iProXProteomicsGuangxu MaOvis Arieshttp://www.iprox.org/page/project.html?id=IPX0009677000gxma1@zju.edu.cnZhejiang UniversityiProXParasitic roundworms are remarkable for their ability to manipulate host immune systems and ameliorate inflammatory diseases. Although much is known about the nature of nematode effectors in immune modulation, little is known about the action mode of these molecules. Here, we report that a serine protease inhibitor SPI-I8 in the extracellular vesicles of blood-feeding nematodes like Ancylostoma ceylanicum, Haemonchus contortus and Nippostrongylus brasiliensis, effectively halts excessive inflammatory responses in vitro and in vivo. We demonstrate that H. contortus SPI-I8 promotes the role of a negative regulator of RACK1 and enhances the effects of RACK1 on tumor necrosis factor (TNF)-α-IκB kinases (IKKs)-nuclear factor kappa beta (NF-κB) axis in mammalian cells, by hijacking E3 ubiquitin protein ligase MKRN1-mediated polyubiquitination of RACK1. Administration of recombinant N. brasiliensis SPI-I8 effectively protects mice from dextran sulfate sodium (DSS)-induced colitis and lipopolysaccharide (LPS)-induced sepsis. Considering the structural and functional conservation of SPI-I8s among Strongylida nematodes and the conservation of interactive mediators (i.e., MKRN1 and RACK1) among mammals, our findings provide insights into the host-parasite interface where parasitic roundworms secret molecules to suppress host inflammatory responses. Harnessing these findings should underpin the exploitation of nematode's immunomodulators to relief excessive inflammation associated diseases in animals and humans.Nematode serine protease inhibitor SPI-I8 negatively regulates host NF-κB signalling by hijacking MKRN1-mediated polyubiquitination of RACK1.Wu Fei F, Chen Yanqiong Y, Chen Xueqiu X, Tong Danni D, Zhou Jingru J, Du Zhendong Z, Yao Chaoqun C, Yang Yi Y, Du Aifang A, Ma Guangxu GfalseProteins of sheep abomasum that interact with Hc-SPI-I8Freshly collected abomasum tissue of the naturally infected sheep was ground, filtered, and centrifuged at 3000 g at 4 °C for cell collection. The collected cells were lysed in NP-40 lysis buffer (Thermo Fisher Scientific) for 0.5 h with shaking, then centrifuged at 12000 g at 4 °C for supernatant collection. Coding sequence of Hc-spi-i8 (HCON_00067680) was amplified and inserted into pcDNA3.1(+) vector to produce FLAG-tagged Hc-SPI-I8 in HEK 293T cells. The recombinant Hc-SPI-I8-FLAG protein was isolated from lysed cells using anti-FLAG-conjugated magnetic beads (Bimake, Shanghai, China), and used to isolate and concentrate interactive proteins from the ovine tissue lysis. The isolated proteins were boiled, separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), from which proteins within a range of molecular weights from 30 to 45 kDa were subjected to digestion with trypsin (Promega), desalination with a sep-Pak C18 (Waters) and LC-MS/MS analysis with a Q-Exactive Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Peptides were identified and annotated using Proteome Discoverer v.2.1.Fri Sep 06 00:00:00 BST 2024PXD055614994040032982