bims-proteo Biomed News
on Proteostasis
Issue of 2024–03–17
28 papers selected by
Eric Chevet, INSERM
  1. Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.
  2. Membrane atg8ylation in canonical and noncanonical autophagy.
  3. Continuous evolution of compact protein degradation tags regulated by selective molecular glues.
  4. Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic reticulum in Saccharomyces cerevisiae.
  5. The Integrated Stress Response in metabolic adaptation.
  6. Coronavirus hijacks STX18-ATG14 axis-regulated lipophagy to evade an anti-viral effect.
  7. Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs.
  8. Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.
  9. dCas13-mediated translational repression for accurate gene silencing in mammalian cells.
  10. p24-Tango1 interactions ensure ER-Golgi interface stability and efficient transport.
  11. Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.
  12. Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.
  13. Exocytic plasma membrane flows remodel endoplasmic reticulum-plasma membrane tethering for septin collar assembly.
  14. A dynamic ubiquitination balance of cell proliferation and endoreduplication regulators determines plant organ size.
  15. Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.
  16. Sorting of secretory proteins at the trans-Golgi network by human TGN46.
  17. Structural transitions modulate the chaperone activities of Grp94.
  18. YTHDF2 governs muscle size through a targeted modulation of proteostasis.
  19. How does the neuronal proteostasis network react to cellular cues?
  20. Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.
  21. Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes.
  22. AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.
  23. Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3.
  24. Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.
  25. Targeting eIF2α in TBI-induced traumatic optic neuropathy: Effects of Salubrinal and the Integrated Stress Response Inhibitor.
  26. Structural Requirements for Activity of Mind bomb1 in Notch Signaling.
  27. Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.
  28. Revealing the grammar of small RNA secretion using interpretable machine learning.
  1. Nat Commun. 2024 Mar 11. 15(1): 2182
    Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.
    Kevin Wu, Samuel Itskanov, Diane L Lynch, Yuanyuan Chen, Aasha Turner, James C Gumbart, Eunyong Park.
      Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel. Our study reveals how extended hydrophobic sequences at the termini of substrate proteins are recognized by Doa10 as a signal for quality control.
    DOI:  https://doi.org/10.1038/s41467-024-46409-2
  2. J Mol Biol. 2024 Mar 11. pii: S0022-2836(24)00119-0. [Epub ahead of print] 168532
    Membrane atg8ylation in canonical and noncanonical autophagy.
    Vojo Deretic, Thabata Duque, Einar Trosdal, Masroor Paddar, Ruheena Javed, Prithvi Akepati.
      Membrane atg8ylation is a homeostatic process responding to membrane remodeling and stress signals. Membranes are atg8ylated by mammalian ATG8 ubiquitin-like proteins through a ubiquitylation-like cascade. A model has recently been put forward which posits that atg8ylation of membranes is conceptually equivalent to ubiquitylation of proteins. Like ubiquitylation, membrane atg8ylation involves E1, E2 and E3 enzymes. The E3 ligases catalyze the final step of atg8ylation of aminophospholipids in membranes. Until recently, the only known E3 ligase for membrane atg8ylation was ATG16L1 in a noncovalent complex with the ATG12-ATG5 conjugate. ATG16L1 was first identified as a factor in canonical autophagy. During canonical autophagy, the ATG16L1-based E3 ligase complex includes WIPI2, which in turn recognizes phosphatidylinositiol 3-phosphate and directs atg8ylation of autophagic phagophores. As an alternative to WIPIs, binding of ATG16L1 to the proton pump V-ATPase guides atg8ylation of endolysosomal and phagosomal membranes in response to lumenal pH changes. Recently, a new E3 complex containing TECPR1 instead of ATG16L1, has been identified that responds to sphingomyelin's presence on the cytofacial side of perturbed endolysosomal membranes. In present review, we cover the principles of membrane atg8ylaton, catalog its various presentations, and provide a perspective on the growing repertoire of E3 ligase complexes directing membrane atg8ylation at diverse locations.
    DOI:  https://doi.org/10.1016/j.jmb.2024.168532
  3. Science. 2024 Mar 15. 383(6688): eadk4422
    Continuous evolution of compact protein degradation tags regulated by selective molecular glues.
    Jaron A M Mercer, Stephan J DeCarlo, Shourya S Roy Burman, Vedagopuram Sreekanth, Andrew T Nelson, Moritz Hunkeler, Peter J Chen, Katherine A Donovan, Praveen Kokkonda, Praveen K Tiwari, Veronika M Shoba, Arghya Deb, Amit Choudhary, Eric S Fischer, David R Liu.
