bims-proteo Biomed News
on Proteostasis
Issue of 2022–11–20
twenty-two papers selected by
Eric Chevet, INSERM
  1. IRE-1 endoribonuclease activity declines early in C. elegans adulthood and is not rescued by reduced reproduction.
  2. Export of polybasic motif-containing secretory proteins BMP8A and SFRP1 from the endoplasmic reticulum is regulated by surfeit locus protein 4 (SURF4): SURF4 regulates ER export of BMP8A and SFRP1.
  3. Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage.
  4. Endoplasmic reticulum stress and lipids in health and diseases.
  5. Glycosylation network mapping and site-specific glycan maturation in vivo.
  6. Identification and structural basis of C-terminal cyclic imides as natural degrons for cereblon.
  7. Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment.
  8. LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.
  9. Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy.
  10. Calnexin reveals a sugar-free taste within the lipid bilayer.
  11. 4-phenylbutyric acid-Identity crisis; can it act as a translation inhibitor?
  12. Organelle homeostasis: from cellular mechanisms to disease.
  13. Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning.
  14. Chaperone-mediated autophagy regulates adipocyte differentiation.
  15. Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein ubiquitination dynamics in dendrites.
  16. Molecular glue CELMoD compounds are regulators of cereblon conformation.
  17. The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain.
  18. The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy.
  19. Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.
  20. Agr2-associated ER stress promotes adherent-invasive E. coli dysbiosis and triggers CD103+ dendritic cell IL-23-dependent ileocolitis.
  21. Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.
  22. Optical Control of Translation with a Puromycin Photoswitch.
  1. Front Aging. 2022 ;3 1044556
    IRE-1 endoribonuclease activity declines early in C. elegans adulthood and is not rescued by reduced reproduction.
    Evandro A De-Souza, Nadia Cummins, Rebecca C Taylor.
      The proteome of a cell helps to define its functional specialization. Most proteins must be translated and properly folded to ensure their biological function, but with aging, animals lose their ability to maintain a correctly folded proteome. This leads to the accumulation of protein aggregates, decreased stress resistance, and the onset of age-related disorders. The unfolded protein response of the endoplasmic reticulum (UPRER) is a central protein quality control mechanism, the function of which is known to decline with age. Here, we show that age-related UPRER decline in Caenorhabditis elegans occurs at the onset of the reproductive period and is caused by a failure in IRE-1 endoribonuclease activities, affecting both the splicing of xbp-1 mRNA and regulated Ire1 dependent decay (RIDD) activity. Animals with a defect in germline development, previously shown to rescue the transcriptional activity of other stress responses during aging, do not show restored UPRER activation with age. This underlines the mechanistic difference between age-associated loss of UPRER activation and that of other stress responses in this system, and uncouples reproductive status from the activity of somatic maintenance pathways. These observations may aid in the development of strategies that aim to overcome the proteostasis decline observed with aging.
    Keywords:  C. elegans; IRE1; UPR; aging; cell stress; proteostasis; stress response; unfolded protein response
    DOI:  https://doi.org/10.3389/fragi.2022.1044556
  2. J Biol Chem. 2022 Nov 09. pii: S0021-9258(22)01130-9. [Epub ahead of print] 102687
    Export of polybasic motif-containing secretory proteins BMP8A and SFRP1 from the endoplasmic reticulum is regulated by surfeit locus protein 4 (SURF4): SURF4 regulates ER export of BMP8A and SFRP1.
    Xiao Tang, Tingxuan Wang, Yusong Guo.
