TWEAKing death

Smac mimetics (inhibitor of apoptosis [IAP] antagonists) are synthetic reagents that kill susceptible tumor cells by inducing degradation of cellular IAP (cIAP) 1 and cIAP2, nuclear factor κB activation, tumor necrosis factor (TNF) α production, TNF receptor 1 occupancy, and caspase-8 activation. In this issue of The Journal of Cell Biology, Vince et al. (see p. 171) report remarkable similarities in the events leading to tumor cell death triggered by the cytokine TWEAK (TNF-like weak inducer of apoptosis) and IAP antagonists. Although the mechanistic details differ, a common and necessary feature that is also shared by TNF receptor 2 signaling is reduction in the level of cIAP1 and, in some cases, cIAP2 and TNF receptor-associated factor 2. These findings not only extend our appreciation of how cell death pathways are kept in check in tumors, they reinforce the possible utility of induced cIDE (cIAP deficiency) in the selective elimination of neoplastic cells

Activation of the Lck Tyrosine Kinase Targets Cell Surface T Cell Antigen Receptors for Lysosomal Degradation

AbstractThe mechanism by which TCR expression is regulated was explored by expressing a constitutively active form of the tyrosine kinase Lck (Lck505F) in T cells. Expression of Lck505F down-regulated TCR levels, an effect that was even more pronounced in CD45− T cells, in which the activity of this tyrosine kinase is further enhanced. Cells expressing Lck505F synthesized all TCR subunits, but lysosomal degradation of assembled receptors was enhanced. TCRs were rapidly internalized and degraded after removal of a tyrosine kinase inhibitor that had permitted cell surface expression. Finally, TCR levels on thymocytes were increased by an Lck inhibitor, and activation- but not phorbol ester–induced internalization of TCRs on Jurkat cells was prevented by inhibition or loss of Lck. These studies identify a regulated nonreceptor tyrosine kinase–mediated pathway for targeting cell surface receptors for lysosomal degradation

Outer Membrane Vesicles of a Human Commensal Mediate Immune Regulation and Disease Protection

Commensal bacteria impact host health and immunity through various mechanisms, including the production of immunomodulatory molecules. Bacteroides fragilis produces a capsular polysaccharide (PSA), which induces regulatory T cells and mucosal tolerance. However, unlike pathogens, which employ secretion systems, the mechanisms by which commensal bacteria deliver molecules to the host remain unknown. We reveal that Bacteroides fragilis releases PSA in outer membrane vesicles (OMVs) that induce immunomodulatory effects and prevent experimental colitis. Dendritic cells (DCs) sense OMV-associated PSA through TLR2, resulting in enhanced regulatory T cells and anti-inflammatory cytokine production. OMV-induced signaling in DCs requires growth arrest and DNA-damage-inducible protein (Gadd45α). DCs treated with PSA-containing OMVs prevent experimental colitis, whereas Gadd45α^(−/−) DCs are unable to promote regulatory T cell responses or suppress proinflammatory cytokine production and host pathology. These findings demonstrate that OMV-mediated delivery of a commensal molecule prevents disease, uncovering a mechanism of interkingdom communication between the microbiota and mammals

Live Cell Imaging Unveils Multiple Domain Requirements for In Vivo Dimerization of the Glucocorticoid Receptor

Glucocorticoids are essential for life, but are also implicated in disease pathogenesis and may produce unwanted effects when given in high doses. Glucocorticoid receptor (GR) transcriptional activity and clinical outcome have been linked to its oligomerization state. Although a point mutation within the GR DNA-binding domain (GRdim mutant) has been reported as crucial for receptor dimerization and DNA binding, this assumption has recently been challenged. Here we have analyzed the GR oligomerization state in vivo using the number and brightness assay. Our results suggest a complete, reversible, and DNA-independent ligand-induced model for GR dimerization. We demonstrate that the GRdim forms dimers in vivo whereas adding another mutation in the ligand-binding domain (I634A) severely compromises homodimer formation. Contrary to dogma, no correlation between the GR monomeric/dimeric state and transcriptional activity was observed. Finally, the state of dimerization affected DNA binding only to a subset of GR binding sites. These results have major implications on future searches for therapeutic glucocorticoids with reduced side effects.Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Ogara, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Stortz, Martin Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Alvarez, Lautaro Damian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Pooley, John R.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. University of Bristol; Reino UnidoFil: Schiltz, R. Louis. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Grøntved, Lars. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Johnson, Thomas A.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Mittelstadt, Paul R.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ashwell, Jonathan D.. National Cancer Institute. Laboratory of Immune Cell Biology; Estados UnidosFil: Ganesan, Sundar. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados Unidos. National Institute of Allergy and Infectious Diseases; Estados UnidosFil: Burton, Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Unidad de Microanálisis y Métodos Físicos en Química Orgánica. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Unidad de Microanálisis y Métodos Físicos en Química Orgánica; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica; ArgentinaFil: Levi, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Hager, Gordon L.. National Cancer Institute. Laboratory of Receptor Biology and Gene Expression; Estados UnidosFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Two Modes of Transcriptional Activation at Native Promoters by NF-κB p65

The NF-κB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The molecular basis by which NF-κB activates endogenous promoters is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-κB–driven transcription at native promoters, we disrupted the interaction between NF-κB p65 and the Mediator complex. We found that expression of many endogenous NF-κB target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that without p65, native NF-κB target promoters cannot be bound by secondary transcription factors. Artificial recruitment of a secondary transcription factor was able to restore transcription of an otherwise NF-κB–dependent target gene in the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-κB

Enzymatic Blockade of the Ubiquitin-Proteasome Pathway

Ubiquitin-dependent processes control much of cellular physiology. We show that expression of a highly active, Epstein-Barr virus-derived deubiquitylating enzyme (EBV-DUB) blocks proteasomal degradation of cytosolic and ER-derived proteins by preemptive removal of ubiquitin from proteasome substrates, a treatment less toxic than the use of proteasome inhibitors. Recognition of misfolded proteins in the ER lumen, their dislocation to the cytosol, and degradation are usually tightly coupled but can be uncoupled by the EBV-DUB: a misfolded glycoprotein that originates in the ER accumulates in association with cytosolic chaperones as a deglycosylated intermediate. Our data underscore the necessity of a DUB activity for completion of the dislocation reaction and provide a new means of inhibition of proteasomal proteolysis with reduced cytotoxicity.National Institutes of Health (U.S.)EMBO (long term Fellowship 2008-379)Boehringer Ingelheim Fond

Non-Canonical NF-κB Activation and Abnormal B Cell Accumulation in Mice Expressing Ubiquitin Protein Ligase-Inactive c-IAP2

Loss of c-IAP2 ubiquitin ligase activity, which occurs in the lymphoma-causing c-IAP2/MALT1 fusion protein, activates non-canonical NF-κB signaling and results in B cell abnormalities characteristic of MALT lymphoma

[Avian cytogenetics goes functional] Third report on chicken genes and chromosomes 2015

High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them