bims-proteo Biomed News
on Proteostasis
Issue of 2024–03–24
forty-two papers selected by
Eric Chevet, INSERM
  1. The emerging roles of non-canonical ubiquitination in proteostasis and beyond.
  2. Integrating the response to stressed mitochondria.
  3. Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.
  4. Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.
  5. Exploring the IRE1 interactome: from canonical signaling functions to unexpected roles.
  6. MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.
  7. CSNK2/CK2 regulates selective autophagy of the endoplasmic reticulum.
  8. A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.
  9. VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates.
  10. Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.
  11. DR5 disulfide bonding as a sensor and effector of protein folding stress.
  12. A conformational-dependent interdomain redox relay at the core of Protein Disulfide Isomerase activity.
  13. DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity.
  14. Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.
  15. Ethnic variation and structure-function analysis of tauopathy-associated PERK alleles.
  16. A SIFI odyssey: Silencing the stress response amid mitochondrial import blockade to safeguard cell survival.
  17. Structural insights into human EMC and its interaction with VDAC.
  18. Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins.
  19. Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease.
  20. Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.
  21. Translational modulator ISRIB alleviates synaptic and behavioral phenotypes in Fragile X syndrome.
  22. Cell non-autonomous control of autophagy and metabolism by glial cells.
  23. Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast.
  24. Proteome-scale discovery of protein degradation and stabilization effectors.
  25. FAM86A methylation of eEF2 links mRNA translation elongation to tumorigenesis.
  26. PROTACable Is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning To Automate the De Novo Design of PROTACs.
  27. Reconstitution of membrane contact by unilamellar vesicles.
  28. Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.
  29. ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum.
  30. Translation-dependent and independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T Cell activation.
  31. Biology of Stress Responses in Aging.
  32. Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.
  33. Computational design of soluble functional analogues of integral membrane proteins.
  34. Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.
  35. A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice.
  36. A fast-acting lipid checkpoint in G1 prevents mitotic defects.
  37. Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals.
  38. Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.
  39. Structure and assembly of a bacterial gasdermin pore.
  40. The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.
  41. RNA damage compartmentalization by DHX9 stress granules.
  42. Native Top-Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies.
  1. J Cell Biol. 2024 May 06. pii: e202311171. [Epub ahead of print]223(5):
    The emerging roles of non-canonical ubiquitination in proteostasis and beyond.
    Yoshino Akizuki, Stephanie Kaypee, Fumiaki Ohtake, Fumiyo Ikeda.
      Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance.
    DOI:  https://doi.org/10.1083/jcb.202311171
  2. Mol Cell. 2024 Mar 21. pii: S1097-2765(24)00168-0. [Epub ahead of print]84(6): 995-997
    Integrating the response to stressed mitochondria.
    Elliot Dine, Richard J Youle.
      Chakrabarty et al.1 demonstrate that phospho-EIF2α (pEIF2α), the translation initiation factor that mediates the integrated stress response (ISR), is necessary and sufficient for the autophagic degradation of mitochondria following the addition of mitochondrial stressors.
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.026
  3. Nat Chem Biol. 2024 Mar 21.
    Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.
    Hai-Tsang Huang, Ryan J Lumpkin, Ryan W Tsai, Shuyao Su, Xu Zhao, Yuan Xiong, James Chen, Nada Mageed, Katherine A Donovan, Eric S Fischer, William R Sellers.
      Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.
    DOI:  https://doi.org/10.1038/s41589-024-01590-9
  4. Mol Cell. 2024 Mar 14. pii: S1097-2765(24)00172-2. [Epub ahead of print]
    Lysosomal damage sensing and lysophagy initiation by SPG20-ITCH.
    Pinki Gahlot, Bojana Kravic, Giulia Rota, Johannes van den Boom, Sophie Levantovsky, Nina Schulze, Elena Maspero, Simona Polo, Christian Behrends, Hemmo Meyer.
      Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis.
    Keywords:  ESCRT; ITCH; Troyer syndrome; lipid sensing; lysophagy; lysosomal membrane permeabilization; lysosomal repair; spartin; spastic paraplegia; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.029
  5. J Biol Chem. 2024 Mar 15. pii: S0021-9258(24)01664-8. [Epub ahead of print] 107169
    Exploring the IRE1 interactome: from canonical signaling functions to unexpected roles.
    Simon Le Goupil, Hadrien Laprade, Marc Aubry, Eric Chevet.
      The unfolded protein response (UPR) is a mechanism aiming at restoring endoplasmic reticulum (ER) homeostasis and is likely involved in other adaptive pathways. The UPR is transduced by three proteins acting as sensors and triggering downstream signaling pathways. Among them, IRE1α (referred to as IRE1 hereafter), an ER-resident type I transmembrane protein, exerts its function through both kinase and endoribonuclease activities, resulting in both XBP1 mRNA splicing and RNA degradation (Regulated IRE1 Dependent Decay, RIDD). An increasing number of studies have reported protein-protein interactions (PPi) as regulators of these signaling mechanisms, and additionally, driving other non-canonical functions. In this review, we deliver evolutive and structural insights on IRE1 and further describe how this protein interaction network (interactome) regulates IRE1 signaling abilities or mediates other cellular processes through catalytic-independent mechanisms. Moreover, we focus on newly discovered targets of IRE1 kinase activity and discuss potentially novel IRE1 functions based on the nature of the interactome, thereby identifying new fields to explore regarding this protein's biological roles.
    Keywords:  Endoplasmic reticulum; IRE1; Unfolded Protein Response; interactome; protein homeostasis; signaling; stress
    DOI:  https://doi.org/10.1016/j.jbc.2024.107169
  6. EMBO J. 2024 Mar 15.
    MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.
    John Reinhard, Leonhard Starke, Christian Klose, Per Haberkant, Henrik Hammarén, Frank Stein, Ofir Klein, Charlotte Berhorst, Heike Stumpf, James P Sáenz, Jochen Hub, Maya Schuldiner, Robert Ernst.
      Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.
    Keywords:  ER Stress; Lipid Bilayer Stress; MemPrep; Organelle Lipidomics; UPR
    DOI:  https://doi.org/10.1038/s44318-024-00063-y
  7. Autophagy. 2024 Mar 21. 1-2
    CSNK2/CK2 regulates selective autophagy of the endoplasmic reticulum.
