Mechanisms of substrate processing during ER-associated protein degradation.

Maintaining proteome integrity is essential for long-term viability of all organisms and is overseen by intrinsic quality control mechanisms. The secretory pathway of eukaryotes poses a challenge for such quality assurance as proteins destined for secretion enter the endoplasmic reticulum (ER) and become spatially segregated from the cytosolic machinery responsible for disposal of aberrant (misfolded or otherwise damaged) or superfluous polypeptides. The elegant solution provided by evolution is ER-membrane-bound ubiquitylation machinery that recognizes misfolded or surplus proteins or by-products of protein biosynthesis in the ER and delivers them to 26S proteasomes for degradation. ER-associated protein degradation (ERAD) collectively describes this specialized arm of protein quality control via the ubiquitin-proteasome system. But, instead of providing a single strategy to remove defective or unwanted proteins, ERAD represents a collection of independent processes that exhibit distinct yet overlapping selectivity for a wide range of substrates. Not surprisingly, ER-membrane-embedded ubiquitin ligases (ER-E3s) act as central hubs for each of these separate ERAD disposal routes. In these processes, ER-E3s cooperate with a plethora of specialized factors, coordinating recognition, transport and ubiquitylation of undesirable secretory, membrane and cytoplasmic proteins. In this Review, we focus on substrate processing during ERAD, highlighting common threads as well as differences between the many routes via ERAD.

KPC1-mediated ubiquitination and proteasomal processing of NF-κB1 p105 to p50 restricts tumor growth.

NF-κB is a key transcriptional regulator involved in inflammation and cell proliferation, survival, and transformation. Several key steps in its activation are mediated by the ubiquitin (Ub) system. One uncharacterized step is limited proteasomal processing of the NF-κB1 precursor p105 to the p50 active subunit. Here, we identify KPC1 as the Ub ligase (E3) that binds to the ankyrin repeats domain of p105, ubiquitinates it, and mediates its processing both under basal conditions and following signaling. Overexpression of KPC1 inhibits tumor growth likely mediated via excessive generation of p50. Also, overabundance of p50 downregulates p65, suggesting that a p50-p50 homodimer may modulate transcription in place of the tumorigenic p50-p65. Transcript analysis reveals increased expression of genes associated with tumor-suppressive signals. Overall, KPC1 regulation of NF-κB1 processing appears to constitute an important balancing step among the stimulatory and inhibitory activities of the transcription factor in cell growth control.

Hexosamine pathway metabolites enhance protein quality control and prolong life.

Aging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases.

The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains.

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.

Who with whom: functional coordination of E2 enzymes by RING E3 ligases during poly-ubiquitylation.

Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system.

The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation.

Ubiquitin conjugation is an essential process modulating protein function in eukaryotic cells. Surprisingly, little is known about how the progressive assembly of ubiquitin chains is managed by the responsible enzymes. Only recently has ubiquitin binding activity emerged as an important factor in chain formation. The Ubc7 activator Cue1 carries a ubiquitin binding CUE domain that substantially stimulates K48-linked polyubiquitination mediated by Ubc7. Our results from NMR-based analysis and in vitro ubiquitination reactions point out that two parameters accelerate ubiquitin chain assembly: the increasing number of CUE binding sites and the position of CUE binding within a growing chain. In particular, interactions with a ubiquitin moiety adjacent to the acceptor ubiquitin facilitate chain elongation. These data indicate a mechanism for ubiquitin binding in which Cue1 positions Ubc7 and the distal acceptor ubiquitin for rapid polyubiquitination. Disrupting this mechanism results in dysfunction of the ERAD pathway by a delayed turnover of substrates.

Efficient determination of the accessible conformation space of multi-domain complexes based on EPR PELDOR data.

Many proteins can adopt multiple conformations which are important for their function. This is also true for proteins and domains that are covalently linked to each other. One important example is ubiquitin, which can form chains of different conformations depending on which of its lysine side chains is used to form an isopeptide bond with the C-terminus of another ubiquitin molecule. Similarly, ubiquitin gets covalently attached to active-site residues of E2 ubiquitin-conjugating enzymes. Due to weak interactions between ubiquitin and its interaction partners, these covalent complexes adopt multiple conformations. Understanding the function of these complexes requires the characterization of the entire accessible conformation space and its modulation by interaction partners. Long-range (1.8-10 nm) distance restraints obtained by EPR spectroscopy in the form of probability distributions are ideally suited for this task as not only the mean distance but also information about the conformation dynamics is encoded in the experimental data. Here we describe a computational method that we have developed based on well-established structure determination software using NMR restraints to calculate the accessible conformation space using PELDOR/DEER data.

DCAF8, a novel MuRF1 interaction partner, promotes muscle atrophy.

The muscle-specific RING-finger protein MuRF1 (also known as TRIM63) constitutes a bona fide ubiquitin ligase that routes proteins like several different myosin heavy chain proteins (MyHC) to proteasomal degradation during muscle atrophy. In two unbiased screens, we identified DCAF8 as a new MuRF1-binding partner. MuRF1 physically interacts with DCAF8 and both proteins localize to overlapping structures in muscle cells. Importantly, similar to what is seen for MuRF1, DCAF8 levels increase during atrophy, and the downregulation of either protein substantially impedes muscle wasting and MyHC degradation in C2C12 myotubes, a model system for muscle differentiation and atrophy. DCAF proteins typically serve as substrate receptors for cullin 4-type (Cul4) ubiquitin ligases (CRL), and we demonstrate that DCAF8 and MuRF1 associate with the subunits of such a protein complex. Because genetic downregulation of DCAF8 and inhibition of cullin activity also impair myotube atrophy in C2C12 cells, our data imply that the DCAF8 promotes muscle wasting by targeting proteins like MyHC as an integral substrate receptor of a Cul4A-containing ring ubiquitin ligase complex (CRL4A).This article has an associated First Person interview with the first author of the paper.

