yGPS-P: A Yeast-Based Peptidome Screen for Studying Quality Control-Associated Proteolysis.

Quality control-associated proteolysis (QCAP) is a fundamental mechanism that maintains cellular homeostasis by eliminating improperly folded proteins. In QCAP, the exposure of normally hidden cis-acting protein sequences, termed degrons, triggers misfolded protein ubiquitination, resulting in their elimination by the proteasome. To identify the landscape of QCAP degrons and learn about their unique features we have developed an unbiased screening method in yeast, termed yGPS-P, which facilitates the determination of thousands of proteome-derived sequences that enhance proteolysis. Here we describe the fundamental features of the yGPS-P method and provide a detailed protocol for its use as a tool for degron search. This includes the cloning of a synthetic peptidome library in a fluorescence-based reporter system, and data acquisition, which entails the combination of Fluorescence-Activated Cell Sorting (FACS) and Next-Generation Sequencing (NGS). We also provide guidelines for data extraction and analysis and for the application of a machine-learning algorithm that established the evolutionarily conserved amino acid preferences and secondary structure propensities of QCAP degrons.

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation.

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

HSP70-binding motifs function as protein quality control degrons.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Prediction of Quality-control Degradation Signals in Yeast Proteins.

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs-degrons-that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

Conserved degronome features governing quality control associated proteolysis.

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identify a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm establishes constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons are present in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of protein half-life.

Disease-linked mutations cause exposure of a protein quality control degron.

More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. Degrons are stretches of amino acids that help mediate recognition by E3 ligases and thus confer protein degradation via the ubiquitin-proteasome system. While degrons that mediate controlled degradation of, for example, signaling components and cell-cycle regulators are well described, so-called protein-quality-control degrons that mediate the degradation of destabilized proteins are poorly understood. Here, we show that disease-linked dihydrofolate reductase (DHFR) missense variants are structurally destabilized and chaperone-dependent proteasome targets. We find two regions in DHFR that act as degrons, and the proteasomal turnover of one of these was dependent on the molecular chaperone Hsp70. Structural analyses by nuclear magnetic resonance (NMR) and hydrogen/deuterium exchange revealed that this degron is buried in wild-type DHFR but becomes transiently exposed in the disease-linked missense variants.

Pls1 Is a Peroxisomal Matrix Protein with a Role in Regulating Lysine Biosynthesis.

Peroxisomes host essential metabolic enzymes and are crucial for human health and survival. Although peroxisomes were first described over 60 years ago, their entire proteome has not yet been identified. As a basis for understanding the variety of peroxisomal functions, we used a high-throughput screen to discover peroxisomal proteins in yeast. To visualize low abundance proteins, we utilized a collection of strains containing a peroxisomal marker in which each protein is expressed from the constitutive and strong TEF2 promoter. Using this approach, we uncovered 18 proteins that were not observed in peroxisomes before and could show their metabolic and targeting factor dependence for peroxisomal localization. We focus on one newly identified and uncharacterized matrix protein, Ynl097c-b, and show that it localizes to peroxisomes upon lysine deprivation and that its localization to peroxisomes depends on the lysine biosynthesis enzyme, Lys1. We demonstrate that Ynl097c-b affects the abundance of Lys1 and the lysine biosynthesis pathway. We have therefore renamed this protein Pls1 for Peroxisomal Lys1 Stabilizing 1. Our work uncovers an additional layer of regulation on the central lysine biosynthesis pathway. More generally it highlights how the discovery of peroxisomal proteins can expand our understanding of cellular metabolism.

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation.

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.

Releasing the Lockdown: An Emerging Role for the Ubiquitin-Proteasome System in the Breakdown of Transient Protein Inclusions.

Intracellular protein inclusions are diverse cellular entities with distinct biological properties. They vary in their protein content, sequestration sites, physiological function, conditions for their generation, and turnover rates. Major distinctions have been recognized between stationary amyloids and dynamic, misfolded protein deposits. The former being a dead end for irreversibly misfolded proteins, hence, cleared predominantly by autophagy, while the latter consists of a protein-quality control mechanism, important for cell endurance, where proteins are sequestered during proteotoxic stress and resolved upon its relief. Accordingly, the disaggregation of transient inclusions is a regulated process consisting of protein solubilization, followed by a triage step to either refolding or to ubiquitin-mediated degradation. Recent studies have demonstrated an indispensable role in disaggregation for components of the chaperone and the ubiquitin-proteasome systems. These include heat-shock chaperones of the 40/70/100 kDa families, the proteasome, proteasome substrate shuttling factors, and deubiquitylating enzymes. Thus, a functional link has been established between the chaperone machinery that extracts proteins from transient deposits and 26S proteasome-dependent disaggregation, indicative of a coordinated process. In this review, we discuss data emanating from these important studies and subsequently consolidate the information in the form of a working model for the disaggregation mechanism.

