SUMO-activated target traps (SATTs) enable the identification of a comprehensive E3-specific SUMO proteome

Abstract

Ubiquitin and ubiquitin-like conjugation cascades consist of dedicated E1, E2, and E3 enzymes with E3s providing substrate specificity. Mass spectrometry–based approaches have enabled the identification of more than 6500 SUMO2/3 target proteins. The limited number of SUMO E3s provides the unique opportunity to systematically study E3 substrate wiring. We developed SUMO-activated target traps (SATTs) and systematically identified substrates for eight different SUMO E3s, PIAS1, PIAS2, PIAS3, PIAS4, NSMCE2, ZNF451, LAZSUL (ZNF451-3), and ZMIZ2. SATTs enabled us to identify 427 SUMO1 and 961 SUMO2/3 targets in an E3-specific manner. We found pronounced E3 substrate preference. Quantitative proteomics enabled us to measure substrate specificity of E3s, quantified using the SATT index. Furthermore, we developed the Polar SATTs web-based tool to browse the dataset in an interactive manner. Overall, we uncover E3-to-target wiring of 1388 SUMO substrates, highlighting unique and overlapping sets of substrates for eight different SUMO E3 ligases.

dsRNAi-mediated silencing of PIAS2beta specifically kills anaplastic carcinomas by mitotic catastrophe

“…Limitations of our study include: i) we performed the preclinical cellular work in vitro. However, we have found the same anticancer results of PIAS2b -dsRNAi in commercial ATC and non-thyroid anaplastic cell lines, as well as in three patient ATC primary cultures, and in the oPDX in vivo; ii) SUMOylation has been studied selecting proteins and not directly in peptides since this required alternative cell-altering approaches 50 , 106 , although we have validated two selected targets; iii) our ATC patients were all women; however, this is also the most affected sex in ATC; and iv) only oPDX from two out of three patients grew in the immune-deficient mouse models; we have tried to compensate this by increasing the number of mice per trial.…”

“…In our work, alpha, beta, and gamma tubulins, and many microtubule-binding proteins, were repeatedly found associated to PIAS2b at mitosis in thyroid anaplastic cells, and the SUMOylation of tubulins altered in PIAS2b -dsRNAi anaplastic cells. Tubulins beta (TUBB and TUBB4B) and alfa (TUBA1C) were also found specifically SUMO1ylated by PIAS2 in a recent high-throughput study using SAAT to delineate E3-SUMO ligases substrate specificity in asynchronous U2OS cells 50 . Tubulins are not considered as simple support filaments, but in the current concept there is a range of post-translational modifications that are becoming known and that control microtubule dynamics 51 , 52 .…”

“…Of note, TUBB3, PSMC3, and PSMC5 present consensus in silico SUMO binding sites and were previously found to be SUMOylated 48 (Supplementary Data 4 ). Five of all these putative PIAS2b SUMOylated proteins (PSMD1, DYNC1H1, TRIM28, EIF4G1, TNPO1) have recently being confirmed as direct PIAS2b targets in non-mitotic U2OS cells using advanced SUMO-activated target traps (SATTS) 50 (Supplementary Data 4 ).…”

BRCA1/BARD1 ubiquitinates PCNA in unperturbed conditions to promote continuous DNA synthesis

“…For the Exploris480, samples were injected as in ref. 64 onto a cartridge precolumn (300 μm × 5 mm, C18 PepMap, 5 μm, 100 A) with a flow of 10 μl/min for 3 min (Thermo, Bremen, Germany) and eluted via a homemade analytical nano-HPLC column (50 cm × 75 μm; Reprosil-Pur C18-AQ 1.9 μm, 120A) (Dr. Maisch, Ammerbuch, Germany). The gradient was run from 2% to 38% solvent B (80% acetonitrile, 0.1% formic acid) in 120 min.…”

Structure and methyl-lysine binding selectivity of the HUSH complex subunit MPP8

“…The PINIT domain of Siz1 is required for SUMOylation of splicing factor Prp45 and the proliferating cell nuclear antigen (PCNA) in yeast (Pfander et al, 2005;Streich & Lima, 2016). In humans, PIAS2 and PIAS3 have been reported to SUMOylate various substrates including Aurora-B kinase, Argonaute-2, and the INO80 chromatin remodeling complex (Ban et al, 2011;Cox et al, 2017;Josa-Prado et al, 2015;Salas-Lloret et al, 2023). The PINIT amino acid sequence motif is not conserved in the PINIT-like domain of MPP8 (Fig.…”