bims-proteo Biomed News
on Proteostasis
Issue of 2025–09–07
53 papers selected by
Eric Chevet, INSERM
  1. Deltex and RING-UIM E3 ligases cooperate to create a ubiquitin-ADP-ribose hybrid mark on tankyrase, promoting its stabilization.
  2. Cotranslational protein folding through non-native structural intermediates.
  3. Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1.
  4. Molecular glues and PROTACs in targeted protein degradation: mechanisms, advances, and therapeutic potential.
  5. Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22.
  6. Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control.
  7. C-terminal tagging, transmembrane domain hydrophobicity, and an ER retention motif influence the secretory trafficking of the inner nuclear membrane protein emerin.
  8. Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22.
  9. Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation.
  10. Processing bodies promote lysosomal quality control and cell survival during recovery from lysosomal damage.
  11. A split-site E3 ligase mechanism enables ZNFX1 to ubiquitinate and cluster single-stranded RNA into ubiquitin-coated nucleoprotein particles.
  12. Structural basis of VCP-VCPIP1-p47 ternary complex in Golgi maintenance.
  13. Potentiating the integrated stress response.
  14. K29-Linked Ubiquitination of Transcription Regulators Controls Cell Proliferation in the Unfolded Protein Response.
  15. Mannose trimming is the dominant signal for the release of misfolded glycoproteins from ER quality control.
  16. The endoplasmic reticulum autophagy receptor TEX264 drives epidermal differentiation and is dysregulated in Darier disease.
  17. Salmonella Typhimurium exploits the reticulophagy/ERphagy receptor RETREG1 to promote infection.
  18. Via Proteins and Lipids - Versatility of VAPs at Dynamic Membrane Contact Sites.
  19. Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID.
  20. UBE3A promotes foam cell formation and counters remyelination by targeting ABCA1 for proteasomal degradation.
  21. The Proteostasis Network in Proteinopathies: Mechanisms and Interconnections.
  22. In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID.
  23. Failure of efficient cardiac proteostatic adaptations to chronic cAMP-stress is associated with accelerated heart aging.
  24. Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation.
  25. VAMP8 stabilization by DRAM1 enables autophagosome-lysosome fusion and promotes metastatic extravasation.
  26. Computational design of potent and selective binders of BAK and BAX.
  27. An alternative pocket for binding the N-degrons by the UBR1 and UBR2 ubiquitin E3 ligases.
  28. In Vivo Tumor Growth Control by General Control Nonderepressible 2-Targeting Agents Results from Kinase Activation.
  29. The cytoplasmic domain of the pseudoprotease iRhom2 mediates distinct signaling mechanisms to control activation of the cell surface protease ADAM17.
  30. Autophagy and exosomes play different roles in the disposal of unwanted cellular materials.
  31. Sequestering sequestosome 1 via S-acylation in autophagy, Huntington disease, and beyond.
  32. E3 ligase RAD18 targets phosphorylated IRF3 to terminate IFNB1 transcription.
  33. MitoTraP: mitochondrial protection for cellular proteostasis.
  34. Multi-omic assessment of mRNA translation dynamics in liver cancer cell lines.
  35. Endocytome profiling uncovers cell-surface protein dynamics underlying neuronal connectivity.
  36. Structural basis for binding of RILPL1 to TMEM55B reveals a lysosomal platform for adaptor assembly through a conserved TBM motif.
  37. Protocol for label-free ribosome-associated protein enrichment from mammalian cells by RAPIDASH.
  38. Rethinking RNA-binding proteins: Riboregulation challenges prevailing views.
  39. FBXO9 mediated the ubiquitination and degradation of YAP in a GSK-3β-dependent manner.
  40. Get4/5-Mediated Remodeling of Get3's Substrate Binding Chamber: Insights into Tail-Anchored Protein Targeting by the GET Pathway.
  41. An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues.
  42. Proteo-transcriptomic reprogramming and resource reallocation define the aging mammalian brain.
  43. Dissecting the Cdc37 Co-chaperone Code: Functional Roles in Chaperone-Mediated Stress Adaptation.
  44. Pp1-87B/PPP1CC-JNK axis integrates apoptosis and ferroptosis-like cell death to regulate cell competition and tumorigenesis.
  45. Reconstitution of autophagic-like membrane tethering reveals that Atg11 can bind and cluster vesicles on cargo mimetics.
  46. Integrated stress response in Behçet disease: expression analyses in peripheral blood and synovial monocytes.
  47. Regulatory role of the N-terminal intrinsically disordered region of the DEAD-box RNA helicase DDX3X in selective RNA recognition.
  48. Conserved interfaces mediate multiple protein-protein interactions in a prokaryotic metabolon.
  49. MERS-CoV antagonizes PKR activation by inhibiting its condensation at viral replication complexes.
  50. Polysomes and mRNA control the biophysical properties of the eukaryotic cytoplasm.
  51. DKC1-mediated pseudouridylation of rRNA targets hnRNP A1 to sustain IRES-dependent translation and ATF4-driven metabolic adaptation.
  52. ATF6 activation alters colonic lipid metabolism causing tumour-associated microbial adaptation.
  53. The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation.
  1. Sci Adv. 2025 Sep 05. 11(36): eadx7172
    Deltex and RING-UIM E3 ligases cooperate to create a ubiquitin-ADP-ribose hybrid mark on tankyrase, promoting its stabilization.
    Jerome Perrard, Kevin Gao, Katherine Ring, Susan Smith.
      ADP-ribosylation can occur as mono-ADP-ribose (MAR) or be extended into poly-ADP-ribose (PAR). Tankyrase, a PAR transferase, adds PAR to itself and other proteins targeting them for proteasomal degradation via the PAR-binding E3 ligase RNF146. This degradation can be counteracted by RING-UIM E3 ligases RNF114 and RNF166, although the process is unclear. Here, we identify a mechanism that can regulate the balance between MAR and PAR on tankyrase to control degradation. We show that Deltex E3 ligases DTX2 and DTX3 catalyze monoubiquitylation of tankyrase in cells. This ubiquitylation occurs, not on a (canonical) lysine, but rather on MAR, creating a monoubiquitin-MAR hybrid mark. RNF114 and RNF166 recognize this mark using a unique hybrid reader domain and further diubiquitylate it. This ubiquitylation of MAR, which occurs near the ADP-ribose addition site, prevents PAR formation, antagonizing the action of the PAR-binding E3 ligase RNF146 and stabilizing tankyrase. These findings reveal an interplay between ubiquitin, ADP-ribose, and E3 ligases in cellular signaling.
    DOI:  https://doi.org/10.1126/sciadv.adx7172
  2. Sci Adv. 2025 Sep 05. 11(36): eady2211
    Cotranslational protein folding through non-native structural intermediates.
    Siyu Wang, Amir Bitran, Ekaterina Samatova, Eugene I Shakhnovich, Marina V Rodnina.
      Cotranslational protein folding follows a distinct pathway shaped by the vectorial emergence of the peptide and spatial constraints of the ribosome exit tunnel. Variations in translation rhythm can cause misfolding linked to disease; however, predicting cotranslational folding pathways remains challenging. Here, we computationally predict and experimentally validate a vectorial hierarchy of folding resolved at the atomistic level, where early intermediates are stabilized through non-native hydrophobic interactions before rearranging into the native-like fold. Disrupting these interactions destabilizes intermediates and impairs folding. The chaperone trigger factor alters the cotranslational folding pathway by keeping the nascent peptide dynamic until the full domain emerges. Our results highlight an unexpected role of surface-exposed residues in protein folding on the ribosome and provide tools to improve folding prediction and protein design.
    DOI:  https://doi.org/10.1126/sciadv.ady2211
  3. Nat Commun. 2025 Sep 02. 16(1): 8182
    Selective ubiquitination of drug-like small molecules by the ubiquitin ligase HUWE1.
    Barbara Orth, Pavel Pohl, Florian Aust, Yanlong Ji, Ayshwarya Seenivasan, Olexandr Dybkov, Xiaojun Julia Liang, Lars Bock, Florian Leidner, Sophie Levantovsky, Patrick Schardey, Pascal Sander, Nathanael J Disch, Masanja L Trautz, Athanasia Mizi, Argyris Papantonis, Christof Lenz, Helmut Grubmüller, Wieland Steinchen, Christian Behrends, Henning Urlaub, Matthias Gehringer, Sonja Lorenz.
      The ubiquitin system regulates eukaryotic physiology by modifying myriad substrate proteins. Substrate specificity and the assembly of ubiquitin signals are determined by ubiquitin ligases, some of which also modify non-protein biomolecules. Here we expand this substrate realm, revealing that the human ligase HUWE1 can target drug-like small molecules. We demonstrate that compounds previously reported as HUWE1 inhibitors present substrates of their target ligase. Compound ubiquitination is driven by the canonical catalytic cascade, linking ubiquitin to the compound's primary amino group. In vitro, the modification is selectively catalyzed by HUWE1, allowing the compounds to compete with protein substrates. We establish cellular detection methods, confirming HUWE1 promotes - but does not exclusively drive - compound ubiquitination in cells. Converting the existing compounds into specific HUWE1 substrates or inhibitors thus requires enhanced specificity. More broadly, our findings open avenues for harnessing the ubiquitin system to transform exogenous small molecules into novel chemical modalities within cells.
    DOI:  https://doi.org/10.1038/s41467-025-63442-x
  4. Biochem Pharmacol. 2025 Sep 02. pii: S0006-2952(25)00562-3. [Epub ahead of print] 117297
    Molecular glues and PROTACs in targeted protein degradation: mechanisms, advances, and therapeutic potential.
    Omar Eladl.
      Targeted protein degradation (TPD) is a transformative approach to drug discovery that enables the modulation of proteins previously considered "undruggable." Unlike traditional inhibitors, which transiently suppress protein activity, TPD harnesses the ubiquitin-proteasome system to selectively eliminate specific proteins and thereby fully abolish their activities. Two prominent approaches within TPD, Molecular Glues and PROteolysis TArgeting Chimeras (PROTACs), differ in both mechanism and therapeutic application. Molecular Glues are small molecules with low molecular weight that act as a molecular bridge, facilitating interaction between a target protein and an E3 ubiquitin ligase to enable degradation without requiring classical binding pockets. PROTACs are heterobifunctional small molecules that simultaneously engage a target protein and an E3 ligase to induce selective degradation through a catalytic mechanism. Both strategies have vastly expanded the druggable proteome and hold great promise for therapeutic interventions. This review provides a comparative overview of Molecular Glues and PROTACs, including their mechanisms, design principles, and therapeutic applications. We highlight their physicochemical properties, advantages, and limitations, as well as recent advances that are fueling the discovery of novel degraders. Through clinical advancements and case studies, we examine how these modalities are reshaping drug discovery and enabling new treatments for a variety of diseases.
