A structural basis for IκB kinase 2 activation via oligomerization-dependent trans auto-phosphorylation.

Activation of the IκB kinase (IKK) is central to NF-κB signaling. However, the precise activation mechanism by which catalytic IKK subunits gain the ability to induce NF-κB transcriptional activity is not well understood. Here we report a 4 Å x-ray crystal structure of human IKK2 (hIKK2) in its catalytically active conformation. The hIKK2 domain architecture closely resembles that of Xenopus IKK2 (xIKK2). However, whereas inactivated xIKK2 displays a closed dimeric structure, hIKK2 dimers adopt open conformations that permit higher order oligomerization within the crystal. Reversible oligomerization of hIKK2 dimers is observed in solution. Mutagenesis confirms that two of the surfaces that mediate oligomerization within the crystal are also critical for the process of hIKK2 activation in cells. We propose that IKK2 dimers transiently associate with one another through these interaction surfaces to promote trans auto-phosphorylation as part of their mechanism of activation. This structure-based model supports recently published structural data that implicate strand exchange as part of a mechanism for IKK2 activation via trans auto-phosphorylation. Moreover, oligomerization through the interfaces identified in this study and subsequent trans auto-phosphorylation account for the rapid amplification of IKK2 phosphorylation observed even in the absence of any upstream kinase

Interleukin-1 and TRAF6-dependent activation of TAK1 in the absence of TAB2 and TAB3

Interleukin-1 (IL-1) signaling induces the formation of Lys63-linked ubiquitin (K63-Ub) chains, which are thought to activate the “master” protein kinase TAK1 by interacting with its TAB2 and TAB3 subunits. Here, we report that IL-1β can also activate the TAB1-TAK1 heterodimer present in TAB2/TAB3 double knock-out (DKO) IL-1 receptor-expressing cells. The IL-1β-dependent activation of the TAB1- TAK1 heterodimer in TAB2/3 DKO cells required the expression and E3 ligase activity of TRAF6 and was reduced by the siRNA knock-down of Ubc13, an E2 conjugating enzyme that directs the formation of K63-Ub chains. IL-1β signaling was restored to TAB1/2/3 triple KO cells by the re-expression of either TAB1 or TAB2, but not by a ubiquitin-binding-defective mutant of TAB2. We conclude that IL-1β can induce the activation of TAK1 in two ways, only one of which requires the binding of K63-Ub chains to TAB2/3. The early IL-1β-stimulated, TAK1-dependent activation of p38α MAP kinase and the canonical IKK complex, as well as the NF-κBdependent transcription of immediate early genes was similar in TAB2/3 DKO and TAB2/3-expressing cells. However, in contrast to TAB2/3-expressing cells, IL-1β signaling was transient in TAB2/3 DKO cells and the activation of JNK1, JNK2 and p38γ was greatly reduced at all times. These observations indicate a role for TAB2/3 in directing the TAK1-dependent activation of MAP kinase kinases that switch on JNK1/2 and p38γ MAP kinases. These observations and the transient activation of the TAB1-TAK1 heterodimer may explain why IL-1β-dependent IL-8 mRNA formation was abolished in TAB2/3 DKO cells

Lentiviral Vpx Accessory Factor Targets VprBP/DCAF1 Substrate Adaptor for Cullin 4 E3 Ubiquitin Ligase to Enable Macrophage Infection

