bims-proteo Biomed News
on Proteostasis
Issue of 2023–02–12
37 papers selected by
Eric Chevet, INSERM
  1. DNAJB12 and Hsp70 Mediate Triage of Misfolded Membrane Proteins for Proteasomal versus Lysosomal Degradation.
  2. Mechanism of signal-anchor triage during early steps of membrane protein insertion.
  3. MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.
  4. The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition.
  5. Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy.
  6. Emerging role of UFMylation in secretory cells involved in the endocrine system by maintaining ER proteostasis.
  7. Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.
  8. High throughput E3 ligase degron binding assays for novel PROTAC ligand discovery.
  9. Calnexin, More Than Just a Molecular Chaperone.
  10. Harnessing nanobodies for target protein degradation through the Affinity-directed PROtein Missile (AdPROM) system.
  11. Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress.
  12. A journey in UPR modeling.
  13. Accelerating inhibitor discovery for deubiquitinating enzymes.
  14. Runx1/3-driven adaptive endoplasmic reticulum stress pathways contribute to neurofibromagenesis.
  15. The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.
  16. Mechanisms of Protein Trafficking and Quality Control in the Kidney and Beyond.
  17. ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling.
  18. Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.
  19. Structural basis for SMAC-mediated antagonism of caspase inhibition by the giant ubiquitin ligase BIRC6.
  20. Control of immune cell function by the unfolded protein response.
  21. The ER Protein Translocation Channel Subunit Sbh1 Controls Virulence of Cryptococcus neoformans.
  22. Structures of BIRC6-client complexes provide a mechanism of Smac-mediated release of caspases.
  23. The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD.
  24. The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain-of-function in cancer.
  25. Metabolomics analysis of an AAA-ATPase Cdc48-deficient yeast strain.
  26. Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.
  27. Systematically characterizing the roles of E3-ligase family members in inflammatory responses with massively parallel Perturb-seq.
  28. Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor.
  29. A molecular device for the redox quality control of GroEL/ES substrates.
  30. Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.
  31. LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site.
  32. Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.
  33. Targeted protein degradation through light-activated E3 ligase recruitment.
  34. Crystallization of VHL-based PROTAC-induced ternary complexes.
  35. The functional role of Ire1 in regulating autophagy and proteasomal degradation under prolonged proteotoxic stress.
  36. G-quadruplex ligands as potent regulators of lysosomes.
  37. AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs.
  1. Autophagy Rep. 2022 ;1(1): 559-562
    DNAJB12 and Hsp70 Mediate Triage of Misfolded Membrane Proteins for Proteasomal versus Lysosomal Degradation.
    Andrew Kennedy, Douglas M Cyr.
      The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation.
    DOI:  https://doi.org/10.1080/27694127.2022.2139335
  2. Mol Cell. 2023 Feb 03. pii: S1097-2765(23)00041-2. [Epub ahead of print]
    Mechanism of signal-anchor triage during early steps of membrane protein insertion.
    Haoxi Wu, Ramanujan S Hegde.
      Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis.
    Keywords:  EMC; Sec61; endoplasmic reticulum; membrane protein insertion; protein homeostasis; protein topology
    DOI:  https://doi.org/10.1016/j.molcel.2023.01.018
  3. Cell Rep. 2023 Feb 03. pii: S2211-1247(23)00077-3. [Epub ahead of print]42(2): 112066
    MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.
    Vera Kovaleva, Li-Ying Yu, Larisa Ivanova, Olesya Shpironok, Jinhan Nam, Ave Eesmaa, Esa-Pekka Kumpula, Sven Sakson, Urve Toots, Mart Ustav, Juha T Huiskonen, Merja H Voutilainen, Päivi Lindholm, Mati Karelson, Mart Saarma.
      Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic β cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.
    Keywords:  BiP; CP: Molecular biology; CP: Neuroscience; ER stress; IRE1α; MANF; PD; Parkinson’s disease; UPR; mesencephalic astrocyte-derived neurotrophic factor; neuronal survival; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2023.112066
  4. Nat Commun. 2023 Feb 06. 14(1): 638
    The p97-UBXD8 complex regulates ER-Mitochondria contact sites by altering membrane lipid saturation and composition.
    Rakesh Ganji, Joao A Paulo, Yuecheng Xi, Ian Kline, Jiang Zhu, Christoph S Clemen, Conrad C Weihl, John G Purdy, Steve P Gygi, Malavika Raman.
      The intimate association between the endoplasmic reticulum (ER) and mitochondrial membranes at ER-Mitochondria contact sites (ERMCS) is a platform for critical cellular processes, particularly lipid synthesis. How contacts are remodeled and the impact of altered contacts on lipid metabolism remains poorly understood. We show that the p97 AAA-ATPase and its adaptor ubiquitin-X domain adaptor 8 (UBXD8) regulate ERMCS. The p97-UBXD8 complex localizes to contacts and its loss increases contacts in a manner that is dependent on p97 catalytic activity. Quantitative proteomics and lipidomics of ERMCS demonstrates alterations in proteins regulating lipid metabolism and a significant change in membrane lipid saturation upon UBXD8 deletion. Loss of p97-UBXD8 increased membrane lipid saturation via SREBP1 and the lipid desaturase SCD1. Aberrant contacts can be rescued by unsaturated fatty acids or overexpression of SCD1. We find that the SREBP1-SCD1 pathway is negatively impacted in the brains of mice with p97 mutations that cause neurodegeneration. We propose that contacts are exquisitely sensitive to alterations to membrane lipid composition and saturation.
    DOI:  https://doi.org/10.1038/s41467-023-36298-2
  5. EMBO J. 2023 Feb 10. e112053
    Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy.
    Lorenzo Picchianti, Víctor Sánchez de Medina Hernández, Ni Zhan, Nicholas At Irwin, Roan Groh, Madlen Stephani, Harald Hornegger, Rebecca Beveridge, Justyna Sawa-Makarska, Thomas Lendl, Nenad Grujic, Christin Naumann, Sascha Martens, Thomas A Richards, Tim Clausen, Silvia Ramundo, G Elif Karagöz, Yasin Dagdas.
      UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.
    Keywords:  ER-phagy; UFMylation; phylogenomics; ribosome stalling; selective autophagy
    DOI:  https://doi.org/10.15252/embj.2022112053
  6. Front Endocrinol (Lausanne). 2022 ;13 1085408
    Emerging role of UFMylation in secretory cells involved in the endocrine system by maintaining ER proteostasis.
    Yun Cheng, Zikang Niu, Yafei Cai, Wei Zhang.
      Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like molecule (UBL) discovered almost two decades ago, but our knowledge about the cellular and molecular mechanisms of this novel protein post-translational modification is still very fragmentary. In this review, we first summarize the core enzymes and factors involved in the UFMylation cascade, which, similar to ubiquitin, is consecutively catalyzed by UFM1-activating enzyme 5 (UBA5), UFM1-conjugating enzyme 1 (UFC1) and UFM1-specific ligase 1 (UFL1). Inspired by the substantial implications of UFM1 machinery in the secretory pathway, we next concentrate on the puzzling role of UFMylation in maintaining ER protein homeostasis, intending to illustrate the underlying mechanisms and future perspectives. At last, given a robust ER network is a hallmark of healthy endocrine secretory cells, we emphasize the function of UFM1 modification in physiology and pathology in the context of endocrine glands pancreas and female ovaries, aiming to provide precise insight into other internal glands of the endocrine system.
