bims-proteo Biomed News
on Proteostasis
Issue of 2025–08–31
fifty papers selected by
Eric Chevet, INSERM
  1. Mechanisms of transmembrane domain recognition during endoplasmic reticulum quality control.
  2. LYMTACs:chimeric small molecules repurpose lysosomal membrane proteins for target protein relocalization and degradation.
  3. UPR deficiency leads to poor growth, aneuploidy, and a trade-off between ER and general proteostasis in yeast.
  4. Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis.
  5. A Novel Small-Molecule GRP94 Modulator Increases PCSK9 Secretion and Promotes LDLR Degradation.
  6. PARP7 is a proteotoxic stress sensor that labels proteins for degradation.
  7. Highly specific intracellular ubiquitination of a small molecule.
  8. NAC couples protein synthesis with nascent polypeptide myristoylation on the ribosome.
  9. Exploring nondegrading molecular glues for protein-protein interactions.
  10. Opportunities in proximity modulation: Bridging academia and industry.
  11. LYVAC/PDZD8 is a lysosomal vacuolator.
  12. Unbiased mapping of cereblon neosubstrate landscape by high-throughput proteomics.
  13. A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy.
  14. YTHDF proteins and m6A-RNA clients undergo autophagic turnover during contact inhibition.
  15. Expanding the druggable zinc-finger proteome defines properties of drug-induced degradation.
  16. CHIP protects lysosomes from CLN4 mutant-induced membrane damage.
  17. LC3 and GABARAP independent autophagy of misfolded procollagen in mouse osteoblasts.
  18. Deacetylation of ATG16L1 is required for LC3-associated lysosomal microautophagy.
  19. Episodic transport of protein aggregates achieves a positive size selectivity in aggresome formation.
  20. Putative PINK1/Parkin activators lower the threshold for mitophagy by sensitizing cells to mitochondrial stress.
  21. Identification of organelle-specific autophagy regulators from tandem CRISPR screens.
  22. Essential role of protein kinase R in the pathogenesis of pulmonary veno-occlusive disease.
  23. DHX8 regulates degradation of RNA by RNautophagy.
  24. Discovering Uncharted Binding Pockets on E3 Ligases Leads to the Identification of FBW7 Allosteric Modulators.
  25. MYC deregulation sensitizes cancer cells to N-myristoyltransferase inhibition.
  26. In Vivo Tumor Growth Control by General Control Nonderepressible 2 Targeting Agents Results from Kinase Activation.
  27. A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia.
  28. The reticulon protein, TtRET1, is required for the initiation of mating in Tetrahymena thermophila.
  29. Design of multi-target peptide modulators for protein chaperone networks.
  30. Unfolded protein response transcription factor XBP1 suppresses necroptosis-induced colitis by reinforcing the mucus barrier.
  31. A repurposed AMP binding domain reveals mitochondrial protein AMPylation as a regulator of cellular metabolism.
  32. Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol.
  33. One-shot design of functional protein binders with BindCraft.
  34. Mutational analysis of Yih1 and IMPACT reveals amino acids required for Gcn2 inhibition.
  35. Dual Nature of Mitochondrial Integrated Stress Response: Molecular Switches from Protection to Pathology.
  36. The integrated stress response promotes macrophage inflammation and migration in autoimmune diabetes.
  37. RABGAP1 is a sensor that facilitates the sorting and processing of amyloid precursor protein.
  38. The functional dynamics of FicD's TPR domain are modulated by the interaction with ATP and BiP.
  39. HerTACs Enable Tumor-Selective Lysosomal Degradation of Membrane and Extracellular Proteins via HER2 Trafficking.
  40. Endocytosis is essential for cysteine-deprivation-induced ferroptosis.
  41. 4D structural biology-quantitative dynamics in the eukaryotic RNA exosome complex.
  42. Non-Canonical, Strongly Selective Protein Disulfide Isomerases as Anticancer Therapeutic Targets.
  43. RTN4IP1 is required for the final stages of mitochondrial complex I assembly and CoQ biosynthesis.
  44. Coordinated protein modules define DNA damage responses to carboplatin at single-cell resolution in human ovarian carcinoma models.
  45. A Functional Assay for Mining Noninhibitory Enzyme Ligands from One Bead One Compound Libraries: Application to E3 Ubiquitin Ligases.
  46. Structural basis for the recognition and ubiquitylation of type-2 N-degron substrate by PRT1 plant N-recognin.
  47. Lysosomal polyamine storage upon ATP13A2 loss impairs β-glucocerebrosidase via altered lysosomal pH and electrostatic hydrolase-lipid interactions.
  48. Cancer-induced nerve injury promotes resistance to anti-PD-1 therapy.
  49. Nascent liver proteome reveals enzymes and transcription regulators under physiological and alcohol exposure conditions.
  50. HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity.
  1. Curr Opin Cell Biol. 2025 Aug 22. pii: S0955-0674(25)00118-8. [Epub ahead of print]96 102580
    Mechanisms of transmembrane domain recognition during endoplasmic reticulum quality control.
    Nikita Sergejevs, Pedro Carvalho.
      Misfolded proteins can be toxic to cells, and their accumulation is a hallmark of diseases such as neurodegeneration. Normally, protein homeostasis is maintained by quality control processes that eliminate misfolded proteins. In the endoplasmic reticulum (ER), misfolded proteins are eliminated through endoplasmic reticulum-associated degradation (ERAD). This process is mediated by ubiquitin ligase complexes that recognize substrates in the membrane and lumen of the ER and retrotranslocate them to the cytosol to mediate their ubiquitination for subsequent degradation by the proteasome. While the recognition of luminal substrates is well understood, how ERAD complexes specifically identify and select aberrant membrane proteins remains poorly defined. Here, we review examples of intramembrane substrate recognition during ERAD and discuss the principles involved.
    DOI:  https://doi.org/10.1016/j.ceb.2025.102580
  2. Nat Commun. 2025 Aug 21. 16(1): 7812
    LYMTACs:chimeric small molecules repurpose lysosomal membrane proteins for target protein relocalization and degradation.
    Dhanusha A Nalawansha, Georgios Mazis, Gitte Husemoen, Kate S Ashton, Weixian Deng, Ryan P Wurz, Anh T Tran, Brian A Lanman, Jiansong Xie, Robert G Guenette, Shiqian Li, Christopher E Smith, Suresh Archunan, Manoj K Agnihotram, Arghya Sadhukhan, Rajiv Kapoor, Chris Wilde, Sajjan Koirala, Felipe De Sousa E Melo, Patrick Ryan Potts.
      Proximity-inducing modalities that co-opt cellular pathways offer new opportunities to regulate oncogenic drivers. Inspired by the success of proximity-based chimeras in both intracellular and extracellular target space, here we describe the development of LYsosome Membrane TArgeting Chimeras (LYMTACs) as a small molecule-based platform that functions intracellularly to modulate the membrane proteome. Conceptually, LYMTACs are heterobifunctional small molecules that co-opt short-lived lysosomal membrane proteins (LMPs) as effectors to deliver targets for lysosomal degradation. We demonstrate that a promiscuous kinase inhibitor-based LYMTAC selectively targets membrane proteins for lysosomal degradation via RNF152, a short-lived LMP. We extend this concept by showing that oncogenic KRASG12D signaling can be potently inhibited by LYMTACs. Mechanistically, LYMTACs display multi-pharmacology and exert their activity through both target relocalization into the lysosome and degradation. We further generalize LYMTACs across various LMPs and thus offer a platform to access challenging membrane proteins through targeted protein relocalization and degradation.
    DOI:  https://doi.org/10.1038/s41467-025-63128-4
  3. Genes Dev. 2025 Aug 22.
    UPR deficiency leads to poor growth, aneuploidy, and a trade-off between ER and general proteostasis in yeast.
    Constantine Bartolutti, Allison J Kim, Yanzhe Ma, Thiago P Fernandes, Charles Boone, Marko Jovanovic, Gloria A Brar.
      The unfolded protein response (UPR) was discovered in budding yeast as a mechanism that allows cells to adapt to endoplasmic reticulum (ER) stressors. Although the UPR is not thought to be necessary for cellular fitness of wild-type cells in the absence of stress, we found that UPR deficiency led to poor growth in cycling mitotic yeast cells. This led to pervasive adaptive aneuploidy of specific chromosomes that was seen in divergent strain backgrounds, indicating an important basal role for this pathway that was missed by studies of the most common laboratory-derived strains. Aneuploid UPR-deficient cells grew better than euploid UPR-deficient cells but exhibited heightened general proteostatic stress, a hallmark of aneuploidy in wild-type cells. Modulation of key genes involved in ER proteostasis that were encoded on aneuploid chromosomes could phenocopy the effects of aneuploidy, indicating that the reason UPR-deficient cells become aneuploid is to counteract protein folding stress in the ER. Proteomic analyses indicate that expression of a small subset of stress-induced UPR targets is supported by basal UPR activity, including the chaperone Kar2/BiP. Together, our results reveal an unexpected role for the UPR in baseline ER folding that is important enough to safeguard cellular fitness that cells tolerate the substantial proteostatic costs that result from aneuploidy to counteract its loss.
    Keywords:  UPR; aneuploidy; yeast
    DOI:  https://doi.org/10.1101/gad.352490.124
  4. Nat Commun. 2025 Aug 25. 16(1): 7929
    Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis.
    Susovan Sarkar, Hao-Yang Liu, Feng Yuan, Brandon T Malady, Liping Wang, Eileen M Lafer, Jessica Perez, Jon M Huibregtse, Jeanne C Stachowiak.
      Clathrin-mediated endocytosis internalizes proteins and lipids from the cell surface. A flexible condensate of initiator proteins catalyzes assembly of clathrin-coated vesicles in diverse organisms. Here we reveal that an endocytic adaptor protein, Epsin1, conditionally stabilizes this network, creating a cargo-dependent endocytic checkpoint. Epsin1 recruits ubiquitylated cargo to endocytic sites. Using in vitro assays, we demonstrate that Epsin1 destabilizes condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, stabilizing condensation. Similarly, in mammalian cells, endocytosis is disrupted by removal of either ubiquitin or Epsin1. When both components are removed simultaneously, endocytic defects are largely rescued, although the ability to preferentially internalize ubiquitylated cargo is lost. These results suggest that Epsin1 tunes protein condensation to internalize ubiquitylated cargo. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions can exert dynamic control over cellular events.
    DOI:  https://doi.org/10.1038/s41467-025-63238-z
  5. Life (Basel). 2025 Aug 20. pii: 1321. [Epub ahead of print]15(8):
    A Novel Small-Molecule GRP94 Modulator Increases PCSK9 Secretion and Promotes LDLR Degradation.
    Wenjing Yan, Yongwang Zhong, Shengyun Fang.