Vpx is a small virion-associated adaptor protein encoded by viruses of the HIV-2/SIVsm lineage of primate lentiviruses that enables these viruses to transduce monocyte-derived cells. This probably reflects the ability of Vpx to overcome an as yet uncharacterized block to an early event in the virus life cycle in these cells, but the underlying mechanism has remained elusive. Using biochemical and proteomic approaches, we have found that Vpx protein of the pathogenic SIVmac 239 strain associates with a ternary protein complex comprising DDB1 and VprBP subunits of Cullin 4–based E3 ubiquitin ligase, and DDA1, which has been implicated in the regulation of E3 catalytic activity, and that Vpx participates in the Cullin 4 E3 complex comprising VprBP. We further demonstrate that the ability of SIVmac as well as HIV-2 Vpx to interact with VprBP and its associated Cullin 4 complex is required for efficient reverse transcription of SIVmac RNA genome in primary macrophages. Strikingly, macrophages in which VprBP levels are depleted by RNA interference resist SIVmac infection. Thus, our observations reveal that Vpx interacts with both catalytic and regulatory components of the ubiquitin proteasome system and demonstrate that these interactions are critical for Vpx ability to enable efficient SIVmac replication in primary macrophages. Furthermore, they identify VprBP/DCAF1 substrate receptor for Cullin 4 E3 ubiquitin ligase and its associated protein complex as immediate downstream effector of Vpx for this function. Together, our findings suggest a model in which Vpx usurps VprBP-associated Cullin 4 ubiquitin ligase to enable efficient reverse transcription and thereby overcome a block to lentivirus replication in monocyte-derived cells, and thus provide novel insights into the underlying molecular mechanism

Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

Inflammatory osteolysis is governed by exacerbated osteoclastogenesis. Ample evidence points to central role of NF-κB in such pathologic responses, yet the precise mechanisms underpinning specificity of these responses remain unclear. We propose that motifs of the scaffold protein IKKγ/NEMO partly facilitate such functions. As proof-of-principle, we used site-specific mutagenesis to examine the role of NEMO in mediating RANKL-induced signaling in mouse bone marrow macrophages, known as osteoclast precursors. We identified lysine (K)270 as a target regulating RANKL signaling as K270A substitution results in exuberant osteoclastogenesis in vitro and murine inflammatory osteolysis in vivo. Mechanistically, we discovered that K270A mutation disrupts autophagy, stabilizes NEMO, and elevates inflammatory burden. Specifically, K270A directly or indirectly hinders binding of NEMO to ISG15, a ubiquitin-like protein, which we show targets the modified proteins to autophagy-mediated lysosomal degradation. Taken together, our findings suggest that NEMO serves as a toolkit to fine-tune specific signals in physiologic and pathologic conditions

Exposed Hydrophobic Residues in Human Immunodeficiency Virus Type 1 Vpr Helix-1 Are Important for Cell Cycle Arrest and Cell Death

The human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein R (Vpr) is a major determinant for virus-induced G2/M cell cycle arrest and cytopathicity. Vpr is thought to perform these functions through the interaction with partner proteins. The NMR structure of Vpr revealed solvent exposed hydrophobic amino acids along helices 1 and 3 of Vpr, which could be putative protein binding domains. We previously showed that the hydrophobic patch along helix-3 was important for G2/M blockade and cytopathicity. Mutations of the exposed hydrophobic residues along helix-1 were found to reduce Vpr-induced cell cycle arrest and cell death as well. The levels of toxicity during virion delivery of Vpr correlated with G2/M arrest. Thus, the exposed hydrophobic amino acids in the amino-terminal helix-1 are important for the cell cycle arrest and cytopathicity functions of Vpr

The red edge excitation shift phenomenon can be used to unmask protein structural ensembles:implications for NEMO-ubiquitin interactions