      Conditional protein degradation tags (degrons) are usually >100 amino acids long or are triggered by small molecules with substantial off-target effects, thwarting their use as specific modulators of endogenous protein levels. We developed a phage-assisted continuous evolution platform for molecular glue complexes (MG-PACE) and evolved a 36-amino acid zinc finger (ZF) degron (SD40) that binds the ubiquitin ligase substrate receptor cereblon in complex with PT-179, an orthogonal thalidomide derivative. Endogenous proteins tagged in-frame with SD40 using prime editing are degraded by otherwise inert PT-179. Cryo-electron microscopy structures of SD40 in complex with ligand-bound cereblon revealed mechanistic insights into the molecular basis of SD40's activity and specificity. Our efforts establish a system for continuous evolution of molecular glue complexes and provide ZF tags that overcome shortcomings associated with existing degrons.
    DOI:  https://doi.org/10.1126/science.adk4422
  4. Autophagy. 2024 Mar 13.
    Quantitative analysis of the spatial distance between autophagy-related membrane structures and the endoplasmic reticulum in Saccharomyces cerevisiae.
    Yang Shi, Kuninori Suzuki.
      Macroautophagy/autophagy is the process by which cells degrade their cytoplasmic proteins or organelles in vacuoles to maintain cellular homeostasis under severe environmental conditions. In the yeast Saccharomyces cerevisiae, autophagy-related (Atg) proteins essential for autophagosome formation accumulate near the vacuole to form the dot-shaped phagophore assembly site/pre-autophagosomal structure (PAS). The PAS then generates the phagophore/isolation membrane (PG), which expands to become a closed double-membrane autophagosome. Hereinafter, we refer to the PAS, PG, and autophagosome as autophagy-related structures (ARSs). During autophagosome formation, Atg2 is responsible for tethering the ARS to the endoplasmic reticulum (ER) via ER exit sites (ERESs), and for transferring phospholipids from the ER to ARSs. Therefore, ARS and the ER are spatially close in the presence of Atg2 but are separated in its absence. Because the contact of an ARS with the ER must be established at the earliest stage of autophagosome formation, it is important to know whether the ARS is tethered to the ER. In this study, we developed a rapid and objective method to estimate tethering of the ARS to the ER by measuring the distance between the ARS and ERES under fluorescence microscopy, and found that tethering of the ARS to the ER was lost without Atg1. This method might be useful to predict the tethering activity of Atg2.
    Keywords:  Autophagy-related 2 (Atg2); Endoplasmic reticulum exit sites (ERES); Fluorescence microscopy; Morphological analysis; Phagophore; Yeast
    DOI:  https://doi.org/10.1080/15548627.2024.2330033
  5. J Biol Chem. 2024 Mar 08. pii: S0021-9258(24)01646-6. [Epub ahead of print] 107151
    The Integrated Stress Response in metabolic adaptation.
    Hyung Don Ryoo.
      The Integrated Stress Response (ISR) refers to signaling pathways initiated by stress-activated eIF2‹ kinases. Distinct eIF2‹ kinases respond to different stress signals, including amino acid deprivation and mitochondrial stress. Such stress-induced eIF2‹ phosphorylation attenuates general mRNA translation and, at the same time, stimulates the preferential translation of specific downstream factors to orchestrate an adaptive gene expression program. In recent years, there have been significant new advances in our understanding of ISR during metabolic stress adaptation. Here, I discuss those advances, reviewing among others the ISR activation mechanisms in response to amino acid deprivation and mitochondrial stress. In addition, I review how ISR regulates the amino acid metabolic pathways and how changes in the ISR impact the physiology and pathology of various disease models.
    Keywords:  ATF4; GCN1; GCN2; HRI; Integrated Stress Response; amino acid deprivation; cysteine; eIF2‹; glutathione; mitochondrial stress; serine biosynthesis
    DOI:  https://doi.org/10.1016/j.jbc.2024.107151
  6. Autophagy. 2024 Mar 13.
    Coronavirus hijacks STX18-ATG14 axis-regulated lipophagy to evade an anti-viral effect.
    Zhen Yuan, Binbin Ding.