      The endoplasmic reticulum (ER) maintains protein homeostasis through chaperone-mediated folding and ER-associated degradation (ERAD). Disruption of this quality control, particularly involving the ER chaperone GRP94, contributes to diseases such as hypercholesterolemia, cancer, and immune disorders, where defective GRP94-dependent folding and the trafficking of client proteins like PCSK9, integrins, and Toll-like receptors drive pathology. Here, we characterize NSC637153 (cp153), a small molecule identified in a drGFP-based ERAD dislocation screen, as a selective probe of GRP94-dependent processes. cp153 inhibits the dislocation of ERAD substrates, preferentially affecting luminal clients, increases PCSK9 secretion, and promotes LDLR degradation. Unlike ATP-competitive HSP90 inhibitors, cp153 does not induce HSP70 or destabilize AKT, suggesting that it perturbs GRP94 function by interfering with client interaction or folding. The identification of cp153 provides a useful tool to for probing GRP94's role in protein folding, trafficking, ER quality control, and disease-relevant signaling pathways, and supports the development of client-selective GRP94-targeted therapies.
    Keywords:  ER-associated degradation (ERAD); GRP94; PCSK9-LDLR; protein quality control
    DOI:  https://doi.org/10.3390/life15081321
  6. EMBO J. 2025 Aug 20.
    PARP7 is a proteotoxic stress sensor that labels proteins for degradation.
    Nonso J Ikenga, Jörg Vervoorts, Bernhard Lüscher, Roko Žaja, Karla L H Feijs-Žaja.
      ADP-ribosylation is a post-translational modification that plays a critical role in cellular stress responses. We have observed that during proteotoxic stress, cellular ADP-ribosylation increases, with ADP-ribosylated proteins accumulating in cytoplasmic foci containing ubiquitin and p62. During prolonged stress, these ADP-ribosylated proteins are transported to aggresomes and subsequently degraded via autophagy. In the absence of ubiquitination, ADP-ribosylated proteins become more prevalent and less soluble, indicating that ubiquitination is indispensable for this process. Upon inhibition of PARP7, accumulation of mono(ADP-ribosyl)ated proteins in response to proteotoxic stress is impeded. PARP7 turnover is very high under normal conditions; however, the protein becomes stabilised following proteotoxic stress and thereby forms an ideal proteotoxic stress sensor. Our findings imply that, contrary to the current paradigm, not all ADP-ribosylation may occur on specific sites to regulate specific protein characteristics. Instead, it may be rather promiscuous to enable efficient protein degradation or segregation to prevent irreversible damage caused by defective proteins.
    Keywords:  ADP-ribosylation; Macrodomain; PARP; Protein Degradation; Ubiquitination
    DOI:  https://doi.org/10.1038/s44318-025-00545-7
  7. Nat Chem Biol. 2025 Aug 21.
    Highly specific intracellular ubiquitination of a small molecule.
    Weicheng Li, Enrique M Garcia-Rivera, Dylan C Mitchell, Joel M Chick, Micah Maetani, Rohit Bhadoria, John M Knapp, Ryosuke Misu, Geoffrey M Matthews, Ryosuke Shirasaki, Ricardo de Matos Simoes, Vasanthi Viswanathan, John L Pulice, Matthew G Rees, Jennifer A Roth, Steven P Gygi, Constantine S Mitsiades, Cigall Kadoch, Stuart L Schreiber, Jonathan M L Ostrem.
      Ubiquitin is a small, highly conserved protein that acts as a posttranslational modification in eukaryotes. Ubiquitination of proteins frequently serves as a degradation signal, marking them for disposal by the proteasome. Here we report a novel small molecule from a diversity-oriented synthesis library, BRD1732, that is directly ubiquitinated in cells, resulting in dramatic accumulation of inactive ubiquitin monomers and polyubiquitin chains, which causes broad inhibition of the ubiquitin-proteasome system. Ubiquitination of BRD1732 and its associated cytotoxicity are stereospecific and dependent on two homologous E3 ubiquitin ligases, RNF19A and RNF19B, and their shared E2 conjugating enzyme, UBE2L3. Our finding opens the possibility for indirect ubiquitination of a target through a ubiquitinated bifunctional small molecule and more broadly raises the potential for posttranslational modification in trans.
    DOI:  https://doi.org/10.1038/s41589-025-02011-1
  8. EMBO J. 2025 Aug 26.
    NAC couples protein synthesis with nascent polypeptide myristoylation on the ribosome.
    Sara Zdancewicz, Emir Maldosevic, Kinga Malezyna, Ahmad Jomaa.
      N-glycine myristoylation allows for reversible association of newly synthesized proteins with membranes to regulate essential functions such as cellular signaling and stress responses. This process can be catalyzed during protein synthesis by N-myristoyltransferases (NMTs), and its dysregulation has been implicated both in cancer and heart disease. Although the nascent polypeptide-associated complex (NAC) orchestrates the binding of several co-translational processing factors on ribosomes, its role in facilitating nascent protein myristoylation by NMT2 remains unclear. Here, we show that NAC mediates binding of NMT2 to translating ribosomes, which together form an extended channel that guides the nascent chain as it emerges from the polypeptide exit tunnel to the catalytic site of NMT2. Furthermore, the ternary ribosome:NMT2:NAC complex is stabilized by a ribosomal RNA clamp that, together with NAC, orients NMT2 on the ribosomal surface for co-translational myristoylation of nascent chains. Our work uncovers the molecular mechanism coupling protein synthesis to nascent protein myristoylation and underscores the role of NAC as a master regulator of protein biogenesis on the ribosome.
    Keywords:  Cryo-EM; N-myristoyltransferases; NAC; NMT2; Nascent Chain; Translating Ribosome
    DOI:  https://doi.org/10.1038/s44318-025-00548-4
  9. Trends Biochem Sci. 2025 Aug 21. pii: S0968-0004(25)00168-9. [Epub ahead of print]
    Exploring nondegrading molecular glues for protein-protein interactions.
    Sitong Yu, Lixin Zhou, Jing Yang, Jian Zhang, Wenchao Lu.
      Protein-protein interactions (PPIs) are central to cellular signaling and represent attractive, yet challenging drug targets. While molecular glue degraders (MGDs) promote target degradation via E3 ligase recruitment, nondegrading molecular glues (MGs) act independently of ubiquitination to stabilize PPIs, enabling modulation of complex assembly, localization, and signaling. In this review, we outline recent progress in nondegrading MGs, highlighting key presenter proteins, such as FKBP12, Cyclophilin A, and 14-3-3, along with emerging case studies beyond these canonical systems. Advances in chemical biology, structural analysis, and computational design are accelerating discovery in this emerging field. Collectively, these insights position nondegrading MGs as a promising therapeutic modality with distinct mechanisms and broad translational potential.
    Keywords:  chemically induced proximity; molecular glues; protein–protein interactions; stabilizers
    DOI:  https://doi.org/10.1016/j.tibs.2025.07.005
  10. Mol Cell. 2025 Aug 21. pii: S1097-2765(25)00619-7. [Epub ahead of print]85(16): 3012-3022
    Opportunities in proximity modulation: Bridging academia and industry.
    Ivan Dikic, Cristina Mayor-Ruiz, Georg E Winter, Kerstin Koch, Alessio Ciulli, Nicolas H Thomä.
      In the past decade, exciting therapeutic strategies to harness the ubiquitin-proteasome system (UPS) for degradation of target proteins have emerged. Proximity-inducing modalities are at the center of these strategies and act by modulating protein-protein interactions. While we are still learning to harvest this approach, it holds tremendous promise for developing treatments for hitherto undruggable proteins. Here, we discuss how academic efforts and academic-industrial collaboration have advanced the development of therapeutic modalities based on the principle of proximity induction. We make a case for forming a global academia-industry alliance to enhance access to training and expertise while accelerating innovation and translation from ground-breaking ideas to proof of concept in the clinic.
    Keywords:  E3 ligases; MGDs; PROTACs; TPD; academic-industrial collaboration; innovation hubs; molecular glue degraders; proximity induction; public-private partnerships; targeted protein degradation; technology transfer
    DOI:  https://doi.org/10.1016/j.molcel.2025.07.018
  11. Science. 2025 Aug 21. 389(6762): eadz0972
    LYVAC/PDZD8 is a lysosomal vacuolator.
    Haoxiang Yang, Jinrui Xun, Yajuan Li, Awishi Mondal, Bo Lv, Simon C Watkins, Lingyan Shi, Jay Xiaojun Tan.
      Lysosomal vacuolation is commonly found in many pathophysiological conditions, but its molecular mechanisms and functions remain largely unknown. Here, we show that the endoplasmic reticulum (ER)-anchored lipid transfer protein PDZ domain-containing 8 (PDZD8), which we propose to be renamed as lysosomal vacuolator (LYVAC), is a general mediator of lysosomal vacuolation. Using human cell lines, we found that diverse lysosomal vacuolation inducers converged on lysosomal osmotic stress, triggering LYVAC recruitment through multivalent interactions. Stress-induced lysosomal lipid signaling contributed to both the recruitment and activation of LYVAC. By directly sensing lysosomal phosphatidylserine and cholesterol, the lipid transfer domain of LYVAC mediated directional ER-to-lysosome lipid movement, leading to osmotic membrane expansion of lysosomes. These findings uncover an essential mechanism for lysosomal vacuolation with broad implications in pathophysiology.
    DOI:  https://doi.org/10.1126/science.adz0972
  12. Nat Commun. 2025 Aug 20. 16(1): 7773
    Unbiased mapping of cereblon neosubstrate landscape by high-throughput proteomics.
    Martin Steger, Gisele Nishiguchi, Qiong Wu, Bjoern Schwalb, Bachuki Shashikadze, Kevin McGowan, Marisa Actis, Anup Aggarwal, Zhe Shi, Jeanine Price, Anand Mayasundari, Lei Yang, Anastasia H Bednarz, Sophie Machata, Tobias Graef, Denis Bartoschek, Vadim Demichev, Uli Ohmayer, Jun Yang, Henrik Daub, Zoran Rankovic.
      Molecular glue degraders (MGDs) are small molecules that co-opt the ubiquitin-proteasome system to induce degradation of target proteins, including those considered undruggable. Their discovery remains challenging due to the lack of rational design strategies and limited throughput of unbiased proteome-wide screening approaches. To address this gap, we develop a high-throughput proteomics platform based on label-free, data-independent acquisition mass spectrometry (DIA-MS), enabling integrated proteomics and ubiquitinomics profiling. Screening a diverse set of 100 cereblon (CRBN)-recruiting ligands on this platform leads to identification of a broad array of novel degraders and neosubstrates. Subsequent hit validation and structure-degradation relationship analyses guided by global proteomics reveal highly selective and potent phenyl glutarimide-based degraders targeting previously uncharacterized neosubstrates such as KDM4B, G3BP2 and VCL; none of which contain the classical CRBN β-hairpin degron. These findings underscore the power of unbiased high-throughput proteomics in MGD drug discovery and reveal a substantially expanded CRBN neosubstrate landscape beyond that defined by classical immunomodulatory imid drugs (IMiDs).