    Alexandra Stolz.
      Tuning and assimilation of endoplasmic reticulum (ER) content in each cell of the human body is an essential part of organismal homeostasis and adaptation to stress. As such, the lysosomal turnover of ER (reticulophagy) needs to be regulated in a spatio-temporal as well as cell-type specific manner. We recently identified CSNK2/CK2 (casein kinase 2) as the enzyme that phosphorylates the reticulophagy receptors RETREG1/FAM134B and RETREG3/FAM134C and regulates their activity. Phosphorylation of the receptors is a prerequisite for their subsequent functional ubiquitination and the formation of high-density clusters, presumably representing active macroautophagy/autophagy sites at the ER membrane. Consistently, treatment with kinase inhibitor SGC-CK2-1, knockdown of endogenous CSNK2, or mutation of respective phospho-sites prevents ubiquitination, the formation of high-density clusters as well as reticulophagy flux. We hypothesize that CSNK2 has a broader impact on ER and Golgi content in a cell-type and context-specific manner by orchestrating the activity of several autophagy receptors and potentially also factors of the ER-associated protein degradation pathway.
    Keywords:  ER-phagy; kinase; kinase signaling; organelle turnover; phosphorylation; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2024.2330037
  8. Mol Cell. 2024 Mar 08. pii: S1097-2765(24)00137-0. [Epub ahead of print]
    A unique chaperoning mechanism in class A JDPs recognizes and stabilizes mutant p53.
    Guy Zoltsman, Thi Lieu Dang, Miriam Kuchersky, Ofrah Faust, Micael S Silva, Tal Ilani, Anne S Wentink, Bernd Bukau, Rina Rosenzweig.
      J-domain proteins (JDPs) constitute a large family of molecular chaperones that bind a broad spectrum of substrates, targeting them to Hsp70, thus determining the specificity of and activating the entire chaperone functional cycle. The malfunction of JDPs is therefore inextricably linked to myriad human disorders. Here, we uncover a unique mechanism by which chaperones recognize misfolded clients, present in human class A JDPs. Through a newly identified β-hairpin site, these chaperones detect changes in protein dynamics at the initial stages of misfolding, prior to exposure of hydrophobic regions or large structural rearrangements. The JDPs then sequester misfolding-prone proteins into large oligomeric assemblies, protecting them from aggregation. Through this mechanism, class A JDPs bind destabilized p53 mutants, preventing clearance of these oncoproteins by Hsp70-mediated degradation, thus promoting cancer progression. Removal of the β-hairpin abrogates this protective activity while minimally affecting other chaperoning functions. This suggests the class A JDP β-hairpin as a highly specific target for cancer therapeutics.
    Keywords:  J-domain proteins; NMR; molecular chaperones; p53; protein aggregation; protein misfolding
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.018
  9. Nat Commun. 2024 Mar 19. 15(1): 2459
    VCF1 is a p97/VCP cofactor promoting recognition of ubiquitylated p97-UFD1-NPL4 substrates.
    Ann Schirin Mirsanaye, Saskia Hoffmann, Melanie Weisser, Andreas Mund, Blanca Lopez Mendez, Dimitris Typas, Johannes van den Boom, Bente Benedict, Ivo A Hendriks, Michael Lund Nielsen, Hemmo Meyer, Julien P Duxin, Guillermo Montoya, Niels Mailand.
      The hexameric AAA+ ATPase p97/VCP functions as an essential mediator of ubiquitin-dependent cellular processes, extracting ubiquitylated proteins from macromolecular complexes or membranes by catalyzing their unfolding. p97 is directed to ubiquitylated client proteins via multiple cofactors, most of which interact with the p97 N-domain. Here, we discover that FAM104A, a protein of unknown function also named VCF1 (VCP/p97 nuclear Cofactor Family member 1), acts as a p97 cofactor in human cells. Detailed structure-function studies reveal that VCF1 directly binds p97 via a conserved α-helical motif that recognizes the p97 N-domain with unusually high affinity, exceeding that of other cofactors. We show that VCF1 engages in joint p97 complex formation with the heterodimeric primary p97 cofactor UFD1-NPL4 and promotes p97-UFD1-NPL4-dependent proteasomal degradation of ubiquitylated substrates in cells. Mechanistically, VCF1 indirectly stimulates UFD1-NPL4 interactions with ubiquitin conjugates via its binding to p97 but has no intrinsic affinity for ubiquitin. Collectively, our findings establish VCF1 as an unconventional p97 cofactor that promotes p97-dependent protein turnover by facilitating p97-UFD1-NPL4 recruitment to ubiquitylated targets.
    DOI:  https://doi.org/10.1038/s41467-024-46760-4
  10. Commun Biol. 2024 Mar 15. 7(1): 334
    Identification of membrane curvature sensing motifs essential for VPS37A phagophore recruitment and autophagosome closure.
    Yansheng Ye, Xinwen Liang, Guifang Wang, Maria C Bewley, Kouta Hamamoto, Xiaoming Liu, John M Flanagan, Hong-Gang Wang, Yoshinori Takahashi, Fang Tian.
      VPS37A, an ESCRT-I complex component, is required for recruiting a subset of ESCRT proteins to the phagophore for autophagosome closure. However, the mechanism by which VPS37A is targeted to the phagophore remains obscure. Here, we demonstrate that the VPS37A N-terminal domain exhibits selective interactions with highly curved membranes, mediated by two membrane-interacting motifs within the disordered regions surrounding its Ubiquitin E2 variant-like (UEVL) domain. Site-directed mutations of residues in these motifs disrupt ESCRT-I localization to the phagophore and result in defective phagophore closure and compromised autophagic flux in vivo, highlighting their essential role during autophagy. In conjunction with the UEVL domain, we postulate that these motifs guide a functional assembly of the ESCRT machinery at the highly curved tip of the phagophore for autophagosome closure. These results advance the notion that the distinctive membrane architecture of the cup-shaped phagophore spatially regulates autophagosome biogenesis.
    DOI:  https://doi.org/10.1038/s42003-024-06026-7
  11. bioRxiv. 2024 Mar 07. pii: 2024.03.04.583390. [Epub ahead of print]
    DR5 disulfide bonding as a sensor and effector of protein folding stress.
