Fig. 1
TRIM72-mediated ubiquitin signaling controls the homeostasis of the p62/SQSTM1 short isoform in regulating selective autophagy. a Schematic domain structure of p62L and p62S, which were derived from alternative splicing of the human p62/SQSTM1 gene. b Endogenous p62S and p62L were mainly degraded by proteasomes, but not by the autophagy pathway in HeLa cells treated with bortezomib (BTZ, 1 μmol/L) or bafilomycin (BAF, 20 nmol/L), as well as cycloheximide (CHX, 100 μg/ml). c Yeast two-hybrid screening identified TRIM72 as an interacting partner for p62S, but not for p62L. SD-2 was deficient in Leu and Trp, and SD-4 was deficient in Ura, His, Leu, and Trp. d Recombinant glutathione-S-transferase (GST)-tagged p62S directly interacted with His6-tagged TRIM72 in vitro as detected by a GST pull-down (PD) assay. Recombinant His6-tagged CYP26A1 acted as a negative control. e TRIM72 promoted the degradation of endogenous p62S, but not p62L, using the proteasome pathway. HeLa cells were ectopically expressed with empty vector or TRIM72, treated with the indicated compounds during different times, and detected by immunoblot analyses. f Wild-type TRIM72 but not the E3 ligase death mutants (TRIM72ΔRING and TRIM72C14A) supported the poly-ubiquitylation of p62S. HEK293T cells were ectopically expressed with the indicated plasmids, and then the cell lysates were immunoprecipitated with anti-Flag affinity gels before being subjected to immunoblotting analysis. g HeLa cells were co-transfected with the indicated plasmids, treated with 2 μmol/L rapamycin for 12 h, then with or without BTZ for 1 h, and subjected to fluorescent microscopy. Puncta formation by GFP-LC3 were counted and calculated. The protein levels of TRIM72 and p62S were also detected by immunoblot analysis. Scale bar = 10 μm. h Salmonella infection assay indicated that TRIM72 mediated the proteasomal degradation of p62S, and facilitated the clearing of Salmonella. HeLa cells were transfected with the indicated plasmids for 24 h, and then treated with the indicated compounds before Salmonella infection for 30 min. The cells were then lysed and one-tenth of the lysate was subjected to plate assays. The Salmonella colony numbers were counted and calculated. The protein levels of TRIM72, p62L, p62S, and LC3 were also detected by immunoblot analysis, and the expression of lipidated LC3 (LC3 II) was quantitated after normalization of control values as 1.00. i TRIM72-mediated proteasomal degradation of p62S facilitated carbonylated protein degradation. HeLa cells were transfected with the indicated plasmids for 24 h, and then treated with the indicated compounds before being subjected to the detection of protein oxidation. Pyocyanin was used as a positive control, and N-acetyl-l-cysteine was used as a negative control. Carbonylated proteins were visualized after derivatization with 2,4-dinitrophenylhydrazine (DNPH), followed by immunoblotting with anti-DNP. The levels of lipidated LC3 (LC3 II) were quantitated after normalization of the control as 1.00. j A model showing how TRIM72-mediated proteasomal degradation of p62S rheostatically regulated selective cellular autophagy. Data are presented as the mean ± SD, and analyzed with one-way analysis of variance using the Bonferroni post-hoc test or the two-tailed unpaired t test. *P < 0.05, **P < 0.01, three independent experiments. CDS coding sequence, CHX cycloheximide, Myc an epitope tag for protein tagged, TRIM72 tripartite motif-containing 72, Ub ubiquitin, UTR Untranslated region