    DOI:  https://doi.org/10.1038/s41467-025-62829-0
  13. Nat Cell Biol. 2025 Aug 27.
    A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy.
    Mario Mauthe, Nicole van de Beek, Muriel Mari, Giel Korsten, Parisa Nobari, Kennith B Castelino, Eduardo P de Mattos, Ibtisam Ouhida, Jesse L Dijkstra, Sabine Schipper-Krom, Laura R de la Ballina, Monja R Mueller, Anne Simonsen, Mark S Hipp, Lukas C Kapitein, Harm H Kampinga, Fulvio Reggiori.
      Perturbations in protein quality control lead to the accumulation of misfolded proteins and protein aggregates, which can compromise health and lifespan. One key mechanism eliminating protein aggregates is aggrephagy, a selective type of autophagy. Here we reveal that fragmentation is required before autophagic clearance of various types of amorphous aggregates. This fragmentation requires both the 19S proteasomal regulatory particle and the DNAJB6-HSP70-HSP110 chaperone module. These two players are also essential for aggregate compaction that leads to the clustering of the selective autophagy receptors, which initiates the autophagic removal of the aggregates. We also found that the same players delay the formation of disease-associated huntingtin inclusions. This study assigns a novel function to the 19S regulatory particle and the DNAJB6-HSP70-HSP110 module, and uncovers that aggrephagy entails a piecemeal process, with relevance for proteinopathies.
    DOI:  https://doi.org/10.1038/s41556-025-01747-1
  14. Cell Rep. 2025 Aug 23. pii: S2211-1247(25)00959-3. [Epub ahead of print]44(9): 116188
    YTHDF proteins and m6A-RNA clients undergo autophagic turnover during contact inhibition.
    Hung Ho-Xuan, Astrid Bruckmann, Lautaro Natali, Cristian Prieto-Garcia, Jan F M Stuke, Lorene Brunello, Alexandre Vicente, Alexander Pfab, Hagen Wesseling, Sara Cano-Franco, Anshu Khatri, Simone Larivera, Pablo Sanz-Martinez, Benjamin de la Cruz-Thea, Anne-Claire Jacomin, Jan Prochazka, Regina Feederle, Volker Dötsch, Radislav Sedlacek, Gerhard Hummer, Stefanie Kaiser, Ivan Dikic, Melina M Musri, Gunter Meister, Alexandra Stolz.
      The YTHDF protein family plays a critical role in cancer development by recognizing and regulating the stability of N6-methyladenosine (m6A)-modified RNA. Here, we reveal an autophagy-dependent mechanism controlling YTHDF protein levels. Using contact inhibition as a cellular model system, we show YTHDF proteins to be rapidly degraded, coinciding with increased autophagy and decreased mTOR activity. Upon pharmacological mTOR inhibition, YTHDF2 is also downregulated via lysosomal degradation. YTHDF2 selectively interacts with the autophagy modifier GABARAP L2 through LC3-interacting region (LIR) motifs in its unstructured N- and C-terminal regions. Autophagic YTHDF2 downregulation results in the co-degradation of its bound m6A-modified RNA clients. While YTHDF depletion induces cell death in contact-inhibition-deficient HCT116 cancer cells, contact-inhibited MRC5 and RPE1 cells remain unaffected. Our findings uncover a regulatory pathway that governs YTHDF protein stability with significant implications for cancer biology and cell fate determination and suggest the existence of an autophagy-mediated degradation pathway for m6A-modified RNA.
    Keywords:  CP: Cell biology; RNA binding; YTHDF; cancer; cellular homeostasis; contact inhibition; lysosomal degradation; m6A; selective autophagy
    DOI:  https://doi.org/10.1016/j.celrep.2025.116188
  15. Mol Cell. 2025 Aug 21. pii: S1097-2765(25)00620-3. [Epub ahead of print]85(16): 3184-3201.e14
    Expanding the druggable zinc-finger proteome defines properties of drug-induced degradation.
    Mikołaj Słabicki, Jiho Park, Radosław P Nowak, Shourya S Roy Burman, Jesse Pellman, Charles Zou, Hlib Razumkov, Jeannie Carreiro, Simran Rastogi, Anna Goldstein, Marek M Nagiec, Katherine A Donovan, Jianwei Che, Moritz Hunkeler, Qixiang Geng, Chi-Lin Hsu, Megha Lakshminarayan, Chelsea Shu, Rebecca L Zon, Zuzanna Kozicka, Paul M C Park, Jonathan M Tsai, Hojong Yoon, Lyn H Jones, Adam S Sperling, Nathanael S Gray, Eric S Fischer, Benjamin L Ebert.
      Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design.
    Keywords:  CRBN; CRL4(CRBN) E3 ligase; ZF; cereblon; degron specificity; flow-based sorting screens; functional genomics; glutarimide analogs; molecular glue; targeted protein degradation; zinc finger proteins
    DOI:  https://doi.org/10.1016/j.molcel.2025.07.019
  16. Nat Cell Biol. 2025 Aug 25.
    CHIP protects lysosomes from CLN4 mutant-induced membrane damage.
    Juhyung Lee, Natalie Chin, Jizhong Zou, Wan Nur Atiqah Binti Mazli, Michal Jarnik, Layla Saidi, Yue Xu, Eutteum Jeong, Jessica Suh, John Replogle, Michael E Ward, Juan S Bonifacino, Wei Zheng, Ling Hao, Yihong Ye.
      Understanding how cells mitigate lysosomal damage is critical for unravelling pathogenic mechanisms of lysosome-related diseases. Here we generate and characterize induced pluripotent stem cell (iPSC)-derived neurons (i3Neuron) bearing ceroid lipofuscinosis neuronal 4 (CLN4)-linked DNAJC5 mutations, which revealed extensive lysosomal abnormality in mutant neurons. In vitro membrane-damaging experiments establish lysosomal damages caused by lysosome-associated CLN4 mutant aggregates, as a critical pathogenic linchpin in CLN4-associated neurodegeneration. Intriguingly, in non-neuronal cells, a ubiquitin-dependent microautophagy mechanism downregulates CLN4 aggregates to counteract CLN4-associated lysotoxicity. Genome-wide CRISPR screens identify the ubiquitin ligase carboxyl terminus of Hsc70-interacting protein (CHIP) as a central microautophagy regulator that confers ubiquitin-dependent lysosome protection. Importantly, CHIP's lysosome protection function is transferrable: ectopic CHIP improves lysosomal function in CLN4 i3Neurons and effectively alleviates lipofuscin accumulation and cell death in a Drosophila CLN4 disease model. Our study establishes CHIP-mediated microautophagy as a key organelle guardian that preserves lysosome integrity, offering new insights into therapeutic development for lysosome-related neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41556-025-01738-2
  17. Autophagy. 2025 Aug 24.
    LC3 and GABARAP independent autophagy of misfolded procollagen in mouse osteoblasts.
    Elena Makareeva, Shakib Omari, Anna M Roberts-Pilgrim, Laura Gorrell, Bella Radant, Muthulakshmi Sellamani, Edward L Mertz, Basma Khoury, Kenneth Kozloff, Sergey Leikin.
      Bone synthesis should depend on autophagy because over 10% of type I procollagen (PC1) - a heterotrimer of COL1A1 and COL1A2 chains and the precursor of the main bone matrix molecule - is misfolded and rerouted from osteoblast endoplasmic reticulum (ER) to lysosomes. However, osteoblast-specific macroautophagy knockouts in mice have produced only mild bone effects. To reconcile these observations, we compared how hypomorphic expression and a conditional knockout (cKO) of Atg5 - encoding a protein required for autophagosome formation - affected Col1a2G610C/+ versus wild-type Col1a2+/+ osteoblasts in vivo and in vitro. The Gly610-to-Cys substitution (G610C) in the triple helical region of the COL1A2/proα2(I) chain increases PC1 misfolding, causing its accumulation in the ER, cell stress, and osteoblast malfunction. Because autophagy reroutes misfolded PC1 from the ER to lysosomes, disruption of PC1 autophagy should significantly increase osteoblast malfunction and bone pathology in Col1a2G610C/+ mice. Nonetheless, the present study revealed only minor effects of the atg5 cKO on osteoblast function and bone formation in the Col1a2G610C/+ mice, like in Col1a2+/+ controls. The cKO did not reduce the autophagy flux of misfolded G610C or wild-type PC1 in primary osteoblast cultures, even though the LC3 and GABARAP lipidation and therefore autophagosome formation were disrupted. Live-cell imaging in atg5 cKO osteoblasts demonstrated that PC1 was efficiently delivered to lysosomes without LC3 via ER exit site (ERES) microautophagy. Taken together, these observations indicate that LC3- and GABARAP-independent ERES microautophagy is the primary pathway of misfolded procollagen degradation in osteoblasts both in culture and in vivo.
    Keywords:  ATG5; ER exit site; ERES microautophagy; G610C mutation; bone; osteogenesis imperfecta
    DOI:  https://doi.org/10.1080/15548627.2025.2551478
  18. Autophagy. 2025 Aug 28. 1-15
    Deacetylation of ATG16L1 is required for LC3-associated lysosomal microautophagy.
    Qian Wang, Wei Wan, Hongtao Zhang, Tianhua Zhou, Han-Ming Shen, Pingtong Huang, Wei Liu.
      Microautophagy is a selective cellular process in which endolysosomes directly engulf cytoplasmic cargo through membrane invagination. The regulatory mechanisms governing microautophagy remain poorly understood. Here, we identified the deacetylation of ATG16L1 as a critical regulator of LC3-associated lysosomal microautophagy. We demonstrate that ATG16L1 acetylation is dynamically controlled by the acetyltransferase KAT2B and the deacetylase HDAC3. Under lysosomal osmotic stress or glucose deprivation, HDAC3-mediated deacetylation of ATG16L1 within its WD40 domain promotes its interaction with V-ATPase, facilitating ATG16L1 recruitment to lysosomal membranes. While dispensable for macroautophagy, this post-translational modification is essential for LC3 lipidation on lysosomes and enables lysosomal recovery, including the restoration of lysosomal size and degradative capacity following stress. Our results reveal a key role for ATG16L1 deacetylation in driving LC3-associated microautophagy to maintain lysosomal homeostasis.
    Keywords:  ATG16L1; Acetylation; LC3 lipidation; LC3-associated microautophagy; V-ATPase; lysosome
    DOI:  https://doi.org/10.1080/15548627.2025.2551669
  19. Nat Commun. 2025 Aug 22. 16(1): 7852
    Episodic transport of protein aggregates achieves a positive size selectivity in aggresome formation.