    Keywords:  ER proteostasis; UFMylation; endocrine; ovaries; pancreas; secretory pathway
    DOI:  https://doi.org/10.3389/fendo.2022.1085408
  7. Mol Biotechnol. 2023 Feb 10.
    Scaffold Hopping and Screening for Potent Small Molecule Agonists for GRP94: Implications to Alleviate ER Stress-Associated Pathogenesis.
    Shoufia Jabeen Mubarak, Surabhi Gupta, Hemamalini Vedagiri.
      Disparity in the activity of Endoplasmic reticulum (ER) leads to degenerative diseases, mainly associated with protein misfolding and aggregation leading to cellular dysfunction and damage, ultimately contributing to ER stress. ER stress activates the complex network of Unfolded Protein Response (UPR) signaling pathways mediated by transmembrane proteins IRE1, ATF6, and PERK. In addition to UPR, many ER chaperones have evolved to optimize the output of properly folded secretory and membrane proteins. Glucose-regulated protein 94 (GRP94), an ER chaperone of heat shock protein HSP90 family, directs protein folding through interaction with other components of the ER protein folding machinery and assists in ER-associated degradation (ERAD). Activation of GRP94 would increase the efficacy of protein folding machinery and regulate the UPR pathway toward homeostasis. The present study aims to screen for novel agonists for GRP94 based on Core hopping, pharmacophore hypothesis, 3D-QSAR, and virtual screening with small-molecule compound libraries in order to improve the efficiency of native protein folding by enhancing GRP94 chaperone activity, therefore to reduce protein misfolding and aggregation. In this study, we have employed the strategy of small molecule-dependent ER programming to enhance the chaperone activity of GRP94 through scaffold hopping-based screening approach to identify specific GRP94 agonists. New scaffolds generated by altering the cores of NECA, the known GRP94 agonist, were validated by employing pharmacophore hypothesis testing, 3D-QSAR modeling, and molecular dynamics simulations. This facilitated the identification of small molecules to improve the efficiency of native protein folding by enhancing GRP94 activity. High-throughput virtual screening of the selected pharmacophore hypothesis against Selleckchem and ZINC databases retrieved a total of 2,27,081 compounds. Further analysis on docking and ADMET properties revealed Epimedin A, Narcissoside, Eriocitrin 1,2,3,4,6-O-Pentagalloylglucose, Secoisolariciresinol diglucoside, ZINC92952357, ZINC67650204, and ZINC72457930 as potential lead molecules. The stability and interaction of these small molecules were far better than the known agonist, NECA indicating their efficacy in selectively alleviating ER stress-associated pathogenesis. These results substantiate the fact that small molecule-dependent ER reprogramming would activate the ER chaperones and therefore reduce the protein misfolding as well as aggregation associated with ER stress in order to restore cellular homeostasis.
    Keywords:  3D-QSAR; ER stress; GRP94; MD simulations; Pharmacophore modeling; Scaffold hopping; UPR
    DOI:  https://doi.org/10.1007/s12033-023-00685-3
  8. Methods Enzymol. 2023 ;pii: S0076-6879(22)00352-4. [Epub ahead of print]681 23-39
    High throughput E3 ligase degron binding assays for novel PROTAC ligand discovery.
    Robert G Guenette, Patrick Ryan Potts.
      The discovery of new small molecule ligands for E3 ligases will enable the creation of novel proteolysis targeting chimeras (PROTACs) and molecular glues to tackle traditionally undruggable proteins. Diversifying both the chemical matter for each E3 ligase and the type of ligases will be important to fully capture the potential of these targeted protein degradation modalities. A key step in this process is to establish an integrated screening platform for the rapid identification and optimization of small molecule ligands for E3 ligases. Here, we provide a method to evaluate E3 ligase ligands using AlphaScreen technology. AlphaScreen allows for the evaluation of a wide array of molecular interactions and is utilized extensively in small molecule screening campaigns. This bead-based proximity technology offers facile development for interactions across a wide range of affinities and can be adapted to interrogate E3 ligase-degron interactions. In this protocol, we demonstrate the development of AlphaScreen for E3 ligase ligand competition assays toward the discovery of new ligands for E3 ligases.
    Keywords:  AlphaScreen; Degron; E3 ligase; High throughput; Targeted protein degradation
    DOI:  https://doi.org/10.1016/bs.mie.2022.08.027
  9. Cells. 2023 Jan 24. pii: 403. [Epub ahead of print]12(3):
    Calnexin, More Than Just a Molecular Chaperone.
    Tautvydas Paskevicius, Rabih Abou Farraj, Marek Michalak, Luis B Agellon.
      Calnexin is a type I integral endoplasmic reticulum (ER) membrane protein with an N-terminal domain that resides in the lumen of the ER and a C-terminal domain that extends into the cytosol. Calnexin is commonly referred to as a molecular chaperone involved in the folding and quality control of membrane-associated and secreted proteins, a function that is attributed to its ER- localized domain with a structure that bears a strong resemblance to another luminal ER chaperone and Ca2+-binding protein known as calreticulin. Studies have discovered that the cytosolic C-terminal domain of calnexin undergoes distinct post-translational modifications and interacts with a variety of proteins. Here, we discuss recent findings and hypothesize that the post-translational modifications of the calnexin C-terminal domain and its interaction with specific cytosolic proteins play a role in coordinating ER functions with events taking place in the cytosol and other cellular compartments.
    Keywords:  calcium binding protein; cell signaling; endoplasmic reticulum; molecular chaperone; protein–protein interactions
    DOI:  https://doi.org/10.3390/cells12030403
  10. Methods Enzymol. 2023 ;pii: S0076-6879(22)00336-6. [Epub ahead of print]681 61-79
    Harnessing nanobodies for target protein degradation through the Affinity-directed PROtein Missile (AdPROM) system.
    Bill Carton, Sascha Röth, Thomas J Macartney, Gopal P Sapkota.
      Targeted protein degradation (TPD) is a useful approach in dissecting protein function and therapeutics. Technologies such as RNA interference or gene knockout that are routinely used rely on protein turnover. However, RNA interference takes a long time to deplete target proteins and is not suitable for long-lived proteins, while a genetic knockout is irreversible, takes a long time to achieve and is not suitable for essential genes. TPD has the potential to overcome the limitations of RNA interference and gene editing approaches. We have established the Affinity directed PROtein Missile (AdPROM) system, which harnesses nanobodies or binders of target proteins to redirect E3 ubiquitin ligase activity to the target protein to induce TPD through the ubiquitin proteasome system. Here we provide a step-by-step protocol for using the AdPROM system for targeted proteolysis of endogenously GFP-tagged K-RAS through an anti-GFP nanobody. This protocol can be amended to target a wide range of different proteins of interest (POIs) either by replacing the anti-GFP nanobody with a nanobody recognising the POI or by endogenously tagging the POI with GFP through CRISPR/Cas9 genome editing.