    Keywords:  Chemical biology; Drug discovery; Molecular glues; PROTACs; Targeted protein degradation; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.bcp.2025.117297
  5. bioRxiv. 2025 Aug 20. pii: 2025.08.19.671158. [Epub ahead of print]
    Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22.
    Tian Qiu, Zhe Zhuang, Woong Sub Byun, Zuzanna Magdalena Kozicka, Kheewoong Baek, Jianing Zhong, Abby M Thornhill, Julia K Ryan, Katherine A Donovan, Eric S Fischer, Benjamin S Ebert, Nathanael S Gray.
      Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands is required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we describe AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioketal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in TPD.
    DOI:  https://doi.org/10.1101/2025.08.19.671158
  6. Science. 2025 Aug 28. 389(6763): 909-914
    Architecture of the UBR4 complex, a giant E4 ligase central to eukaryotic protein quality control.
    Daniel B Grabarczyk, Julian F Ehrmann, Paul Murphy, Woo Seok Yang, Robert Kurzbauer, Lillie E Bell, Luiza Deszcz, Jana Neuhold, Alexander Schleiffer, Alexandra Shulkina, Juyeon Lee, Jin Seok Shin, Anton Meinhart, Gijs A Versteeg, Eszter Zavodszky, Hyun Kyu Song, Ramanujan S Hegde, Tim Clausen.
      Eukaryotic cells have evolved sophisticated quality control mechanisms to eliminate aggregation-prone proteins that compromise cellular health. Central to this defense is the ubiquitin-proteasome system, where UBR4 acts as an essential E4 ubiquitin ligase, amplifying degradation marks on defective proteins. Cryo-electron microscopy analysis of UBR4 in complex with its cofactors KCMF1 and CALM1 reveals a massive 1.3-megadalton ring structure, featuring a central substrate-binding arena and flexibly attached catalytic units. Our structure shows how UBR4 binds substrate and extends lysine-48-specific ubiquitin chains. Efficient substrate targeting depends on both preubiquitination and specific N-degrons, with KCMF1 acting as a key substrate filter. The architecture of the E4 megacomplex is conserved across eukaryotes, but species-specific adaptations allow UBR4 to perform its precisely tuned quality control function in diverse cellular environments.
    DOI:  https://doi.org/10.1126/science.adv9309
  7. Elife. 2025 Aug 28. pii: RP105937. [Epub ahead of print]14
    C-terminal tagging, transmembrane domain hydrophobicity, and an ER retention motif influence the secretory trafficking of the inner nuclear membrane protein emerin.
    Jessica Mella, Regan F Volk, Balyn W Zaro, Abigail Buchwalter.
      The inner nuclear membrane (INM), a subdomain of the endoplasmic reticulum (ER), sequesters hundreds of transmembrane proteins within the nucleus. We previously found that one INM protein, emerin, can evade the INM by secretory transport to the lysosome, where it is degraded (Buchwalter et al., 2019). In this work, we used targeted mutagenesis to identify intrinsic sequences that promote or inhibit emerin's secretory trafficking. By manipulating these sequences across several tag and expression level combinations, we now find that emerin's localization is sensitive to C-terminal GFP tagging. While emerin's long, hydrophobic C-terminal transmembrane domain facilitates trafficking to the lysosome, extending its lumenal terminus with a GFP tag biases the protein toward this pathway. In contrast, we identify a conserved ER retention sequence that stabilizes N- and C-terminally tagged emerin by limiting its lysosomal flux. These findings underscore long-standing concerns about tagging artifacts and reveal novel determinants of tail-anchored INM protein targeting.
    Keywords:  cell biology; human; inner nuclear membrane; membrane protein; mouse; protein tagging; secretory trafficking
    DOI:  https://doi.org/10.7554/eLife.105937
  8. bioRxiv. 2025 Aug 30. pii: 2025.08.29.673087. [Epub ahead of print]
    Structural basis of NSD2 degradation via targeted recruitment of SCF-FBXO22.
    Kevin C Robertson, Sascha J Amann, Tongkun Liu, Adam V Funk, Xianxi Wang, Irina Grishkovskaya, John R Tabor, Jacqueline L Norris-Drouin, Cheryl H Arrowsmith, Jon L Collins, Michael J Emanuele, David Haselbach, Lindsey I James, Nicholas Gene Brown.
      Targeted protein degradation (TPD) through the ubiquitin-proteasome system is driven by compound-mediated polyubiquitination of a protein-of-interest by an E3 ubiquitin (Ub) ligase. To date, relatively few E3s have been successfully utilized for TPD and the governing principles of functional ternary complex formation between the E3, degrader, and protein target remain elusive. FBXO22 has recently been harnessed by several groups to target different proteins for degradation. FBXO22 recruitment has been enabled through degraders that covalently modify its cysteine residues. Here, we reveal that the aldehyde derivative of UNC10088 promotes cooperative binding of FBXO22 to NSD2, a histone methyltransferase and oncogenic protein, leading to a cryo-EM structure of the full SKP1-CUL1-F-box (SCF)-FBXO22 complex with NSD2. This structure revealed a conformational change in the FBXO22 loop surrounding C326, further exposing the cysteine for covalent recruitment. Additional medicinal chemistry efforts led to the discovery of benzaldehyde-based non-prodrug degraders that similarly engage C326 of FBXO22 and potently degrade NSD2. Furthermore, unlike many degraders, our molecules recruit NSD2 to a different surface of FBXO22 than the known FBXO22 substrate BACH1, allowing for concurrent complex formation and degradation of both the neosubstrate and endogenous substrates. Overall, we demonstrate the biochemical and structural basis for NSD2 degradation, revealing key principles for efficient and selective TPD by SCF-FBXO22.
    DOI:  https://doi.org/10.1101/2025.08.29.673087
  9. bioRxiv. 2025 Aug 22. pii: 2025.08.20.671298. [Epub ahead of print]
    Single-Assay Characterization of Ternary Complex Assembly and Activity in Targeted Protein Degradation.
    Corey H Yu, Vi Dougherty, Dongwen Lv, Pirouz Ebadi, Shikha Dubey, Digant Nayak, Anindita Nayak, Daohong Zhou, Shaun K Olsen, Dmitri N Ivanov.
      Targeted protein degradation (TPD) is a rapidly advancing therapeutic strategy that selectively eliminates disease-associated proteins by co-opting the cell's protein degradation machinery. Covalent modification of proteins with ubiquitin is a critical event in TPD, yet the analytical tools for quantifying the ubiquitination kinetics have been limited. Here, we present a real-time, high-throughput fluorescent assay utilizing purified, FRET-active E2-Ub conjugates to monitor ubiquitin transfer. This assay is highly versatile, requiring no engineering of the target protein or ligase, thereby accelerating assay development and minimizing the risk of artifacts. The single-step, single-turnover nature of the monitored reaction enables rigorous and quantitative analysis of ubiquitination kinetics. We show that this assay can be used to measure key degrader characteristics such as degrader affinity for the target protein, degrader affinity for the ligase, affinity of ternary complex assembly, and catalytic efficiency of the ternary complex. The high sensitivity and accuracy of this comprehensive, single-assay approach to ternary complex characterization will empower the discovery and optimization of heterobifunctional degraders and molecular glues.
    DOI:  https://doi.org/10.1101/2025.08.20.671298
  10. bioRxiv. 2025 Aug 30. pii: 2025.08.27.672666. [Epub ahead of print]
    Processing bodies promote lysosomal quality control and cell survival during recovery from lysosomal damage.
    Jingyue Jia, Jacob Duran, Li Chen, Jing Pu, Pavel Ivanov.
      Lysosomes are essential for cell survival but are highly susceptible to diverse physical and pathological stressors. Thus, the ability to initiate an acute damage response and promote recovery after stressor resolution is critical for maintaining cellular homeostasis and viability. Although recent studies have advanced our understanding of acute responses to lysosomal injury, the molecular mechanisms governing the recovery stage and distinguishing it from the acute phase remain poorly defined. Here, we delineate a key difference between these two stages in translational regulation and uncover lysosomal recovery from acute damage as a novel trigger for processing body (PB) formation. PBs are membraneless biomolecular condensates involved in RNA metabolism and translational reprogramming. We provide the first evidence that PBs are critical for lysosomal quality control and cell survival during recovery. Mechanistically, PBs are induced selectively during the recovery phase, but not during the acute damage response, through interactions with stress granules (SGs), distinct membraneless biomolecular condensates formed upon acute injury to stabilize damaged lysosomal membranes for repair. Functional analyses reveal that PBs promote lysosomal quality control by collaborating with SG-mediated membrane stabilization, while independently recruiting released cathepsins, thereby collectively supporting cell survival. Together, these findings establish PBs as central effectors of the lysosomal recovery program and underscore the broader relevance of biomolecular condensates in cellular responses to lysosomal damage and related disease processes.
    DOI:  https://doi.org/10.1101/2025.08.27.672666
  11. Cell. 2025 Aug 22. pii: S0092-8674(25)00920-1. [Epub ahead of print]
    A split-site E3 ligase mechanism enables ZNFX1 to ubiquitinate and cluster single-stranded RNA into ubiquitin-coated nucleoprotein particles.
    Daniel B Grabarczyk, Eric J Aird, Vanessa Reznikow, Paul C Kirchgatterer, Julian F Ehrmann, Robert Kurzbauer, Lillie E Bell, Max J Kellner, Ritika Aggarwal, Alexander Schleiffer, Victoria Faas, Luiza Deszcz, Anton Meinhart, Gijs A Versteeg, Josef M Penninger, Lukas S Stelzl, Moritz M Gaidt, Ingrid Tessmer, Jacob E Corn, Tim Clausen.
