Browsing by Author "Skaug, Brian"
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Item Mechanisms Governing NF-κB Regulation by the Anti-Inflammatory Protein A20(2013-05-31) Skaug, Brian; Chen, Zhijian J.; Cobb, Melanie H.; Sternweis, Paul C.; Yarovinsky, FelixA20 is a potent anti-inflammatory protein that inhibits NF-ΚB, and A20 dysfunction is associated with autoimmunity and B-cell lymphoma. A20 harbors a deubiquitination enzyme domain and can employ multiple mechanisms to antagonize ubiquitination upstream of NEMO, a regulatory subunit of the IκB kinase complex (IKK). However, direct evidence of IKK inhibition by A20 is lacking, and the inhibitory mechanism remains poorly understood. Here we show that A20 can directly impair IKK activation without deubiquitination or impairment of ubiquitination enzymes. We find that polyubiquitin binding by A20, which is largely dependent on A20’s 7th zinc finger motif (ZnF7), induces specific binding to NEMO. Remarkably, this ubiquitin-induced recruitment of A20 to NEMO is sufficient to block IKK phosphorylation by its upstream kinase TAK1. Our results suggest a novel mechanism of IKK inhibition and a means by which polyubiquitin chains can specify a signaling outcome.Item The Role of Foxo3 in B Cell Tolerance(2017-01-17) Barrios, Evan; Ottens, Kristina; Hinman, Rochelle; Skaug, Brian; Castrillion, Diego; Satterthwaite, AnneB cells secrete antibodies in order to help defend the body against pathogens. Due to the enormous diversity of B cell receptors required to recognize pathogens, some self-reactive receptors are generated. It is important for the immune system to prevent autoimmune cells from attacking one's self. This can be done at the immature B cell stage through receptor editing, deletion of the B cell, or anergy (inactivation) of the B cell. During editing, the receptor recombines its components in order to generate a new receptor, which is tested for auto-reactivity. If the receptor is strongly auto-reactive after editing, the cell dies by apoptosis. Foxo3 is a transcription factor that participates in pro-apoptotic pathways in several cell types. Previous work in our lab showed that apoptosis is reduced in immature B cells from Foxo3-/- mice, and others have observed decreased levels of Foxo3 in B cells from mouse models of lupus (an autoimmune disease in which B cells produce antibodies reactive against the body's own DNA). It is hypothesized that edited cells that remain auto-reactive may survive inappropriately in the absence of Foxo3. B cells that have undergone receptor editing are more likely to express the Igλ light chain and to have undergone a process called "RS recombination." Foxo3-/- mice were found by flow cytometry to exhibit increased Igλ+ B cells in the immature B cells of the bone marrow, as well as in the transitional B cells of the spleen. PCR demonstrated increased RS recombination in Igλ+ B cells from Foxo3-/- mice compared to wild-type mice. Anti-double-stranded DNA ELISAs were run on supernatants from total B cells stimulated by LPS to secrete antibodies. These showed no difference in auto-reactivity between wild-type mice and Foxo3-/- mice. Thus, there appears to be an increase in receptor editing in the knockout mice, but not an overall increase in the auto-reactivity of the total population of B cells. This suggests that the reduced apoptosis of Foxo3-/- immature B cells allows cells that were originally auto-reactive a longer window of time in which to edit their receptors away from auto-reactivity.