    Mary E Law, Zaafir M Dulloo, Samantha R Eggleston, Gregory P Takacs, Grace M Alexandrow, Mengxiong Wang, Hanyu Su, Bianca Forsyth, Chi-Wu Chiang, Abhisheak Sharma, Siva Rama Raju Kanumuri, Olga A Guryanova, Jeffrey K Harrison, Boaz Tirosh, Ronald K Castellano, Brian K Law.
      New agents are needed that selectively kill cancer cells without harming normal tissues. The TRAIL ligand and its receptors, DR5 and DR4, exhibit cancer-selective toxicity, but TRAIL analogs or agonistic antibodies targeting these receptors have not received FDA approval for cancer therapy. Small molecules for activating DR5 or DR4 independently of protein ligands may bypass some of the pharmacological limitations of these protein drugs. Previously described Disulfide bond Disrupting Agents (DDAs) activate DR5 by altering its disulfide bonding through inhibition of the Protein Disulfide Isomerases (PDIs) ERp44, AGR2, and PDIA1. Work presented here extends these findings by showing that disruption of single DR5 disulfide bonds causes high-level DR5 expression, disulfide-mediated clustering, and activation of Caspase 8-Caspase 3 mediated pro-apoptotic signaling. Recognition of the extracellular domain of DR5 by various antibodies is strongly influenced by the pattern of DR5 disulfide bonding, which has important implications for the use of agonistic DR5 antibodies for cancer therapy. Disulfide-defective DR5 mutants do not activate the ER stress response or stimulate autophagy, indicating that these DDA-mediated responses are separable from DR5 activation and pro-apoptotic signaling. Importantly, other ER stressors, including Thapsigargin and Tunicamycin also alter DR5 disulfide bonding in various cancer cell lines and in some instances, DR5 mis-disulfide bonding is potentiated by overriding the Integrated Stress Response (ISR) with inhibitors of the PERK kinase or the ISR inhibitor ISRIB. These observations indicate that the pattern of DR5 disulfide bonding functions as a sensor of ER stress and serves as an effector of proteotoxic stress by driving extrinsic apoptosis independently of extracellular ligands.
    DOI:  https://doi.org/10.1101/2024.03.04.583390
  12. Antioxid Redox Signal. 2024 Mar 18.
    A conformational-dependent interdomain redox relay at the core of Protein Disulfide Isomerase activity.
    Eduardo José Xavier Rodrigues Pinho Melo, Soukaina El-Guendouz, Catia Correia, Fernando Teodoro, Carlos Lopes, Paulo J Martel.
       AIMS: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to an holdase function, some members catalyse disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical PDIA1 organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a´, at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER.
    RESULTS: Using two fluorescent redox sensors, roGFP2 and HyPer, either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a´ active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a´ active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds.
    INNOVATION AND CONCLUSIONS: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.
    DOI:  https://doi.org/10.1089/ars.2023.0288
  13. Structure. 2024 Mar 08. pii: S0969-2126(24)00055-8. [Epub ahead of print]
    DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity.
    Bryan D Ryder, Elizaveta Ustyantseva, David R Boyer, Ayde Mendoza-Oliva, Mikołaj I Kuska, Paweł M Wydorski, Paulina Macierzyńska, Nabil Morgan, Michael R Sawaya, Marc I Diamond, Harm H Kampinga, Lukasz A Joachimiak.
      J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.
    Keywords:  Alzheimer‘s disease; DNAJB6; DNAJB8; J-domain proteins; LARKS; chaperone; neurodegeneration; polyQ; proteostasis; tau
    DOI:  https://doi.org/10.1016/j.str.2024.02.015
  14. Cell Chem Biol. 2024 Mar 13. pii: S2451-9456(24)00082-5. [Epub ahead of print]
    Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.
    Christopher R Mansfield, Baiyi Quan, Michael E Chirgwin, Benjamin Eduful, Philip F Hughes, Gaëlle Neveu, Kayla Sylvester, Daniel H Ryan, Björn F C Kafsack, Timothy A J Haystead, James W Leahy, Michael C Fitzgerald, Emily R Derbyshire.
      The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.
    Keywords:  Hsp90; Plasmodium; chemoproteomics; heat shock protein 90; proteasome; proteostasis
    DOI:  https://doi.org/10.1016/j.chembiol.2024.02.008
  15. medRxiv. 2024 Mar 04. pii: 2024.03.03.24303689. [Epub ahead of print]
    Ethnic variation and structure-function analysis of tauopathy-associated PERK alleles.
    Goonho Park, Angela Galdamez, Keon-Hyoung Song, Masako Le, Kyle Kim, Jonathan H Lin.
      EIF2AK3, also known as PERK, plays a pivotal role in cellular proteostasis, orchestrating the Unfolded Protein Response (UPR) and Integrated Stress Response (ISR) pathways. In addition to its central position in intracellular stress regulation, human GWAS identify EIF2AK3 as a risk factor in tauopathies, neurodegenerative diseases caused by aberrant tau protein accumulation. Guided by these genomic indicators, our investigation systematically analyzed human PERK variants, focusing on those with potential tauopathy linkages. We assembled a comprehensive data set of human PERK variants associated with Wolcott Rallison Syndrome (WRS), tauopathies, and bioinformatically predicted loss-of-function, referencing the gnomAD, Ensembl, and NCBI databases. We found extensive racial/ethnic variation in the prevalence of common PERK polymorphisms linked to tauopathies. Using SWISS-MODEL, we identified structural perturbations in the ER stress-sensing luminal domain dimers/oligomers of tauopathy-associated PERK variants, Haplotypes A and B, in combination with another tauopathy-linked R240H mutation. Recombinant expression of disease-associated variants in vitro revealed altered PERK signal transduction kinetics in response to ER stress compared to the predominant non-disease variant. In summary, our data further substantiates that human PERK variants identified in tauopathy genetic studies negatively impact PERK structure, function, and downstream signaling with significant variations in prevalence among different racial and ethnic groups.