    Rui Fang, Luolan Bai, Bert M Verheijen, Boyan Li, Kevin Dong, Joao A Paulo, Mengying Zhou, Yi-Chi Chu, Yuyu Song, Michael Y Sherman, Steven Gygi, Christine M Field, Timothy J Mitchison, Ying Lu.
      Eukaryotic cells direct toxic misfolded proteins to various quality control pathways based on their chemical properties and aggregation status. Aggregated proteins are targeted to selective autophagy or specifically sequestered into the "aggresome", a perinuclear inclusion at the microtubule-organizing center (MTOC). However, the mechanism for selective aggresome recruitment remains unclear. To investigate this process, here we reconstitute MTOC-directed aggregate transport in Xenopus laevis egg extract using AgDD, a chemically inducible aggregation system. High-resolution single-particle tracking reveals that dynein-mediated aggregate transport is highly episodic, with average velocity positively correlating with aggregate size. Mechanistic modeling suggests that recurrent formation of the dynein transport complex biases larger aggregates towards active transport, compensating for the slowdown due to viscosity. Both episodic transport and positive size selectivity are conferred by aggresome-specific dynein adapters. Coupling an aggresome adapter to polystyrene beads recapitulates positive size selectivity in transport, while recruiting conventional dynein adapters to protein aggregates perturbs aggresome formation and reverses the size selectivity.
    DOI:  https://doi.org/10.1038/s41467-025-62751-5
  20. Sci Adv. 2025 Aug 29. 11(35): eady0240
    Putative PINK1/Parkin activators lower the threshold for mitophagy by sensitizing cells to mitochondrial stress.
    William M Rosencrans, Ryan W Lee, Logan McGraw, Ian Horsburgh, Ting-Yu Wang, Baiyi Quan, Diana Huynh, Jennifer A Johnston, David C Chan, Tsui-Fen Chou.
      The PINK1/Parkin pathway targets damaged mitochondria for degradation via mitophagy. Genetic evidence implicates impaired mitophagy in Parkinson's disease, making its pharmacological enhancement a promising therapeutic strategy. Here, we characterize two mitophagy activators: a novel Parkin activator, FB231, and the reported PINK1 activator MTK458. Both compounds lower the threshold for mitochondrial toxins to induce PINK1/Parkin-mediated mitophagy. However, global proteomics revealed that FB231 and MTK458 independently induce mild mitochondrial stress, resulting in impaired mitochondrial function and activation of the integrated stress response, effects that result from PINK1/Parkin-independent off-target activities. We find that these compounds impair mitochondria by distinct mechanisms and synergistically decrease mitochondrial function and cell viability in combination with classical mitochondrial toxins. Our findings support a model whereby weak or "silent" mitochondrial toxins potentiate other mitochondrial stressors, enhancing PINK1/Parkin-mediated mitophagy. These insights highlight important considerations for therapeutic strategies targeting mitophagy activation in Parkinson's disease.
    DOI:  https://doi.org/10.1126/sciadv.ady0240
  21. J Cell Biol. 2025 Oct 06. pii: e202405138. [Epub ahead of print]224(10):
    Identification of organelle-specific autophagy regulators from tandem CRISPR screens.
    Truc T Losier, Karyn E King, Maxime W C Rousseaux, Ryan C Russell.
      Autophagy is a conserved degradative process that promotes cellular homeostasis under stress conditions. Under nutrient starvation, autophagy is nonselective, promoting indiscriminate breakdown of cytosolic components. Conversely, selective autophagy is responsible for the specific turnover of damaged organelles. We hypothesized that selective autophagy may be regulated by signaling pathways distinct from those controlling starvation-induced autophagy, thereby promoting organelle turnover. To address this question, we conducted kinome-wide CRISPR screens to identify distinct signaling pathways responsible for the regulation of basal autophagy, starvation-induced autophagy, and two types of selective autophagy, ER-phagy and pexophagy. These parallel screens identified both known and novel autophagy regulators, some common to all conditions and others specific to selective autophagy. More specifically, CDK11A and NME3 were further characterized to be selective ER-phagy regulators. Meanwhile, PAN3 and CDC42BPG were identified as an activator and inhibitor of pexophagy, respectively. Collectively, these datasets provide the first comparative description of the kinase signaling that defines the regulation of selective autophagy and bulk autophagy.
    DOI:  https://doi.org/10.1083/jcb.202405138
  22. JCI Insight. 2025 Aug 21. pii: e193495. [Epub ahead of print]
    Essential role of protein kinase R in the pathogenesis of pulmonary veno-occlusive disease.
    Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Abhilash Barpanda, Joseph Lyons, Asavari S Gowda, Simren P Gupta, Ananyaa Arvind, Prajakta Ghatpande, Arun P Wiita, Brian B Graham, Giorgio Lagna, Akiko Hata.
      Pulmonary veno-occlusive disease (PVOD) is a rare and severe subtype of pulmonary arterial hypertension, characterized by progressive remodeling of small pulmonary arteries and veins with no therapies. Using a mitomycin C (MMC)-induced rat model, we previously demonstrated that protein kinase R (PKR)-mediated integrated stress response (ISR) drives endothelial dysfunction and vascular remodeling. To determine if PKR is the primary mediator of ISR and the pathogenesis, we treated control (Ctrl) and PKR knockout (KO) mice with the same dose of MMC. Consistent with rat data, Ctrl mice displayed ISR activation, vascular remodeling, and pulmonary hypertension after MMC treatment, while KO mice showed none of these phenotypes. Proteomic analysis revealed that MMC-mediated ISR activation attenuates protein synthesis in Ctrl but not in KO mice. These findings underscore the critical role of PKR-dependent ISR activation and subsequent perturbation of proteostasis as central mechanisms driving PVOD pathogenesis and identifying PKR as a promising therapeutic target.
    Keywords:  Cell biology; Cell stress; Vascular biology
    DOI:  https://doi.org/10.1172/jci.insight.193495
  23. Nucleic Acids Res. 2025 Aug 11. pii: gkaf801. [Epub ahead of print]53(15):
    DHX8 regulates degradation of RNA by RNautophagy.
    Ryohei Sakai, Eigo Takeda, Chihana Kabuta, Viorica Raluca Contu, Yuuki Fujiwara, Nobuhiro Fujikake, Tadafumi Hashimoto, Yoshinori Ohsumi, Tomohiro Kabuta.
      RNautophagy is an intracellular degradation pathway in which RNA is directly taken up by lysosomes. The cytoplasmic regions of the lysosomal membrane proteins, LAMP2C and SIDT2, can interact with consecutive guanine sequences in RNA, mediating the uptake of RNA during RNautophagy. RNautophagy has also been implicated in the clearance of expanded CAG-repeat mRNA and RNA foci associated with polyQ disease. However, the mechanisms of RNA uptake during RNautophagy remain unclear. Here, we screened for proteins that bind consecutive guanine sequences and identified RNA helicase DHX8 as a binding partner. DHX8 interacts with SIDT2 and is partially localized to the cytoplasmic side of the lysosomal membrane. We found that DHX8 regulates intracellular RNA degradation via SIDT2-dependent RNautophagy but not via macroautophagy. RNA binding, but not ATPase activity, of DHX8 is likely to be important for regulating RNA degradation. DHX8 also contributes to the clearance of pathogenic CAG repeat mRNA and RNA foci, and the levels of both soluble protein and insoluble high-molecular-weight aggregates of expanded polyQ tracts. Our findings provide insights into the mechanisms underlying the regulation of intracellular RNA degradation, autophagic pathways, and possibly the pathogenesis of repeat RNA-related disorders.
    DOI:  https://doi.org/10.1093/nar/gkaf801
  24. Adv Sci (Weinh). 2025 Aug 20. e06068
    Discovering Uncharted Binding Pockets on E3 Ligases Leads to the Identification of FBW7 Allosteric Modulators.
    Míriam Martínez-Cartró, Álvaro Serrano-Morrás, Andrea Bertran-Mostazo, Roger Castaño-Muñiz, Salvatore Scaffidi, Varbina Ivanova, Noémi Csorba, József Simon, Péter Ábrányi-Balogh, Yunfeng Li, György M Keserü, Bing Hao, Xavier Barril, Carles Galdeano.
      E3 ligases are key regulators of the ubiquitin-proteasome system (UPS) and have emerged as attractive drug target candidates for precise therapeutic intervention. Additionally, their ligands are extremely valuable as handles for Targeted Protein Degradation (TPD). However, only a limited number of E3 ligases have been targeted with small molecules. An efficient approach to identify ligandable surfaces on 22 structurally diverse E3 ligases has been developed, revealing that they offer significant binding opportunities through allosteric pockets. As a proof of concept, an allosteric pocket identified in FBW7 has been targeted, leading to the discovery of the first potent and reversible small-molecule binders of this E3 ligase. Biophysical and structural studies have confirmed the binding site, while functional cell assays have showed that some of these molecules act as allosteric enhancers of c-MYC and c-JUN degradation in an FBW7-dependent manner. These allosteric modulators of E3 ligases represent a novel mechanism of action in the TPD landscape and could be used as PROTAC handles.
    Keywords:  Allosterism; E3 ligases; Ligandability/druggability; drug discovery; targeted protein degradation
    DOI:  https://doi.org/10.1002/advs.202506068
  25. Cell Rep. 2025 Aug 22. pii: S2211-1247(25)00951-9. [Epub ahead of print]44(9): 116180
    MYC deregulation sensitizes cancer cells to N-myristoyltransferase inhibition.
    Gregor A Lueg, James Zhang, Monica Faronato, Andrii Gorelik, Wouter W Kallemeijn, Francesco Falciani, Josephine Walton, Jack W Houghton, Silvia Vannini, Evon Poon, Barbara M Costa, Roberto Solari, Robin Carr, Andrew S Bell, Edward J Bartlett, Bernadette Brzezicha, Martin Janz, Louis Chesler, Dinis P Calado, Edward W Tate.
      Human N-myristoyltransferases (NMTs) catalyze N-terminal protein N-myristoylation and are promising targets in cancer, with an emerging mechanistic rationale for targeted therapy. Here, we screened 245 cancer cell lines against IMP-1320, a potent NMT inhibitor (NMTi), and conducted pathway-level analyses to identify that deregulated MYC increases cancer cell sensitivity to NMTis. Proteomics on detergent-enriched membrane fractions in MYC or MYCN-deregulated cancer cell models revealed that cell death is associated at least in part with loss of membrane association of mitochondrial respiratory complex I. This is concurrent with loss of myristoylation and degradation of the complex I assembly factor NDUFAF4, and induction of mitochondrial dysfunction, driven by MYC or MYCN-deregulation. NMTis eliminated or suppressed MYC- and MYCN-driven tumors in vivo without overt toxicity, suggesting that this constitutive co-translational protein modification can be targeted in MYC-driven cancers.
