bims-proteo Biomed News
on Proteostasis
Issue of 2023‒07‒09
thirty-one papers selected by
Eric Chevet
INSERM
  1. HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates.
  2. Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5.
  3. Cryo-EM reveals important regulatory mechanism of the workhorses of targeted protein degradation.
  4. Endoplasmic reticulum network heterogeneity guides diffusive transport and kinetics.
  5. TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.
  6. CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.
  7. ER Ca2+ overload activates the IRE1α signaling and promotes cell survival.
  8. The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2.
  9. Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.
  10. How host ER membrane chaperones and morphogenic proteins support virus infection.
  11. The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice.
  12. Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.
  13. Multiple E3 ligases control tankyrase stability and function.
  14. Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.
  15. Global and tissue-specific aging effects on murine proteomes.
  16. Inducible protein degradation as a strategy to identify Phosphoprotein Phosphatase 6 substrates in RAS-mutant colorectal cancer cells.
  17. A strategy for small-molecule RNA degraders.
  18. Near atmospheric carbon dioxide activates plant ubiquitin cross-linking.
  19. A Modular Chemistry Platform for the Development of a Cereblon E3 Ligase-based Partial PROTAC Library.
  20. ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.
  21. Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.
  22. A degron blocking strategy towards improved CRL4CRBN recruiting PROTAC selectivity.
  23. Principles of human pre-60S biogenesis.
  24. Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis.
  25. Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling.
  26. N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2.
  27. Translation dynamics in human cells visualized at high resolution reveal cancer drug action.
  28. AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.
  29. PERK-mediated antioxidant response is key for pathogen persistence in ticks.
  30. SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.
  31. The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).
  1. bioRxiv. 2023 May 30. pii: 2023.05.30.542866. [Epub ahead of print]
    HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates.
    Mengying Zhou, Rui Fang, Louis Colson, Katherine A Donovan, Moritz Hunkeler, Yuyu Song, Can Zhang, Siyi Chen, Dong-Hoon Lee, Gary A Bradshaw, Robyn Eisert, Yihong Ye, Marian Kalocsay, Alfred Goldberg, Eric S Fischer, Ying Lu.
      Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.
    DOI:  https://doi.org/10.1101/2023.05.30.542866
  2. EMBO J. 2023 Jul 06. e113348
    Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5.
    Zuzana Hodáková, Irina Grishkovskaya, Hanna L Brunner, Derek L Bolhuis, Katarina Belačić, Alexander Schleiffer, Harald Kotisch, Nicholas G Brown, David Haselbach.
      UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo-EM structure of human UBR5, revealing an α-solenoid scaffold with numerous protein-protein interacting motifs, assembled into an antiparallel dimer that adopts further oligomeric states. Using cryo-EM processing tools, we observe the dynamic nature of the UBR5 catalytic domain, which we postulate is important for its enzymatic activity. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates and several distinct domains for protein-protein interactions may explain how UBR5 is linked to several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3 ligases.
    Keywords:  HECT; UBR5; Ubiquitin-Proteasome system; cryo-EM
    DOI:  https://doi.org/10.15252/embj.2022113348
  3. Mol Cell. 2023 Jul 06. pii: S1097-2765(23)00464-1. [Epub ahead of print]83(13): 2159-2160
    Cryo-EM reveals important regulatory mechanism of the workhorses of targeted protein degradation.
    Roland Hjerpe, Thimo Kurz.
      Most methods for targeted protein degradation (TPD) deliver targets to E3 ubiquitin ligases, leading to proteasomal degradation. In this issue of Molecular Cell, Shaaban et al.1 illuminate cullin-RING ubiquitin ligase (CRL) modulation by CAND1, which can be utilized for TPD.
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.017
  4. Biophys J. 2023 Jul 03. pii: S0006-3495(23)00410-1. [Epub ahead of print]
    Endoplasmic reticulum network heterogeneity guides diffusive transport and kinetics.
    Zubenelgenubi C Scott, Katherine Koning, Molly Vanderwerp, Lorna Cohen, Laura M Westrate, Elena F Koslover.
      The endoplasmic reticulum (ER) is a dynamic network of interconnected sheets and tubules that orchestrates the distribution of lipids, ions, and proteins throughout the cell. The impact of its complex, dynamic morphology on its function as an intracellular transport hub remains poorly understood. To elucidate the functional consequences of ER network structure and dynamics, we quantify how the heterogeneity of the peripheral ER in COS7 cells affects diffusive protein transport. In vivo imaging of photoactivated ER membrane proteins demonstrates their non-uniform spreading to adjacent regions, in a manner consistent with simulations of diffusing particles on extracted network structures. Using a minimal network model to represent tubule rearrangements, we demonstrate that ER network dynamics are sufficiently slow to have little effect on diffusive protein transport. Furthermore, stochastic simulations reveal a novel consequence of ER network heterogeneity: the existence of 'hot spots' where sparse diffusive reactants are more likely to find one another. ER exit sites, specialized domains regulating cargo export from the ER, are shown to be disproportionately located in highly accessible regions, further from the outer boundary of the cell. Combining in vivo experiments with analytic calculations, quantitative image analysis, and computational modeling, we demonstrate how structure guides diffusive protein transport and reactions in the ER.