    DOI:  https://doi.org/10.1101/2024.03.03.24303689
  16. Mol Cell. 2024 Mar 21. pii: S1097-2765(24)00174-6. [Epub ahead of print]84(6): 1000-1002
    A SIFI odyssey: Silencing the stress response amid mitochondrial import blockade to safeguard cell survival.
    Yuleika G Martinez Castillo, Chantell S Evans.
      In a recent study in Nature, Haakonsen et al.1 identify the SIFI complex as a stress response silencer via its E3 ligase activity to target unimported mitochondrial proteins and stress response components for degradation via the proteasome.
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.034
  17. Aging (Albany NY). 2024 Mar 15. 16
    Structural insights into human EMC and its interaction with VDAC.
    Mingyue Li, Chunli Zhang, Yuntao Xu, Shaobai Li, Chenhui Huang, Jian Wu, Ming Lei.
      The endoplasmic reticulum (ER) membrane protein complex (EMC) is a conserved, multi-subunit complex acting as an insertase at the ER membrane. Growing evidence shows that the EMC is also involved in stabilizing and trafficking membrane proteins. However, the structural basis and regulation of its multifunctionality remain elusive. Here, we report cryo-electron microscopy structures of human EMC in apo- and voltage-dependent anion channel (VDAC)-bound states at resolutions of 3.47 Å and 3.32 Å, respectively. We discovered a specific interaction between VDAC proteins and the EMC at mitochondria-ER contact sites, which is conserved from yeast to humans. Moreover, we identified a gating plug located inside the EMC hydrophilic vestibule, the substrate-binding pocket for client insertion. Conformation changes of this gating plug during the apo-to-VDAC-bound transition reveal that the EMC unlikely acts as an insertase in the VDAC1-bound state. Based on the data analysis, the gating plug may regulate EMC functions by modifying the hydrophilic vestibule in different states. Our discovery offers valuable insights into the structural basis of EMC's multifunctionality.
    Keywords:  EMC; VDAC; cryo-electron microscopy; gating plug; membrane proteins biogenesis
    DOI:  https://doi.org/10.18632/aging.205660
  18. J Neurochem. 2024 Mar 15.
    Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins.
    Amanda M Dossat, Kathleen A Trychta, Elliot J Glotfelty, Joshua J Hinkle, Lowella V Fortuno, Lana N Gore, Christopher T Richie, Brandon K Harvey.
      Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity.
    Keywords:  ER calcium; KDEL receptor; endoplasmic reticulum; excitotoxicity; glutamate; proteostasis
    DOI:  https://doi.org/10.1111/jnc.16093
  19. Nat Commun. 2024 Mar 16. 15(1): 2378
    Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease.
    Janina Lara Gerber, Suria Itzel Morales Guzmán, Lorenz Worf, Petra Hubbe, Jürgen Kopp, Jirka Peschek.
      RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.
    DOI:  https://doi.org/10.1038/s41467-024-46568-2
  20. Cell Rep. 2024 Mar 19. pii: S2211-1247(24)00304-8. [Epub ahead of print]43(4): 113976
    Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control.
    Anna M Smirnova, Vladislava Hronová, Mahabub Pasha Mohammad, Anna Herrmannová, Stanislava Gunišová, Denisa Petráčková, Petr Halada, Štěpán Coufal, Michał Świrski, Justin Rendleman, Kristína Jendruchová, Maria Hatzoglou, Petra Beznosková, Christine Vogel, Leoš Shivaya Valášek.
      Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.
    Keywords:  ATF4; CP: Molecular biology; integrated stress response; ribosome; ribosome queuing; translation reinitiation; translational control; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2024.113976
  21. iScience. 2024 Apr 19. 27(4): 109259
    Translational modulator ISRIB alleviates synaptic and behavioral phenotypes in Fragile X syndrome.
    Rochelle L Coulson, Valentina Frattini, Caitlin E Moyer, Jennifer Hodges, Peter Walter, Philippe Mourrain, Yi Zuo, Gordon X Wang.
      Fragile X syndrome (FXS) is caused by the loss of fragile X messenger ribonucleoprotein (FMRP), a translational regulator that binds the transcripts of proteins involved in synaptic function and plasticity. Dysregulated protein synthesis is a central effect of FMRP loss, however, direct translational modulation has not been leveraged in the treatment of FXS. Thus, we examined the effect of the translational modulator integrated stress response inhibitor (ISRIB) in treating synaptic and behavioral symptoms of FXS. We show that FMRP loss dysregulates synaptic protein abundance, stabilizing dendritic spines through increased PSD-95 levels while preventing spine maturation through reduced glutamate receptor accumulation, thus leading to the formation of dense, immature dendritic spines, characteristic of FXS patients and Fmr1 knockout (KO) mice. ISRIB rescues these deficits and improves social recognition in Fmr1 KO mice. These findings highlight the therapeutic potential of targeting core translational mechanisms in FXS and neurodevelopmental disorders more broadly.
    Keywords:  Cell biology; Cellular neuroscience; Model organism; Molecular neuroscience; Small molecule
    DOI:  https://doi.org/10.1016/j.isci.2024.109259
  22. iScience. 2024 Apr 19. 27(4): 109354
    Cell non-autonomous control of autophagy and metabolism by glial cells.
    Melissa G Metcalf, Samira Monshietehadi, Arushi Sahay, Jenni Durieux, Ashley E Frakes, Martina Velichkovska, Cesar Mena, Amelia Farinas, Melissa Sanchez, Andrew Dillin.
      Glia are the protectors of the nervous system, providing neurons with support and protection from cytotoxic insults. We previously discovered that four astrocyte-like glia can regulate organismal proteostasis and longevity in C. elegans. Expression of the UPRER transcription factor, XBP-1s, in these glia increases stress resistance, and longevity, and activates the UPRER in intestinal cells via neuropeptides. Autophagy, a key regulator of metabolism and aging, has been described as a cell autonomous process. Surprisingly, we find that glial XBP-1s enhances proteostasis and longevity by cell non-autonomously reprogramming organismal lipid metabolism and activating autophagy. Glial XBP-1s regulates the activation of another transcription factor, HLH-30/TFEB, in the intestine. HLH-30 activates intestinal autophagy, increases intestinal lipid catabolism, and upregulates a robust transcriptional program. Our study reveals a novel role for glia in regulating peripheral lipid metabolism, autophagy, and organellar health through peripheral activation of HLH-30 and autophagy.
