[Abstract unavailable

The inactivation of the prototype NF-B inhibitor, IB␣, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. We identify valosincontaining protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, that co-precipitates with IB␣ immune complexes. The ubiquitinated IB␣ conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-B. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IB␣ complexes sediment in the 19 S fractions, while the unmodified IB␣ sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IB␣ are critical for VCP binding, which in turn is necessary but not sufficient for IB␣ degradation; while the N-terminal domain of IB␣ is required in all three reactions, both N-and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. Our results suggest that VCP may provide a physical and functional link between IB␣ and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IB␣.Transcription factor NF-B is involved in a large variety of processes, such as cell growth, transcriptional regulation, immune, inflammatory, and stress responses (reviewed in Refs. 1-4). NF-B is a homo-or heterodimer consisting of various combinations of the family members, including NFB1 (p50 and precursor p105), c-Rel, RelA, NFB2 (p52 and precursor p100), RelB, and Drosophila proteins Dorsal and Dif. Unlike many other transcription factors that are localized in the nucleus, the NF-B dimeric factor is sequestered in the cytoplasm of most cells through binding to a family of inhibitor proteins, IB. In response to extracellular stimuli, the inhibitors are partially or entirely degraded, thus liberating the DNA-binding dimer for translocation to the nucleus. The I6B family contains IB␣, IB␤, IB␥, Bcl-3, the precursor proteins p105 and p100, and the Drosophila protein Cactus. All members of the IB family contain 5-8 ankyrin motifs, thought to be involved in protein-protein interactions. It has been shown that when the precursor protein p105 is involved as the inhibitor, the processing from p105 to the active p50 occurs through the ubiquitindependent proteasome (Ub-Pr) 1 pathway, which degrades the C-terminal ankyrin-containing domain of p105 (5, 6).2 For the prototype complex that contains p50, p65, and IB␣, upon stimulation the entire NF-B complex becomes hyperphosphorylated. The induced phosphorylation of IB␣ does not lead to its immediate dissociation from the complex; rather, it signals for rapid IB␣ degradation, thus liberating the active p50⅐p65 dimer for translocation to the nucleus (7-15). We and others showed that the degradation of IB␣ is sensitive to proteasome inhibitors and is ubiquitin-dependent. Recently, it was further shown that signal-ind...

Cell cycle progression is monitored by checkpoint mechanisms that ensure faithful duplication and accurate segregation of the genome. Defects in spindle assembly or spindle-kinetochore attachment activate the mitotic checkpoint. Once activated, this checkpoint arrests cells prior to the metaphase-anaphase transition with unsegregated chromosomes, stable cyclin B, and elevated M phase promoting factor activity. However, the mechanisms underlying this process remain obscure. Here we report that upon activation of the mitotic checkpoint, MAD2, an essential component of the mitotic checkpoint, associates with the cyclin B-ubiquitin ligase, known as the cyclosome or anaphase-promoting complex. Moreover, purified MAD2 causes a metaphase arrest in cycling Xenopus laevis egg extracts and prevents cyclin B proteolysis by blocking its ubiquitination, indicating that MAD2 functions as an inhibitor of the cyclosome. Thus, MAD2 links the mitotic checkpoint pathway to the cyclin B destruction machinery which is critical in controlling the metaphase-anaphase transition.

The innate antiviral response is mediated, at least in part, by Toll-like receptors (TLRs). TLR3 signaling is activated in response to viral infection, and the absence of TLR3 in mice significantly increases mortality after infection with enteroviruses that cause myocarditis and/or dilated cardiomyopathy. We screened TLR3 in patients diagnosed with enteroviral myocarditis/cardiomyopathy and identified a rare variant in one patient as well as a significantly increased occurrence of a common polymorphism compared with controls. Expression of either variant resulted in significantly reduced TLR3-mediated signaling after stimulation with synthetic double-stranded RNA. Furthermore, Coxsackievirus B3 infection of cell lines expressing mutated TLR3 abrogated activation of the type I interferon pathway, leading to increased viral replication. TLR3-mediated type I interferon signaling required cellular autophagy and was suppressed by 3-methyladenine and bafilomycin A1, by inhibitors of lysosomal proteolysis, and by reduced expression of Beclin 1, Atg5, or microtubule-associated protein 1 light chain 3␤ (MAP1LC3␤). However, TLR3-mediated signaling was restored upon exogenous expression of Beclin 1 or a variant MAP1LC3␤ fusion protein refractory to RNA interference. These data suggest that individuals harboring these variants may have a blunted innate immune response to enteroviral infection, leading to reduced viral clearance and an increased risk of cardiac pathology.Virus-induced myocarditis is an important cause of morbidity and mortality (1, 2). The enteroviruses have been considered the most common etiologic agents; however, other viruses, such as the adenoviruses, have also been implicated (3). The advent of molecular hybridization and PCR techniques has directly demonstrated infection of the myocardium with these viruses and has provided evidence of a viral etiology for dilated cardiomyopathy (DCM) 5 (1, 3). These findings further support the hypothesis that DCM is in some cases a long term sequela of acute or chronic myocarditis (4, 5), either due to the pathogenic effect of persistent viral replication or to an ongoing autoimmune process secondary to the viral infection.The recognition of viruses or viral particles by the host triggers the activation of an innate immune response that is characterized by the production of mediators such as tumor necrosis factor-␣, interleukins, interferons, and nitric oxide, all of which are toxic to replicating viruses (6). This initial antiviral response by the host is now known to be mediated at least in part by Toll-like receptors (TLRs) (5, 6). It has been shown that double-stranded (ds) RNA is the primary ligand for TLR3 (7). Although most TLR signaling pathways utilize the adaptor molecule MyD88 (myeloid differentiation primary response gene 88), TLR3 signals through the adaptor molecule called Toll-like receptor adaptor molecule 1 (Ticam1) or Toll/Interleukin 1 receptor domain-containing adapter-inducing interferon ␤ (TRIF) (8, 9). Activation of this pathway triggers produ...