bims-proteo 2023-06-18 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–06–18
thirty papers selected by
Eric Chevet, INSERM

C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.
PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.
Co-translational binding of importins to nascent proteins.
Syntaxin 5-dependent phosphorylation of the small heat shock protein Hsp42 and its role in protein quality control.
The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions.
TMX4-driven LINC complex disassembly and asymmetric autophagy of the nuclear envelope upon acute ER stress.
Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.
Reducing oxidative protein folding alleviates senescence by minimizing ER-to-nucleus H2 O2 release.
Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.
Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.
ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation.
A mitochondrial iron-responsive pathway regulated by DELE1.
LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.
Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic "integrated stress response".
The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection.
Expanding EMC foldopathies: Topogenesis deficits alter the neural crest.
Astrocytic deletion of protein kinase R-like ER kinase (PERK) does not affect learning and memory in aged mice but worsens outcome from experimental stroke.
Chemically induced degradation of epigenetic targets.
The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation.
Dynamic interactome of the MHC I peptide loading complex in human dendritic cells.
Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation.
Targeting the ATF6-mediated ER stress response and autophagy blocks integrin-driven prostate cancer progression.
An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.
Aggregation prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity.
Nascent Proteomics: Chemical Tools for Monitoring Newly Synthesized Proteins.
Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.
Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.
Bioorthogonal PROTAC Prodrugs Enabled by On-Target Activation.
Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.
Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

EMBO Rep. 2023 Jun 15. e55895

C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.

Julia Jülg, Dieter Edbauer, Christian Behrends.

  Hexanucleotide repeat expansions within C9orf72 are a frequent cause of amyotrophic lateral sclerosis and frontotemporal dementia. Haploinsufficiency leading to reduced C9orf72 protein contributes to disease pathogenesis. C9orf72 binds SMCR8 to form a robust complex that regulates small GTPases, lysosomal integrity, and autophagy. In contrast to this functional understanding, we know far less about the assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ablation of the respective partner. However, the molecular mechanism underlying this interdependence remains elusive. Here, we identify C9orf72 as a substrate of branched ubiquitin chain-dependent protein quality control. We find that SMCR8 prevents C9orf72 from rapid degradation by the proteasome. Mass spectrometry and biochemical analyses reveal the E3 ligase UBR5 and the BAG6 chaperone complex as C9orf72-interacting proteins, which are components of the machinery that modifies proteins with K11/K48-linked heterotypic ubiquitin chains. Depletion of UBR5 results in reduced K11/K48 ubiquitination and increased C9orf72 when SMCR8 is absent. Our data provide novel insights into C9orf72 regulation with potential implication for strategies to antagonize C9orf72 loss during disease progression.

Keywords:  BAG6 complex; C9orf72; K11/K48-linked ubiquitin; UBR5; heterotypic ubiquitin chains

DOI:  https://doi.org/10.15252/embr.202255895
EMBO J. 2023 Jun 12. e113908

PERK signaling promotes mitochondrial elongation by remodeling membrane phosphatidic acid.

Valerie Perea, Christian Cole, Justine Lebeau, Vivian Dolina, Kelsey R Baron, Aparajita Madhavan, Jeffery W Kelly, Danielle A Grotjahn, R Luke Wiseman.

  Endoplasmic reticulum (ER) stress and mitochondrial dysfunction are linked in the onset and pathogenesis of numerous diseases. This has led to considerable interest in defining the mechanisms responsible for regulating mitochondria during ER stress. The PERK signaling arm of the unfolded protein response (UPR) has emerged as a prominent ER stress-responsive signaling pathway that regulates diverse aspects of mitochondrial biology. Here, we show that PERK activity promotes adaptive remodeling of mitochondrial membrane phosphatidic acid (PA) to induce protective mitochondrial elongation during acute ER stress. We find that PERK activity is required for ER stress-dependent increases in both cellular PA and YME1L-dependent degradation of the intramitochondrial PA transporter PRELID1. These two processes lead to the accumulation of PA on the outer mitochondrial membrane where it can induce mitochondrial elongation by inhibiting mitochondrial fission. Our results establish a new role for PERK in the adaptive remodeling of mitochondrial phospholipids and demonstrate that PERK-dependent PA regulation adapts organellar shape in response to ER stress.

Keywords:  endoplasmic reticulum (ER) stress; mitochondrial morphology; phosphatidic acid; unfolded protein response (UPR)

DOI:  https://doi.org/10.15252/embj.2023113908
Nat Commun. 2023 Jun 09. 14(1): 3418

Co-translational binding of importins to nascent proteins.

Maximilian Seidel, Natalie Romanov, Agnieszka Obarska-Kosinska, Anja Becker, Nayara Trevisan Doimo de Azevedo, Jan Provaznik, Sankarshana R Nagaraja, Jonathan J M Landry, Vladimir Benes, Martin Beck.