    Keywords:  Biological sciences; Cell biology; Functional aspects of cell biology; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2024.109354
  23. Elife. 2024 Mar 19. pii: e92900. [Epub ahead of print]13
    Spatiotemporal dissection of the Golgi apparatus and the ER-Golgi intermediate compartment in budding yeast.
    Takuro Tojima, Yasuyuki Suda, Natsuko Jin, Kazuo Kurokawa, Akihiko Nakano.
      Cargo traffic through the Golgi apparatus is mediated by cisternal maturation, but it remains largely unclear how the cis-cisternae, the earliest Golgi sub-compartment, is generated and how the Golgi matures into the trans-Golgi network (TGN). Here, we use high-speed and high-resolution confocal microscopy to analyze the spatiotemporal dynamics of a diverse set of proteins that reside in and around the Golgi in budding yeast. We find many mobile punctate structures that harbor yeast counterparts of mammalian endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) proteins, which we term 'yeast ERGIC'. It occasionally exhibits approach and contact behavior toward the ER exit sites and gradually matures into the cis-Golgi. Upon treatment with the Golgi-disrupting agent brefeldin A, the ERGIC proteins form larger aggregates corresponding to the Golgi entry core compartment in plants, while cis- and medial-Golgi proteins are absorbed into the ER. We further analyze the dynamics of several late Golgi proteins to better understand the Golgi-TGN transition. Together with our previous studies, we demonstrate a detailed spatiotemporal profile of the entire cisternal maturation process from the ERGIC to the Golgi and further to the TGN.
    Keywords:  ERGIC; GECCO; Golgi apparatus; S. cerevisiae; cell biology; cisternal maturation; membrane traffic; super-resolution live imaging
    DOI:  https://doi.org/10.7554/eLife.92900
  24. Nature. 2024 Mar 20.
    Proteome-scale discovery of protein degradation and stabilization effectors.
    Juline Poirson, Hanna Cho, Akashdeep Dhillon, Shahan Haider, Ahmad Zoheyr Imrit, Mandy Hiu Yi Lam, Nader Alerasool, Jessica Lacoste, Lamisa Mizan, Cassandra Wong, Anne-Claude Gingras, Daniel Schramek, Mikko Taipale.
      Targeted protein degradation and stabilization are promising therapeutic modalities because of their potency, versatility and their potential to expand the druggable target space1,2. However, only a few of the hundreds of E3 ligases and deubiquitinases in the human proteome have been harnessed for this purpose, which substantially limits the potential of the approach. Moreover, there may be other protein classes that could be exploited for protein stabilization or degradation3-5, but there are currently no methods that can identify such effector proteins in a scalable and unbiased manner. Here we established a synthetic proteome-scale platform to functionally identify human proteins that can promote the degradation or stabilization of a target protein in a proximity-dependent manner. Our results reveal that the human proteome contains a large cache of effectors of protein stability. The approach further enabled us to comprehensively compare the activities of human E3 ligases and deubiquitinases, identify and characterize non-canonical protein degraders and stabilizers and establish that effectors have vastly different activities against diverse targets. Notably, the top degraders were more potent against multiple therapeutically relevant targets than the currently used E3 ligases cereblon and VHL. Our study provides a functional catalogue of stability effectors for targeted protein degradation and stabilization and highlights the potential of induced proximity screens for the discovery of new proximity-dependent protein modulators.
    DOI:  https://doi.org/10.1038/s41586-024-07224-3
  25. Mol Cell. 2024 Mar 12. pii: S1097-2765(24)00180-1. [Epub ahead of print]
    FAM86A methylation of eEF2 links mRNA translation elongation to tumorigenesis.
    Joel William Francis, Simone Hausmann, Sabeen Ikram, Kunlun Yin, Robert Mealey-Farr, Natasha Mahealani Flores, Annie Truc Trinh, Tourkian Chasan, Julia Thompson, Pawel Karol Mazur, Or Gozani.
      eEF2 post-translational modifications (PTMs) can profoundly affect mRNA translation dynamics. However, the physiologic function of eEF2K525 trimethylation (eEF2K525me3), a PTM catalyzed by the enzyme FAM86A, is unknown. Here, we find that FAM86A methylation of eEF2 regulates nascent elongation to promote protein synthesis and lung adenocarcinoma (LUAD) pathogenesis. The principal physiologic substrate of FAM86A is eEF2, with K525me3 modeled to facilitate productive eEF2-ribosome engagement during translocation. FAM86A depletion in LUAD cells causes 80S monosome accumulation and mRNA translation inhibition. FAM86A is overexpressed in LUAD and eEF2K525me3 levels increase through advancing LUAD disease stages. FAM86A knockdown attenuates LUAD cell proliferation and suppression of the FAM86A-eEF2K525me3 axis inhibits cancer cell and patient-derived LUAD xenograft growth in vivo. Finally, FAM86A ablation strongly attenuates tumor growth and extends survival in KRASG12C-driven LUAD mouse models. Thus, our work uncovers an eEF2 methylation-mediated mRNA translation elongation regulatory node and nominates FAM86A as an etiologic agent in LUAD.
    Keywords:  FAM86A; KRAS; cancer; eEF2; elongation; lung; lysine methylation; mRNA translation; protein synthesis
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.037
  26. J Chem Inf Model. 2024 Mar 19.
    PROTACable Is an Integrative Computational Pipeline of 3-D Modeling and Deep Learning To Automate the De Novo Design of PROTACs.
    Hazem Mslati, Francesco Gentile, Mohit Pandey, Fuqiang Ban, Artem Cherkasov.
      Proteolysis-targeting chimeras (PROTACs) that engage two biological targets at once are a promising technology in degrading clinically relevant protein targets. Since factors that influence the biological activities of PROTACs are more complex than those of a small molecule drug, we explored a combination of computational chemistry and deep learning strategies to forecast PROTAC activity and enable automated design. A new method named PROTACable was developed for the de novo design of PROTACs, which includes a robust 3-D modeling workflow to model PROTAC ternary complexes using a library of E3 ligase and linker and an SE(3)-equivariant graph transformer network to predict the activity of newly designed PROTACs. PROTACable is available at https://github.com/giaguaro/PROTACable/.