    DOI:  https://doi.org/10.1016/j.bpj.2023.06.022
  5. EMBO J. 2023 Jul 06. e113012
    TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.
    Keith B Boyle, Cara J Ellison, Paul R Elliott, Martina Schuschnig, Krista Grimes, Marc S Dionne, Chihiro Sasakawa, Sean Munro, Sascha Martens, Felix Randow.
      Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases.
    Keywords:  ATG5-ATG12 E3 ligase; DysF; autophagy; membrane damage; sphingomyelin
    DOI:  https://doi.org/10.15252/embj.2022113012
  6. bioRxiv. 2023 Jun 26. pii: 2023.06.16.545386. [Epub ahead of print]
    CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.
    Avi J Samelson, Nabeela Ariqat, Justin McKetney, Gita Rohanitazangi, Celeste Parra Bravo, Darrin Goodness, Ruilin Tian, Parker Grosjean, Romany Abskharon, David Eisenberg, Nicholas M Kanaan, Li Gan, Carlo Condello, Danielle L Swaney, Martin Kampmann.
      A hallmark of age-associated neurodegenerative diseases is the aggregation of proteins. Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to the accumulation of tau aggregates, and subsequent dysfunction and death. The mechanisms underlying cell type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways including UFMylation and GPI anchor synthesis, that control tau oligomer levels. We identify the E3 ubiquitin ligase CUL5 as a tau interactor and potent modifier of tau levels. In addition, disruption of mitochondrial function increases tau oligomer levels and promotes proteasomal misprocessing of tau. These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.
    DOI:  https://doi.org/10.1101/2023.06.16.545386
  7. Cell Biosci. 2023 Jul 03. 13(1): 123
    ER Ca2+ overload activates the IRE1α signaling and promotes cell survival.
    Song Zhao, Haiping Feng, Dongfang Jiang, Keyan Yang, Si-Tong Wang, Yu-Xin Zhang, Yun Wang, Hongmei Liu, Caixia Guo, Tie-Shan Tang.
      BACKGROUND: Maintaining homeostasis of Ca2+ stores in the endoplasmic reticulum (ER) is crucial for proper Ca2+ signaling and key cellular functions. Although Ca2+ depletion has been known to cause ER stress which in turn activates the unfolded protein response (UPR), how UPR sensors/transducers respond to excess Ca2+ when ER stores are overloaded remain largely unclear.RESULTS: Here, we report for the first time that overloading of ER Ca2+ can directly sensitize the IRE1α-XBP1 axis. The overloaded ER Ca2+ in TMCO1-deficient cells can cause BiP dissociation from IRE1α, promote the dimerization and stability of the IRE1α protein, and boost IRE1α activation. Intriguingly, attenuation of the over-activated IRE1α-XBP1s signaling by a IRE1α inhibitor can cause a significant cell death in TMCO1-deficient cells.
    CONCLUSIONS: Our data establish a causal link between excess Ca2+ in ER stores and the selective activation of IRE1α-XBP1 axis, underscoring an unexpected role of overload of ER Ca2+ in IRE1α activation and in preventing cell death.
    Keywords:  ER Ca2+ overload; ER stress; IRE1α; TMCO1
    DOI:  https://doi.org/10.1186/s13578-023-01062-y
  8. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00735-0. [Epub ahead of print]42(7): 112724
    The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2.
    Julia Katharina Charlotte Kreß, Christina Jessen, Anita Hufnagel, Werner Schmitz, Thamara Nishida Xavier da Silva, Ancély Ferreira Dos Santos, Laura Mosteo, Colin R Goding, José Pedro Friedmann Angeli, Svenja Meierjohann.
      The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.
    Keywords:  ATF4; CHAC1; CP: Cell biology; GSH; NRF2; SLC7A11; integrated stress response; melanoma
    DOI:  https://doi.org/10.1016/j.celrep.2023.112724
  9. J Am Chem Soc. 2023 Jul 06.
    Human Small Heat Shock Protein B8 Inhibits Protein Aggregation without Affecting the Native Folding Process.
    Dhawal Choudhary, Laura Mediani, Mario J Avellaneda, Sveinn Bjarnason, Simon Alberti, Edgar E Boczek, Pétur O Heidarsson, Alessandro Mossa, Serena Carra, Sander J Tans, Ciro Cecconi.
