP13 in cancer is context-dependent, as suggested by others in the literature. 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