Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach

Abstract

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein–protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

Human USP18 protects diverse cancer lineages from Type I Interferon independently of its canonical catalytic function

“…Nonetheless, I60N could disrupt additional functions of USP18 that have yet to be described (Martin-Fernandez et al, 2022; Vasou et al, 2021). For example, recent studies have described additional protein-protein interactions with NEMO in the absence of exogenous IFN and with STAT1, OAS3, MX1, and ROBO1 upon IFN stimulation (Singh et al, 2022; Yang et al, 2015). In leukemia-derived THP-1 monocytic cells, nuclear USP18 interacts with IRF9 and STAT2 to regulate non-canonical ISG expression and pyroptosis (Arimoto et al, 2023).…”

In Vitro Cross-Linking MS Reveals SMG1–UPF2–SMG7 Assembly as Molecular Partners within the NMD Surveillance

“…Detailed understanding of the structure and dynamics of these multimeric and functional entities is critical towards understanding their biological functions. In our previous study [ 21 ]/we cross-linked the Flo-1 treated IFNα14 cancer cells with DSS (disuccinimidyl suberate) and identified crucial PPIs with validating co-immunoprecipitation (Co-IP; Figure 1 A). Detailed parameters about sample preparation and processing in mass spectrometry were described in our previous work [ 21 ].…”

“…Therefore, this suggests that a detailed understanding of the protein–protein network is crucial and investigating the mutational effect on the stability and binding of protein–protein can provide new insights into the molecular mechanisms of this complex process and assist in the development of novel therapeutic approaches. Implementing a cross-linking mass spectrometry (CLMS [ 20 , 21 ]) approach and using the non-cleavable DSS (disuccinimidyl suberate) cross-linker, we identified novel interaction sites within the NMD pathway components SMG1–UPF2–SMG7. The DSS cross-linker, with a spacer arm length of 11.4 Å, undergoes covalent bond formation targeting Lysine (K) or Serine (S) residues [ 20 , 22 ].…”

“…The DSS cross-linker, with a spacer arm length of 11.4 Å, undergoes covalent bond formation targeting Lysine (K) or Serine (S) residues [ 20 , 22 ]. In our previous study [ 21 ], we validated crucial interacting proteins by applying a similar method by co-immunoprecipitation (Co-IP), preferentially often using a cross-linker range of 10–12.5 Å space length for the cleavable (DSS, BS3, etc.) and non-cleavable (DSSO, DSBU, etc.)…”

Type I interferon regulation by USP18 is a key vulnerability in cancer

“…For example, recent studies have described additional protein-protein interactions with NEMO in the absence of exogenous IFN and with STAT1, OAS3, MX1, and ROBO1 upon IFN stimulation. 63 , 64 In leukemia-derived THP-1 monocytic cells, nuclear hUSP18 interacts with IRF9 and STAT2 to regulate non-canonical ISG expression and pyroptosis. 41 It is unknown if these interactions are conserved across cancer cell lines.…”