      Eukaryotic cells use a multi-layered immune response to combat intracellular pathogens. The ubiquitin ligase ZNFX1 has emerged as a crucial yet little understood player that regulates the immune response while protecting against RNA viruses. Our study unveils the molecular mechanism of ZNFX1, mediated by the joint activity of a helicase serving as a nucleic acid sensor and a non-conventional E3 module featuring a split active site. We demonstrate that single-stranded RNA stimulates E3 activity by fostering dimerization of ZNFX1 subunits that translocate along nucleic acid tracks. Juxtaposed E3 domains complement each other, leading to the ubiquitination of ZNFX1 itself and engaged RNA molecules, while clustering nucleic acids into dense nucleoprotein particles. We show that the E3 ligase activity of ZNFX1 protects cells during an immune response and propose that ubiquitin-coated particles formed by ZNFX1 represent part of an ancient mechanism to regulate both foreign and host RNA in the cell.
    Keywords:  E3 ubiquitin ligase; ZNFX1; helicase; innate immunity; non-canonical ubiquitination; nucleic acid sensor
    DOI:  https://doi.org/10.1016/j.cell.2025.08.006
  12. Nat Commun. 2025 Aug 28. 16(1): 8025
    Structural basis of VCP-VCPIP1-p47 ternary complex in Golgi maintenance.
    Binita Shah, Moritz Hunkeler, Ariana Bratt, Hong Yue, Isabella Jaen Maisonet, Eric S Fischer, Sara J Buhrlage.
      VCP/p97 regulates a wide range of cellular processes, including post-mitotic Golgi reassembly. In this context, VCP is assisted by p47, an adapter protein, and VCPIP1, a deubiquitylase (DUB). However, how they organize into a functional ternary complex to promote Golgi assembly remains unknown. Here, we use cryo-EM to characterize both VCP-VCPIP1 and VCP-VCPIP1-p47 complexes. We show that VCPIP1 engages VCP through two interfaces: one involving the N-domain of VCP and the UBX domain of VCPIP1, and the other involving the VCP D2 domains and a region of VCPIP1 we refer to as VCPID. The p47 UBX domain competitively binds to the VCP N-domain, while not affecting VCPID binding. We show that VCPID is critical for VCP-mediated enhancement of DUB activity and proper Golgi assembly. The ternary structure along with biochemical and cellular data provides new insights into the complex interplay of VCP with its co-factors.
    DOI:  https://doi.org/10.1038/s41467-025-63161-3
  13. Nat Rev Drug Discov. 2025 Sep 03.
    Potentiating the integrated stress response.
    Sarah Crunkhorn.
      
    DOI:  https://doi.org/10.1038/d41573-025-00147-2
  14. Adv Sci (Weinh). 2025 Aug 30. e09817
    K29-Linked Ubiquitination of Transcription Regulators Controls Cell Proliferation in the Unfolded Protein Response.
    Qiushuang Zhang, Xucong Teng, Yicong Dai, Yuncong Wu, Hongwei Hou, Jinghong Li.
      The ubiquitin chains perform diverse biological functions through different linkages. However, the understanding of non-canonical K29-linked ubiquitin chains is relatively limited. Exploring the physiological functions of K29-linked ubiquitin chains beyond degradation is crucial for deciphering the ubiquitin chain code, which is essential for understanding cellular physiology. The unfolded protein response (UPR) serves as a crucial mechanism for cells to cope with endoplasmic reticulum stress and involves comprehensive and precise regulation. Ubiquitin, as a regulator of protein function, has potential regulatory functions other than guiding protein degradation in the UPR. Here, a close association is revealed between K29-linked ubiquitin chains and transcriptional regulation during the UPR. After UPR induction, the K29-linked ubiquitination of the SMC1A and SMC3 proteins in the cohesin complex increases. The transcription of cell proliferation-related genes, such as SERTAD1 and NUDT16L1, is regulated by the K29-linked ubiquitination of cohesin. Overall, the upregulation of K29-linked ubiquitination of cohesin during the UPR disrupts the formation of the transcription initiation complex, resulting in the transcriptional downregulation of cell proliferation-related genes.
    Keywords:  K29‐linked ubiquitination; cohesin complex; transcription regulation; unfolded protein response
    DOI:  https://doi.org/10.1002/advs.202509817
  15. J Biol Chem. 2025 Aug 28. pii: S0021-9258(25)02501-3. [Epub ahead of print] 110649
    Mannose trimming is the dominant signal for the release of misfolded glycoproteins from ER quality control.
    Yun-Ji Shin, Ulrike Vavra, Daniel Maresch, Clemens Grünwald-Gruber, Richard Strasser.
      N-glycosylation is essential for protein folding in the endoplasmic reticulum (ER). Glycan attachment facilitates the binding of newly synthesised polypeptides to calnexin and calreticulin, two ER-resident lectins that act as chaperones and promote folding. The regulatory mechanism underlying this process is dictated by the glycan composition, and this study has elucidated the function of mannose trimming in the release of misfolded glycoprotein from ER quality control and subsequent transfer to ER-associated degradation (ERAD) in plants. Co-immunoprecipitation experiments were performed to investigate the interaction between the ERAD substrate SUBEX-C57Y and ER-resident lectins, providing evidence for a specific interaction with calnexin. Furthermore, the results show that the expression of calnexin and calreticulin can specifically inhibit the degradation of glycosylated ERAD substrates. However, overexpression of the α-mannosidase MNS4 was shown to overcome lectin-dependent accumulation and promote degradation via ERAD. MS-based analysis of released N-glycans and jack bean α-mannosidase-digested glycopeptides revealed the presence of monoglucosylated N-glycans even after mannose trimming. This finding is consistent with the observation that calnexin remained associated with SUBEX-C57Y after mannose removal from the C-branch of the N-glycan. In contrast, specific removal of the first α1,2-linked mannose from the A-branch or a block of glucose trimming of Glc3Man9GlcNAc2 prevented the interaction with calnexin. In conclusion, the study provides fundamental insights into the specific role of glycan residues for the interdependence between lectin-based ER quality control and glycan-dependent ERAD in plants.
    Keywords:  Cell biology; N-linked glycosylation; endoplasmic reticulum (ER); endoplasmic reticulum‐associated protein degradation (ERAD); glycoprotein; plant; protein degradation
    DOI:  https://doi.org/10.1016/j.jbc.2025.110649
  16. bioRxiv. 2025 Aug 07. pii: 2025.08.05.668774. [Epub ahead of print]
    The endoplasmic reticulum autophagy receptor TEX264 drives epidermal differentiation and is dysregulated in Darier disease.
    Christopher J Johnson, Afua Tiwaa, Arti Parihar, Edwin Antonio, Brooke D Lorenz, Reeteka Kudallur, Aaron Ramonett, Anthony Coon, Mrinal K Sarkar, Johann E Gudjonsson, Cory L Simpson.
      Differentiating keratinocytes break down their organelles and nuclei to become the compacted cornified layers of the epidermal barrier in a poorly understood catabolic process. Live confocal imaging of stratified human organotypic epidermis revealed endoplasmic reticulum (ER) fragmentation and lysosomal engulfment in the cornifying layers, where we found up-regulation of TEX264, a receptor that mediates selective autophagy of the ER (reticulophagy). TEX264 expression was increased by ER stress, which caused precocious cornification of organotypic epidermis. In undifferentiated keratinocytes, ectopic TEX264 was sufficient to fragment the ER, while in highly differentiated keratinocytes, it accelerated ER elimination and induced nuclear shrinkage; these effects were abolished by mutating the LC3 interacting region required for its autophagic function. Knockout of TEX264 or inhibiting its activation disrupted maturation of organotypic cultures, pointing to a critical role for reticulophagy in cornification. Finally, in patient biopsies and an organotypic model of Darier disease, a genetic cornification disorder linked to ER dysfunction, we found increased TEX264 in areas of premature cornification (dyskeratosis). Our results identified TEX264 as a key driver of epidermal differentiation and led us to propose a novel model of cornification in which keratinocytes activate selective autophagy receptors to orchestrate orderly organelle elimination during cutaneous barrier formation.
    GRAPHICAL ABSTRACT:
    DOI:  https://doi.org/10.1101/2025.08.05.668774
  17. Autophagy. 2025 Aug 27. 1-3
    Salmonella Typhimurium exploits the reticulophagy/ERphagy receptor RETREG1 to promote infection.
    Damian Gatica, Reham Alsaadi, Ryan C Russell.
      Macroautophagy/autophagy is a key catabolic-recycling pathway that can selectively target damaged organelles or invading pathogens for degradation. The selective autophagic degradation of the endoplasmic reticulum, called reticulophagy/ERphagy, controls ER size and degradation of misfolded protein aggregates. RETREG1/FAM134B is an ERphagy receptor that acts by inducing ER membrane curvature and scission through oligomerization. Interestingly, RETREG1 has also been implicated in the cellular response against pathogen infection. Multiple microbes have developed strategies to inhibit ERphagy by targeting RETREG1. In a recent study, we characterized an unidentified mechanism of bacterial-mediated inhibition of ERphagy. Specifically, we found that Salmonella enterica Serovar Typhimurium, a well-known intracellular pathogen that continues to be a major cause of foodborne infections worldwide, inhibits ERphagy by specifically targeting the activity of RETREG1, leading to a pronounced increase in Salmonella burden. We show that Salmonella prevents RETREG1 oligomerization, which is required for efficient ERphagy. Conversely, Salmonella-mediated ERphagy blockage can be bypassed by promoting RETREG1 oligomerization, which recovers ERphagy levels. Salmonella infection also decreases RETREG1 phosphorylation and acetylation, previously reported to be requisite steps in RETREG1-driven ERphagy. Furthermore, in vivo analysis of retreg1 knockout mice infected with Salmonella reveals increased intestinal damage and bacterial levels. Our results provide insights into the interplay between ERphagy and bacterial infection, highlighting a key role for RETREG1 in innate immunity.
    Keywords:  Bacteria; FAM134B; SopF; endoplasmic reticulum; xenophagy
    DOI:  https://doi.org/10.1080/15548627.2025.2551672
  18. Contact (Thousand Oaks). 2025 Jan-Dec;8:8 25152564251372673
    Via Proteins and Lipids - Versatility of VAPs at Dynamic Membrane Contact Sites.