    DOI:  https://doi.org/10.1021/acs.jcim.3c01878
  27. Biophys Rep. 2023 Aug 31. 9(4): 188-194
    Reconstitution of membrane contact by unilamellar vesicles.
    Shulin Li, Min Zhang, Liang Ge.
      Eukaryotic cells compartmentalize diverse biochemical functions within organelles defined by intracellular membranes. Recent focus has intensified on studying the interactions among organelles and the role of membrane contacts in maintaining cellular balance. While analyzing these contacts mainly involves fluorescence and electron microscopy, as well as biochemical cell fractionation, understanding their mechanisms and responses to genetic and environmental changes remains challenging. Here we describe an approach employing in vitro reconstitution of membrane contacts using unilamellar vesicles. This technique offers insights into contact mechanisms when combined with established methods like fluorescence imaging and mass spectrometry, potentially deepening our understanding of membrane contacts and organelle networks.
    Keywords:  Autophagosome; Autophagy; COPII; ER-Golgi intermediate compartment (ERGIC); ER-exit sites (ERES); Giant unilamellar vesicle (GUV); Liposome; Membrane contacts; Organelle interaction network; SEC12; TMED9
    DOI:  https://doi.org/10.52601/bpr.2023.230011
  28. Nat Commun. 2024 Mar 16. 15(1): 2404
    Structure of full-length ERGIC-53 in complex with MCFD2 for cargo transport.
    Satoshi Watanabe, Yoshiaki Kise, Kento Yonezawa, Mariko Inoue, Nobutaka Shimizu, Osamu Nureki, Kenji Inaba.
      ERGIC-53 transports certain subsets of newly synthesized secretory proteins and membrane proteins from the endoplasmic reticulum to the Golgi apparatus. Despite numerous structural and functional studies since its identification, the overall architecture and mechanism of action of ERGIC-53 remain unclear. Here we present cryo-EM structures of full-length ERGIC-53 in complex with its functional partner MCFD2. These structures reveal that ERGIC-53 exists as a homotetramer, not a homohexamer as previously suggested, and comprises a four-leaf clover-like head and a long stalk composed of three sets of four-helix coiled-coil followed by a transmembrane domain. 3D variability analysis visualizes the flexible motion of the long stalk and local plasticity of the head region. Notably, MCFD2 is shown to possess a Zn2+-binding site in its N-terminal lid, which appears to modulate cargo binding. Altogether, distinct mechanisms of cargo capture and release by ERGIC- 53 via the stalk bending and metal binding are proposed.
    DOI:  https://doi.org/10.1038/s41467-024-46747-1
  29. Mol Cell. 2024 Mar 13. pii: S1097-2765(24)00177-1. [Epub ahead of print]
    ERMA (TMEM94) is a P-type ATPase transporter for Mg2+ uptake in the endoplasmic reticulum.
    Neelanjan Vishnu, Manigandan Venkatesan, Travis R Madaris, Mridula K Venkateswaran, Kristen Stanley, Karthik Ramachandran, Adhishree Chidambaram, Abitha K Madesh, Wenli Yang, Jyotsna Nair, Melanie Narkunan, Tharani Muthukumar, Varsha Karanam, Leroy C Joseph, Amy Le, Ayodeji Osidele, M Imran Aslam, John P Morrow, May C Malicdan, Peter B Stathopulos, Muniswamy Madesh.
      Intracellular Mg2+ (iMg2+) is bound with phosphometabolites, nucleic acids, and proteins in eukaryotes. Little is known about the intracellular compartmentalization and molecular details of Mg2+ transport into/from cellular organelles such as the endoplasmic reticulum (ER). We found that the ER is a major iMg2+ compartment refilled by a largely uncharacterized ER-localized protein, TMEM94. Conventional and AlphaFold2 predictions suggest that ERMA (TMEM94) is a multi-pass transmembrane protein with large cytosolic headpiece actuator, nucleotide, and phosphorylation domains, analogous to P-type ATPases. However, ERMA uniquely combines a P-type ATPase domain and a GMN motif for ERMg2+ uptake. Experiments reveal that a tyrosine residue is crucial for Mg2+ binding and activity in a mechanism conserved in both prokaryotic (mgtB and mgtA) and eukaryotic Mg2+ ATPases. Cardiac dysfunction by haploinsufficiency, abnormal Ca2+ cycling in mouse Erma+/- cardiomyocytes, and ERMA mRNA silencing in human iPSC-cardiomyocytes collectively define ERMA as an essential component of ERMg2+ uptake in eukaryotes.
    Keywords:  AlphaFold2; ERMA; GMN motif; P-type ATPase; RNAi screen; TMEM94; calcium; endoplasmic reticulum; hepatocytes; human cardiomyocytes; lactate; magnesium; mitochondria; mutations; refill; uptake
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.033
  30. Genome Res. 2024 Mar 20. pii: gr.277863.123. [Epub ahead of print]
    Translation-dependent and independent mRNA decay occur through mutually exclusive pathways defined by ribosome density during T Cell activation.
    Blandine C Mercier, Emmanuel Labaronne, David Cluet, Laura Guiguettaz, Nicolas Fontrodona, Alicia Bicknell, Antoine Corbin, Melanie Wencker, Fabien Aube, Laurent Modolo, Karina Jouravleva, Didier Auboeuf, Melissa J Moore, Emiliano P Ricci.
      mRNA translation and decay are tightly interconnected processes both in the context of mRNA quality control pathways and for the degradation of functional mRNAs. Cotranslational mRNA degradation through codon usage, ribosome collisions and through the recruitment of specific proteins to ribosomes are important determinants of mRNA turnover. However, the extent to which translation-dependent (TDD) and independent (TID) mRNA decay pathways participate in the degradation of mRNAs has not been studied yet. Here we describe a comprehensive analysis of basal and signal-induced TDD and TID in mouse primary CD4+ T cells. Our results indicate that most cellular transcripts are decayed to some extent in a translation-dependent manner. Our analysis further identifies the length of untranslated regions, the density of ribosomes and GC3 content as important determinants of TDD magnitude. Consistently, all transcripts that undergo changes in ribosome density within their coding sequence upon T cell activation display a corresponding change in their TDD level. Moreover, we reveal a dynamic modulation in the relationship between GC3 content and TDD upon T cell activation, with a reversal in the impact of GC3 and AU3 rich codons. Altogether, our data demonstrate a strong and dynamic interconnection between mRNA translation and decay in mammalian primary cells.
