A survey of the interactome of Kaposi's sarcoma-associated herpesvirus ORF45 revealed its binding to viral ORF33 and cellular USP7, resulting in stabilization of ORF33 that is required for production of progeny viruses.

UNLABELLED: The ORF45 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus-specific immediate-early tegument protein. Our previous studies have revealed its crucial roles in both early and late stages of KSHV infection. In this study, we surveyed the interactome of ORF45 using a panel of monoclonal antibodies. In addition to the previously identified extracellular regulated kinase (ERK) and p90 ribosomal S6 kinase (RSK) proteins, we found several other copurified proteins, including prominent ones of ∼38 kDa and ∼130 kDa. Mass spectrometry revealed that the 38-kDa protein is viral ORF33 and the 130-kDa protein is cellular USP7 (ubiquitin-specific protease 7). We mapped the ORF33-binding domain to the highly conserved carboxyl-terminal 19 amino acids (aa) of ORF45 and the USP7-binding domain to the reported consensus motif in the central region of ORF45. Using immunofluorescence staining, we observed colocalization of ORF45 with ORF33 or USP7 both under transfected conditions and in KSHV-infected cells. Moreover, we noticed ORF45-dependent relocalization of a portion of ORF33/USP7 from the nucleus to the cytoplasm. We found that ORF45 caused an increase in ORF33 protein accumulation that was abolished if either the ORF33- or USP7-binding domain in ORF45 was deleted. Furthermore, deletion of the conserved carboxyl terminus of ORF45 in the KSHV genome drastically reduced the level of ORF33 protein in KSHV-infected cells and abolished production of progeny virions. Collectively, our results not only reveal new components of the ORF45 interactome, but also demonstrate that the interactions among these proteins are crucial for KSHV lytic replication. IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human cancers. KSHV ORF45 is a multifunctional protein that is required for KSHV lytic replication, but the exact mechanisms by which ORF45 performs its critical functions are unclear. Our previous studies revealed that all ORF45 protein in cells exists in high-molecular-weight complexes. We therefore sought to characterize the interactome of ORF45 to provide insights into its roles during lytic replication. Using a panel of monoclonal antibodies, we surveyed the ORF45 interactome in KSHV-infected cells. We identified two new binding partners of ORF45: the viral protein ORF33 and cellular ubiquitin-specific protease 7 (USP7). We further demonstrate that the interaction between ORF45 and ORF33 is crucial for the efficient production of KSHV viral particles, suggesting that the targeted interference with this interaction may represent a novel strategy to inhibit KSHV lytic replication.

Novel interaction between the co-chaperone Cdc37 and Rho GTPase exchange factor Vav3 promotes androgen receptor activity and prostate cancer growth.

Elevated androgen receptor (AR) activity in castration-resistant prostate cancer may occur through increased levels of AR co-activator proteins. Vav3, a guanine nucleotide exchange factor, is up-regulated following progression to castration resistance in preclinical models and is overexpressed in a significant number of human prostate cancers. Vav3 is a novel co-activator of the AR. We sought to identify Vav3 binding partners in an effort to understand the molecular mechanisms underlying Vav3 enhancement of AR activity and to identify new therapeutic targets. The cell division cycle 37 homolog (Cdc37), a protein kinase-specific co-chaperone for Hsp90, was identified as a Vav3 interacting protein by yeast two-hybrid screening. Vav3-Cdc37 interaction was confirmed by GST pulldown and, for native proteins, by co-immunoprecipitation experiments in prostate cancer cells. Cdc37 potentiated Vav3 co-activation of AR transcriptional activity and Vav3 enhancement of AR N-terminal-C-terminal interaction, which is essential for optimal receptor transcriptional activity. Cdc37 increased prostate cancer cell proliferation selectively in Vav3-expressing cells. Cdc37 did not affect Vav3 nucleotide exchange activity, Vav3 protein levels, or subcellular localization. Disruption of Vav3-Cdc37 interaction inhibited Vav3 enhancement of AR transcriptional activity and AR N-C interaction. Diminished Vav3-Cdc37 interaction also caused decreased prostate cancer cell proliferation selectively in Vav3-expressing cells. Taken together, we identified a novel Vav3 interacting protein that enhances Vav3 co-activation of AR and prostate cancer cell proliferation. Vav3-Cdc37 interaction may provide a new therapeutic target in prostate cancer.

ORF45 of Kaposi's sarcoma-associated herpesvirus inhibits phosphorylation of interferon regulatory factor 7 by IKKε and TBK1 as an alternative substrate.

Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early and tegument protein that plays critical roles in antagonizing host antiviral responses. We have previously shown (Zhu et al, Proc. Natl. Acad. Sci. U. S. A., 99:5573-5578, 2002) that ORF45 suppresses activation of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression, by blocking its virus-induced phosphorylation and nuclear accumulation. We report here further characterization of the mechanisms by which ORF45 inhibits IRF7 phosphorylation. In most cell types, IRF7 is phosphorylated and activated by IKKε and TBK1 after viral infection. We found that phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1 is inhibited by ORF45. The inhibition is specific to IRF7 because phosphorylation of its close relative IRF3 is not affected by ORF45, implying that ORF45 does not inactivate the kinases directly. In fact, we found that ORF45 is phosphorylated efficiently on Ser41 and Ser162 by IKKε and TBK1. We demonstrated that ORF45 competes with the associated IRF7 and inhibits its phosphorylation by IKKε or TBK1 by acting as an alternative substrate.

Locating a lipid at the portal to the lipoxygenase active site.

Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.

In vivo 17ß-estradiol treatment contributes to podocyte actin stabilization in female db/db mice.

We recently showed that 17ß-estradiol (E(2)) treatment ameliorated type 2 diabetic glomerulosclerosis in mice in part by protecting podocyte structure and function. Progressive podocyte damage is characterized by foot process effacement, vacuolization, detachment of podocytes from the glomerular basement membrane, and apoptosis. In addition, podocytes are highly dependent on the preservation of their actin cytoskeleton to ensure proper function and survival. Because E(2) administration prevented podocyte damage in our study on diabetic db/db mice and has been shown to regulate both actin cytoskeleton and apoptosis in other cell types and tissues, we investigated whether actin remodeling and apoptosis were prevented in podocytes isolated from E(2)-treated diabetic db/db mice. We performed G-actin/F-actin assays, Western analysis for Hsp25 expression, Ras-related C(3) botulinum toxin substrate 1 (Rac1) activity, and apoptosis assays on previously characterized podocytes isolated from both in vivo-treated placebo and E(2) female db/db mice. We found that in vivo E(2) protects against a phenotype change in the cultured podocytes characterized by a percent increase of F-actin vs. G-actin, suppression of Hsp25 expression and transcriptional activation, increase of Rac1 activity, and decreased apoptotic intermediates. We conclude from these studies that E(2) treatment protects against podocyte damage and may prevent/reduce diabetes-induced kidney disease.

Mechanism of sustained activation of ribosomal S6 kinase (RSK) and ERK by kaposi sarcoma-associated herpesvirus ORF45: multiprotein complexes retain active phosphorylated ERK AND RSK and protect them from dephosphorylation.

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities ( Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82, 1838-1850 ). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.

Dynamic behavior of fatty acid spin labels within a binding site of soybean lipoxygenase-1.

The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity to position themselves correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin-labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a DeltaDeltaG of -190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin-labeled at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a K(i) of 9 muM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty-acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction.

Role of radical formation at tyrosine 193 in the allene oxide synthase domain of a lipoxygenase-AOS fusion protein from coral.

Coral allene oxide synthase (cAOS), a fusion protein with 8R-lipoxygenase in Plexaura homomalla, is a hemoprotein with sequence similarity to catalases. cAOS reacts rapidly with the oxidant peracetic acid to form heme compound I and intermediate II. Concomitantly, an electron paramagnetic resonance (EPR) signal with tyrosyl radical-like features, centered at a g-value of 2.004-2.005, is formed. The radical is identified as tyrosyl by changes in EPR spectra when deuterated tyrosine is incorporated in cAOS. The radical location in cAOS is determined by mutagenesis of Y193 and Y209. Upon oxidation, native cAOS and mutant Y209F exhibit the same radical spectrum, but no significant tyrosine radical forms in mutant Y193H, implicating Y193 as the radical site in native cAOS. Estimates of the side chain torsion angles for the radical at Y193, based on the beta-proton isotropic EPR hyperfine splitting, A(iso), are theta(1) = 21 to 30 degrees and theta(2) = -99 to -90 degrees. The results show that cAOS can cleave nonsubstrate hydroperoxides by a heterolytic path, although a homolytic course is likely taken in converting the normal substrate, 8R-hydroperoxyeicosatetraenoic acid (8R-HpETE), to product. Coral AOS achieves specificity for the allene oxide formed by selection of the homolytic pathway normally, while it inactivates by the heterolytic path with nonoptimal substrates. Accordingly, with the nonoptimal substrate, 13R-hydroperoxyoctadecadienoic acid (13R-HpODE), mutant Y193H is inactivated after turning over significantly fewer substrate molecules than required to inactivate native cAOS or the Y209F mutant because it cannot absorb oxidizing equivalents by forming a radical at Y193.