    Dan Zhang.
      VAMP-associated proteins (VAPs) are highly conserved, endoplasmic reticulum (ER)-resident receptors that tether the ER to various membrane compartments in eukaryotic cells. Each VAP contains a transmembrane helix at its extreme C-terminus and a conserved N-terminal major sperm protein (MSP) domain that mediates various cytosolic interactions via both protein and lipid binding. Here, I question the fundamental difference between protein- and lipid-based associations in VAP-driven membrane contact site (MCS) formation and function - could the lipid affinity of VAPs be an overlooked factor in MCS dynamic regulation?
    Keywords:  FFAT-related motif; MSP domain; VAP; anionic phospholipid; membrane contact plasticity; membrane contact site
    DOI:  https://doi.org/10.1177/25152564251372673
  19. Nat Commun. 2025 Aug 29. 16(1): 8089
    Characterization of PROTAC specificity and endogenous protein interactomes using ProtacID.
    Suman Shrestha, Matthew E R Maitland, Laili Jing, Shili Duan, David Y Nie, Jonathan St-Germain, Michael Kanaris, Dalia Barsyte-Lovejoy, Cheryl H Arrowsmith, Brian Raught.
      Here we describe ProtacID, a flexible BioID (proximity-dependent biotinylation)-based approach to identify PROTAC-proximal proteins in living cells. ProtacID analysis of VHL- and CRBN-recruiting PROTACs targeting a number of different proteins (localized to chromatin or cellular membranes, and tested across six different human cell lines) demonstrates how this technique can be used to validate PROTAC degradation targets and identify non-productive (i.e. non-degraded) PROTAC-interacting proteins, addressing a critical need in the field of PROTAC development. We also demonstrate that ProtacID can be used to characterize native, endogenous multiprotein complexes without the use of antibodies, or modification of the protein of interest with epitope tags or biotin ligase tagging.
    DOI:  https://doi.org/10.1038/s41467-025-63357-7
  20. Nat Commun. 2025 Aug 29. 16(1): 8077
    UBE3A promotes foam cell formation and counters remyelination by targeting ABCA1 for proteasomal degradation.
    Melanie Loix, Sam Vanherle, Laura Bolkaerts, Sanne G S Verberk, Mattijs Punt, Flore Wouters, Brecht Moonen, Rob Verhagen, Suzanne A E Van Wouw, Aldo Jongejan, Ben Distel, Ype Elgersma, Mansour Haidar, Noam Zelcer, Jerome J A Hendriks, Jeroen F J Bogie.
      The accumulation of foamy macrophages is a pathological hallmark of demyelinating brain disorders. Perturbed metabolism and efflux of intracellular lipids underlie the development of a harmful foamy macrophage phenotype in these disorders, yet, the molecular mechanisms underlying this dysregulation are poorly understood. Here, we show that the ubiquitin-proteasome system controls the turnover of the cholesterol efflux transporter ATP-binding cassette A1 (ABCA1) in lipid-loaded macrophages in the brain. We report that accumulation of myelin-derived lipids promotes the abundance and activity of ubiquitin-protein E3 ligase A (UBE3A) in macrophages, which stimulates ABCA1 ubiquitination and subsequent degradation. This boosts cellular lipid accumulation and induces an inflammatory macrophage phenotype that impairs remyelination. We further establish Tat-interacting protein 30 (TIP30), an inhibitor of importin β-mediated nuclear import, as an essential regulator of cytosolic UBE3A levels. Together, our findings identify UBE3A as a driver of foam cell formation and indicate that targeting UBE3A-mediated ABCA1 degradation is a promising strategy to enhance central nervous system repair.
    DOI:  https://doi.org/10.1038/s41467-025-62053-w
  21. Am J Pathol. 2025 Aug 26. pii: S0002-9440(25)00300-1. [Epub ahead of print]
    The Proteostasis Network in Proteinopathies: Mechanisms and Interconnections.
    Dariusz Pytel, Jody Fromm Longo.
      Proteinopathies are neurodegenerative disorders that are characterized by accumulation of misfolded toxic protein aggregates that lead to synaptic and neuronal dysfunction. Though genetically, clinically and pathologically distinct, a common feature of these diseases is disruption of protein homeostasis (proteostasis), which causes accumulation of misfolded proteins. The machinery mediating proteostasis exquisitely balances and interlaces protein synthesis, protein folding and trafficking, and protein degradation processes within the proteostasis network to maintain homeostasis. The proteostasis network governs a functional and dynamic proteome by modulating the timing, location, and stoichiometry of protein expression, surveillance and maintenance of protein folding and removal of misfolded or excess proteins. Although a functional proteome is essential for the health of all cell types, this is especially true for neurons which are prone to enhanced cellular stress. Aging is the most important risk factor for proteostasis decline and the development of proteinopathies. However, germline and somatic mutations can also functionally impair components of the proteostasis network. Post-mitotic cells, particularly neurons, are rendered further susceptible to proteostasis dysfunction due to their extended lifespan. This review discusses the interconnections between the functional components mediating proteostasis in neuronal cells and how aberrations in proteostasis contribute to neuronal dysfunction and disease.
    Keywords:  Alzheimer’s Disease; Amyotrophic Lateral Sclerosis; ER stress; ERAD; Frontotemporal Dementia; Huntington’s Disease; Parkinson’s Disease; UPR; aggregates; autophagy; protein homeostasis; proteinopathies
    DOI:  https://doi.org/10.1016/j.ajpath.2025.07.011
  22. Commun Biol. 2025 Aug 30. 8(1): 1323
    In-cell proximity target validation methods for heterobifunctional molecules with CRBN- or VHL-binder using AirID.
    Kohdai Yamada, Satoshi Yamanaka, Hiroyuki Yamakoshi, Aki Kohyama, Yoshiharu Iwabuchi, Hidetaka Kosako, Tatsuya Sawasaki.
      Heterobifunctional molecules, such as proteolysis-targeting and autophagy-targeting chimera, represent new drug concepts. They are composed of two protein binders that can induce proximity interactions between two proteins and protein catalysis. Currently, cereblon (CRBN)- and von Hippel-Lindau (VHL)-binders with thalidomide- and VH032-backbones are widely used as E3 ligase binders. Here, we developed a method to validate proteins that interact with heterobifunctional molecules in cells using AirID, a proximity biotinylation enzyme. Interactome of target proteins was validated for six heterobifunctional molecules. ThBD-AirID, a fusion of the thalidomide-binding domain (ThBD) of CRBN and AirID, effectively biotinylated the target proteins. AirID fused to full-length VHL also exhibited highly effective biotinylation. Heterobifunctional molecules with the same target binder but different E3 binders showed different proximity interactome profiles in cells. Analysis using ThBD-AirID revealed a nuclear interaction between androgen receptor and ARV-110. AirID-fused ThBD and VHL could be useful for validating the heterobifunctional molecular interactome in cells.
    DOI:  https://doi.org/10.1038/s42003-025-08761-x
  23. Geroscience. 2025 Sep 01.
    Failure of efficient cardiac proteostatic adaptations to chronic cAMP-stress is associated with accelerated heart aging.
    Maria Grazia Perino, Miguel Calvo-Rubio Barrera, Daniel R Riordon, Giulio Agnetti, Alexander Maltsev, Admira Parveen, Christopher H Morrell, Ismayil Ahmet, Khalid Chakir, Yelena S Tarasova, Jia-Hua Qu, Kirill V Tarasov, Alexey E Lyashkov, Yevgeniya O Lukyanenko, Hikmet Kadioglu, Mark Ranek, Rafael De Cabo, Edward G Lakatta.
      Dysregulated proteostasis is a hallmark of aging. We investigated how efficiently proteostatic adaptations to chronic cardiac cyclic-adenosine-monophosphate (cAMP)-dependent stress change with aging in mice harboring marked cardiac-specific over-expression of adenylyl cyclase VIII (TGAC8). We assessed protein quality control mechanisms (PQC) (ubiquitin proteasome system, autophagic flux via macroautophagy, and mitophagy) in left ventricles of TGAC8 and wild-type littermates (WT) at 3-4 and 17-21 months of age. At 3-4 months, TGAC8 exhibited markers of increased autophagic flux (microtubule-associated protein 1A/1B light chain 3B (LC3), p62, and their phospho-forms) and enhanced canonical mitophagy signaling (PARKIN, p62S405 and p62S349 receptors), confirming a more efficient proteostasis, vs WT. In aged TGAC8, however, the PQC mechanisms were overwhelmed by proteotoxic stress, manifested in insufficient proteasome activity, slower autophagic flux, and increased mitochondrial dysfunction (network fragmentation). The accumulation of protein aggregates (increased ratio of insoluble/soluble protein fractions), of lipofuscin bodies and of desmin cardiac preamyloid oligomers, and of LC3+- and p62+-inclusions of aberrant sizes was increased in aged TGAC8 compared to young TGAC8. Thus, while increased proteostatic mechanisms maintain cardiac health in TGAC8 in youth (3-4 months), long-term exposure to sustained activation of the AC/cAMP/PKA/Ca2+ signaling axis results in severe proteostasis insufficiency in aged TGAC8, leading to cardiomyopathy and accelerated cardiac aging.
    Keywords:  Aging; Dysregulated proteostasis; Protein quality control
    DOI:  https://doi.org/10.1007/s11357-025-01851-y
  24. Cell. 2025 Aug 25. pii: S0092-8674(25)00916-X. [Epub ahead of print]
    Proximity-specific ribosome profiling reveals the logic of localized mitochondrial translation.
    Jingchuan Luo, Stuti Khandwala, Jingjie Hu, Song-Yi Lee, Kelsey L Hickey, Zebulon G Levine, J Wade Harper, Alice Y Ting, Jonathan S Weissman.