    Keywords:  CP: Cancer; CP: Molecular biology; Complex I; MYC; MYCN; N-myristoylation; N-myristoyltransferase; NDUFAF4; NMT
    DOI:  https://doi.org/10.1016/j.celrep.2025.116180
  26. Mol Cancer Ther. 2025 Aug 20.
    In Vivo Tumor Growth Control by General Control Nonderepressible 2 Targeting Agents Results from Kinase Activation.
    Feven Tameire, Paulina Wojnarowicz, Crissy Dudgeon, Kathryn T Bieging-Rolett, Sho Fujisawa, Savi Ramurthy, Owen Reilly, Christopher G Thomson, Bradley S Sherborne, Simon J Taylor, Fang He, Pengwei Pan, Baozhong Li, Earl May, Alan C Rigby, Mark J Mulvihill, Nandita Bose, David Surguladze, Eric S Lightcap.
      General Control Nonderepressible 2 (GCN2; EIF2AK4) is a serine-threonine kinase in the integrated stress response (ISR) signaling pathway that initiates adaptive responses during nutrient stress conditions. While pharmacological inhibition of GCN2 under nutrient stress conditions induces apoptosis and inhibits tumor growth, GCN2 inhibition without nutrient stress has been reported to have no effect on tumor growth. By exploring an array of GCN2 inhibitors, we demonstrate that multiple agents in fact activate GCN2 in biochemical and cell-based assays at low concentrations and inhibit GCN2 at higher concentrations. Unexpectedly, it is this activation, and not inhibition, of the GCN2 pathway that is associated with decreased viability in vitro and tumor growth inhibition in vivo across multiple models. Knockdown and knockout experiments show that activation of the ISR by GCN2-targeting agents is dependent on GCN2. ISRIB, a modulator of eIF2B, ablates the viability effect, demonstrating the dependence on translation initiation. Activating doses result in the induction of cleaved caspase 3 and cleaved PARP. In contrast, a nonactivating GCN2-targeting agent does not impact viability. These results provide a clearer understanding of the challenges and opportunities for the clinical development of compounds targeting GCN2.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-24-0960
  27. Cell. 2025 Aug 18. pii: S0092-8674(25)00865-7. [Epub ahead of print]
    A conserved mechanism for the retrieval of polyubiquitinated proteins from cilia.
    Sven M Lange, Jeremy A Bennett, Robyn J Eisert, Alan Brown.
      The temporospatial distribution of proteins within cilia is regulated by intraflagellar transport (IFT), wherein molecular trains shuttle between the cell body and cilium. Defects in this process impair various signal-transduction pathways and cause ciliopathies. Although K63-linked ubiquitination appears to trigger protein export from cilia, the mechanisms coupling polyubiquitinated proteins to IFT remain unclear. Using a multidisciplinary approach, we demonstrate that a complex of CFAP36, a conserved ciliary protein of previously unknown function, and ARL3, a GTPase involved in ciliary import, binds polyubiquitinated proteins and links them to retrograde IFT trains. CFAP36 uses a coincidence detection mechanism to simultaneously bind two IFT subunits accessible only in retrograde trains. Depleting CFAP36 accumulates K63-linked ubiquitin in cilia and disrupts hedgehog signaling, a pathway reliant on the retrieval of ubiquitinated receptors. These findings advance our understanding of ubiquitin-mediated protein transport and ciliary homeostasis and demonstrate how structural changes in IFT trains achieve cargo selectivity.
    Keywords:  cilia; flagella; hedgehog signaling; intraflagellar transport; ubiquitin
    DOI:  https://doi.org/10.1016/j.cell.2025.07.043
  28. MicroPubl Biol. 2025 ;2025
    The reticulon protein, TtRET1, is required for the initiation of mating in Tetrahymena thermophila.
    Sabrice Guerrier, Jordon Williams, Brandon Garcia.
      We recently identified Tetrahymena thermophila reticulon (TtRET1) as a novel marker of the endoplasmic reticulum (ER) that reveals ER morphology during the conjugation phase of mating, but its functional role was previously unknown. Here, we show that TtRET1 is required for the early initiation of mating, prior to conjugation. Furthermore, TtRET1 relocalizes during the mating reaction, suggesting it may regulate ER remodeling events necessary to initiate the mating program.
    DOI:  https://doi.org/10.17912/micropub.biology.001763
  29. Structure. 2025 Aug 20. pii: S0969-2126(25)00269-2. [Epub ahead of print]
    Design of multi-target peptide modulators for protein chaperone networks.
    Luca Torielli, Matteo Castelli, Francesca Milani, Jennifer A Heritz, Sara J Cayaban, Jason Hernandez, Stefano A Serapian, Andrea Magni, Elena Frasnetti, Filippo Doria, Valentina Pirota, Laura A Wengert, Mark R Woodford, Giulia Lodigiani, Greta Bergamaschi, Marina Veronesi, Tiziano Bandiera, Stefania Girotto, Antonella Paladino, Chrisostomos Prodromou, Sarah J Backe, Dimitra Bourboulia, Anselmo Canciani, Cristina Arrigoni, Marco Lolicato, Jason E Gestwicki, Mehdi Mollapour, Giorgio Colombo.
      Essential chaperones heat shock protein 70 (Hsp70) and heat shock protein 90 (Hsp90) collaborate in oncoprotein folding. Dual inhibition of these chaperones has shown synergy in preclinical studies but remains challenging to achieve. Using a computational approach, we designed peptides mimicking the predicted unfolding regions of Kinase CDK4, a client protein of both Hsp70 and Hsp90. Peptide Cdk4-2 is shown to simultaneously bind Hsp70, Hsp90, and co-chaperone Cdc37. Cdk4-2 is membrane permeable, inhibits CDK4-mediated retinoblastoma phosphorylation, and induces apoptosis in renal carcinoma cells. Structure-function studies identified a minimal pharmacophore for Hsp70 binding and critical interactions for peptide affinity. These findings demonstrate the feasibility of rationally designing multi-target modulators of chaperone networks. Cdk4-2 is a promising lead for therapeutic development, expanding the molecular space of modulators of cancer-associated multiprotein machineries. While focused on chaperones, the idea behind our strategy is general and immediately transferable to other multiprotein targets and networks.
    Keywords:  anticancer molecules; drug design; folding blockers; molecular dynamics; molecular recognition; oncogenes; protein degraders; protein folding; protein levels
    DOI:  https://doi.org/10.1016/j.str.2025.07.021
  30. Immunity. 2025 Aug 22. pii: S1074-7613(25)00332-2. [Epub ahead of print]
    Unfolded protein response transcription factor XBP1 suppresses necroptosis-induced colitis by reinforcing the mucus barrier.
    Göksu Gökberk Kaya, Robin Schwarzer, Marius Dannappel, Katerina Vlantis, Ulrike Göbel, Vangelis Kondylis, Sergei A Nedospasov, Manolis Pasparakis.
      Endoplasmic reticulum (ER) stress and necroptosis are associated with the pathogenesis of inflammatory bowel disease (IBD); however, the potential crosstalk between these pathways is unclear. Here, we show that intestinal epithelial cell (IEC)-specific X-box binding protein 1 (XBP1) deficiency strongly aggravates the development of necroptosis-induced colitis, but not ileitis, in mice lacking caspase-8 or its adapter Fas associated with death domain (FADD) in IECs. Mechanistically, XBP1 ablation led to diminished mucin 2 (MUC2) expression and impaired mucus layer formation in the colon, which allowed bacteria to penetrate and reach the epithelial surface. This was not sufficient to trigger colitis in the presence of an intact epithelial monolayer but synergized with IEC necroptosis to induce severe colon inflammation. Our results revealed that XBP1 and caspase-8 control different components of the intestinal barrier that synergize to maintain mucosal immune homeostasis and prevent colon inflammation. This could be relevant for the better understanding of the mechanisms causing IBD.
    Keywords:  FADD; IRE1; XBP1; caspase-8; impaired mucus layer; inflammatory bowel disease; intestinal epithelial barrier; mucin 2; necroptosis; unfolded protein response
    DOI:  https://doi.org/10.1016/j.immuni.2025.07.023
  31. Nat Commun. 2025 Aug 23. 16(1): 7863
    A repurposed AMP binding domain reveals mitochondrial protein AMPylation as a regulator of cellular metabolism.
    Abner Gonzalez, Alex Pon, Kelly Servage, Krzysztof Pawłowski, Yan Han, Anju Sreelatha.
      Protein AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates, has been known as a post translational modification for over 50 years. Research in this field is largely underdeveloped due to the lack of tools that enable the systematic identification of AMPylated substrates. Here, we address this gap by developing an enrichment technique to isolate and study AMPylated proteins using a nucleotide-binding protein, hinT. Cryo-EM reconstruction of an AMPylated protein bound to hinT provides a structural basis for AMP selectivity. Using structure guided mutagenesis, we optimize enrichment to identify novel substrates of the evolutionarily conserved AMPylase, Selenoprotein O. We show that mammalian Selenoprotein O regulates metabolic flux through AMPylation of key mitochondrial proteins including glutamate dehydrogenase and pyruvate dehydrogenase. Our findings highlight the broader significance of AMPylation, an emerging post translational modification with critical roles in signal transduction and disease pathology. Furthermore, we establish a powerful enrichment platform for the discovery of novel AMPylated proteins to study the mechanisms and significance of protein AMPylation in cellular function.
    DOI:  https://doi.org/10.1038/s41467-025-63014-z
  32. Nat Commun. 2025 Aug 21. 16(1): 7808
    Control of Golgi- V-ATPase through Sac1-dependent co-regulation of PI(4)P and cholesterol.
    Xin Zhou, Miesje M van der Stoel, Shreyas Kaptan, Haoran Li, Shiqian Li, Maarit Hölttä, Helena Vihinen, Eija Jokitalo, Christoph Thiele, Olli Pietiläinen, Shin Morioka, Junko Sasaki, Takehiko Sasaki, Ilpo Vattulainen, Elina Ikonen.
      Sac1 is a conserved phosphoinositide phosphatase, whose loss-of-function compromises cell and organism viability. Here, we employ acute auxin-inducible Sac1 degradation to identify its immediate downstream effectors in human cells. Most of Sac1 is degraded in ~1 h, paralleled by increased PI(4)P and decreased cholesterol in the trans-Golgi network (TGN) during the following hour, and superseded by Golgi fragmentation, impaired glycosylation, and selective degradation of TGN proteins by ~4 h. The TGN disintegration results from its acute deacidification caused by disassembly of the Golgi V-ATPase. Mechanistically, Sac1 mediated TGN membrane composition maintains an assembly-promoting conformation of the V0a2 subunit. Key phenotypes of acute Sac1 degradation are recapitulated in human differentiated trophoblasts, causing processing defects of chorionic gonadotropin, in line with loss-of-function intolerance of the human SACM1L gene. Collectively, our findings reveal that the assembly of the Golgi V-ATPase is controlled by the TGN membrane via Sac1 fuelled lipid exchange.
