The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance

Gallbladder Volvulus: Diagnosis and Management of a Rare Cause of Acalculous Cholecystitis

Gallbladder volvulus is a rare cause of acalculous cholecystitis which clinically presents as acute abdomen and can rapidly evolve in a severe life-threatening sepsis. Awareness is crucial for differential diagnosis leading to the early and effective treatment. We present the case of an elderly woman who underwent laparoscopic cholecystectomy after pre-operative ultrasound diagnosis of gallbladder volvulus.</jats:p

p62 deletion mutants interacting with TRIM50.

<p>(A) Schematic representation of p62 deletion mutants with the minimal region of interaction with TRIM50. Lysates from FLAG-TRIM50#3 expressing a number of p62 deletion mutants were immunoprecipitated with anti-FLAG and immunoblotted with anti-GFP (B) and anti-GST (C).</p

TRIM50 colocalizes with p62 into aggresome.

<p>(A) SH-SY5Y cells were stained with anti-HDAC6 and with anti-p62 antibodies. The cells were treated with 25 µM MG132 for 6 h (d,e,f). (B) SH-SY5Y cells were stained with anti-TRIM50 and anti-p62 antibodies. The cells were treated with 25 µM MG132 for 6 h (d,e,f). (C) Lysates from FLAG-TRIM50#3 and FLAG#3 cell lines, treated with vehicle (–) or MG132 (+) were separated in RIPA detergent-soluble (S) and detergent insoluble (I) fractions and immunoblotting with anti-FLAG and anti-ubiquitin antibodies. (D) Lysates from MEF <i>Trim50</i> cell lines with different genotype (+/+, −/−), treated with vehicle (–) or MG132 (+) were separated in RIPA detergent-soluble (S) and detergent insoluble (I) fractions, analyzed by Western Blot and immunoblotting with anti-ubiquitin antibody.</p

TRIM50 promotes the accumulation of polyubiquitinated proteins into the aggresome.

<p>(A) MEF <i>Trim50</i> cell lines were stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (B) MEF <i>Trim50</i> cell lines were treated with 10 µM MG132 over night and stained with anti-FK2 antibody. The area, perimeter and intensity of signal of three independent experiments of each genotype were estimated using imageJ program. (C) MEF <i>Trim50</i> cell lines were treated with 10 µM MG132 over night, incubated o and 48 h with DMEM after MG132 wash out and stained with anti-FK2 antibody. The diagram shows the percentage of aggresome-positive cells.</p

TRIM50 interacts with HDAC6 and p62.

<p>(A) The interaction between TRIM50 and HDAC6 was assayed in HEK293 FLAG-TRIM50#3 cell line, treated with MG132. The cell lysates were immunoprecipitated (IP) with anti-FLAG and Immunoblot (IB) with HDAC6 antibody. Asterisk indicate IgG aspecific band. (B) Schematic representation of TRIM50 deletion mutants used, with the minimal TRIM50 interaction region. (C) The interaction between TRIM50 and p62 was assayed in FLAG-TRIM50#3 transfected with Myc-p62. The cell lysates were immunoprecipitated with anti-FLAG (left) and with anti-Myc (right) antibodies, respectively. Immunoblot were done as indicated (asterisk indicates FLAG-TRIM50 coming from the first FLAG blotting). (D) Total lysates of HEK293 cells transfected both with TRIM50 deletion mutants and Myc-p62 were immunoprecipitated with anti-FLAG and immunoblotted with anti-Myc.</p