      In the conventional secretory pathway, cargo receptors play important roles in exporting newly synthesized secretory proteins from the endoplasmic reticulum (ER). We previously showed that a cargo receptor, surfeit locus protein 4 (SURF4), promotes ER export of a soluble signaling molecule, sonic hedgehog (Shh), via recognizing the polybasic residues within its Cardin-Weintraub motif. In addition to Shh, we found 30 more secretory proteins containing the polybasic motif (K/R)(K/R)(K/R)XX(K/R)(K/R), but whether SURF4 plays a general role in mediating ER export of these secretory proteins is unclear. Here, we analyzed the trafficking of four of these secretory proteins: desert hedgehog (Dhh), Indian hedgehog (Ihh), bone morphogenetic protein 8A (BMP8A), and secreted frizzled-related protein 1 (SFRP1). We found that the polybasic motifs contained in these cargo proteins are important for their ER export. Further analyses indicated that the polybasic motifs of BMP8A and SFRP1 interact with the tri-acidic motif on the predicted first luminal domain of SURF4. These interactions with SURF4 are essential and sufficient for the ER-to-Golgi trafficking of BMP8A and SFRP1. Moreover, we demonstrate that SURF4 localizes at a subpopulation of ER exit sites (ERES) to regulate ER export of its clients. Taken together, these results suggest that SURF4 is recruited to specific ERESs and plays a general role in capturing polybasic motif-containing secretory cargo proteins through electrostatic interactions.
    Keywords:  COPII; Cargo sorting; ER export; SURF4; Secretion
    DOI:  https://doi.org/10.1016/j.jbc.2022.102687
  3. Autophagy. 2022 Nov 17.
    Membrane Atg8ylation, stress granule formation, and MTOR regulation during lysosomal damage.
    Jingyue Jia, Fulong Wang, Zambarlal Bhujabal, Ryan Peters, Michal Mudd, Thabata Duque, Lee Allers, Ruheena Javed, Michelle Salemi, Christian Behrends, Brett Phinney, Terje Johansen, Vojo Deretic.
      The functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosis infection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.
    Keywords:  Atg8ylation; MTOR; Mycobacterium tuberculosis; NUFIP2; PKR; SARS-CoV-2 ORF3a; integrated stress response; lysosomal damage; proteopathic tau; stress granules
    DOI:  https://doi.org/10.1080/15548627.2022.2148900
  4. Prog Lipid Res. 2022 Nov 13. pii: S0163-7827(22)00053-4. [Epub ahead of print]89 101198
    Endoplasmic reticulum stress and lipids in health and diseases.
    Cenk Celik, Stella Yue Ting Lee, Wei Sheng Yap, Guillaume Thibault.
      The endoplasmic reticulum (ER) is a complex and dynamic organelle that regulates many cellular pathways, including protein synthesis, protein quality control, and lipid synthesis. When one or multiple ER roles are dysregulated and saturated, the ER enters a stress state, which, in turn, activates the highly conserved unfolded protein response (UPR). By sensing the accumulation of unfolded proteins or lipid bilayer stress (LBS) at the ER, the UPR triggers pathways to restore ER homeostasis and eventually induces apoptosis if the stress remains unresolved. In recent years, it has emerged that the UPR works intimately with other cellular pathways to maintain lipid homeostasis at the ER, and so does at cellular levels. Lipid distribution, along with lipid anabolism and catabolism, are tightly regulated, in part, by the ER. Dysfunctional and overwhelmed lipid-related pathways, independently or in combination with ER stress, can have reciprocal effects on other cellular functions, contributing to the development of diseases. In this review, we summarize the current understanding of the UPR in response to proteotoxic stress and LBS and the breadth of the functions mitigated by the UPR in different tissues and in the context of diseases.
    Keywords:  Cancer; Diabetes; Endoplasmic reticulum stress; Fatty liver; Immune response; Lipid bilayer stress; Lipid homeostasis; Lipotoxicity; Metabolic diseases; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.plipres.2022.101198
  5. iScience. 2022 Nov 18. 25(11): 105417
    Glycosylation network mapping and site-specific glycan maturation in vivo.
    Marie-Estelle Losfeld, Ernesto Scibona, Chia-Wei Lin, Markus Aebi.
      Glycoprotein processing along a complex highly compartmentalized pathway is a hallmark of eukaryotic cells. We followed the kinetics of intracellular, site-specific glycan processing of a model protein with five distinct N-glycosylation sites and deduced a mathematical model of the secretory pathway that describes a complex set of processing reactions localized in defined intracellular compartments such as the endoplasmic reticulum the Golgi, or the lysosome. The model was able to accommodate site-specific N-glycan processing and we identified phosphorylated glycan structures of the mannose-6-phosphate pathway responsible for the lysosomal sorting of the glycoprotein. Importantly, our model protein can take different routes of the cellular secretory pathway, resulting in an increased glycan complexity of the secreted protein.