Various cellular quality control mechanisms support proteostasis. While, ribosome-associated chaperones prevent the misfolding of nascent chains during translation, importins were shown to prevent the aggregation of specific cargoes in a post-translational mechanism prior the import into the nucleoplasm. Here, we hypothesize that importins may already bind ribosome-associated cargo in a co-translational manner. We systematically measure the nascent chain association of all importins in Saccharomyces cerevisiae by selective ribosome profiling. We identify a subset of importins that bind to a wide range of nascent, often uncharacterized cargoes. This includes ribosomal proteins, chromatin remodelers and RNA binding proteins that are aggregation prone in the cytosol. We show that importins act consecutively with other ribosome-associated chaperones. Thus, the nuclear import system is directly intertwined with nascent chain folding and chaperoning.

DOI: https://doi.org/10.1038/s41467-023-39150-9
FEBS J. 2023 Jun 12.

Syntaxin 5-dependent phosphorylation of the small heat shock protein Hsp42 and its role in protein quality control.

Doryaneh Ahmadpour, Navinder Kumar, Arthur Fischbach, Srishti Chawla, Per O Widlund, Thomas Nyström.

  The small heat shock protein Hsp42 and the t-SNARE protein Sed5 have central roles in the sequestration of misfolded proteins into insoluble protein deposits in the yeast Saccharomyces cerevisiae. However, whether these proteins/processes interact in protein quality control (PQC) is not known. Here, we show that Sed5 and anterograde trafficking modulates phosphorylation of Hsp42 partially via the MAPK kinase Hog1. Such phosphorylation, specifically at residue S215, abrogated the co-localization of Hsp42 with the Hsp104 disaggregase, aggregate clearance, chaperone activity, and sequestration of aggregates to IPOD and mitochondria. Further, we found that Hsp42 is hyper-phosphorylated in old cells leading to a drastic failure in disaggregation. Old cells also displayed a retarded anterograde trafficking, which, together with slow aggregate clearance and hyper-phosphorylation of Hsp42, could be counteracted by Sed5 overproduction. We hypothesize that the breakdown of proper PQC during yeast aging may, in part, be due to a retarded anterograde trafficking leading to hyper-phosphorylation of Hsp42.

Keywords:  Small Heat shock protein Hsp42; Syntaxin 5; ageing; phosphorylation; protein quality control

DOI:  https://doi.org/10.1111/febs.16886
Mol Cell. 2023 Jun 15. pii: S1097-2765(23)00377-5. [Epub ahead of print]83(12): 2035-2044.e7

The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions.

Janhavi A Kolhe, Neethu L Babu, Brian C Freeman.

  Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach, we characterized the features driving the Hsp90 physical interactome. We found that Hsp90 associated with ∼20% of the yeast proteome using its three domains to preferentially target intrinsically disordered regions (IDRs) of client proteins. Hsp90 selectively utilized an IDR to regulate client activity as well as maintained IDR-protein health by preventing the transition to stress granules or P-bodies at physiological temperatures. We also discovered that Hsp90 controls the fidelity of ribosome initiation that triggers a heat shock response when disrupted. Our study provides insights into how this abundant molecular chaperone supports a dynamic and healthy native protein landscape.

Keywords:  Hsp90; fidelity of translation initiation; intrinsically disordered regions; molecular chaperone; proteostasis

DOI:  https://doi.org/10.1016/j.molcel.2023.05.021
Nat Commun. 2023 Jun 13. 14(1): 3497

TMX4-driven LINC complex disassembly and asymmetric autophagy of the nuclear envelope upon acute ER stress.

Marika K Kucińska, Juliette Fedry, Carmela Galli, Diego Morone, Andrea Raimondi, Tatiana Soldà, Friedrich Förster, Maurizio Molinari.

The endoplasmic reticulum (ER) is an organelle of nucleated cells that produces proteins, lipids and oligosaccharides. ER volume and activity are increased upon induction of unfolded protein responses (UPR) and are reduced upon activation of ER-phagy programs. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM) separated by the perinuclear space (PNS). Here we report that expansion of the mammalian ER upon homeostatic perturbations results in TMX4 reductase-driven disassembly of the LINC complexes connecting INM and ONM and in ONM swelling. The physiologic distance between ONM and INM is restored, upon resolution of the ER stress, by asymmetric autophagy of the NE, which involves the LC3 lipidation machinery, the autophagy receptor SEC62 and the direct capture of ONM-derived vesicles by degradative LAMP1/RAB7-positive endolysosomes in a catabolic pathway mechanistically defined as micro-ONM-phagy.

DOI: https://doi.org/10.1038/s41467-023-39172-3
Sci Adv. 2023 Jun 16. 9(24): eadd8910

Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.

Dmitry Zenko, Jade Marsh, Andrew R Castle, Rahel Lewin, Roman Fischer, George K Tofaris.

While defective α-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

DOI: https://doi.org/10.1126/sciadv.add8910
EMBO Rep. 2023 Jun 12. e56439

Reducing oxidative protein folding alleviates senescence by minimizing ER-to-nucleus H2 O2 release.

Fang Cheng, Qianzhao Ji, Lu Wang, Chih-Chen Wang, Guang-Hui Liu, Lei Wang.