      ATG14 is a core subunit of the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) for macroautophagy/autophagy initiation and also binds to the STX17 to promote autophagosome-lysosome fusion. Our recent work found that ATG14 also targets lipid droplets (LDs) and interacts with mammalian Atg8-family proteins (ATG8s) to mediate lipophagy (selective autophagic degradation of lipid droplets). We also demonstrated that STX18 (syntaxin 18) acts as a negative regulator that disrupts the interactions of ATG14-ATG8s and the formation of the PtdIns3K-C1 through binding to ATG14. Furthermore, we found that knockdown of STX18 induces LD-associated anti-viral protein RSAD2/Viperin degradation dependent on ATG14-mediated lipophagy. Additionally, coronavirus M protein hijacks STX18 to induce lipophagy and degrade RSAD2, facilitating virus production. In summary, our findings reveal new roles of ATG14 in lipid metabolism and viral replication as an autophagic receptor.
    Keywords:  ATG14; SARS-CoV-2; STX18; Viperin; lipid droplets; selective autophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2330039
  7. bioRxiv. 2024 Mar 03. pii: 2024.02.28.582532. [Epub ahead of print]
    Quantitative profiling of human translation initiation reveals regulatory elements that potently affect endogenous and therapeutically modified mRNAs.
    Cole J T Lewis, Li Xie, Shivani Bhandarkar, Danni Jin, Kyrillos S Abdallah, Austin S Draycott, Yixuan Chen, Carson C Thoreen, Wendy V Gilbert.
      mRNA therapeutics offer a potentially universal strategy for the efficient development and delivery of therapeutic proteins. Current mRNA vaccines include chemically modified nucleotides to reduce cellular immunogenicity. Here, we develop an efficient, high-throughput method to measure human translation initiation on therapeutically modified as well as endogenous RNAs. Using systems-level biochemistry, we quantify ribosome recruitment to tens of thousands of human 5' untranslated regions and identify sequences that mediate 250-fold effects. We observe widespread effects of coding sequences on translation initiation and identify small regulatory elements of 3-6 nucleotides that are sufficient to potently affect translational output. Incorporation of N1-methylpseudouridine (m1Ψ) selectively enhances translation by specific 5' UTRs that we demonstrate surpass those of current mRNA vaccines. Our approach is broadly applicable to dissect mechanisms of human translation initiation and engineer more potent therapeutic mRNAs.
    Highlights: Measurement of >30,000 human 5' UTRs reveals a 250-fold range of translation outputSystematic mutagenesis demonstrates the causality of short (3-6nt) regulatory elementsN1-methylpseudouridine alters translation initiation in a sequence-specific mannerOptimal modified 5' UTRs outperform those in the current class of mRNA vaccines.
    DOI:  https://doi.org/10.1101/2024.02.28.582532
  8. J Med Chem. 2024 Mar 13.
    Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.
    Rishi R Shah, Elena De Vita, Preethi S Sathyamurthi, Daniel Conole, Xinyue Zhang, Elliot Fellows, Eleanor R Dickinson, Carlos M Fleites, Markus A Queisser, John D Harling, Edward W Tate.
      Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c02123
  9. Nat Commun. 2024 Mar 11. 15(1): 2205
    dCas13-mediated translational repression for accurate gene silencing in mammalian cells.
    Antonios Apostolopoulos, Naohiro Kawamoto, Siu Yu A Chow, Hitomi Tsuiji, Yoshiho Ikeuchi, Yuichi Shichino, Shintaro Iwasaki.
      Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.
    DOI:  https://doi.org/10.1038/s41467-024-46412-7
  10. J Cell Biol. 2024 May 06. pii: e202309045. [Epub ahead of print]223(5):
    p24-Tango1 interactions ensure ER-Golgi interface stability and efficient transport.
    Ke Yang, Zhi Feng, José Carlos Pastor-Pareja.
      The eukaryotic p24 family, consisting of α-, β-, γ- and δ-p24 subfamilies, has long been known to be involved in regulating secretion. Despite increasing interest in these proteins, fundamental questions remain about their role. Here, we systematically investigated Drosophila p24 proteins. We discovered that members of all four p24 subfamilies are required for general secretion and that their localizations between ER exit site (ERES) and Golgi are interdependent in an α→βδ→γ sequence. We also found that localization of p24 proteins and ERES determinant Tango1 requires interaction through their respective GOLD and SH3 lumenal domains, with Tango1 loss sending p24 proteins to the plasma membrane and vice versa. Finally, we show that p24 loss expands the COPII zone at ERES and increases the number of ER-Golgi vesicles, supporting a restrictive role of p24 proteins on vesicle budding for efficient transport. Our results reveal Tango1-p24 interplay as central to the generation of a stable ER-Golgi interface.
    DOI:  https://doi.org/10.1083/jcb.202309045
  11. Nat Commun. 2024 Mar 11. 15(1): 2207
    Simultaneous proteome localization and turnover analysis reveals spatiotemporal features of protein homeostasis disruptions.