    Keywords:  Cell biology; Integrative aspects of cell biology; Mathematical biosciences
    DOI:  https://doi.org/10.1016/j.isci.2022.105417
  6. Biochem Biophys Res Commun. 2022 Nov 05. pii: S0006-291X(22)01529-7. [Epub ahead of print]637 66-72
    Identification and structural basis of C-terminal cyclic imides as natural degrons for cereblon.
    Christopher Heim, Anna-Katharina Spring, Sören Kirchgäßner, Dirk Schwarzer, Marcus D Hartmann.
      Cereblon (CRBN) is a ubiquitously expressed E3 ligase substrate receptor and a key player in pharmaceutical targeted protein degradation. Despite substantial insight gained into its chemical ligand space that is exploited in small-molecule protein degraders, its cellular role and native mechanism of substrate recognition remained elusive so far. In this communication, we report the discovery of C-terminal aspartimide and aminoglutarimide residues as natural degron motifs that are recognized by CRBN with high specificity. These C-terminal cyclic imides are known to form in ageing proteins as a result of spontaneous chain breaks after an attack of an asparagine or glutamine side chain amide on the adjacent peptide bond, and thereby mark potentially malfunctional protein fragments. In crystal structures, we uncover that these C-terminal cyclic imides are bound in the same fashion as small-molecule CRBN modulators, and that the residues preceding the cyclic terminus contribute to the interaction with a sequence-unspecific backbone hydrogen bonding pattern with strictly conserved residues in CRBN. We postulate that C-terminal aspartimide and aminoglutarimide residues resulting from chain breaks are largely underappreciated protein damages and represent the native degrons of CRBN.
    Keywords:  Aspartimide; E3 ubiquitin ligase; Protein ageing; Protein chain break; Protein damage; Targeted protein degradation
    DOI:  https://doi.org/10.1016/j.bbrc.2022.11.001
  7. J Biol Chem. 2022 Nov 14. pii: S0021-9258(22)01143-7. [Epub ahead of print] 102700
    Diptoindonesin G is a middle domain HSP90 modulator for cancer treatment.
    Kristine Donahue, Haibo Xie, Miyang Li, Ang Gao, Min Ma, Yidan Wang, Rose Tipton, Nicole Semanik, Tina Primeau, Shunqiang Li, Lingjun Li, Weiping Tang, Wei Xu.
      HSP90 inhibitors can target many oncoproteins simultaneously, but none have made it through clinical trials due to dose-limiting toxicity and induction of heat shock response, leading to clinical resistance. We identified diptoindonesin G (dip G) as an HSP90 modulator that can promote degradation of HSP90 clients by binding to the middle domain of HSP90 (Kd =0.13 ± 0.02 μM) without inducing heat shock response. This is likely because dip G does not interfere with the HSP90-HSF1 interaction like N-terminal inhibitors, maintaining HSF1 in a transcriptionally silent state. We found that binding of dip G to HSP90 promotes degradation of HSP90 client protein estrogen receptor α (ER), a major oncogenic driver protein in most breast cancers. Mutations in the ER ligand-binding domain (LBD) are an established mechanism of endocrine resistance, and decrease the binding affinity of mainstay endocrine therapies targeting ER, reducing their ability to promote ER degradation or transcriptionally silence ER. Because dip G binds to HSP90 and does not bind to the LBD of ER, unlike endocrine therapies, it is insensitive to ER LBD mutations that drive endocrine resistance. Additionally, we determined that dip G promoted degradation of wild type and mutant ER with similar efficacy, downregulated ER- and mutant ER-regulated gene expression, and inhibited WT and mutant cell proliferation. Our data suggest that dip G is not only a molecular probe to study HSP90 biology and the HSP90 conformation cycle, but also a new therapeutic avenue for various cancers, particularly endocrine-resistant breast cancer harboring ER LBD mutations.