    Keywords:  AdPROM; E3; Proteasome; TPD; Target protein degradation; Ubiquitin; Ubiquitination
    DOI:  https://doi.org/10.1016/bs.mie.2022.08.011
  11. Cell Host Microbe. 2023 Jan 31. pii: S1931-3128(23)00031-8. [Epub ahead of print]
    Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress.
    Maria Naama, Shahar Telpaz, Aya Awad, Shira Ben-Simon, Sarina Harshuk-Shabso, Sonia Modilevsky, Elad Rubin, Jasmin Sawaed, Lilach Zelik, Mor Zigdon, Nofar Asulin, Sondra Turjeman, Michal Werbner, Supapit Wongkuna, Rachel Feeney, Bjoern O Schroeder, Abraham Nyska, Meital Nuriel-Ohayon, Shai Bel.
      Colonic goblet cells are specialized epithelial cells that secrete mucus to physically separate the host and its microbiota, thus preventing bacterial invasion and inflammation. How goblet cells control the amount of mucus they secrete is unclear. We found that constitutive activation of autophagy in mice via Beclin 1 enables the production of a thicker and less penetrable mucus layer by reducing endoplasmic reticulum (ER) stress. Accordingly, genetically inhibiting Beclin 1-induced autophagy impairs mucus secretion, while pharmacologically alleviating ER stress results in excessive mucus production. This ER-stress-mediated regulation of mucus secretion is microbiota dependent and requires the Crohn's-disease-risk gene Nod2. Overproduction of mucus alters the gut microbiome, specifically expanding mucus-utilizing bacteria, such as Akkermansia muciniphila, and protects against chemical and microbial-driven intestinal inflammation. Thus, ER stress is a cell-intrinsic switch that limits mucus secretion, whereas autophagy maintains intestinal homeostasis by relieving ER stress.
    Keywords:  Beclin 1; ER stress; Nod2; autophagy; colitis; goblet cell; inflammatory bowel diseases; microbiota; mucus; unfolded protein response
    DOI:  https://doi.org/10.1016/j.chom.2023.01.006
  12. Biol Cell. 2023 Feb 07.
    A journey in UPR modeling.
    Ilaria Pontisso, Roberto Ornelas Guevara, Laurent Combettes, Geneviève Dupont.
      Protein folding and protein maturation largely occur in the controlled environment of the Endoplasmic Reticulum (ER). Perturbation to the correct functioning of this organelle leads to altered proteostasis and accumulation of misfolded proteins in the ER lumen. This condition is commonly known as ER stress and is appearing as an important contributor in the pathogenesis of several human diseases. Monitoring of the quality control processes is mediated by the Unfolded Protein Response (UPR). This response consists in a complex network of signaling pathways that aim to restore protein folding and ER homeostasis. Conditions in which UPR is not able to overcome ER stress lead to a switch of the UPR signaling program from an adaptive to a pro-apoptotic one, revealing a key role of UPR in modulating cell fate decisions. Because of its high complexity and its involvement in the regulation of different cellular outcomes, UPR has been the center of the development of computational models, which tried to better dissect the role of UPR or of its specific components in several contexts. In this review, we go through the existing mathematical models of UPR. We emphasize how their study contributed to an improved characterization of the role of this intricate response in the modulation of cellular functions. This article is protected by copyright. All rights reserved.
    Keywords:  ATF6; ER stress; IRE1; PERK; Unfolded Protein Response; computational model; mathematical model; signaling
    DOI:  https://doi.org/10.1111/boc.202200111
  13. Nat Commun. 2023 Feb 08. 14(1): 686
    Accelerating inhibitor discovery for deubiquitinating enzymes.
    Wai Cheung Chan, Xiaoxi Liu, Robert S Magin, Nicholas M Girardi, Scott B Ficarro, Wanyi Hu, Maria I Tarazona Guzman, Cara A Starnbach, Alejandra Felix, Guillaume Adelmant, Anthony C Varca, Bin Hu, Ariana S Bratt, Ethan DaSilva, Nathan J Schauer, Isabella Jaen Maisonet, Emma K Dolen, Anthony X Ayala, Jarrod A Marto, Sara J Buhrlage.
      Deubiquitinating enzymes (DUBs) are an emerging drug target class of ~100 proteases that cleave ubiquitin from protein substrates to regulate many cellular processes. A lack of selective chemical probes impedes pharmacologic interrogation of this important gene family. DUBs engage their cognate ligands through a myriad of interactions. We embrace this structural complexity to tailor a chemical diversification strategy for a DUB-focused covalent library. Pairing our library with activity-based protein profiling as a high-density primary screen, we identify selective hits against 23 endogenous DUBs spanning four subfamilies. Optimization of an azetidine hit yields a probe for the understudied DUB VCPIP1 with nanomolar potency and in-family selectivity. Our success in identifying good chemical starting points as well as structure-activity relationships across the gene family from a modest but purpose-build library challenges current paradigms that emphasize ultrahigh throughput in vitro or virtual screens against an ever-increasing scope of chemical space.
    DOI:  https://doi.org/10.1038/s41467-023-36246-0
  14. Oncogene. 2023 Feb 09.
    Runx1/3-driven adaptive endoplasmic reticulum stress pathways contribute to neurofibromagenesis.
    Youjin Na, Ashley Hall, Yanan Yu, Liang Hu, Kwangmin Choi, Jake A Burgard, Sara Szabo, Gang Huang, Nancy Ratner, Jianqiang Wu.
      Neurofibromatosis type 1 (NF1) patients are predisposed to develop plexiform neurofibromas (PNFs). Three endoplasmic reticulum (ER) stress response pathways restore cellular homeostasis. The unfolded protein response (UPR) sensors contribute to tumor initiation in many cancers. We found that all three UPR pathways were activated in mouse and human PNFs, with protein kinase RNA [PKR]-like ER kinase (PERK) the most highly expressed. We tested if neurofibroma cells adapt to ER stress, leading to their growth. Pharmacological or genetic inhibition of PERK reduced mouse neurofibroma-sphere number, and genetic inhibition in PERK in Schwann cell precursors (SCPs) decreased tumor-like lesion numbers in a cell transplantation model. Further, in a PNF mouse model, deletion of PERK in Schwann cells (SCs) and SCPs reduced tumor size, number, and increased survival. Mechanistically, loss of Nf1 activated PERK-eIF2α-ATF4 signaling and increased ATF4 downstream target gene p21 translocation from nucleus to cytoplasm. This nucleus-cytoplasm translocation was mediated by exportin-1. Runx transcriptionally activated ribosome gene expression and increased protein synthesis to allow SCs to adapt to ER stress and tumor formation. We propose that targeting proteostasis might provide cytotoxic and/or potentially durable novel therapy for PNFs.
    DOI:  https://doi.org/10.1038/s41388-023-02620-x
  15. bioRxiv. 2023 Jan 27. pii: 2023.01.26.525608. [Epub ahead of print]
    The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.
    Lukas A Fuentes, Zach Marin, Jonathan Tyson, David Baddeley, Joerg Bewersdorf.
