Proteomic Analysis of ISGylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon.

Chengbo Zhu,Guanggang Qu,Jingrui Li,Mengmeng Qin,Zhenni Wang,Bingjun Shi,Chaonan Tian,Yuchen Nan,Chunyan Wu

doi:10.3390/vaccines9020164

Abstract

Interferon-stimulated gene product 15 (ISG15), a ubiquitin-like molecule, can be conjugated to protein substrates through a reversible process known as ISGylation. ISG15 and ISGylation are both strongly upregulated by type I interferons and play putative key roles in host innate immunity against viral infection. However, the function of ISGylation and identities of ISGylation substrates are largely unknown. Here, a novel monoclonal antibody (Mab) that specifically recognizes porcine ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation candidates; several candidates contained more than one ISGylation-modifiable lysine residue, including pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates were mainly associated with the host metabolism, especially nucleotide metabolic pathways that ultimately may participate in cellular antiviral defenses. Notably, several ISGs (MX1, IFIT1, OAS1, ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics analysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses.

Highlights

Interferon-stimulated gene product 15 (ISG15)-antagonistic function effect of porcine reproductive and respiratory syndrome vivirus (PRRSV) non-structural protein 2 (NSP 2) ovarian tumor (OTU) protease domain rus (PRRSV) non-structural protein 2 (NSP 2) ovarian tumor (OTU) protease domain proproduced a recombinant virus with reduced virulence in vivo [34]
Host ISG15 may conjugate to viral proteins during different stages of viral replication in a virusmay conjugate to viral proteins during different stages of viral replication in a virus-spespecific manner [35,36], but no common structural or primary motif associated with ISG15 cific manner [35,36], but no common structural or primary motif associated with ISG15 conjugation to viral protein as well [31]
Gene Ontology (GO) annotation and enrichment analyses, along with protein-protein interaction (PPI) network analysis of these proteins, demonstrated that ISGylation occurring within porcine cells mainly impacted host metabolic pathways

Summary

Introduction

Interferons (IFNs), a group of secreted cytokines that play key roles in host antiviral immunity, are defined by their abilities to inhibit virus replication in vitro and in vivo. Three types of IFNs (type I, II and III) have been characterized [1]. Type I IFNs, the most well-known IFN type, constitute the largest of the three IFN families [2] and include IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω [3,4]. All type I IFNs bind to a ubiquitously expressed heterodimeric receptor complex composed of IFN-α receptors IFNAR1 and IFNAR2 to activate Janus kinase and signal transducer and activator of transcription (JAK/STAT)

Methods

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Discussion

Conclusion