bims-proteo Biomed News
on Proteostasis
Issue of 2024–12–29
forty-five papers selected by
Eric Chevet, INSERM
  1. Impairment of lipid homeostasis causes lysosomal accumulation of endogenous protein aggregates through ESCRT disruption.
  2. Reconsidering the selectivity of bulk autophagy: cargo hitchhiking specifies cargo for degradation.
  3. Formyl-methionine-mediated eukaryotic ribosome quality control pathway for cold adaptation.
  4. Investigating protein degradability through site-specific ubiquitin ligase recruitment.
  5. Light-Activatable Ubiquitin for Studying Linkage-Specific Ubiquitin Chain Formation Kinetics.
  6. Regulation of N-Degron Recognin-Mediated Autophagy by the sars-cov-2 plpro ubiquitin deconjugase.
  7. NOVEL APPROACHES IN TARGETING MEMBRANE AND SECRETED PROTEINS FOR LYSOSOMAL DEGRADATION.
  8. Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs.
  9. Structural determinants of co-translational protein complex assembly.
  10. Nuclear ESCRT is involved in intranuclear protein quality control by micronucleophagy.
  11. Antibody-mediated clearance of an ER-resident aggregate that causes glaucoma.
  12. The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation.
  13. Ubiquitin-mediated recruitment of the ATG9A-ATG2 lipid transfer complex drives clearance of phosphorylated p62 aggregates.
  14. HSP90 stabilizes visual cycle retinol dehydrogenase 5 in the endoplasmic reticulum by inhibiting its degradation during autophagy.
  15. G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
  16. Buffering of nuclear membrane tension and mechanotransduction by the endoplasmic reticulum revealed by quantitative ALPIN imaging.
  17. Leucine Aminopeptidase LyLAP enables lysosomal degradation of membrane proteins.
  18. Inhibition of the Integrated Stress Response Prevents Natural Forgetting and Corrects Accelerated Forgetting Associated with Epilepsy.
  19. A neurodegenerative cellular stress response linked to dark microglia and toxic lipid secretion.
  20. PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.
  21. Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples.
  22. Interrogation of RNA-bound proteome with XRNAX illuminates molecular alterations in the mouse brain affected with dysmyelination.
  23. Targeted protein degradation comes of age.
  24. Targeting host integrated stress response: lead discovery of flavonoid compounds active against coronaviruses PEDV and PDCoV.
  25. Chaperone-dependent and chaperone-independent functions of carboxylate clamp tetratricopeptide repeat (CC-TPR) proteins.
  26. Baicalein tethers CD274/PD-L1 for autophagic degradation to boost antitumor immunity.
  27. Imaging-Based Quantitative Assessment of Biomolecular Condensates in vitro and in Cells.
  28. TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.
  29. The endoplasmic reticulum as a cradle for virus and extracellular vesicle secretion.
  30. The N-degron pathway mediates the autophagic degradation of cytosolic mitochondrial DNA during sterile innate immune responses.
  31. Temporal dissection of the roles of Atg4 and ESCRT in autophagosome formation in yeast.
  32. Identification of Polycystin 2 Missense Mutants Targeted for Endoplasmic Reticulum-Associated Degradation.
  33. G3BP1/2-Targeting PROTAC Disrupts Stress Granules Dependent ATF4 Migracytosis as Cancer Therapy.
  34. SETD7 promotes LC3B methylation and degradation in ovarian cancer.
  35. Neuronal constitutive endolysosomal perforations enable α-synuclein aggregation by internalized PFFs.
  36. Decreased Hsp90 activity protects against TDP-43 neurotoxicity in a C. elegans model of amyotrophic lateral sclerosis.
  37. Molecular tag for promoting N-glycan maturation in the cargo receptor-mediated secretion pathway.
  38. Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction.
  39. Heterozygous UBR5 variants result in a neurodevelopmental syndrome with developmental delay, autism, and intellectual disability.
  40. Preventing excessive autophagy protects from the pathology of mtDNA mutations in Drosophila melanogaster.
  41. Systematic characterization of indel variants using a yeast-based protein folding sensor.
  42. Discovery and biological evaluation of potent 2-trifluoromethyl acrylamide warhead-containing inhibitors of protein disulfide isomerase.
  43. Development and Discovery of a Selective Degrader of Casein Kinases 1 δ/ε.
  44. Targeting UBE2T suppresses breast cancer stemness through CBX6-mediated transcriptional repression of SOX2 and NANOG.
  45. Targeted degradation of oncogenic KRASG12V triggers antitumor immunity in lung cancer models.
  1. Elife. 2024 Dec 23. pii: RP86194. [Epub ahead of print]12
    Impairment of lipid homeostasis causes lysosomal accumulation of endogenous protein aggregates through ESCRT disruption.
    John Yong, Jacqueline E Villalta, Ngoc Vu, Matthew A Kukurugya, Niclas Olsson, Magdalena Preciado López, Julia R Lazzari-Dean, Kayley Hake, Fiona E McAllister, Bryson D Bennett, Calvin H Jan.
      Protein aggregation increases during aging and is a pathological hallmark of many age-related diseases. Protein homeostasis (proteostasis) depends on a core network of factors directly influencing protein production, folding, trafficking, and degradation. Cellular proteostasis also depends on the overall composition of the proteome and numerous environmental variables. Modulating this cellular proteostasis state can influence the stability of multiple endogenous proteins, yet the factors contributing to this state remain incompletely characterized. Here, we performed genome-wide CRISPRi screens to elucidate the modulators of proteostasis state in mammalian cells, using a fluorescent dye to monitor endogenous protein aggregation. These screens identified known components of the proteostasis network and uncovered a novel link between protein and lipid homeostasis. Increasing lipid uptake and/or disrupting lipid metabolism promotes the accumulation of sphingomyelins and cholesterol esters and drives the formation of detergent-insoluble protein aggregates at the lysosome. Proteome profiling of lysosomes revealed ESCRT accumulation, suggesting disruption of ESCRT disassembly, lysosomal membrane repair, and microautophagy. Lipid dysregulation leads to lysosomal membrane permeabilization but does not otherwise impact fundamental aspects of lysosomal and proteasomal functions. Together, these results demonstrate that lipid dysregulation disrupts ESCRT function and impairs proteostasis.
    Keywords:  CRISPR; ESCRT; aggregation; cell biology; human; lipid dysregulation; lysosome; proteostasis
    DOI:  https://doi.org/10.7554/eLife.86194
  2. Autophagy. 2024 Dec 27.
    Reconsidering the selectivity of bulk autophagy: cargo hitchhiking specifies cargo for degradation.
    Eigo Takeda, Alexander I May, Yoshinori Ohsumi.
      Bulk macroautophagy/autophagy, typically induced by starvation, is generally thought to non-selectively isolate cytosolic components for degradation. However, a detailed analysis of bulk autophagy cargo has not been conducted. We recently employed mass spectrometry to analyze the contents of isolated autophagic bodies. In this process, we uncovered Hab1 (Highly enriched in Autophagic Bodies 1), a novel protein, that is preferentially delivered via autophagy. Hab1 is a receptor protein that binds Atg8-PE and ribosomes at its N- and C-termini, respectively. We found that ribosome-bound Hab1 is preferentially delivered to the vacuole by "'hitchhiking'" on phagophores/isolation membranes formed during bulk autophagy. This hitchhiking mechanism confers selectivity to bulk autophagy.
    Keywords:  Atg8; Hab1; Saccharomyces cerevisiae; hitchhiking; ribosome
    DOI:  https://doi.org/10.1080/15548627.2024.2447209
  3. Mol Cell. 2024 Dec 17. pii: S1097-2765(24)00990-0. [Epub ahead of print]
    Formyl-methionine-mediated eukaryotic ribosome quality control pathway for cold adaptation.
    Chang-Seok Lee, Jaehwan Sim, Sang-Yoon Kim, Hyeonji Lee, Tae-Young Roh, Cheol-Sang Hwang.
      Protein synthesis in the eukaryotic cytosol can start using both conventional methionine and formyl-methionine (fMet). However, a mechanism, if such exists, for detecting and regulating the incorporation of fMet (instead of Met) during translation, thereby preventing cellular toxicity of nascent fMet-bearing (fMet-) polypeptides, remains unknown. Here, we describe the fMet-mediated ribosome quality control (fMet-RQC) pathway in Saccharomyces cerevisiae. A eukaryotic translation initiation factor 3 subunit c, Nip1, specifically recognizes N-terminal fMet in nascent polypeptides, recruiting a small GTPase, Arf1, to induce ribosome stalling, largely with 41-residue fMet-peptidyl tRNAs. This leads to ribosome dissociation and subsequent stress granule formation. Loss of the fMet-RQC pathway causes the continued synthesis of fMet polypeptides, which inhibits essential N-terminal Met modifications and promotes their coaggregation with ribosomes. This fMet-RQC pathway is important for the adaptation of yeast cells to cold stress by promoting stress granule formation and preventing a buildup of toxic fMet polypeptides.
    Keywords:  Arf1; Nip1; cellular adaptation; cold stress; formyl-methionine; proteotoxicity; ribosome quality control; stress granule
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.035
  4. RSC Chem Biol. 2024 Dec 13.
    Investigating protein degradability through site-specific ubiquitin ligase recruitment.
    Olivia Shade, Amy Ryan, Gabriella Belsito, Alexander Deiters.
      We report targeted protein degradation through the site-specific recruitment of native ubiquitin ligases to a protein of interest via conjugation of E3 ligase ligands. Direct comparison of degradation ability of proteins displaying the corresponding bioconjugation handle at different regions of protein surfaces was explored. We demonstrate the benefit of proximal lysine residues and investigate flexibility in linker length for the design of optimal degraders. Two proteins without known small molecule ligands, EGFP and DUSP6, were differentially degraded when modified at different locations on their protein surfaces. Further, the cereblon-mediated degradation of the known PROTAC target ERRα was improved through the recruitment of the E3 ligase to regions different from the known ligand binding site. This new methodology will provide insight into overall protein degradability, even in the absence of a known small molecule ligand and inform the process of new ligand and PROTAC development to achieve optimal protein degradation. Furthermore, this approach represents a new, small molecule-based conditional OFF switch of protein function with complete genetic specificity. Importantly, the protein of interest is only modified with a minimal surface modification (<200 Da) and does not require any protein domain fusions.
