The host restriction factor Apobec3G is a cytidine deaminase that incorporates into HIV-1 virions and interferes with viral replication. The HIV-1 accessory protein Vif subverts Apobec3G by targeting it for proteasomal degradation. We propose a model in which Apobec3G N-terminal domains symmetrically interact via a head-to-head interface containing residues 122 RLYYFW 127. To validate this model and to characterize the Apobec3G–Apobec3G and the Apobec3G–Vif interactions, the mammalian protein–protein interaction trap two-hybrid technique was used. Mutations in the head-to-head interface abrogate the Apobec3G–Apobec3G interaction. All mutations that inhibit Apobec3G–Apobec3G binding also inhibit the Apobec3G–Vif interaction, indicating that the head-to head interface plays an important role in the interaction with Vif. Only the D128K, P129A and T32Q mutations specifically affect the Apobec3G–Vif association. In our model, D128, P129 and T32 cluster at the edge of the head-to-head interface, possibly forming a Vif binding site composed of two Apobec3G molecules. We propose that Vif either binds at the Apobec3G head-to-head interface or associates with an RNA-stabilized Apobec3G oligomer.
Binding of GH to its receptor induces rapid phosphorylation of conserved tyrosine motifs that function as recruitment sites for downstream signaling molecules. Using mammalian protein-protein interaction trap (MAPPIT), a mammalian two-hybrid method, we mapped the binding sites in the GH receptor for signal transducer and activator of transcription 5 (STAT5) a and b and for the negative regulators of cytokine signaling cytokine-inducible Src-homology 2 (SH2)-containing protein (CIS) and suppressor of cytokine signaling 2 (SOCS2). Y534, Y566, and Y627 are the major recruitment sites for STAT5. A non-overlapping recruitment pattern is observed for SOCS2 and CIS with positions Y487 and Y595 as major binding sites, ruling out SOCS-mediated inhibition of STAT5 activation by competition for shared binding sites. More detailed analysis revealed that CIS binding to the Y595, but not to the Y487 motif, depends on both its SH2 domain and the C-terminal part of its SOCS box, with a critical role for the CIS Y253 residue. This functional divergence of the two CIS/SOCS2 recruitment sites is also observed upon substitution of the Y+1 residue by leucine, turning the Y487, but not the Y595 motif into a functional STAT5 recruitment site.
BackgroundISG15 is an Ubiquitin-like protein, highly induced by Type I Interferons. Upon the cooperative activity of specific Ubiquitinating enzymes, ISG15 can be conjugated to its substrates. Increasing evidence points to a role for protein ISGylation in anti-viral and anti-tumoral defense.Principal FindingsWe identified ISG15 from Old World Monkeys (OWm) as a hyper-efficient protein modifier. Western blot analysis visualized more efficient conjugation of OWmISG15 relative to HuISG15 in human (Hu), monkey and mouse (Mo) cell-lines. Moreover, the substrates of OWmISG15 identified upon Tandem Affinity Purification followed by LC-MS/MS identification largely outnumbered these of HuISG15 itself. Several Ubiquitin-Conjugating enzymes were identified as novel ISGylated substrates. Introduction of a N89D mutation in HuISG15 improved its ISGylation capacity, and additional Q31K/T33A/D133N mutations yielded a HuISG15 variant with an ISGylation efficiency comparable to OWmISG15. Homology modeling and structural superposition situate N89 in the interaction interface with the Activating enzyme. Analysis of the UbE1L residues in this interface revealed a striking homology between OWmUbE1L and HuUbE1, the Activating enzyme of Ubiquitin. In line with this observation, we found efficient activation of AgmISG15, but not HuISG15 or MoISG15, by HuUbE1, thus providing a likely explanation for OWm hyperISGylation.ConclusionsThis study discloses the poor conjugation competence of HuISG15 compared to OWmISG15 and maps the critical determinants for efficient conjugation. HyperISGylation may greatly assist ISGylation studies and may enhance its function as positive regulator of Interferon-related immune responses or as anti-tumoral modulator.
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