    Jordan Currie, Vyshnavi Manda, Sean K Robinson, Celine Lai, Vertica Agnihotri, Veronica Hidalgo, R W Ludwig, Kai Zhang, Jay Pavelka, Zhao V Wang, June-Wha Rhee, Maggie P Y Lam, Edward Lau.
      The spatial and temporal distributions of proteins are critical to protein function, but cannot be directly assessed by measuring protein bundance. Here we describe a mass spectrometry-based proteomics strategy, Simultaneous Proteome Localization and Turnover (SPLAT), to measure concurrently protein turnover rates and subcellular localization in the same experiment. Applying the method, we find that unfolded protein response (UPR) has different effects on protein turnover dependent on their subcellular location in human AC16 cells, with proteome-wide slowdown but acceleration among stress response proteins in the ER and Golgi. In parallel, UPR triggers broad differential localization of proteins including RNA-binding proteins and amino acid transporters. Moreover, we observe newly synthesized proteins including EGFR that show a differential localization under stress than the existing protein pools, reminiscent of protein trafficking disruptions. We next applied SPLAT to an induced pluripotent stem cell derived cardiomyocyte (iPSC-CM) model of cancer drug cardiotoxicity upon treatment with the proteasome inhibitor carfilzomib. Paradoxically, carfilzomib has little effect on global average protein half-life, but may instead selectively disrupt sarcomere protein homeostasis. This study provides a view into the interactions of protein spatial and temporal dynamics and demonstrates a method to examine protein homeostasis regulations in stress and drug response.
    DOI:  https://doi.org/10.1038/s41467-024-46600-5
  12. PLoS Biol. 2024 Mar 12. 22(3): e3002567
    Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membrane.
    Tânia Francisco, Ana G Pedrosa, Tony A Rodrigues, Tarad Abalkhail, Hongli Li, Maria J Ferreira, Gerbrand J van der Heden van Noort, Marc Fransen, Ewald H Hettema, Jorge E Azevedo.
      PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.
    DOI:  https://doi.org/10.1371/journal.pbio.3002567
  13. Sci Adv. 2024 Mar 15. 10(11): eadj1512
    Exocytic plasma membrane flows remodel endoplasmic reticulum-plasma membrane tethering for septin collar assembly.
    Shinju Sugiyama, Keiko Kono.
      Endoplasmic reticulum (ER)-plasma membrane (PM) tethering is crucial for the non-vesicular lipid transport between the ER membrane and the PM. However, the PM-associated ER can impede the PM binding of cytoskeletons and other organelles. It is poorly understood how the competition between the ER and cytoskeletons/organelles on the PM is resolved. Here, we show that, upon septin collar assembly, ER-PM tethering proteins are excluded from the yeast bud sites, and the PM-associated ER is locally detached from the PM. Our results suggest that PM flows by polarized exocytosis extrude PM proteins, including ER-PM tethering proteins, from the bud sites. When the reorganization of the ER-PM tethering was inhibited by exocytosis repression, septin localization was restricted to the PM sites poor in ER-PM tethering proteins. This study proposes machinery reconciling ER-septin competition on the PM, providing mechanistic insights into the spatial organization of PM-associated organelles and cytoskeletons.
    DOI:  https://doi.org/10.1126/sciadv.adj1512
  14. Sci Adv. 2024 Mar 15. 10(11): eadj2570
    A dynamic ubiquitination balance of cell proliferation and endoreduplication regulators determines plant organ size.
    Ying Chen, Mattias Vermeersch, Jelle Van Leene, Geert De Jaeger, Yunhai Li, Hannes Vanhaeren.
      Ubiquitination plays a crucial role throughout plant growth and development. The E3 ligase DA2 has been reported to activate the peptidase DA1 by ubiquitination, hereby limiting cell proliferation. However, the molecular mechanisms that regulate DA2 remain elusive. Here, we demonstrate that DA2 has a very high turnover and auto-ubiquitinates with K48-linkage polyubiquitin chains, which is counteracted by two deubiquitinating enzymes, UBIQUITIN-SPECIFIC PROTEASE 12 (UBP12) and UBP13. Unexpectedly, we found that auto-ubiquitination of DA2 does not influence its stability but determines its E3 ligase activity. We also demonstrate that impairing the protease activity of DA1 abolishes the growth-reducing effect of DA2. Last, we show that synthetic, constitutively activated DA1-ubiquitin fusion proteins overrule this complex balance of ubiquitination and deubiquitination and strongly restrict growth and promote endoreduplication. Our findings highlight a nonproteolytic function of K48-linked polyubiquitination and reveal a mechanism by which DA2 auto-ubiquitination levels, in concert with UBP12 and UBP13, precisely monitor the activity of DA1 and fine-tune plant organ size.
