Proteomic Analysis of Isgylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon
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Article Proteomic Analysis of ISGylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon Chengbo Zhu 1,†, Jingrui Li 2,†, Chaonan Tian 1, Mengmeng Qin 1, Zhenni Wang 1, Bingjun Shi 1,3, Guanggang Qu 4,*, Chunyan Wu 1,* and Yuchen Nan 1,* 1 Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China; [email protected] (C.Z.); [email protected] (C.T.); [email protected] (M.Q.); [email protected] (Z.W.); [email protected] (B.S.) 2 State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193, China; [email protected] 3 Shaanxi Animal Disease Control Center, Xi’an 710000, China 4 Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, China * Correspondence: [email protected] (G.Q.); [email protected] (C.W.); [email protected] (Y.N.) † These authors contributed equally to this article. 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 Citation: Zhu, C.; Li, J.; Tian, C.; Qin, M.; ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine Wang, Z.; Shi, B.; Qu, G.; Wu, C.; Nan, Y. cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify Proteomic Analysis of ISGylation in potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine Immortalized Porcine Alveolar cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation Macrophage Cell Lines Induced by candidates; several candidates contained more than one ISGylation-modifiable lysine residue, includ- Type I Interferon. Vaccines 2021, 9, ing pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate 164. https://doi.org/10.3390/vaccines9 bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene 020164 Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates Academic Editor: Giampiero Girolomoni 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, Received: 7 January 2021 ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates Accepted: 13 February 2021 within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics anal- Published: 17 February 2021 ysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: interferons; interferons-stimulated genes (ISGs), ISG15; ISGylation; ubiquitination published maps and institutional affil- iations. 1. Introduction Interferons (IFNs), a group of secreted cytokines that play key roles in host antiviral im- Copyright: © 2021 by the authors. munity, are defined by their abilities to inhibit virus replication in vitro and in vivo. So far, Licensee MDPI, Basel, Switzerland. three types of IFNs (type I, II and III) have been characterized [1]. Type I IFNs, the most This article is an open access article well-known IFN type, constitute the largest of the three IFN families [2] and include IFN-α, distributed under the terms and IFN-β, IFN-", IFN-κ and IFN-! [3,4]. All type I IFNs bind to a ubiquitously expressed conditions of the Creative Commons heterodimeric receptor complex composed of IFN-α receptors IFNAR1 and IFNAR2 to Attribution (CC BY) license (https:// activate Janus kinase and signal transducer and activator of transcription (JAK/STAT) creativecommons.org/licenses/by/ pathways. The Type II IFN family, which is solely comprised of IFN-γ, is distinct from type 4.0/). Vaccines 2021, 9, 164. https://doi.org/10.3390/vaccines9020164 https://www.mdpi.com/journal/vaccines Vaccines 2021, 9, 164 2 of 14 I IFNs, due to its different receptors and distinct chromosomal location [2,5]. IFN-γ signal transduction depends on participation of a broadly expressed IFN-γ receptor complex (IFNGR) [6–8]. IFN-γ plays a central role in establishing cellular immunity, while also inducing expression of a specific group of antiviral genes which apparently also respond to type I IFNs as well [9,10]. The Type III IFN family, the most recently discovered family, is comprised of IFN-λs 1 through 4 [5,11]. IFN-λ signaling is mediated by a heterodimeric receptor composed of IFNLR1 (or IL-28R) and IL-10R2 that is quite distinct from receptors triggered by type I and type II IFNs. Nevertheless, IFN-λ activates the same downstream pathways as those triggered by type I IFNs and induces expression of similar downstream genes. Thus, all IFNs stimulate cells via binding of their corresponding receptors to trigger JAK/STAT pathway activation [12] that subsequently evokes a sequential cascade of events leading to increased expression of IFN-stimulated genes (ISGs) [13]. Generally, more than 300 ISGs have been shown to be upregulated by type I IFNs [14]. IFN-stimulated gene product 15 (ISG15) was identified soon after the discovery of ubiquitin and is recognized as a ubiquitin homologue [15]. Mature human ISG15 protein is shorter than its precursor, due to the removal of 8 C-terminal amino acids, and retains a shared C-terminal amino acid motif LRLRGG with mature ubiquitin [16], which is capable of undergoing conjugation with protein substrates. However, unlike other ubiquitin homo- logues, such as small ubiquitin-like modifier (SUMO) or neural precursor cell-expressed developmentally down-regulated gene 8 (Nedd8), ISG15 orthologues have not been de- tected in yeast, insects, plants or other lower organisms, implying that ISG15 performs a highly specialized function in vertebrates [17]. Moreover, sequence analysis has demon- strated that ISG15 is not conserved among mammalian species, suggesting that ISG15 plays distinct species-specific antiviral roles in different hosts [18]. As a ubiquitin-like molecule, ISG15 can be conjugated to protein substrates through a reversible process known as ISGylation [15]. While it has been demonstrated that protein ISGylation does not lead to substrate degradation, as occurs after K48-linked ubiquitination, no universal role of protein ISGylation has been identified. Indeed, it is still unclear whether ISGylated proteins share a common fate similar to ubiquitination or if fates of individual ISGylated proteins vary [19,20]. Nevertheless, ISG15 expression is strongly upregulated after IFN treatment, supporting an important role for ISG15 in the host innate immune response. Moreover, it has been reported that ISG15-deficient mice exhibit increased susceptibility to influenza A and B viruses, Sindbis virus and HSV-1 [21,22]. Early studies using proteomics-based approaches have also identified 76 total ISGylation candidates in IFNs-treated mouse and human cells, with only 21 of these putative targets overlapping between species [23], evidence that ISG15-conjugated substrates may be species-specific as well. However, natural substrates of porcine ISG15 have not yet been investigated, due to the lack of antibodies that specifically bind to porcine ISG15. In our previous report, we described development of a novel monoclonal antibody (Mab) that specifically recognized endogenous porcine ISG15 (pISG15) produced by IFNs- stimulated porcine cells but did not cross-react with ISG15 from other species [24]. Here we utilized this Mab to capture pISG15-conjugated substrates from IFNs-treated porcine cells then conducted proteomics analyses of potential targets of porcine ISGylation to reveal antiviral roles played by ISG15-conjugated proteins in porcine cells. Our data here demonstrated that ISG15 and ISG15-conjugated targets were mainly associated with host metabolic pathways, while several antiviral proteins, such as MX1, IFIT1, OAS1 and ISG15, were also identified as ISGylation candidates. In summary, our data reveal a novel function of ISG15 in porcine cells that is tied to cellular antiviral responses. 2. Material and Methods 2.1. Cells and Chemicals Immortalized porcine alveolar macrophage cell line 3D4/21 ATCC® CRL-2843 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Biological Industries, Vaccines 2021, 9, 164 3 of 14 Israel) supplemented with 10% FBS (Biological Industries). Recombinant porcine IFN-α (pIFN-α) was purchased from R&D Systems (Minneapolis, MN, USA) and used to treat CRL-2843 cells at a concentration of 10 ng/mL. 2.2. Western Blot (WB) Analysis Whole cell lysates of CRL-2843 cells with or without pIFN-α treatment were pre- pared by addition to cells of 1× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA). Lysate