    DOI:  https://doi.org/10.1039/d4cb00273c
  5. Adv Sci (Weinh). 2024 Dec 24. e2406570
    Light-Activatable Ubiquitin for Studying Linkage-Specific Ubiquitin Chain Formation Kinetics.
    Sudakshina Banerjee, Zeyneb Vildan Cakil, Kai Gallant, Johannes van den Boom, Shubhendu Palei, Hemmo Meyer, Malte Gersch, Daniel Summerer.
      Ubiquitination is a dynamic post-translational modification governing protein abundance, function, and localization in eukaryotes. The Ubiquitin protein is conjugated to lysine residues of target proteins, but can also repeatedly be ubiquitinated itself, giving rise to a complex code of ubiquitin chains with different linkage types. To enable studying the cellular dynamics of linkage-specific ubiquitination, light-activatable polyubiquitin chain formation is reported here. By incorporating a photocaged lysine at specific sites within ubiquitin through amber codon suppression, light-dependent activation of ubiquitin chain extension is enabled for the monitoring of linkage-specific polyubiquitination. The studies reveal rapid, minute-scale ubiquitination kinetics for K11, K48, and K63 linkages. The role of individual components of the ubiquitin-proteasome system in K48-initiated chain synthesis is further studied by small molecule inhibition. The approach expands current perturbation strategies with the ability to control linkage-specific ubiquitination with high temporal resolution and should find broad application for studying ubiquitinome dynamics.
    Keywords:  genetic code expansion; optochemical biology; small molecule inhibitors; ubiquitin
    DOI:  https://doi.org/10.1002/advs.202406570
  6. Autophagy. 2024 Dec 26.
    Regulation of N-Degron Recognin-Mediated Autophagy by the sars-cov-2 plpro ubiquitin deconjugase.
    Carlos Ayala Torres, Jiangnan Liu, Nico P Dantuma, Maria G Masucci.
      Viral proteases play critical roles in the host cell and immune remodeling that allows virus production. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) papain-like protease (PLpro) encoded in the large nonstructural protein 3 (Nsp3) also possesses isopeptidase activity with specificity for ubiquitin and ISG15 conjugates. Here, we interrogated the cellular interactome of the SARS-CoV-2 PLpro catalytic domain to gain insight into the putative substrates and cellular functions affected by the viral deubiquitinase. PLpro was detected in protein complexes that control multiple ubiquitin and ubiquitin-like (UbL) regulated signaling and effector pathways. By restricting the analysis to cytosolic and membrane-associated ubiquitin ligases, we found that PLpro interacts with N-recognin ubiquitin ligases and preferentially rescues type I N-degron substrates from proteasomal degradation. PLpro stabilized N-degron carrying HSPA5/BiP/GRP78, which is arginylated in the cytosol upon release from the endoplasmic reticulum (ER) upon ER stress and enhanced the Arg-HSPA5-driven oligomerization of the N-recognin SQSTM1/p62 that serves as a platform for phagophore assembly. However, while in addition to Arg-HSPA5 and SQSTM1/p62, ATG9A, WIPI2, and BECN1/Beclin 1 were detected in PLpro immunoprecipitates, other components of the autophagosome biogenesis machinery, such as the ATG12-ATG5-ATG16L1 complex and MAP1LC3/LC3 were absent, which correlated with proteolytic inactivation of ULK1, impaired production of lipidated LC3-II, and inhibition of reticulophagy. The findings highlight a novel mechanism by which, through the reprogramming of autophagy, the PLpro deubiquitinase may contribute to the remodeling of intracellular membranes in coronavirus-infected cells.
    Keywords:  HSPA5/BiP/GRP78; N-degron; Plpro; SARS-CoV-2; SQSTM1/p62; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2442849
  7. Chembiochem. 2024 Dec 23. e202400887
    NOVEL APPROACHES IN TARGETING MEMBRANE AND SECRETED PROTEINS FOR LYSOSOMAL DEGRADATION.
    Mohamed Eldeeb, Grace Hohman, Michael Shahid.
      Protein degradation is pivotal for all biochemical aspects of cellular function. In mammalian cells, protein degradation is mediated mainly by the ubiquitin proteasome system (UPS) and the autophagic-lysosomal system (ALS). Over the last two decades, different types of targeted protein degradation approaches have been developed including proteolysis targeting chimeras (PROTACs) and lysosome targeting chimeras (LYTACs), which employ the UPS to degrade intracellular proteins and the ALS to degrade extracellular and membrane proteins respectively. Nevertheless, current targeted membrane protein degradation approaches face some inherent challenges including limited target protein degradation efficacy and cell type specific applicability. Herein, we highlight a recent development of novel targeted membrane protein degradation modalities that exhibit wide-applicability and high protein degradation efficiency. These novel membrane protein degraders hold tremendous promise as new pharmacological and biochemical tools in targeting membrane and secretory proteins for lysosomal degradation.
    Keywords:  Lysosome; Protein degradation; Proteolysis; autophagy; membrane proteins
    DOI:  https://doi.org/10.1002/cbic.202400887
  8. Mol Cell. 2024 Dec 12. pii: S1097-2765(24)00955-9. [Epub ahead of print]
    Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs.
    Cole J T Lewis, Li H Xie, Shivani Milind Bhandarkar, Danni Jin, Kyrillos 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 (UTRs) including alternative isoforms and identify sequences that mediate 200-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 dissecting mechanisms of human translation initiation and engineering more potent therapeutic mRNAs.
    Keywords:  5′ untranslated region; N1-methylpseudouridine; RNA modification; high-throughput screening; ribosome; therapeutic mRNA; translation initiation
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.030
  9. Cell. 2024 Dec 18. pii: S0092-8674(24)01330-8. [Epub ahead of print]
    Structural determinants of co-translational protein complex assembly.
    Saurav Mallik, Johannes Venezian, Arseniy Lobov, Meta Heidenreich, Hector Garcia-Seisdedos, Todd O Yeates, Ayala Shiber, Emmanuel D Levy.
      Protein assembly into functional complexes is critical to life's processes. While complex assembly is classically described as occurring between fully synthesized proteins, recent work showed that co-translational assembly is prevalent in human cells. However, the biological basis for the existence of this process and the identity of protein pairs that assemble co-translationally remain unknown. We show that co-translational assembly is governed by structural characteristics of complexes and involves mutually stabilized subunits. Accordingly, co-translationally assembling subunits are unstable in isolation and exhibit synchronized proteostasis with their partner. By leveraging structural signatures and AlphaFold2-based predictions, we accurately predicted co-translational assembly, including pair identities, at proteome scale and across species. We validated our predictions by ribosome profiling, stoichiometry perturbations, and single-molecule RNA-fluorescence in situ hybridization (smFISH) experiments that revealed co-localized mRNAs. This work establishes a fundamental connection between protein structure and the translation process, highlighting the overarching impact of three-dimensional structure on gene expression, mRNA localization, and proteostasis.
    Keywords:  AlphaFold; co-translational assembly; mRNA localization; protein complexes; protein interactions; protein structure; proteostasis; ribosome profiling; single-molecule FISH; translational regulation
    DOI:  https://doi.org/10.1016/j.cell.2024.11.013
  10. Biochem Biophys Res Commun. 2024 Dec 19. pii: S0006-291X(24)01755-8. [Epub ahead of print]744 151219
    Nuclear ESCRT is involved in intranuclear protein quality control by micronucleophagy.
    Most Naoshia Tasnin, Yuka Takahashi, Tsuneyuki Takuma, Takashi Ushimaru.
      Intranuclear protein quality control (PQC) is critical for protein homeostasis (or proteostasis) in non-dividing cells including brain nerve cells, but its molecular mechanism remains unresolved. In nutrient-starved conditions, elimination of nucleolar proteins is critical for cell viability in budding yeast, providing a model system to study the mechanisms involved in intranuclear PQC. The nuclear-specific endosomal sorting complex required for transport (ESCRT) CHMP7/Chm7 is linked to neurodegenerative diseases, but its known role is limited. Here, we show a novel role of nuclear ESCRT in intranuclear PQC. Chm7 and its recruiter protein Heh1 were critical for micronucleophagic degradation of nucleolar proteins and for rDNA condensation and nucleolar remodeling, which is prerequisite for proper micronucleophagic degradation of nucleolar proteins. By contrast, Chm7 was dispensable for macronucleophagy. Finally, not only authentic ESCRT but also Chm7 was crucial for the survival of quiescent cells in prolonged nutrient-starved conditions. This study uncovered that nuclear ESCRT together with authentic ESCRT orchestrate micronucleophagic degradation of nucleolar proteins, contributing to intranuclear protein homeostasis.
    Keywords:  Autophagy; Chm7; ESCRT; Nucleophagy; Protein quality control; TORC1
    DOI:  https://doi.org/10.1016/j.bbrc.2024.151219
  11. PNAS Nexus. 2025 Jan;4(1): pgae556
    Antibody-mediated clearance of an ER-resident aggregate that causes glaucoma.
    Minh Thu Ma, Ahlam N Qerqez, Kamisha R Hill, Laura R Azouz, Hannah A Youngblood, Shannon E Hill, Yemo Ku, Donna M Peters, Jennifer A Maynard, Raquel L Lieberman.