    DOI:  https://doi.org/10.1126/sciadv.adj2570
  15. Cell Host Microbe. 2024 Mar 05. pii: S1931-3128(24)00055-6. [Epub ahead of print]
    Human cytomegalovirus degrades DMXL1 to inhibit autophagy, lysosomal acidification, and viral assembly.
    Hanqi Li, Alice Fletcher-Etherington, Leah M Hunter, Swati Keshri, Ceri A Fielding, Katie Nightingale, Benjamin Ravenhill, Luis Nobre, Martin Potts, Robin Antrobus, Colin M Crump, David C Rubinsztein, Richard J Stanton, Michael P Weekes.
      Human cytomegalovirus (HCMV) is an important human pathogen that regulates host immunity and hijacks host compartments, including lysosomes, to assemble virions. We combined a quantitative proteomic analysis of HCMV infection with a database of proteins involved in vacuolar acidification, revealing Dmx-like protein-1 (DMXL1) as the only protein that acidifies vacuoles yet is degraded by HCMV. Systematic comparison of viral deletion mutants reveals the uncharacterized 7 kDa US33A protein as necessary and sufficient for DMXL1 degradation, which occurs via recruitment of the E3 ubiquitin ligase Kip1 ubiquitination-promoting complex (KPC). US33A-mediated DMXL1 degradation inhibits lysosome acidification and autophagic cargo degradation. Formation of the virion assembly compartment, which requires lysosomes, occurs significantly later with US33A-expressing virus infection, with reduced viral replication. These data thus identify a viral strategy for cellular remodeling, with the potential to employ US33A in therapies for viral infection or rheumatic conditions, in which inhibition of lysosome acidification can attenuate disease.
    Keywords:  autophagy; human cytomegalovirus; lysosome; pH; proteomics; systems virology; viral assembly; viral replication
    DOI:  https://doi.org/10.1016/j.chom.2024.02.013
  16. Elife. 2024 Mar 11. pii: RP91708. [Epub ahead of print]12
    Sorting of secretory proteins at the trans-Golgi network by human TGN46.
    Pablo Lujan, Carla Garcia-Cabau, Yuichi Wakana, Javier Vera Lillo, Carmen Rodilla-Ramírez, Hideaki Sugiura, Vivek Malhotra, Xavier Salvatella, Maria F Garcia-Parajo, Felix Campelo.
      Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.
    Keywords:  Golgi apparatus; cell biology; human; membrane trafficking; protein secretion
    DOI:  https://doi.org/10.7554/eLife.91708
  17. Proc Natl Acad Sci U S A. 2024 Mar 19. 121(12): e2309326121
    Structural transitions modulate the chaperone activities of Grp94.
    Yaa S Amankwah, Yasmeen Fleifil, Erin Unruh, Preston Collins, Yi Wang, Katherine Vitou, Alison Bates, Ikponwmosa Obaseki, Meghana Sugoor, John Paul Alao, Robert M McCarrick, Daniel T Gewirth, Indra D Sahu, Zihai Li, Gary A Lorigan, Andrea N Kravats.
      Hsp90s are ATP-dependent chaperones that collaborate with co-chaperones and Hsp70s to remodel client proteins. Grp94 is the ER Hsp90 homolog essential for folding multiple secretory and membrane proteins. Grp94 interacts with the ER Hsp70, BiP, although the collaboration of the ER chaperones in protein remodeling is not well understood. Grp94 undergoes large-scale conformational changes that are coupled to chaperone activity. Within Grp94, a region called the pre-N domain suppresses ATP hydrolysis and conformational transitions to the active chaperone conformation. In this work, we combined in vivo and in vitro functional assays and structural studies to characterize the chaperone mechanism of Grp94. We show that Grp94 directly collaborates with the BiP chaperone system to fold clients. Grp94's pre-N domain is not necessary for Grp94-client interactions. The folding of some Grp94 clients does not require direct interactions between Grp94 and BiP in vivo, suggesting that the canonical collaboration may not be a general chaperone mechanism for Grp94. The BiP co-chaperone DnaJB11 promotes the interaction between Grp94 and BiP, relieving the pre-N domain suppression of Grp94's ATP hydrolysis activity. In structural studies, we find that ATP binding by Grp94 alters the ATP lid conformation, while BiP binding stabilizes a partially closed Grp94 intermediate. Together, BiP and ATP push Grp94 into the active closed conformation for client folding. We also find that nucleotide binding reduces Grp94's affinity for clients, which is important for productive client folding. Alteration of client affinity by nucleotide binding may be a conserved chaperone mechanism for a subset of ER chaperones.
