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Although antibodies targeting specific tumor-expressed antigens are the standard of care for some cancers, the identification of cancer-specific targets amenable to antibody binding has remained a bottleneck in development of new therapeutics. To overcome this challenge, we developed a high-throughput platform that allows for the unbiased, simultaneous discovery of antibodies and targets based on phenotypic binding profiles. Applying this platform to ovarian cancer, we identified a wide diversity of cancer targets including receptor tyrosine kinases, adhesion and migration proteins, proteases and proteins regulating angiogenesis in a single round of screening using genomics, flow cytometry, and mass spectrometry. In particular, we identified BCAM as a promising candidate for targeted therapy in high-grade serous ovarian cancers. More generally, this approach provides a rapid and flexible framework to identify cancer targets and antibodies.

Tumor necrosis factor receptor (TNFR) superfamily members mediate the cellular response to a wide variety of biological inputs. The responses range from cell death, survival, differentiation, proliferation, to the regulation of immunity. All these physiological responses are regulated by a limited number of highly pleiotropic kinases. The fact that the same signaling molecules are involved in transducing signals from TNFR superfamily members that regulate different and even opposing processes raises the question of how their specificity is determined. Regulatory strategies that can contribute to signaling specificity include scaffolding to control kinase specificity, combinatorial use of several signal transducers, and temporal control of signaling. In this review, we discuss these strategies in the context of TNFR superfamily member signaling.

The Vpr accessory protein of HIV-1 induces a response similar to that of DNA damage. In cells expressing Vpr, the DNA damage sensing kinase, ATR, is activated, resulting in G 2 arrest and apoptosis. In addition, Vpr causes rapid degradation of the uracil-DNA glycosylases UNG2 and SMUG1. Although several cellular proteins have been reported to bind to Vpr, the mechanism by which Vpr mediates its biological effects is unknown. Using tandem affinity purification and mass spectrometry, we identified a predominant cellular protein that binds to Vpr as the damage-specific DNA-binding protein 1 (DDB1). In addition to its role in the repair of damaged DNA, DDB1 is a component of an E3 ubiquitin ligase that degrades numerous cellular substrates. Interestingly, DDB1 is targeted by specific regulatory proteins of other viruses, including simian virus 5 and hepatitis B. We show that the interaction with DDB1 mediates Vpr-induced apoptosis and UNG2/SMUG1 degradation and impairs the repair of UV-damaged DNA, which could account for G 2 arrest and apoptosis. The interaction with DDB1 may explain several of the diverse biological functions of Vpr and suggests potential roles for Vpr in HIV-1 replication.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr has previously been shown to bind to the cellular uracil DNA glycosylase UNG. We show here that the binding of Vpr to UNG and to the related enzyme SMUG induces their proteasomal degradation. UNG and SMUG were found to be encapsidated in Δ vpr HIV-1 virions but were significantly less abundant in vpr + virions. Δ vpr virions contained readily detectable uracil-DNA glycosylase enzymatic activity, while the activity was reduced to undetectable levels in vpr + virions. Consistent with proteasomal degradation, complexes that contained Vpr and the E3 ubiquitin ligase components Cul1 and Cul4 were detected in cell lysates. We hypothesized that the interaction of Vpr might be a means for the virus to reduce the frequency of abasic sites in viral reverse transcripts at uracil residues caused by APOBEC3-catalyzed deamination of cytosine residues. Although APOBEC3 is largely neutralized by the Vif accessory protein, residual enzyme could remain in virions that would generate uracils. In support of this, Δ vif vpr + HIV-1 produced in the presence of limited amounts of APOBEC3G was significantly more infectious than Δ vif Δ vpr virus. In Addition, vpr + HIV-1 replicated more efficiently than vpr − virus in cells that expressed limited amounts of APOBEC3G. The findings highlight the importance of cytidine deamination in the virus replication cycle and present a novel function for Vpr.

The virion infectivity factor (Vif) accessory protein of HIV-1 forms a complex with the cellular cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) to block its antiviral activity. The antiviral property of APOBEC3G is conserved in several mammalian species, but the ability of Vif to block this activity is species-specific. HIV-1 Vif blocks human APOBEC3G but does not block the mouse or African green monkey (AGM) enzyme. Conversely, SIV AGM Vif blocks the antiviral activity of AGM but not human APOBEC3G. We demonstrate that the species specificity is caused by a single amino acid difference in APOBEC3G. Replacement of Asp-128 in human APOBEC3G with the Lys-128 of AGM APOBEC3G caused the enzyme to switch its interaction, becoming sensitive to SIV AGM Vif and resistant to HIV-1 Vif. Conversely, the reciprocal Lys to Asp switch in AGM APOBEC3G reversed its specificity for Vif. The reversal of biological activity was accompanied by the corresponding switch in the species specificity with which the enzyme physically associated with Vif and was excluded from virions. The charge of the amino acid at position 128 was a critical determinant of species specificity. Based on the crystal structure of the distantly related Escherichia coli cytidine deaminase, we propose that this amino acid is positioned on a solvent-exposed loop of APOBEC3G on the same face of the protein as the catalytic site.