      Localized translation broadly enables spatiotemporal control of gene expression. Here, we present LOV-domain-controlled ligase for translation localization (LOCL-TL), an optogenetic approach for monitoring translation with codon resolution at any defined subcellular location under physiological conditions. Application of LOCL-TL to mitochondrially localized translation revealed that ∼20% of human nuclear-encoded mitochondrial genes are translated on the outer mitochondrial membrane (OMM). Mitochondrially translated messages form two classes distinguished by encoded protein length, recruitment mechanism, and cellular function. An evolutionarily ancient mechanism allows nascent chains to drive cotranslational recruitment of long proteins via an unanticipated bipartite targeting signal. Conversely, mRNAs of short proteins, especially eukaryotic-origin electron transport chain (ETC) components, are specifically recruited by the OMM protein A-kinase anchoring protein 1 (AKAP1) in a translation-independent manner that depends on mRNA splicing. AKAP1 loss lowers ETC levels. LOCL-TL thus reveals a hierarchical strategy that enables preferential translation of a subset of proteins on the OMM.
    Keywords:  AKAP1; OXPHOS; cis-element analysis; cotranslational targeting; localized translation; mitochondrial bipartite targeting signal; outer mitochondrial membrane; oxidative phosphorylation; translation-independent mRNA targeting
    DOI:  https://doi.org/10.1016/j.cell.2025.08.002
  25. Autophagy. 2025 Sep 02. 1-3
    VAMP8 stabilization by DRAM1 enables autophagosome-lysosome fusion and promotes metastatic extravasation.
    Rui Zhang, Vincenzo Torraca, Chaojun Yan, Hao Lyu, Shuai Xiao, Dong Guo, Qi Zhang, Cefan Zhou, Jingfeng Tang.
      Autophagosome-lysosome fusion, essential for macroautophagy/autophagy completion, requires the STX17-SNAP29-VAMP8 SNARE complex. While VAMP8 is crucial, its regulatory mechanisms remain incompletely understood. Here, we identify DRAM1 (DNA damage regulated autophagy modulator 1) as a key interactor and stabilizer of VAMP8 on lysosomes. In this study, we demonstrated that DRAM1 directly binds VAMP8, and this interaction is enhanced during autophagy induction. Mechanistically, DRAM1 inhibits ubiquitin-mediated degradation of lysosomal VAMP8 by the E3 ligase STUB1/CHIP to enhance autolysosome formation. DRAM1 competitively binds VAMP8 within residues 66-100 aa, shielding lysines 68, 72, and 75 from STUB1-mediated ubiquitination. This stabilization promotes assembly of the STX17-SNAP29-VAMP8 complex, enhancing autophagosome-lysosome fusion. Functionally, DRAM1-mediated VAMP8 stabilization and autophagic flux promote the extravasation and metastasis of hepatocellular carcinoma (HCC) cells in vitro and in vivo (mouse and zebrafish models). Depletion of core autophagy genes (ATG5 or ATG7) abolishes DRAM1's pro-metastatic effects. Our findings reveal a novel DRAM1-VAMP8 axis that regulates autophagic flux and identify DRAM1 as a potential therapeutic target for inhibiting autophagy-dependent HCC metastasis. Here, we summarize our findings and discuss their implications for our understanding of autophagy regulation.
    Keywords:  Autophagosome-lysosome fusion; DRAM1; STUB1; VAMP8; extravasation; ubiquitination
    DOI:  https://doi.org/10.1080/15548627.2025.2554794
  26. Sci Adv. 2025 Sep 05. 11(36): eadt4170
    Computational design of potent and selective binders of BAK and BAX.
    Stephanie Berger, Erinna F Lee, Tiffany J Harris, Sharon Tran, Asim K Bera, Lauren Arguinchona, Alex Kang, Banumathi Sankaran, Sila Kasapgil, Michelle S Miller, Sean Smyth, Mariam Lutfi, Rachel T Uren, Ruth M Kluck, Peter M Colman, Walter D Fairlie, Peter E Czabotar, David Baker, Richard W Birkinshaw.
      Potent and selective binders of the key proapoptotic proteins BAK and BAX have not been described. We use computational protein design to generate high affinity binders of BAK and BAX with greater than 100-fold specificity for their target. Both binders activate their targets when at low concentration, driving pore formation, but inhibit membrane permeabilization when in excess. Crystallography shows that the BAK binder induces BAK unfolding, exposing the α6 helix and BH3 domain. Together, these data suggest that upon binding, BAK or BAX unfold; at high binder concentrations, self-association of the partially folded BAK or BAX proteins is blocked and the membrane remains intact, whereas at low concentrations, dimers form, and the membrane ruptures. Our designed binders modulate apoptosis via direct, specific interactions with BAK and BAX and reveal that for therapeutic strategies targeting BAK and BAX, inhibition requires saturating binder concentrations at the site of action.
    DOI:  https://doi.org/10.1126/sciadv.adt4170
  27. Protein Sci. 2025 Aug;34(8): e70248
    An alternative pocket for binding the N-degrons by the UBR1 and UBR2 ubiquitin E3 ligases.
    Shih-Ting Huang, Dai-Hua Chen, Tianchen Ren, Nicole Thomas, Jian Wu, Banumathi Sankaran, Renee Jones, Susan Taylor, Yuan Chen.
      The UBR family of ubiquitin ligases binds to N-termini of their targets (known as N-degron) to induce their ubiquitination and degradation via a conserved domain known as UBR-box. UBR1 and UBR2 share the highest sequence homology among the family, and substantial structural studies were previously performed for substrate binding by the UBR-boxes of UBR1 and UBR2. Here, we describe a new pocket in the UBR-boxes of UBR1 and UBR2 for binding the second residues of N-degrons through determining five co-crystal structures of the UBR-boxes with various N-degron peptides. Together with binding affinities measured by fluorescence polarization, we show that the two highly homologous UBR-boxes can interact with the second residue of an N-degron differently. In addition, the UBR-boxes undergo different conformational changes when binding N-degrons. Furthermore, we demonstrate that the sidechain of the third amino acid of an N-degron has no contribution to binding the UBR-boxes. These findings represent a new conceptual advancement for the UBR E3 ligases and the new insights described here can be leveraged for developing their selective ligands for research and potential therapies.
    Keywords:  N‐end rule; UBR1; UBR2; crystal structure; fluorescence polarization; ubiquitin
    DOI:  https://doi.org/10.1002/pro.70248
  28. Mol Cancer Ther. 2025 Sep 05. OF1-OF13
    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 signaling pathway that initiates adaptive responses during nutrient stress conditions. Although pharmacologic 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 integrated stress response 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 affect 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
  29. J Biol Chem. 2025 Aug 28. pii: S0021-9258(25)02495-0. [Epub ahead of print] 110643
    The cytoplasmic domain of the pseudoprotease iRhom2 mediates distinct signaling mechanisms to control activation of the cell surface protease ADAM17.
    Fangfang Lu, Marjorie Fournier, Matthew Freeman.
      ADAM17 is a cell surface protease that controls the release of the ectodomains of signaling proteins including EGFR ligands and the primary inflammatory cytokine TNF. Reflecting this important role in signaling, dysregulated ADAM17 activity is linked to many human diseases including immunodeficiency, inflammatory bowel disease (IBD), rheumatic arthritis, cancer, and Alzheimer's disease. iRhom2, a pseudoprotease of the rhomboid-like superfamily, has evolved to be a multifunctional regulatory co-factor of ADAM17. Recent structural and functional work has begun to reveal how the iRhom2 transmembrane and extracellular domains act to control ADAM17 activity. The cytoplasmic domain, however, remains less explored. Here, using a combination of proteomic, genetic and biochemical approaches, we report three distinct mechanisms by which the cytoplasmic domain of iRhom2 contributes to ADAM17 regulation. First, upon oncogenic KRAS signaling, the serine/threonine kinase RSK2 is recruited to the iRhom2 cytoplasmic N-terminus, and coordinates with phosphorylated ERK to activate the iRhom2/ADAM17 sheddase complex. Second, we show that iRhom2 may have an inhibitory function on ADAM17 at the cell surface: stabilising iRhom2 at cell surface by overexpressing iRhom2's cytoplasmic binding partner, FRMD8, inhibits PMA-stimulated ADAM17 activity. Third, we have identified a previously undefined motif (RKR) in the iRhom2 cytoplasmic domain that represses unstimulated ADAM17 activity. Overall, these findings reveal the complex regulatory system by which the iRhom2 cytoplasmic tail transduces cellular signals to regulate ADAM17 activation, potentially paving the way towards understanding and possibly manipulating the iRhom2/ADAM17 complex in health and disease.
    Keywords:  ADAM17; Cell Surface; FRMD8; KRAS; Membrane protein; RSK; Shedding; iRhom2
    DOI:  https://doi.org/10.1016/j.jbc.2025.110643
  30. FEBS J. 2025 Sep 02.
    Autophagy and exosomes play different roles in the disposal of unwanted cellular materials.
    Kedan Mao, Lin Wei, Fangfang Huo, Sidong Xiong, Yuxuan Fu.
      Degradative autophagy supplies a source of nutrients and energy by digesting cytoplasmic components. Additionally, it eliminates toxic protein aggregates and defective organelles from cells. Exosomes are small vesicles that are released by cells into the extracellular environment and are also involved in maintenance of homeostasis by removing unwanted materials and intracellular pathogens. Nevertheless, it remains unclear how these two processes may differ or are alike in their roles in maintaining intracellular homeostasis. In this study, we found that secretory exosomes served as a quality control mechanism, maintaining intracellular RNA homeostasis by facilitating both the selective packaging of endogenous and exogenous RNA species. Conversely, autophagic degradation primarily functions to dispose of both endogenous and exogenous proteins, resulting in controlling intracellular proteostasis. The depletion of exosome secretion resulted in prolonged accumulation of exogenous RNA within the cells, whereas it had no significant effect on the accumulation of exogenous proteins. Viral infection not only induced the host autophagy response, but also impacted secretion of exosomes. Our data showed that secretory exosomes contributed to the clearing of increased intracellular microRNAs induced by enterovirus infection, thereby weakening viral replication. Furthermore, the secretory exosomes were essential for the disposal of viral RNA replicon rather than autophagic degradation, thereby facilitating host survival. Our results collectively revealed that both secretory exosome and autophagic degradation were crucial for maintaining cellular homeostasis, but that they operate through distinct mechanisms and dispose of different types of unwanted materials.
