Tollip Is a Mediator of Protein Sumoylation

Tollip is an interactor of the interleukin-1 receptor involved in its activation. The endosomal turnover of ubiquitylated IL-1RI is also controlled by Tollip. Furthermore, together with Tom1, Tollip has a general role in endosomal protein traffic. This work shows that Tollip is involved in the sumoylation process. Using the yeast two-hybrid technique, we have isolated new Tollip partners including two sumoylation enzymes, SUMO-1 and the transcriptional repressor Daxx. The interactions were confirmed by GST-pull down experiments and immunoprecipitation of the co-expressed recombinants. More specifically, we show that the TIR domain of the cytoplasmic region of IL-1RI is a sumoylation target of Tollip. The sumoylated and unsumoylated RanGAP-1 protein also interacts with Tollip, suggesting a possible role in RanGAP-1 modification and nuclear-cytoplasmic protein translocation. In fact, Tollip is found in the nuclear bodies of SAOS-2/IL-1RI cells where it colocalizes with SUMO-1 and the Daxx repressor. We conclude that Tollip is involved in the control of both nuclear and cytoplasmic protein traffic, through two different and often contrasting processes: ubiquitylation and sumoylation

Insights in progressive myoclonus epilepsy: HSP70 promotes cystatin B polymerization

Cystatin B (CSTB) is an anti-protease frequently mutated in progressive myoclonus epilepsy (EPM1), a devastating degenerative disease. This work shows that rat CSTB is an unstable protein that undergoes structural changes following the interaction with a chaperone, either prokaryotic or eukaryotic. Both the prokaryotic DnaK and eukaryotic HSP70 promote CSTB polymerization. Denaturated CSTB is polymerized by the chaperone alone. Native CSTB monomers are more stable than denatured monomers and require Cu(2+) for chaperone-dependent polymerization. Cu(2+) interacts with at least two conserved histidines, at positions 72 and 95 modifying the structure of native monomeric CSTB. Subsequently, CSTB becomes unstable and readily responds to the addition of DnaK or HSP70, generating polymers. This reaction depends strictly on the presence of this divalent metal ion and on the presence of one cysteine in the protein chain. The cysteine deletion mutant does not polymerize. We propose that Cu(2+) modifies the redox environment of the protein, allowing the oxidation of the cysteine residue of CSTB that triggers polymerization. These polymers are sensitive to reducing agents while polymers obtained from denatured CSTB monomers are DTT resistant. We propose that the Cu(2+)/HSP70 dependent polymers are physiological and functional in eukaryotic cells. Furthermore, while monomeric CSTB has anti-protease function, it seems likely that polymeric CSTB fulfils different function(s)

Sumoylation of Tollip.

<p>A) Western blot analysis of protein extracts from 293T cells transfected as indicated above the figure and stained for the proteins indicated on the right. B) Western blot analysis of protein extracts from 293T cells transfected with HA-Tollip and SUMO-1 (lane 1) and SUMO-1 only (lane 2) stained with anti-SUMO-1 abs. The same protein extracts were immunoprecipitated with anti-HA abs and stained with anti-SUMO-1 abs (lanes 3 and 4) and, after stripping of the membrane, they were stained with anti-HA abs (lanes 5 and 6). These samples were electrophoresed in 8% SDS-PAGE. C) Western blot of protein extracts from 293T cells transfected and stained as indicated.</p

Tollip interactions.

<p>A) GST-Tollip interaction with ³⁵S-methionine labelled proteins isolated by the yeast two hybrid technique. Lane 1 contains 1/10 input of the ³⁵S-methionine protein used for each interaction; lanes 2 and 3 contain the elution product from incubation of the ³⁵S-proteins with the GST-Tollip and GST-protein alone, respectively.³⁵S-methionine labelled proteins were visualized by autoradiography. Western blot analysis of 293T cells transfected with Ubc9 and HA-Tollip (B), Flag-ARIP3 and HA-Tollip (D), HA-Cystatin B and Ubc9 (C), HA-Cystatin B and Flag-ARIP3 (E). HA-Cystatin B is a ubiquitous protein with antiprotease function, unrelated to Tollip, nor to the inflammatory pathway <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004404#pone.0004404-DiGiaimo1" target="_blank">[28]</a>. Lane 1 contains the protein extract; lane 2 contains the proteins immunoprecipitated with anti-HA abs. In this and in the following figures, “pe.” refers to the protein extract and “ip.” to the immunoprecipitated protein. Staining carried out as indicated under the figures.</p