The extent of Ssa1/Ssa2 Hsp70 chaperone involvement in nuclear protein quality control degradation varies with the substrate.

Protein misfolding is a recurring phenomenon that cells must manage; otherwise misfolded proteins can aggregate and become toxic should they persist. To counter this burden, cells have evolved protein quality control (PQC) mechanisms that manage misfolded proteins. Two classes of systems that function in PQC are chaperones that aid in protein folding and ubiquitin-protein ligases that ubiquitinate misfolded proteins for proteasomal degradation. How folding and degradative PQC systems interact and coordinate their respective functions is not yet fully understood. Previous studies of PQC degradation pathways in the endoplasmic reticulum and cytosol have led to the prevailing idea that these pathways require the activity of Hsp70 chaperones. Here, we find that involvement of the budding yeast Hsp70 chaperones Ssa1 and Ssa2 in nuclear PQC degradation varies with the substrate. In particular, nuclear PQC degradation mediated by the yeast ubiquitin-protein ligase San1 often involves Ssa1/Ssa2, but San1 substrate recognition and ubiquitination can proceed without these Hsp70 chaperone functions in vivo and in vitro. Our studies provide new insights into the variability of Hsp70 chaperone involvement with a nuclear PQC degradation pathway.

The Hunt for Degrons of the 26S Proteasome.

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.

Assays for dissecting the in vitro enzymatic activity of yeast Ubc7.

Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation (ERAD). Yeast ERAD is composed of three Ub-protein conjugation complexes, named according to their E3 Ub-protein ligase components, Hrd1, Doa10, and the Asi complex, which resides at the nuclear envelope (NE). These ER/NE membrane-associated RING-type E3 ligases recognize and ubiquitylate defective proteins in cooperation with the E2 conjugating enzyme Ubc7 and the obligatory Ubc7 cofactor Cue1. Interaction of Ubc7 with the RING domains of its cognate E3 Ub-protein ligases stimulates the formation of isopeptide (amide) Ub-Ub linkages. Each isopeptide bond is formed by transfer of an Ubc7-linked activated Ub to a lysine side chain of an acceptor Ub. Multiple Ub transfer reactions form a poly-Ub chain that targets the conjugated protein for degradation by the proteasome. To study the mechanism of Ub-Ub bond formation, this reaction is reconstituted in a cell-free system consisting of recombinant E1, Ub, Ubc7, its cofactor Cue1, and the RING domain of either Doa10 or Hrd1. Here we provide detailed protocols for the purification of the required recombinant proteins and for the reactions that produce an Ub-Ub bond, specifically, the formation of an Ubc7~Ub thiolester (Ub charging) and subsequent formation of the isopeptide Ub-Ub linkage (Ub transfer). These protocols also provide a useful guideline for similar in vitro ubiquitylation reactions intended to explore the mechanism of other Ub-conjugation systems.

The insulin/IGF signaling cascade modulates SUMOylation to regulate aging and proteostasis in Caenorhabditis elegans.

Although aging-regulating pathways were discovered a few decades ago, it is not entirely clear how their activities are orchestrated, to govern lifespan and proteostasis at the organismal level. Here, we utilized the nematode Caenorhabditis elegans to examine whether the alteration of aging, by reducing the activity of the Insulin/IGF signaling (IIS) cascade, affects protein SUMOylation. We found that IIS activity promotes the SUMOylation of the germline protein, CAR-1, thereby shortening lifespan and impairing proteostasis. In contrast, the expression of mutated CAR-1, that cannot be SUMOylated at residue 185, extends lifespan and enhances proteostasis. A mechanistic analysis indicated that CAR-1 mediates its aging-altering functions, at least partially, through the notch-like receptor glp-1. Our findings unveil a novel regulatory axis in which SUMOylation is utilized to integrate the aging-controlling functions of the IIS and of the germline and provide new insights into the roles of SUMOylation in the regulation of organismal aging.

Integrated Proteogenomic Approach for Identifying Degradation Motifs in Eukaryotic Cells.