    Keywords:  ESCRT pathway; autophagic degradation; exosomes; homeostasis; virus infection
    DOI:  https://doi.org/10.1111/febs.70244
  31. Autophagy Rep. 2025 ;4(1): 2547975
    Sequestering sequestosome 1 via S-acylation in autophagy, Huntington disease, and beyond.
    Y Alshehabi, F Abrar, D D O Martin.
      Protein mislocalization and aggregation are hallmark features in neurodegeneration. As proteins mislocalize, proteostasis deficiency and protein aggregation typically follow. Autophagy is a crucial pathway for the removal of protein aggregates to maintain neuronal health, but is impaired in various neurodegenerative diseases, including Huntington disease (HD). We identified S-acylation, a reversible lipid modification of proteins, as an important regulator in protein trafficking and autophagy. SQSTM1 (sequestosome 1/p62) is an essential selective autophagy receptor for the sequestration of ubiquitinated cargoes within autophagosomes and subsequent delivery into lysosomes for degradation. Recently, we reported that S-acylation of SQSTM1 at the di-cysteine motif C289,290 directs SQSTM1 to lysosomes. We further showed that SQSTM1 S-acylation is significantly reduced in brains from both HD patients and mouse HD model, which may result in the cargo sequestration defect within autophagosomes in HD. Treatment with palmostatin B, a deacylation inhibitor, significantly increases SQSTM1 localization to lysosomes. Our work highlights SQSTM1 S-acylation as a novel potential therapeutic strategy in HD. As a crucial autophagy component, our work suggests S-acylation of SQSTM1 may have a broader role in neurodegeneration.
    Keywords:  Autophagy; Huntington disease; S-acylation; fasting; huntingtin; localization; mouse model; palmitoylation; palmostatin B; sequestosome 1
    DOI:  https://doi.org/10.1080/27694127.2025.2547975
  32. Nat Immunol. 2025 Sep;26(9): 1581-1595
    E3 ligase RAD18 targets phosphorylated IRF3 to terminate IFNB1 transcription.
    Yiting Cai, Jiaqi Zheng, Linlin Zhao, Xue Wang, Lu Zhang, Jun Wang, Yuchuan Zhang, Rui Shi, Jin Han, Wei Han, Wei Chen.
      The transcription factor interferon regulatory factor 3 (IRF3) initiates type I interferon transcription, which is required for host defense. Here, we identify RAD18 as a central E3 ubiquitin ligase that selectively targets phosphorylated IRF3 (p-IRF3) for autophagic degradation. RAD18 specifically promotes the dissociation of p-IRF3 from the IFNB promoter and in turn terminates its transcriptional activity. Mechanistically, RAD18 binds the p-IRF3 dimer located on the IFNB promoter and triggers K63 polyubiquitylation of p-IRF3 at Lys 193. The ubiquitylated p-IRF3 dimer consequently dissociates from the IFNB promoter, translocates out of the nucleus and undergoes OPTN-mediated autophagic degradation. Rad18fl/fl Lysm-cre mice resist lethal vesicular stomatitis virus infection in vivo due to IFNβ overproduction. In H1N1-infected human macrophages or monocytes from individuals with active systemic lupus erythematosus, RAD18 protein levels negatively correlate with p-IRF3 and IFNB1 mRNA levels. Thus, RAD18 functions as a break to terminate IRF3-driven IFNB1 transcription and may be a potential therapeutic target for RNA virus infection or autoimmune diseases.
    DOI:  https://doi.org/10.1038/s41590-025-02256-x
  33. Trends Biochem Sci. 2025 Aug 27. pii: S0968-0004(25)00193-8. [Epub ahead of print]
    MitoTraP: mitochondrial protection for cellular proteostasis.
    Michaela Oborská-Oplová, Michael A Ruoss, Vikram G Panse.
      Cells depend on the efficient import of thousands of nuclear-encoded mitochondrial proteins to maintain mitochondrial function. A new study by Flohr et al. reveals a quality control strategy that traps a subset of mitochondrial precursors in the intermembrane space during energy stress, preventing their toxic accumulation in the cytosol or nucleus.
    Keywords:  mitochondrial import; mitochondrial intermembrane space; mitochondrial quality control; mitochondrial ribosomal proteins (MRPs); mitochondrial stress; proteotoxic stress
    DOI:  https://doi.org/10.1016/j.tibs.2025.08.004
  34. Sci Data. 2025 Aug 30. 12(1): 1520
    Multi-omic assessment of mRNA translation dynamics in liver cancer cell lines.
    Asier González, Muskan Pandey, Niels Schlusser, Sayanur Rahaman, Meric Ataman, Nitish Mittal, Alexander Schmidt, Attila Becskei, Mihaela Zavolan.
      The limited correlation between mRNA and protein levels within cells highlighted the need to study mechanisms of translational control. To decipher the factors that determine the rates of individual steps in mRNA translation, machine learning approaches are currently applied to large libraries of synthetic constructs, whose properties are generally different from those of endogenous mRNAs. To fill this gap and thus enable the discovery of elements driving the translation of individual endogenous mRNAs, we here report steady-state and dynamic multi-omics data from human liver cancer cell lines, specifically (i) ribosome profiling data from unperturbed cells as well as following the block of translation initiation (ribosome run-off, to trace translation elongation), (ii) protein synthesis rates estimated by pulsed stable isotope labeled amino acids in cell culture (pSILAC), and (iii) mean ribosome load on individual mRNAs determined by mRNA sequencing of polysome fractions (polysome profiling). These data will enable improved predictions of mRNA sequence-dependent protein output, which is crucial for engineering protein expression and for the design of mRNA vaccines.
    DOI:  https://doi.org/10.1038/s41597-025-05861-5
  35. bioRxiv. 2025 Aug 29. pii: 2025.08.28.672852. [Epub ahead of print]
    Endocytome profiling uncovers cell-surface protein dynamics underlying neuronal connectivity.
    Colleen N McLaughlin, Hui Ji, Katherine X Dong, Chuanyun Xu, Kenneth Kin Lam Wong, Zhuoran Li, David J Luginbuhl, Charles Xu, Cheng Lyu, Wei Qin, Jiefu Li, Namrata D Udeshi, Steven A Carr, Alice Y Ting, Liqun Luo.
      Endocytosis actively remodels the neuronal surface proteome to drive diverse cellular processes, yet its global extent and circuit-level consequences have defied comprehensive interrogation. Here, we introduce endocytome profiling: a systematic, cell-type-specific approach for mapping cell-surface protein (CSP) dynamics in situ. Quantitative proteomic analysis of developing olfactory receptor neuron (ORN) axons generated an endocytic atlas comprising over 1,100 proteins and revealed the extent to which the surface proteome is remodeled to meet distinct developmental demands. Targeted interrogation of a junctional CSP showed that its endosome-to-surface ratio is precisely balanced to enable developmental axon pruning while preserving mature axon integrity. Multi-omic integration uncovered wide-spread transcellular signaling and identified a growth factor secreted by neighboring neurons to direct ORN axon targeting via endocytic regulation of its receptor. Endocytome profiling thus provides unprecedented access to cell-surface proteome dynamics and offers a powerful platform for dissecting proteome remodeling across diverse cell types and contexts.
    DOI:  https://doi.org/10.1101/2025.08.28.672852
  36. bioRxiv. 2025 Aug 24. pii: 2025.08.19.670962. [Epub ahead of print]
    Structural basis for binding of RILPL1 to TMEM55B reveals a lysosomal platform for adaptor assembly through a conserved TBM motif.
    Dieter Waschbüsch, Prosenjit Pal, Raja S Nirujogi, Melanie Cavin, Jaijeet Singh, Dario R Alessi, Amir R Khan.
      Inherited mutations in VPS35 and the kinase LRRK2 lead to hyperphosphorylation of Rab GTPases and promote the formation of phospho-Rab signalling complexes. A subset of RH2 domain-containing proteins from the RILP-homology family, including RILP, RILPL1, RILPL2, JIP3, and JIP4 are Rab effectors that recognize the LRRK2-phosphorylated switch 2 threonine of phospho-Rab8A and phospho-Rab10. More recently, phospho-Rabs have been found on lysosomal membranes within multi-protein assemblies involving TMEM55B and RILPL1. TMEM55B is a 284-residue lysosomal membrane protein with no homology to known proteins. It comprises a 218-residue cytosolic N-terminal region and two predicted transmembrane α-helices. Residues 80-160, which face the cytosol, mediate binding to a C-terminal motif of RILPL1, formed after RILPL1 associates with phospho-Rab8A. Here, we report the crystal structures of TMEM55B alone and in complex with a C-terminal RILPL1 peptide, encompassing the TMEM55B interaction region, which we define as the TMEM55B Binding Motif (TBM). The cytosolic domain of TMEM55B adopts a rigid architecture of two tandem RING-like domains, each forming a Zn2+-stabilized 40-residue β-sandwich. TBM binding is mediated primarily by backbone hydrogen bonding and anchored by two glutamate residues from RILPL1. These findings support a model in which RILPL1 is recruited to phospho-Rab8A-positive lysosomes prior to TMEM55B engagement. Further co-immunoprecipitation and mutational analyses indicate that TMEM55B forms complexes independently of phospho-Rabs with proteins containing a conserved TBM, like that of RILPL1, including JIP3, JIP4, OCRL, WDR81, and TBC1D9B. Together, these findings uncover previously unrecognized regulatory networks associated with TMEM55B and lysosomal function and suggest that TMEM55B serves as a central hub for adaptor recruitment at the lysosomal membrane.
    DOI:  https://doi.org/10.1101/2025.08.19.670962
  37. STAR Protoc. 2025 Sep 01. pii: S2666-1667(25)00458-7. [Epub ahead of print]6(3): 104052
    Protocol for label-free ribosome-associated protein enrichment from mammalian cells by RAPIDASH.
    Victoria Hung, Teodorus Theo Susanto, Maria Barna.
      Ribosome-associated proteins (RAPs) enable modulation of gene expression at the level of mRNA translation in response to cellular needs. Here, we describe a method called ribosome-associated protein identification by affinity to sulfhydryl-charged resin (RAPIDASH) for tag-free isolation of RAP-bound ribosomes from mammalian samples for mass spectrometry-based proteomics. Samples are first lysed and then undergo sucrose cushion ultracentrifugation and subsequent chromatography using a sulfhydryl-charged resin. While RAPIDASH is optimized for mammalian samples, we expect that it can be adapted for non-mammalian samples. For complete details on the use and execution of this protocol, please refer to Susanto et al.1.