To understand complex molecular interactions it is necessary to account for molecular flexibility and the available equilibrium of conformational states. Only a small number of experimental approaches can access such information. Potentially steady-state red edge excitation shift (REES) spectroscopy can act as a qualitative metric of changes to the protein free energy landscape (FEL) and the equilibrium of conformational states. First we validate this hypothesis using a single Trp containing protein, NF-κB essential modulator (NEMO). We provide detailed evidence from chemical denaturation studies, macromolecular crowding studies and the first report of the pressure-dependence of the REES effect. Combined these data demonstrate that the REES effect can report on the 'ruggedness' of the FEL and we present a phenomenological model, based on realistic physical interpretations, for fitting steady-state REES data to allow quantification of this aspect of the REES effect. We test the conceptual framework we have developed by correlating findings from NEMO ligand binding studies with the REES data in a range of NEMO-ligand binary complexes. Our findings shed light on the nature of the interaction between NEMO and poly-ubiquitin, suggesting that NEMO is differentially regulated by poly-ubiqutin chain length and that this regulation occurs via a modulation of the available equilibrium of conformational states, rather than gross structural change. This study therefore demonstrates the potential of REES as a powerful tool for tackling contemporary issues in structural biology and biophysics and elucidates novel information on the structure-function relationship of NEMO and key interaction partners. This article is protected by copyright. All rights reserved.</p

Multi-Scale Modeling of HIV Infection in vitro and APOBEC3G-Based Anti-Retroviral Therapy

The human APOBEC3G is an innate restriction factor that, in the absence of Vif, restricts HIV-1 replication by inducing excessive deamination of cytidine residues in nascent reverse transcripts and inhibiting reverse transcription and integration. To shed light on impact of A3G-Vif interactions on HIV replication, we developed a multi-scale computational system consisting of intracellular (single-cell), cellular and extracellular (multicellular) events by using ordinary differential equations. The single-cell model describes molecular-level events within individual cells (such as production and degradation of host and viral proteins, and assembly and release of new virions), whereas the multicellular model describes the viral dynamics and multiple cycles of infection within a population of cells. We estimated the model parameters either directly from previously published experimental data or by running simulations to find the optimum values. We validated our integrated model by reproducing the results of in vitro T cell culture experiments. Crucially, both downstream effects of A3G (hypermutation and reduction of viral burst size) were necessary to replicate the experimental results in silico. We also used the model to study anti-HIV capability of several possible therapeutic strategies including: an antibody to Vif; upregulation of A3G; and mutated forms of A3G. According to our simulations, A3G with a mutated Vif binding site is predicted to be significantly more effective than other molecules at the same dose. Ultimately, we performed sensitivity analysis to identify important model parameters. The results showed that the timing of particle formation and virus release had the highest impacts on HIV replication. The model also predicted that the degradation of A3G by Vif is not a crucial step in HIV pathogenesis

Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIVmac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIVsmm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts

APOBEC3G-Augmented Stem Cell Therapy to Modulate HIV Replication: A Computational Study

PMC3661658The interplay between the innate immune system restriction factor APOBEC3G and the HIV protein Vif is a key host-retrovirus interaction. APOBEC3G can counteract HIV infection in at least two ways: by inducing lethal mutations on the viral cDNA; and by blocking steps in reverse transcription and viral integration into the host genome. HIV-Vif blocks these antiviral functions of APOBEC3G by impeding its encapsulation. Nonetheless, it has been shown that overexpression of APOBEC3G, or interfering with APOBEC3G-Vif binding, can efficiently block in vitro HIV replication. Some clinical studies have also suggested that high levels of APOBEC3G expression in HIV patients are correlated with increased CD4+ T cell count and low levels of viral load; however, other studies have reported contradictory results and challenged this observation. Stem cell therapy to replace a patient's immune cells with cells that are more HIV-resistant is a promising approach. Pre-implantation gene transfection of these stem cells can augment the HIV-resistance of progeny CD4+ T cells. As a protein, APOBEC3G has the advantage that it can be genetically encoded, while small molecules cannot. We have developed a mathematical model to quantitatively study the effects on in vivo HIV replication of therapeutic delivery of CD34+ stem cells transfected to overexpress APOBEC3G. Our model suggests that stem cell therapy resulting in a high fraction of APOBEC3G-overexpressing CD4+ T cells can effectively inhibit in vivo HIV replication. We extended our model to simulate the combination of APOBEC3G therapy with other biological activities, to estimate the likelihood of improved outcomes.JH Libraries Open Access Fun