Mechanism of degrader-targeted protein ubiquitinability.

Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.

Concerted SUMO-targeted ubiquitin ligase activities of TOPORS and RNF4 are essential for stress management and cell proliferation.

Protein SUMOylation provides a principal driving force for cellular stress responses, including DNA-protein crosslink (DPC) repair and arsenic-induced PML body degradation. In this study, using genome-scale screens, we identified the human E3 ligase TOPORS as a key effector of SUMO-dependent DPC resolution. We demonstrate that TOPORS promotes DPC repair by functioning as a SUMO-targeted ubiquitin ligase (STUbL), combining ubiquitin ligase activity through its RING domain with poly-SUMO binding via SUMO-interacting motifs, analogous to the STUbL RNF4. Mechanistically, TOPORS is a SUMO1-selective STUbL that complements RNF4 in generating complex ubiquitin landscapes on SUMOylated targets, including DPCs and PML, stimulating efficient p97/VCP unfoldase recruitment and proteasomal degradation. Combined loss of TOPORS and RNF4 is synthetic lethal even in unstressed cells, involving defective clearance of SUMOylated proteins from chromatin accompanied by cell cycle arrest and apoptosis. Our findings establish TOPORS as a STUbL whose parallel action with RNF4 defines a general mechanistic principle in crucial cellular processes governed by direct SUMO-ubiquitin crosstalk.

Adenovirus E1B-55K controls SUMO-dependent degradation of antiviral cellular restriction factors.

IMPORTANCE: Human adenoviruses (HAdVs) generally cause mild and self-limiting diseases of the upper respiratory and gastrointestinal tracts but pose a serious risk to immunocompromised patients and children. Moreover, they are widely used as vectors for vaccines and vector-based gene therapy approaches. It is therefore vital to thoroughly characterize HAdV gene products and especially HAdV virulence factors. Early region 1B 55 kDa protein (E1B-55K) is a multifunctional HAdV-encoded oncoprotein involved in various viral and cellular pathways that promote viral replication and cell transformation. We analyzed the E1B-55K dependency of SUMOylation, a post-translational protein modification, in infected cells using quantitative proteomics. We found that HAdV increases overall cellular SUMOylation and that this increased SUMOylation can target antiviral cellular pathways that impact HAdV replication. Moreover, we showed that E1B-55K orchestrates the SUMO-dependent degradation of certain cellular antiviral factors. These results once more emphasize the key role of E1B-55K in the regulation of viral and cellular proteins in productive HAdV infections.

SUMO protease SENP6 protects the nucleus from hyperSUMOylation-induced laminopathy-like alterations.

The small ubiquitin-like modifier (SUMO) protease SENP6 disassembles SUMO chains from cellular substrate proteins. We use a proteomic method to identify putative SENP6 substrates based on increased apparent molecular weight after SENP6 depletion. Proteins of the lamin family of intermediate filaments show substantially increased SUMO modification after SENP6 depletion. This is accompanied by nuclear structural changes remarkably like those associated with laminopathies. Two SUMO attachment sites on lamin A/C are close to sites of mutations in Emery-Driefuss and limb girdle muscular dystrophy. To establish a direct link between lamin SUMOylation and the observed phenotype, we developed proximity-induced SUMO modification (PISM), which fuses a lamin A/C targeting DARPin to a SUMO E3 ligase domain. This directly targets lamin A/C for SUMO conjugation and demonstrates that enhanced lamin SUMO modification recapitulates the altered nuclear structure manifest after SENP6 depletion. This shows SENP6 activity protects the nucleus against hyperSUMOylation-induced laminopathy-like alterations.

Changes in SUMO-modified proteins in Epstein-Barr virus infection identifies reciprocal regulation of TRIM24/28/33 complexes and the lytic switch BZLF1.

SUMO modifications regulate the function of many proteins and are important in controlling herpesvirus infections. We performed a site-specific proteomic analysis of SUMO1- and SUMO2-modified proteins in Epstein-Barr virus (EBV) latent and lytic infection to identify proteins that change in SUMO modification status in response to EBV reactivation. Major changes were identified in all three components of the TRIM24/TRIM28/TRIM33 complex, with TRIM24 being rapidly degraded and TRIM33 being phosphorylated and SUMOylated in response to EBV lytic infection. Further experiments revealed TRIM24 and TRIM33 repress expression of the EBV BZLF1 lytic switch gene, suppressing EBV reactivation. However, BZLF1 was shown to interact with TRIM24 and TRIM33, resulting in disruption of TRIM24/TRIM28/TRIM33 complexes, degradation of TRIM24 and modification followed by degradation of TRIM33. Therefore, we have identified TRIM24 and TRIM33 as cellular antiviral defence factors against EBV lytic infection and established the mechanism by which BZLF1 disables this defence.

Lysine deserts prevent adventitious ubiquitylation of ubiquitin-proteasome components.

In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic speckles/inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to nonspecific ubiquitylation.