    DOI:  https://doi.org/10.1038/s41467-025-63125-7
  33. Nature. 2025 Aug 27.
    One-shot design of functional protein binders with BindCraft.
    Martin Pacesa, Lennart Nickel, Christian Schellhaas, Joseph Schmidt, Ekaterina Pyatova, Lucas Kissling, Patrick Barendse, Jagrity Choudhury, Srajan Kapoor, Ana Alcaraz-Serna, Yehlin Cho, Kourosh H Ghamary, Laura Vinué, Brahm J Yachnin, Andrew M Wollacott, Stephen Buckley, Adrie H Westphal, Simon Lindhoud, Sandrine Georgeon, Casper A Goverde, Georgios N Hatzopoulos, Pierre Gönczy, Yannick D Muller, Gerald Schwank, Daan C Swarts, Alex J Vecchio, Bernard L Schneider, Sergey Ovchinnikov, Bruno E Correia.
      Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. 1) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.
    DOI:  https://doi.org/10.1038/s41586-025-09429-6
  34. FEBS Lett. 2025 Aug 27.
    Mutational analysis of Yih1 and IMPACT reveals amino acids required for Gcn2 inhibition.
    Bianca J M Sansom, Victoria J Gibbs, Anja H Schiemann, Evelyn Sattlegger.
      In response to amino acid starvation, the protein kinase Gcn2 phosphorylates the eukaryotic translation initiation factor eIF2α, allowing cells to adapt to adverse conditions. Gcn2 function requires direct binding to effector protein Gcn1 via the Gcn2 RWD-domain. The orthologues yeast Yih1 and mammalian IMPACT also contain an RWD-domain that can bind Gcn1, thereby impairing the Gcn2-Gcn1 interaction. In yeast, overexpressed Yih1/IMPACT impairs eIF2α phosphorylation, visible by reduced growth under starvation conditions. We found that Yih1 D102A and D108A substitutions each revert this defect, suggesting that Yih1-mediated Gcn2 inhibition is impaired. Similar effects were found for at least the D111A substitution in IMPACT. The respective amino acids are located in a common helix, suggesting this helix is a conserved determinant for Gcn1 binding.
    Keywords:  Gcn1; Gcn2; IMPACT; RWD domain; Yih1; eIF2α
    DOI:  https://doi.org/10.1002/1873-3468.70148
  35. Genes (Basel). 2025 Aug 13. pii: 957. [Epub ahead of print]16(8):
    Dual Nature of Mitochondrial Integrated Stress Response: Molecular Switches from Protection to Pathology.
    Jisu Jeong, Junghyun Kim, Man S Kim.
       BACKGROUND: The mitochondrial integrated stress response (ISR) represents a fundamental cellular adaptation mechanism with dual protective and pathological roles. We critically analyzed current literature on ISR mechanisms, focusing on recent paradigm shifts including the 2020 discovery of the OMA1-DELE1-HRI axis, emerging controversies over context-dependent activation patterns, and the January 2025 clinical trial failures that have reshaped the therapeutic landscape.
    METHODS: We reviewed recent literature (2020-2025) examining ISR mechanisms, clinical trials, and therapeutic developments through comprehensive database searches.
    RESULTS: The field has evolved from simple linear pathway models to recognition of complex, context-dependent networks. Recent findings reveal that ISR activation mechanisms vary dramatically based on cellular metabolic state, with distinct pathways operating in proliferating versus differentiated cells. The "dark microglia" phenotype in neurodegeneration and DR5-mediated apoptotic switches exemplify pathological ISR manifestations, while adaptive responses include metabolic reprogramming and quality control enhancement.
    CONCLUSIONS: The 2025 failures of DNL343 and ABBV-CLS-7262 in ALS trials underscore the need for precision medicine approaches that account for context-dependent ISR functions, temporal dynamics, and disease-specific mechanisms.
    Keywords:  cellular adaptation; eIF2α phosphorylation; integrated stress response; mitochondrial dysfunction; neurodegeneration; precision medicine
    DOI:  https://doi.org/10.3390/genes16080957
  36. Cell Commun Signal. 2025 Aug 20. 23(1): 374
    The integrated stress response promotes macrophage inflammation and migration in autoimmune diabetes.
    Jiayi E Wang, Charanya Muralidharan, Armando A Puente, Titli Nargis, Jacob R Enriquez, Ryan M Anderson, Raghavendra G Mirmira, Sarah A Tersey.
      Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing pancreatic β-cells. Macrophages infiltrate islets early in T1D pathogenesis, preceding the influx of T- and B-lymphocytes. The integrated stress response (ISR), a cellular pathway activated during stress, coordinates adaptive changes in gene expression to maintain cell function and survival. To study the ISR in macrophages, bone-marrow-derived macrophages were treated with a pharmacological inhibitor of the ISR (ISRIB) and polarized to a proinflammatory M1-like state. We observed a reduction in the number of proinflammatory macrophages, as well as a decrease in iNOS mRNA and protein levels, following ISRIB treatment. RNA-sequencing revealed a reduction in pathways related to stress responses, including ER stress, reactive oxygen species (ROS) regulation, and autophagy, as well as migration pathway genes. ISRIB treatment led to decreased macrophage migration after stimulation in vitro and reduced migration of macrophages to the site of injury after tailfin injury in zebrafish in vivo. Interestingly, ISRIB mediated reduction of M1-like macrophages and reduction of migration was recapitulated by the inhibition of PKR but not PERK, both upstream ISR kinases, highlighting PKR as a key mediator of the ISR in macrophages. Pre-diabetic female non-obese diabetic (NOD) mice administered ISRIB demonstrated an overall reduction in the macrophage numbers in the pancreatic islets. Additionally, the insulitic area of pancreata from ISRIB treated NOD mice had increased PD-L1 levels. PD-L1 protein but not mRNA levels were increased in M1-like macrophages after ISR and PKR inhibition. Our findings identify the ISR, particularly via PKR, as a critical regulator of macrophage driven inflammation and migration in T1D. Our study offers new insights into ISR signaling in macrophages, demonstrating that the ISR may serve as a potential target for intervention in macrophages during early T1D pathogenesis.
    Keywords:  Diabetes; Inflammation; Integrated stress response (ISR); Macrophages; Migration; Pancreatic islet; Unfolded protein response (UPR)
    DOI:  https://doi.org/10.1186/s12964-025-02372-z
  37. EMBO J. 2025 Aug 26.
    RABGAP1 is a sensor that facilitates the sorting and processing of amyloid precursor protein.
    Jessica Eden, Jonathan G G Kaufman, Conceição Pereira, Eleanor Fox, Jerome Cattin-Ortolá, Lorena Benedetti, Bart Nieuwenhuis, David J Owen, Jennifer Lippincott-Schwartz, Sean Munro, David C Gershlick.
      A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β plaques in the brain. Amyloid-β is a 40-42 amino acid peptide generated by proteolytic processing of amyloid precursor protein (APP) via membrane-bound proteases. APP is a transmembrane protein, and its trafficking to sites of proteolysis represents a rate-limiting step in AD progression. Although APP processing has been well-studied, its trafficking itinerary and machinery remain incompletely understood. To address this, we performed an unbiased interaction screen for interactors of the APP cytosolic tail. We identified previously characterised APP binders as well as novel interactors, including RABGAP1. We demonstrated that RABGAP1 partially co-localises with APP and directly interacts with a YENPTY motif in the APP cytosolic tail. Depletion or overexpression of RABGAP1 caused mistrafficking and misprocessing of endogenous APP in human and rodent neurons. This effect is dependent on the GAP activity of RABGAP1, demonstrating that RABGAP1 affects the trafficking of APP by modulating RAB activity on endosomal subdomains. This novel trafficking mechanism has implications for other NPXY cargoes and presents a possible therapeutic avenue to explore.
    Keywords:  APP; Alzheimer’s Disease; RABGAP1; Trafficking
    DOI:  https://doi.org/10.1038/s44318-025-00530-0
  38. Proc Natl Acad Sci U S A. 2025 Aug 26. 122(34): e2500079122
    The functional dynamics of FicD's TPR domain are modulated by the interaction with ATP and BiP.
    Svenja Runge, Ecenaz Bilgen, Andrea Magni, Giorgio Bonollo, Giorgio Colombo, Aymelt Itzen, Don C Lamb.
      The human Fic enzyme FicD plays an important role in regulating the Hsp70 homolog BiP in the endoplasmic reticulum: FicD reversibly modulates BiP's activity through attaching an adenosine monophosphate to the substrate binding domain. This reduces BiP's chaperone activity by shifting it into a conformation with reduced substrate affinity. Crystal structures of FicD in the apo, adenosine triphosphate (ATP)-bound, and BiP-bound states suggested significant conformational variability in the tetratricopeptide repeat (TPR) motifs. However, nothing is known about the underlying dynamics. In this study, we investigate the conformational dynamics of FicD's TPR motifs using two-color, single-molecule Förster resonance energy transfer (smFRET) experiments. We demonstrate that the TPR motifs exhibit conformational dynamics between a TPR-out and a TPR-in conformation on timescales ranging from microseconds to milliseconds. In addition, we extend our investigation on multiple labeling positions within FicD, revealing how conformational dynamics vary depending on the location within the TPR motif. We quantify the motions with dynamic photon distribution analysis for the FRET constructs and generate an ensemble of structures for the different states consistent with the smFRET data using molecular dynamic simulations. We propose a conformational landscape model for FicD where the TPR-in/out states exist in equilibrium and the fraction of dynamic population is altered due to the presence of ATP and BiP. These results indicate that not only is FicD dynamic, but the dynamics are linked to the functionality and interactions of FicD with BiP.
    Keywords:  AMPylation; BiP; FicD; Hsp70; single-molecule FRET
    DOI:  https://doi.org/10.1073/pnas.2500079122
  39. Angew Chem Int Ed Engl. 2025 Aug 21. e202511467
    HerTACs Enable Tumor-Selective Lysosomal Degradation of Membrane and Extracellular Proteins via HER2 Trafficking.
    Shipeng He, Wenjing Huang, Yaojin Zhu, Fei Gao, Yuxin Fang, Jiayi Zhu, Yuhan Bao, Guoqiang Dong, Chunquan Sheng.
