key: cord-0994398-idyzp3os
authors: Chasapis, Christos T.; Georgiopoulou, Athanasia K.; Perlepes, Spyros P.; Bjørklund, Geir; Peana, Massimiliano
title: A SARS-CoV-2 –human metalloproteome interaction map
date: 2021-03-13
journal: J Inorg Biochem
DOI: 10.1016/j.jinorgbio.2021.111423
sha: ddd7aa8f304f0b6ca8c323dee074fe0a7a40b70b
doc_id: 994398
cord_uid: idyzp3os

The recent pandemic caused by the novel coronavirus resulted in the greatest global health crisis since the Spanish flu pandemic of 1918. There is limited knowledge of whether SARS-CoV-2 is physically associated with human metalloproteins. Recently, high-confidence, experimentally supported protein-protein interactions between SARS-CoV-2 and human proteins were reported. In this work, 58 metalloproteins among these human targets have been identified by a structure-based approach. This study reveals that most human metalloproteins interact with the recently discovered SARS-CoV-2 orf8 protein, whose antibodies are one of the principal markers of SARS-CoV-2 infections. Furthermore, this work provides sufficient evidence to conclude that Zn(2+) plays an important role in the interplay between the novel coronavirus and humans. First, the content of Zn-binding proteins in the involved human metalloproteome is significantly higher than that of the other metal ions. Second, a molecular linkage between the identified human Zn-binding proteome with underlying medical conditions that might increase the risk of severe illness from the SARS-CoV-2 virus has been found. Likely perturbations of host cellular metal homeostasis by SARS-CoV-2 infection are highlighted.

Since the beginning of the 21st century, the world has faced severe crises due to deadly viruses such as Ebola, Zika, avian influenza, AIDS, SARS, and MERS, to name a few. The large-scale explosion of these diseases' viral epidemics resulted from the evolution of pre-existing viruses or the appearance of new viral species, causing enormous damage to society in terms of health, economic and social crisis. Since December 2019, another virus named Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) has spread worldwide. The resulting pandemic is severely affecting global healthcare systems. Around a year after the pandemic began, about 110 emerging, and vaccination campaigns for mass immunization are just beginning. Therefore, the search for effective antiviral drugs must continue together with the need to contain the spread of the virus by proper everyday preventive actions following the advice provided by the health authorities, which includes social distancing, wearing a mask, and cleaning the hands), and a rational immunomodulatory approach for host immunity-boosting [3] [4] [5] .

In COVID-19 infection, like other coronavirus infections, SARS-CoV-2 needs to interact with the host cells and replicate its genome [6] . Consequently, virus-host protein-protein interaction (PPI) identification can help us to understand the virus invasion mechanism better and to design proper therapeutic strategies. Experimental and computational studies have revealed PPIs between human targets and SARS-CoV-2 [7, 8] and other viruses [9] [10] [11] involved in important host biological processes. Specifically, SARS-CoV-2 interacts with i) innate immune pathways (the interferon and the NF-κB pathway and E3 ubiquitin ligases that regulate antiviral innate immune signaling: TRIM59 and MIB1), ii) bromodomain proteins (BRD2 and BRD4 members of the extra-terminal (BET) domain family of epigenetic readers that bind to acetylated histones to regulate gene transcription), and iii) hijacks ubiquitination pathways for replication and pathogenesis (interacts with members of a cullin-2 RING E3 ligase complex) [7] .

In this work, metal-binding molecular targets of COVID-19 infection have been identified by applying a systematic structure-based approach, used previously by our group, to propose putative metalloproteomes in various organisms and viruses [11] [12] [13] . Furthermore, possible perturbations in cellular metal homeostasis and molecular linkage between the involved human metalloproteome with several disorders were in silico investigated.