      Small Heat Shock Proteins (sHSPs) are key components of our Protein Quality Control system and are thought to act as reservoirs that neutralize irreversible protein aggregation. Yet, sHSPs can also act as sequestrases, promoting protein sequestration into aggregates, thus challenging our understanding of their exact mechanisms of action. Here, we employ optical tweezers to explore the mechanisms of action of the human small heat shock protein HSPB8 and its pathogenic mutant K141E, which is associated with neuromuscular disease. Through single-molecule manipulation experiments, we studied how HSPB8 and its K141E mutant affect the refolding and aggregation processes of the maltose binding protein. Our data show that HSPB8 selectively suppresses protein aggregation without affecting the native folding process. This anti-aggregation mechanism is distinct from previous models that rely on the stabilization of unfolded polypeptide chains or partially folded structures, as has been reported for other chaperones. Rather, it appears that HSPB8 selectively recognizes and binds to aggregated species formed at the early stages of aggregation, preventing them from growing into larger aggregated structures. Consistently, the K141E mutation specifically targets the affinity for aggregated structures without impacting native folding, and hence impairs its anti-aggregation activity.
    DOI:  https://doi.org/10.1021/jacs.3c02022
  10. J Cell Sci. 2023 07 01. pii: jcs261121. [Epub ahead of print]136(13):
    How host ER membrane chaperones and morphogenic proteins support virus infection.
    Tai-Ting Woo, Jeffrey M Williams, Billy Tsai.
      The multi-functional endoplasmic reticulum (ER) is exploited by viruses to cause infection. Morphologically, this organelle is a highly interconnected membranous network consisting of sheets and tubules whose levels are dynamic, changing in response to cellular conditions. Functionally, the ER is responsible for protein synthesis, folding, secretion and degradation, as well as Ca2+ homeostasis and lipid biosynthesis, with each event catalyzed by defined ER factors. Strikingly, these ER host factors are hijacked by viruses to support different infection steps, including entry, translation, replication, assembly and egress. Although the full repertoire of these ER factors that are hijacked is unknown, recent studies have uncovered several ER membrane machineries that are exploited by viruses - ranging from polyomavirus to flavivirus and coronavirus - to facilitate different steps of their life cycle. These discoveries should provide better understanding of virus infection mechanisms, potentially leading to the development of more effective anti-viral therapies.
    Keywords:  Coronavirus; ER membrane complex; ER morphogenesis; Endoplasmic reticulum; Flavivirus; Polyomavirus
    DOI:  https://doi.org/10.1242/jcs.261121
  11. Biochem Biophys Res Commun. 2023 Jun 28. pii: S0006-291X(23)00840-9. [Epub ahead of print]674 102-108
    The UFMylated ribosome-recognition protein SAYSD1 is predominantly expressed in spermatids but is dispensable for fertility in mice.
    Lei Chang, Wataru Fujii, Keiichiro Yogo.
      SAYSVFN domain-containing protein 1 (SAYSD1) is an evolutionarily conserved membrane protein that has recently been identified as a ubiquitin-fold modifier 1 (UFM1)-conjugated ribosome-recognition protein that plays a critical role in translocation-associated quality control (TAQC). However, its expression and roles in mammals in vivo remain largely unknown. We found that SAYSD1 is predominantly expressed in round and elongating spermatids and localizes in the endoplasmic reticulum (ER) of mouse testes, but not in differentiated spermatozoa. Mice deficient in Saysd1 developed normally post-partum. Furthermore, Saysd1-deficient mice were fertile, with no apparent differences in sperm morphology or motility compared with wild-type sperm, although the cauda epididymis contained slightly less sperm. Expression of the ER stress markers spliced X-box binding protein 1s (XBP1s) and CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP) in the testes was comparable between Saysd1-deficient and wild-type mice. These results suggested that SAYSD1 is involved in sperm production in mice but is dispensable for their development and fertility.
    Keywords:  Endoplasmic reticulum stress; Male fertility; Protein quality control; Spermatogenesis
    DOI:  https://doi.org/10.1016/j.bbrc.2023.06.085
  12. bioRxiv. 2023 Jun 01. pii: 2023.05.30.542896. [Epub ahead of print]
    Lysine 117 on ataxin-3 modulates toxicity in Drosophila models of Spinocerebellar Ataxia Type 3.
    Jessica R Blount, Nikhil C Patel, Kozeta Libohova, Autumn L Harris, Wei-Ling Tsou, Alyson Sujkowski, Sokol V Todi.