    Keywords:  BiP; DEER; DnaJB11; EPR; Grp94
    DOI:  https://doi.org/10.1073/pnas.2309326121
  18. Nat Commun. 2024 Mar 11. 15(1): 2176
    YTHDF2 governs muscle size through a targeted modulation of proteostasis.
    Christopher J Gilbert, Charles P Rabolli, Volha A Golubeva, Kristina M Sattler, Meifang Wang, Arsh Ketabforoush, W David Arnold, Christoph Lepper, Federica Accornero.
      The regulation of proteostasis is fundamental for maintenance of muscle mass and function. Activation of the TGF-β pathway drives wasting and premature aging by favoring the proteasomal degradation of structural muscle proteins. Yet, how this critical post-translational mechanism is kept in check to preserve muscle health remains unclear. Here, we reveal the molecular link between the post-transcriptional regulation of m6A-modified mRNA and the modulation of SMAD-dependent TGF-β signaling. We show that the m6A-binding protein YTHDF2 is essential to determining postnatal muscle size. Indeed, muscle-specific genetic deletion of YTHDF2 impairs skeletal muscle growth and abrogates the response to hypertrophic stimuli. We report that YTHDF2 controls the mRNA stability of the ubiquitin ligase ASB2 with consequences on anti-growth gene program activation through SMAD3. Our study identifies a post-transcriptional to post-translational mechanism for the coordination of gene expression in muscle.
    DOI:  https://doi.org/10.1038/s41467-024-46546-8
  19. Biochem Soc Trans. 2024 Mar 15. pii: BST20230316. [Epub ahead of print]
    How does the neuronal proteostasis network react to cellular cues?
    Ki Hong Nam, Alban Ordureau.
      Even though neurons are post-mitotic cells, they still engage in protein synthesis to uphold their cellular content balance, including for organelles, such as the endoplasmic reticulum or mitochondria. Additionally, they expend significant energy on tasks like neurotransmitter production and maintaining redox homeostasis. This cellular homeostasis is upheld through a delicate interplay between mRNA transcription-translation and protein degradative pathways, such as autophagy and proteasome degradation. When faced with cues such as nutrient stress, neurons must adapt by altering their proteome to survive. However, in many neurodegenerative disorders, such as Parkinson's disease, the pathway and processes for coping with cellular stress are impaired. This review explores neuronal proteome adaptation in response to cellular stress, such as nutrient stress, with a focus on proteins associated with autophagy, stress response pathways, and neurotransmitters.
    Keywords:  ISR; UPR; autophagy; neurons; nutrient stress; proteome
    DOI:  https://doi.org/10.1042/BST20230316
  20. bioRxiv. 2024 Feb 26. pii: 2024.02.22.579698. [Epub ahead of print]
    Loss of the APP regulator RHBDL4 preserves memory in an Alzheimer's disease mouse model.
    Ylauna Christine Megane Penalva, Sandra Paschkowsky, Jingyun Yang, Sherilyn Junelle Recinto, Jessica Cinkorpumin, Bin Xiao, Albert Nitu, Helen Wu, Hans Markus Munter, Bernadeta Michalski, Margaret Fahnestock, William Pastor, David A Bennett, Lisa Marie Munter.
      Characteristic cerebral pathological changes of Alzheimer's disease (AD) such as glucose hypometabolism or the accumulation of cleavage products of the amyloid precursor protein (APP), known as Aβ peptides, lead to sustained endoplasmic reticulum (ER) stress and neurodegeneration. To preserve ER homeostasis, cells activate their unfolded protein response (UPR). The rhomboid-like-protease 4 (RHBDL4) is an enzyme that participates in the UPR by targeting proteins for proteasomal degradation. We demonstrated previously that RHBLD4 cleaves APP in HEK293T cells, leading to decreased total APP and Aβ. More recently, we showed that RHBDL4 processes APP in mouse primary mixed cortical cultures as well. Here, we aim to examine the physiological relevance of RHBDL4 in the brain. We first found that brain samples from AD patients and an AD mouse model (APPtg) showed increased RHBDL4 mRNA and protein expression. To determine the effects of RHBDL4's absence on APP physiology in vivo , we crossed APPtg mice to a RHBDL4 knockout (R4 KO) model. RHBDL4 deficiency in APPtg mice led to increased total cerebral APP and Aβ levels when compared to APPtg controls. Contrary to expectations, as assessed by cognitive tests, RHBDL4 absence rescued cognition in 5-month-old female APPtg mice. Informed by unbiased RNAseq data, we demonstrated in vitro and in vivo that RHBDL4 absence leads to greater levels of active β-catenin due to decreased proteasomal clearance. Decreased β-catenin activity is known to underlie cognitive defects in APPtg mice and AD. Our work suggests that RHBDL4's increased expression in AD, in addition to regulating APP levels, leads to aberrant degradation of β-catenin, contributing to cognitive impairment.