  Oxidative protein folding occurs in the endoplasmic reticulum (ER) to generate disulfide bonds, and the by-product is hydrogen peroxide (H2 O2 ). However, the relationship between oxidative protein folding and senescence remains uncharacterized. Here, we find that the protein disulfide isomerase (PDI), a key oxidoreductase that catalyzes oxidative protein folding, accumulated in aged human mesenchymal stem cells (hMSCs) and deletion of PDI alleviated hMSCs senescence. Mechanistically, knocking out PDI slows the rate of oxidative protein folding and decreases the leakage of ER-derived H2 O2 into the nucleus, thereby decreasing the expression of SERPINE1, which was identified as a key driver of cell senescence. Furthermore, we show that depletion of PDI alleviated senescence in various cell models of aging. Our findings reveal a previously unrecognized role of oxidative protein folding in promoting cell aging, providing a potential target for aging and aging-related disease intervention.

Keywords:  human mesenchymal stem cells (hMSCs); hydrogen peroxide (H2O2); oxidative protein folding; protein disulfide isomerase (PDI); senescence

DOI:  https://doi.org/10.15252/embr.202256439
J Biol Chem. 2023 Jun 12. pii: S0021-9258(23)01943-9. [Epub ahead of print] 104915

Ubiquitin-like protein 5 is a novel player in the UPR-PERK arm and ER stress-induced cell death.

Wei Wang, Adam M Hawkridge, Yibao Ma, Bei Zhang, John B Mangrum, Zaneera H Hassan, Tianhai He, Sofiya Blat, Chunqing Guo, Huiping Zhou, Jinze Liu, Xiang-Yang Wang, Xianjun Fang.

  Biological functions of the highly conserved ubiquitin-like protein 5 (UBL5) are not well understood. In C. elegans, UBL5 is induced under mitochondrial stress to mount the mitochondrial unfolded protein response (UPRmt). However, the role of UBL5 in the more prevalent endoplasmic reticulum (ER) stress-unfolded protein response (UPR) in the mammalian system is unknown. In the present work, we demonstrated that UBL5 was an ER stress-responsive protein, undergoing rapid depletion in mammalian cells and livers of mice. The ER stress-induced UBL5 depletion was mediated by proteasome-dependent yet ubiquitin-independent proteolysis. Activation of the protein kinase R-like endoplasmic reticulum kinase (PERK) arm of the UPR was essential and sufficient for inducing UBL5 degradation. RNA-Seq analysis of UBL5-regulated transcriptome revealed that multiple death pathways were activated in UBL5-silenced cells. In agreement with this, UBL5 knockdown induced severe apoptosis in culture and suppressed tumorigenicity of cancer cells in vivo. Furthermore, overexpression of UBL5 protected specifically against ER stress-induced apoptosis. These results identify UBL5 as a physiologically relevant survival regulator that is proteolytically depleted by the UPR-PERK pathway, linking ER stress to cell death.

Keywords:  ER stress; PERK; UBL5; UPR; apoptosis; cell survival; proteasome degradation; ubiquitin-independent proteasome system

DOI:  https://doi.org/10.1016/j.jbc.2023.104915
Autophagy Rep. 2023 ;pii: 2213541. [Epub ahead of print]2(1):

Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.

Damián Lobato-Márquez, José Javier Conesa, Ana Teresa López-Jiménez, Michael E Divine, Jonathan N Pruneda, Serge Mostowy.

  Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.

Keywords:  Shigella; autophagy; cryo-SXT; cytoskeleton; septins; ubiquitin

DOI:  https://doi.org/10.1080/27694127.2023.2213541
Cell Chem Biol. 2023 Jun 08. pii: S2451-9456(23)00153-8. [Epub ahead of print]

ROTACs leverage signaling-incompetent R-spondin for targeted protein degradation.

Rui Sun, Zibo Meng, Hyeyoon Lee, Rienk Offringa, Christof Niehrs.

  Proteolysis-targeting chimeras (PROTACs) are an emerging technology for therapeutic intervention, but options to target cell surface proteins and receptors remain limited. Here we introduce ROTACs, bispecific WNT- and BMP-signaling-disabled R-spondin (RSPO) chimeras, which leverage the specificity of these stem cell growth factors for ZNRF3/RNF43 E3 transmembrane ligases, to target degradation of transmembrane proteins. As a proof-of-concept, we targeted the immune checkpoint protein, programmed death ligand 1 (PD-L1), a prominent cancer therapeutic target, with a bispecific RSPO2 chimera, R2PD1. The R2PD1 chimeric protein binds to PD-L1 and at picomolar concentration induces its lysosomal degradation. In three melanoma cell lines, R2PD1 induced between 50 and 90% PD-L1 protein degradation. PD-L1 degradation was strictly dependent on ZNRF3/RNF43. Moreover, R2PD1 reactivates cytotoxic T cells and inhibits tumor cell proliferation more potently than Atezolizumab. We suggest that signaling-disabled ROTACs represent a paradigm to target cell surface proteins for degradation in a range of applications.

