Definition of the interacting interfaces of Apobec3G and HIV-1 Vif using MAPPIT mutagenesis analysis

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

The many faces of the SOCS box

The suppressors of cytokine signalling (SOCS) box is a structural domain found at the C-terminus of over 70 human proteins. It is usually coupled to a protein interaction module such as an SH2 domain in case of SOCS proteins, a family of modulators of cytokine signaling. The SOCS box participates in the formation of E3 ligase complexes, marking activated cytokine receptor complexes for proteasomal degradation. A similar mechanism was recently uncovered for controlling SOCS activity itself, since SOCS2 was found to enhance the turnover of other SOCS proteins. The SOCS box can also add unique features to individual SOCS proteins: it can function as an adaptor domain as was demonstrated for SOCS3, or as a modulator of substrate binding in case of CIS. In this review we discuss these multiple roles of the SOCS box, which emerges as a versatile module controlling cytokine signaling via multiple mechanisms

MAPPIT (Mammalian Protein-Protein Interaction Trap) analysis of early steps in Toll-like receptor signalling

The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid technique founded on type I cytokine signal transduction. Thereby, bait and prey proteins are linked to signalling deficient cytokine receptor chimeras. Interaction of bait and prey and ligand stimulation restores functional JAK (Janus kinase)-STAT (signal transducers and activators of transcription) signalling, which ultimately leads to the transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In the subsequent protocol, we describe the use of MAPPIT to study early events in Toll-like receptor (TLR) signalling. We here demonstrate a "signalling interaction cascade" from TLR4 to IRAK-1

The ins and outs of leptin receptor activation

AbstractThe adipocyte-derived hormone leptin signals the status of body energy stores by activating its receptor in hypothalamic nuclei. In contrast to the initial expectations, leptin treatment of human obesity was largely unsuccessful. One explanation for this is the marked leptin resistance, which likely operates in part at the receptor level. The leptin receptor is a member of the class I cytokine receptor family, which uses the Janus kinase/signal transducer and activator of transcription pathway as a major signaling route. In this review, we focus on the molecular mechanisms underlying leptin receptor activation. Different modes of leptin-induced clustering of the ectodomains and the subsequent signaling events will be discussed

Comparison of leptin- and interleukin-6-regulated expression of the rPAP gene family: evidence for differential co-regulatory signals

The pancreatitis-associated protein (PAP)/regenerating protein (REG) family represents a complex group of small secretory proteins, which can function as acute phase reactants, lectins, antiapoptotic factors or growth factors for pancreatic beta-cells and neural cells. Transcriptional induction of rPAP/Reg genes was studied here in PC12 cells made responsive to leptin. Northern-blots showed quantitative differences in induction of four major family members by leptin and IL-6. Surprisingly, induction by leptin was strongly enhanced upon forskolin co-treatment whereas induction by IL-6 was counteracted. Functional studies involving progressive rPAP I promoter deletions showed, in the case of leptin, a clear correlation with predicted cis-regulatory elements. Leptin-induced stimulation was dependent on STAT3, since over-expression of dominant-negative STAT3, but not of dominant-negative STAT1, completely blocked transcriptional activation. In case of IL-6, an enhancer element outside the cloned promoter fragment is required for full stimulation. The effects of forskolin in a leptin and IL-6 context could not be explained at the promoter level, but rather events occurring upstream in the signalling cascade must be postulated to explain the differential co-regulatory effects

The C-terminus of CIS defines its interaction pattern

Proteins of the SOCS (suppressors of cytokine signalling) family are characterized by a conserved modular structure with pre-SH2 (Src homology 2), SH2 and SOCS-box domains. Several members, including CIS (cytokine-inducible SH2 protein), SOCS1 and SOCS3, are induced rapidly upon cytokine receptor activation and function in a negative-feedback loop, attenuating signalling at the receptor level. We used a recently developed mammalian two-hybrid system [MAPPIT (mammalian protein–protein interaction trap)] to analyse SOCS protein-interaction patterns in intact cells, allowing direct comparison with biological function. We find that, besides the SH2 domain, the C-terminal part of the CIS SOCS-box is required for functional interaction with the cytokine receptor motifs examined, but not with the N-terminal death domain of the TLR (Toll-like receptor) adaptor MyD88. Mutagenesis revealed that one single tyrosine residue at position 253 is a critical binding determinant. In contrast, substrate binding by the highly related SOCS2 protein, and also by SOCS1 and SOCS3, does not require their SOCS-box