      Targeting extracellular and membrane proteins for degradation remains a frontier challenge in the field of targeted protein degradation (TPD), largely due to the intracellular confinement of existing proteolysis systems and reliance on bulky biologics. Here, we develop a novel TPD platform, human epidermal growth factor receptor 2 (HER2)-targeted lysosome-tethering chimeras (HerTACs), which co-opts the tumor overexpressed, endocytic, and lysosomal trafficking capability of HER2. Starting from the HER2-binding peptide LTVSPWY, we engineered the first-generation HerTAC (LP), a conjugate of the HER2-binding peptide and a PD-L1 ligand, to degrade programmed death ligand 1 (PD-L1) in HER2-positive cells. Guided by AlphaFold modeling and alanine scanning, we developed a stapled peptide-based HerTAC (L2,5P) with enhanced degradative efficacy (DC50 = 156 nM), stability, and pharmacokinetics. HerTAC L2,5P showed potent antitumor activity and low systemic toxicity in HER2+ breast cancer animal models. The HerTAC strategy was further extended to other clinically relevant inaccessible membrane and extracellular targets (i.e., V-domain Ig suppressor of T cell activation [VISTA] and macrophage migration inhibitory factor [MIF]), highlighting its generality and broad applicability. This work establishes a tumor-selective, lysosome-directed TPD strategy that expands the druggable proteome and offers a clinically transformable approach for precision oncology.
    Keywords:  Extracellular and membrane proteins; Human epidermal growth factor receptor 2; Lysosome‐targeting chimera; Targeted protein degradation
    DOI:  https://doi.org/10.1002/anie.202511467
  40. Mol Cell. 2025 Aug 19. pii: S1097-2765(25)00656-2. [Epub ahead of print]
    Endocytosis is essential for cysteine-deprivation-induced ferroptosis.
    Xuesong Liu, Zechuan Zhao, Zhixuan Bian, Fahad A Benthani, Yingying Hu, Deguang Liang, Xuejun Jiang.
      Ferroptosis is a form of cell death caused by iron-dependent phospholipid peroxidation and subsequent membrane rupture. Autophagic degradation of the iron-storage protein ferritin promotes ferroptosis by increasing cytosolic bioactive iron, presumably explaining how lysosomal inhibitors suppress ferroptosis. Surprisingly, we found that lysosomal inhibitors suppress cysteine-deprivation-induced (CDI) ferroptosis, even in autophagy-defective cells, and subsequently discovered that clathrin-mediated endocytosis (CME) of transferrin is essential for CDI ferroptosis. Blocking lysosomal proteolytic activity failed to inhibit ferroptosis, whereas disrupting endosomal acidification and eliminating the endocytic protein AP2M1 both impeded ferroptosis. Conversely, replenishing cellular iron with ferric ammonium citrate, but not with transferrin, restored CDI ferroptosis in endocytosis-deficient cells. Unexpectedly, abolishing endosomal acidification, CME, and the associated increase in cellular labile iron could not prevent ferroptosis triggered by direct inhibition of the ferroptosis-suppressing enzyme glutathione peroxidase-4 (GPX4). Together, this study reveals the essential role of endocytosis, specifically for CDI ferroptosis.
    Keywords:  AP2M1; GPX4; autophagy; cysteine deprivation; endocytosis; endosome; ferroptosis; iron; lysosome; transferrin
    DOI:  https://doi.org/10.1016/j.molcel.2025.08.006
  41. Nat Commun. 2025 Aug 24. 16(1): 7896
    4D structural biology-quantitative dynamics in the eukaryotic RNA exosome complex.
    Jobst Liebau, Daniela Lazzaretti, Torben Fürtges, Anna Bichler, Michael Pilsl, Till Rudack, Remco Sprangers.
      Molecular machines play pivotal roles in all biological processes. Most structural methods, however, are unable to directly probe molecular motions. Here, we demonstrate that dedicated NMR experiments can provide quantitative insights into functionally important dynamic regions in very large asymmetric protein complexes. We establish this for the 410 kDa eukaryotic RNA exosome complex that contains ten distinct protein chains. Methyl-group and fluorine NMR experiments reveal site-specific interactions among subunits and with an RNA substrate. Furthermore, we extract quantitative insights into conformational changes within the complex in response to substrate and subunit binding for regions that are invisible in static cryo-EM and crystal structures. In particular, we identify a flexible plug region that can block an aberrant route for RNA towards the active site. Based on molecular dynamics simulations and NMR data, we provide a model that shows how the flexible plug is structured in the open and closed conformations. Our work thus demonstrates that a combination of state-of-the-art structural biology methods can provide quantitative insights into large molecular machines that go significantly beyond the well-resolved and static images of biomolecular complexes, thereby adding the time domain to structural biology.
    DOI:  https://doi.org/10.1038/s41467-025-62982-6
  42. Biomolecules. 2025 Aug 08. pii: 1146. [Epub ahead of print]15(8):
    Non-Canonical, Strongly Selective Protein Disulfide Isomerases as Anticancer Therapeutic Targets.
    Mary E Law, Zaafir M Dulloo, Brian Hardy, Ania Kelegama, Reagan Clark, Mariana Rivas Montbrun, Gabriella Antmann, Srihith Nooka, Ronald K Castellano, Brian K Law.
      Protein Disulfide Isomerases (PDIs) are emerging targets in anticancer therapy, with several PDI inhibitors demonstrating anticancer efficacy in preclinical models. Research has largely focused on "canonical" PDIs, such as PDIA1, which contain CXXC active site motifs where C represents Cysteine. Canonical PDIs have well-studied, critical roles in forming, breaking, and exchanging/scrambling disulfide bonds during protein folding. In contrast, non-canonical PDIs, which harbor CXXS active site motifs, remain less well-studied despite their role as sensors or effectors of protein folding quality control during protein trafficking in the secretory pathway. Here, we provide a review of the literature relating to the non-canonical PDIs ERp44, AGR2, and AGR3, which have been identified as strong dependencies in specific cancer subtypes according to the DepMap database. The biological and biochemical functions of ERp44, AGR2, and AGR3 are discussed, highlighting the role of ERp44 in two mechanisms of protein folding quality control, AGR2 as a selective sensor of mucin protein misfolding, and a unique role for AGR3 in cilia. Finally, we discuss recent efforts to develop small molecule inhibitors of ERp44, AGR2, and AGR3 as tool compounds and experimental therapeutics.
    Keywords:  AGR2; AGR3; ERp44; PDIA1; protein disulfide isomerases
    DOI:  https://doi.org/10.3390/biom15081146
  43. EMBO J. 2025 Aug 26.
    RTN4IP1 is required for the final stages of mitochondrial complex I assembly and CoQ biosynthesis.
    Monika Oláhová, Rachel M Guerra, Jack J Collier, Juliana Heidler, Kyle Thompson, Chelsea R White, Paulina Castañeda-Tamez, Alfredo Cabrera-Orefice, Robert N Lightowlers, Zofia M A Chrzanowska-Lightowlers, Alexander Galkin, Ilka Wittig, David J Pagliarini, Robert W Taylor.
      A biochemical deficiency of mitochondrial complex I (CI) underlies approximately 30% of cases of primary mitochondrial disease, yet the inventory of molecular machinery required for CI assembly remains incomplete. We previously characterised patients with isolated CI deficiency caused by segregating variants in RTN4IP1, a gene that encodes a mitochondrial NAD(P)H oxidoreductase. Here, we demonstrate that RTN4IP1 deficiency causes a CI assembly defect in both patient fibroblasts and knockout cells, and report that RTN4IP1 is a bona fide CI assembly factor. Complexome profiling revealed accumulation of unincorporated ND5-module and impaired N-module production. RTN4IP1 patient fibroblasts also exhibited defective coenzyme Q biosynthesis, substantiating a second function of RTN4IP1. Thus, our data reveal RTN4IP1 plays necessary and independent roles in both the terminal stages of CI assembly and in coenzyme Q metabolism, and that pathogenic RTN4IP1 variants impair both functions in patients with mitochondrial disease.
    Keywords:  Coenzyme Q; Complex I Assembly; Complexome Profiling; Mitochondria; RTN4IP1
    DOI:  https://doi.org/10.1038/s44318-025-00533-x
  44. Cell Rep Med. 2025 Aug 19. pii: S2666-3791(25)00368-4. [Epub ahead of print] 102295
    Coordinated protein modules define DNA damage responses to carboplatin at single-cell resolution in human ovarian carcinoma models.
    Jacob S Bedia, Antonio Delgado-Gonzalez, Ying-Wen Huang, Veronica D Gonzalez, Ionut-Gabriel Funingana, Zainab Rahil, Alyssa Mike, Alexis Lowber, Maria Vias, Alan Ashworth, James D Brenton, Wendy J Fantl.
      Tubo-ovarian high-grade serous carcinoma (HGSC), the most lethal gynecologic malignancy, initially responds to platinum-based chemotherapy, but due to frequent defects in the DNA damage response (DDR), most tumors develop resistance. The molecular mechanisms underlying clinical platinum resistance remain poorly defined with no biomarkers or targeted therapies to improve outcomes. Here, applying mass cytometry, we quantify phosphorylation and abundance of DDR proteins in carboplatin-treated HGSC cell line models. Despite similar levels of intranuclear platinum, a proxy for carboplatin uptake, cells follow divergent fates, reflecting DDR heterogeneity. Unsupervised analysis reveals a continuum of DDR states, and matrix factorization identifies eight protein modules. The activity of one module, containing canonical DDR proteins, increases in carboplatin-sensitive cells. Resistant cells engage a broader DDR protein module. These findings demonstrate the ability of single-cell proteomics to identify functional DDR states and reveal a DDR sensitivity module as a promising biomarker for clinical stratification and therapeutic decisions in HGSC.
    Keywords:  CIOV1; CIOV2; CIOV3; DNA damage response; UWB1.289; Uniform ManifoldApproximation and Projection/UMAP; carboplatin resistance; mass cytometry/CyTOF; non-negative matrix factorization; partition-based graph abstraction; single cells; tubo-ovarian high grade carcinoma
    DOI:  https://doi.org/10.1016/j.xcrm.2025.102295
  45. J Am Chem Soc. 2025 Sep 03. 147(35): 31630-31638
    A Functional Assay for Mining Noninhibitory Enzyme Ligands from One Bead One Compound Libraries: Application to E3 Ubiquitin Ligases.
    Weijun Gui, Allison Goss, Thomas Kodadek.
      Chemical dimerizers are synthetic molecules that bring into proximity two or more proteins that do not normally interact with one another. A major application of this technology is to recruit an enzyme to a target protein, resulting in its post-translational modification (PTM). In particular, chemical dimerizer-mediated polyubiquitylation of proteins has garnered an enormous amount of interest as a new drug modality. A fundamental requirement for the construction of new PTM-driving dimerizers is an enzyme ligand that does not inhibit its activity. Traditional activity-based high-throughput screening platforms are not suited for this purpose. Here we describe a novel platform for screening libraries of bead-displayed compounds that links a requirement for small-molecule binding to the enzyme with enzyme-mediated modification of a nearby substrate. This system ensures that the enzyme-recruiting small molecules do not interfere with the catalytic function of the enzyme. We demonstrate the utility of this system in the context of E3 ubiquitin ligase-recruiting molecules and report the discovery of a novel low-molecular-mass ligand for the von Hippel-Lindau (VHL) protein.