      Ataxin-3 (Atxn3) is a deubiquitinase with a polyglutamine (polyQ) repeat tract whose abnormal expansion causes the neurodegenerative disease, Spinocerebellar Ataxia Type 3 (SCA3; also known as Machado-Joseph Disease). The ubiquitin chain cleavage properties of Atxn3 are enhanced when it is ubiquitinated at lysine (K) at position 117. K117-ubiqutinated Atxn3 cleaves poly-ubiquitin more rapidly in vitro compared to its unmodified counterpart and this residue is also important for Atxn3 roles in cell culture and in Drosophila melanogaster . How polyQ expansion causes SCA3 remains unclear. To gather insight into the biology of disease of SCA3, here we posited the question: is K117 important for toxicity caused by Atxn3? We generated transgenic Drosophila lines that express full-length, human, pathogenic Atxn3 with 80 polyQ with an intact or mutated K117. We found that K117 mutation mildly enhances the toxicity and aggregation of pathogenic Atxn3 in Drosophila . An additional transgenic line that expresses Atxn3 without any K residues confirms increased aggregation of pathogenic Atxn3 whose ubiquitination is perturbed. These findings suggest Atxn3 ubiquitination as a regulatory step of SCA3, in part by modulating its aggregation.
    DOI:  https://doi.org/10.1101/2023.05.30.542896
  13. bioRxiv. 2023 Jun 01. pii: 2023.05.31.543093. [Epub ahead of print]
    Multiple E3 ligases control tankyrase stability and function.
    Jerome Perrard, Susan Smith.
      Tankyrase 1 and 2 are ADP-ribosyltransferases that use NAD + as a substrate to catalyze polyADP-Ribose (PAR) onto themselves and their protein binding partners. Tankyrases have diverse cellular functions, ranging from resolution of telomere cohesion to activation of the Wnt/β-catenin signaling pathway. Robust and specific small molecule tankyrase inhibitors have been developed and are being investigated for cancer therapies. Tankyrase is regulated by the PAR-binding E3 ligase RNF146, which promotes K48-linked polyubiquitylation and proteasomal degradation of PARylated tankyrases and their PARylated partners. We have identified a novel interaction between tankyrase and a distinct class of E3 ligases: the RING-UIM (Ubiquitin-Interacting Motif) family. We show that RING-UIM E3 ligases (specifically RNF114 and RNF166) bind and stabilize monoubiquitylated tankyrase and promote K11-linked diubiquitylation. This action competes with RNF146-mediated K48-linked polyubiquitylation and degradation, leading to stabilization of tankyrase and to a subset of its binding partners, including Angiomotin, a protein that functions in cancer signaling pathways. Moreover, we identify multiple PAR-binding E3 ligases (in addition to RNF146) that promote ubiquitylation of tankyrase and induce stabilization or degradation. Discovery of this novel K11 ubiquitylation of tankyrase that opposes K48-mediated degradation along with identification of multiple PAR-binding E3 ligases that ubiquitylate tankyrase, provide new insights into mechanisms of tankyrase regulation and may offer new uses for tankyrase inhibitors in cancer therapy.
    DOI:  https://doi.org/10.1101/2023.05.31.543093
  14. bioRxiv. 2023 May 31. pii: 2023.05.30.542711. [Epub ahead of print]
    Rescue of secretion of a rare-disease associated mis-folded mutant glycoprotein in UGGT1 knock-out mammalian cells.
    Gábor Tax, Kevin P Guay, Tatiana Soldà, Charlie J Hitchman, Johan C Hill, Snežana Vasiljević, Andrea Lia, Carlos P Modenutti, Kees R Straatman, Angelo Santino, Maurizio Molinari, Nicole Zitzmann, Daniel N Hebert, Pietro Roversi, Marco Trerotola.
      Endoplasmic reticulum (ER) retention of mis-folded glycoproteins is mediated by the ER- localised eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognises a mis-folded glycoprotein and flags it for ER retention by reglucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease even if the mutant glycoprotein retains activity ("responsive mutant"). Here, we investigated the subcellular localisation of the human Trop-2 Q118E variant, which causes gelatinous drop- like corneal dystrophy (GDLD). Compared with the wild type Trop-2, which is correctly localised at the plasma membrane, the Trop-2-Q118E variant is found to be heavily retained in the ER. Using Trop-2-Q118E, we tested UGGT modulation as a rescue-of-secretion therapeutic strategy for congenital rare disease caused by responsive mutations in genes encoding secreted glycoproteins. We investigated secretion of a EYFP-fusion of Trop-2-Q118E by confocal laser scanning microscopy. As a limiting case of UGGT inhibition, mammalian cells harbouring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 gene expressions were used. The membrane localisation of the Trop-2-Q118E-EYFP mutant was successfully rescued in UGGT1 -/- and UGGT1/2 -/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula . The study supports the hypothesis that UGGT1 modulation constitutes a novel therapeutic strategy for the treatment of Trop-2-Q118E associated GDLD, and it encourages the testing of modulators of ER glycoprotein folding Quality Control (ERQC) as broad-spectrum rescue- of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.Synopsis: Deletion of the UGGT1 and UGGT1/2 genes in HEK 293T cells rescues secretion of an EYFP-fusion of the human Trop-2-Q118E glycoprotein mutant. The mutant is retained in the secretory pathway in wild type cells and it localises to the cell membrane in UGGT1 -/- single and UGGT1/2 -/- double knock-out cells. The Trop-2-Q118E glycoprotein disease mutant is efficiently glucosylated by UGGT1 in human cells demonstrating that it is a bona fide cellular UGGT1 substrate.
