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Project description:Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the β-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.
| S-EPMC7710139 | biostudies-literature
Project description:To check whether mRNA level change for the changed proteins in protein level.
2022-12-31 | GSE181513 | GEO
Project description:Proteome-wide analysis reveals substrates of E3 ligase RNF146 targeted for degradation
| PRJNA656776 | ENA
Project description:The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded.
| S-EPMC2709196 | biostudies-literature
Project description:Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.
| S-EPMC7422721 | biostudies-literature
Project description:The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL-LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL-LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.
| S-EPMC3250164 | biostudies-literature
Project description:The HIB-Cul3 complex E3 ligase regulates physiological homeostasis through regulating its substrate stability and its activity can be modulated by changing HIB abundance. However, regulation of HIB remains elusive. Here we provide evidence that HIB is degraded through the proteasome by Cul3-mediated polyubiquitination in K48 manner in Drosophila. Strikingly, HIB is targeted for degradation by itself. We further identify that three degrons ((52)LKSS(56)T, (76)LDEE(80)S and (117)MESQ(121)R) and K185 and K198 of HIB are essential for its auto-degradation. Finally, we demonstrate that HIB-Cul3 substrates, Ci and Puc, can effectively protect HIB from HIB-Cul3-mediated degradation. Taken together, our study indicates that there is an exquisite equilibrium between the adaptor and targets to achieve the tight control of the HIB, which is essential for maintaining suitable Hh and JNK signaling. And the mechanism of adaptor self-degradation and reciprocal control of the abundance between adaptor and its substrates is also applied to BTB-Cul3 E3 ligase adaptor dKeap1, dDiablo and dKLHL18.
| S-EPMC4533009 | biostudies-literature
Project description:SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance.
| S-EPMC5062998 | biostudies-literature
Project description:Conventional dendritic cells (cDCs) comprise distinct populations with specialized immune functions that are mediators of innate and adaptive immune responses. Transcriptomic and proteomic approaches have been used so far to identify transcripts and proteins that are differentially expressed in these subsets to understand the respective functions of cDCs subsets. Here, we showed that the Cullin 5-RING E3 ubiquitin ligase (E3) ASB2?, by driving degradation of filamin A (FLNa) and filamin B (FLNb), is responsible for the difference in FLNa and FLNb abundance in the different spleen cDC subsets. Importantly, the ability of these cDC subsets to migrate correlates with the level of FLNa. Furthermore, our results strongly point to CD4 positive and double negative cDCs as distinct populations. Finally, we develop quantitative global proteomic approaches to identify ASB2? substrates in DCs using ASB2 conditional knockout mice. As component of the ubiquitin-proteasome system (UPS) are amenable to pharmacological manipulation, these approaches aimed to the identification of E3 substrates in physiological relevant settings could potentially lead to novel targets for therapeutic strategies.
| S-EPMC4633680 | biostudies-literature
Project description:The Slx5/Slx8 heterodimer constitutes a SUMO-targeted ubiquitin ligase (STUbL) with an important role in SUMO-targeted degradation and SUMO-dependent signaling. This STUbL relies on SUMO-interacting motifs in Slx5 to aid in substrate targeting and carboxy-terminal RING domains in both Slx5 and Slx8 for substrate ubiquitylation. In budding yeast cells, Slx5 resides in the nucleus, forms distinct foci, and can associate with double-stranded DNA breaks. However, it remains unclear how STUbLs interact with other proteins and their substrates. To examine the targeting and functions of the Slx5/Slx8 STUbL, we constructed and analyzed truncations of the Slx5 protein. Our structure-function analysis reveals a domain of Slx5 involved in nuclear localization and in the interaction with Slx5, SUMO, Slx8, and a novel interactor, the SUMO E3 ligase Siz1. We further analyzed the functional interaction of Slx5 and Siz1 in vitro and in vivo. We found that a recombinant Siz1 fragment is an in vitro ubiquitylation target of the Slx5/Slx8 STUbL. Furthermore, slx5 cells accumulate phosphorylated and sumoylated adducts of Siz1 in vivo. Specifically, we show that Siz1 can be ubiquitylated in vivo and is degraded in an Slx5-dependent manner when its nuclear egress is prevented in mitosis. In conclusion, our data provide a first look into the STUbL-mediated regulation of a SUMO E3 ligase.
| S-EPMC3873881 | biostudies-other