NK4/IL-32 is a novel type of GEF for Rap1, specifically tailored to inflammatory conditions. We discovered NK4 during a screening of human endothelial cells upon activation of NF-kB. We showed NK4 was induced by inflammatory cytokines, and it is a positive regulator that propagates/amplifies vascular inflammation. However, regarding the identity of this protein has a storied history as complex as its biology. It was originally cloned from activated NK cells, and subsequently rediscovered many years later as IL-32, although it shares no structural similarity with any known interleukin nor does it has a secretory signal peptide. No receptor has been identified despite efforts by many labs. Several labs including ours have reported that NK4 is an intracellular protein that binds with lipid. Recently, we demonstrate NK4/IL-32 is actually a novel GEF for Rap1 and created by retrotransposon. Therefore, our study clarifies a confusion in the field. A central goal of biology is to understand the evolution of new genes and species. Natural selection and random DNA mutation are ubiquitous. But speciation occurs in rapid bursts with complex changes in linked protein networks that are difficult to reconcile with a process driven by uncorrelated genetic changes. Large assemblies of genes have no apparent similarity with other vertebrates, clouding interpretations of ancestry and interaction. Transposable elements and endogenous retroviruses offer a resolution of this paradox. Their genetic information encodes pre-existing interactions with the host and other transposons, and their exaptation is at the root of the development of whole systems, such as the placenta and adaptive immunity. Retrotransposons now account for approximately one-third of the human genome. In this study, we reveal that the NK4 gene is created by retrotransposon in evolution. NK4 represents a novel type of GEF for Rap. Although the active site of NK4 exhibits sequence homology to other Rap GEFs, the remainder of the enzyme is notably distinct. Importantly, NK4 is induced by inflammatory cytokines. Hence, it may have evolved to allow a distinct pathway to Rap1 activation, specifically tailored to inflammatory conditions, thereby bridges the network between inflammation/infection and the activity of small GTPases. Furthermore, we performed a serious of studies to identify novel NK4 interactants and found that NK4 is constitutively ubiquitinated. Moreover, NK4 was co-translationally ubiquitinated via a splicing regulated ribosome fusion protein UBA52. Intriguingly, ubiquitination of NK4 did not promote degradation. Rather, ubiquitination was required to traffic NK4 to the cell's sorting center, the mutivesicular body or MVB. Our results indicate that a genetically encoded motif within NK4 induces co-translational ubiquitination and NK4 is the first factor that is specifically ubiquitinated by a ribosome fusion protein. That may be particularly crucial for a GEF protein like NK4. The uncontrolled activation of small GTPases is a powerful initiator of tumorigenesis. Co-translational ubiquitination restricts the activity of NK4 to a small portion of the cell and may limit its interaction with substrate. Consistent with the role of Rap1 activation, we show that overexpression of NK4 promotes the development of autoimmune disorders. Autoimmune disorders are the third most common diseases in the United States, and affect the daily lives of millions of people. In this study, we analyzed patient samples, utilized a transgenic mouse model and human B cells to reveal IL-32 as a novel regulator that promotes the development of autoimmune disorders. IL-32 was significantly elevated in samples from patients with Sjogren's Syndrome (SS). The serum levels of IL-32 positively correlated with the duration of the disease. Interestingly, transgenic expression of IL-32 in a mouse model led to the development of autoantibodies and lymphocytic infiltration in salivary glands similar to those in SS patients. Those phenotypes were associated with increased B1a cells in the peritoneum, plasma cells in the spleen, and increased IgM, IgA, and IgG2a in serum of the IL-32 transgenic mice. The autoimmune phenotypes became more severe in older mice. Consistent with women having much higher incidence of developing autoimmune conditions than men, the majority of the autoimmune IL-32 mice were female. Remarkably, estrogen increased the production of IL-32, thus providing a mechanistic link to the high tendency of females to develop autoimmune disorders. Moreover, IL-32 efficiently stimulated CD19+ human B cell differentiation into IgG and IgA-plasmablasts, resulting in an increased production of autoantibodies. IL-32 also promoted B cell migration in a paracrine fashion through an induction of CXCL13 in endothelial cells, and activated B cells through T cell-dependent and -independent pathways. Collectively, these findings identify IL-32 as a promoter of the development of autoimmune disorders through its effect on B cells. Thus IL-32 may be a novel therapeutic target for the treatment of autoimmune diseases.
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