Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System.
ABSTRACT: The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.
Project description:Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation.
Project description:The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins. The conjugation of a polyubiquitin chain, or polyubiquitination, to a target protein requires an increasingly diverse cascade of enzymes culminating with the E3 ubiquitin ligases. Protein recognition by an E3 ligase occurs through a specific sequence of amino acids, termed a degradation sequence or degron. Recently, degrons have been incorporated into novel reporters to monitor proteasome activity; however only a limited few degrons have successfully been incorporated into such reporters. The goal of this work was to evaluate the ubiquitination kinetics of a small library of portable degrons that could eventually be incorporated into novel single cell reporters to assess proteasome activity. After an intensive literary search, eight degrons were identified from proteins recognized by a variety of E3 ubiquitin ligases and incorporated into a four component degron-based substrate to comparatively calculate ubiquitination kinetics. The mechanism of placement of multiple ubiquitins on the different degron-based substrates was assessed by comparing the data to computational models incorporating first order reaction kinetics using either multi-monoubiquitination or polyubiquitination of the degron-based substrates. A subset of three degrons was further characterized to determine the importance of the location and proximity of the ubiquitination site lysine with respect to the degron. Ultimately, this work identified three candidate portable degrons that exhibit a higher rate of ubiquitination compared to peptidase-dependent degradation, a desired trait for a proteasomal targeting motif.
Project description:Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.
Project description:Since the discovery of ubiquitin conjugation as a cellular mechanism that triggers proteasomal degradation, the mode of substrate recognition by the ubiquitin-ligation system has been the holy grail of research in the field. This entails the discovery of recognition determinants within protein substrates, which are part of a degron, and explicit E3 ubiquitin (Ub)-protein ligases that trigger their degradation. Indeed, many protein substrates and their cognate E3's have been discovered in the past 40 years. In the course of these studies, various degrons have been randomly identified, most of which are acquired through post-translational modification, typically, but not exclusively, protein phosphorylation. Nevertheless, acquired degrons cannot account for the vast diversity in cellular protein half-life times. Obviously, regulation of the proteome is largely determined by inherent degrons, that is, determinants integral to the protein structure. Inherent degrons are difficult to predict since they consist of diverse sequence and secondary structure features. Therefore, unbiased methods have been employed for their discovery. This review describes the history of degron discovery methods, including the development of high throughput screening methods, state of the art data acquisition and data analysis. Additionally, it summarizes major discoveries that led to the identification of cognate E3 ligases and hitherto unrecognized complexities of degron function. Finally, we discuss future perspectives and what still needs to be accomplished towards achieving the goal of understanding how the eukaryotic proteome is regulated via coordinated action of components of the ubiquitin-proteasome system.
Project description:Selective protein degradation by the ubiquitin-proteasome system (UPS) is thought to be governed primarily by the recognition of specific motifs - degrons - present in substrate proteins. The ends of proteins - the N- and C-termini - have unique properties, and an important subset of protein-protein interactions involve the recognition of free termini. The first degrons to be discovered were located at the extreme N-terminus of proteins, a finding which initiated the study of the N-degron (formerly N-end rule) pathways, but only in the last few years has it emerged that a diverse set of C-degron pathways target analogous degron motifs located at the extreme C-terminus of proteins. In this minireview we summarise the N-degron and C-degron pathways currently known to operate in human cells, focussing primarily on those that have been discovered in recent years. In each case we describe the cellular machinery responsible for terminal degron recognition, and then consider some of the functional roles of terminal degron pathways. Altogether, a broad spectrum of E3 ubiquitin ligases mediate the recognition of a diverse array of terminal degron motifs; these degradative pathways have the potential to influence a wide variety of cellular functions.
Project description:The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.