Keywords:  LYTAC; PD-L1; PROTAC; R-spondin; RNF43; ZNRF3; lysosome; protein degradation; transmembrane E3 ligase

DOI:  https://doi.org/10.1016/j.chembiol.2023.05.010
Mol Cell. 2023 Jun 15. pii: S1097-2765(23)00413-6. [Epub ahead of print]83(12): 2059-2076.e6

A mitochondrial iron-responsive pathway regulated by DELE1.

Yusuke Sekine, Ryan Houston, Eva-Maria Eckl, Evelyn Fessler, Derek P Narendra, Lucas T Jae, Shiori Sekine.

  The heme-regulated kinase HRI is activated under heme/iron deficient conditions; however, the underlying molecular mechanism is incompletely understood. Here, we show that iron-deficiency-induced HRI activation requires the mitochondrial protein DELE1. Notably, mitochondrial import of DELE1 and its subsequent protein stability are regulated by iron availability. Under steady-state conditions, DELE1 is degraded by the mitochondrial matrix-resident protease LONP1 soon after mitochondrial import. Upon iron chelation, DELE1 import is arrested, thereby stabilizing DELE1 on the mitochondrial surface to activate the HRI-mediated integrated stress response (ISR). Ablation of this DELE1-HRI-ISR pathway in an erythroid cell model enhances cell death under iron-limited conditions, suggesting a cell-protective role for this pathway in iron-demanding cell lineages. Our findings highlight mitochondrial import regulation of DELE1 as the core component of a previously unrecognized mitochondrial iron responsive pathway that elicits stress signaling following perturbation of iron homeostasis.

Keywords:  DELE1; HRI; LONP1; erythroid cells; integrated stress response; iron; mitochondria; mitochondrial import; mitochondrial proteostasis

DOI:  https://doi.org/10.1016/j.molcel.2023.05.031
Dev Cell. 2023 Jun 08. pii: S1534-5807(23)00242-3. [Epub ahead of print]

LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

Mohyeddine Omrane, Kalthoum Ben M'Barek, Alexandre Santinho, Nathan Nguyen, Shanta Nag, Thomas J Melia, Abdou Rachid Thiam.

  Lipid droplets (LDs) store lipids that can be utilized during times of scarcity via autophagic and lysosomal pathways, but how LDs and autophagosomes interact remained unclear. Here, we discovered that the E2 autophagic enzyme, ATG3, localizes to the surface of certain ultra-large LDs in differentiated murine 3T3-L1 adipocytes or Huh7 human liver cells undergoing prolonged starvation. Subsequently, ATG3 lipidates microtubule-associated protein 1 light-chain 3B (LC3B) to these LDs. In vitro, ATG3 could bind alone to purified and artificial LDs to mediate this lipidation reaction. We observed that LC3B-lipidated LDs were consistently in close proximity to collections of LC3B-membranes and were lacking Plin1. This phenotype is distinct from macrolipophagy, but it required autophagy because it disappeared following ATG5 or Beclin1 knockout. Our data suggest that extended starvation triggers a noncanonical autophagy mechanism, similar to LC3B-associated phagocytosis, in which the surface of large LDs serves as an LC3B lipidation platform for autophagic processes.

Keywords:  Atg3; LC3B; lipid droplets; noncanonical autophagy; organelle biogenesis; prolonged starvation

DOI:  https://doi.org/10.1016/j.devcel.2023.05.009
Cell Rep. 2023 Jun 13. pii: S2211-1247(23)00643-5. [Epub ahead of print]42(6): 112632

Nuclear translocation of an aminoacyl-tRNA synthetase may mediate a chronic "integrated stress response".

Julia A Jones, Na Wei, Haissi Cui, Yi Shi, Guangsen Fu, Navin Rauniyar, Ryan Shapiro, Yosuke Morodomi, Nadine Berenst, Calin Dan Dumitru, Sachiko Kanaji, John R Yates, Taisuke Kanaji, Xiang-Lei Yang.

  Various stress conditions are signaled through phosphorylation of translation initiation factor eukaryotic initiation factor 2α (eIF2α) to inhibit global translation while selectively activating transcription factor ATF4 to aid cell survival and recovery. However, this integrated stress response is acute and cannot resolve lasting stress. Here, we report that tyrosyl-tRNA synthetase (TyrRS), a member of the aminoacyl-tRNA synthetase family that responds to diverse stress conditions through cytosol-nucleus translocation to activate stress-response genes, also inhibits global translation. However, it occurs at a later stage than eIF2α/ATF4 and mammalian target of rapamycin (mTOR) responses. Excluding TyrRS from the nucleus over-activates translation and increases apoptosis in cells under prolonged oxidative stress. Nuclear TyrRS transcriptionally represses translation genes by recruiting TRIM28 and/or NuRD complex. We propose that TyrRS, possibly along with other family members, can sense a variety of stress signals through intrinsic properties of this enzyme and strategically located nuclear localization signal and integrate them by nucleus translocation to effect protective responses against chronic stress.

Keywords:  CP: Cell biology; CP: Molecular biology; aminoacyl-tRNA synthetase; cell survival; oxidative stress; stress response; transcriptional regulation; translation inhibition

DOI:  https://doi.org/10.1016/j.celrep.2023.112632
mBio. 2023 Jun 12. e0054023

The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection.