    DOI:  https://doi.org/10.1101/2023.05.30.542711
  15. Cell Rep. 2023 Jul 04. pii: S2211-1247(23)00726-X. [Epub ahead of print]42(7): 112715
    Global and tissue-specific aging effects on murine proteomes.
    Gregory R Keele, Ji-Gang Zhang, John Szpyt, Ron Korstanje, Steven P Gygi, Gary A Churchill, Devin K Schweppe.
      Maintenance of protein homeostasis degrades with age, contributing to aging-related decline and disease. Previous studies have primarily surveyed transcriptional aging changes. To define the effects of age directly at the protein level, we perform discovery-based proteomics in 10 tissues from 20 C57BL/6J mice, representing both sexes at adult and late midlife ages (8 and 18 months). Consistent with previous studies, age-related changes in protein abundance often have no corresponding transcriptional change. Aging results in increases in immune proteins across all tissues, consistent with a global pattern of immune infiltration with age. Our protein-centric data reveal tissue-specific aging changes with functional consequences, including altered endoplasmic reticulum and protein trafficking in the spleen. We further observe changes in the stoichiometry of protein complexes with important roles in protein homeostasis, including the CCT/TriC complex and large ribosomal subunit. These data provide a foundation for understanding how proteins contribute to systemic aging across tissues.
    Keywords:  B6; C57BL/6J; CP: Genomics; CP: Metabolism; TMT; multitissue; organismal aging; protein complex; protein homeostasis; proteomics; proteostasis; tandem mass tag
    DOI:  https://doi.org/10.1016/j.celrep.2023.112715
  16. Mol Cell Proteomics. 2023 Jun 29. pii: S1535-9476(23)00125-1. [Epub ahead of print] 100614
    Inducible protein degradation as a strategy to identify Phosphoprotein Phosphatase 6 substrates in RAS-mutant colorectal cancer cells.
    Natasha C Mariano, Scott F Rusin, Isha Nasa, Arminja N Kettenbach.
      Protein phosphorylation is an essential regulatory mechanism that controls most cellular processes, including cell cycle progression, cell division, and response to extracellular stimuli, among many others, and is deregulated in many diseases. Protein phosphorylation is coordinated by the opposing activities of protein kinases and protein phosphatases. In eukaryotic cells, most serine/threonine phosphorylation sites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, we only know for a few phosphorylation sites which specific PPP dephosphorylates them. Although natural compounds such as calyculin A and okadaic acid inhibit PPPs at low nanomolar concentrations, no selective chemical PPP inhibitors exist. Here, we demonstrate the utility of endogenous tagging of genomic loci with an auxin-inducible degron (AID) as a strategy to investigate specific PPP signaling. Using Protein Phosphatase 6 (PP6) as an example, we demonstrate how rapidly inducible protein degradation can be employed to identify dephosphorylation sites and elucidate PP6 biology. Using genome editing, we introduce AID-tags into each allele of the PP6 catalytic subunit (PP6c) in DLD-1 cells expressing the auxin receptor Tir1. Upon rapid auxin-induced degradation of PP6c, we perform quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in mitosis. PP6 is an essential enzyme with conserved roles in mitosis and growth signaling. Consistently, we identify candidate PP6c-dependent phosphorylation sites on proteins implicated in coordinating the mitotic cell cycle, cytoskeleton, gene expression, and mitogen-activated protein kinase (MAPK) and Hippo signaling. Finally, we demonstrate that PP6c opposes the activation of large tumor suppressor 1 (LATS1) by dephosphorylating Threonine 35 (T35) on Mps One Binder (MOB1), thereby blocking the interaction of MOB1 and LATS1. Our analyses highlight the utility of combining genome engineering, inducible degradation, and multiplexed phosphoproteomics to investigate signaling by individual PPPs on a global level, which is currently limited by the lack of tools for specific interrogation.
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100614
  17. Nat Rev Drug Discov. 2023 Jul 05.
    A strategy for small-molecule RNA degraders.
    Alex Eccleston.
      
    DOI:  https://doi.org/10.1038/d41573-023-00112-x
  18. BBA Adv. 2023 ;4 100096
    Near atmospheric carbon dioxide activates plant ubiquitin cross-linking.
    Harry G Gannon, Martin J Cann.