The p97/VCP segregase is essential for arsenic-induced degradation of PML and PML-RARA.

Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)-Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

Identification of SUMO Targets Associated With the Pluripotent State in Human Stem Cells.

To investigate the role of SUMO modification in the maintenance of pluripotent stem cells, we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 resulted in the loss of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with C-terminal TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created three networks of proteins with functions in regulation of gene expression, ribosome biogenesis, and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The rest have roles in transcription and the regulation of chromatin structure. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centered on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins, there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO influences the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

The SUMOylation pathway suppresses arbovirus replication in Aedes aegypti cells.

Mosquitoes are responsible for the transmission of many clinically important arboviruses that cause significant levels of annual mortality and socioeconomic health burden worldwide. Deciphering the mechanisms by which mosquitoes modulate arbovirus infection is crucial to understand how viral-host interactions promote vector transmission and human disease. SUMOylation is a post-translational modification that leads to the covalent attachment of the Small Ubiquitin-like MOdifier (SUMO) protein to host factors, which in turn can modulate their stability, interaction networks, sub-cellular localisation, and biochemical function. While the SUMOylation pathway is known to play a key role in the regulation of host immune defences to virus infection in humans, the importance of this pathway during arbovirus infection in mosquito vectors, such as Aedes aegypti (Ae. aegypti), remains unknown. Here we characterise the sequence, structure, biochemical properties, and tissue-specific expression profiles of component proteins of the Ae. aegypti SUMOylation pathway. We demonstrate significant biochemical differences between Ae. aegypti and Homo sapiens SUMOylation pathways and identify cell-type specific patterns of SUMO expression in Ae. aegypti tissues known to support arbovirus replication. Importantly, depletion of core SUMOylation effector proteins (SUMO, Ubc9 and PIAS) in Ae. aegypti cells led to enhanced levels of arbovirus replication from three different families; Zika (Flaviviridae), Semliki Forest (Togaviridae), and Bunyamwera (Bunyaviridae) viruses. Our findings identify an important role for mosquito SUMOylation in the cellular restriction of arboviruses that may directly influence vector competence and transmission of clinically important arboviruses.

Downregulation of Keap1 Confers Features of a Fasted Metabolic State.

Transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator, Kelch-like ECH-associated protein 1 (Keap1), are at the interface between redox and intermediary metabolism, allowing adaptation and survival under conditions of oxidative, inflammatory, and metabolic stress. Nrf2 is the principal determinant of redox homeostasis, and contributes to mitochondrial function and integrity and cellular bioenergetics. Using proteomics and lipidomics, we show that genetic downregulation of Keap1 in mice, and the consequent Nrf2 activation to pharmacologically relevant levels, leads to upregulation of carboxylesterase 1 (Ces1) and acyl-CoA oxidase 2 (Acox2), decreases triglyceride levels, and alters the lipidome. This is accompanied by downregulation of hepatic ATP-citrate lyase (Acly) and decreased levels of acetyl-CoA, a trigger for autophagy. These findings suggest that downregulation of Keap1 confers features of a fasted metabolic state, which is an important consideration in the drug development of Keap1-targeting pharmacologic Nrf2 activators.

Antibody RING-Mediated Destruction of Endogenous Proteins.

To understand gene function, the encoding DNA or mRNA transcript can be manipulated and the consequences observed. However, these approaches do not have a direct effect on the protein product of the gene, which is either permanently abrogated or depleted at a rate defined by the half-life of the protein. We therefore developed a single-component system that could induce the rapid degradation of the specific endogenous protein itself. A construct combining the RING domain of ubiquitin E3 ligase RNF4 with a protein-specific camelid nanobody mediates target destruction by the ubiquitin proteasome system, a process we describe as antibody RING-mediated destruction (ARMeD). The technique is highly specific because we observed no off-target protein destruction. Furthermore, bacterially produced nanobody-RING fusion proteins electroporated into cells induce degradation of target within minutes. With increasing availability of protein-specific nanobodies, this method will allow rapid and specific degradation of a wide range of endogenous proteins.

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε.

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.

An influenza virus-triggered SUMO switch orchestrates co-opted endogenous retroviruses to stimulate host antiviral immunity.