The Centers for Disease Control and Prevention (CDC) provided a list of underlying medical conditions that might increase the risk of severe illness caused by the SARS-CoV-2 virus [2, 21] . All established alleles (at-risk genes) associated with humans disorders were retrieved from the Genome-Wide Association Studies (GWAS) online catalog (https://www.ebi.ac.uk/gwas/) and are shown in Table 1 . Then, the retrieved genetic loci employed were used to identify gene networks using the random walk algorithm with restart, previously described [11] , and the Gene Prioritization and Evidence Collection (GPEC) plug-in of Cytoscape [22] . Using this algorithm, GPEC finds any neighboring interactants of at-risk genes with a topological distance of 1. The genes in the developed networks were defined as the "neighboring genes". Then, each neurological disorder's neighboring genes were individually Venn-analyzed compared to the genes that encode the human targets.

Functional annotation of human targets was performed by the DAVID tool (http://david.abcc.ncifcrf.gov/) [23] . Subcellular localization was predicted based on the UniProt catalog's information (https://www.uniprot.org/) [24] . Their protein abundance information was taken from PaxDb (https://pax-db.org/) [25] .

12.3%, 3%, 1.8%, 0.3%, and 0.3% of the studied human proteome interacting with SARS-CoV-2-proteins contained Zn, Mg, Fe, Cu, and Mn metalloproteins, respectively (Fig. 1) . The complete list of metalloproteins is collected in Supplementary Table S1 and is illustrated in Fig. 2 . The majority of these metalloproteins (17%) interact with accessory protein SARS-CoV-2 orf8, and 8.6% interact with structural membrane protein SARS-CoV-2 M and the non-structural proteins SARS-CoV-2 nsp12 and nsp13 ( Table 2 ). The majority of the human metalloproteins that interact with the viral proteins of SARS-CoV-2 localized in membranes represent 58.36%, followed by those in the cytoplasm (23.6%), nucleus (8.7%), and mitochondria (0.6%). According to the protein abundance database (paxdb.org), the human Mg-binding targets are abundantly expressed compared with those of Fe-, Mn-Zn-, and Cu-binders (Fig. 3) . The most significant molecular function gene ontology terms represented in human metalloproteins refer to binding (85%), catalytic (46%), transporter (7%), and transcription (3%) regulator activity. Venn analysis revealed shared genes between human targeted metalloproteome and certain underlying medical conditions that increase the risk for severe illness from the virus that causes COVID-19. Table 3 .

The human metalloproteome targeted by SARS-CoV-2 proteins has been identified from the experimentally supported, high-confidence, SARS-CoV-2 protein-human protein interactions available in the literature [7] . The results show that the content of Zn-binding proteins (12.3%) in the human proteome is significantly higher than that of the other metal ions, followed by the contents of Mg-, Fe-, Cu-and Mn-binding proteins. Based on the present analysis, the metalloproteome has a notable number of zinc transporters [26] [27] [28] [29] [30] [31] , indicating that the SARS-CoV-2 infection may cause strong intracellular zinc homeostasis disturbances.

Additionally, 27% of the zinc-binding domains of the virus-targeted human metalloproteome are of the zinc fingertype known by many reports [32] [33] [34] [35] [36] to be crucial for the structural stability and catalytic activity of many enzymes [37] . Specifically, four human zinc-binding RING (Really Interesting New Gene) E3 ligases can be identified: E3 ubiquitin-protein ligase MIB, MYCBP2, NRDP1, and RBX1. This result is expected since RING E3s are considered acting as hubs in the immunity-regulating network connecting different signaling pathways or different systems [38] [39] [40] . One of the Mg-binding proteins is the cation-transporting ATPase 13A3 [41] , which is involved in cation transmembrane transfer through channels, indicating that SARS-CoV-2 infection could interfere with metal uptake and efflux, and sensing pathways.

Interestingly, the present study reveals that most metalloproteins identified (17%) interact with the accessory protein SARS-CoV-2 orf8. The orf8 region is poorly conserved among related coronaviruses and is prone to mutations or deletions during interspecies transmission. Although the function of orf8 remains to be elucidated, its structural plasticity and high diversity suggest an important role in SARS-CoV-2 pathogenicity and the virus's ability to spread [42] .

