Dipeptidyl peptidases and E3 ligases of N-degron pathways cooperate to regulate protein stability.

N-degrons are short sequences located at protein N-terminus that mediate the interaction of E3 ligases (E3s) with substrates to promote their proteolysis. It is well established that N-degrons can be exposed following protease cleavage to allow recognition by E3s. However, our knowledge regarding how proteases and E3s cooperate in protein quality control mechanisms remains minimal. Using a systematic approach to monitor the protein stability of an N-terminome library, we found that proline residue at the third N-terminal position (hereafter "P+3") promotes instability. Genetic perturbations identified the dipeptidyl peptidases DPP8 and DPP9 and the primary E3s of N-degron pathways, UBR proteins, as regulators of P+3 bearing substrate turnover. Interestingly, P+3 UBR substrates are significantly enriched for secretory proteins. We found that secretory proteins relying on a signal peptide (SP) for their targeting contain a "built-in" N-degron within their SP. This degron becomes exposed by DPP8/9 upon translocation failure to the designated compartments, thus enabling clearance of mislocalized proteins by UBRs to maintain proteostasis.

Mechanism of Ψ-Pro/C-degron recognition by the CRL2FEM1B ubiquitin ligase.

The E3 ligase-degron interaction determines the specificity of the ubiquitin‒proteasome system. We recently discovered that FEM1B, a substrate receptor of Cullin 2-RING ligase (CRL2), recognizes C-degrons containing a C-terminal proline. By solving several cryo-EM structures of CRL2FEM1B bound to different C-degrons, we elucidate the dimeric assembly of the complex. Furthermore, we reveal distinct dimerization states of unmodified and neddylated CRL2FEM1B to uncover the NEDD8-mediated activation mechanism of CRL2FEM1B. Our research also indicates that, FEM1B utilizes a bipartite mechanism to recognize both the C-terminal proline and an upstream aromatic residue within the substrate. These structural findings, complemented by in vitro ubiquitination and in vivo cell-based assays, demonstrate that CRL2FEM1B-mediated polyubiquitination and subsequent protein turnover depend on both FEM1B-degron interactions and the dimerization state of the E3 ligase complex. Overall, this study deepens our molecular understanding of how Cullin-RING E3 ligase substrate selection mediates protein turnover.

Defining E3 ligase-substrate relationships through multiplex CRISPR screening.

Specificity within the ubiquitin-proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates-and in particular the molecular features (degrons) that they recognize-remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin-proteasome system.

Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping.

The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Germline-encoded amino acid-binding motifs drive immunodominant public antibody responses.

Despite the vast diversity of the antibody repertoire, infected individuals often mount antibody responses to precisely the same epitopes within antigens. The immunological mechanisms underpinning this phenomenon remain unknown. By mapping 376 immunodominant "public epitopes" at high resolution and characterizing several of their cognate antibodies, we concluded that germline-encoded sequences in antibodies drive recurrent recognition. Systematic analysis of antibody-antigen structures uncovered 18 human and 21 partially overlapping mouse germline-encoded amino acid-binding (GRAB) motifs within heavy and light V gene segments that in case studies proved critical for public epitope recognition. GRAB motifs represent a fundamental component of the immune system's architecture that promotes recognition of pathogens and leads to species-specific public antibody responses that can exert selective pressure on pathogens.

A central role for regulated protein stability in the control of TFE3 and MITF by nutrients.

The TFE3 and MITF master transcription factors maintain metabolic homeostasis by regulating lysosomal, melanocytic, and autophagy genes. Previous studies posited that their cytosolic retention by 14-3-3, mediated by the Rag GTPases-mTORC1, was key for suppressing transcriptional activity in the presence of nutrients. Here, we demonstrate using mammalian cells that regulated protein stability plays a fundamental role in their control. Amino acids promote the recruitment of TFE3 and MITF to the lysosomal surface via the Rag GTPases, activating an evolutionarily conserved phospho-degron and leading to ubiquitination by CUL1ß-TrCP and degradation. Elucidation of the minimal functional degron revealed a conserved alpha-helix required for interaction with RagA, illuminating the molecular basis for a severe neurodevelopmental syndrome caused by missense mutations in TFE3 within the RagA-TFE3 interface. Additionally, the phospho-degron is recurrently lost in TFE3 genomic translocations that cause kidney cancer. Therefore, two divergent pathologies converge on the loss of protein stability regulation by nutrients.

High-throughput, targeted MHC class I immunopeptidomics using a functional genetics screening platform.