      Recombinant antibodies are a promising class of therapeutics to treat protein misfolding associated with neurodegenerative diseases, and several antibodies that inhibit aggregation are approved or in clinical trials to treat Alzheimer's disease. Here, we developed antibodies targeting the aggregation-prone β-propeller olfactomedin (OLF) domain of myocilin, variants of which comprise the strongest genetic link to glaucoma and cause early onset vision loss for several million individuals worldwide. Mutant myocilin aggregates intracellularly in the endoplasmic reticulum (ER). Subsequent ER stress causes cytotoxicity that hastens dysregulation of intraocular pressure, the primary risk factor for most forms of glaucoma. Our antibody discovery campaign yielded two recombinant antibodies: anti-OLF1 recognizes a linear epitope, while anti-OLF2 is selective for natively folded OLF and inhibits aggregation in vitro. By binding OLF, these antibodies engage autophagy/lysosomal degradation to promote degradation of two pathogenic mutant myocilins. This work demonstrates the potential for therapeutic antibodies to disrupt ER-localized protein aggregates by altering the fate of folding intermediates. This approach could be translated as a precision medicine to treat myocilin-associated glaucoma with in situ antibody expression. More generally, the study supports the approach of enhancing lysosomal degradation to treat proteostasis decline in glaucoma and other diseases.
    Keywords:  autophagy; molecular recognition; myocilin; protein misfolding; proteostasis
    DOI:  https://doi.org/10.1093/pnasnexus/pgae556
  12. Cell Rep. 2024 Dec 20. pii: S2211-1247(24)01452-9. [Epub ahead of print]44(1): 115101
    The Cullin3-Rbx1-KLHL9 E3 ubiquitin ligase complex ubiquitinates Rheb and supports amino acid-induced mTORC1 activation.
    Yao Yao, Sungki Hong, Shota Yoshida, Vinamra Swaroop, Brady Curtin, Ken Inoki.
      Mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosomal membrane by the active Rag heterodimer, where mTORC1 interacts with active Rheb for its activation. It has been shown that polyubiquitination of Rheb is crucial for enhancing its interaction with mTORC1 on the lysosome. However, the specific ubiquitin ligases for Rheb, which promotes mTORC1 activation, remain elusive. We report that the CUL3-RBX1-KLHL9 E3 ubiquitin ligase complex is translocated to the lysosome and ubiquitinates Rheb in response to amino acid stimulation. KLHL9 serves as an essential adaptor for CUL3-RBX1 to target Rheb on the lysosome. Deleting either CUL3, RBX1, or KLHL9 diminishes Rheb ubiquitination and reduces amino acid-induced mTORC1 activation without impacting lysosomal mTORC1 localization or Akt activity. Thus, the CUL3-RBX1-KLHL9 complex functions as a mTORC1 activator by acting as an E3 ubiquitin ligase for Rheb and supports amino acid-induced mTORC1 activation.
    Keywords:  CP: Cell biology; CUL3; KLHL9; Rheb; lysosome; mTORC1; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2024.115101
  13. Mol Biol Cell. 2024 Dec 24. mbcE24030101
    Ubiquitin-mediated recruitment of the ATG9A-ATG2 lipid transfer complex drives clearance of phosphorylated p62 aggregates.
    D G Broadbent, C M McEwan, D Jayatunge, E G Kaminsky, T M Tsang, D M Poole, B C Naylor, J C Price, J C Schmidt, J L Andersen.
      Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A, or the lipid transfer protein ATG2, leads to the accumulation of phosphorylated p62 aggregates in nutrient replete conditions. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-03-0101
  14. J Biol Chem. 2024 Dec 24. pii: S0021-9258(24)02628-0. [Epub ahead of print] 108126
    HSP90 stabilizes visual cycle retinol dehydrogenase 5 in the endoplasmic reticulum by inhibiting its degradation during autophagy.
    Xiaolin Jia, Yuxuan Wang, Mingjun Jiang, Dan-Dan Chen, Guohui Shang, Baixue Liu, Mengjiao Xue, Youfei Lang, Guiling Zhou, Yichen Dong, Fengyan Zhang, Xuyan Peng, Yanzhong Hu.
      Genetic mutations in retinol dehydrogenase 5 (RDH5), a rate-limiting enzyme of the visual cycle, is associated with nyctalopia, AMD and stationary congenital fundus albipunctatus (FA). A majority of these mutations impair RDH5 protein expression and intracellular localization. However, the regulatory mechanisms underlying RDH5 metabolism remain unclear. Here, we find that RDH5 undergoes degradation via the autophagy-lysosomal pathway, and its stability is regulated by interacting with HSP90. Deletion of HSP90α or HSP90β by CRISPR-Cas9 or inhibition of HSP90 activity by IPI-504 down-regulates RDH5 protein level, but not its mRNA expression, and this downregulation is restored by autophagic inhibitors (3-MA, CQ and Baf-A1) and siRNA of ATG5 or ATG7, but not by the proteasome inhibitor MG132. RDH5 can physically interact with SQSTM1/P62, and this interaction is enhanced in HSP90-deficient cells as well as in CQ-treated cells. Knocking down SQSTM1/P62 by siRNA induces RDH5 protein accumulation. Moreover, HSP90, RDH5 and Calnexin form a complex through intermolecular interactions. Deficiency of HSP90α or HSP90β dissociates RDH5 from Calnexin, and increases RDH5 translocation from the endoplasmic reticulum (ER) to the cytosol. Taken together, we propose that dysfunction of HSP90 leads to RDH5 release from Calnexin in the ER into the cytosol, where it binds to the adaptor SQSTM1/P62 for degradation in the autolysosome. RDH5 is a novel client candidate of HSP90. The downregulation of RDH5 may be responsible for the nyctalopia side effect noted in cancer patients receiving HSP90 inhibitor treatment currently in the clinical trial.
    Keywords:  autophagy and endoplasmic reticulum; calnexin; heat shock protein 90; retinol dehydrogenase 5; visual cycle
    DOI:  https://doi.org/10.1016/j.jbc.2024.108126
  15. Mol Cell. 2024 Dec 19. pii: S1097-2765(24)00994-8. [Epub ahead of print]
    G3BP-driven RNP granules promote inhibitory RNA-RNA interactions resolved by DDX3X to regulate mRNA translatability.
    Irmela R E A Trussina, Andreas Hartmann, Christine Desroches Altamirano, Janani Natarajan, Charlotte M Fischer, Marta Aleksejczuk, Hannes Ausserwöger, Tuomas P J Knowles, Michael Schlierf, Titus M Franzmann, Simon Alberti.
      Ribonucleoprotein (RNP) granules have been linked to translation regulation and disease, but their assembly and regulatory mechanisms are not well understood. Here, we show that the RNA-binding protein G3BP1 preferentially interacts with unfolded RNA, driving the assembly of RNP granule-like condensates that establish RNA-RNA interactions. These RNA-RNA interactions limit the mobility and translatability of sequestered mRNAs and stabilize the condensates. The DEAD-box RNA helicase DDX3X attenuates RNA-RNA interactions inside RNP granule-like condensates, rendering the condensates dynamic and enabling mRNA translation. Importantly, disease-associated and catalytically inactive DDX3X variants fail to resolve such RNA-RNA interactions. Inhibiting DDX3X in cultured cells accelerates RNP granule assembly and delays their disassembly, indicating that RNA-RNA interactions contribute to RNP granule stability in cells. Our findings reveal how RNP granules generate inhibitory RNA-RNA interactions that are modulated by DEAD-box RNA helicases to ensure RNA availability and translatability.
    Keywords:  DDX3X; DEAD-box helicase; G3BP1; RNA-RNA interactions; RNP granule; biomolecular condensate; stress granule
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.039
  16. Res Sq. 2024 Dec 09. pii: rs.3.rs-5530637. [Epub ahead of print]
    Buffering of nuclear membrane tension and mechanotransduction by the endoplasmic reticulum revealed by quantitative ALPIN imaging.
    Philipp Niethammer, Zhouyang Shen, Zaza Gelashvili.
      Nuclear deformation by osmotic shock or necrosis activates the cytosolic phospholipase A2 (cPla2) nuclear shape sensing pathway, a key regulator of tissue inflammation and repair. Ca²⁺ and inner nuclear membrane (INM) tension (T INM ) are believed to mediate nucleoplasmic cPla2 activation. The concept implies that T INM persists long enough to stimulate cPla2-INM adsorption. However, T INM may instead be rapidly dissipated by the contiguous endoplasmic reticulum (ER), with cPla2-INM adsorption reporting rather on changes in Ca²⁺ than T INM . The impact of T INM and ER contiguity on nuclear shape sensing and mechanotransduction remains unknown. To address this gap, we developed the Ca 2+ insensitive, T INM -only biosensor ALPIN (Amphipathic Lipid Packing sensor domain Inside the Nucleus). By quantitative ALPIN imaging, we found that stress-induced ER fragmentation increases T INM and nuclear membrane mechanotransduction in osmotically shocked or ferroptotic cells, permeabilized cell corpses, and at zebrafish wounds in vivo. Our findings reveal critical roles for the ER and T INM in nuclear shape sensing and introduce ALPIN as promising tool for studying organelle membrane mechanotransduction in health and disease.
    DOI:  https://doi.org/10.21203/rs.3.rs-5530637/v1
  17. bioRxiv. 2024 Dec 14. pii: 2024.12.13.628212. [Epub ahead of print]
    Leucine Aminopeptidase LyLAP enables lysosomal degradation of membrane proteins.
    Aakriti Jain, Isaac Heremans, Gilles Rademaker, Tyler C Detomasi, Grace A Hernandez, Justin Zhang, Suprit Gupta, Teresa von Linde, Mike Lange, Martina Spacci, Peter Rohweder, Dashiell Anderson, Y Rose Citron, James A Olzmann, David W Dawson, Charles S Craik, Guido Bommer, Rushika M Perera, Roberto Zoncu.
