The transcription factor NF-B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein IB␣ maintains NF-B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, IB␣ is rapidly degraded, leading to nuclear translocation of free NF-B. However, NF-B-dependent transcription of the IB␣ gene leads to rapid resynthesis of the IB␣ protein and inhibition of NF-B-dependent transcription. Here we demonstrate a new regulatory function of IB␣ exerted on NF-B in the nuclear compartment. Although normally found in the cytoplasm, IB␣, newly synthesized in response to tumor necrosis factor or interleukin 1, is transported to the nucleus. In the nucleus IB␣ associates with the p50 and p65 subunits of NF-B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of IB␣ correlates with transcription termination of transfected NF-B-dependent luciferase genes. Following the appearance of IB␣ in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-B-IB␣ complexes are cleared from the nucleus.Nuclear factor B (NF-B) is a sequence-specific DNAbinding protein complex which regulates the expression of viral genomes, including the human immunodeficiency virus (HIV), and a wide variety of cellular genes, particularly those involved in immune and inflammatory responses (for a review, see reference 6). NF-B is composed of two polypeptide species with molecular weights of 50,000 (p50) and 65,000 (p65) (7,27). Cloning of the p50 (21, 29) and p65 (40, 44) subunits of the NF-B heterodimer showed that they belong to a multigene family (the rel family) of proteins, all of which are implicated in transcriptionally regulated processes, such as the expression of cytokines, acute-phase response genes, and the determination of the dorsoventral polarity in early insect embryos (for a review, see reference 22).A major component of the regulation of p50-p65 complex activity is the control of the intracellular localization of the heterodimer. Indeed, in most cell types, NF-B is retained in an inactive form in the cytoplasm by the inhibitor protein IB␣ (for a review, see reference 6). Induction of NF-B by extracellular or intracellular stimuli has been demonstrated in a large number of cell types. Thus, it has been shown that following cell stimulation by a number of inducers, such as specific antigen recognition, tumor necrosis factor (TNF), interleukin 1 (IL-1), UV light, phorbol esters, bacterial lipids, oxygen radicals, or DNA and RNA virus infections (for a review, see reference 6), the inhibitory capacity of IB␣ is lost and transcriptionally active NF-B is translocated into the nucleus. IB␣, or MAD-3 (23), belongs to a family of proteins including IB␤ (50), Bcl-3 (41), cactus (18, 28), and the carboxy-terminal region of p105 IB␥ (11,26,33,43), which are c...

SummaryDuring Caenorhabditis elegans oocyte meiosis, a multi-protein ring complex (RC) localized between homologous chromosomes, promotes chromosome congression through the action of the chromokinesin KLP-19. While some RC components are known, the mechanism of RC assembly has remained obscure. We show that SUMO E3 ligase GEI-17/PIAS is required for KLP-19 recruitment to the RC, and proteomic analysis identified KLP-19 as a SUMO substrate in vivo. In vitro analysis revealed that KLP-19 is efficiently sumoylated in a GEI-17-dependent manner, while GEI-17 undergoes extensive auto-sumoylation. GEI-17 and another RC component, the kinase BUB-1, contain functional SUMO interaction motifs (SIMs), allowing them to recruit SUMO modified proteins, including KLP-19, into the RC. Thus, dynamic SUMO modification and the presence of SIMs in RC components generate a SUMO-SIM network that facilitates assembly of the RC. Our results highlight the importance of SUMO-SIM networks in regulating the assembly of dynamic protein complexes.

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of “non-self” nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of “self” double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.

After exposure of cells to tumor necrosis factor (TNF), I kappa B alpha is rapidly degraded by a proteolytic activity that is required for nuclear localization and activation of transcription factor NF-kappa B. To investigate this problem, we have developed a cell-free system to study the degradation of I kappa B alpha initiated in vivo. In this in vitro system, characteristics of endogenous I kappa B alpha degradation were comparable to those observed in vivo. Recombinant I kappa B alpha, when added to lysates from cells exposed to TNF, was specifically degraded by a cellular proteolytic activity; however, it was stable in extracts from unstimulated cells. Inhibition characteristics of the proteolytic activity responsible for I kappa B alpha degradation suggest the involvement of a serine protease. Analysis of mutated forms of I kappa B alpha in the in vitro system demonstrated that an I kappa B alpha species which was unable to interact with NF-kappa B was still efficiently degraded. In contrast, deletion of the C-terminal 61 amino acids from I kappa B alpha rendered the protein resistant to proteolytic degradation. Expression of I kappa B alpha mutated forms in COS-7 cells confirmed the importance of the C-terminal domain for the degradation of the protein in vivo following cell activation. Thus, it is likely that the acidic, negatively charged region represented by the C-terminal 61 amino acids of the protein contains residues critical for TNF-inducible degradation of I kappa B alpha.

SummaryX-linked intellectual disability (XLID) is a heterogeneous syndrome affecting mainly males. Human genetics has identified >100 XLID genes, although the molecular and developmental mechanisms underpinning this disorder remain unclear. Here, we employ an embryonic stem cell model to explore developmental functions of a recently identified XLID gene, the RNF12/RLIM E3 ubiquitin ligase. We show that RNF12 catalytic activity is required for proper stem cell maintenance and neural differentiation, and this is disrupted by patient-associated XLID mutation. We further demonstrate that RNF12 XLID mutations specifically impair ubiquitylation of developmentally relevant substrates. XLID mutants disrupt distinct RNF12 functional modules by either inactivating the catalytic RING domain or interfering with a distal regulatory region required for efficient ubiquitin transfer. Our data thereby uncover a key function for RNF12 E3 ubiquitin ligase activity in stem cell and neural development and identify mechanisms by which this is disrupted in intellectual disability.