      The endoplasmic reticulum’s (ER) structure is directly linked to the many functions of the ER but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear-shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4.
    Summary: Rtn4 forms linear-shaped oligomers that contain an average of five Rtn4 proteins, localize to the sides of elliptical tubules, prefer orientations near parallel to the tubule axis, and increase local curvature of the ER membrane by increasing local Rtn4 density.
    DOI:  https://doi.org/10.1101/2023.01.26.525608
  16. Annu Rev Physiol. 2023 Feb 10. 85 407-423
    Mechanisms of Protein Trafficking and Quality Control in the Kidney and Beyond.
    Jillian L Shaw, Juan Lorenzo Pablo, Anna Greka.
      Numerous trafficking and quality control pathways evolved to handle the diversity of proteins made by eukaryotic cells. However, at every step along the biosynthetic pathway, there is the potential for quality control system failure. This review focuses on the mechanisms of disrupted proteostasis. Inspired by diseases caused by misfolded proteins in the kidney (mucin 1 and uromodulin), we outline the general principles of protein biosynthesis, delineate the recognition and degradation pathways targeting misfolded proteins, and discuss the role of cargo receptors in protein trafficking and lipid homeostasis. We also discuss technical approaches including live-cell fluorescent microscopy, chemical screens to elucidate trafficking mechanisms, multiplexed single-cell CRISPR screening platforms to systematically delineate mechanisms of proteostasis, and the advancement of novel tools to degrade secretory and membrane-associated proteins. By focusing on components of trafficking that go awry, we highlight ongoing efforts to understand fundamental mechanisms of disrupted proteostasis and implications for the treatment of human proteinopathies.
    Keywords:  Golgi; TMED; cargo; endoplasmic reticulum; kidney disease; lysosome; proteome; secretory pathway; trafficking; vesicle
    DOI:  https://doi.org/10.1146/annurev-physiol-031522-100639
  17. Nat Commun. 2023 Feb 04. 14(1): 611
    ESCRT-dependent STING degradation inhibits steady-state and cGAMP-induced signalling.
    Matteo Gentili, Bingxu Liu, Malvina Papanastasiou, Deborah Dele-Oni, Marc A Schwartz, Rebecca J Carlson, Aziz M Al'Khafaji, Karsten Krug, Adam Brown, John G Doench, Steven A Carr, Nir Hacohen.
      Stimulator of interferon genes (STING) is an intracellular sensor of cyclic di-nucleotides involved in the innate immune response against pathogen- or self-derived DNA. STING trafficking is tightly linked to its function, and its dysregulation can lead to disease. Here, we systematically characterize genes regulating STING trafficking and examine their impact on STING-mediated responses. Using proximity-ligation proteomics and genetic screens, we demonstrate that an endosomal sorting complex required for transport (ESCRT) complex containing HGS, VPS37A and UBAP1 promotes STING degradation, thereby terminating STING-mediated signaling. Mechanistically, STING oligomerization increases its ubiquitination by UBE2N, forming a platform for ESCRT recruitment at the endosome that terminates STING signaling via sorting in the lysosome. Finally, we show that expression of a UBAP1 mutant identified in patients with hereditary spastic paraplegia and associated with disrupted ESCRT function, increases steady-state STING-dependent type I IFN responses in healthy primary monocyte-derived dendritic cells and fibroblasts. Based on these findings, we propose that STING is subject to a tonic degradative flux and that the ESCRT complex acts as a homeostatic regulator of STING signaling.
    DOI:  https://doi.org/10.1038/s41467-023-36132-9
  18. Nucleic Acids Res. 2023 Feb 11. pii: gkad077. [Epub ahead of print]
    Genomic targets of the IRE1-XBP1s pathway in mediating metabolic adaptation in epithelial plasticity.
    Dianhua Qiao, Melissa Skibba, Xiaofang Xu, Allan R Brasier.
      Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis. Recently we observed the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1α)-X-box-binding protein 1 spliced (XBP1s) axis, enhancing glucose shunting to protein N glycosylation. To better understand the genomic targets of XBP1s, we identified its genomic targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1s in RSV infection. CUT&RUN identified 7086 binding sites in chromatin that were enriched in AP-1 motifs and GC-sequences. Of these binding sites, XBP1s peaks mapped to 4827 genes controlling Rho-GTPase signaling, N-linked glycosylation and ER-Golgi transport. Strikingly, XBP1s peaks were within 1 kb of transcription start sites of 2119 promoters. In addition to binding core mesenchymal transcription factors SNAI1 and ZEB1, we observed that hexosamine biosynthetic pathway (HBP) enzymes were induced and contained proximal XBP1s peaks. We demonstrate that IRE1α -XBP1s signaling is necessary and sufficient to activate core enzymes by recruiting elongation-competent phospho-Ser2 CTD modified RNA Pol II. We conclude that the IRE1α-XBP1s pathway coordinately regulates mesenchymal transcription factors and hexosamine biosynthesis in EMP by a mechanism involving recruitment of activated pSer2-Pol II to GC-rich promoters.
    DOI:  https://doi.org/10.1093/nar/gkad077
  19. Science. 2023 Feb 09. eade8840
    Structural basis for SMAC-mediated antagonism of caspase inhibition by the giant ubiquitin ligase BIRC6.
    Larissa Dietz, Cara J Ellison, Carlos Riechmann, C Keith Cassidy, F Daniel Felfoldi, Adán Pinto-Fernández, Benedikt M Kessler, Paul R Elliott.
      Certain Inhibitors of apoptosis (IAP) family members are sentinel proteins preventing untimely cell death by inhibiting caspases. Antagonists including second mitochondria-derived activator of caspase (SMAC) regulate IAPs, driving cell death. Baculoviral IAP repeat-containing protein 6 (BIRC6), a giant IAP with dual E2/E3 ubiquitin ligase activity, regulates programmed cell death through unknown mechanisms. We show BIRC6 directly restricts executioner caspases-3 and -7, and ubiquitinates caspases-3, -7 and -9 working exclusively with non-canonical E1, UBA6. Importantly, we show SMAC suppresses both mechanisms. Cryo-electron microscopy structures of BIRC6 alone and in complex with SMAC reveals BIRC6 is an anti-parallel dimer juxtaposing the substrate-binding module against the catalytic domain. Furthermore, we discover SMAC multi-site binding to BIRC6 results in a sub-nanomolar affinity interaction, enabling SMAC to competitively displace caspases thus antagonizing BIRC6 anti-caspase function.
    DOI:  https://doi.org/10.1126/science.ade8840
  20. Nat Rev Immunol. 2023 Feb 08.
    Control of immune cell function by the unfolded protein response.
    Giusy Di Conza, Ping-Chih Ho, Juan R Cubillos-Ruiz, Stanley Ching-Cheng Huang.