    Keywords:  ESR1; Heat shock factor protein 1 (HSF1); Heat shock protein 90 (HSP90); LBD mutations; breast cancer; estrogen receptor; inhibitor
    DOI:  https://doi.org/10.1016/j.jbc.2022.102700
  8. EMBO J. 2022 Nov 18. e112006
    LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus.
    Zhixiao Wu, Lena A Berlemann, Verian Bader, Dominik A Sehr, Eva Dawin, Alberto Covallero, Jens Meschede, Lena Angersbach, Cathrin Showkat, Jonas B Michaelis, Christian Münch, Bettina Rieger, Dmitry Namgaladze, Maria Georgina Herrera, Fabienne C Fiesel, Wolfdieter Springer, Marta Mendes, Jennifer Stepien, Katalin Barkovits, Katrin Marcus, Albert Sickmann, Gunnar Dittmar, Karin B Busch, Dietmar Riedel, Marisa Brini, Jörg Tatzelt, Tito Cali, Konstanze F Winklhofer.
      Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear.
    Keywords:  HOIP; NEMO; OTULIN; PINK1; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2022112006
  9. EBioMedicine. 2022 Nov 11. pii: S2352-3964(22)00535-7. [Epub ahead of print]86 104353
    Atypical E3 ligase ZFP91 promotes small-molecule-induced E2F2 transcription factor degradation for cancer therapy.
    Ting-Ting Liu, Heng Yang, Fang-Fang Zhuo, Zhuo Yang, Mei-Mei Zhao, Qiang Guo, Yang Liu, Dan Liu, Ke-Wu Zeng, Peng-Fei Tu.
       BACKGROUND: The E2F family of transcription factors play a crucial role in the development of various cancers. However, E2F members lack targetable binding pockets and are typically considered "undruggable". Unlike canonical small-molecule therapeutics, molecular glues mediate new E3 ligase-protein interactions to induce selective proteasomal degradation, which represents an attractive option to overcome these limitations.
    METHODS: Human proteome microarray was utilized to identify a natural product-derived molecular glue for targeting E2F2 degradation. Co-IP analysis with stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative proteomics was carried out to further explore the E3 ligase for E2F2 degradation.
    FINDINGS: In this study, we identified a molecular glue bufalin, which significantly promoted E2F2 degradation. Unexpectedly, E2F2 underwent ubiquitination and proteasomal degradation via a previously undisclosed atypical E3 ligase, zinc finger protein 91 (ZFP91). In particular, we observed that bufalin markedly promoted E2F2-ZFP91 complex formation, thereby leading to E2F2 polyubiquitination via K48-linked ubiquitin chains for degradation. E2F2 degradation subsequently caused transcriptional suppression of multiple oncogenes including c-Myc, CCNE1, CCNE2, MCM5 and CDK1, and inhibited hepatocellular carcinoma growth in vitro and in vivo.
    INTERPRETATION: Collectively, our findings open up a new direction for transcription factors degradation by targeting atypical E3 ligase ZFP91. Meanwhile, the chemical knockdown strategy with molecular glue may promote innovative transcription factor degrader development in cancer therapy.
    FUNDING: This work was financially supported by the National Key Research and Development Project of China (2022YFC3501601), National Natural Sciences Foundation of China (81973505, 82174008, 82030114), and China Postdoctoral Science Foundation (2019M650396), the Fundamental Research Funds for the Central Universities.
    Keywords:  Cancer therapy; E2F2 transcription factor; E3 ligase ZFP91; Molecular glue; Proteasomal degradation
    DOI:  https://doi.org/10.1016/j.ebiom.2022.104353
  10. EMBO J. 2022 Nov 15. e113003
    Calnexin reveals a sugar-free taste within the lipid bilayer.
    Kevin P Guay, Robert V Williams, Daniel N Hebert.
      Maturation of membrane proteins is complicated by the need to fold in three distinct environments. While much is known about folding in the two aqueous milieus constituted by cytoplasm and ER lumen, our knowledge of the folding, arrangement, and quality control of transmembrane regions within the lipid bilayer, and its facilitation by molecular chaperones, is limited. New work by Bloemeke et al now reveals an expanded role of the ER chaperone calnexin acting within the lipid bilayer in a carbohydrate-independent manner.