Dynamic small ubiquitin-like modifier (SUMO) linkages to diverse cellular protein groups are critical to orchestrate resolution of stresses such as genome damage, hypoxia, or proteotoxicity. Defense against pathogen insult (often reliant upon host recognition of "non-self" nucleic acids) is also modulated by SUMO, but the underlying mechanisms are incompletely understood. Here, we used quantitative SILAC-based proteomics to survey pan-viral host SUMOylation responses, creating a resource of almost 600 common and unique SUMO remodeling events that are mounted during influenza A and B virus infections, as well as during viral innate immune stimulation. Subsequent mechanistic profiling focused on a common infection-induced loss of the SUMO-modified form of TRIM28/KAP1, a host transcriptional repressor. By integrating knockout and reconstitution models with system-wide transcriptomics, we provide evidence that influenza virus-triggered loss of SUMO-modified TRIM28 leads to derepression of endogenous retroviral (ERV) elements, unmasking this cellular source of "self" double-stranded (ds)RNA. Consequently, loss of SUMO-modified TRIM28 potentiates canonical cytosolic dsRNA-activated IFN-mediated defenses that rely on RIG-I, MAVS, TBK1, and JAK1. Intriguingly, although wild-type influenza A virus robustly triggers this SUMO switch in TRIM28, the induction of IFN-stimulated genes is limited unless expression of the viral dsRNA-binding protein NS1 is abrogated. This may imply a viral strategy to antagonize such a host response by sequestration of induced immunostimulatory ERV dsRNAs. Overall, our data reveal that a key nuclear mechanism that normally prevents aberrant expression of ERV elements (ERVs) has been functionally co-opted via a stress-induced SUMO switch to augment antiviral immunity.

Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and ß-Catenin-Independent Targets by Proteomics.

Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of ß-catenin, but the extent to which APC exerts Wnt/ß-catenin-independent tumor-suppressive activity is unclear. To identify interaction partners and ß-catenin-independent targets of endogenous, full-length APC, we applied label-free and multiplexed tandem mass tag-based mass spectrometry. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, our global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of ß-catenin. Combining these two analyses, we identified Misshapen-like kinase 1 (MINK1) as a putative substrate of an APC-containing destruction complex. We validated the interaction between endogenous MINK1 and APC and further confirmed the negative, and ß-catenin-independent, regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared with wild-type tissue/cells. Collectively, our results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. IMPLICATIONS: The tumor-suppressive function of APC, the most frequently mutated gene in colorectal cancer, is mainly attributed to its role in ß-catenin/Wnt signaling. Our study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by ß-catenin-independent mechanisms.

Multiomics Analyses of HNF4α Protein Domain Function during Human Pluripotent Stem Cell Differentiation.

During mammalian development, liver differentiation is driven by signals that converge on multiple transcription factor networks. The hepatocyte nuclear factor signaling network is known to be essential for hepatocyte specification and maintenance. In this study, we have generated deletion and point mutants of hepatocyte nuclear factor-4alpha (HNF4α) to precisely evaluate the function of protein domains during hepatocyte specification from human pluripotent stem cells. We demonstrate that nuclear HNF4α is essential for hepatic progenitor specification, and the introduction of point mutations in HNF4α's Small Ubiquitin-like Modifier (SUMO) consensus motif leads to disrupted hepatocyte differentiation. Taking a multiomics approach, we identified key deficiencies in cell biology, which included dysfunctional metabolism, substrate adhesion, tricarboxylic acid cycle flux, microRNA transport, and mRNA processing. In summary, the combination of genome editing and multiomics analyses has provided valuable insight into the diverse functions of HNF4α during pluripotent stem cell entry into the hepatic lineage and during hepatocellular differentiation.

Expanded Interactome of the Intrinsically Disordered Protein Dss1.

Dss1 (also known as Sem1) is a conserved, intrinsically disordered protein with a remarkably broad functional diversity. It is a proteasome subunit but also associates with the BRCA2, RPA, Csn12-Thp1, and TREX-2 complexes. Accordingly, Dss1 functions in protein degradation, DNA repair, transcription, and mRNA export. Here in Schizosaccharomyces pombe, we expand its interactome further to include eIF3, the COP9 signalosome, and the mitotic septins. Within its intrinsically disordered ensemble, Dss1 forms a transiently populated C-terminal helix that dynamically interacts with and shields a central binding region. The helix interfered with the interaction to ATP-citrate lyase but was required for septin binding, and in strains lacking Dss1, ATP-citrate lyase solubility was reduced and septin rings were more persistent. Thus, even weak, transient interactions within Dss1 may dynamically rewire its interactome.

Predicting the impact of Lynch syndrome-causing missense mutations from structural calculations.

Accurate methods to assess the pathogenicity of mutations are needed to fully leverage the possibilities of genome sequencing in diagnosis. Current data-driven and bioinformatics approaches are, however, limited by the large number of new variations found in each newly sequenced genome, and often do not provide direct mechanistic insight. Here we demonstrate, for the first time, that saturation mutagenesis, biophysical modeling and co-variation analysis, performed in silico, can predict the abundance, metabolic stability, and function of proteins inside living cells. As a model system, we selected the human mismatch repair protein, MSH2, where missense variants are known to cause the hereditary cancer predisposition disease, known as Lynch syndrome. We show that the majority of disease-causing MSH2 mutations give rise to folding defects and proteasome-dependent degradation rather than inherent loss of function, and accordingly our in silico modeling data accurately identifies disease-causing mutations and outperforms the traditionally used genetic disease predictors. Thus, in conclusion, in silico biophysical modeling should be considered for making genotype-phenotype predictions and for diagnosis of Lynch syndrome, and perhaps other hereditary diseases.

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication.

Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 "segregase". Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway.

The ubiquitin-proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.