A Label-free Quantitative Proteomics Strategy to Identify E3 Ubiquitin Ligase Substrates Targeted to Proteasome Degradation

Abstract

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. The ubiquitin-proteasome system (UPS) 1 plays an essential role in the regulation of protein stability in eukaryotic cells.Degradation of a protein by the UPS entails two successive steps: the covalent attachment of multiple ubiquitin molecules to the protein substrate and its degradation by the 26 S proteasome (1, 2). Ubiquitylation of protein substrates occurs through the sequential action of distinct enzymes: a ubiquitinactivating enzyme, E1; a ubiquitin-conjugating enzyme, E2; and a ubiquitin ligase, E3, responsible for the specific recognition of substrates. Increasing attention has been recently given to the UPS leading to the identification of hundreds of E3 ubiquitin ligases (E3s). Two major classes of E3s have been described: (i) E3s of the HECT (homologous to the E6-associated protein carboxyl terminus) domain family that function as ubiquitin carriers (3, 4) and (ii) E3s of the RING (really interesting new gene) or of the U box families that have no inherent catalytic activity but recruit an E2 enzyme toward substrates (5-7).Classical approaches to identify substrates of E3s are based on the identification of interacting proteins. Although these have successfully led to the identification of a number of substrates of monomeric E3s, identification of substrates of multimeric E3s is very challenging because of the weak affinity of substrates for their requisite specificity subunit and because of the labile nature of the substrate complexed with the specificity subunit (8).Acute promyelocytic leukemia (APL) is associated with six reciprocal translocations always involving the retinoic acid receptor ␣ (RAR␣) gene (9 -11). The RAR␣ protein is a member of the nuclear receptor superfamily that stimulates myeloid differentiation in the presence of its ligand, all-transretinoic acid (RA). In more than 95% of APL, the t(15;17) translocation between the promyelocytic leukemia (PML) gene on chromosome 15 and the RAR␣ gene on chromosome 17 produces the PML-RAR␣ fusion protein (12). The PML-RAR␣ protein enhances the rep...

Ubiquitin ligase Siah2 regulates RevErbα degradation and the mammalian circadian clock

“…As discussed above, physical interaction screens require strong physical interactions that may depend on posttranslational modifications. Recently, cell-based functional screening approaches have been developed to identify substrates based on differential ubiquitination (14,15) or stability proteins expressed as GFP fusions (13,16) in cells where a specific E3 ligase has been mutated or inhibited. Although these approaches are immensely useful for determining the function of specific E3 ligases, they are not directly amenable for identifying the E3 ligases of a specific substrate protein of interest.…”

“…However, this approach is most applicable when the E3 ligase-substrate interactions are strong and stable and may not work for transient enzyme-substrate interactions. Recently, a couple of promising large-scale approaches have been developed to identify substrates of particular E3 complexes (13)(14)(15)(16), but these approaches are not designed to identify the E3 ligases that ubiquitinate a specific protein for degradation. In fact, aside from protein interaction screening, large-scale approaches to identify how an individual protein is degraded are limited.…”

The E3 ubiquitin ligase specificity subunit ASB2α targets filamins for proteasomal degradation by interacting with the filamin actin-binding domain

“…We previously showed that ASB2 serves as the substrate recognition subunit of the E3 ubiquitin ligase complex that mediates polyubiquitylation and proteasomal-mediated degradation of FLNs (Baldassarre et al, 2009;Bello et al, 2009;Burande et al, 2009;Heuze et al, 2008). In order for this process to occur, FLNs and ASB2 must, at least transiently, interact.…”

“…We previously showed that ASB2 targets FLNa, FLNb and FLNc for proteasomal degradation (Baldassarre et al, 2009;Bello et al, 2009;Burande et al, 2009;Heuze et al, 2008) and have now mapped the ABD of FLNa as the minimal fragment sufficient for ASB2-mediated degradation. The structure of FLNaABD is highly similar to the structure of FLNbABD [root mean square deviation (r.m.s.d.)…”

Label-free Quantitative Proteomics and N-Glycoproteomics Analysis of KRAS-activated Human Bronchial Epithelial Cells

“…Quantitative mass spectrometry (MS)-based proteomics is one of the most powerful tools to study the expression profiles of activated genes of interest (13,26,27). In this study, we established the proteomic and N-glycoproteomic profiles of human bronchial epithelial cell (HBEC) 1 lines that are isogenic and differ only by the presence or absence of activated KRAS.…”