    DOI:  https://doi.org/10.15252/embj.2022113003
  11. Aging Cell. 2022 Nov 14. e13738
    4-phenylbutyric acid-Identity crisis; can it act as a translation inhibitor?
    Daniel Stein, Zeev Slobodnik, Benjamin Tam, Monica Einav, Barak Akabayov, Shimon Berstein, Debra Toiber.
      Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4-phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore-it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases.
    Keywords:  4PBA; Proteostasis; chemical chaperone; protein synthesis; translation inhibition; unfolded protein response
    DOI:  https://doi.org/10.1111/acel.13738
  12. FEBS J. 2022 Nov;289(22): 6822-6831
    Organelle homeostasis: from cellular mechanisms to disease.
    Emma Burbridge, Colin Adrain.
      The major criterion that distinguishes eukaryotes from prokaryotes is the presence of organelles in the former. Organelles provide a compartment in which biochemical processes are corralled within bespoke biophysical conditions and act as storage depots, powerhouses, waste storage/recycling units and innate immune signalling hubs. A key challenge faced by organelles is to define, and then retain, their identity; this is mediated by complex proteostasis mechanisms including the import of an organelle-specific proteome, the exclusion of non-organellar proteins and the removal of misfolded proteins via dedicated quality control mechanisms. This Special Issue on Organelle Homeostasis provides an engaging, eclectic, yet integrative, perspective on organelle homeostasis in a range of organelles including those from the secretory and endocytic pathways, mitochondria, the autophagy-lysosomal pathway and the nucleus and its sub-compartments. Some lesser-known organelles including migrasomes (organelles that are released by migrating cells) and GOMED (a Golgi-specific form of autophagy) are also introduced. In the spirit of the principles of organelle biology, we hope you find the reviews in this Issue both encapsulating and captivating, and we thank the authors for their excellent contributions.
    Keywords:  endoplasmic reticulum; mitochondria; nucleus; organelle homeostasis; quality control
    DOI:  https://doi.org/10.1111/febs.16667
  13. Traffic. 2022 Nov 18.
    Cab45 deficiency leads to the mistargeting of progranulin and prosaposin and aberrant lysosomal positioning.
    Mai Ly Tran, Johanna Tüshaus, Yeongho Kim, Bulat R Ramazanov, Swathi Devireddy, Stefan F Lichtenthaler, Shawn M Ferguson, Julia von Blume.
      The trans-Golgi Network (TGN) sorts molecular "addresses" and sends newly synthesized proteins to their destination via vesicular transport carriers. Despite the functional significance of packaging processes at the TGN, the sorting of soluble proteins remains poorly understood. Recent research has shown that the Golgi resident protein Cab45 is a significant regulator of secretory cargo sorting at the TGN. Cab45 oligomerizes upon transient Ca2+ influx, recruits soluble cargo molecules (clients), and packs them in sphingomyelin-rich transport carriers. However, the identity of client molecules packed into Cab45 vesicles is scarce. Therefore, we used a precise and highly efficient secretome analysis technology called hiSPECs. Intriguingly, we observed that Cab45 deficient cells manifest hypersecretion of lysosomal hydrolases. Specifically, Cab45 deficient cells secrete the unprocessed precursors of prosaposin (PSAP) and progranulin (PGRN). In addition, lysosomes in these cells show an aberrant perinuclear accumulation suggesting a new role of Cab45 in lysosomal positioning. This work uncovers a yet unknown function of Cab45 in regulating lysosomal function.
    Keywords:  Cab45; TGN export; lysosomal hydrolases; progranulin; prosaposin
    DOI:  https://doi.org/10.1111/tra.12873
  14. Sci Adv. 2022 Nov 18. 8(46): eabq2733
    Chaperone-mediated autophagy regulates adipocyte differentiation.
