Covalent linkage of distinct substrate degrons controls assembly and disassembly of DegP proteolytic cages.
ABSTRACT: Protein quality control requires careful regulation of intracellular proteolysis. For DegP, a periplasmic protease, substrates promote assembly of inactive hexamers into proteolytically active cages with 12, 18, 24, or 30 subunits. Here, we show that sensitive activation and cage assembly require covalent linkage of distinct substrate sequences that affect degradation (degrons). One degron binds the DegP active site, and another degron binds a separate tethering site in PDZ1 in the crystal structure of a substrate-bound DegP dodecamer. FRET experiments demonstrate that active cages assemble rapidly in a reaction that is positively cooperative in substrate concentration, remain stably assembled while uncleaved substrate is present, and dissociate once degradation is complete. Thus, the energy of binding of linked substrate degrons drives assembly of the proteolytic machine responsible for subsequent degradation. Substrate cleavage and depletion results in disassembly, ensuring that DegP is proteolytically active only when sufficient quantities of protein substrates are present.
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:DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be uncoupled. Indeed, DegP variants that remain trimeric, hexameric, or dodecameric in the presence or absence of substrate still display robust and positively cooperative substrate degradation in vitro and, most importantly, sustain high-temperature bacterial growth as well as the wild-type enzyme. Our results support a model in which substrate binding converts inactive trimers into proteolytically active trimers, and simultaneously leads to cage assembly by enhancing binding of PDZ1 domains in one trimer to PDZ2' domains in neighboring trimers. Thus, both processes depend on substrate binding, but they can be uncoupled without loss of biological function. We discuss potential coupling mechanisms and why cage formation may have evolved if it is not required for DegP proteolysis.
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: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:The anaphase-promoting complex/cyclosome (APC/C) controls a variety of cellular processes through its ability to target numerous protein substrates for timely degradation. Substrate selection by this ubiquitin ligase depends on related activator proteins, Cdc20 and Cdh1, which bind and activate the APC/C at distinct cell cycle stages. Biochemical and structural studies revealed that Cdc20 and Cdh1 carry conserved receptor domains to recognize specific sequence motifs in substrates, such as D and KEN boxes. The mechanisms for ordered degradation of APC/C substrates, however, remain incompletely understood. Here we describe minimal degradation sequences (degrons) sufficient for rapid APC/C-Cdh1-specific in vivo degradation. The polo kinase Cdc5-derived degron contained an essential KEN motif, whereas a single RxxL-type D box was the relevant signal in the Cdc20-derived degradation domain, indicating that either motif may support specific recognition by Cdh1. In both degrons, the APC/C recognition motif was flanked by a nuclear localization sequence. Forced localization of the degron constructs revealed that proteolysis mediated by APC/C-Cdh1 is restricted to the nucleus and maximally active in the nucleoplasm. Levels of Iqg1, a cytoplasmic Cdh1 substrate, decreased detectably later than the nucleus-localized Cdh1 substrate Ase1, indicating that confinement to the nucleus may allow for temporal control of APC/C-Cdh1-mediated proteolysis.
Project description:Recently, controllable, targeted proteolysis has emerged as one of the most promising new strategies to study essential genes and otherwise toxic mutations. One of the principal limitations preventing the wider adoption of this approach is due to the lack of easily identifiable species-specific degrons that can be used to trigger the degradation of target proteins. Here, we report new advancements in the targeted proteolysis concept by creating the first prokaryotic N-terminal targeted proteolysis system. We demonstrate how proteins from the LexA-like protein superfamily can be exploited as species-specific reservoirs of N- and/or C-degrons, which are easily identifiable due to their proximity to strictly conserved residues found among LexA-like proteins. Using the LexA-like regulator HdiR of Streptococcus mutans, we identified two separate N-degrons derived from HdiR that confer highly efficient constitutive proteolysis upon target proteins when added as N-terminal peptide tags. Both degrons mediate degradation via AAA+ family housekeeping proteases with one degron primarily targeting FtsH and the other targeting the ClpP-dependent proteases. To modulate degron activity, our approach incorporates a hybrid N-terminal protein tag consisting of the ubiquitin-like protein NEDD8 fused to an HdiR degron. The NEDD8 fusion inhibits degron function until the NEDD8-specific endopeptidase NEDP1 is heterologously expressed to expose the N-degron. By fusing the NEDD8-degron tag onto GFP, luciferase, and the pleiotropic regulator RNase J2, we demonstrate that the N-terminal proteolysis approach exhibits far superior performance compared to the classic transcriptional depletion approach and is similarly applicable for the study of highly toxic mutations.