Jessica M Oda, Andreas B den Hartigh, Shoen M Jackson, Ana R Tronco, Susan L Fink.

  The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that induces the expression of ER-related targets. Activation of the IRE1α-XBP1 pathway occurs in association with risk factors for severe human coronavirus infection. In this study, we found that the human coronaviruses HCoV-OC43 (human coronavirus OC43) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) both robustly activate the IRE1α-XBP1 branch of the unfolded protein response in cultured cells. Using IRE1α nuclease inhibitors and genetic knockdown of IRE1α and XBP1, we found that these host factors are required for optimal replication of both viruses. Our data suggest that IRE1α supports infection downstream of initial viral attachment and entry. In addition, we found that ER stress-inducing conditions are sufficient to enhance human coronavirus replication. Furthermore, we found markedly increased XBP1 in circulation in human patients with severe coronavirus disease 2019 (COVID-19). Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.IMPORTANCEThere is a critical need to understand the cellular processes co-opted during human coronavirus replication, with an emphasis on identifying mechanisms underlying severe disease and potential therapeutic targets. Here, we demonstrate that the host proteins IRE1α and XBP1 are required for robust infection by the human coronaviruses, SARS-CoV-2 and HCoV-OC43. IRE1α and XBP1 participate in the cellular response to ER stress and are activated during conditions that predispose to severe COVID-19. We found enhanced viral replication with exogenous IRE1α activation, and evidence that this pathway is activated in humans during severe COVID-19. Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.

Keywords:  ER stress; HCoV-OC43; IRE1α; SARS-CoV-2; XBP1; coronavirus; endoplasmic reticulum; unfolded protein response

DOI:  https://doi.org/10.1128/mbio.00540-23
Genesis. 2023 Jun 15. e23520

Expanding EMC foldopathies: Topogenesis deficits alter the neural crest.

Jonathan Marquez, Faiza Aslam, Mustafa K Khokha.

  The endoplasmic reticulum (ER) membrane protein complex (EMC) is essential for the insertion of a wide variety of transmembrane proteins into the plasma membrane across cell types. Each EMC is composed of Emc1-7, Emc10, and either Emc8 or Emc9. Recent human genetics studies have implicated variants in EMC genes as the basis for a group of human congenital diseases. The patient phenotypes are varied but appear to affect a subset of tissues more prominently than others. Namely, craniofacial development seems to be commonly affected. We previously developed an array of assays in Xenopus tropicalis to assess the effects of emc1 depletion on the neural crest, craniofacial cartilage, and neuromuscular function. We sought to extend this approach to additional EMC components identified in patients with congenital malformations. Through this approach, we determine that EMC9 and EMC10 are important for neural crest development and the development of craniofacial structures. The phenotypes observed in patients and our Xenopus model phenotypes similar to EMC1 loss of function likely due to a similar mechanism of dysfunction in transmembrane protein topogenesis.

Keywords:  CHD; EMC; NDD; Xenopus tropicalis; craniofacial cartilage; development; neural crest

DOI:  https://doi.org/10.1002/dvg.23520
J Neurosci Res. 2023 Jun 14.

Astrocytic deletion of protein kinase R-like ER kinase (PERK) does not affect learning and memory in aged mice but worsens outcome from experimental stroke.

Anirudhya Lahiri, James C Walton, Ning Zhang, Neil Billington, A Courtney DeVries, Gordon P Meares.

  Aging is associated with cognitive decline and is the main risk factor for a myriad of conditions including neurodegeneration and stroke. Concomitant with aging is the progressive accumulation of misfolded proteins and loss of proteostasis. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). The UPR is mediated, in part, by the eukaryotic initiation factor 2α (eIF2α) kinase protein kinase R-like ER kinase (PERK). Phosphorylation of eIF2α reduces protein translation as an adaptive mechanism but this also opposes synaptic plasticity. PERK, and other eIF2α kinases, have been widely studied in neurons where they modulate both cognitive function and response to injury. The impact of astrocytic PERK signaling in cognitive processes was previously unknown. To examine this, we deleted PERK from astrocytes (AstroPERKKO ) and examined the impact on cognitive functions in middle-aged and old mice of both sexes. Additionally, we tested the outcome following experimental stroke using the transient middle cerebral artery occlusion (MCAO) model. Tests of short-term and long-term learning and memory as well as of cognitive flexibility in middle-aged and old mice revealed that astrocytic PERK does not regulate these processes. Following MCAO, AstroPERKKO had increased morbidity and mortality. Collectively, our data demonstrate that astrocytic PERK has limited impact on cognitive function and has a more prominent role in the response to neural injury.

Keywords:  MCAO; RRID:AB_10692650; RRID:AB_2095847; RRID:AB_2096481; RRID:AB_2107445; RRID:AB_2631098; RRID:AB_304334; RRID:Addgene_28306; RRID:IMSR_JAX:023066; RRID:IMSR_JAX:024098; RRID:SCR_002798; RRID:SCR_014210; RRID:SCR_014289; RRID:nif-0000-00280; aging; astrocytes; glia; learning and memory; protein kinase R-like ER kinase (PERK); stroke; unfolded protein response

DOI:  https://doi.org/10.1002/jnr.25224
Chem Soc Rev. 2023 Jun 14.