      Proteolysis of hydrophobic helices is required for complete breakdown of every transmembrane protein trafficked to the lysosome and sustains high rates of endocytosis. However, the lysosomal mechanisms for degrading hydrophobic domains remain unknown. Combining lysosomal proteomics with functional genomic data mining, we identify Lysosomal Leucine Aminopeptidase (LyLAP; formerly Phospholipase B Domain-Containing 1) as the hydrolase most tightly associated with elevated endocytic activity. Untargeted metabolomics and biochemical reconstitution demonstrate that LyLAP is not a phospholipase, but a processive monoaminopeptidase with strong preference for N-terminal leucine - an activity necessary and sufficient for breakdown of hydrophobic transmembrane domains. LyLAP is upregulated in pancreatic ductal adenocarcinoma (PDA), which relies on macropinocytosis for nutrient uptake, and its ablation led to buildup of undigested hydrophobic peptides, which compromised lysosomal membrane integrity and inhibited PDA cell growth. Thus, LyLAP enables lysosomal degradation of membrane proteins, and may represent a vulnerability in highly endocytic cancer cells.
    One sentence summary: LyLAP degrades transmembrane proteins to sustain high endocytosis and lysosomal membrane stability in pancreatic cancer.
    DOI:  https://doi.org/10.1101/2024.12.13.628212
  18. Mol Neurobiol. 2024 Dec 21.
    Inhibition of the Integrated Stress Response Prevents Natural Forgetting and Corrects Accelerated Forgetting Associated with Epilepsy.
    Shi-Yi Wang, Bo Wang, Lu-Yao Li, Yi Zuo, Xin Jin, Bo Zhang, Shao-Wen Tian.
      The neural mechanisms underlying the natural and maladaptive forgetting of established memory remain largely unknown. Brain disease states might hijack the physiological forgetting mechanisms, resulting in maladaptive forgetting such as accelerated forgetting that contributes to cognitive decline in various neurologic conditions including epilepsy. Based on the key role of the integrated stress response (ISR) in memory storage and maintenance, we determined whether the ISR underpins natural and accelerated forgetting. Here, based on the object location recognition (OLR) and novel object recognition (NOR) paradigms in mice, we found that the ISR was activated while an established memory was naturally forgotten, which was denoted by increased levels of phosphorylated eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4), and reduced general protein synthesis. Multiple administrations of ISRIB, a small molecule ISR inhibitor, during the memory retention interval attenuated the ISR activation, and prevented the natural forgetting of established OLR and NOR memories. At the same time, a single injection of ISRIB has no effect on natural forgetting and memory retrieval. Moreover, administration of pentylenetetrazole (PTZ), an inducer of epileptic seizures, during the memory retention interval provoked the ISR activation and accelerated forgetting, which was corrected by ISRIB treatment. Together, our findings suggest that the ISR is critically involved in natural forgetting and accelerated forgetting associated with epilepsy, and pharmacological inhibition of the ISR may emerge as a novel intervention strategy for accelerated forgetting in patients with epilepsy.
    Keywords:  Epilepsy; Forgetting; ISRIB; Integrated stress response; Memory
    DOI:  https://doi.org/10.1007/s12035-024-04669-5
  19. Neuron. 2024 Dec 19. pii: S0896-6273(24)00875-4. [Epub ahead of print]
    A neurodegenerative cellular stress response linked to dark microglia and toxic lipid secretion.
    Anna Flury, Leen Aljayousi, Hye-Jin Park, Mohammadparsa Khakpour, Jack Mechler, Siaresh Aziz, Jackson D McGrath, Pragney Deme, Colby Sandberg, Fernando González Ibáñez, Olivia Braniff, Thi Ngo, Simira Smith, Matthew Velez, Denice Moran Ramirez, Dvir Avnon-Klein, John W Murray, Jia Liu, Martin Parent, Susana Mingote, Norman J Haughey, Sebastian Werneburg, Marie-Ève Tremblay, Pinar Ayata.
      The brain's primary immune cells, microglia, are a leading causal cell type in Alzheimer's disease (AD). Yet, the mechanisms by which microglia can drive neurodegeneration remain unresolved. Here, we discover that a conserved stress signaling pathway, the integrated stress response (ISR), characterizes a microglia subset with neurodegenerative outcomes. Autonomous activation of ISR in microglia is sufficient to induce early features of the ultrastructurally distinct "dark microglia" linked to pathological synapse loss. In AD models, microglial ISR activation exacerbates neurodegenerative pathologies and synapse loss while its inhibition ameliorates them. Mechanistically, we present evidence that ISR activation promotes the secretion of toxic lipids by microglia, impairing neuron homeostasis and survival in vitro. Accordingly, pharmacological inhibition of ISR or lipid synthesis mitigates synapse loss in AD models. Our results demonstrate that microglial ISR activation represents a neurodegenerative phenotype, which may be sustained, at least in part, by the secretion of toxic lipids.
    Keywords:  Alzheimer’s disease; ISR; dark microglia; integrated stress response; lipid secretion; lipotoxicity; microglia; neurodegeneration; neurotoxic microglia; non-cell-autonomous stress
    DOI:  https://doi.org/10.1016/j.neuron.2024.11.018
  20. bioRxiv. 2024 Dec 13. pii: 2024.12.10.627778. [Epub ahead of print]
    PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.
    Connor J Sheedy, Soham P Chowdhury, Bashir A Ali, Julia Miyamoto, Eric Z Pang, Julien Bacal, Katherine U Tavasoli, Chris D Richardson, Brooke M Gardner.
      The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1's active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1's affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.
    Keywords:  ATPases associated with diverse cellular activities (AAA); Peroxisome; neurodegenerative disease; proteasome; protein assembly; protein degradation; protein stability; ubiquitin thioesterase (OTUB1)
    DOI:  https://doi.org/10.1101/2024.12.10.627778
  21. J Clin Invest. 2024 Dec 26. pii: e183592. [Epub ahead of print]
    Tagless LysoIP for immunoaffinity enrichment of native lysosomes from clinical samples.
    Daniel Saarela, Pawel Lis, Sara Gomes, Raja S Nirujogi, Wentao Dong, Eshaan S Rawat, Sophie Glendinning, Karolina Zeneviciute, Enrico Bagnoli, Rotimi Fasimoye, Cindy Lin, Kwamina Nyame, Fanni A Boros, Friederike Zunke, Frederic Lamoliatte, Sadik Elshani, Matthew Jaconelli, Judith Jm Jans, Margriet A Huisman, Christian Posern, Lena M Westermann, Angela Schulz, Peter M van Hasselt, Dario R Alessi, Monther Abu-Remaileh, Esther M Sammler.
      Lysosomes are implicated in a wide spectrum of human diseases including monogenic lysosomal storage disorders (LSDs), age-associated neurodegeneration and cancer. Profiling lysosomal content using tag-based lysosomal immunoprecipitation (LysoTagIP) in cell and animal models has substantially moved the field forward, but studying lysosomal dysfunction in human patients remains challenging. Here, we report the development of the 'tagless LysoIP' method, designed to enable the rapid enrichment of lysosomes, via immunoprecipitation, using the endogenous integral lysosomal membrane protein TMEM192, directly from clinical samples and human cell lines (e.g., induced pluripotent stem cell derived neurons). Isolated lysosomes were intact and suitable for subsequent multimodal omics analyses. To validate our approach, we applied the tagless LysoIP to enrich lysosomes from peripheral blood mononuclear cells derived from fresh blood of healthy donors and patients with CLN3 disease, an autosomal recessive neurodegenerative LSD. Metabolic profiling of isolated lysosomes revealed massive accumulation of glycerophosphodiesters (GPDs) in patients' lysosomes. Interestingly, a patient with a milder phenotype and genotype displayed lower accumulation of lysosomal GPDs, consistent with their potential role as disease biomarkers. Altogether, the tagless LysoIP provides a framework to study native lysosomes from patient samples, identify disease biomarkers, and discover human-relevant disease mechanisms.
    Keywords:  Cell biology; Genetic diseases; Lysosomes; Neurodegeneration; Neuroscience
    DOI:  https://doi.org/10.1172/JCI183592
  22. Cell Rep. 2024 Dec 21. pii: S2211-1247(24)01446-3. [Epub ahead of print]44(1): 115095
    Interrogation of RNA-bound proteome with XRNAX illuminates molecular alterations in the mouse brain affected with dysmyelination.
    Marta Sztachera, Weronika Wendlandt-Stanek, Remigiusz A Serwa, Luiza Stanaszek, Michał Smuszkiewicz, Dorota Wronka, Monika Piwecka.
      RNA-protein interactions orchestrate hundreds of pathways in homeostatic and stressed cells. We applied an RNA-protein interactome capture method called protein cross-linked RNA extraction (XRNAX) to shed light on the RNA-bound proteome in dysmyelination. We found sets of canonical RNA-binding proteins (RBPs) regulating alternative splicing and engaged in the cytoplasmic granules to be perturbed at the level of their RNA interactome. We validated these observations for PCBP1 and MBNL1. We show that the number of PCBP1 bodies is markedly increased in the mossy cells of the hippocampus and that the pattern of MBNL1-regulated alternatively spliced exons differs between the myelin-deficient and the wild-type brain, which is likely associated with Mbnl1 splicing perturbation and circular RNA generation from this locus. In the broader perspective, our results demonstrate that, with the application of the RNA-protein interactome approach, we can uncover alterations in RBP functioning in the disease context that are not always directly visible from their mRNA or protein levels.
    Keywords:  CP: Molecular biology; CP: Neuroscience; MBNL1; PCBP1; RNA processing; RNA-binding proteins; RNA-protein interactions; alternative splicing; circRNA; dysmyelination
    DOI:  https://doi.org/10.1016/j.celrep.2024.115095
  23. Nat Chem Biol. 2025 Jan;21(1): 22-23
    Targeted protein degradation comes of age.
    Mary E Matyskiela.