Besides activating NFκB by phosphorylating IκBs, IKKα/IKKβ kinases are also involved in regulating metabolic insulin signaling, the mTOR pathway, Wnt signaling, and autophagy. How IKKβ enzymatic activity is targeted to stimulus-specific substrates has remained unclear. We show here that NEMO, known to be essential for IKKβ activation by inflammatory stimuli, is also a specificity factor that directs IKKβ activity toward IκBα. Physical interaction and functional competition studies with mutant NEMO and IκB proteins indicate that NEMO functions as a scaffold to recruit IκBα to IKKβ. Interestingly, expression of NEMO mutants that allow for IKKβ activation by the cytokine IL-1, but fail to recruit IκBs, results in hyperphosphorylation of alternative IKKβ substrates. Furthermore IKK's function in autophagy, which is independent of NFκB, is significantly enhanced without NEMO as IκB scaffold. Our work establishes a role for scaffolds such as NEMO in determining stimulus-specific signal transduction via the pleiotropic signaling hub IKK.

Human APOBEC3F (hA3F) and APOBEC3G (hA3G) are antiretroviral cytidine deaminases that can be encapsidated during virus assembly to catalyze C→U deamination of the viral reverse transcripts in the next round of infection. Lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have evolved the accessory protein Vif to induce their degradation before packaging. HIV type 1 (HIV-1) Vif counteracts hA3G but not rhesus macaque APOBEC3G (rhA3G) or African green monkey (AGM) APOBEC3G (agmA3G) because of a failure to bind the nonhuman primate proteins. The species specificity of the interaction is controlled by amino acid 128, which is aspartate in hA3G and lysine in rhA3G. With the objective of overcoming this species restriction, mutations were introduced into HIV-1 Vif at amino acid positions that differed in charge between HIV-1 Vif and SIV Vif. The mutant proteins were tested for the ability to counteract hA3G, rhA3G, and agmA3G. Alteration of the conserved sequence at positions 14 to 17 from DRMR to SERQ, which is the sequence in AGM Vif, caused HIV-1 Vif to functionally interact with rhA3G and agmA3G. Mutation of three residues to the sequence SEMQ allowed interaction with rhA3G. SEMQ Vif also counteracted D128K mutant hA3G and wild-type hA3G. Introduction of the sequence into an infectious molecular HIV-1 clone allowed the virus to replicate productively in human cells that expressed rhA3G or hA3G. These findings provide insight into the interaction of Vif with A3G and are a step toward the development of a novel primate model for AIDS.

HIV-1 and most of the other lentiviruses encode Vif (virion infectivity factor), an accessory protein that the virus requires to replicate in primary CD4+ T-cells and monocytes. The host cell factor with which Vif interacts was recently identified as APOBEC3G, a cytidine deaminase related to the RNA-editing enzymes. Identification of this key host protein has allowed for dramatic leaps in our understanding of how Vif functions. Vif prevents the encapsidation of APOBEC3G into HIV-1 virions during virus assembly. If not for Vif, the encapsidated APOBEC3G would damage the virus reverse transcripts, causing their degradation and closing the open reading frames of its genes.

The HIV-1 accessory protein Vif (virion infectivity factor) is required for the production of infectious virions by CD4 + lymphocytes. Vif facilitates particle infectivity by blocking the inhibitory activity of APOBEC3G (CEM15), a virion-encapsidated cellular protein that deaminates minus-strand reverse transcript cytosines to uracils. We report that HIV-1 Vif forms a complex with human APOBEC3G that prevents its virion encapsidation. HIV-1 Vif did not efficiently form a complex with mouse APOBEC3G. Vif dramatically reduced the amount of human APOBEC3G encapsidated in HIV-1 virions but did not prevent encapsidation of mouse or AGM APOBEC3G. As a result, these enzymes are potent inhibitors of wild-type HIV-1 replication. The species-specificity of this interaction may play a role in restricting HIV-1 infection to humans. Together these findings suggest that therapeutic intervention that either induced APOBEC3G or blocked its interaction with Vif could be clinically beneficial.