      Initiating and maintaining optimal immune responses requires high levels of protein synthesis, folding, modification and trafficking in leukocytes, which are processes orchestrated by the endoplasmic reticulum. Importantly, diverse extracellular and intracellular conditions can compromise the protein-handling capacity of this organelle, inducing a state of 'endoplasmic reticulum stress' that activates the unfolded protein response (UPR). Emerging evidence shows that physiological or pathological activation of the UPR can have effects on immune cell survival, metabolism, function and fate. In this Review, we discuss the canonical role of the adaptive UPR in immune cells and how dysregulation of this pathway in leukocytes contributes to diverse pathologies such as cancer, autoimmunity and metabolic disorders. Furthermore, we provide an overview as to how pharmacological approaches that modulate the UPR could be harnessed to control or activate immune cell function in disease.
    DOI:  https://doi.org/10.1038/s41577-023-00838-0
  21. mBio. 2023 Feb 07. e0338422
    The ER Protein Translocation Channel Subunit Sbh1 Controls Virulence of Cryptococcus neoformans.
    Felipe H Santiago-Tirado, Thomas Hurtaux, Jennifer Geddes-McAlister, Duy Nguyen, Volkhard Helms, Tamara L Doering, Karin Römisch.
      The fungal pathogen Cryptococcus neoformans is distinguished by a cell-wall-anchored polysaccharide capsule that is critical for virulence. Biogenesis of both cell wall and capsule relies on the secretory pathway. Protein secretion begins with polypeptide translocation across the endoplasmic reticulum (ER) membrane through a highly conserved channel formed by three proteins: Sec61, Sbh1, and Sss1. Sbh1, the most divergent, contains multiple phosphorylation sites, which may allow it to regulate entry into the secretory pathway in a species- and protein-specific manner. Absence of SBH1 causes a cell-wall defect in both Saccharomyces cerevisiae and C. neoformans, although other phenotypes differ. Notably, proteomic analysis showed that when cryptococci are grown in conditions that mimic aspects of the mammalian host environment (tissue culture medium, 37°C, 5% CO2), a set of secretory and transmembrane proteins is upregulated in wild-type, but not in Δsbh1 mutant cells. The Sbh1-dependent proteins show specific features of their ER targeting sequences that likely cause them to transit less efficiently into the secretory pathway. Many also act in cell-wall biogenesis, while several are known virulence factors. Consistent with these observations, the C. neoformans Δsbh1 mutant is avirulent in a mouse infection model. We conclude that, in the context of conditions encountered during infection, Sbh1 controls the entry of virulence factors into the secretory pathway of C. neoformans, and thereby regulates fungal pathogenicity. IMPORTANCE Cryptococcus neoformans is a yeast that causes almost 200,000 deaths worldwide each year, mainly of immunocompromised individuals. The surface structures of this pathogen, a protective cell wall surrounded by a polysaccharide capsule, are made and maintained by proteins that are synthesized inside the cell and travel outwards through the secretory pathway. A protein called Sbh1 is part of the machinery that determines which polypeptides enter this export pathway. We found that when Sbh1 is absent, both C. neoformans and the model yeast S. cerevisiae show cell-wall defects. Lack of Sbh1 also changes the pattern of secretion of both transmembrane and soluble proteins, in a manner that depends on characteristics of their sequences. Notably, multiple proteins that are normally upregulated in conditions similar to those encountered during infection, including several needed for cryptococcal virulence, are no longer increased. Sbh1 thereby regulates the ability of this important pathogen to cause disease.
    Keywords:  Cryptococcus neoformans; Saccharomyces cerevisiae; Sbh1; cell wall; protein translocation; virulence
    DOI:  https://doi.org/10.1128/mbio.03384-22
  22. Science. 2023 Feb 09. eade5750
    Structures of BIRC6-client complexes provide a mechanism of Smac-mediated release of caspases.
    Moritz Hunkeler, Cyrus Y Jin, Eric S Fischer.
      Tight regulation of apoptosis is essential for metazoan development and prevents diseases such as cancer and neurodegeneration. Caspase activation is central to apoptosis and inhibitor of apoptosis (IAP) proteins are the principal actors that restrain caspase activity and are therefore attractive therapeutic targets. IAPs, in turn, are regulated by mitochondria-derived pro-apoptotic factors such as Smac and HtrA2. Through a series of cryo-electron microscopy (cryo-EM) structures of full-length human baculoviral IAP repeat-containing protein 6 (BIRC6) bound to Smac, caspases, and HtrA2, we provide a molecular understanding for BIRC6-mediated caspase inhibition and its release by Smac. The architecture of BIRC6, together with near-irreversible binding of Smac, elucidates how the IAP inhibitor Smac can effectively control a processive ubiquitin ligase to respond to apoptotic stimuli.
    DOI:  https://doi.org/10.1126/science.ade5750
  23. J Biol Chem. 2023 Feb 08. pii: S0021-9258(23)00127-8. [Epub ahead of print] 102995
    The mTORC2/AKT/VCP axis is associated with quality control of the stalled translation of poly(GR) dipeptide repeats in C9-ALS/FTD.
    Yu Li, Ji Geng, Suman Rimal, Haochuan Wang, Xiangguo Liu, Bingwei Lu, Shuangxi Li.
      Expansion of G4C2 hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis with frontotemporal dementia (C9-ALS/FTD). Dipeptide repeats (DPRs) generated by unconventional translation, especially the R-containing poly(GR), have been implicated in C9-ALS/FTD pathogenesis. Mutations in other genes, including TAR DNA-binding protein 43 KD (TDP-43), fused in sarcoma (FUS), and valosin-containing protein (VCP), have also been linked to ALS/FTD, and upregulation of amyloid precursor protein (APP) is observed at the early stage of ALS and FTD. Fundamental questions remain as to the relationships between these ALS/FTD genes and whether they converge on similar cellular pathways. Here, using biochemical, cell biological, and genetic analyses in Drosophila disease models, patient-derived fibroblasts, and mammalian cell culture, we show that mechanistic target of rapamycin complex 2 (mTORC2)/AKT signaling is activated by APP, TDP-43, and FUS, and that mTORC2/AKT and its downstream target VCP mediate the effect of APP, TDP-43, and FUS on the quality control of C9-ALS/FTD-associated poly(GR) translation. We also find that poly(GR) expression results in reduction of global translation, and that the co-expression of APP, TDP-43, and FUS results in further reduction of global translation, presumably through the GCN2/eIF2α integrated stress response pathway. Together, our results implicate mTORC2/AKT signaling and GCN2/eIF2α integrated stress response as common signaling pathways underlying ALS/FTD pathogenesis.
    Keywords:  APP; C9-ALS/FTD; FUS; TDP-43; mTORC2/AKT/VCP axis; poly(GR)
    DOI:  https://doi.org/10.1016/j.jbc.2023.102995
  24. J Clin Invest. 2023 Feb 07. pii: e164354. [Epub ahead of print]
    The ubiquitin ligase TRIM21 regulates mutant p53 accumulation and gain-of-function in cancer.
    Juan Liu, Cen Zhang, Dandan Xu, Tianliang Zhang, Chun-Yuan Chang, Jianming Wang, Jie Liu, Lanjing Zhang, Bruce G Haffty, Wei-Xing Zong, Wenwei Hu, Zhaohui Feng.