      
    DOI:  https://doi.org/10.1038/s41589-024-01789-w
  24. RSC Med Chem. 2024 Dec 23.
    Targeting host integrated stress response: lead discovery of flavonoid compounds active against coronaviruses PEDV and PDCoV.
    Liang Yi, Yishuai Wang, Jiehuang Wang, Yihan Chen, Weixue Huang, Ying Liao, Qingwen Zhang.
      Viral infections trigger the integrated stress response (ISR) in eukaryotic cells that leads to the activation of eIF2α kinases, the elevation of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation, and thereby the shutdown of global protein synthesis that viruses rely on to replicate. Coronaviruses and other viruses have evolved various subversion mechanisms to counteract the antiviral ISR. These intricate host-virus interactions may be exploited by pharmacologically activating the host ISR for the development of host-directed antivirals (HDAs), an increasingly relevant area of research. In this study, we have discovered a new class of flavonoid-based ISR activators that exhibit potent antiviral activity against porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV). PEDV and PDCoV are animal coronaviruses of great veterinary and economic importance, for which there are currently no effective therapeutics. The mechanistic study indicated that lead compounds 1-B and 1-C inhibit PEDV and PDCoV replication via upregulating eIF2α phosphorylation and thereby downregulating global protein synthesis in host cells, suggesting they are HDA antivirals.
    DOI:  https://doi.org/10.1039/d4md00846d
  25. Trends Biochem Sci. 2024 Dec 19. pii: S0968-0004(24)00256-1. [Epub ahead of print]
    Chaperone-dependent and chaperone-independent functions of carboxylate clamp tetratricopeptide repeat (CC-TPR) proteins.
    Saugat Pokhrel, Shweta Devi, Jason E Gestwicki.
      The molecular chaperones HSP70 and HSP90 play key roles in proteostasis by acting as adapters; they bind to a 'client' protein, often with the assistance of cochaperones, and then recruit additional cochaperones that promote specific fates (e.g., folding or degradation). One family of cochaperones contains a region termed the tetratricopeptide repeat with carboxylate clamps (CC-TPRs) domain. These domains bind to an EEVD motif at the C-termini of cytoplasmic HSP70 and HSP90 proteins, bringing them into proximity to chaperone-bound clients. It has recently become clear that CC-TPR proteins also bind to 'EEVD-like' motifs in non-chaperone proteins, circumventing the need for HSP70s or HSP90s. We provide an overview of the chaperone-dependent and -independent roles of CC-TPR proteins and discuss how, together, they shape proteostasis.
    Keywords:  C-end rule, degrons; cochaperones; microtubule-associated protein tau; proteostasis; short linear motifs (SLiMs)
    DOI:  https://doi.org/10.1016/j.tibs.2024.11.004
  26. Autophagy. 2024 Dec 22. 1-17
    Baicalein tethers CD274/PD-L1 for autophagic degradation to boost antitumor immunity.
    Bingjie Hao, Shumeng Lin, Haipeng Liu, Junfang Xu, Li Chen, Tiansheng Zheng, Wen Zhang, Yifang Dang, Russel J Reiter, Chaoqun Li, Hong Zhai, Qing Xia, Lihong Fan.
      Immune checkpoint inhibitors, especially those targeting CD274/PD-L1yield powerful clinical therapeutic efficacy. Thoughmuch progress has been made in the development of antibody-basedCD274 drugs, chemical compounds applied for CD274degradation remain largely unavailable. Herein,baicalein, a monomer of traditional Chinese medicine, isscreened and validated to target CD274 and induces itsmacroautophagic/autophagic degradation. Moreover, we demonstrate thatCD274 directly interacts with MAP1LC3B (microtubule associatedprotein 1 light chain 3 beta). Intriguingly, baicalein potentiatesCD274-LC3 interaction to facilitate autophagic-lysosomal degradationof CD274. Importantly, targeted CD274. degradation via baicaleininhibits tumor development by boosting T-cell-mediated antitumorimmunity. Thus, we elucidate a critical role of autophagy-lysosomalpathway in mediating CD274 degradation, and conceptually demonstratethat the design of a molecular "glue" that tethers the CD274-LC3interaction is an appealing strategy to develop CD274 inhibitors incancer therapy.Abbreviations: ATTECs: autophagy-tethering compounds; AUTACs: AUtophagy-TArgeting Chimeras; AUTOTACs: AUTOphagy-TArgeting Chimeras; AMPK: adenosine 5'-monophosphate (AMP)-activated protein kinase; BiFC: bimolecular fluorescence complementation; BafA1: bafilomycin A1; CD274/PD-L1/B7-H1: CD274 molecule; CQ: chloroquine; CGAS: cyclic GMP-AMP synthase; DAPI: 4'6-diamino-2-phenylindole; FITC: fluorescein isothiocyanate isomer; GFP: green fluorescent protein; GZMB: granzyme B; IHC: immunohistochemistry; ICB: immune checkpoint blockade; KO: knockout; KD: equilibrium dissociation constant; LYTAC: LYsosome-TArgeting Chimera; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MST: microscale thermophoresis; NFAT: nuclear factor of activated T cells; NFKB/NF-kB: nuclear factor kappa B; NSCLC: non-small-cell lung cancer; PDCD1: programmed cell death 1; PROTACs: PROteolysis TArgeting Chimeras; PRF1: perforin 1; PE: phosphatidylethanolamine; PHA: phytohemagglutinin; PMA: phorbol 12-myristate 13-acetate; STAT: signal transducer and activator of transcription; SPR: surface plasmon resonance; TILs: tumor-infiltrating lymphocyte; TME: tumor microenvironment.
    Keywords:  Antitumor immunity; CD274; MAP1LC3B; autophagy; baicalein; protein degradation
    DOI:  https://doi.org/10.1080/15548627.2024.2439657
  27. J Biol Chem. 2024 Dec 24. pii: S0021-9258(24)02632-2. [Epub ahead of print] 108130
    Imaging-Based Quantitative Assessment of Biomolecular Condensates in vitro and in Cells.
    Tessa Bergsma, Anton Steen, Julia L Kamenz, Tegan Otto, Paola Gallardo, Liesbeth M Veenhoff.
      The formation of biomolecular condensates contributes to intracellular compartmentalization, and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including particles formed in vitro in chemically defined buffers or in Xenopus egg extracts, and in cellular systems. Comparing the results with biochemical assays, we conclude that PhaseMetrics reliably detects changes induced by various conditions, including the presence of polyethylene glycol, 1,6-hexanediol, or a salt gradient, as well as the activity of the molecular chaperone DNAJB6b and the protein disaggregase Hsp104. Given the flexibility in its analysis parameters, the pipeline should also be applicable to other condensate-forming systems and we show it application for detecting TDP-43 particles. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single-condensate level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.
    Keywords:  Biomolecular condensates; FG-nucleoporin; aggregation; image-based analysis; intrinsically disordered protein; phase separation
    DOI:  https://doi.org/10.1016/j.jbc.2024.108130
  28. Mol Cell. 2024 Dec 19. pii: S1097-2765(24)00912-2. [Epub ahead of print]84(24): 4929-4944.e8
    TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.
    Bercin K Cenik, Yuki Aoi, Marta Iwanaszko, Benjamin C Howard, Marc A Morgan, Grant D Andersen, Elizabeth T Bartom, Ali Shilatifard.
      Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.
    Keywords:  BRD4; FOS; FUBP3; MYC; chromatin-binding proteins; dCas9; gene regulation; heat shock; proximity labeling
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.007
  29. Trends Cell Biol. 2024 Dec 26. pii: S0962-8924(24)00250-2. [Epub ahead of print]
    The endoplasmic reticulum as a cradle for virus and extracellular vesicle secretion.
    Yonis Bare, Kyra Defourny, Marine Bretou, Guillaume Van Niel, Esther Nolte-'t Hoen, Raphael Gaudin.
      Extracellular vesicles (EVs) are small membranous carriers of protein, lipid, and nucleic acid cargoes and play a key role in intercellular communication. Recent work has revealed the previously under-recognized participation of endoplasmic reticulum (ER)-associated proteins (ERAPs) during EV secretion, using pathways reminiscent of viral replication and secretion. Here, we present highlights of the literature involving ER/ERAPs in EV biogenesis and propose mechanistic parallels with ERAPs exploited during viral infections. We propose that ERAPs play an active role in the release of EVs and viral particles, and we present views on whether viruses hijack or enhance pre-existing ERAP-dependent secretory machineries or whether they repurpose ERAPs to create new secretory pathways.
    Keywords:  exocytosis; membrane contact sites; secretory autophagy; viral infection; virus assembly; virus release
    DOI:  https://doi.org/10.1016/j.tcb.2024.11.008
  30. Cell Rep. 2024 Dec 21. pii: S2211-1247(24)01445-1. [Epub ahead of print]44(1): 115094
    The N-degron pathway mediates the autophagic degradation of cytosolic mitochondrial DNA during sterile innate immune responses.
    Chan Hoon Jung, Yoon Jee Lee, Eun Hye Cho, Gee Eun Lee, Sung Tae Kim, Ki Sa Sung, Daeho Kim, Dong Hyun Kim, Yeon Sung Son, Jin-Hyun Ahn, Dohyun Han, Yong Tae Kwon.
      The human body reacts to tissue damage by generating damage-associated molecular patterns (DAMPs) that activate sterile immune responses. To date, little is known about how DAMPs are removed to avoid excessive immune responses. Here, we show that proteasomal dysfunction induces the release of mitochondrial DNA (mtDNA) as a DAMP that activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon gene (STING) pathway and is subsequently degraded through the N-degron pathway. In the resolution phase of sterile immune responses, DNA-dependent protein kinase (DNA-PK) senses cytosolic mtDNA and activates N-terminal (Nt-) arginylation by ATE1 R-transferases. The substrates of Nt-arginylation include the molecular chaperone BiP/GRP78 retrotranslocated from the endoplasmic reticulum (ER). R-BiP, the Nt-arginylated species of BiP, is associated with cytosolic mtDNA to accelerate its targeting to autophagic membranes for lysosomal degradation. Thus, cytosolic mtDNA activates the N-degron pathway to facilitate its own degradation and form a negative feedback loop, by which the cell can turn off sterile immune responses at the right time.