Since its discovery nearly 40 years ago, many components of the ubiquitin-proteasome system (UPS) have been identified and characterized in detail. However, a key aspect of the UPS that remains largely obscure is the signals that initiate the interaction of a substrate with enzymes of the UPS machinery. Understanding these signals is of particular interest for studies that examine the mechanism of substrate recognition for proteins that have adopted a non-native structure, as part of the cellular protein quality control (PQC) defense mechanism. Such studies are quite salient as the entire proteome makes up the potential battery of PQC substrates, and yet only a limited number of ubiquitination pathways are known to handle misfolded proteins. Our current research aims at understanding how a small number of PQC ubiquitin-protein ligases specifically recognize and ubiquitinate the overwhelming assortment of misfolded proteins. Here, we present a new proteogenomic approach for identifying and characterizing recognition motifs within degradation elements (degrons) in a high-throughput manner. The method utilizes yeast growth under restrictive conditions for selecting protein fragments that confer instability. The corresponding cDNA fragments are analyzed by next-generation sequencing (NGS) that provides information about each fragment's identity, reading frame, and abundance over time. This method was used by us to identify PQC-specific and compartment-specific degrons. It can readily be modified to study protein degradation signals and pathways in other organisms and in various settings, such as different strain backgrounds and under various cell conditions, all of which can be sequenced and analyzed simultaneously.

Protein Quality Control Degradation in the Nucleus.

Nuclear proteins participate in diverse cellular processes, many of which are essential for cell survival and viability. To maintain optimal nuclear physiology, the cell employs the ubiquitin-proteasome system to eliminate damaged and misfolded proteins in the nucleus that could otherwise harm the cell. In this review, we highlight the current knowledge about the major ubiquitin-protein ligases involved in protein quality control degradation (PQCD) in the nucleus and how they orchestrate their functions to eliminate misfolded proteins in different nuclear subcompartments. Many human disorders are causally linked to protein misfolding in the nucleus, hence we discuss major concepts that still need to be clarified to better understand the basis of the nuclear misfolded proteins' toxic effects. Additionally, we touch upon potential strategies for manipulating nuclear PQCD pathways to ameliorate diseases associated with protein misfolding and aggregation in the nucleus.

From Precise Slicing to General SHREDding: The Ubiquitin Ligase Ubr1 Roqs as a Multipurpose Protein Terminator.

In the current issue of Molecular Cell, Szoradi et al. (2018) present compelling data demonstrating how the newly identified SHRED pathway in yeast selectively shifts the E3 ligase Ubr1 specificity from N-end rule substrates to misfolded proteins in cells under proteostatic stress.

Temporal profiling of redox-dependent heterogeneity in single cells.

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

Mapping the Landscape of a Eukaryotic Degronome.

The ubiquitin-proteasome system (UPS) for protein degradation has been under intensive study, and yet, we have only partial understanding of mechanisms by which proteins are selected to be targeted for proteolysis. One of the obstacles in studying these recognition pathways is the limited repertoire of known degradation signals (degrons). To better understand what determines the susceptibility of intracellular proteins to degradation by the UPS, we developed an unbiased method for large-scale identification of eukaryotic degrons. Using a reporter-based high-throughput competition assay, followed by deep sequencing, we measured a degradation potency index for thousands of native polypeptides in a single experiment. We further used this method to identify protein quality control (PQC)-specific and compartment-specific degrons. Our method provides an unprecedented insight into the yeast degronome, and it can readily be modified to study protein degradation signals and pathways in other organisms and in various settings.

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.

Degradation of Ndd1 by APC/C(Cdh1) generates a feed forward loop that times mitotic protein accumulation.

Ndd1 activates the Mcm1-Fkh2 transcription factor to transcribe mitotic regulators. The anaphase-promoting complex/cyclosome activated by Cdh1 (APC/C(Cdh1)) mediates the degradation of proteins throughout G1. Here we show that the APC/C(Cdh1) ubiquitinates Ndd1 and mediates its degradation, and that APC/C(Cdh1) activity suppresses accumulation of Ndd1 targets. We confirm putative Ndd1 targets and identify novel ones, many of them APC/C(Cdh1) substrates. The APC/C(Cdh1) thus regulates these proteins in a dual manner­both pretranscriptionally and post-translationally, forming a multi-layered feedforward loop (FFL). We predict by mathematical modelling and verify experimentally that this FFL introduces a lag between APC/C(Cdh1) inactivation at the end of G1 and accumulation of genes transcribed by Ndd1 in G2. This regulation generates two classes of APC/C(Cdh1) substrates, early ones that accumulate in S and late ones that accumulate in G2. Our results show how the dual state APC/C(Cdh1) activity is converted into multiple outputs by interactions between its substrates.