    Keywords:  Cell Biology; Molecular Biology; Protein Biochemistry
    DOI:  https://doi.org/10.1016/j.xpro.2025.104052
  38. Cell. 2025 Sep 04. pii: S0092-8674(25)00687-7. [Epub ahead of print]188(18): 4811-4827
    Rethinking RNA-binding proteins: Riboregulation challenges prevailing views.
    Matthias W Hentze, Pia Sommerkamp, Venkatraman Ravi, Fátima Gebauer.
      RNA-binding proteins (RBPs) are best known as effectors along the entire gene expression pathway and as constituents of RNA-protein machines such as the ribosome and the spliceosome. Around 1,000 RBPs account for these functions in mammalian cells. The total number of RBPs has recently more than tripled to include many "well-known" proteins such as metabolic enzymes or membrane proteins, sparking debate about the biological relevance of their RNA binding. We examine the experimental basis underlying the dramatic expansion of the RBPome, consider arguments that challenge its relevance, and discuss recent data that describe new RBP and RNA functions. We suggest that the scope of interplay between RNA and proteins is underexplored and that riboregulation of proteins represents an emerging theme in cell biology and translational medicine.
    Keywords:  ◼◼◼
    DOI:  https://doi.org/10.1016/j.cell.2025.06.021
  39. J Biol Chem. 2025 Sep 01. pii: S0021-9258(25)02504-9. [Epub ahead of print] 110652
    FBXO9 mediated the ubiquitination and degradation of YAP in a GSK-3β-dependent manner.
    Yili Jin, Xue Yun, Jiatao Yao, Chengyun Xu, Rui Yu.
      The Hippo signaling pathway effector YAP (Yes-associated protein) serves as a critical transcriptional regulator involved in a wide range of biological processes, including oncogenesis. Despite its potential as a therapeutic target, pharmacologically targeting the Hippo/YAP axis remains challenging, necessitating further exploration of the mechanisms governing YAP regulation. In this study, we identify the Cullin-RING E3 ligase complex SCF-FBXO9-CRL1 as a novel posttranslational regulator of YAP stability. Mechanistically, FBXO9 recognizes YAP through a conserved degron motif and facilitates its K48-linked polyubiquitination at lysine 76 (K76), thereby promoting proteasomal degradation. Notably, We demonstrate that phosphorylation of YAP at Ser338 and Thr342 by GSK-3β primes YAP for FBXO9 recognition, leading to subsequent ubiquitination. Furthermore, our analysis of the signaling cascade reveals that Akt kinase activity modulates this regulatory axis by influencing the phosphorylation status of GSK-3β. Pharmacological inhibition of Akt signaling leads to YAP degradation in a GSK-3β/FBXO9-dependent manner, significantly enhancing chemosensitivity in cancer models. These findings establish a previously unrecognized regulatory axis involving Akt, GSK-3β, FBXO9, and YAP that controls YAP protein turnover, providing a mechanistic basis for therapeutic strategies that combine Akt inhibitors with conventional chemotherapeutics. Our work advances the understanding of posttranslational YAP regulation and identifies several potential therapeutic targets for YAP-driven malignancies.
    Keywords:  FBXO9; GSK-3β; Hippo signaling pathway; YAP; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2025.110652
  40. J Biol Chem. 2025 Sep 01. pii: S0021-9258(25)02519-0. [Epub ahead of print] 110667
    Get4/5-Mediated Remodeling of Get3's Substrate Binding Chamber: Insights into Tail-Anchored Protein Targeting by the GET Pathway.
    Diego Granados-Villanueva, Andrew Rossow, Kelly H Kim.
      The Guided Entry of Tail-Anchored Proteins (GET) pathway ensures accurate targeting of Tail-Anchored proteins (TAs) - a diverse class of membrane proteins - to the endoplasmic reticulum (ER) membrane. In yeast, newly synthesized TAs are captured by Sgt2 and transferred to Get3 for delivery to the ER, where they undergo subsequent membrane insertion. Efficient and protected handoff of hydrophobic TAs from Sgt2 to Get3 is facilitated by the Get4/5 complex, which is thought to act as a scaffold to position TA-bound Sgt2 and Get3 in proximity while trapping Get3 in an ATP-bound conformation necessary for TA binding. To define the molecular basis for this process, we determined the cryo-EM structure of the Saccharomyces cerevisiae Get3-Get4/5 complex at 3.2 Å resolution. Our structure shows that Get4/5 remodels Get3's TA-binding chamber by unfolding helices that form the lateral walls of the chamber. We termed this region the 'lateral gate', as its helix-to-coil transition makes the TA-binding chamber more solvent accessible. Molecular dynamics simulations highlighted the flexibility of the lateral gate, indicating it is structurally dynamic and prone to conformational changes. Mutagenesis studies showed that the lateral gate residues influence both the binding affinity of Get3 for Get4/5 and its ATPase activity. Additionally, our cryo-EM map shows that the Sgt2-binding domain of Get5 is positioned near the lateral gate opening of Get3's TA-binding chamber. Based on these findings, we propose a model in which Get4/5 opens Get3's TA-binding chamber to form a lateral opening, enabling protected, lateral transfer of TAs from Sgt2 to Get3.
    Keywords:  GET pathway; chaperone; cryo-EM; membrane protein; protein complex; protein targeting; protein-protein interaction; tail-anchored protein
    DOI:  https://doi.org/10.1016/j.jbc.2025.110667
  41. Bio Protoc. 2025 Aug 20. 15(16): e5414
    An Ex Vivo Protocol to Assess IRE1α-Dependent RNA Cleavage Using Total RNA Isolated from Mouse Tissues.
    Mohammad Mamun Ur Rashid, So-Young Rah, Se-Woong Ko, Han-Jung Chae.
      Regulated IRE1-dependent decay (RIDD) is a critical cellular mechanism mediated by the endoplasmic reticulum (ER) stress sensor IRE1α, which cleaves a variety of RNA targets to regulate ER homeostasis. Current in vitro assays to study IRE1α activity largely rely on synthetic or in vitro transcribed RNA substrates, which may not fully replicate the physiological complexities of native RNA molecules. Here, we present a comprehensive protocol to assess IRE1α-dependent RNA cleavage activity using total RNA isolated directly from mouse tissues. This protocol provides a step-by-step guide for tissue collection, RNA isolation, an ex vivo RIDD assay, cDNA synthesis, and subsequent RT-PCR analysis of target mRNA cleavage products. Key reagents include active IRE1α protein, the RIDD-specific inhibitor 4μ8C, and target-specific primers for RIDD-regulated genes such as Bloc1s1 and Col6a1. Quantitative assessment is achieved using agarose gel electrophoresis and imaging software. This methodology enables the study of IRE1α's RNA cleavage activity under conditions that closely mimic in vivo environments, providing a more physiologically relevant approach to understanding the role of RIDD in cellular and tissue-specific contexts. Key features • Uses total RNA from mouse tissues instead of synthetic RNA to better reflect in vivo conditions. • Includes RIDD-specific controls such as IRE1α inhibitor (4μ8C) and RNase A to confirm targeted RNA cleavage. • Combines agarose gel electrophoresis and ImageJ quantification for both qualitative and statistical validation. • Allows comparative studies of IRE1α activity across multiple mouse tissues in different biological contexts.
    Keywords:  ER stress; Endoplasmic reticulum; Ex vivo; IRE1α; RIDD; RNA Cleavage
    DOI:  https://doi.org/10.21769/BioProtoc.5414
  42. bioRxiv. 2025 Aug 19. pii: 2025.08.14.669896. [Epub ahead of print]
    Proteo-transcriptomic reprogramming and resource reallocation define the aging mammalian brain.
    Nisha Hemandhar-Kumar, Verena Kluever, Svenja V Kaufmann, Cornelius Bergmann, Kanaan Mousaei, Miguel Tomas, Miguel Correa Marrero, Avika Chopra, Misa Hirose, Mercè Pallas, Coral Sanfeliu, Saleh M Ibrahim, Andre Fischer, Tiago F Outeiro, Henning Urlaub, Tatjana Tchumatchenko, Carlos López Otín, Eugenio F Fornasiero.
      Brain aging is a major risk for neurodegeneration, yet the underlying molecular mechanisms remain poorly understood. Here we performed an integrative proteo-transcriptomic analysis of the aging mouse brain, uncovering molecular signatures of aging through the assessment of protein aggregation, mRNA relocalization, and comparative proteomics across eight models of premature aging and neurodegeneration. We identified dynamic changes in physiological aging highlighting differences in synaptic maintenance and energy-allocation. These were linked to changes associated with fundamental protein biochemical properties such as size and net charge. Network analysis highlighted a decrease in mitochondrial complex I proteins not compensated at the mRNA level. Aggregation of 60S ribosome subunits indicated deteriorating translation efficiency and was accompanied by mitochondrial and proteasomal imbalance. The analysis of the nine models revealed key similarities and differences between physiological aging and pathology. Overall, our study provides an extensive resource on molecular aging, and offers insights into mechanisms predisposing to neurodegeneration, easily accessible at our Brain Aging and Molecular Atlas Project (BrainAging-MAP) website.
    DOI:  https://doi.org/10.1101/2025.08.14.669896
  43. J Biol Chem. 2025 Sep 01. pii: S0021-9258(25)02524-4. [Epub ahead of print] 110672
    Dissecting the Cdc37 Co-chaperone Code: Functional Roles in Chaperone-Mediated Stress Adaptation.
    Megan M Mitchem, Ashley Choi, Duhita A Mirikar, Rajlekha Deb, Andrew W Truman.