Project description:Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.
Project description:This perspective is partly review and partly proposal. N-degrons and C-degrons are degradation signals whose main determinants are, respectively, the N-terminal and C-terminal residues of cellular proteins. N-degrons and C-degrons include, to varying extents, adjoining sequence motifs, and also internal lysine residues that function as polyubiquitylation sites. Discovered in 1986, N-degrons were the first degradation signals in short-lived proteins. A particularly large set of C-degrons was discovered in 2018. We describe multifunctional proteolytic systems that target N-degrons and C-degrons. We also propose to denote these systems as "N-degron pathways" and "C-degron pathways." The former notation replaces the earlier name "N-end rule pathways." The term "N-end rule" was introduced 33 years ago, when only some N-terminal residues were thought to be destabilizing. However, studies over the last three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing N-terminal residues. Advantages of the proposed terms include their brevity and semantic uniformity for N-degrons and C-degrons. In addition to being topologically analogous, N-degrons and C-degrons are related functionally. A proteolytic cleavage of a subunit in a multisubunit complex can create, at the same time, an N-degron (in a C-terminal fragment) and a spatially adjacent C-degron (in an N-terminal fragment). Consequently, both fragments of a subunit can be selectively destroyed through attacks by the N-degron and C-degron pathways.
Project description:Inhibitor-of-apoptosis (IAP) proteins are key regulators of the innate antiviral response by virtue of their capacity to respond to signals affecting cell survival. In insects, wherein the host IAP provides a primary restriction to apoptosis, diverse viruses trigger rapid IAP depletion that initiates caspase-mediated apoptosis, thereby limiting virus multiplication. We report here that the N-terminal leader of two insect IAPs, Spodoptera frugiperda SfIAP and Drosophila melanogaster DIAP1, contain distinct instability motifs that regulate IAP turnover and apoptotic consequences. Functioning as a protein degron, the cellular IAP leader dramatically shortened the life span of a long-lived viral IAP (Op-IAP3) when fused to its N terminus. The SfIAP degron contains mitogen-activated kinase (MAPK)-like regulatory sites, responsible for MAPK inhibitor-sensitive phosphorylation of SfIAP. Hyperphosphorylation correlated with increased SfIAP turnover independent of the E3 ubiquitin-ligase activity of the SfIAP RING, which also regulated IAP stability. Together, our findings suggest that the SfIAP phospho-degron responds rapidly to a signal-activated kinase cascade, which regulates SfIAP levels and thus apoptosis. The N-terminal leader of dipteran DIAP1 also conferred virus-induced IAP depletion by a caspase-independent mechanism. DIAP1 instability mapped to previously unrecognized motifs that are not found in lepidopteran IAPs. Thus, the leaders of cellular IAPs from diverse insects carry unique signal-responsive degrons that control IAP turnover. Rapid response pathways that trigger IAP degradation and initiate apoptosis independent of canonical prodeath gene (Reaper-Grim-Hid) expression may provide important innate immune advantages. Furthermore, the elimination of these response motifs within viral IAPs, including those of baculoviruses, explains their unusual stability and their potent antiapoptotic activity.Apoptosis is an effective means by which a host controls virus infection. In insects, inhibitor-of-apoptosis (IAP) proteins act as regulatory sentinels by responding to cellular signals that determine the fate of infected cells. We discovered that lepidopteran (moth and butterfly) IAPs, which are degraded upon baculovirus infection, are controlled by a conserved phosphorylation-sensitive degron within the IAP N-terminal leader. The degron likely responds to virus-induced kinase-specific signals for degradation through SKP1/Cullin/F-box complex-mediated ubiquitination. Such signal-induced destruction of cellular IAPs is distinct from degradation caused by well-known IAP antagonists, which act to expel IAP-bound caspases. The major implication of this study is that insects have multiple signal-responsive mechanisms by which the sentinel IAPs are actively degraded to initiate host apoptosis. Such diversity of pathways likely provides insects with rapid and efficient strategies for pathogen control. Furthermore, the absence of analogous degrons in virus-encoded IAPs explains their relative stability and antiapoptotic potency.
Project description:The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.