      Tumor suppressor TP53 is the most frequently mutated gene in human cancers. Mutant p53 (mutp53) proteins often accumulate to very high levels in human cancers to promote cancer progression through the gain-of-function (GOF) mechanism. Currently, the mechanism underlying mutp53 accumulation and GOF is incompletely understood. Here, we identified TRIM21 as a critical E3 ubiquitin ligase of mutp53 by screening for specific mutp53-interacting proteins. TRIM21 directly interacted with mutp53 but not wild-type p53, resulting in ubiquitination and degradation of mutp53 to suppress mutp53 GOF in tumorigenesis. TRIM21 deficiency in cancer cells promoted mutp53 accumulation and GOF in tumorigenesis. Compared with p53R172H knock-in mice, which displayed mutp53 accumulation specifically in tumors but not normal tissues, TRIM21 deletion in p53R172H knock-in mice resulted in mutp53 accumulation in normal tissues, an earlier tumor onset, and a shortened lifespan of mice. Furthermore, TRIM21 was frequently downregulated in some human cancers, including colorectal and breast cancers, and low TRIM21 expression was associated with poor prognosis in patients with cancers carrying mutp53. Our results revealed a critical mechanism underlying mutp53 accumulation in cancers, and also uncovered an important tumor-suppressive function of TRIM21 and its mechanism in cancers carrying mutp53.
    Keywords:  Cancer; Cell Biology; Oncology; Tumor suppressors; p53
    DOI:  https://doi.org/10.1172/JCI164354
  25. Heliyon. 2023 Feb;9(2): e13219
    Metabolomics analysis of an AAA-ATPase Cdc48-deficient yeast strain.
    Tomoyuki Kawarasaki, Kunio Nakatsukasa.
      The ubiquitin-specific chaperone AAA-ATPase Cdc48 and its orthologs p97/valosin-containing protein (VCP) in mammals play crucial roles in regulating numerous intracellular pathways via segregase activity, which separates polyubiquitinated targets from membranes or binding partners. Interestingly, high-throughput experiments show that a vast number of metabolic enzymes are modified with ubiquitin. Therefore, Cdc48 may regulate metabolic pathways, for example by acting on the polyubiquitin chains of metabolic enzymes; however, the role of Cdc48 in metabolic regulation remains largely unknown. To begin to analyze the role of Cdc48 in metabolic regulation in yeast, we performed a metabolomics analysis of temperature-sensitive cdc48-3 mutant cells. We found that the amount of metabolites in the glycolytic pathway was altered. Moreover, the pool of nucleotides, as well as the levels of metabolites involved in the tricarboxylic acid cycle and oxidative phosphorylation, increased, whereas the pool of amino acids decreased. These results suggest the involvement of Cdc48 in metabolic regulation in yeast. In addition, because of the roles of p97/VCP in regulating multiple cellular pathways, its inhibition is being considered as a promising anticancer drug target. We propose that the metabolomics study of Cdc48-deficient yeast will be useful as a complement to p97/VCP-related pathological and therapeutic studies.
    Keywords:  Cdc48/p97; Citrate synthase; Glyoxylate cycle; Metabolome analysis; Saccharomyces cerevisiae; Ubiquitin proteasome system
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e13219
  26. Science. 2023 Feb 09. eade8873
    Structural basis for regulation of apoptosis and autophagy by the BIRC6/SMAC complex.
    Julian F Ehrmann, Daniel B Grabarczyk, Maria Heinke, Luiza Deszcz, Robert Kurzbauer, Otto Hudecz, Alexandra Shulkina, Rebeca Gogova, Anton Meinhart, Gijs A Versteeg, Tim Clausen.
      Inhibitor of apoptosis proteins (IAPs) bind to pro-apoptotic proteases, keeping them inactive and preventing cell death. The atypical ubiquitin ligase BIRC6 is the only essential IAP, additionally functioning as a suppressor of autophagy. We performed a structure-function analysis of BIRC6 in complex with caspase-9, HTRA2, SMAC, and LC3B which are critical apoptosis and autophagy proteins. Cryo-electron microscopy structures show that BIRC6 forms a megadalton crescent shape that arcs around a spacious cavity containing receptor sites for client proteins. Multivalent binding of SMAC obstructs client binding, impeding ubiquitination of both autophagy and apoptotic substrates. Based on these data, we discuss how the BIRC6/SMAC complex can act as a stress-induced hub to regulate apoptosis and autophagy drivers.
    DOI:  https://doi.org/10.1126/science.ade8873
  27. bioRxiv. 2023 Jan 24. pii: 2023.01.23.525198. [Epub ahead of print]
    Systematically characterizing the roles of E3-ligase family members in inflammatory responses with massively parallel Perturb-seq.
    Kathryn Geiger-Schuller, Basak Eraslan, Olena Kuksenko, Kushal K Dey, Karthik A Jagadeesh, Pratiksha I Thakore, Ozge Karayel, Andrea R Yung, Anugraha Rajagopalan, Ana M Meireles, Karren Dai Yang, Liat Amir-Zilberstein, Toni Delorey, Devan Phillips, Raktima Raychowdhury, Christine Moussion, Alkes L Price, Nir Hacohen, John G Doench, Caroline Uhler, Orit Rozenblatt-Rosen, Aviv Regev.
      E3 ligases regulate key processes, but many of their roles remain unknown. Using Perturb-seq, we interrogated the function of 1,130 E3 ligases, partners and substrates in the inflammatory response in primary dendritic cells (DCs). Dozens impacted the balance of DC1, DC2, migratory DC and macrophage states and a gradient of DC maturation. Family members grouped into co-functional modules that were enriched for physical interactions and impacted specific programs through substrate transcription factors. E3s and their adaptors co-regulated the same processes, but partnered with different substrate recognition adaptors to impact distinct aspects of the DC life cycle. Genetic interactions were more prevalent within than between modules, and a deep learning model, comβVAE, predicts the outcome of new combinations by leveraging modularity. The E3 regulatory network was associated with heritable variation and aberrant gene expression in immune cells in human inflammatory diseases. Our study provides a general approach to dissect gene function.
    DOI:  https://doi.org/10.1101/2023.01.23.525198
  28. EMBO J. 2023 Feb 10. e112351
    Structural mechanism of CRL4-instructed STAT2 degradation via a novel cytomegaloviral DCAF receptor.
    Vu Thuy Khanh Le-Trilling, Sofia Banchenko, Darius Paydar, Pia Madeleine Leipe, Lukas Binting, Simon Lauer, Andrea Graziadei, Robin Klingen, Christine Gotthold, Jörg Bürger, Thilo Bracht, Barbara Sitek, Robert Jan Lebbink, Anna Malyshkina, Thorsten Mielke, Juri Rappsilber, Christian Mt Spahn, Sebastian Voigt, Mirko Trilling, David Schwefel.
