title: Nucleotide-binding oligomerization domain (NOD) signaling defects and cell death susceptibility cannot be uncoupled in X-linked inhibitor of apoptosis (XIAP)-driven inflammatory disease.
abstract: The X-linked inhibitor of apoptosis (XIAP) protein has been identified as a key genetic driver of two distinct inflammatory disorders, X-linked lymphoproliferative syndrome 2 (XLP-2) and very-early-onset inflammatory bowel disease (VEO-IBD). Molecularly, the role of XIAP mutations in the pathogenesis of these disorders is unclear. Recent work has consistently shown XIAP to be critical for signaling downstream of the Crohn's disease susceptibility protein nucleotide-binding oligomerization domain-containing 2 (NOD2); however, the reported effects of XLP-2 and VEO-IBD XIAP mutations on cell death have been inconsistent. In this manuscript, we describe a CRISPR-mediated genetic system for cells of the myeloid lineage in which XIAP alleles can be replaced with disease-associated XIAP variants expressed at endogenous levels to simultaneously study inflammation-related cell death and NOD2 signaling. We show that, consistent with previous studies, NOD2 signaling is critically dependent on the BIR2 domain of XIAP. We further used this system to reconcile the aforementioned inconsistent XIAP cell death data to show that XLP-2 and VEO-IBD XIAP mutations that exhibit a loss-of-function NOD2 phenotype also lower the threshold for inflammatory cell death. Last, we identified and studied three novel patient XIAP mutations and used this system to characterize NOD2 and cell death phenotypes driven by XIAP. The results of this work support the role of XIAP in mediating NOD2 signaling while reconciling the role of XLP-2 and VEO-IBD XIAP mutations in inflammatory cell death and provide a set of tools and framework to rapidly test newly discovered XIAP variants.
authors.affiliation: From the Departments of Pathology and.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Chirieleison
authors.affiliation: the Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children's Hospital, Cincinnati, Ohio 45229.
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Marsh
authors.affiliation: From the Departments of Pathology and.
authors.forename: Prathna
authors.initials: P
authors.lastname: Kumar
authors.affiliation: From the Departments of Pathology and.
authors.forename: Joseph K
authors.initials: JK
authors.lastname: Rathkey
authors.affiliation: Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106 and.
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
authors.affiliation: From the Departments of Pathology and dwa4@case.edu.
authors.forename: Derek W
authors.initials: DW
authors.lastname: Abbott
mesh_terms: D002460:Cell Line; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008232:Lymphoproliferative Disorders / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D015398:Signal Transduction; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human
keywords: CRISPR/Cas; X-linked inhibitor of apoptosis protein (XIAP); cell death; dual specificity kinase; ubiquitylation (ubiquitination)