      Background Identifying CO2-binding proteins is vital for our knowledge of CO2-regulated molecular processes. The carbamate post-translational modification is a reversible CO2-mediated adduct that can form on neutral N-terminal α-amino or lysine ε-amino groups. Methods We have developed triethyloxonium ion (TEO) as a chemical proteomics tool to trap the carbamate post-translational modification on protein covalently. We use 13C-NMR and TEO and identify ubiquitin as a plant CO2-binding protein. Results We observe the carbamate post-translational modification on the Arabidopsis thaliana ubiquitin ε-amino groups of lysines 6, 33, and 48. We show that biologically relevant near atmospheric PCO2 levels increase ubiquitin conjugation dependent on lysine 6. We further demonstrate that CO2 increases the ubiquitin E2 ligase (AtUBC5) charging step via the transthioesterification reaction in which Ub is transferred from the E1 ligase active site to the E2 active site. Conclusions and general significance Therefore, plant ubiquitin is a CO2-binding protein, and the carbamate post-translational modification represents a potential mechanism through which plant cells can respond to fluctuating PCO2.
    Keywords:  Arabidopsis; Carbamate; Carbon dioxide; Ubiquitin
    DOI:  https://doi.org/10.1016/j.bbadva.2023.100096
  19. Chembiochem. 2023 Jul 07. e202300482
    A Modular Chemistry Platform for the Development of a Cereblon E3 Ligase-based Partial PROTAC Library.
    Chelsi M Almodóvar-Rivera, Zhen Zhang, Jingyao Li, Haibo Xie, Yu Zhao, Le Guo, Marissa G Mannhardt, Weiping Tang.
      Proteolysis Targeting Chimeras (PROTACs) are a promising therapeutic strategy to selectively promote the degradation of protein targets by exploiting the ubiquitin-proteasome system. Among the limited number of E3 ligase ligands discovered for the PROTAC technology, ligands of cereblon (CRBN) E3 ligase such as pomalidomide, thalidomide, or lenalidomide are the most frequently used for the development of PROTACs. Our group previously reported that a phenyl group could be tolerated on the C4-position of lenalidomide as the ligand of CRBN to develop PROTACs. Herein, we report a modular chemistry platform for the efficient attachment of various ortho, meta, and para substituted phenyls to the C4-position of the lenalidomide via Suzuki cross-coupling reaction, which allows the systematic investigation of the linker effect for the development of PROTACs against any target. We examined the substrate scope by preparing twelve lenalidomide-derived CRBN E3 ligase ligands with different linkers.
    Keywords:  Cereblon ligands; Suzuki cross-coupling; lenalidomide; partial PROTAC; phthalimide
    DOI:  https://doi.org/10.1002/cbic.202300482
  20. EMBO Rep. 2023 Jul 04. e57499
    ER-mitochondria contacts and cholesterol metabolism are disrupted by disease-associated tau protein.
    Leonora Szabo, Nadia Cummins, Paolo Paganetti, Alex Odermatt, Andreas Papassotiropoulos, Celeste Karch, Jürgen Götz, Anne Eckert, Amandine Grimm.
      Abnormal tau protein impairs mitochondrial function, including transport, dynamics, and bioenergetics. Mitochondria interact with the endoplasmic reticulum (ER) via mitochondria-associated ER membranes (MAMs), which coordinate and modulate many cellular functions, including mitochondrial cholesterol metabolism. Here, we show that abnormal tau loosens the association between the ER and mitochondria in vivo and in vitro. Especially, ER-mitochondria interactions via vesicle-associated membrane protein-associated protein (VAPB)-protein tyrosine phosphatase-interacting protein 51 (PTPIP51) are decreased in the presence of abnormal tau. Disruption of MAMs in cells with abnormal tau alters the levels of mitochondrial cholesterol and pregnenolone, indicating that conversion of cholesterol into pregnenolone is impaired. Opposite effects are observed in the absence of tau. Besides, targeted metabolomics reveals overall alterations in cholesterol-related metabolites by tau. The inhibition of GSK3β decreases abnormal tau hyperphosphorylation and increases VAPB-PTPIP51 interactions, restoring mitochondrial cholesterol and pregnenolone levels. This study is the first to highlight a link between tau-induced impairments in the ER-mitochondria interaction and cholesterol metabolism.
    Keywords:  GSK3β; cholesterol; endoplasmic reticulum; mitochondria; tau protein
    DOI:  https://doi.org/10.15252/embr.202357499
  21. PLoS Genet. 2023 Jul 03. 19(7): e1010793
    Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.
    Lucy Granat, Debbra Y Knorr, Daniel C Ranson, Emma L Hamer, Ram Prosad Chakrabarty, Francesca Mattedi, Laura Fort-Aznar, Frank Hirth, Sean T Sweeney, Alessio Vagnoni, Navdeep S Chandel, Joseph M Bateman.
      Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.
    DOI:  https://doi.org/10.1371/journal.pgen.1010793
  22. Chembiochem. 2023 Jul 07. e202300351
    A degron blocking strategy towards improved CRL4CRBN recruiting PROTAC selectivity.