This study shows a significant molecular association between certain underlying medical conditions that might increase the risk for severe illness from the virus that causes COVID-19 and the involved human It is involved in different processes such as regulation of neuronal growth, synaptogenesis, and synaptic plasticity by modulating several signaling pathways, including the p38 MAPK signaling cascade [42] .

ii) Vacuolar protein sorting-associated protein 11 homolog (encoded by the VPS11 gene) plays a role in vesicle-mediated protein trafficking to lysosomal compartments, including the endocytic membrane transport and autophagic pathways [43, 44] . C846G mutation in its RING-H 2 domain causes aberrant ubiquitination and accelerates the turnover of VPS11 protein. Zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrates a significant reduction in central nervous system (CNS) myelination following extensive neuronal death in the hindbrain and midbrain [45] .

iii) Casein kinase II subunit beta (encoded by the CSNK2B gene) is a ubiquitous protein kinase that regulates metabolic pathways, signal transduction, transcription, translation, and replication. The enzyme is localized at the endoplasmic reticulum and the Golgi apparatus [46] . CK2 is present in high levels in the brain, and mutations encoding the α-subunit of CK2 were previously identified in patients with neurodevelopmental disorders and dysmorphic features [47] . iv) NAD-dependent protein deacetylase sirtuin-5, mitochondrial (encoded by the SIRT5 gene) activates CPS1 and contributes to the regulation of blood ammonia levels during prolonged fasting: it acts by mediating desuccinylation and deglutarylation of CPS1, thereby increasing CPS1 activity in response to elevated NAD levels during fasting [48, 49] . SIRT5 plays a significant role in inhibiting mitochondrial ROS levels. SIRT5 alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced nigrostriatal dopaminergic degeneration by suppressing mitochondrial-derived ROS levels. Given the crucial roles of mitochondria in neuronal viability, SIRT5 seems well-positioned to suppress the onset and/or pace of neurodegenerative disease [50] .

Journal Pre-proof v) DNA methyltransferase 1 (encoded by the DNMT1 gene) is an enzyme that transfers a methyl group to cytosine nucleotides of genomic DNA. This protein is the major enzyme responsible for maintaining methylation patterns following DNA replication and shows a preference for hemimethylated DNA. DNMTs may be a potential target for enhancing HNSCC chemotherapy by using inhibitors of DNMTs and reversal of gene methylation [51, 52] . It has been reported that DNMT1 enhances the radiosensitivity of neck squamous cell carcinomas via downregulating SMG1 [53] .

vi) The zinc finger protein 318 (encoded by the ZNF318 gene) was recognized previously as a blood pressure regulator [54] .

The Fe-binding Cytochrome b5 (Cb5) is encoded by the CYB5B gene shared with Parkinson's disease and smoking behavior-related traits. This protein is a membrane-bound hemoprotein functioning as an electron carrier for several membrane-bound oxygenases [55] . Cb5 has a high potential as a biomarker of health and disease in the brain because it regulates metabolic pathways essential to maintain normal neuronal function, like lipid biosynthesis, steroid and xenobiotics metabolism, neuronal bioenergetics, and production of reactive oxygen species. Cb5 is highly expressed in pyramidal neurons of the primary and secondary motor areas of the frontoparietal cerebral cortex, hippocampus, vestibular, reticular, and motor nuclei of the cerebellum and brain stem, and also in Purkinje and granule neurons of the cerebellum cortex [56] . Two Mg-binding proteins are encoded by shared genes with Multiple Sclerosis, Alzheimer's Disease, Parkinson's Disease, Glaucoma, and KIRC: i) integrin beta-1 (encoded by the ITGB1 gene) is a cell surface receptor associated with integrin alpha 1 and integrin alpha 2 to form integrin complexes that function as collagen receptors [57] .

