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

Heart Function and Myocardial Tissue Characterization in Patients with HCV Related Cirrhosis: Diastolic Dysfunction and Cardiac Hypertrophy

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Indexing cardiac parameters in echocardiographic practice: do estimates depend on how weight and height have been assessed? A study on left atrial dilatation

We examined the difference between self-reported and measured height and weight in detecting echocardiographic left atrial dilatation (LAD), as defined by LA diameter indexed to body size parameters in an outpatient population referred to echocardiographic laboratories for routine examination. LAD was defined by 2 criteria: (1) LA diameter indexed to height greater than 24 mm/m; (2) LA diameter indexed to body surface area greater than 23 mm/m2. Prevalence of LAD was calculated by indexing LA diameter to both self-reported and measured anthropometric values. In the whole population, LAD tended to be underestimated when LA diameter was indexed to self-reported compared with measured values, by 3.6% according to criterion 1 (26.4% versus 30.0%, P < .001) and by 0.6% according to criterion 2 (21.1% versus 21.6%, P = not significant). The difference between LAD estimates was more pronounced in older than in younger patients, either by criterion 1 (6.4% versus 1.6 %, P < .001) or by criterion 2 (2.1% versus 0.1%, P < .001). The error is related to demographic characteristics of patients and is more pronounced when LA diameter is normalized to height

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

Cellular localization of Tollip.

<p>A–B) HeLa cells transiently expressing HA-Tollip and Flag-SUMO-1 stained with anti−ΗΑ (Cy5-red) and anti-Flag (FITC-green) abs. E–F) SAOS-2/IL-1RI cells transfected with HA-Tollip only and stained with anti−HA (Cy5 -red) and anti-SUMO-1 abs (FITC- green). I–J) SAOS-2/IL-1RI cells transfected with HA-Tollip only and stained with anti-HA (Cy5 -red) and anti-Daxx (FITC- green) abs. C,D,G,H,K,L) Merged reconstructed images of extended focus projections are shown. The selected areas (white rectangles) are reconstructed as three-dimensional imaging using the surface-shaded algorithm operating above a defined threshold of fluorescence intensity. The detail allows a precise evaluation of localization of the two fluorescent signals. Magnification bar: 5 µm.</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

Tollip and ARIP3 interacting domains.

<p>A) Schematic representation of HA-Tollip deletion mutants. The black box represents the C2 domain (aa 54–179), the grey box represents the CUE domain (aa 229–274). HA-Tollip wt: aa 1–274; Δ1: aa 1–229; Δ2; aa 1–179: Δ3: aa 54–274; Δ4: aa 94–274; Δ5: aa 134–274; Δ6: aa 179–274; Δ7: aa 54–179. In all western blots described in this figure, Lane 1 contains the protein extract and lane 2 the immunoprecipitated proteins. B) Western blot of protein extracts from 293T cells, transfected with Flag-ARIP3 cDNA and with the indicated HA-Tollip deletion mutants, before (lane 1) and after (lane 2) immunoprecipitation with anti-HA abs. The staining is with anti-Flag abs. C) Western blot from the same protein extracts as in B stained with anti-HA abs. D) Schematic representation of the Flag-ARIP3 deletion mutants. The circle represents the SAP domain (aa 11–45), the white box with black stripes represents the RING domain (aa 347–388) and the black box with white stripes represents the AR-ID domain (aa 443–548). Flag-ARIP3 wt: aa 1–572; Δa: aa 1–467; Δb: aa 1–347; Δc: aa 1–169. E) Western blot of protein extracts from 293T cells transfected with HA-Tollip and the indicated Flag-ARIP3 deletion mutants. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag antibodies. F) Western blot of protein extracts from 293T cells transfected with the Flag-PIAS-1 cDNA together with HA-Tollip wt and mutants as indicated. Immunoprecipitation with anti-HA abs; staining with anti-Flag abs. G) Western blot of protein extracts from 293T cells transfected with the Flag-PIASxβ cDNA together with the HA-Tollip wt and mutants as indicated. The protein extract was immunoprecipitated with anti-HA abs and stained with anti-Flag abs. H, I) Western blots of the same samples as in F and G respectively, stained with anti–HA abs.</p