    DOI:  https://doi.org/10.1101/gr.277863.123
  31. Aging Biol. 2023 ;pii: 20230002. [Epub ahead of print]1
    Biology of Stress Responses in Aging.
    Manolis Maragkakis, Sulochan Malla, Maria Hatzoglou, Aleksandra Trifunovic, Adam B Glick, Toren Finkel, Valter D Longo, Susmita Kaushik, Pura Muñoz-Cánoves, Gordon J Lithgow, Nirinjini Naidoo, Lauren N Booth, Matthew J Payea, Allison B Herman, Rafael de Cabo, David M Wilson, Luigi Ferrucci, Myriam Gorospe.
      On April 28th, 2022, a group of scientific leaders gathered virtually to discuss molecular and cellular mechanisms of responses to stress. Conditions of acute, high-intensity stress are well documented to induce a series of adaptive responses that aim to promote survival until the stress has dissipated and then guide recovery. However, high-intensity or persistent stress that goes beyond the cell's compensatory capacity are countered with resilience strategies that are not completely understood. These adaptative strategies, which are an essential component of the study of aging biology, were the theme of the meeting. Specific topics discussed included mechanisms of proteostasis, such as the unfolded protein response (UPR) and the integrated stress response (ISR), as well as mitochondrial stress and lysosomal stress responses. Attention was also given to regulatory mechanisms and associated biological processes linked to age-related conditions, such as muscle loss and regeneration, cancer, senescence, sleep quality, and degenerative disease, with a general focus on the relevance of stress responses to frailty. We summarize the concepts and potential future directions that emerged from the discussion and highlight their relevance to the study of aging and age-related chronic diseases.
    DOI:  https://doi.org/10.59368/agingbio.20230001
  32. Nat Commun. 2024 Mar 20. 15(1): 2488
    Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.
    Lijun Shi, Chenguang Yang, Mingyuan Zhang, Kangning Li, Keying Wang, Li Jiao, Ruming Liu, Yunyun Wang, Ming Li, Yong Wang, Lu Ma, Shuxin Hu, Xin Bian.
      Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATLcyto), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATLcyto can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATLcyto monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.
    DOI:  https://doi.org/10.1038/s41467-024-46919-z
  33. bioRxiv. 2024 Mar 07. pii: 2023.05.09.540044. [Epub ahead of print]
    Computational design of soluble functional analogues of integral membrane proteins.
    Casper A Goverde, Martin Pacesa, Nicolas Goldbach, Lars J Dornfeld, Petra E M Balbi, Sandrine Georgeon, Stéphane Rosset, Srajan Kapoor, Jagrity Choudhury, Justas Dauparas, Christian Schellhaas, Simon Kozlov, David Baker, Sergey Ovchinnikov, Alex J Vecchio, Bruno E Correia.
      De novo design of complex protein folds using solely computational means remains a significant challenge. Here, we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from GPCRs, are not found in the soluble proteome and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses reveal high thermal stability of the designs and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, standing as a proof-of-concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
    DOI:  https://doi.org/10.1101/2023.05.09.540044
  34. iScience. 2024 Mar 15. 27(3): 109173
    Serine metabolism is crucial for cGAS-STING signaling and viral defense control in the gut.
    Björn Becker, Felix Wottawa, Mohamed Bakr, Eric Koncina, Lisa Mayr, Julia Kugler, Guang Yang, Samuel J Windross, Laura Neises, Neha Mishra, Danielle Harris, Florian Tran, Lina Welz, Julian Schwärzler, Zoltán Bánki, Stephanie T Stengel, Go Ito, Christina Krötz, Olivia I Coleman, Christian Jaeger, Dirk Haller, Søren R Paludan, Richard Blumberg, Arthur Kaser, Luka Cicin-Sain, Stefan Schreiber, Timon E Adolph, Elisabeth Letellier, Philip Rosenstiel, Johannes Meiser, Konrad Aden.
      Inflammatory bowel diseases are characterized by the chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER stress on the pathophysiology of IBD remain unclear. By using in vitro, in vivo models, and patient datasets, we identified a distinct polarization of the mitochondrial one-carbon metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon ER stress. This metabolic phenotype strongly correlates with IBD severity and therapy response. Mechanistically, we uncover that both chronic ER stress and serine limitation disrupt cGAS-STING signaling, impairing the epithelial response against viral and bacterial infection and fueling experimental enteritis. Consequently, the antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of serine metabolism to allow proper cGAS-STING signaling and innate immune responses upon gut inflammation.
    Keywords:  Microbial metabolism; Virology
    DOI:  https://doi.org/10.1016/j.isci.2024.109173
  35. bioRxiv. 2024 Mar 06. pii: 2024.03.03.583191. [Epub ahead of print]
    A transient brain endothelial translatome response to endotoxin is associated with mild cognitive changes post-shock in young mice.
    Shuhan Lu, Iria Di John Portela, Nina Martino, Ramon Bossardi Ramos, Abigail E Salinero, Rachel M Smith, Kristen L Zuloaga, Alejandro P Adam.
      Sepsis-associated encephalopathy (SAE) is a common manifestation in septic patients that is associated with increased risk of long-term cognitive impairment. SAE is driven, at least in part, by brain endothelial dysfunction in response to systemic cytokine signaling. However, the mechanisms driving SAE and its consequences remain largely unknown. Here, we performed translating ribosome affinity purification (TRAP) and RNA-sequencing (TRAP-seq) from the brain endothelium to determine the transcriptional changes after an acute endotoxemic (LPS) challenge. We found that LPS induces a strong acute transcriptional response in the brain endothelium that partially correlates with the whole brain transcriptional response and suggested an endothelial-specific hypoxia response. Consistent with a critical role for the IL-6 pathway, loss of the main regulator of this pathway, SOCS3, leads to a broadening of the population of genes responsive to LPS, suggesting that an overactivation of the IL-6/JAK/STAT3 pathway leads to an increased transcriptional response that could explain our prior findings of severe brain injury in these mice. To identify any potential sequelae of this acute response, we performed brain TRAP-seq following a battery of behavioral tests in mice after apparent recovery. We found that the transcriptional response returns to baseline within days post-challenge. Despite the transient nature of the response, we observed that mice that recovered from the endotoxemic shock showed mild, sex-dependent cognitive impairment, suggesting that the acute brain injury led to sustained, non-transcriptional effects. A better understanding of the transcriptional and non-transcriptional changes in response to shock is needed in order to prevent and/or revert the devastating consequences of septic shock.