Project description:Increasing amounts of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. It selectively recruits substrates for their ubiquitination and subsequent degradation. Recently, several exome-sequencing studies of endometrial cancer revealed high frequency somatic mutations in SPOP (5.7-10%). However, how SPOP mutations contribute to endometrial cancer remains unknown. Here, we identified estrogen receptor-? (ER?), a major endometrial cancer promoter, as a substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes multiple Ser/Thr (S/T)-rich degrons located in the AF2 domain of ER?, and triggers ER? degradation via the ubiquitin-proteasome pathway. SPOP depletion by siRNAs promotes endometrial cells growth. Strikingly, endometrial cancer-associated mutants of SPOP are defective in regulating ER? degradation and ubiquitination. Furthermore, we found that SPOP participates in estrogen-induced ER? degradation and transactivation. Our study revealed novel molecular mechanisms underlying the regulation of ER? protein homeostasis in physiological and pathological conditions, and provided insights in understanding the relationship between SPOP mutations and the development of endometrial cancer.
Project description:Dysregulation of the ubiquitin-proteasome pathway is closely associated with cancer initiation and progression. SPOP is an adapter protein of the CUL3-based E3 ubiquitin ligase complexes. Several whole genome/exome sequencing studies on endometrial cancers (ECs) revealed that the SPOP gene is frequently mutated. However, how SPOP mutations contribute to EC remains poorly understood. In this study, transcription factor ZBTB3 was identified as a proteolytic substrate for the SPOP-CUL3-RBX1 E3 ubiquitin ligase complex. SPOP specifically recognizes two Ser/Thr (S/T)-rich degrons located in ZBTB3 and triggers the degradation of ZBTB3 via the ubiquitin-proteasome pathway. By contrast, EC-associated SPOP mutants are defective in regulating ZBTB3 stability. SPOP inactivation promotes endometrial cell proliferation, migration, and invasion partly through ZBTB3 accumulation. Sonic hedgehog (SHH) was found to be a transcriptional target of ZBTB3. SPOP inactivation leads to ZBTB3-dependent SHH upregulation in EC cells. RUSKI-43, a small molecule inhibitor of SHH, suppresses cell proliferation, migration, and invasion in SPOP-depleted or EC-associated SPOP mutant-overexpressed EC cells. Our data indicate that pharmacological inhibition of SHH represents a possible treatment strategy for SPOP-mutated ECs.
Project description:Regulated proteolysis by proteasomes involves ~800 enzymes for substrate modification with ubiquitin, including ~600 E3 ligases. We report here that E6AP/UBE3A is distinguished from other E3 ligases by having a 12?nM binding site at the proteasome contributed by substrate receptor hRpn10/PSMD4/S5a. Intrinsically disordered by itself, and previously uncharacterized, the E6AP-binding domain in hRpn10 locks into a well-defined helical structure to form an intermolecular 4-helix bundle with the E6AP AZUL, which is unique to this E3. We thus name the hRpn10 AZUL-binding domain RAZUL. We further find in human cells that loss of RAZUL by CRISPR-based gene editing leads to loss of E6AP at proteasomes. Moreover, proteasome-associated ubiquitin is reduced following E6AP knockdown or displacement from proteasomes, suggesting that E6AP ubiquitinates substrates at or for the proteasome. Altogether, our findings indicate E6AP to be a privileged E3 for the proteasome, with a dedicated, high affinity binding site contributed by hRpn10.
Project description:Eukaryotic cell cycle transitions are driven by E3 ubiquitin ligases that catalyze the ubiquitylation and destruction of specific protein targets. For example, the anaphase-promoting complex/cyclosome (APC/C) promotes the exit from mitosis via destruction of securin and mitotic cyclins, whereas CRL1(Skp2) allows entry into S phase by targeting the destruction of the cyclin-dependent kinase (CDK) inhibitor p27. Recently, an E3 ubiquitin ligase called CRL4(Cdt2) has been characterized, which couples proteolysis to DNA synthesis via an unusual mechanism that involves display of substrate degrons on the DNA polymerase processivity factor PCNA. Through its destruction of Cdt1, p21, and Set8, CRL4(Cdt2) has emerged as a master regulator that prevents rereplication in S phase. In addition, it also targets other factors such as E2F and DNA polymerase ?. In this review, we discuss our current understanding of the molecular mechanism of substrate recognition by CRL4(Cdt2) and how this E3 ligase helps to maintain genome integrity.