Chemically induced degradation of epigenetic targets.

Md Kabir, Xufen Yu, H Ümit Kaniskan, Jian Jin.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional small molecules that induce the ternary complex formation between a protein-of-interest (POI) and an E3 ligase, leading to targeted polyubiquitination and degradation of the POI. Particularly, PROTACs have the distinct advantage of targeting both canonical and noncanonical functions of epigenetic targets over traditional inhibitors, which typically target canonical functions only, resulting in greater therapeutic efficacy. In this review, we methodically analyze published PROTAC degraders of epigenetic writer, reader, and eraser proteins and their in vitro and in vivo effects. We highlight the mechanism of action of these degraders and their advantages in targeting both canonical and noncanonical functions of epigenetic targets in the context of cancer treatment. Furthermore, we present a future outlook for this exciting field. Overall, pharmacological degradation of epigenetic targets has emerged as an effective and attractive strategy to thwart cancer progression and growth.

DOI: https://doi.org/10.1039/d3cs00100h
Structure. 2023 Jun 02. pii: S0969-2126(23)00169-7. [Epub ahead of print]

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

Tal Oppenheim, Meytal Radzinski, Merav Braitbard, Esther S Brielle, Ohad Yogev, Eliya Goldberger, Yarden Yesharim, Tommer Ravid, Dina Schneidman-Duhovny, Dana Reichmann.

  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.

Keywords:  CDC48; CL-MS; NPL4; UFD1; crosslinking coupled with MS (XL-MS); integrative modeling; protein quality control; structural mass spectrometry

DOI:  https://doi.org/10.1016/j.str.2023.05.014
Proc Natl Acad Sci U S A. 2023 Jun 20. 120(25): e2219790120

Dynamic interactome of the MHC I peptide loading complex in human dendritic cells.

Martina Barends, Nicole Koller, Christian Schölz, Verónica Durán, Berislav Bošnjak, Jennifer Becker, Marius Döring, Hanna Blees, Reinhold Förster, Ulrich Kalinke, Robert Tampé.

  Dendritic cells (DCs) orchestrate immune responses by presenting antigenic peptides on major histocompatibility complex (MHC) molecules to T cells. Antigen processing and presentation via MHC I rely on the peptide-loading complex (PLC), a supramolecular machinery assembled around the transporter associated with antigen processing (TAP), which is the peptide transporter in the endoplasmic reticulum (ER) membrane. We studied antigen presentation in human DCs by isolating monocytes from blood and differentiating them into immature and mature DCs. We uncovered that during DC differentiation and maturation, additional proteins are recruited to the PLC, including B-cell receptor-associated protein 31 (BAP31), vesicle-associated membrane protein-associated protein A (VAPA), and extended synaptotagmin-1 (ESYT1). We demonstrated that these ER cargo export and contact site-tethering proteins colocalize with TAP and are within 40 nm proximity of the PLC, suggesting that the antigen processing machinery is located near ER exit- and membrane contact sites. While CRISPR/Cas9-mediated deletion of TAP and tapasin significantly reduced MHC I surface expression, single-gene deletions of the identified PLC interaction partners revealed a redundant role of BAP31, VAPA, and ESYT1 in MHC I antigen processing in DCs. These data highlight the dynamics and plasticity of PLC composition in DCs that previously was not recognized by the analysis of cell lines.

Keywords:  antigen presentation; antigen processing; membrane organization; membrane proteins; primary cells

DOI:  https://doi.org/10.1073/pnas.2219790120
Mol Cell. 2023 Jun 01. pii: S1097-2765(23)00376-3. [Epub ahead of print]

Mechanism of ribosome-associated mRNA degradation during tubulin autoregulation.

Markus Höpfler, Eva Absmeier, Sew-Yeu Peak-Chew, Evangelia Vartholomaiou, Lori A Passmore, Ivana Gasic, Ramanujan S Hegde.

  Microtubules play crucial roles in cellular architecture, intracellular transport, and mitosis. The availability of free tubulin subunits affects polymerization dynamics and microtubule function. When cells sense excess free tubulin, they trigger degradation of the encoding mRNAs, which requires recognition of the nascent polypeptide by the tubulin-specific ribosome-binding factor TTC5. How TTC5 initiates the decay of tubulin mRNAs is unknown. Here, our biochemical and structural analysis reveals that TTC5 recruits the poorly studied protein SCAPER to the ribosome. SCAPER, in turn, engages the CCR4-NOT deadenylase complex through its CNOT11 subunit to trigger tubulin mRNA decay. SCAPER mutants that cause intellectual disability and retinitis pigmentosa in humans are impaired in CCR4-NOT recruitment, tubulin mRNA degradation, and microtubule-dependent chromosome segregation. Our findings demonstrate how recognition of a nascent polypeptide on the ribosome is physically linked to mRNA decay factors via a relay of protein-protein interactions, providing a paradigm for specificity in cytoplasmic gene regulation.

