ABSTRACT
Constitutive activation of the canonical NF-κB signaling pathway is a major factor in Kaposi's sarcoma-associated herpes virus pathogenesis where it is essential for the survival of primary effusion lymphoma. Central to this process is persistent upregulation of the inhibitor of κB kinase (IKK) complex by the virally encoded oncoprotein vFLIP. Although the physical interaction between vFLIP and the IKK kinase regulatory component essential for persistent activation, IKKγ, has been well characterized, it remains unclear how the kinase subunits are rendered active mechanistically. Using a combination of cell-based assays, biophysical techniques, and structural biology, we demonstrate here that vFLIP alone is sufficient to activate the IKK kinase complex. Furthermore, we identify weakly stabilized, high molecular weight vFLIP-IKKγ assemblies that are key to the activation process. Taken together, our results are the first to reveal that vFLIP-induced NF-κB activation pivots on the formation of structurally specific vFLIP-IKKγ multimers which have an important role in rendering the kinase subunits active through a process of autophosphorylation. This mechanism of NF-κB activation is in contrast to those utilized by endogenous cytokines and cellular FLIP homologues.
Subject(s)
Herpesvirus 8, Human , Sarcoma, Kaposi , Enzyme Activation/genetics , Herpesvirus 8, Human/genetics , Herpesvirus 8, Human/metabolism , Humans , I-kappa B Kinase/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Oncogene Proteins/metabolism , Sarcoma, Kaposi/enzymology , Sarcoma, Kaposi/virology , Viral Proteins/metabolismABSTRACT
Primary effusion lymphoma (PEL) is a lymphogenic disorder associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection. Key to the survival and proliferation of PEL is the canonical NF-κB pathway, which becomes constitutively activated following overexpression of the viral oncoprotein KSHV vFLIP (ks-vFLIP). This arises from its capacity to form a complex with the modulatory subunit of the IκB kinase (IKK) kinase, IKKγ (or NEMO), resulting in the overproduction of proteins that promote cellular survival and prevent apoptosis, both of which are important drivers of tumorigenesis. Using a combination of cell-based and biophysical assays together with structural techniques, we showed that the observed resistance to cell death is largely independent of autophagy or major death receptor signaling pathways and demonstrated that direct targeting of the ks-vFLIP-IKKγ interaction both in cells and in vitro can be achieved using IKKγ-mimetic peptides. Our results further reveal that these peptides not only induce cell killing but also potently sensitize PEL to the proapoptotic agents tumor necrosis factor alpha and etoposide and are the first to confirm ks-vFLIP as a tractable target for the treatment of PEL and related disorders.IMPORTANCE KSHV vFLIP (ks-vFLIP) has been shown to have a crucial role in cellular transformation, in which it is vital for the survival and proliferation of primary effusion lymphoma (PEL), an aggressive malignancy associated with infection that is resistant to the majority of chemotherapeutic drugs. It operates via subversion of the canonical NF-κB pathway, which requires a physical interaction between ks-vFLIP and the IKK kinase modulatory subunit IKKγ. While this interaction has been directly linked to protection against apoptosis, it is unclear whether the suppression of other cell death pathways implicated in ks-vFLIP pathogenesis is an additional contributor. We demonstrate that the interaction between ks-vFLIP and IKKγ is pivotal in conferring resistance to apoptosis. Additionally, we show that the ks-vFLIP-IKKγ complex can be disrupted using peptides leading to direct killing and the sensitization of PEL cells to proapoptotic agents. Our studies thus provide a framework for future therapeutic interventions.
Subject(s)
Apoptosis , Herpesvirus 8, Human/physiology , I-kappa B Kinase/chemistry , Peptides/metabolism , Peptides/pharmacology , Sarcoma, Kaposi/virology , Autophagy , Etoposide/pharmacology , Herpesvirus 8, Human/chemistry , Humans , I-kappa B Kinase/metabolism , Jurkat Cells , Molecular Mimicry , Peptides/chemistry , Protein Binding , Sarcoma, Kaposi/physiopathology , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Viral Proteins/metabolismABSTRACT
Onset of the lytic phase in the KSHV life cycle is accompanied by the rapid, global degradation of host (and viral) mRNA transcripts in a process termed host shutoff. Key to this destruction is the virally encoded alkaline exonuclease SOX. While SOX has been shown to possess an intrinsic RNase activity and a potential consensus sequence for endonucleolytic cleavage identified, the structures of the RNA substrates targeted remained unclear. Based on an analysis of three reported target transcripts, we were able to identify common structures and confirm that these are indeed degraded by SOX in vitro as well as predict the presence of such elements in the KSHV pre-microRNA transcript K12-2. From these studies, we were able to determine the crystal structure of SOX productively bound to a 31 nucleotide K12-2 fragment. This complex not only reveals the structural determinants required for RNA recognition and degradation but, together with biochemical and biophysical studies, reveals distinct roles for residues implicated in host shutoff. Our results further confirm that SOX and the host exoribonuclease Xrn1 act in concert to elicit the rapid degradation of mRNA substrates observed in vivo, and that the activities of the two ribonucleases are co-ordinated.