Identification of CD8+ T cell epitopes is critical for the development of immunotherapeutics. Existing methods for major histocompatibility complex class I (MHC class I) ligand discovery are time intensive, specialized and unable to interrogate specific proteins on a large scale. Here, we present EpiScan, which uses surface MHC class I levels as a readout for whether a genetically encoded peptide is an MHC class I ligand. Predetermined starting pools composed of >100,000 peptides can be designed using oligonucleotide synthesis, permitting large-scale MHC class I screening. We exploit this programmability of EpiScan to uncover an unappreciated role for cysteine that increases the number of predicted ligands by 9-21%, reveal affinity hierarchies by analysis of biased anchor peptide libraries and screen viral proteomes for MHC class I ligands. Using these data, we generate and iteratively refine peptide binding predictions to create EpiScan Predictor. EpiScan Predictor performs comparably to other state-of-the-art MHC class I peptide binding prediction algorithms without suffering from underrepresentation of cysteine-containing peptides. Thus, targeted immunopeptidomics using EpiScan will accelerate CD8+ T cell epitope discovery toward the goal of individual-specific immunotherapeutics.

Systematic characterization of mutations altering protein degradation in human cancers.

The ubiquitin-proteasome system (UPS) is the primary route for selective protein degradation in human cells. The UPS is an attractive target for novel cancer therapies, but the precise UPS genes and substrates important for cancer growth are incompletely understood. Leveraging multi-omics data across more than 9,000 human tumors and 33 cancer types, we found that over 19% of all cancer driver genes affect UPS function. We implicate transcription factors as important substrates and show that c-Myc stability is modulated by CUL3. Moreover, we developed a deep learning model (deepDegron) to identify mutations that result in degron loss and experimentally validated the prediction that gain-of-function truncating mutations in GATA3 and PPM1D result in increased protein stability. Last, we identified UPS driver genes associated with prognosis and the tumor microenvironment. This study demonstrates the important role of UPS dysregulation in human cancer and underscores the potential therapeutic utility of targeting the UPS.

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control.

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity.

Understanding humoral responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics, therapeutics, and vaccines. Deep serological profiling of 232 coronavirus disease 2019 (COVID-19) patients and 190 pre-COVID-19 era controls using VirScan revealed more than 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies. Preexisting antibodies in controls recognized SARS-CoV-2 ORF1, whereas only COVID-19 patient antibodies primarily recognized spike protein and nucleoprotein. A machine learning model trained on VirScan data predicted SARS-CoV-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-CoV-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-CoV-2 responses, weaker antibody responses to prior infections, and higher incidence of cytomegalovirus and herpes simplex virus 1, possibly influenced by demographic covariates. Among hospitalized patients, males produce stronger SARS-CoV-2 antibody responses than females.

Periphilin self-association underpins epigenetic silencing by the HUSH complex.

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.

Tying up loose ends: the N-degron and C-degron pathways of protein degradation.

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.

A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation.

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.

Differential viral accessibility (DIVA) identifies alterations in chromatin architecture through large-scale mapping of lentiviral integration sites.

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis.

The sterol-responsive RNF145 E3 ubiquitin ligase mediates the degradation of HMG-CoA reductase together with gp78 and Hrd1.

Mammalian HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the cholesterol biosynthetic pathway and the therapeutic target of statins, is post-transcriptionally regulated by sterol-accelerated degradation. Under cholesterol-replete conditions, HMGCR is ubiquitinated and degraded, but the identity of the E3 ubiquitin ligase(s) responsible for mammalian HMGCR turnover remains controversial. Using systematic, unbiased CRISPR/Cas9 genome-wide screens with a sterol-sensitive endogenous HMGCR reporter, we comprehensively map the E3 ligase landscape required for sterol-accelerated HMGCR degradation. We find that RNF145 and gp78 independently co-ordinate HMGCR ubiquitination and degradation. RNF145, a sterol-responsive ER-resident E3 ligase, is unstable but accumulates following sterol depletion. Sterol addition triggers RNF145 recruitment to HMGCR via Insigs, promoting HMGCR ubiquitination and proteasome-mediated degradation. In the absence of both RNF145 and gp78, Hrd1, a third UBE2G2-dependent E3 ligase, partially regulates HMGCR activity. Our findings reveal a critical role for the sterol-responsive RNF145 in HMGCR regulation and elucidate the complexity of sterol-accelerated HMGCR degradation. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

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.

The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes.

Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of an SVA retrotransposon reporter. By using naïve versus primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. Although the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages <5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s, and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon noncoding DNA.