β1 integrin subunit has been implicated in neuronal migration and in the laminar organization of the brain. Formation, maturation, and function of synaptic connections require the engagement of several integrins. Presynaptic β1 integrins serve as counter-receptors for postsynaptic intercellular adhesion molecule-5 (ICAM-5; aka telencephalin) [58] . Integrin beta-1 is mainly expressed in normal cells and in tumor-associated cells, where they control various developing processes, including angiogenesis, tumor progression, apoptosis, and metastasis [59] . Recently, flow cytometry showed that ITGB1 was expressed at high levels in clear cell renal cell carcinoma [60] .

Journal Pre-proof ii) Transforming protein RhoA (encoded by RHOA gene) is a small GTPase protein primarily associated with cytoskeleton regulation, mostly actin stress fibers formation and actomyosin contractility [61] .

Rho GTPases dysregulated in various neurological diseases/disorders show synaptic irregularity, such as

Huntington's Disease, Parkinson's Disease, Amyotrophic Lateral Sclerosis, and Schizophrenia. They are also characterized as key regulators in Alzheimer's disease (AD)-related signals and studied as AD targets [62] . The Cubinding Protein-lysine 6-oxidase is an enzyme encoded by the LOX gene linked to multiple sclerosis and glaucoma.

It catalyzes the conversion of lysine molecules into highly reactive aldehydes that form cross-links in extracellular matrix proteins [63] . Localization of LOX at the CNS has been reported previously. In these studies, LOX is localized at the vascular wall and intracellularly in the brain matrix's cortical and subcortical neurons. Also, LOX is one of the genes known to be up-regulated in Amyotrophic Lateral Sclerosis (ALS) patients [64] .

In this work, the human metalloproteome, which is physically associated with SARS-CoV-2 proteins, has been mined from available experimental data and highlighted for the first time. This work strongly indicates the important role of Zn 2+ in the interplay between COVID-19 and human for two main reasons:

i) The content of Zn-binding proteins (12.3%) is significantly higher than that of the other metal ions.

ii binding to the active-site Cys 145 thiolate would be lethal to enzyme function. Finally, we hope that this study opens the doors for developing metallodrugs as alternatives in the fight against SARS-CoV-2. This is a topic of current intense interest [66, 67] , and the well-established abilities of coordination complexes to inhibit different stages of the replicative cycles of several viruses may be a good starting point. Furthermore, we believe that this type of study could be useful for developing more efficient techniques for the reliable detection of SARS-CoV-2, which is also an area of intense research efforts [68] [69] [70] , in order to control, prevent and therefore reduce its pathogenic spread.

The authors declare no conflict of interest. Table 2 Distribution of the metallo-binding interactors of SARS-CoV-2 proteins. 

An interactive web-based dashboard to track COVID-19 in real time

Interrelations between COVID-19 and other disorders

Reduction of secondary transmission of SARS-CoV-2 in households by face mask use, disinfection and social distancing: a cohort study

Micronutrients as immunomodulatory tools for COVID-19 management

The role of diet and supplementation of natural products in COVID-19 prevention

Coronavirus biology and replication: implications for SARS-CoV-2

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

SARS-CoV-2-human protein-protein interaction network

Human-virus interactome atlas

A Structure-Informed Atlas of Human-Virus Interactions

Interactions between metal binding viral proteins and human targets as revealed by network-based bioinformatics

Vlamis-Gardikas, Identification of the zinc, copper and cadmium metalloproteome of the protozoon Tetrahymena thermophila by systematic bioinformatics

A Model for Manganese interaction with Deinococcus radiodurans proteome network involved in ROS response and defense

A simple protocol for the comparative analysis of the structure and occurrence of biochemical pathways across superkingdoms

Shared gene-network signatures between the human heavy metal proteome and neurological disorders and cancer types

Building Bridges Between Structural and Network-Based Systems Biology

Pfam: The protein families database in 2021

The Protein Data Bank

MetalPDB in 2018: a database of metal sites in biological macromolecular structures

nhmmer: DNA homology search with profile HMMs

GPEC: A Cytoscape plug-in for random walk-based gene prioritization and biomedical evidence collection

DAVID-WS: a stateful web service to facilitate gene/protein list analysis

The UniProt Consortium

Version 4.0 of PaxDb: Protein abundance data, integrated across model organisms, tissues, and cell-lines