Ubiquitin binding by a CUE domain regulates ubiquitin chain formation by ERAD E3 ligases.

Ubiquitin-binding domains (UBDs) differentially recognize ubiquitin (ub) modifications. Some of them specifically bind mono-ub, as has been shown for the CUE domain. Interestingly, so far no significant ubiquitin binding has been observed for the CUE domain of yeast Cue1p. Cue1p is receptor and activator of the ubiquitin-conjugating enzyme Ubc7p. It integrates Ubc7p into endoplasmic reticulum (ER) membrane-bound ubiquitin ligase complexes, and thus, it is crucial for ER-associated protein degradation (ERAD). Here we show that the CUE domain of Cue1p binds ubiquitin chains, which is pivotal for the efficient formation of K48-linked polyubiquitin chains in vitro. Mutations that abolish ubiquitin binding by Cue1p affect the turnover of ERAD substrates in vivo. Our data strongly imply that the CUE domain facilitates substrate ubiquitylation by stabilizing growing ubiquitin chains at the ERAD ubiquitin ligases. Hence, we demonstrate an unexpected function of a UBD in the regulation of ubiquitin chain synthesis.

The size of the proteasomal substrate determines whether its degradation will be mediated by mono- or polyubiquitylation.

A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to ∼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.

Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.

The "canonical" proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established-in both human and yeast cells-a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes.

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9.

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between ß-strands performing an inward movement and that this movement also affects the entire ß-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.

Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms.

In cells, both newly synthesized and pre-existing proteins are constantly endangered by misfolding and aggregation. The accumulation of damaged proteins can perturb cellular homeostasis and provoke aging, pathological states, and even cell death. To avert these dangers, cells have developed powerful quality control strategies that counteract protein damage in a compartment-specific way. Here, we compare the protein quality control systems of the eukaryotic cytosol and the endoplasmic reticulum, focusing on the principles of damage recognition, the triage decisions between chaperone-mediated refolding and proteolytic elimination of damaged proteins, the repair of misfolded and aggregated protein species, and the mechanisms by which perturbations of protein homeostasis are sensed to induce compartment-specific stress responses.

A Complex of Htm1 and the Oxidoreductase Pdi1 Accelerates Degradation of Misfolded Glycoproteins.

A quality control system in the endoplasmic reticulum (ER) efficiently discriminates polypeptides that are in the process of productive folding from conformers that are trapped in an aberrant state. Only the latter are transported into the cytoplasm and degraded in a process termed ER-associated protein degradation (ERAD). In the ER, an enzymatic cascade generates a specific N-glycan structure of seven mannosyl and two N-acetylglucosamine residues (Man7GlcNAc2) on misfolded glycoproteins to facilitate their disposal. We show that a complex encompassing the yeast lectin-like protein Htm1 and the oxidoreductase Pdi1 converts Man8GlcNAc2 on glycoproteins into the Man7GlcNAc2 signal. In vitro the Htm1-Pdi1 complex processes both unfolded and native proteins albeit with a preference for the former. In vivo, elevated expression of HTM1 causes glycan trimming on misfolded and folded proteins, but only degradation of the non-native species is accelerated. Thus, modification with a Man7GlcNAc2 structure does not inevitably commit a protein for ER-associated protein degradation. The function of Htm1 in ERAD relies on its association with Pdi1, which appears to regulate the access to substrates. Our data support a model in which the balanced activities of Pdi1 and Htm1 are crucial determinants for the efficient removal of misfolded secretory glycoproteins.

Usa1 functions as a scaffold of the HRD-ubiquitin ligase.

Protein quality control in the endoplasmic reticulum is of central importance for cellular homeostasis in eukaryotes. Crucial for this process is the HRD-ubiquitin ligase (HMG-CoA reductase degradation), which singles out terminally misfolded proteins and routes them for degradation to cytoplasmic 26S-proteasomes. Certain functions of this enzyme complex are allocated to defined subunits. However, it remains unclear how these components act in a concerted manner. Here, we show that Usa1 functions as a major scaffold protein of the HRD-ligase. For the turnover of soluble substrates, Der1 binding to the C terminus of Usa1 is required. The N terminus of Usa1 associates with Hrd1 and thus bridges Der1 to Hrd1. Strikingly, the Usa1 N terminus also induces oligomerization of the HRD complex, which is an exclusive prerequisite for the degradation of membrane proteins. Our data demonstrate that scaffold proteins are required to adapt ubiquitin ligase activities toward different classes of substrates.

Disassembly of Lys11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6.

Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly 2 orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the autoinhibitory role of its C terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared with homogeneous Lys(48)-linked chains. The reduced ability to disassemble homogeneous Lys(48)-linked chains longer than 4 Ub units may prolong residency time on the proteasome.

Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system.

Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

The ubiquitylation machinery of the endoplasmic reticulum.

As proteins travel through the endoplasmic reticulum (ER), a quality-control system retains newly synthesized polypeptides and supports their maturation. Only properly folded proteins are released to their designated destinations. Proteins that cannot mature are left to accumulate, impairing the function of the ER. To maintain homeostasis, the protein-quality-control system singles out aberrant polypeptides and delivers them to the cytosol, where they are destroyed by the proteasome. The importance of this pathway is evident from the growing list of pathologies associated with quality-control defects in the ER.

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types-lysine 48-linked (K48) and lysine 63-linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors-chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.