Subject(s)
Herpesvirus 8, Human/chemistry , RNA-Binding Proteins/chemistry , RNA/chemistry , SOXB1 Transcription Factors/chemistry , Crystallography, X-Ray , Gene Expression , Herpesvirus 8, Human/genetics , Host-Pathogen Interactions/genetics , Humans , Life Cycle Stages/genetics , Protein Conformation , RNA, Messenger/genetics , SOXB1 Transcription Factors/geneticsABSTRACT
Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/ß subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKß is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site.
Subject(s)
Herpesvirus 8, Human/metabolism , I-kappa B Kinase/chemistry , I-kappa B Kinase/metabolism , Sarcoma, Kaposi/enzymology , Viral Proteins/metabolism , Amino Acid Motifs , Binding Sites , Electron Spin Resonance Spectroscopy , Herpesvirus 8, Human/chemistry , Herpesvirus 8, Human/genetics , Humans , I-kappa B Kinase/genetics , Protein Binding , Sarcoma, Kaposi/genetics , Sarcoma, Kaposi/virology , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
In conformational diseases, native protein conformers convert to pathological intermediates that polymerize. Structural characterization of these key intermediates is challenging. They are unstable and minimally populated in dynamic equilibria that may be perturbed by many analytical techniques. We have characterized a forme fruste deficiency variant of α(1)-antitrypsin (Lys154Asn) that forms polymers recapitulating the conformer-specific neo-epitope observed in polymers that form in vivo. Lys154Asn α(1)-antitrypsin populates an intermediate ensemble along the polymerization pathway at physiological temperatures. Nuclear magnetic resonance spectroscopy was used to report the structural and dynamic changes associated with this. Our data highlight an interaction network likely to regulate conformational change and do not support the recent contention that the disease-relevant intermediate is substantially unfolded. Conformational disease intermediates may best be defined using powerful but minimally perturbing techniques, mild disease mutants, and physiological conditions.
Subject(s)
Epitopes/genetics , Models, Molecular , Protein Conformation , Proteostasis Deficiencies/genetics , alpha 1-Antitrypsin/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Polymerization , Proteostasis Deficiencies/pathology , alpha 1-Antitrypsin/geneticsABSTRACT
The intrinsic propensity of α(1)-antitrypsin to undergo conformational transitions from its metastable native state to hyperstable forms provides a motive force for its antiprotease function. However, aberrant conformational change can also occur via an intermolecular linkage that results in polymerization. This has both loss-of-function and gain-of-function effects that lead to deficiency of the protein in human circulation, emphysema and hepatic cirrhosis. One of the most promising therapeutic strategies being developed to treat this disease targets small molecules to an allosteric site in the α(1)-antitrypsin molecule. Partial filling of this site impedes polymerization without abolishing function. Drug development can be improved by optimizing data on the structure and dynamics of this site. A new 1.8 Å resolution structure of α(1)-antitrypsin demonstrates structural variability within this site, with associated fluctuations in its upper and lower entrance grooves and ligand-binding characteristics around the innermost stable enclosed hydrophobic recess. These data will allow a broader selection of chemotypes and derivatives to be tested in silico and in vitro when screening and developing compounds to modulate conformational change to block the pathological mechanism while preserving function.
Subject(s)
alpha 1-Antitrypsin/chemistry , Crystallography, X-Ray , Humans , Models, Molecular , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The early lytic phase of Kaposi's sarcoma herpesvirus infection is characterized by viral replication and the global degradation (shutoff) of host mRNA. Key to both activities is the virally encoded alkaline exonuclease KSHV SOX. While the DNase activity of KSHV SOX is required for the resolution of viral genomic DNA as a precursor to encapsidation, its exact involvement in host shutoff remains to be determined. We present the first crystal structure of a KSHV SOX-DNA complex that has illuminated the catalytic mechanism underpinning both its endo and exonuclease activities. We further illustrate that KSHV SOX, similar to its Epstein-Barr virus homologue, has an intrinsic RNase activity in vitro that although an element of host shutoff, cannot solely account for the phenomenon.