    Habib Bouguenina, Andrea Scarpino, Jack O'Hanlon, Justin Warne, Hannah Z Wang, Laura Chan Wah Hak, Amine Sadok, P Craig McAndrew, Mark Stubbs, Olivier A Pierrat, Tamas Hahner, Marc P Cabry, Yann-Vai Le Bihan, Costas Mitsopoulos, Fernando Jr Sialana, Theodoros I Roumeliotis, Rosemary Burke, Rob L M Van Montfort, Jyoti Choudhary, Rajesh Chopra, John J Caldwell, Ian Collins.
      Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation.
    Keywords:  Cereblon; IMiDs; PROTAC; Zinc finger proteins; targeted protein degradation
    DOI:  https://doi.org/10.1002/cbic.202300351
  23. Science. 2023 Jul 07. 381(6653): eadh3892
    Principles of human pre-60S biogenesis.
    Arnaud Vanden Broeck, Sebastian Klinge.
      During the early stages of human large ribosomal subunit (60S) biogenesis, an ensemble of assembly factors establishes and fine-tunes the essential RNA functional centers of pre-60S particles by an unknown mechanism. Here, we report a series of cryo-electron microscopy structures of human nucleolar and nuclear pre-60S assembly intermediates at resolutions of 2.5 to 3.2 angstroms. These structures show how protein interaction hubs tether assembly factor complexes to nucleolar particles and how guanosine triphosphatases and adenosine triphosphatase couple irreversible nucleotide hydrolysis steps to the installation of functional centers. Nuclear stages highlight how a conserved RNA-processing complex, the rixosome, couples large-scale RNA conformational changes with pre-ribosomal RNA processing by the RNA degradation machinery. Our ensemble of human pre-60S particles provides a rich foundation with which to elucidate the molecular principles of ribosome formation.
    DOI:  https://doi.org/10.1126/science.adh3892
  24. PLoS Genet. 2023 Jul 03. 19(7): e1010827
    Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis.
    Huanfeng Hao, Li Song, Luoying Zhang.
      Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, we aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. We found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, we propose a role for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations.
    DOI:  https://doi.org/10.1371/journal.pgen.1010827
  25. PLoS Genet. 2023 Jul 07. 19(7): e1010344
    Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling.
    Luca Tadini, Nicolaj Jeran, Guido Domingo, Federico Zambelli, Simona Masiero, Anna Calabritto, Elena Costantini, Sara Forlani, Milena Marsoni, Federica Briani, Candida Vannini, Paolo Pesaresi.
      The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insight into cellular responses to impaired plastid protein homeostasis.
    DOI:  https://doi.org/10.1371/journal.pgen.1010344
  26. Nucleic Acids Res. 2023 Jul 07. pii: gkad528. [Epub ahead of print]
    N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2.
    Roser Buscà, Cercina Onesto, Mylène Egensperger, Jacques Pouysségur, Gilles Pagès, Philippe Lenormand.
      We report the discovery of N-terminal alanine-rich sequences, which we term NTARs, that act in concert with their native 5'-untranslated regions to promote selection of the proper start codon. NTARs also facilitate efficient translation initiation while limiting the production of non-functional polypeptides through leaky scanning. We first identified NTARs in the ERK1/2 kinases, which are among the most important signaling molecules in mammals. Analysis of the human proteome reveals that hundreds of proteins possess NTARs, with housekeeping proteins showing a particularly high prevalence. Our data indicate that several of these NTARs act in a manner similar to those found in the ERKs and suggest a mechanism involving some or all of the following features: alanine richness, codon rarity, a repeated amino acid stretch and a nearby second AUG. These features may help slow down the leading ribosome, causing trailing pre-initiation complexes (PICs) to pause near the native AUG, thereby facilitating accurate translation initiation. Amplification of erk genes is frequently observed in cancer, and we show that NTAR-dependent ERK protein levels are a rate-limiting step for signal output. Thus, NTAR-mediated control of translation may reflect a cellular need to precisely control translation of key transcripts such as potential oncogenes. By preventing translation in alternative reading frames, NTAR sequences may be useful in synthetic biology applications, e.g. translation from RNA vaccines.
    DOI:  https://doi.org/10.1093/nar/gkad528
  27. Science. 2023 Jul 07. 381(6653): 70-75
    Translation dynamics in human cells visualized at high resolution reveal cancer drug action.
    Huaipeng Xing, Reiya Taniguchi, Iskander Khusainov, Jan Philipp Kreysing, Sonja Welsch, Beata Turoňová, Martin Beck.
      Ribosomes catalyze protein synthesis by cycling through various functional states. These states have been extensively characterized in vitro, but their distribution in actively translating human cells remains elusive. We used a cryo-electron tomography-based approach and resolved ribosome structures inside human cells with high resolution. These structures revealed the distribution of functional states of the elongation cycle, a Z transfer RNA binding site, and the dynamics of ribosome expansion segments. Ribosome structures from cells treated with Homoharringtonine, a drug used against chronic myeloid leukemia, revealed how translation dynamics were altered in situ and resolve the small molecules within the active site of the ribosome. Thus, structural dynamics and drug effects can be assessed at high resolution within human cells.