    Susmita Kaushik, Yves R Juste, Kristen Lindenau, Shuxian Dong, Adrián Macho-González, Olaya Santiago-Fernández, Mericka McCabe, Rajat Singh, Evripidis Gavathiotis, Ana Maria Cuervo.
      Adipogenesis is a tightly orchestrated multistep process wherein preadipocytes differentiate into adipocytes. The most studied aspect of adipogenesis is its transcriptional regulation through timely expression and silencing of a vast number of genes. However, whether turnover of key regulatory proteins per se controls adipogenesis remains largely understudied. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal protein degradation that, in response to diverse cues, remodels the proteome for regulatory purposes. We report here the activation of CMA during adipocyte differentiation and show that CMA regulates adipogenesis at different steps through timely degradation of key regulatory signaling proteins and transcription factors that dictate proliferation, energetic adaptation, and signaling changes required for adipogenesis.
    DOI:  https://doi.org/10.1126/sciadv.abq2733
  15. Cell Rep. 2022 Nov 15. pii: S2211-1247(22)01529-7. [Epub ahead of print]41(7): 111658
    Development of single-molecule ubiquitination mediated fluorescence complementation to visualize protein ubiquitination dynamics in dendrites.
    Marius F Ifrim, Aleksandra Janusz-Kaminska, Gary J Bassell.
      The ubiquitination/proteasome system is important for the spatiotemporal control of protein synthesis and degradation at synapses, while dysregulation may underlie autism spectrum disorders (ASDs). However, methods allowing direct visualization of the subcellular localization and temporal dynamics of protein ubiquitination are lacking. Here we report the development of Single-Molecule Ubiquitin Mediated Fluorescence Complementation (SM-UbFC) as a method to visualize and quantify the dynamics of protein ubiquitination in dendrites of live neurons in culture. Using SM-UbFC, we demonstrate that the rate of PSD-95 ubiquitination is elevated in dendrites of FMR1 KO neurons compared with wild-type controls. We further demonstrate the rapid ubiquitination of the fragile X messenger ribonucleoprotein, FMRP, and the AMPA receptor subunit, GluA1, which are known to be key events in the regulation of synaptic protein synthesis and plasticity. SM-UbFC will be useful for future studies on the regulation of synaptic protein homeostasis.
    Keywords:  BiFC; CP: Neuroscience; FMRP; Fragile X syndrome; GluA1; Mdm2; PSD-95; UbFC; mGluR; single-molecule imaging; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2022.111658
  16. Science. 2022 11 04. 378(6619): 549-553
    Molecular glue CELMoD compounds are regulators of cereblon conformation.
    Edmond R Watson, Scott Novick, Mary E Matyskiela, Philip P Chamberlain, Andres H de la Peña, Jinyi Zhu, Eileen Tran, Patrick R Griffin, Ingrid E Wertz, Gabriel C Lander.
      Cereblon (CRBN) is a ubiquitin ligase (E3) substrate receptor protein co-opted by CRBN E3 ligase modulatory drug (CELMoD) agents that target therapeutically relevant proteins for degradation. Prior crystallographic studies defined the drug-binding site within CRBN's thalidomide-binding domain (TBD), but the allostery of drug-induced neosubstrate binding remains unclear. We performed cryo-electron microscopy analyses of the DNA damage-binding protein 1 (DDB1)-CRBN apo complex and compared these structures with DDB1-CRBN in the presence of CELMoD compounds alone and complexed with neosubstrates. Association of CELMoD compounds to the TBD is necessary and sufficient for triggering CRBN allosteric rearrangement from an open conformation to the canonical closed conformation. The neosubstrate Ikaros only stably associates with the closed CRBN conformation, illustrating the importance of allostery for CELMoD compound efficacy and informing structure-guided design strategies to improve therapeutic efficacy.
    DOI:  https://doi.org/10.1126/science.add7574
  17. iScience. 2022 Nov 18. 25(11): 105425
    The RNA repair proteins RtcAB regulate transcription activator RtcR via its CRISPR-associated Rossmann fold domain.
    Ioly Kotta-Loizou, Maria Grazia Giuliano, Milija Jovanovic, Jorrit Schaefer, Fuzhou Ye, Nan Zhang, Danai Athina Irakleidi, Xiaojiao Liu, Xiaodong Zhang, Martin Buck, Christoph Engl.