    Keywords:  ATE1; CP: Immunology; DNA-PK; KU70; R-BiP; autophagy; mitochondrial DNA; proteasomal dysfunction; sterile immune response; the N-degron pathway; type I interferon
    DOI:  https://doi.org/10.1016/j.celrep.2024.115094
  31. Cell Death Differ. 2024 Dec 23.
    Temporal dissection of the roles of Atg4 and ESCRT in autophagosome formation in yeast.
    Hui Li, Jing-Zhen Song, Cheng-Wen He, Meng-Xi Xie, Zheng-Tan Zhang, You Zhou, Xin-Jing Li, Li Cui, Jing Zhu, Qingqiu Gong, Zhiping Xie.
      Autophagosomes are formed by the enlargement and sealing of phagophores. This is accompanied by the recruitment and release of autophagy-related (Atg) proteins that function therein. Presently, the relationship among factors that act after the initial emergence of the phagophore is unclear. The endosomal sorting complexes required for transport (ESCRT) machinery and Atg4 are known to function in phagophore sealing and Atg8 release, respectively. Here we show that biochemically, both Atg4 and ESCRT promoted phagophore sealing. Intriguingly, Atg4-mediated release of Atg8 from the phagophore promoted phagophore sealing even in the absence of ESCRT. This sealing activity could be reconstituted in vitro using cell lysate and purified Atg4. To elucidate the temporal relationship between Atg4 and ESCRT, we charted a timeline of the autophagosome formation cycle based on the trafficking of Atg proteins and mapped the actions of Atg4 and ESCRT to specific stages. The temporal impact of Atg4-mediated release of Atg8 from phagophore was mapped to the stage after the assembly of phagophore assembly site (PAS) scaffold and phosphatidylinositol-3-kinase (PtdIns-3-K) complex; its retardation only extended the duration of Atg8 release stage, leading to delayed phagophore sealing and accumulation of multiple phagophores. The impacts of ESCRT were mapped to two stages. In addition to promoting phagophore sealing, it also dictates whether PtdIns-3-K recruitment can occur by controlling Atg9 trafficking, thereby determining the incidence of autophagosome formation. Accordingly, ESCRT deficiency led to a combination of reduced autophagosome frequency and extended autophagosome formation duration, manifesting as reduced autophagic flux but normal apparent Atg8 puncta number. Our study thus identifies Atg4-mediated Atg8 shedding as a novel membrane scission mechanism and reveals a new early-stage role for ESCRT in autophagy.
    DOI:  https://doi.org/10.1038/s41418-024-01438-8
  32. Am J Physiol Cell Physiol. 2024 Dec 23.
    Identification of Polycystin 2 Missense Mutants Targeted for Endoplasmic Reticulum-Associated Degradation.
    Christopher J Guerriero, Marcelo D Carattino, Katherine G Sharp, Luke J Kantz, Nikolay P Gresko, Michael J Caplan, Jeffrey L Brodsky.
      Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder leading to end-stage renal disease. ADPKD arises from mutations in the PKD1 and PKD2 genes, which encode polycystin 1 (PC1) and polycystin 2 (PC2), respectively. PC2 is a non-selective cation channel, and disease-linked mutations disrupt normal cellular processes, including signaling and fluid secretion. In this study, we investigate whether disease-causing missense mutations compromise PC2 folding, an event which can lead to endoplasmic reticulum-associated degradation (ERAD). To this end, we first developed a new yeast PC2 expression system. We show that the yeast system provides a tractable model to investigate PC2 biogenesis and that a disease-associated PC2 mutant, D511V, exhibits increased polyubiquitination and accelerated proteasome-dependent degradation compared to wild-type PC2. In contrast to wild-type PC2, the PC2 variant also failed to improve the growth of yeast strains that lack endogenous potassium transporters, highlighting a loss of channel function at the cell surface and a new assay for loss-of-function PKD2 variants. In HEK293 cells, both D511V along with another disease-associated mutant, R322Q, were targeted for ERAD. Consistent with defects in protein folding, the surface localization of these PC2 variants was increased by incubation at low-temperature in HEK293 cells, underscoring the potential to pharmacologically rescue these and perhaps other misfolded PC2 alleles. Together, our study supports the hypothesis that select PC2 missense variants are degraded by ERAD, the potential for screening PKD2 alleles in a new genetic system, and that chemical chaperone-based therapeutic interventions might be used to treat ADPKD.
    Keywords:  autosomal dominant polycystic kidney disease (ADPKD); endoplasmic reticulum-associated degradation (ERAD); polycystin 2 (PC2)
    DOI:  https://doi.org/10.1152/ajpcell.00776.2024
  33. J Am Chem Soc. 2024 Dec 22.
    G3BP1/2-Targeting PROTAC Disrupts Stress Granules Dependent ATF4 Migracytosis as Cancer Therapy.
    Ting Dong, Fabao Zhao, Mengmeng Wang, Kaige Lyu, Jiayu Zhu, Wenru Zhang, Wenzhe Li, Yixuan An, Na Liu, Akhand Pratap Singh, Yue Yang, Dongwei Kang, Xiaohui Liu.
      Stress granules (SGs) are membraneless cytoplasmic compartments that form in response to stress stimuli. In these compartments, most translation factors stall, except for activating transcription factor 4 (ATF4), which is preferentially translated to ensure cell survival under stressful conditions. Cancer cells encounter various stress conditions in the tumor microenvironment during tumorigenesis; however, how they exploit the pro-survival effects of ATF4 in SGs remains unclear. G3BP1/2 are central nodes of the SG network, regulating SG dynamics. In this study, we designed two small molecules, #129 and PROTAC (Proteolysis Targeting Chimera) degrader 129 (PT-129), which specifically target the NTF2L domain of G3BP1/2, a crucial hub for protein and RNA interactions. These compounds inhibit the formation of stress granules in stressed cells and disassemble pre-existing stress granules. Furthermore, pharmacological inhibition by PT-129 suppressed fibroblast-mediated cancer cell growth in vitro and reduced tumor growth in vivo. Mechanistically, SG facilitates the delivery of ATF4 from fibroblasts to tumor cells via migracytosis, a primary mediator of fibroblast-associated tumor growth. PT-129-mediated disassembly of stress granules disrupts ATF4 delivery, thereby preventing cancer cell proliferation. These compounds, therefore, represent powerful tools for gaining molecular insights into SGs and hold promise for cancer therapeutic interventions by modulating stress granule dynamics.
    DOI:  https://doi.org/10.1021/jacs.4c11146
  34. J Biol Chem. 2024 Dec 24. pii: S0021-9258(24)02636-X. [Epub ahead of print] 108134
    SETD7 promotes LC3B methylation and degradation in ovarian cancer.
    Ziwei Zhang, Mingyang Li, Yanan Hou, Ting Huang, Bowen Zhang, Qiong Lin, Genbao Shao.
      Microtubule-associated protein 1 light chain 3 (LC3) is a key autophagy-related protein involved in regulating autophagosome formation and autophagy activity. Post-translational modifications of LC3 are necessary to modulate its function. However, LC3 protein methylation and its physiological significance have not yet been elucidated. Here, we show that SET domain containing lysine methyltransferase 7 (SETD7) interacts with LC3B, a common isoform of LC3, and methylates LC3B at lysine 51 (K51). SETD7-mediated methylation of LC3B promotes ubiquitination and degradation of LC3B, resulting in reduced autophagosome formation. Furthermore, SETD7 exerts a tumor-promotive function in ovarian cancer (OC) cells in a K51 methylation-dependent manner. Collectively, our data define a novel modification of LC3B and highlight the oncogenic effect of SETD7 via mediating LC3B methylation and degradation.
    Keywords:  Autophagy; LC3B; Ovarian cancer; Posttranslational modification; SETD7
    DOI:  https://doi.org/10.1016/j.jbc.2024.108134
  35. J Cell Biol. 2025 Feb 03. pii: e202401136. [Epub ahead of print]224(2):
    Neuronal constitutive endolysosomal perforations enable α-synuclein aggregation by internalized PFFs.
    Anwesha Sanyal, Gustavo Scanavachi, Elliott Somerville, Anand Saminathan, Athul Nair, Ricardo F Bango Da Cunha Correia, Beren Aylan, Ewa Sitarska, Athanasios Oikonomou, Nikos S Hatzakis, Tom Kirchhausen.
      Endocytosis, required for the uptake of receptors and their ligands, can also introduce pathological aggregates such as α-synuclein (α-syn) in Parkinson's Disease. We show here the unexpected presence of intrinsically perforated endolysosomes in neurons, suggesting involvement in the genesis of toxic α-syn aggregates induced by internalized preformed fibrils (PFFs). Aggregation of endogenous α-syn in late endosomes and lysosomes of human iPSC-derived neurons (iNs), seeded by internalized α-syn PFFs, caused the death of the iNs but not of the parental iPSCs and non-neuronal cells. Live-cell imaging of iNs showed constitutive perforations in ∼5% of their endolysosomes. These perforations, identified by 3D electron microscopy in iNs and CA1 pyramidal neurons and absent in non-neuronal cells, may facilitate cytosolic access of endogenous α-syn to PFFs in the lumen of endolysosomes, triggering aggregation. Inhibiting the PIKfyve phosphoinositol kinase reduced α-syn aggregation and associated iN death, even with ongoing PFF endolysosomal entry, suggesting that maintaining endolysosomal integrity might afford a therapeutic strategy to counteract synucleinopathies.