      Human cytomegalovirus (CMV) is a ubiquitously distributed pathogen whose rodent counterparts such as mouse and rat CMV serve as common infection models. Here, we conducted global proteome profiling of rat CMV-infected cells and uncovered a pronounced loss of the transcription factor STAT2, which is crucial for antiviral interferon signalling. Via deletion mutagenesis, we found that the viral protein E27 is required for CMV-induced STAT2 depletion. Cellular and in vitro analyses showed that E27 exploits host-cell Cullin4-RING ubiquitin ligase (CRL4) complexes to induce poly-ubiquitylation and proteasomal degradation of STAT2. Cryo-electron microscopy revealed how E27 mimics molecular surface properties of cellular CRL4 substrate receptors called DCAFs (DDB1- and Cullin4-associated factors), thereby displacing them from the catalytic core of CRL4. Moreover, structural analyses showed that E27 recruits STAT2 through a bipartite binding interface, which partially overlaps with the IRF9 binding site. Structure-based mutations in M27, the murine CMV homologue of E27, impair the interferon-suppressing capacity and virus replication in mouse models, supporting the conserved importance of DCAF mimicry for CMV immune evasion.
    Keywords:  cullin-RING ubiquitin ligases; cytomegalovirus; interferon; ubiquitin-proteasome system; viral DCAF
    DOI:  https://doi.org/10.15252/embj.2022112351
  29. Cell. 2023 Feb 01. pii: S0092-8674(23)00043-0. [Epub ahead of print]
    A molecular device for the redox quality control of GroEL/ES substrates.
    Emile Dupuy, Sander Egbert Van der Verren, Jiusheng Lin, Mark Alan Wilson, Alix Vincent Dachsbeck, Felipe Viela, Emmanuelle Latour, Alexandra Gennaris, Didier Vertommen, Yves Frédéric Dufrêne, Bogdan Iuliu Iorga, Camille Véronique Goemans, Han Remaut, Jean-François Collet.
      Hsp60 chaperonins and their Hsp10 cofactors assist protein folding in all living cells, constituting the paradigmatic example of molecular chaperones. Despite extensive investigations of their structure and mechanism, crucial questions regarding how these chaperonins promote folding remain unsolved. Here, we report that the bacterial Hsp60 chaperonin GroEL forms a stable, functionally relevant complex with the chaperedoxin CnoX, a protein combining a chaperone and a redox function. Binding of GroES (Hsp10 cofactor) to GroEL induces CnoX release. Cryoelectron microscopy provided crucial structural information on the GroEL-CnoX complex, showing that CnoX binds GroEL outside the substrate-binding site via a highly conserved C-terminal α-helix. Furthermore, we identified complexes in which CnoX, bound to GroEL, forms mixed disulfides with GroEL substrates, indicating that CnoX likely functions as a redox quality-control plugin for GroEL. Proteins sharing structural features with CnoX exist in eukaryotes, suggesting that Hsp60 molecular plugins have been conserved through evolution.
    Keywords:  TPR; chaperones; chaperonin; protein folding; proteostasis; redox; thioredoxin
    DOI:  https://doi.org/10.1016/j.cell.2023.01.013
  30. J Med Chem. 2023 Feb 07.
    Discovery of Small-Molecule Autophagy Inhibitors by Disrupting the Protein-Protein Interactions Involving Autophagy-Related 5.
    Honggang Xiang, Ruiqi Liu, Xiangying Zhang, Ran An, Mi Zhou, Chang Tan, Qing Li, Minyi Su, Chun Guo, Lu Zhou, Yingxia Li, Renxiao Wang.
      One possible strategy for modulating autophagy is to disrupt the critical protein-protein interactions (PPIs) formed during this process. Our attention is on the autophagy-related 12 (ATG12)-autophagy-related 5 (ATG5)-autophagy-related 16-like 1 (ATG16L1) heterotrimer complex, which is responsible for ATG8 translocation from ATG3 to phosphatidylethanolamine. In this work, we discovered a compound with an (E)-3-(2-furanylmethylene)-2-pyrrolidinone core moiety (T1742) that blocked the ATG5-ATG16L1 and ATG5-TECAIR interactions in the in vitro binding assay (IC50 = 1-2 μM) and also exhibited autophagy inhibition in cellular assays. The possible binding mode of T1742 to ATG5 was predicted through molecular modeling, and a batch of derivatives sharing essentially the same core moiety were synthesized and tested. The outcomes of the in vitro binding assay and the flow cytometry assay of those newly synthesized compounds were generally consistent. This work has validated our central hypothesis that small-molecule inhibitors of the PPIs involving ATG5 can tune down autophagy effectively, and their pharmaceutical potential may be further explored.
    DOI:  https://doi.org/10.1021/acs.jmedchem.2c01233
  31. J Cell Sci. 2023 Feb 06. pii: jcs.260566. [Epub ahead of print]
    LRRK1 functions as a scaffold for PTP1B-mediated EGFR sorting into ILVs at the ER-endosome contact site.
    Hiroshi Hanafusa, Keitaro Fujita, Misa Kamio, Shiori Iida, Yasushi Tamura, Naoki Hisamoto, Kunihiro Matsumoto.
      Proper control of epidermal growth factor receptor (EGFR) signaling is important for maintaining cellular homeostasis. Since EGFR signaling occurs at the plasma membrane and endosomes following internalization, endosomal trafficking of EGFR regulates EGFR signaling spatiotemporally. In this process, leucine-rich repeat kinase 1 (LRRK1) has multiple roles in kinase activity-dependent transport of EGFR-containing endosomes and kinase-independent sorting of EGFR into the intraluminal vesicles (ILVs) of multivesicular bodies. Active EGFR inactivates the LRRK1 kinase activity by phosphorylating Tyr-944. In this study, we demonstrate that LRRK1 facilitates EGFR dephosphorylation by PTP1B, an endoplasmic reticulum (ER)-localized protein tyrosine phosphatase, at the ER-endosome contact site, after which EGFR is sorted into the ILVs of endosomes. LRRK1 is required for the PTP1B-EGFR interaction in response to EGF stimulation, resulting in the down-regulation of EGFR signaling. Furthermore, PTP1B activates LRRK1 by dephosphorylating pTyr-944 on the contact site, which promotes the transport of EGFR-containing endosomes. These findings provide evidence that the ER-endosome contact site functions as a hub for LRRK1-dependent signaling that regulates EGFR trafficking.
    Keywords:  EGFR; LRRK1; PTP1B
    DOI:  https://doi.org/10.1242/jcs.260566
  32. bioRxiv. 2023 Jan 28. pii: 2023.01.23.525251. [Epub ahead of print]
    Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.
    Taiasean Wu, Joshua C Yu, Arundhati Suresh, Zachary J Gale-Day, Matthew G Alteen, Amanda S Woo, Zoe Millbern, Oleta T Johnson, Emma C Carroll, Carrie L Partch, Denis Fourches, Nelson R Vinueza, David J Vocadlo, Jason E Gestwicki.
      Flexible in vitro methods alter the course of biological discoveries. Differential Scanning Fluorimetry (DSF) is a particularly versatile technique which reports protein thermal unfolding via fluorogenic dye. However, applications of DSF are limited by widespread protein incompatibilities with the available DSF dyes. Here, we enable DSF applications for 66 of 70 tested proteins (94%) including 10 from the SARS-CoV2 virus using a chemically diverse dye library, Aurora, to identify compatible dye-protein pairs in high throughput. We find that this protein-adaptive DSF platform (paDSF) not only triples the previous protein compatibility, but also fundamentally extends the processes observable by DSF, including interdomain allostery in O-GlcNAc Transferase (OGT). paDSF enables routine measurement of protein stability, dynamics, and ligand binding.