      CRISPR-associated Rossmann fold (CARF) domain signaling underpins modulation of CRISPR-Cas nucleases; however, the RtcR CARF domain controls expression of two conserved RNA repair enzymes, cyclase RtcA and ligase RtcB. Here, we demonstrate that RtcAB are required for RtcR-dependent transcription activation and directly bind to RtcR CARF. RtcAB catalytic activity is not required for complex formation with CARF, but is essential yet not sufficient for RtcRAB-dependent transcription activation, implying the need for an additional RNA repair-dependent activating signal. This signal differs from oligoadenylates, a known ligand of CARF domains, and instead appears to originate from the translation apparatus: RtcB repairs a tmRNA that rescues stalled ribosomes and increases translation elongation speed. Taken together, our data provide evidence for an expanded range for CARF domain signaling, including the first evidence of its control via in trans protein-protein interactions, and a feed-forward mechanism to regulate RNA repair required for a functioning translation apparatus.
    Keywords:  Cell biology; Molecular biology; Molecular mechanism of gene regulation
    DOI:  https://doi.org/10.1016/j.isci.2022.105425
  18. J Biol Chem. 2022 Nov 12. pii: S0021-9258(22)01147-4. [Epub ahead of print] 102704
    The interaction between E3 ubiquitin ligase Parkin and mitophagy receptor PHB2 links inner mitochondrial membrane ubiquitination to efficient mitophagy.
    Shan Sun, Hongyu Hou, Guoqiang Ma, Qilian Ma, Ningning Li, Li Zhang, Chunsheng Dong, Mian Cao, Kin Yip Tam, Zheng Ying, Hongfeng Wang.
      The autophagic clearance of mitochondria has been defined as mitophagy, which is triggered by mitochondrial damage and serves as a major pathway for mitochondrial homeostasis and cellular quality control. PINK1 and Parkin-mediated mitophagy is the most extensively studied form of mitophagy, which has been linked to the pathogenesis of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The current paradigm of this particular mitophagy pathway is that the ubiquitination of the outer mitochondrial membrane is the key step to enable the recognition of damaged mitochondria by the core autophagic component autophagosome. However, whether the inner mitochondrial membrane (IMM) is ubiquitinated by Parkin and its contribution to sufficient mitophagy remain unclear. Here, using molecular, cellular, and biochemical approaches, we report that prohibitin 2 (PHB2), an essential IMM receptor for mitophagy, is ubiquitinated by Parkin and thereby gains higher affinity to the autophagosome during mitophagy. Our findings suggest that Parkin directly binds to PHB2 through its RING1 domain and promotes K11- and K33-linked ubiquitination on K142/K200 sites of PHB2, thereby enhancing the interaction between PHB2 and MAP1LC3B/LC3B. Interestingly and importantly, our study allows us to propose a novel model in which IMM protein PHB2 serves as both a receptor and a ubiquitin-mediated base for autophagosome recruitment to ensure efficient mitophagy.
    Keywords:  MAP1LC3B/LC3B; PHB2; Parkin; mitophagy; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2022.102704
  19. Nat Commun. 2022 Nov 12. 13(1): 6897
    Lipid-mediated activation of plasma membrane-localized deubiquitylating enzymes modulate endosomal trafficking.
    Karin Vogel, Tobias Bläske, Marie-Kristin Nagel, Christoph Globisch, Shane Maguire, Lorenz Mattes, Christian Gude, Michael Kovermann, Karin Hauser, Christine Peter, Erika Isono.
      The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.
    DOI:  https://doi.org/10.1038/s41467-022-34637-3
  20. Cell Rep. 2022 Nov 15. pii: S2211-1247(22)01508-X. [Epub ahead of print]41(7): 111637
    Agr2-associated ER stress promotes adherent-invasive E. coli dysbiosis and triggers CD103+ dendritic cell IL-23-dependent ileocolitis.