    DOI:  https://doi.org/10.1101/2024.03.03.583191
  36. Nat Commun. 2024 Mar 18. 15(1): 2441
    A fast-acting lipid checkpoint in G1 prevents mitotic defects.
    Marielle S Köberlin, Yilin Fan, Chad Liu, Mingyu Chung, Antonio F M Pinto, Peter K Jackson, Alan Saghatelian, Tobias Meyer.
      Lipid synthesis increases during the cell cycle to ensure sufficient membrane mass, but how insufficient synthesis restricts cell-cycle entry is not understood. Here, we identify a lipid checkpoint in G1 phase of the mammalian cell cycle by using live single-cell imaging, lipidome, and transcriptome analysis of a non-transformed cell. We show that synthesis of fatty acids in G1 not only increases lipid mass but extensively shifts the lipid composition to unsaturated phospholipids and neutral lipids. Strikingly, acute lowering of lipid synthesis rapidly activates the PERK/ATF4 endoplasmic reticulum (ER) stress pathway that blocks cell-cycle entry by increasing p21 levels, decreasing Cyclin D levels, and suppressing Retinoblastoma protein phosphorylation. Together, our study identifies a rapid anticipatory ER lipid checkpoint in G1 that prevents cells from starting the cell cycle as long as lipid synthesis is low, thereby preventing mitotic defects, which are triggered by low lipid synthesis much later in mitosis.
    DOI:  https://doi.org/10.1038/s41467-024-46696-9
  37. Proc Natl Acad Sci U S A. 2024 Mar 26. 121(13): e2312172121
    Luminal transport through intact endoplasmic reticulum limits the magnitude of localized Ca2+ signals.
    Cécile C Crapart, Zubenelgenubi C Scott, Tasuku Konno, Aman Sharma, Pierre Parutto, David M D Bailey, Laura M Westrate, Edward Avezov, Elena F Koslover.
      The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.
    Keywords:  calcium signaling; endoplasmic reticulum; intracellular transport; mathematical modeling; organelles
    DOI:  https://doi.org/10.1073/pnas.2312172121
  38. J Proteome Res. 2024 Mar 20.
    Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.
    Karl J Schreiber, Eileigh Kadijk, Ji-Young Youn.
      Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.
    Keywords:  APEX2; BioID; MagReSyn MS; SAINTexpress; miniTurbo; proximal interactions; proximity labeling; proximity-dependent biotinylation
    DOI:  https://doi.org/10.1021/acs.jproteome.3c00908
  39. Nature. 2024 Mar 20.
    Structure and assembly of a bacterial gasdermin pore.
    Alex G Johnson, Megan L Mayer, Stefan L Schaefer, Nora K McNamara-Bordewick, Gerhard Hummer, Philip J Kranzusch.
      In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning β-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.
    DOI:  https://doi.org/10.1038/s41586-024-07216-3
  40. EMBO Mol Med. 2024 Mar 21.
    The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.
    Carlos Costas-Insua, Alba Hermoso-López, Estefanía Moreno, Carlos Montero-Fernández, Alicia Álvaro-Blázquez, Irene B Maroto, Andrea Sánchez-Ruiz, Rebeca Diez-Alarcia, Cristina Blázquez, Paula Morales, Enric I Canela, Vicent Casadó, Leyre Urigüen, Gertrudis Perea, Luigi Bellocchio, Ignacio Rodríguez-Crespo, Manuel Guzmán.
      Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.
    Keywords:  Cannabinoid; Cereblon; Hippocampus; Memory; Rimonabant
    DOI:  https://doi.org/10.1038/s44321-024-00054-w
  41. Cell. 2024 Mar 07. pii: S0092-8674(24)00231-9. [Epub ahead of print]
    RNA damage compartmentalization by DHX9 stress granules.
    Yilong Zhou, Amol Panhale, Maria Shvedunova, Mirela Balan, Alejandro Gomez-Auli, Herbert Holz, Janine Seyfferth, Martin Helmstädter, Séverine Kayser, Yuling Zhao, Niyazi Umut Erdogdu, Iga Grzadzielewska, Gerhard Mittler, Thomas Manke, Asifa Akhtar.
      Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.
    Keywords:  DHX9; DNA damage; RNA damage; autophagy; condensate; mitosis; p62; stress granules
    DOI:  https://doi.org/10.1016/j.cell.2024.02.028
  42. Angew Chem Weinheim Bergstr Ger. 2022 Dec 05. 134(49): e202213170
    Native Top-Down Mass Spectrometry Reveals a Role for Interfacial Glycans on Therapeutic Cytokine and Hormone Assemblies.
    Di Wu, Carol V Robinson.
      Oligomerization and glycosylation modulate therapeutic glycoprotein stability and efficacy. The interplay between these two critical attributes on therapeutic glycoproteins, is however often hard to define. Here, we present a native top-down mass spectrometry (MS) approach to assess the glycosylation status of therapeutic cytokine and hormone assemblies and relate interfacial glycan occupancy to complex stability. We found that interfacial O-glycan stabilizes tumor necrosis factor-α trimer. On the contrary, interferon-β1a dimerization is independent of glycosylation. Moreover, we discovered a unique distribution of N-glycans on the follicle-stimulating hormone α subunit. We found that the interfacial N-glycan, at Asn52 of the α subunit, interacts extensively with the β subunit to regulate the dimer assembly. Overall, we have exemplified a method to link glycosylation with assembly status, for cytokines and hormones, critical for informing optimal stability and bioavailability.
    Keywords:  Biopharmaceuticals; Glycan; Glycoprotein; Native Mass Spectrometry; Oligomerization
    DOI:  https://doi.org/10.1002/ange.202213170
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