Keywords:  CCR4-NOT complex; RNA degradation; co-translational regulation; microtubules; ribosome; tubulin

DOI:  https://doi.org/10.1016/j.molcel.2023.05.020
Mol Cancer Res. 2023 Jun 14. pii: MCR-23-0108. [Epub ahead of print]

Targeting the ATF6-mediated ER stress response and autophagy blocks integrin-driven prostate cancer progression.

Amanda J Macke, Artem N Pachikov, Taylor E Divita, Mary E Morris, Chad A LaGrange, Melissa S Holzapfel, Anatoly V Kubyshkin, Evgeniya Y Zyablitskaya, Tatiana P Makalish, Sergey N Eremenko, Haowen Qiu, Jean-Jack M Riethoven, George P Hemstreet, Armen Petrosyan.

Prostate cancer (PCa) progression to the lethal metastatic castration-resistant phenotype (mCRPC) is driven by αv integrins and is associated with Golgi disorganization and activation of the ATF6 branch of unfolded protein response (UPR). Overexpression of integrins requires N-acetylglucosaminyltransferase-V (MGAT5)-mediated glycosylation and subsequent cluster formation with Galectin-3 (Gal-3). However, the mechanism underlying this altered glycosylation is missing. For the first time, using HALO analysis of immunohistochemistry, we found a strong association of Integrin αv and Gal-3 at the plasma membrane (PM) in primary PCa and mCRPC samples. We discovered that MGAT5 activation is caused by Golgi fragmentation and mislocalization of its competitor, N-acetylglucosaminyltransferase-III, MGAT3, from Golgi to the endoplasmic reticulum (ER). This was validated in an ethanol-induced model of ER stress, where alcohol treatment in androgen-refractory PC-3 and DU145 cells or alcohol consumption in PCa patient samples aggravates Golgi scattering, activates MGAT5, and enhances integrin expression at PM. This explains known link between alcohol consumption and PCa mortality. ATF6 depletion significantly blocks UPR and reduces the number of Golgi fragments in both PC-3 and DU145 cells. Inhibition of autophagy by hydroxychloroquine (HCQ) restores compact Golgi, rescues MGAT3 intra-Golgi localization, blocks glycan modification via MGAT5, and abrogates delivery of Gal-3 to the cell surface. Importantly, the loss of Gal-3 leads to reduced integrins at PM and their accelerated internalization. ATF6 depletion and HCQ treatment synergistically decrease Integrin αv and Gal-3 expression and temper orthotopic tumor growth and metastasis. Implications: Combined ablation of ATF6 and autophagy can serve as new mCRPC therapeutic.

DOI: https://doi.org/10.1158/1541-7786.MCR-23-0108
Mol Cell. 2023 Jun 08. pii: S1097-2765(23)00374-X. [Epub ahead of print]

An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation.

Stephanie Pei Tung Yiu, Cassie Zerbe, David Vanderwall, Edward L Huttlin, Michael P Weekes, Benjamin E Gewurz.

  Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Keywords:  MAVS; NLRP3 inflammasome; UFMylation; antiviral defense; gamma-herpesvirus; herpesvirus; interactome; mitochondrial-derived vesicles; viral evasion; virus/host interaction

DOI:  https://doi.org/10.1016/j.molcel.2023.05.018
J Cell Sci. 2023 Jun 12. pii: jcs.260993. [Epub ahead of print]

Aggregation prone Tau impairs mitochondrial import, which affects organelle morphology and neuronal complexity.

Hope I Needs, Kevin A Wilkinson, Jeremy M Henley, Ian Collinson.

  Mitochondrial protein import is essential for organellar biogenesis, and thereby for the sufficient supply of cytosolic ATP-particularly important for cells with high energy demands like neurons. This study explores the prospect of import machinery perturbation as a cause of neurodegeneration instigated by the accumulation of aggregating proteins linked to disease. The aggregation-prone Tau variant (TauP301L) reduces the levels of components of the import machinery of the outer (TOM20) and inner membrane (TIM23) while associating with TOM40. Intriguingly, this interaction affects mitochondrial morphology, but not protein import or respiratory function; raising the prospect of an intrinsic rescue mechanism. Indeed, TauP301L induced the formation of tunnelling nanotubes (TNTs), potentially for the recruitment of healthy mitochondria from neighbouring cells and/or the disposal of mitochondria incapacitated by aggregated Tau. Consistent with this, inhibition of TNT formation (and rescue) reveals Tau-induced import impairment. In primary neuronal cultures, TauP301L induced morphological changes characteristic of neurodegeneration. Interestingly, these effects were mirrored in cells where the import sites were blocked artificially. Our results reveal a link between aggregation-prone Tau and defective mitochondrial import machinery relevant to disease.

Keywords:  Mitochondria; Mitochondrial import; Mitochondrial morphology.; Neurodegeneration; Neuronal complexity; Tau

DOI:  https://doi.org/10.1242/jcs.260993
Angew Chem Int Ed Engl. 2023 Jun 12. e202305866

Nascent Proteomics: Chemical Tools for Monitoring Newly Synthesized Proteins.