    Monica Viladomiu, Manirath Khounlotham, Belgin Dogan, Svetlana F Lima, Ahmed Elsaadi, Emre Cardakli, Jim G Castellanos, Charles Ng, Jeremy Herzog, Alexi A Schoenborn, Melissa Ellermann, Bo Liu, Shiying Zhang, Ajay S Gulati, R Balfour Sartor, Kenneth W Simpson, Steven M Lipkin, Randy S Longman.
      Endoplasmic reticulum (ER) stress is associated with Crohn's disease (CD), but its impact on host-microbe interaction in disease pathogenesis is not well defined. Functional deficiency in the protein disulfide isomerase anterior gradient 2 (AGR2) has been linked with CD and leads to epithelial cell ER stress and ileocolitis in mice and humans. Here, we show that ileal expression of AGR2 correlates with mucosal Enterobactericeae abundance in human inflammatory bowel disease (IBD) and that Agr2 deletion leads to ER-stress-dependent expansion of mucosal-associated adherent-invasive Escherichia coli (AIEC), which drives Th17 cell ileocolitis in mice. Mechanistically, our data reveal that AIEC-induced epithelial cell ER stress triggers CD103+ dendritic cell production of interleukin-23 (IL-23) and that IL-23R is required for ileocolitis in Agr2-/- mice. Overall, these data reveal a specific and reciprocal interaction of the expansion of the CD pathobiont AIEC with ER-stress-associated ileocolitis and highlight a distinct cellular mechanism for IL-23-dependent ileocolitis.
    Keywords:  AIEC; Agr2; CP: Immunology; CP: Microbiology; ER stress; IL-23; Th17; ileitis
    DOI:  https://doi.org/10.1016/j.celrep.2022.111637
  21. Cell Metab. 2022 Nov 08. pii: S1550-4131(22)00489-2. [Epub ahead of print]
    Ribosome stalling is a signal for metabolic regulation by the ribotoxic stress response.
    Goda Snieckute, Aitana Victoria Genzor, Anna Constance Vind, Laura Ryder, Mark Stoneley, Sébastien Chamois, René Dreos, Cathrine Nordgaard, Frederike Sass, Melanie Blasius, Aida Rodríguez López, Sólveig Hlín Brynjólfsdóttir, Kasper Langebjerg Andersen, Anne E Willis, Lisa B Frankel, Steen Seier Poulsen, David Gatfield, Zachary Gerhart-Hines, Christoffer Clemmensen, Simon Bekker-Jensen.
      Impairment of translation can lead to collisions of ribosomes, which constitute an activation platform for several ribosomal stress-surveillance pathways. Among these is the ribotoxic stress response (RSR), where ribosomal sensing by the MAP3K ZAKα leads to activation of p38 and JNK kinases. Despite these insights, the physiological ramifications of ribosomal impairment and downstream RSR signaling remain elusive. Here, we show that stalling of ribosomes is sufficient to activate ZAKα. In response to amino acid deprivation and full nutrient starvation, RSR impacts on the ensuing metabolic responses in cells, nematodes, and mice. The RSR-regulated responses in these model systems include regulation of AMPK and mTOR signaling, survival under starvation conditions, stress hormone production, and regulation of blood sugar control. In addition, ZAK-/- male mice present a lean phenotype. Our work highlights impaired ribosomes as metabolic signals and demonstrates a role for RSR signaling in metabolic regulation.
    Keywords:  AMPK; FGF21; ZAK-alpha; amino acid starvation; mTOR; metabolic regulation; mouse models; ribosome collision; ribotoxic stress response
    DOI:  https://doi.org/10.1016/j.cmet.2022.10.011
  22. J Am Chem Soc. 2022 Nov 17.
    Optical Control of Translation with a Puromycin Photoswitch.
    Tongil Ko, Mauricio M Oliveira, Jessica M Alapin, Johannes Morstein, Eric Klann, Dirk Trauner.
      Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed puroswitch, features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. Puroswitch shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that puroswitch inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, puroswitch reacts with standard puromycin antibodies, which allows for tracking de novo protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, puroswitch can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision puroswitch as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.
    DOI:  https://doi.org/10.1021/jacs.2c07374
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