MDM2 is a novel E3 ligase for HIV-1 Vif

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug

APOBEC3B can impair genomic stability by inducing base substitutions in genomic DNA in human cells

新規発癌遺伝子アポベック3による新たな発癌機構. 京都大学プレスリリース. 2012-11-14.Human APOBEC3 proteins play pivotal roles in intracellular defense against viral infection by catalyzing deamination of cytidine residues, leading to base substitutions in viral DNA. Activation-induced cytidine deaminase (AID), another member of the APOBEC family, is capable of editing immunoglobulin (Ig) and non-Ig genes, and aberrant expression of AID leads to tumorigenesis. However, it remains unclear whether APOBEC3 (A3) proteins affect stability of human genome. Here we demonstrate that both A3A and A3B can induce base substitutions into human genome as AID can. A3B is highly expressed in several lymphoma cells and somatic mutations occur in some oncogenes of the cells highly expressing A3B. Furthermore, transfection of A3B gene into lymphoma cells induces base substitutions incMYC gene. These data suggest that aberrant expression of A3B can evoke genomic instability by inducing base substitutions into human genome, which might lead to tumorigenesis in human cells

Defining HIV-1 Vif residues that interact with CBFβ by site-directed mutagenesis

AbstractVif is essential for HIV-1 replication in T cells and macrophages. Vif recruits a host ubiquitin ligase complex to promote proteasomal degradation of the APOBEC3 restriction factors by poly-ubiquitination. The cellular transcription cofactor CBFβ is required for Vif function by stabilizing the Vif protein and promoting recruitment of a cellular Cullin5-RING ubiquitin ligase complex. Interaction between Vif and CBFβ is a promising therapeutic target, but little is known about the interfacial residues. We now demonstrate that Vif conserved residues E88/W89 are crucial for CBFβ binding. Substitution of E88/W89 to alanines impaired binding to CBFβ, degradation of APOBEC3, and virus infectivity in the presence of APOBEC3 in single-cycle infection. In spreading infection, NL4-3 with Vif E88A/W89A mutation replicated comparably to wild-type virus in permissive CEM-SS cells, but not in multiple APOBEC3 expressing non-permissive CEM cells. These results support a model in which HIV-1 Vif residues E88/W89 may participate in binding CBFβ

CKIP-1 Is an Intrinsic Negative Regulator of T-Cell Activation through an Interaction with CARMA1

<div><p>The transcription factor NF-κB plays a key regulatory role in lymphocyte activation and generation of immune response. Stimulation of T cell receptor (TCR) induces phosphorylation of CARMA1 by PKCθ, resulting in formation of CARMA1-Bcl10-MALT1 (CBM) complex at lipid rafts and subsequently leading to NF-κB activation. While many molecular events leading to NF-κB activation have been reported, it is less understood how this activation is negatively regulated. We performed a cell-based screening for negative regulators of TCR-mediated NF-κB activation, using mutagenesis and complementation cloning strategies. Here we show that casein kinase-2 interacting protein-1 (CKIP-1) suppresses PKCθ-CBM-NF-κB signaling. We found that CKIP-1 interacts with CARMA1 and competes with PKCθ for association. We further confirmed that a PH domain of CKIP-1 is required for association with CARMA1 and its inhibitory effect. CKIP-1 represses NF-κB activity in unstimulated cells, and inhibits NF-κB activation induced by stimulation with PMA or constitutively active PKCθ, but not by stimulation with TNFα. Interestingly, CKIP-1 does not inhibit NF-κB activation induced by CD3/CD28 costimulation, which caused dissociation of CKIP-1 from lipid rafts. These data suggest that CKIP-1 contributes maintenance of a resting state on NF-κB activity or prevents T cells from being activated by inadequate signaling. In conclusion, we demonstrate that CKIP-1 interacts with CARMA1 and has an inhibitory effect on PKCθ-CBM-NF-κB signaling.</p></div

Lipid rafts accumulated by CD3/CD28 costimulation do not contain CKIP-1.

<p>Jurkat T cells were stimulated for 15-CD3 (10 µg/ml) and anti-CD28 (5 µg/ml), together with 15 µg of mouse IgG. The cells were then lysed and subjected to OptiPrep density gradient centrifugation to isolate lipid rafts. Lysates were subjected to SDS-PAGE and analyzed by Western blotting.</p

PH domain of CKIP-1 is essential not only for the interaction with CARMA1 but also for the inhibitory effect on NF-κB activation.

<p>(A) Jurkat T cells were electroporated with 5 µg of each CKIP-1 truncated form together with 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. Nineteen hours later, cells were stimulated for 5 hr upon PMA (10 ng/ml) or CD3/CD28 (2 µg/ml each). The expressed protein levels were analyzed by Western blotting. (B) Jurkat T cells were electroporated with 5 µg of each CKIP-1 truncated form together with 5 µg of PKCθ AE or Myc-CARMA1, 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. After 24 hr, cells were lysed and luciferase activity was assessed. The expressed protein levels were analyzed by Western blotting. Values represent the average of three independent experiments and error bars represent the SD from the average.</p

CKIP-1 inhibits the interaction between PKCθ and CARMA1.

<p>(A) HEK293T cells were transfected with CKIP-1 or empty vector (mock) together with PKCθ and FLAG-CARMA1 (left panel), or together with HA-Bcl10 and FLAG-CARMA1 (right panel). Cell lysates were immunoprecipitated by anti-FLAG antibody, followed by Western blotting with indicated antibodies. (B) HEK293T cells were transfected with CKIP-1 truncated form together with PKCθ and FLAG-CARMA1. Cell lysates were immunoprecipitated by anti-FLAG antibody, followed by Western blotting with indicated antibodies.</p

Identification of CKIP-1 as a negative regulator in NF-κB activation.

<p>(A) 400 pmol of human CKIP-1-specific siRNA or non-targeting siRNA together with 5 µg of κB-Luc, 0.1 µg of <i>Renilla</i>-Luc were electroporated into Jurkat T cells. Luciferase activity was assayed after 48 hr. The reduction of endogenous CKIP-1 protein levels was analyzed by Western blotting. (B) Jurkat T cells were electroporated with human CKIP-1-specific siRNA or non-targeting siRNA using AMAXA Nucleofector System (Lonza). Thirty hours later, nuclear protein extracts were harvested and NF-κB activity was measured by TransAM NF-κB p65 chemi kit (Active Motif). The reduction of endogenous CKIP-1 protein levels was analyzed by Western blotting. (C) Jurkat T cells were transfected with 5 µg of CKIP-1 or empty vector (mock) together with 5 µg of κB-Luc and 0.1 µg of <i>Renilla</i>-Luc. Nineteen hours later, cells were stimulated for 5 hr upon CD3 (2 µg/ml), CD3/CD28 (2 µg/ml each), TNFα (20 ng/ml), PMA (10 ng/ml) or PMA (10 ng/ml) + CD28 (2 µg/ml). The expressed protein levels were analyzed by Western blotting. (D) Jurkat T cells were transfected with 5 µg of CKIP-1 or empty vector (mock). Twenty-four hours later, cells were stimulated for 30 min upon PMA (10 ng/ml). Then cells were harvested and NF-κB activity was measured by TransAM NF-κB p65 chemi kit. The expressed protein levels were analyzed by Western blotting. Values represent the average of three independent experiments and error bars represent the SD from the average.</p