    DOI:  https://doi.org/10.1126/science.adh1411
  28. bioRxiv. 2023 Jun 14. pii: 2023.06.14.544560. [Epub ahead of print]
    AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.
    Philipp Trepte, Christopher Secker, Simona Kostova, Sibusiso B Maseko, Soon Gang Choi, Jeremy Blavier, Igor Minia, Eduardo Silva Ramos, Patricia Cassonnet, Sabrina Golusik, Martina Zenkner, Stephanie Beetz, Mara J Liebich, Nadine Scharek, Anja Schütz, Marcel Sperling, Michael Lisurek, Yang Wang, Kerstin Spirohn, Tong Hao, Michael A Calderwood, David E Hill, Markus Landthaler, Julien Olivet, Jean-Claude Twizere, Marc Vidal, Erich E Wanker.
      Protein-protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays and AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways.
    DOI:  https://doi.org/10.1101/2023.06.14.544560
  29. bioRxiv. 2023 Jun 01. pii: 2023.05.30.542958. [Epub ahead of print]
    PERK-mediated antioxidant response is key for pathogen persistence in ticks.
    Kristin L Rosche, Joanna Hurtado, Elis A Fisk, Kaylee A Vosbigian, Ashley L Warren, Lindsay C Sidak-Loftis, Sarah J Wright, Elisabeth Ramirez-Zepp, Jason M Park, Dana K Shaw.
      A crucial phase in the lifecycle of tick-borne pathogens is the time spent colonizing and persisting within the arthropod. Tick immunity is emerging as a key force shaping how transmissible pathogens interact with the vector. How pathogens remain in the tick despite immunological pressure remains unknown. In persistently infected Ixodes scapularis , we found that Borrelia burgdorferi (Lyme disease) and Anaplasma phagocytophilum (granulocytic anaplasmosis) activate a cellular stress pathway mediated by the endoplasmic reticulum receptor PERK and the central regulatory molecule, eIF2α. Disabling the PERK pathway through pharmacological inhibition and RNAi significantly decreased microbial numbers. In vivo RNA interference of the PERK pathway not only reduced the number of A. phagocytophilum and B. burgdorferi colonizing larvae after a bloodmeal, but also significantly reduced the number of bacteria that survive the molt. An investigation into PERK pathway-regulated targets revealed that A. phagocytophilum and B. burgdorferi induce activity of the antioxidant response regulator, Nrf2. Tick cells deficient for nrf2 expression or PERK signaling showed accumulation of reactive oxygen and nitrogen species in addition to reduced microbial survival. Supplementation with antioxidants rescued the microbicidal phenotype caused by blocking the PERK pathway. Altogether, our study demonstrates that the Ixodes PERK pathway is activated by transmissible microbes and facilitates persistence in the arthropod by potentiating an Nrf2-regulated antioxidant environment.
    DOI:  https://doi.org/10.1101/2023.05.30.542958
  30. J Cell Biol. 2023 08 07. pii: e202208137. [Epub ahead of print]222(8):
    SUMOylation at the inner nuclear membrane facilitates nuclear envelope biogenesis during mitosis.
    Natasha O Saik, Christopher Ptak, Saif Rehman, John D Aitchison, Ben Montpetit, Richard W Wozniak.
      As eukaryotic cells progress through cell division, the nuclear envelope (NE) membrane must expand to accommodate the formation of progeny nuclei. In Saccharomyces cerevisiae, closed mitosis allows visualization of NE biogenesis during mitosis. During this period, the SUMO E3 ligase Siz2 binds the inner nuclear membrane (INM) and initiates a wave of INM protein SUMOylation. Here, we show these events increase INM levels of phosphatidic acid (PA), an intermediate of phospholipid biogenesis, and are necessary for normal mitotic NE membrane expansion. The increase in INM PA is driven by the Siz2-mediated inhibition of the PA phosphatase Pah1. During mitosis, this results from the binding of Siz2 to the INM and dissociation of Spo7 and Nem1, a complex required for the activation of Pah1. As cells enter interphase, the process is then reversed by the deSUMOylase Ulp1. This work further establishes a central role for temporally controlled INM SUMOylation in coordinating processes, including membrane expansion, that regulate NE biogenesis during mitosis.
    DOI:  https://doi.org/10.1083/jcb.202208137
  31. Autophagy. 2023 Jul 07. 1-19
    The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).
    Valeria Coppola, Ilaria Marino, Uwe Warnken, Mario Falchi, Luca Pasquini, Mauro Biffoni, Ruggero De Maria, Tobias Longin Haas.
      Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers.Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.
    Keywords:  FYCO1; TRAIL; apoptosis; autophagy; caspase 8; lysosomal degradation
    DOI:  https://doi.org/10.1080/15548627.2023.2229656
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