Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response

Temporal profiling of the coding and noncoding murine cytomegalovirus transcriptomes

The global transcriptional program of murine cytomegalovirus (MCMV), involving coding, noncoding, and antisense transcription, remains unknown. Here we report an oligonucleotide custom microarray platform capable of measuring both coding and noncoding transcription on a genome-wide scale. By profiling MCMV wild-type and immediate-early mutant strains in fibroblasts, we found rapid activation of the transcriptome by 6.5 h postinfection, with absolute dependency on ie3, but not ie1 or ie2, for genomic programming of viral gene expression. Evidence is also presented to show, for the first time, genome-wide noncoding and bidirectional transcription at late stages of MCMV infection

TRAF6 regulates proliferation and differentiation of skeletal myoblasts

We could recently demonstrate an important role of receptor interacting protein-2 (RIP2), an activator of nuclear factor kappa B (NF-\u3baB) and a target of activated receptors of the tumor necrosis factor receptor (TNFR) type, in myogenic differentiation and regeneration. Here, we analyze a potential role of TNFR associated factor 6 (TRAF6), which also associates with the cytoplasmic domain of TNFR type, but also IL-1-R and TLR type receptors, and activates NF-\u3baB, in these processes. Specifically, we show that during myogenic differentiation in vitro, traf6 gene expression is downregulated in normal myoblasts, but not in rhabdomyosarcoma cells, suggesting a role of the TRAF6 protein in this process. Inhibition of traf6 expression using specific siRNAs led to an inhibition of both myoblast proliferation and differentiation, whereas inhibition of the TRAF6 effector NF-\u3baB alone in our system only blocked proliferation. Finally, we demonstrate that the traf6 gene is downregulated in skeletal muscle tissue of the dystrophic mdx mouse. Taken together, these data argue for a role of TRAF6 in the regulation of skeletal muscle differentiation and regeneration. \ua9 2010 International Society of Differentiation

Zinc Inhibition of Bacterial Cytochrome bc1 Reveals the Role of Cytochrome b E295 in Proton Release at the Qo Site

The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 +/- 0.2 and 3.1 +/- 0.4 μM, respectively), whereas their KI values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2\ufe. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Q o site of cyt bc1