Subject(s)
DNA/chemistry , Exodeoxyribonucleases/chemistry , Herpesvirus 8, Human/enzymology , Viral Proteins/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography , DNA/metabolism , DNA-Binding Proteins/chemistry , Endodeoxyribonucleases/chemistry , Endodeoxyribonucleases/metabolism , Models, Molecular , Molecular Sequence Data , Ribonucleases/metabolism , Sequence Alignment , Viral Proteins/metabolismABSTRACT
The common Z mutant (Glu342Lys) of alpha(1)-antitrypsin results in the formation of polymers that are retained within hepatocytes. This causes liver disease whilst the plasma deficiency of an important proteinase inhibitor predisposes to emphysema. The Thr114Phe and Gly117Phe mutations border a surface cavity identified as a target for rational drug design. These mutations preserve inhibitory activity but reduce the polymerisation of wild-type native alpha(1)-antitrypsin in vitro and increase secretion in a Xenopus oocyte model of disease. To understand these effects, we have crystallised both mutants and solved their structures. The 2.2 A structure of Thr114Phe alpha(1)-antitrypsin demonstrates that the effects of the mutation are mediated entirely by well-defined partial cavity blockade and allows in silico screening of fragments capable of mimicking the effects of the mutation. The Gly117Phe mutation operates differently, repacking aromatic side chains in the helix F-beta-sheet A interface to induce a half-turn downward shift of the adjacent F helix. We have further characterised the effects of these two mutations in combination with the Z mutation in a eukaryotic cell model of disease. Both mutations increase the secretion of Z alpha(1)-antitrypsin in the native conformation, but the double mutants remain more polymerogenic than the wild-type (M) protein. Taken together, these data support different mechanisms by which the Thr114Phe and Gly117Phe mutations stabilise the native fold of alpha(1)-antitrypsin and increase secretion of monomeric protein in cell models of disease.
Subject(s)
alpha 1-Antitrypsin/chemistry , Amino Acid Substitution , Animals , COS Cells , Chlorocebus aethiops , Crystallography, X-Ray , Drug Design , Female , Hepatocytes/metabolism , Humans , In Vitro Techniques , Models, Molecular , Mutation, Missense , Oocytes/metabolism , Protein Conformation , Protein Folding , Protein Structure, Quaternary , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity , Xenopus , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/metabolismABSTRACT
UvrB is the damage recognition element of the highly conserved UvrABC pathway that functions in the removal of bulky DNA adducts. Pivotal to this is the formation of a damage detection complex that relies on the ability of UvrB to locate and sequester diverse lesions. Whilst structures of UvrB bound to DNA have recently been reported, none address the issue of lesion recognition. Here, we describe the crystal structure of UvrB bound to a pentanucleotide containing a single fluorescein-adducted thymine that reveals a unique mechanism for damage detection entirely dependent on the exclusion of lesions larger than an undamaged nucleotide.
Subject(s)
DNA Damage , DNA Helicases/metabolism , Endodeoxyribonucleases/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Models, Molecular , Oligodeoxyribonucleotides/metabolism , Crystallography, X-Ray/methods , DNA Adducts/chemistry , DNA Adducts/metabolism , DNA Helicases/chemistry , Endodeoxyribonucleases/chemistry , Escherichia coli Proteins/chemistry , Oligodeoxyribonucleotides/chemistry , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The UvrABC pathway is a ubiquitously occurring mechanism targeted towards the repair of bulky base damage. Key to this process is UvrB, a DNA-dependent limited helicase that acts as a lesion recognition element whilst part of a tracking complex involving UvrA, and as a DNA-binding platform required for the presentation of damage to UvrC for subsequent processing. We have been able to determine the structure of a ternary complex involving UvrB* (a C-terminal truncation of full-length UvrB), a polythymine trinucleotide and ADP. This structure has highlighted the roles of key conserved residues in DNA binding distinct from those of the beta-hairpin, where most of the attention in previous studies has been focussed. We are also the first to report the structural basis underlying conformational re-modelling of the beta-hairpin that is absolutely required for DNA binding and how this event results in an ATPase primed for catalysis. Our data provide the first insights at the molecular level into the transformation of UvrB into an active helicase.
Subject(s)
Adenosine Triphosphatases/chemistry , Bacillus subtilis/enzymology , Bacillus/enzymology , Bacterial Proteins/chemistry , DNA Helicases/chemistry , Protein Structure, Tertiary , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Bacillus/genetics , Bacillus subtilis/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Crystallography, X-Ray , DNA/chemistry , DNA/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Repair , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , Thermus thermophilus/enzymology , Thermus thermophilus/geneticsABSTRACT
Both 8oxo-guanine and formamidopyrimidines are major products of oxidative DNA damage that can result in the fixation of transversion mutations following replication if left unrepaired. These lesions are targeted by the N-DNA glycosylase hOgg1, which catalyses excision of the aberrant base followed by cleavage of the phosphate backbone directly 5' to the resultant abasic site in a context, dependent manner. We present the crystal structure of native hOgg1 refined to 2.15 A resolution that reveals a number of highly significant conformational changes on association with DNA that are clearly required for substrate recognition and specificity. Changes of this magnitude appear to be unique to hOgg1 and have not been observed in any of the DNA-glycosylase structures analysed to date where both native and DNA-bound forms are available. It has been possible to identify a mechanism whereby the catalytic residue Lys 249 is "primed" for nucleophilic attack of the N-glycosidic bond.