The emerging role of zinc transporters in cellular homeostasis and cancer

Current understanding of ZIP and ZnT zinc transporters in human health and diseases

Zinc through the Three Domains of Life

Zip6 Transporter Is an Essential Component of the Lymphocyte Activation Machinery

An essential role for the Zn2+ transporter ZIP7 in B cell development

ZIP9, a novel membrane androgen receptor and zinc transporter protein

A Residue Specific Insight into the Arkadia E3 Ubiquitin Ligase Activity and Conformational Plasticity

NMR-based insights into the conformational and interaction properties of Arkadia RING-H2 E3 Ub ligase

High yield expression and NMR characterization of Arkadia E3 ubiquitin ligase RING-H2 finger domain

Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

Hierarchical core decomposition of RING structure as a method to capture novel functional residues within RING-type E3 ligases: a structural systems biology approach

Recent aspects of the effects of zinc on human health

Assessing the Direct Binding of Ark-Like E3 RING Ligases to Ubiquitin and Its Implication on Their Protein Interaction Network

Comprehensive database of human E3 ubiquitin ligases: application to aquaporin-2 regulation

An integrated bioinformatics platform for investigating the human E3 ubiquitin ligase-substrate interaction network

Comprehensive comparison of two protein family of P-ATPases (13A1 and 13A3) in insects

Evolutionary dynamics of the SARS-CoV-2 ORF8 accessory gene

Recruitment of VPS33A to HOPS by VPS16 Is Required for Lysosome Fusion with Endosomes and Autophagosomes, Traffic

The ERM proteins interact with the HOPS complex to regulate the maturation of endosomes

A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects

Casein kinase 2-dependent serine phosphorylation of MuSK regulates acetylcholine receptor aggregation at the neuromuscular junction

CSNK2B splice site mutations in patients cause intellectual disability with or without myoclonic epilepsy

Sirt5 Is a NAD-Dependent Protein Lysine Demalonylase and Desuccinylase

Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

Functions of the sirtuin deacylase SIRT5 in normal physiology and pathobiology

Zebularine suppresses the apoptotic potential of 5-fluorouracil via cAMP/PKA/CREB pathway against human oral squamous cell carcinoma cells

Epigenetic Modifications in Head and Neck Cancer

DNMT1 Enhances the Radiosensitivity of HPV-Positive Head and Neck Squamous Cell Carcinomas via Downregulating SMG1

The many roles of cytochrome b5

Biochemical and anatomical basis of brain dysfunctions caused by cytochrome b5 reductase deficiency or dysregulation

Integrin-mediated Cell Adhesion to Type I Collagen Fibrils

Integrin adhesion in brain assembly: From molecular structure to neuropsychiatric disorders

β1 integrin modulates tumor growth and apoptosis of human colorectal cancer

Stem/progenitor cell marker expression in clear cell renal cell carcinoma: a potential relationship with the immune microenvironment to be explored

Postzygotic inactivating mutations of RHOA cause a mosaic neuroectodermal syndrome

Rho GTPases as therapeutic targets in Alzheimer's disease, Alzheimer's Res

LOX Mutations Predispose to Thoracic Aortic Aneurysms and Dissections

Up-regulation and altered distribution of lysyl oxidase in the central nervous system of mutant SOD1 transgenic mouse model of amyotrophic lateral sclerosis

Structural stability of the SARS-CoV-2 main protease: Can metal ions affect function?

What is holding back the development of antiviral metallodrugs? A literature overview and implications for SARS-CoV-2 therapeutics and future viral outbreaks

Novel Small-Molecule Scaffolds as Candidates against the SARS Coronavirus 2 Main Protease: A Fragment-Guided in Silico Approach

Detecting the Coronavirus (COVID-19)

Opportunities and Challenges for Biosensors and Nanoscale Analytical Tools for Pandemics: COVID-19

Nano-and biosensors for the detection of SARS-CoV-2: challenges and opportunities