Qi Tang, Xing Chen.

  Cellular proteins are dynamically regulated in response to environmental stimuli. Conventional proteomics compares the entire proteome in different cellular states to identify differentially expressed proteins, which suffers from limited sensitivity for analyzing acute and subtle changes. To address this challenge, nascent proteomics has been developed, which selectively analyze the newly synthesized proteins, thus offering a more sensitive and timely insight into the dynamic changes of the proteome. In this Minireview, we discuss recent advancements in nascent proteomics, with an emphasis on methodological developments. Also, we delve into the current challenges and provide an outlook on the future prospects of this exciting field.

Keywords:  chemoproteomics; metabolic protein labeling; nascent proteomics; newly synthesized proteins; noncanonical amino acids

DOI:  https://doi.org/10.1002/anie.202305866
Nat Chem Biol. 2023 Jun 15.

Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.

Mijin Kim, Chen Chen, Zvi Yaari, Rune Frederiksen, Ewelina Randall, Jaina Wollowitz, Christian Cupo, Xiaojian Wu, Janki Shah, Daniel Worroll, Rachel E Lagenbacher, Dana Goerzen, Yue-Ming Li, Heeseon An, YuHuang Wang, Daniel A Heller.

Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.

DOI: https://doi.org/10.1038/s41589-023-01364-9
Life Sci Alliance. 2023 Sep;pii: e202302127. [Epub ahead of print]6(9):

Time-resolved proteomic analyses of senescence highlight metabolic rewiring of mitochondria.

Jun Yong Kim, Ilian Atanassov, Frederik Dethloff, Lara Kroczek, Thomas Langer.

Mitochondrial dysfunction and cellular senescence are hallmarks of aging. However, the relationship between these two phenomena remains incompletely understood. In this study, we investigated the rewiring of mitochondria upon development of the senescent state in human IMR90 fibroblasts. Determining the bioenergetic activities and abundance of mitochondria, we demonstrate that senescent cells accumulate mitochondria with reduced OXPHOS activity, resulting in an overall increase of mitochondrial activities in senescent cells. Time-resolved proteomic analyses revealed extensive reprogramming of the mitochondrial proteome upon senescence development and allowed the identification of metabolic pathways that are rewired with different kinetics upon establishment of the senescent state. Among the early responding pathways, the degradation of branched-chain amino acid was increased, whereas the one carbon folate metabolism was decreased. Late-responding pathways include lipid metabolism and mitochondrial translation. These signatures were confirmed by metabolic flux analyses, highlighting metabolic rewiring as a central feature of mitochondria in cellular senescence. Together, our data provide a comprehensive view on the changes in mitochondrial proteome in senescent cells and reveal how the mitochondrial metabolism is rewired in senescent cells.

DOI: https://doi.org/10.26508/lsa.202302127
J Am Chem Soc. 2023 Jun 16.

Bioorthogonal PROTAC Prodrugs Enabled by On-Target Activation.

Mengyang Chang, Feng Gao, Devin Pontigon, Giri Gnawali, Hang Xu, Wei Wang.

Although proteolysis targeting chimeras (PROTACs) have become promising therapeutic modalities, important concerns exist about the potential toxicity of the approach owing to uncontrolled degradation of proteins and undesirable ligase-mediated off-target effects. Precision manipulation of degradation activity of PROTACs could minimize potential toxicity and side effects. As a result, extensive efforts have been devoted to developing cancer biomarker activating prodrugs of PROTACs. In this investigation, we developed a bioorthogonal on-demand prodrug strategy (termed click-release "crPROTACs") that enables on-target activation of PROTAC prodrugs and release of PROTACs in cancer cells selectively. Inactive PROTAC prodrugs TCO-ARV-771 and TCO-DT2216 are rationally designed by conjugating a bioorthogonal trans-cyclooctenes (TCO) group into the ligand of the VHL E3 ubiquitin ligase. The tetrazine (Tz)-modified RGD peptide, c(RGDyK)-Tz, which targets integrin αvβ3 biomarker in cancer cells, serves as the activation component for click-release of the PROTAC prodrugs to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. The results of studies accessing the viability of this strategy show that the PROTAC prodrugs are selectively activated in an integrin αvβ3-dependent manner to produce PROTACs, which degrade POIs in cancer cells. The crPROTAC strategy might be a general, abiotic approach to induce selective cancer cell death through the ubiquitin-proteasome pathway.

DOI: https://doi.org/10.1021/jacs.3c05159
PLoS Biol. 2023 Jun 15. 21(6): e3002159

Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.

Rachel L Kinsella, Jacqueline M Kimmey, Asya Smirnov, Reilly Woodson, Margaret R Gaggioli, Sthefany M Chavez, Darren Kreamalmeyer, Christina L Stallings.

The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.

DOI: https://doi.org/10.1371/journal.pbio.3002159
EMBO J. 2023 Jun 12. e113349

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda, Masaaki Komatsu.

  NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.

Keywords:  KEAP1; NRF2/NFE2L2; ULK1; liquid-liquid phase separation; p62/SQSTM1

DOI:  https://doi.org/10.15252/embj.2022113349