    One-Sentence Summary: Next generation protein-adaptive DSF (paDSF) enables rapid and general measurements of protein stability and dynamics.
    DOI:  https://doi.org/10.1101/2023.01.23.525251
  33. Methods Enzymol. 2023 ;pii: S0076-6879(22)00418-9. [Epub ahead of print]681 265-286
    Targeted protein degradation through light-activated E3 ligase recruitment.
    Olivia Shade, Amy Ryan, Alexander Deiters.
      Optical control of protein function through proteasomal degradation benefits from the noninvasive nature and spatiotemporal precision of light as a trigger. In this chapter, light activation of protein degradation with an optically controlled degron, termed optoDeg, is discussed. This method utilizes genetic code expansion to insert a photocaged analog of lysine at the N-terminal position of a protein of interest for spatial and temporal control of the N-end pathway, inducing proteasomal degradation. Methods for the use of optoDeg for degradation of the fluorescent reporter EGFP and the kinase MEK1 are described. The system is fast, with complete degradation of proteins within minutes following irradiation, and highly specific, with genetically directed introduction of the light-activated degron.
    Keywords:  Degron; E3 ligase; Genetic code expansion; Optogenetics; Photocaged amino acid; Protein degradation
    DOI:  https://doi.org/10.1016/bs.mie.2022.10.001
  34. Methods Enzymol. 2023 ;pii: S0076-6879(22)00442-6. [Epub ahead of print]681 241-263
    Crystallization of VHL-based PROTAC-induced ternary complexes.
    Andre J Wijaya, William Farnaby, Alessio Ciulli.
      X-ray crystal structures of PROTAC-induced ternary complexes provide invaluable insights into the critical species underpinning PROTAC mode of action, explain protein degradation selectivity profiles, and can guide rational degrader design. Nevertheless, crystallization of the ternary complexes formed by PROTACs remains an important bottleneck in employing this method. This is mainly due to the potential flexibility and heterogeneity that is inherent to a non-native protein-protein complex mediated by a small molecule, which together can hamper crystallization of the desired species. To overcome this limitation, selecting PROTAC compounds that enable the formation of stable, high-affinity and preferably cooperative ternary complexes in stoichiometric amount is, in our experience, critical to the success of co-crystallization studies. In this chapter, examples of stable PROTAC-mediated ternary complexes are illustrated. Learnings from biophysical & biochemical data are used as a guideline in achieving the highest "crystallizability" of ternary complexes. A case study of VHL-based SMARCA2 PROTAC degrader ternary complex crystallization is described. The procedure includes over-expression and purification of the E3 ligase and target protein, forming (and sometimes isolating) the ternary complex, and crystallizing it. The protocols can be applied for other combinations of E3 ligase, PROTAC and target protein.
    Keywords:  Cereblon; Crystallization; E3 ligase; PROTAC; Structural biology; VHL; X-ray crystallography
    DOI:  https://doi.org/10.1016/bs.mie.2022.10.005
  35. FEBS J. 2023 Feb 09.
    The functional role of Ire1 in regulating autophagy and proteasomal degradation under prolonged proteotoxic stress.
    Eshita Das, Kiran Kumari Sahu, Ipsita Roy.
      Inhibition of endoribonuclease/kinase Ire1 has shown beneficial effects in many proteotoxicity-induced pathology models. The mechanism by which this occurs has not been elucidated completely. Using a proteotoxic yeast model of Huntington's disease, we show that deletion of Ire1 led to lower protein aggregation at longer time points. The rate of protein degradation was higher in ΔIre1 cells. We monitored the two major protein degradation mechanisms in the cell. The increase in expression of Rpn4, coding for the transcription factor controlling proteasome biogenesis, was higher in ΔIre1 cells. The chymotrypsin-like proteasomal activity was also significantly enhanced in these cells at later time points of aggregation. The gene and protein expression levels of the autophagy gene Atg8 were higher in ΔIre1 than in wild-type cells. Significant increase in autophagy flux was also seen in ΔIre1 cells at later time points of aggregation. The results suggest that deletion of Ire1 activates UPR-independent arms of the proteostasis network, especially under conditions of aggravated stress. Thus, inhibition of Ire1 may regulate UPR-independent cellular stress-response pathways under prolonged stress.
    Keywords:  Atg8; Autophagy flux; Proteasome; Protein degradation; Rpn4
    DOI:  https://doi.org/10.1111/febs.16747
  36. Autophagy. 2023 Feb 05. 1-15
    G-quadruplex ligands as potent regulators of lysosomes.
    Lucille Ferret, Karla Alvarez-Valadez, Jennifer Rivière, Alexandra Muller, Natalia Bohálová, Luo Yu, Lionel Guittat, Vaclav Brázda, Guido Kroemer, Jean-Louis Mergny, Mojgan Djavaheri-Mergny.
      Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.
    Keywords:  Autophagy; TFEB; guanine-quadruplex; lysosome membrane permeabilization; transcriptional regulation
    DOI:  https://doi.org/10.1080/15548627.2023.2170071
  37. PLoS Biol. 2023 Feb;21(2): e3001962
    AlphaFold2-multimer guided high-accuracy prediction of typical and atypical ATG8-binding motifs.
    Tarhan Ibrahim, Virendrasinh Khandare, Federico Gabriel Mirkin, Yasin Tumtas, Doryen Bubeck, Tolga O Bozkurt.
      Macroautophagy/autophagy is an intracellular degradation process central to cellular homeostasis and defense against pathogens in eukaryotic cells. Regulation of autophagy relies on hierarchical binding of autophagy cargo receptors and adaptors to ATG8/LC3 protein family members. Interactions with ATG8/LC3 are typically facilitated by a conserved, short linear sequence, referred to as the ATG8/LC3 interacting motif/region (AIM/LIR), present in autophagy adaptors and receptors as well as pathogen virulence factors targeting host autophagy machinery. Since the canonical AIM/LIR sequence can be found in many proteins, identifying functional AIM/LIR motifs has proven challenging. Here, we show that protein modelling using Alphafold-Multimer (AF2-multimer) identifies both canonical and atypical AIM/LIR motifs with a high level of accuracy. AF2-multimer can be modified to detect additional functional AIM/LIR motifs by using protein sequences with mutations in primary AIM/LIR residues. By combining protein modelling data from AF2-multimer with phylogenetic analysis of protein sequences and protein-protein interaction assays, we demonstrate that AF2-multimer predicts the physiologically relevant AIM motif in the ATG8-interacting protein 2 (ATI-2) as well as the previously uncharacterized noncanonical AIM motif in ATG3 from potato (Solanum tuberosum). AF2-multimer also identified the AIM/LIR motifs in pathogen-encoded virulence factors that target ATG8 members in their plant and human hosts, revealing that cross-kingdom ATG8-LIR/AIM associations can also be predicted by AF2-multimer. We conclude that the AF2-guided discovery of autophagy adaptors/receptors will substantially accelerate our understanding of the molecular basis of autophagy in all biological kingdoms.
    DOI:  https://doi.org/10.1371/journal.pbio.3001962
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