    DOI:  https://doi.org/10.1101/2024.02.22.579698
  21. bioRxiv. 2024 Feb 29. pii: 2024.02.27.582206. [Epub ahead of print]
    Chemical tools to expand the ligandable proteome: diversity-oriented synthesis-based photoreactive stereoprobes.
    Daisuke Ogasawara, David B Konrad, Zher Yin Tan, Kimberly L Carey, Jessica Luo, Sang Joon Won, Haoxin Li, Trever Carter, Kristen E DeMeester, Evert Njomen, Stuart L Schreiber, Ramnik J Xavier, Bruno Melillo, Benjamin F Cravatt.
      Chemical proteomics enables the global assessment of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, been limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically-defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these 'photo-stereoprobes' interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible nanoBRET assays. Integrated phenotypic analysis and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and discovering and characterizing bioactive small molecules by cell-based screening.
    DOI:  https://doi.org/10.1101/2024.02.27.582206
  22. Mol Syst Biol. 2024 Mar 11.
    AI-guided pipeline for protein-protein interaction drug discovery identifies a SARS-CoV-2 inhibitor.
    Philipp Trepte, Christopher Secker, Julien Olivet, Jeremy Blavier, Simona Kostova, Sibusiso B Maseko, 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, Soon Gang Choi, 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 or 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.
    Keywords:  AlphaFold; Machine Learning; Protein–Protein Interactions; SARS-CoV-2; VirtualFlow
    DOI:  https://doi.org/10.1038/s44320-024-00019-8
  23. J Med Chem. 2024 Mar 11.
    Uncovering PROTAC Sensitivity and Efficacy by Multidimensional Proteome Profiling: A Case for STAT3.
    Yuying Suo, Daohai Du, Chao Chen, Hongwen Zhu, Xiongjun Wang, Nixue Song, Dayun Lu, Yaxi Yang, Jiacheng Li, Jun Wang, Zhongyuan Luo, Bing Zhou, Cheng Luo, Hu Zhou.
      Proteolysis-targeting chimera (PROTAC) is a powerful technology that can effectively trigger the degradation of target proteins. The intricate interplay among various factors leads to a heterogeneous drug response, bringing about significant challenges in comprehending drug mechanisms. Our study applied data-independent acquisition-based mass spectrometry to multidimensional proteome profiling of PROTAC (DIA-MPP) to uncover the efficacy and sensitivity of the PROTAC compound. We profiled the signal transducer and activator of transcription 3 (STAT3) PROTAC degrader in six leukemia and lymphoma cell lines under multiple conditions, demonstrating the pharmacodynamic properties and downstream biological responses. Through comparison between sensitive and insensitive cell lines, we revealed that STAT1 can be regarded as a biomarker for STAT3 PROTAC degrader, which was validated in cells, patient-derived organoids, and mouse models. These results set an example for a comprehensive description of the multidimensional PROTAC pharmacodynamic response and PROTAC drug sensitivity biomarker exploration.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c02371
  24. bioRxiv. 2024 Feb 29. pii: 2024.02.27.582310. [Epub ahead of print]
    Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.
    Vi T Tang, Jie Xiang, Zhimin Chen, Joseph McCormick, Prabhodh S Abbineni, Xiao-Wei Chen, Mark Hoenerhoff, Brian T Emmer, Rami Khoriaty, Jiandie D Lin, David Ginsburg.
      Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during mid-embryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these 2 paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans.
    DOI:  https://doi.org/10.1101/2024.02.27.582310
  25. bioRxiv. 2024 Feb 19. pii: 2024.02.15.580435. [Epub ahead of print]
    Targeting eIF2α in TBI-induced traumatic optic neuropathy: Effects of Salubrinal and the Integrated Stress Response Inhibitor.
    Shelby Hetzer, Rohan Bellary, Jordyn N Torrens, Robert F Grimaldi, Nathan K Evanson.