    DOI:  https://doi.org/10.1083/jcb.202401136
  36. PLoS Genet. 2024 Dec 26. 20(12): e1011518
    Decreased Hsp90 activity protects against TDP-43 neurotoxicity in a C. elegans model of amyotrophic lateral sclerosis.
    Laura Garcia-Toscano, Heather N Currey, Joshua C Hincks, Jade G Stair, Nicolas J Lehrbach, Nicole F Liachko.
      Neuronal inclusions of hyperphosphorylated TDP-43 are hallmarks of disease for most patients with amyotrophic lateral sclerosis (ALS). Mutations in TARDBP, the gene coding for TDP-43, can cause some cases of familial inherited ALS (fALS), indicating dysfunction of TDP-43 drives disease. Aggregated, phosphorylated TDP-43 may contribute to disease phenotypes; alternatively, TDP-43 aggregation may be a protective cellular response sequestering toxic protein away from the rest of the cell. The heat shock responsive chaperone Hsp90 has been shown to interact with TDP-43 and stabilize its normal conformation; however, it is not known whether this interaction contributes to neurotoxicity in vivo. Using a C. elegans model of fALS mutant TDP-43 proteinopathy, we find that loss of function of HSP-90 protects against TDP-43 neurotoxicity and subsequent neurodegeneration in adult animals. This protection is accompanied by a decrease in both total and phosphorylated TDP-43 protein. We also find that hsp-90 mutation or inhibition upregulates key stress responsive heat shock pathway gene expression, including hsp-70 and hsp-16.1, and we demonstrate that normal levels of hsp-16.1 are required for hsp-90 mutation effects on TDP-43. We also observe that the neuroprotective effect due to HSP-90 dysfunction does not involve direct regulation of proteasome activity in C. elegans. Our data demonstrate for the first time that Hsp90 chaperone activity contributes to adverse outcomes in TDP-43 proteinopathies in vivo using a whole animal model of ALS.
    DOI:  https://doi.org/10.1371/journal.pgen.1011518
  37. iScience. 2024 Dec 20. 27(12): 111457
    Molecular tag for promoting N-glycan maturation in the cargo receptor-mediated secretion pathway.
    Hirokazu Yagi, Rino Yamada, Taiki Saito, Rena Honda, Rio Nakano, Kengo Inutsuka, Seigo Tateo, Hideo Kusano, Kumiko Nishimura, Saeko Yanaka, Takuro Tojima, Akihiko Nakano, Jun-Ichi Furukawa, Maho Yagi-Utsumi, Shungo Adachi, Koichi Kato.
      MCFD2 and ERGIC-53 form a cargo receptor complex that plays a crucial role in transporting specific glycoproteins, including blood coagulation factor VIII, from the endoplasmic reticulum to the Golgi apparatus. We have demonstrated that MCFD2 recognizes a 10-amino-acid sequence in factor VIII, thereby facilitating its efficient transport. Moreover, the secretion of biopharmaceutical recombinant glycoproteins, such as erythropoietin, can be enhanced by tagging them with this sequence, which we have termed the "passport sequence" (PS). Here, we found that the PS promotes the galactosylation and sialylation of N-glycans on glycoproteins. Furthermore, we discovered that glycoproteins tagged with the PS follow a unique route in the Golgi, where they encounter NUCB1. NUCB1 also recognizes the PS and mediates its interaction with the galactosylation enzyme B4GALT1. These findings offer a promising strategy for controlling the glycosylation of recombinant glycoproteins of biopharmaceutical interest.
    Keywords:  Biochemistry; Glycobiology; Molecular biology; Properties of biomolecules; Protein
    DOI:  https://doi.org/10.1016/j.isci.2024.111457
  38. Protein Sci. 2025 Jan;34(1): e70016
    Allosteric regulation of the tyrosine phosphatase PTP1B by a protein-protein interaction.
    Cassandra A Chartier, Virgil A Woods, Yunyao Xu, Anne E van Vlimmeren, Andrew C Johns, Marko Jovanovic, Ann E McDermott, Daniel A Keedy, Neel H Shah.
      The rapid identification of protein-protein interactions has been significantly enabled by mass spectrometry (MS) proteomics-based methods, including affinity purification-MS, crosslinking-MS, and proximity-labeling proteomics. While these methods can reveal networks of interacting proteins, they cannot reveal how specific protein-protein interactions alter protein function or cell signaling. For instance, when two proteins interact, there can be emergent signaling processes driven purely by the individual activities of those proteins being co-localized. Alternatively, protein-protein interactions can allosterically regulate function, enhancing or suppressing activity in response to binding. In this work, we investigate the interaction between the tyrosine phosphatase PTP1B and the adaptor protein Grb2, which have been annotated as binding partners in a number of proteomics studies. This interaction has been postulated to co-localize PTP1B with its substrate IRS-1 by forming a ternary complex, thereby enhancing the dephosphorylation of IRS-1 to suppress insulin signaling. Here, we report that Grb2 binding to PTP1B also allosterically enhances PTP1B catalytic activity. We show that this interaction is dependent on the proline-rich region of PTP1B, which interacts with the C-terminal SH3 domain of Grb2. Using NMR spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) we show that Grb2 binding alters PTP1B structure and/or dynamics. Finally, we use MS proteomics to identify other interactors of the PTP1B proline-rich region that may also regulate PTP1B function similarly to Grb2. This work presents one of the first examples of a protein allosterically regulating the enzymatic activity of PTP1B and lays the foundation for discovering new mechanisms of PTP1B regulation in cell signaling.
    Keywords:  Grb2; PTP1B; SH3 domain; allostery; mass spectrometry; protein tyrosine phosphatase; protein–protein interaction
    DOI:  https://doi.org/10.1002/pro.70016
  39. Am J Hum Genet. 2024 Dec 24. pii: S0002-9297(24)00418-X. [Epub ahead of print]
    Heterozygous UBR5 variants result in a neurodevelopmental syndrome with developmental delay, autism, and intellectual disability.
    Pascale Sabeh, Samantha A Dumas, Claudia Maios, Hiba Daghar, Marek Korzeniowski, Justine Rousseau, Matthew Lines, Andrea Guerin, John J Millichap, Megan Landsverk, Theresa Grebe, Kristin Lindstrom, Jonathan Strober, Tarik Ait Mouhoub, Christiane Zweier, Michelle Steinraths, Moritz Hebebrand, Bert Callewaert, Rami Abou Jamra, Monika Kautza-Lucht, Meret Wegler, Paul Kruszka, Candy Kumps, Ehud Banne, Marta Biderman Waberski, Anne Dieux, Sarah Raible, Ian Krantz, Livija Medne, Kieran Pechter, Laurent Villard, Renzo Guerrini, Claudia Bianchini, Carmen Barba, Davide Mei, Xavier Blanc, Christine Kallay, Emmanuelle Ranza, Xiao-Ru Yang, Emily O'Heir, Kirsten A Donald, Serini Murugasen, Zandre Bruwer, Muge Calikoglu, Jennifer M Mathews, Marion Lesieur-Sebellin, Geneviève Baujat, Nicolas Derive, Tyler Mark Pierson, Jill R Murrell, Amelle Shillington, Clothilde Ormieres, Sophie Rondeau, André Reis, Alberto Fernandez-Jaen, Ping Yee Billie Au, David A Sweetser, Lauren C Briere, Nathalie Couque, Laurence Perrin, Jennifer Schymick, Paul Gueguen, Mathilde Lefebvre, Michael Van Andel, Jane Juusola, Stylianos E Antonarakis, J Alex Parker, Barrington G Burnett, Philippe M Campeau.
      E3 ubiquitin ligases have been linked to developmental diseases including autism, Angelman syndrome (UBE3A), and Johanson-Blizzard syndrome (JBS) (UBR1). Here, we report variants in the E3 ligase UBR5 in 29 individuals presenting with a neurodevelopmental syndrome that includes developmental delay, autism, intellectual disability, epilepsy, movement disorders, and/or genital anomalies. Their phenotype is distinct from JBS due to the absence of exocrine pancreatic insufficiency and the presence of autism, epilepsy, and, in some probands, a movement disorder. E3 ubiquitin ligases are responsible for transferring ubiquitin to substrate proteins to regulate a variety of cellular functions, including protein degradation, protein-protein interactions, and protein localization. Knocking out ubr-5 in C. elegans resulted in a lower movement score compared to the wild type, supporting a role for UBR5 in neurodevelopment. Using an in vitro autoubiquitination assay and confocal microscopy for the human protein, we found decreased ubiquitination activity and altered cellular localization in several variants found in our cohort compared to the wild type. In conclusion, we found that variants in UBR5 cause a neurodevelopmental syndrome that can be associated with a movement disorder, reinforcing the role of the UBR protein family in a neurodevelopmental disease that differs from previously described ubiquitin-ligase-related syndromes. We also provide evidence for the pathogenic potential loss of UBR5 function with functional experiments in C. elegans and in vitro ubiquitination assays.
    Keywords:  Mendelian phenotype; UBR5; autism; autosomal dominant; developmental disease; epilepsy; intellectual disability; movement disorders; ubiquitin
    DOI:  https://doi.org/10.1016/j.ajhg.2024.11.009
  40. Nat Commun. 2024 Dec 23. 15(1): 10719
    Preventing excessive autophagy protects from the pathology of mtDNA mutations in Drosophila melanogaster.
    Najla El Fissi, Florian A Rosenberger, Kai Chang, Alissa Wilhalm, Tom Barton-Owen, Fynn M Hansen, Zoe Golder, David Alsina, Anna Wedell, Matthias Mann, Patrick F Chinnery, Christoph Freyer, Anna Wredenberg.