    DOI:  https://doi.org/10.1021/jacs.5c07307
  46. Nat Commun. 2025 Aug 21. 16(1): 7817
    Structural basis for the recognition and ubiquitylation of type-2 N-degron substrate by PRT1 plant N-recognin.
    Woo Seok Yang, Seu Ha Kim, Minsang Kim, Hejeong Shin, Juyeon Lee, Alexander Sandmann, Ohkmae K Park, Nico Dissmeyer, Hyun Kyu Song.
      PROTEOLYSIS1 (PRT1), an N-recognin of Arabidopsis thaliana, recognizes the N-terminal aromatic hydrophobic residue (Tyr/Phe/Trp) of its substrates and ubiquitylates them for degradation by the ubiquitin-proteasome system. Herein, we report the structures of the ZZ domain of PRT1 (PRT1ZZ) in complex with bulky hydrophobic N-degron peptides. Unlike other ZZ domains, PRT1ZZ has an unusual binding site with two hydrophobic regions. The N-terminal aromatic residues of N-degrons interact with Ile333 and Phe352 in the flexible loops, which undergo a conformational change. Notably, we identify a third residue from the N-terminus of the substrate that participates in the hydrophobic network with PRT1ZZ. Moreover, AlphaFold prediction and biochemical assays revealed that the tandem RING1 and RING2 domains of PRT1 interact intramolecularly. The dimeric RING domains in a single protein represent a unique feature among the RING-type E3 ligases. The biochemical assays using the N-terminal tyrosine-exposed substrate, BIG BROTHER, show that the intramolecular RING dimer is essential for PRT1's robust activity. Therefore, this study expands our knowledge of the structural repertoire in the N-degron pathway and provides insights into the regulation of E3 ligases containing tandem RING domains.
    DOI:  https://doi.org/10.1038/s41467-025-63282-9
  47. Cell Rep. 2025 Aug 22. pii: S2211-1247(25)00950-7. [Epub ahead of print]44(9): 116179
    Lysosomal polyamine storage upon ATP13A2 loss impairs β-glucocerebrosidase via altered lysosomal pH and electrostatic hydrolase-lipid interactions.
    Madhuja Samaddar, Gabriel A Fitzgerald, Ann Hong Nguyen, Sonnet S Davis, Shourya Jain, Jian Guo, Nicholas E Propson, Bettina van Lengerich, Yajuan Shi, Srijana Balasundar, Lionel Rougé, Jamal Alkabsh, Anil Rana, Julie Yi, Marcus Y Chin, Isabel A Becerra, Annie Arguello, Brian M Fox, Todd Logan, Jung H Suh, Anastasia G Henry, Gilbert Di Paolo.
      ATP13A2 is an endolysosomal polyamine transporter mutated in several neurodegenerative conditions involving lysosomal defects, including Parkinson's disease (PD). While polyamines are polybasic and polycationic molecules that play pleiotropic cellular roles, their specific impact on lysosomal health is unknown. Here, we demonstrate lysosomal polyamine accumulation in ATP13A2 knockout (KO) cell lines and human induced pluripotent stem cell (iPSC)-derived neurons. Primary polyamine storage caused secondary storage of lysosomal anionic phospholipid bis(monoacylglycero)phosphate (BMP) and an age-dependent increase in the β-glucocerebrosidase (GCase) substrate glucosylsphingosine in Atp13a2 KO brains. Polyamine accumulation inhibited lysosomal GCase activity in cells, and this was reversed by lysosome reacidification or BMP supplementation. A liposome-based GCase assay utilizing physiological substrates demonstrated dose-dependent inhibition of BMP-stimulated GCase activity by polyamines, in part via a pH-independent, electrostatics-based mechanism. Therefore, excess polyamine compromises lysosomes by disrupting pH and electrostatic interactions between GCase and BMP that enable efficient substrate hydrolysis, potentially clarifying pathogenic mechanisms and suggesting convergence on PD-relevant pathways.
    Keywords:  CP: Neuroscience; Kufor-Rakeb syndrome; P-type ATPase; Parkinson’s disease; glucocerebrosidase; glycosphingolipid; lysosomal storage disorder; neuronal ceroid lipofuscinosis; polyamine; spermine
    DOI:  https://doi.org/10.1016/j.celrep.2025.116179
  48. Nature. 2025 Aug 20.
    Cancer-induced nerve injury promotes resistance to anti-PD-1 therapy.
    Erez N Baruch, Frederico O Gleber-Netto, Priyadharsini Nagarajan, Xiayu Rao, Shamima Akhter, Tuany Eichwald, Tongxin Xie, Mohammad Balood, Adebayo Adewale, Shorook Naara, Hinduja N Sathishkumar, Shajedul Islam, William McCarthy, Brandi J Mattson, Renata Ferrarotto, Michael K Wong, Michael A Davies, Sonali Jindal, Sreyashi Basu, Karine Roversi, Amin Reza Nikpoor, Maryam Ahmadi, Ali Ahmadi, Catherine Harwood, Irene Leigh, Dennis Gong, Paulino Tallón de Lara, Derrick L Tao, Tara M Davidson, Nadim J Ajami, Andrew Futreal, Kunal Rai, Veena Kochat, Micah Castillo, Preethi Gunaratne, Ryan P Goepfert, Sharia D Hernandez, Nikhil I Khushalani, Jing Wang, Stephanie S Watowich, George A Calin, Michael R Migden, Mona Yuan, Naijiang Liu, Yi Ye, William L Hwang, Paola D Vermeer, Nisha J D'Silva, Yuri L Bunimovich, Dan Yaniv, Jared K Burks, Javier Gomez, Patrick M Dougherty, Kenneth Y Tsai, James P Allison, Padmanee Sharma, Jennifer A Wargo, Jeffrey N Myers, Sebastien Talbot, Neil D Gross, Moran Amit.
      Perineural invasion (PNI) is a well-established factor of poor prognosis in multiple cancer types1, yet its mechanism remains unclear. Here we provide clinical and mechanistic insights into the role of PNI and cancer-induced nerve injury (CINI) in resistance to anti-PD-1 therapy. Our study demonstrates that PNI and CINI of tumour-associated nerves are associated with poor response to anti-PD-1 therapy among patients with cutaneous squamous cell carcinoma, melanoma and gastric cancer. Electron microscopy and electrical conduction analyses reveal that cancer cells degrade the nerve fibre myelin sheets. The injured neurons respond by autonomously initiating IL-6- and type I interferon-mediated inflammation to promote nerve healing and regeneration. As the tumour grows, the CINI burden increases, and its associated inflammation becomes chronic and skews the general immune tone within the tumour microenvironment into a suppressive and exhaustive state. The CINI-driven anti-PD-1 resistance can be reversed by targeting multiple steps in the CINI signalling process: denervating the tumour, conditional knockout of the transcription factor mediating the injury signal within neurons (Atf3), knockout of interferon-α receptor signalling (Ifnar1-/-) or by combining anti-PD-1 and anti-IL-6-receptor blockade. Our findings demonstrate the direct immunoregulatory roles of CINI and its therapeutic potential.
    DOI:  https://doi.org/10.1038/s41586-025-09370-8
  49. Nat Commun. 2025 Aug 26. 16(1): 7945
    Nascent liver proteome reveals enzymes and transcription regulators under physiological and alcohol exposure conditions.
    Jiayu Gu, Lihui Lao, Linzhen Hu, Jia Zang, Chao Liu, Ruixi Wan, Ling Tang, Ying Yuan, Yulin Chen, Shixian Lin.
      The liver proteome undergoes dynamic changes while performing hundreds of essential biological functions. Dysregulation of the liver proteome under alcoholic conditions leads to alcohol-associated liver disease (ALD), a major health challenge worldwide. There is an urgent need for quantitative and liver-specific proteome information in living animals to understand the pathophysiological dynamics of this largest solid organ. Here, we develop a comprehensive approach that specifically identifies the nascent proteome and preferentially enriches membrane proteins in living mouse hepatocytes and is broadly applicable to studies of the liver under various physiological and pathological conditions. In the ethanol-induced liver injury mouse model, the nascent proteome successfully identifies and validates a number of transcription regulators, enzymes, and protective chaperones involved in the molecular regulation of hepatic steatosis, in addition to almost all known regulatory proteins and pathways related to alcohol metabolism. We discover that Phb1/2 is an important transcription coregulator in the process of ethanol metabolism, and one identified fatty acid metabolism enzyme Acsl1/5, whose inhibition protects cells and mice from lipid accumulation, a key symptom of hepatic steatosis.
    DOI:  https://doi.org/10.1038/s41467-025-63212-9
  50. EMBO J. 2025 Aug 20.
    HDAC6-dependent deacetylation of SAE2 enhances SUMO1 conjugation for mitotic integrity.
    Alexander J Lanz, Alexandra K Walker, Mohammed Jamshad, Alexander J Garvin, Matthew Stewart, Peter Wotherspoon, Benjamin F Cooper, Matthew Mackintosh, Oliver Crutchley, Timothy J Knowles, Joanna R Morris.
      Mammalian cells express three conjugatable SUMO variants: SUMO1 and the closely related SUMO2 and SUMO3 (together referred to as SUMO2/3). While some substrates are modified by both, others show a clear preference, though the basis for this selectivity remains unclear. Here, we examine a modification of the catalytic component of the human SUMO activation enzyme, SAE2. We find that lysine 164 of SAE2 undergoes HDAC6-dependent deacetylation during mitosis. A non-deacetylatable acetyl-mimetic mutant, SAE2-K164Q, selectively enhances SUMO2 over SUMO1 activation and conjugation, and distinguishes between SUMO1 and SUMO2/3 based on differences in their C-terminal tails. Complementation of SAE2-deficient or inhibited cells with SAE2-K164Q suppresses mitotic SUMO1 conjugation and promotes multipolar spindle formation. We identify NuMA as a SUMO E1-dependent substrate and demonstrate that mitotic defects caused by SAE2-K164Q or HDAC6 inhibition are rescued by SUMO1 overexpression or expression of a GFP-SUMO1-NuMA-K1766R fusion. These results support a model in which SAE1:SAE2 deacetylation during early mitosis promotes SUMO1 conjugation to ensure mitotic fidelity, highlighting a regulatory role for the SUMO-activating enzyme in the selection of SUMO proteins.
    Keywords:  Acetylation; E1; HDAC; Mitosis; SUMOylation
    DOI:  https://doi.org/10.1038/s44318-025-00532-y
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