Proteomic Approaches to Dissect Host SUMOylation during Innate Antiviral Immune Responses

Abstract

SUMOylation is a highly dynamic ubiquitin-like post-translational modification that is essential for cells to respond to and resolve various genotoxic and proteotoxic stresses. Virus infections also constitute a considerable stress scenario for cells, and recent research has started to uncover the diverse roles of SUMOylation in regulating virus replication, not least by impacting antiviral defenses. Here, we review some of the key findings of this virus-host interplay, and discuss the increasingly important contribution that large-scale, unbiased, proteomic methodologies are making to discoveries in this field. We highlight the latest proteomic technologies that have been specifically developed to understand SUMOylation dynamics in response to cellular stresses, and comment on how these techniques might be best applied to dissect the biology of SUMOylation during innate immunity. Furthermore, we showcase a selection of studies that have already used SUMO proteomics to reveal novel aspects of host innate defense against viruses, such as functional cross-talk between SUMO proteins and other ubiquitin-like modifiers, viral antagonism of SUMO-modified antiviral restriction factors, and an infection-triggered SUMO-switch that releases endogenous retroelement RNAs to stimulate antiviral interferon responses. Future research in this area has the potential to provide new and diverse mechanistic insights into host immune defenses.

Keywords: ISG15; SUMO; TRIM28; endogenous retroelements; influenza; innate immunity; interferon; proteomics; ubiquitin-like modification; virus infection.

Figure 1

The SUMOylation machinery. Small ubiquitin-like modifiers (SUMOs; S in the cartoon) are covalently attached to lysine (K) residues in target proteins through the concerted action of dimeric E1 activating enzymes (SAE1/SAE2) and an E2 conjugating enzyme (UBC9). SUMOylation is usually aided by specific SUMO E3 ligases. SUMO specific proteases (e.g., SENP family members) can de-conjugate SUMO from its targets (known as deSUMOylation) and are also essential for the proteolytic maturation of SUMO precursors by cleaving off C-terminal residues to expose the di-glycine motif that is necessary for conjugation.

Figure 2

The C-terminal amino-acid sequences of ubiquitin and SUMO paralogs. Scissors indicate trypsin cleavage sites. Amino acid remnants that remain on UBL-modified peptides after tryptic digest are highlighted in red.

Figure 3

Strategies for the enrichment of SUMOylated target proteins. Ectopically expressed, epitope-tagged SUMO can be purified using tag-specific antibodies (A) or affinity matrices that bind specific tags (e.g., His6-tag and Ni-NTA under denaturing conditions) (B). Endogenous SUMO can also be enriched by engineered SUMO-traps that consist of multiple SUMO-interacting motifs (SIMs) immobilized onto an affinity matrix (C) or by immunoaffinity purification using SUMO-specific antibodies (D).

Figure 4

MS-based strategies to identify specific SUMOylation sites. (A) His6-SUMO1-T95R or HisSUMO2-T91R modified proteins are purified by Ni-NTA, digested with trypsin, and peptides containing the di-glycine remnant are enriched using a specific α-KεGG antibody. (B) His6-SUMO2 T91K modified proteins are purified by Ni-NTA, digested with Lys-C, and peptides containing the diglycine remnant are enriched using a specific α-KεGG antibody. (C) Proteins modified with His10-SUMO2-K0-Q88R (K0 = all K residues mutated to R) are purified by Ni-NTA and digested with Lys-C. Peptides containing the intact His10-SUMO modification are then enriched by a second Ni-NTA purification step before trypsin digest. (D) His6-SUMO1 Q92R, His6-SUMO2 Q88R and His6-SUMO3 QQ87/88RN modified proteins are purified by Ni-NTA, digested with trypsin, and peptides containing a (X)QTGG remnant are enriched using a specific α-NQTGG antibody. (E) Target proteins modified with endogenous SUMO are digested with Lys-C. Peptides containing a SUMO fragment (with the intact SUMO2/3 antibody epitope) are then immunoaffinity-purified before a second digestion step with Asp-N. (F) Target proteins modified with endogenous SUMO are digested with the endoproteinase WaLP, and peptides containing the di-glycine remnant are enriched using a specific α-KεGG antibody. For all methods (A–F), enriched peptides are analyzed by mass spectrometry (MS) for identification and quantification.

Figure 5

Identification of SUMO-interacting proteins by SUMO-ID. The N-terminal Split-TurboID construct is fused to the SUMO of interest, and the C-terminal fragment is fused to a potentially SUMOylated (or SIM-containing) target. Target SUMOylation, or SUMO-SIM interaction, results in reconstitution of the Split-TurboID and permits biotin labeling of proximal proteins. Biotinylated proteins can then be purified on streptavidin matrices and analyzed by mass spectrometry (MS). Concept developed in [68].

Figure 6

Example of how SUMO proteomics has identified a new type of innate antiviral response. Model of the infection-triggered TRIM28 SUMO-switch identified by proteomics that leads to increased antiviral responses. Influenza A virus (IAV) infection causes loss of SUMOylated TRIM28, destabilizing a multi-protein transcriptional repression complex and permitting endogenous retroelement (e.g., endogenous retrovirus, ERV) release. ERV expression can lead to the formation of endogenous ‘self’ double-stranded (ds) RNA that may be sensed by cellular Pattern Recognition Receptors (PRRs) that normally detect exogenous Pathogen-Associated Molecular Patterns (PAMPs), such as viral dsRNA, and trigger antiviral immunity via IFN production.