9nn9
From Proteopedia
ISG15 complexed with nanobody
Structural highlights
FunctionISG15_HUMAN Ubiquitin-like protein that is conjugated to intracellular target proteins after IFN-alpha or IFN-beta stimulation. Its enzymatic pathway is partially distinct from that of ubiquitin, differing in substrate specificity and interaction with ligating enzymes. ISG15 conjugation pathway uses a dedicated E1 enzyme, but seems to converge with the Ub conjugation pathway at the level of a specific E2 enzyme. Targets include STAT1, SERPINA3G/SPI2A, JAK1, MAPK3/ERK1, PLCG1, EIF2AK2/PKR, MX1/MxA, and RIG-1. Deconjugated by USP18/UBP43. Shows specific chemotactic activity towards neutrophils and activates them to induce release of eosinophil chemotactic factors. May serve as a trans-acting binding factor directing the association of ligated target proteins to intermediate filaments. May also be involved in autocrine, paracrine and endocrine mechanisms, as in cell-to-cell signaling, possibly partly by inducing IFN-gamma secretion by monocytes and macrophages. Seems to display antiviral activity during viral infections.[1] [2] [3] [4] [5] [6] In response to IFN-tau secreted by the conceptus, may ligate to and regulate proteins involved in the release of prostaglandin F2-alpha (PGF), and thus prevent lysis of the corpus luteum and maintain the pregnancy (By similarity).[7] [8] [9] [10] [11] [12] Publication Abstract from PubMedInterferon-induced ubiquitin (Ub)-like modifier Interferon Stimulated Gene 15 (ISG15) functions both intracellularly and as a secreted protein with cytokine-like properties. The ISG15 pathway is implicated in various diseases, including cancer and inflammatory disorders, but understanding its precise roles has been challenging because of limited availability of tools to study ISG15 biology. Here, we report the development of two novel nanobodies that target human ISG15, obtained through alpaca immunization and phage display. These nanobodies, VHH(ISG15-A) and VHH(ISG15-B), exhibit nanomolar binding affinities and recognize distinct epitopes on ISG15's C- and N-terminal domains, respectively, as demonstrated by NMR and X-ray structural analyses. Both nanobodies enable the immunoprecipitation and proteomic identification of ISGylated substrates with minimal background contamination. VHH(ISG15-A) is compatible with immunoblotting and recognizes unconjugated ISG15 under denaturing conditions. Functional assays showed that VHH(ISG15-A), but not VHH(ISG15-B), inhibits ubiquitin-specific peptidase 16-mediated deISGylation, likely by steric hindrance at the ISG15-binding interface. These results underscore the utility of VHH(ISG15-A) and VHH(ISG15-B) as tools to study ISG15 biology. Identification and characterization of nanobodies specific for the human ubiquitin-like ISG15 protein.,Gan J, Dabhade P, Wijne C, McKibben W, Draganov SD, Alrawili H, Sun ZJ, Houghton JW, Tate EW, Le Gall C, Suresh P, Pishesha N, Pinto-Fernandez A, Schwartz TU, Ploegh HL J Biol Chem. 2025 Aug 6;301(9):110564. doi: 10.1016/j.jbc.2025.110564. PMID:40774387[13] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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