      Traumatic brain injury (TBI) can induce traumatic axonal injury in the optic nerve, which is referred to as traumatic optic neuropathy (TON). TON occurs in up to 5% of TBI cases and leads to irreversible visual deficits. TON-induced phosphorylation of eIF2α, a downstream ER stress activator in the PERK pathway presents a potential point for therapeutic intervention. For eIF2α phosphorylation can lead to apoptosis or adaptation to stress. We hypothesized that dephosphorylation, rather than phosphorylation, of eIF2α would lead to reduced apoptosis and improved visual performance and retinal cell survival. Adult male mice were injected with Salubrinal (increases p-eIF2α) or ISRIB (decreases p-eIF2α) 60 minutes post-injury. Contrary to literature, both drugs hindered control animal visual function with minimal improvements in injured mice. Additionally, differences in eIF2α phosphorylation, antioxidant responses, and protein folding chaperones were different when examining protein expression between the retina and its axons in the optic nerve. These results reveal important compartmentalized ER stress responses to axon injury and suggest that interventions in the PERK pathway may alter necessary homeostatic regulation of the UPR in the retina.
    DOI:  https://doi.org/10.1101/2024.02.15.580435
  26. bioRxiv. 2024 Mar 03. pii: 2024.03.01.582834. [Epub ahead of print]
    Structural Requirements for Activity of Mind bomb1 in Notch Signaling.
    Ruili Cao, Oren Gozlan, Lena Tveriakhina, Haixia Zhou, Hanjie Jiang, Philip A Cole, Jon C Aster, David Sprinzak, Stephen C Blacklow.
      Mind bomb 1 (MIB1) is a RING E3 ligase that ubiquitinates Notch ligands, a necessary step for induction of Notch signaling. The structural basis for binding of the JAG1 ligand by the N-terminal region of MIB1 is known, yet how the ankyrin (ANK) and RING domains of MIB1 cooperate to catalyze ubiquitin transfer from E2∼Ub to Notch ligands remains unclear. Here, we show that the third RING domain and adjacent coiled coil region of MIB1 (ccRING3) drives MIB1 dimerization and that ubiquitin transfer activity of MIB1 relies solely on RING3. We report x-ray crystal structures of a UbcH5B-ccRING3 complex as a fusion protein and of the ANK region. Directly tethering the N-terminal region to ccRING3 forms a minimal MIB1 protein, which is sufficient to induce a Notch response in receiver cells. Together, these studies define the functional elements of an E3 ligase needed for ligands to induce a Notch signaling response.
    DOI:  https://doi.org/10.1101/2024.03.01.582834
  27. PLoS Biol. 2024 Mar 11. 22(3): e3002543
    Proteasome inhibition triggers tissue-specific immune responses against different pathogens in C. elegans.
    Manish Grover, Spencer S Gang, Emily R Troemel, Michalis Barkoulas.
      Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.
    DOI:  https://doi.org/10.1371/journal.pbio.3002543
  28. Cell Genom. 2024 Mar 02. pii: S2666-979X(24)00064-8. [Epub ahead of print] 100522
    Revealing the grammar of small RNA secretion using interpretable machine learning.
    Bahar Zirak, Mohsen Naghipourfar, Ali Saberi, Delaram Pouyabahar, Amirhossein Zarezadeh, Lixi Luo, Lisa Fish, Doowon Huh, Albertas Navickas, Ali Sharifi-Zarchi, Hani Goodarzi.
      Small non-coding RNAs can be secreted through a variety of mechanisms, including exosomal sorting, in small extracellular vesicles, and within lipoprotein complexes. However, the mechanisms that govern their sorting and secretion are not well understood. Here, we present ExoGRU, a machine learning model that predicts small RNA secretion probabilities from primary RNA sequences. We experimentally validated the performance of this model through ExoGRU-guided mutagenesis and synthetic RNA sequence analysis. Additionally, we used ExoGRU to reveal cis and trans factors that underlie small RNA secretion, including known and novel RNA-binding proteins (RBPs), e.g., YBX1, HNRNPA2B1, and RBM24. We also developed a novel technique called exoCLIP, which reveals the RNA interactome of RBPs within the cell-free space. Together, our results demonstrate the power of machine learning in revealing novel biological mechanisms. In addition to providing deeper insight into small RNA secretion, this knowledge can be leveraged in therapeutic and synthetic biology applications.
    Keywords:  ExoCLIP; ExoGRU; extracellular RNA; machine learning; small RNA; small RNA secretion
    DOI:  https://doi.org/10.1016/j.xgen.2024.100522
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