      Aberration of mitochondrial function is a shared feature of many human pathologies, characterised by changes in metabolic flux, cellular energetics, morphology, composition, and dynamics of the mitochondrial network. While some of these changes serve as compensatory mechanisms to maintain cellular homeostasis, their chronic activation can permanently affect cellular metabolism and signalling, ultimately impairing cell function. Here, we use a Drosophila melanogaster model expressing a proofreading-deficient mtDNA polymerase (POLγexo-) in a genetic screen to find genes that mitigate the harmful accumulation of mtDNA mutations. We identify critical pathways associated with nutrient sensing, insulin signalling, mitochondrial protein import, and autophagy that can rescue the lethal phenotype of the POLγexo- flies. Rescued flies, hemizygous for dilp1, atg2, tim14 or melted, normalise their autophagic flux and proteasome function and adapt their metabolism. Mutation frequencies remain high with the exception of melted-rescued flies, suggesting that melted may act early in development. Treating POLγexo- larvae with the autophagy activator rapamycin aggravates their lethal phenotype, highlighting that excessive autophagy can significantly contribute to the pathophysiology of mitochondrial diseases. Moreover, we show that the nucleation process of autophagy is a critical target for intervention.
    DOI:  https://doi.org/10.1038/s41467-024-55559-2
  41. Structure. 2024 Dec 13. pii: S0969-2126(24)00530-6. [Epub ahead of print]
    Systematic characterization of indel variants using a yeast-based protein folding sensor.
    Sven Larsen-Ledet, Søren Lindemose, Aleksandra Panfilova, Sarah Gersing, Caroline H Suhr, Aitana Victoria Genzor, Heleen Lanters, Sofie V Nielsen, Kresten Lindorff-Larsen, Jakob R Winther, Amelie Stein, Rasmus Hartmann-Petersen.
      Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.
    Keywords:  AlphaFold2; DMS; MAVE; Rosetta; deep mutational scanning; multiplexed assays of variant effects; predictions; protein folding; protein stability; proteostasis
    DOI:  https://doi.org/10.1016/j.str.2024.11.017
  42. Eur J Med Chem. 2024 Dec 16. pii: S0223-5234(24)01051-1. [Epub ahead of print]283 117169
    Discovery and biological evaluation of potent 2-trifluoromethyl acrylamide warhead-containing inhibitors of protein disulfide isomerase.
    Jung-Chun Chu, Keng-Chang Tsai, Ting-Yu Wang, Tzu-Yin Chen, Ju-Ying Tsai, Tien Lee, Mei-Hsiang Lin, Yves S Y Hsieh, Chin-Chung Wu, Wei-Jan Huang.
      Protein disulfide isomerase (PDI) regulates multiple protein functions by catalyzing the oxidation, reduction, and isomerization of disulfide bonds. The enzyme is considered a potential target for treating thrombosis. We previously developed a potent PDI inhibitor, CPD, which contains the propiolamide as a warhead targeting cysteine residue in PDI. To address its issues with undesirable off-target effects and weak metabolic stability, we replaced the propiolamide group with various electrophiles. Among these, compound 2d, which contains 2-trifluoromethyl acrylamide exhibited potent PDI inhibition compared to the reference PACMA31. Further structural optimization of compound 2d led to a novel series of 2-trifluoromethyl acrylamide derivatives. Several of these compounds displayed substantially improved enzyme inhibition. Notably, compound 14d demonstrated the strongest inhibition against PDI, with an IC50 value of 0.48 ± 0.004 μM. Additionally, compound 14d was found to exhibit a reversible binding mode with PDI enzyme. Further biological evaluations show that 14d suppressed platelet aggregation and thrombus formation by attenuating GPIIb/IIIa activation without significantly causing cytotoxicity. Altogether, these findings suggest PDI inhibitors could be a potential strategy for anti-thrombosis.
    Keywords:  2-Trifluoromethyl acrylamide; Protein disulfide isomerases; Thrombosis; Warhead
    DOI:  https://doi.org/10.1016/j.ejmech.2024.117169
  43. J Med Chem. 2024 Dec 27.
    Development and Discovery of a Selective Degrader of Casein Kinases 1 δ/ε.
    Adrian Haag, Václav Němec, Pavlína Janovská, Jana Bartošíková, Bikash Adhikari, Juliane Müller, Martin P Schwalm, Štěpán Čada, Uli Ohmayer, Henrik Daub, Yeojin Kim, Florian Born, Elmar Wolf, Vítězslav Bryja, Stefan Knapp.
      Members of the casein kinase 1 (CK1) family have emerged as key regulators of cellular signaling and as potential drug targets. Functional annotation of the 7 human isoforms would benefit from isoform-selective inhibitors, allowing studies on the role of these enzymes in normal physiology and disease pathogenesis. However, due to significant sequence homology within the catalytic domain, isoform selectivity is difficult to achieve with conventional small molecules. Here, we used a PROTAC (Proteolysis TArgeting Chimeras) approach to develop a highly selective degrader AH078 (37) targeting CK1δ and CK1ε with excellent selectivity over the highly related CK1α isoform. The developed PROTAC, AH078 (37) selectively degraded CK1δ and CK1ε with a DC50 of 200 nM. Characterization of AH078 (37) revealed a VHL and Ubiquitin-dependent degradation mechanism. Thus, AH078 (37) represents a versatile chemical tool to study CK1δ and CK1ε function in cellular systems.
    DOI:  https://doi.org/10.1021/acs.jmedchem.4c02201
  44. Cancer Lett. 2024 Dec 21. pii: S0304-3835(24)00804-8. [Epub ahead of print] 217409
    Targeting UBE2T suppresses breast cancer stemness through CBX6-mediated transcriptional repression of SOX2 and NANOG.
    Keshen Wang, Qichen He, Xiangyan Jiang, Tao Wang, Zhigang Li, Huiguo Qing, Yuman Dong, Yong Ma, Bin Zhao, Junchang Zhang, Haonan Sun, Zongrui Xing, Yuxia Wu, Wenbo Liu, Junhong Guan, Ailin Song, Yan Wang, Peng Zhao, Long Qin, Wengui Shi, Zeyuan Yu, Huinian Zhou, Zuoyi Jiao.
      Breast cancer stem cells (BCSCs) are the main cause of breast cancer recurrence and metastasis. While the ubiquitin-proteasome system contributes to the regulation of BCSC stemness, the underlying mechanisms remain unclear. Here, we identified ubiquitin-conjugating enzyme E2T (UBE2T) as a pivotal ubiquitin enzyme regulating BCSC stemness through systemic screening assays, including single-cell RNA sequencing (scRNA-seq) and stemness-index analysis. We found that patients with high UBE2T expression exhibited worse prognosis than those with low expression (10-year PFS: 55.95% vs. 85.08%), which are consistent across various subtypes of breast cancers. Genetic ablation of UBE2T suppresses BCSC stemness and tumor progression in organoids and spontaneous MMTV-PyMT mice, dependent on the transcriptional inactivation of pluripotency genes SOX2 and NANOG. Mechanically, UBE2T collaborates with the E3 ligase TRIM25 to perform K48-linked polyubiquitination and degradation of CBX6 at K214, which deficiency helps to promote the transcription of SOX2 and NANOG and enhances BCSC stemness. The pharmacological inhibitor of UBE2T significantly reduced the expression of NANOG and SOX2, suppressed tumor progression, and demonstrated synergistic effects when combined with chemotherapeutics, but not with other treatments. Collectively, our study revealed that the UBE2T-TRIM25-CBX6 axis can regulate BCSC stemness and offers a potentially therapeutic strategy to combat breast cancer in a clinical translation setting.
    Keywords:  Breast cancer; CBX6; UBE2T; stemness; ubiquitination
    DOI:  https://doi.org/10.1016/j.canlet.2024.217409
  45. J Clin Invest. 2024 Dec 24. pii: e174249. [Epub ahead of print]
    Targeted degradation of oncogenic KRASG12V triggers antitumor immunity in lung cancer models.
    Dezhi Li, Ke Geng, Yuan Hao, Jiajia Gu, Saurav Kumar, Annabel T Olson, Christina C Kuismi, Hye Mi Kim, Yuanwang Pan, Fiona Sherman, Asia M Williams, Yiting Li, Fei Li, Ting Chen, Cassandra Thakurdin, Michela Ranieri, Mary Meynardie, Daniel S Levin, Janaye Stephens, Alison Chafitz, Joy Chen, Mia S Donald-Paladino, Jaylen M Powell, Ze-Yan Zhang, Wei Chen, Magdalena Ploszaj, Han Han, Shengqing Gu, Tinghu Zhang, Baoli Hu, Benjamin A Nacev, Medard Ernest Kaiza, Alice H Berger, Xuerui Wang, Jing Li, Xuejiao Sun, Yang Liu, Xiaoyang Zhang, Tullia C Bruno, Nathanael S Gray, Behnam Nabet, Kwok-Kin Wong, Hua Zhang.
      KRAS is the most frequently mutated oncogene in lung adenocarcinoma, with G12C and G12V being the most predominant forms. Recent breakthroughs in KRASG12C inhibitors have transformed the clinical management of patients with G12C mutation and advanced our understanding of its function. However, little is known about the targeted disruption of KRASG12V, partly due to a lack of specific inhibitors. Here, we leverage the degradation tag (dTAG) system to develop a KRASG12V transgenic mouse model. We explore the therapeutic potential of KRASG12V degradation and characterize its impact on the tumor microenvironment (TME). Our study reveals that degrading KRASG12V abolishes lung and pancreatic tumors in mice and causes a robust inhibition of KRAS-regulated cancer intrinsic signaling. Importantly, targeted degradation of KRASG12V reprograms the TME towards a stimulatory milieu and drives antitumor immunity, elicited mainly by effector and cytotoxic CD8+ T cells. Our work provides important insights into the impact of degrading KRASG12V on both tumor progression and immune response, highlighting degraders as a powerful strategy for targeting KRAS mutant cancers.
    Keywords:  Immunology; Lung cancer; Oncology
    DOI:  https://doi.org/10.1172/JCI174249
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