      Cdc37 is a kinase-specific co-chaperone that scaffolds protein kinase clients to the Hsp90 chaperone system. Although phosphorylation at residues S14 and S17 is known to regulate Cdc37 function, the broader role of phosphorylation across the protein remains unclear. To systematically investigate this, we created a "Cdc37 code collection," a set of 46 yeast strains expressing single phospho-site mutants of Cdc37, and performed phenotypic profiling across a wide panel of environmental and chemical stressors. While canonical sites like S14 and S17 were essential for stress tolerance, 34 additional phospho-mutants exhibited distinct phenotypes, often in a stress-specific manner. Notably, the mutations displayed little overlap in their stress responses, suggesting a modular and context-dependent regulation of Cdc37. Our data reveal that Cdc37 function is intricately modulated by site-specific phosphorylation, which shapes its capacity to maintain proteostasis under diverse cellular conditions. This study provides a comprehensive resource for dissecting the functional landscape of Cdc37 post-translational regulation and highlights new regulatory sites with potential relevance to chaperone-kinase network dysregulation in disease.
    Keywords:  Cdc37 (cell division cycle 37); Hsp90 (heat shock 90); chaperone; post-translational modification (PTM); proteostasis; stress responses; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2025.110672
  44. Cell Rep. 2025 Sep 03. pii: S2211-1247(25)01011-3. [Epub ahead of print]44(9): 116240
    Pp1-87B/PPP1CC-JNK axis integrates apoptosis and ferroptosis-like cell death to regulate cell competition and tumorigenesis.
    Du Kong, Zhongbo Li, Sihua Zhao, Xueliang Yang, Jiadong Zheng, Yifan Guo, Wenyan Xu, Han Gao, Hanqing Guo, Qiang Zhang, Jian Zhu, Xianjue Ma, Bin Jin.
      Cell competition, an evolutionarily conserved quality control mechanism, selectively removes unfit or pre-malignant cells via cell-cell interactions. Through a genetic screen in Drosophila, we identify the phosphatase Pp1-87B as an essential regulator of JNK signaling crucial for eliminating scrib-deficient precancerous cells during tumor-suppressive cell competition. Mechanistically, impaired Pp1-87B activates JNK signaling via the Moe-Rho1 axis. Subsequently, JNK signaling acts as a central hub, integrating apoptosis and ferroptosis-like cell death by activating the Hippo signaling pathway. Critically, we demonstrate that the human ortholog, PPP1CC (protein phosphatase 1 catalytic subunit gamma), functions similarly to drive apoptosis and ferroptosis in human liver tumor cells through JNK activation. Collectively, our findings provide insights into the molecular integration of distinct cell death pathways during premalignant cell elimination in cell competition and identify PPP1CC as a promising therapeutic target for liver cancer treatment.
    Keywords:  CP: Cancer; Drosophila; Hippo pathway; JNK pathway; PPP1CC; Pp1-87B; TSCC; ferroptosis; liver cancer; scrib; tumor-suppressive cell competition
    DOI:  https://doi.org/10.1016/j.celrep.2025.116240
  45. Autophagy. 2025 Sep 03. 1-20
    Reconstitution of autophagic-like membrane tethering reveals that Atg11 can bind and cluster vesicles on cargo mimetics.
    Devika Andhare, Sarah Katzenell, Sarah I Najera, Sylvie C Mauras, Katherine M Bauer, Michael J Ragusa.
      Macroautophagy (hereafter, autophagy) is essential for the degradation of mitochondria from yeast to humans. Mitochondrial autophagy in yeast is initiated when the selective autophagy scaffolding protein Atg11 is recruited to mitochondria through its interaction with the selective autophagy receptor Atg32. This also results in the recruitment of small 30-nm vesicles that fuse to generate the initial phagophore membrane. We demonstrate that Atg11 can bind to autophagic-like membranes in vitro in a curvature-dependent manner in part via a predicted amphipathic helix. Deletion of the amphipathic helix from Atg11 results in a delay in the formation of mitophagy initiation sites in yeast. Furthermore, using a novel biochemical approach, we demonstrate that the interaction between Atg11 and Atg32 results in the tethering of autophagic-like vesicles in clusters to giant unilamellar vesicles containing a lipid composition designed to mimic the outer mitochondrial membrane. We also demonstrate that the N-terminal region of Atg11 is an important mediator of vesicle tethering to cargo mimetics and that clustering of autophagic-like vesicles requires the C-terminal region of Atg11. Taken together, our results reveal that Atg11 clusters into high-order oligomers, can tether autophagic-like membranes and due to its ability to oligomerize can cluster vesicles on the surface of cargo mimetics. This work provides new insight into the mechanisms of protein and membrane clustering by Atg11. Given the increasing importance of protein oligomerization and clustering in autophagy, these results have important implications in the initiation of mitochondrial autophagy.Abbreviations Atg11: autophagy related 11; Atg11-Cterm: C-terminal region of Atg11; Atg11-Nterm: N-terminal region of Atg11; Atg32: autophagy related 32; COV: coefficient of variance; DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DOPS: 1,2-dioleoyl-sn-glycero-3-phospho-L-serine; FRAP: fluorescence recovery after photobleaching; GLT: GUV and liposome tethering; GUV: giant unilamellar vesicle; MKO: multiple knockout; OMM: outer mitochondrial membrane; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; RhPE: rhodamine phosphatidylethanolamine; SAR: selective autophagy receptor; SEC: size-exclusion chromatography; SLB: supported lipid bilayers; SMrT: supported membrane templates; YPL: yeast polar lipids.
    Keywords:  Biochemistry; membrane tethering; mitophagy; reconstitution; selective autophagy; yeast
    DOI:  https://doi.org/10.1080/15548627.2025.2551678
  46. Rheumatol Int. 2025 Sep 06. 45(9): 219
    Integrated stress response in Behçet disease: expression analyses in peripheral blood and synovial monocytes.
    İrem Coşkuntan, Ferda Paçal, Fulya Coşan, Esin Çetin Aktaş, Günnur Deniz, Duran Üstek, Sema Sırma Ekmekci, Neslihan Abacı, Ahmet Gül.
      Behçet disease (BD) is a chronic, relapsing inflammatory disorder, and human leukocyte antigen (HLA)-B*51 is considered to be the strongest genetic susceptibility factor. The integrated stress response (ISR), defined by the eIF2α/ATF4 axis, is a signaling network that maintains protein homeostasis and regulates innate immunity in eukaryotic cells; pathological activation of this pathway can affect the immune response and cause various diseases. In this study, we aimed to investigate the role of the ISR signaling pathway in the pathogenesis of BD. The study group comprised 83 BD patients and 33 matched healthy controls. eIF2α, PERK, HRI, HSPB8, and ATF4 mRNA expression analysis was performed by real-time quantitative polymerase chain reaction (RTq-PCR) using cDNA prepared from monocytes isolated from peripheral blood and synovial fluid. HLA-B*51 status was available for 73 BD patients. ISR analysis revealed decreased expression of eIF2α and increased expression of PERK, HRI, HSPB8, and ATF4 genes in BD compared to controls (all p < 0.05). Findings of synovial fluid monocytes of active BD patients with arthritis showed a consistent (p = 0.0417, one-tailed) increase in HSPB8 expression and strong correlation with those in corresponding blood monocytes. BD patients exhibit dysregulated ISR signatures regardless of HLA-B*51 status. Despite limitations in sample size and in vivo validation, our findings implicate these signaling pathways as potential contributors to the pathogenesis of BD and possibly other the major histocompatibility complex (MHC) class I-associated diseases.
    Keywords:  Behçet disease; Inflammation; Monocytes; Proteotoxic stress; Small heat-shock protein; eIF-2 kinase
    DOI:  https://doi.org/10.1007/s00296-025-05972-7
  47. Nat Commun. 2025 Aug 28. 16(1): 7762
    Regulatory role of the N-terminal intrinsically disordered region of the DEAD-box RNA helicase DDX3X in selective RNA recognition.
    Yuki Toyama, Koh Takeuchi, Ichio Shimada.
      DDX3X, a member of the DEAD-box RNA helicase family, plays a central role in the translational regulation of gene expression through its unwinding activity toward complex RNA structures in messenger RNAs (mRNAs). Although DDX3X is known to selectively stimulate the translation of a subset of genes, a specific sequence motif has not been identified; thus, the molecular mechanism underlying this selectivity remains elusive. Using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that the N-terminal intrinsically disordered region (IDR) of DDX3X plays a critical role in the binding and unwinding of structured RNAs. We propose that the selectivity toward target transcripts is mediated by its preferential binding to structured motifs, particularly the G-quadruplex structure, through arginine-rich segments within the N-terminal IDR. Our results provide a molecular basis for understanding translational regulation by DDX3X and highlight the remarkable role of the flexible IDR in controlling the cellular translational landscape.
    DOI:  https://doi.org/10.1038/s41467-025-62806-7
  48. Mol Syst Biol. 2025 Sep 03.
    Conserved interfaces mediate multiple protein-protein interactions in a prokaryotic metabolon.
    Sanchari Bhattacharyya, Srivastav Ranganathan, Sourav Chowdhury, Bharat V Adkar, Mark Khrapko, Eugene I Shakhnovich.
      Enzymes in a pathway often form metabolons through weak protein-protein interactions (PPI) that localize and protect labile metabolites. Due to their transient nature, the structural architecture of these enzyme assemblies has largely remained elusive, limiting our abilities to re-engineer novel metabolic pathways. Here, we delineate a complete PPI map of 1225 interactions in the E. coli 1-carbon metabolism pathway using bimolecular fluorescence complementation that can capture transient interactions in vivo and show strong intra- and inter-pathway clusters within the folate and purine biosynthesis pathways. Scanning mutagenesis experiments along with AlphaFold predictions and metadynamics simulations reveal that most proteins use conserved "dedicated" interfaces distant from their active sites to interact with multiple partners. Diffusion-reaction simulations with shared interaction surfaces and realistic PPI networks reveal a dramatic speedup in metabolic pathway fluxes. Overall, this study sheds light on the fundamental features of metabolon biophysics and structural aspects of transient binary complexes.
    Keywords:  AlphaFold; Enzyme Flux; Metabolon; Split YFP; Transient PPI
    DOI:  https://doi.org/10.1038/s44320-025-00139-9
  49. bioRxiv. 2025 Aug 22. pii: 2025.08.18.670656. [Epub ahead of print]
    MERS-CoV antagonizes PKR activation by inhibiting its condensation at viral replication complexes.
    Ebba K Blomqvist, Nicole Bracci, Helena Winstone, J Monty Watkins, Susan R Weiss, James M Burke.
      Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that antagonizes innate immune responses, including the protein kinase R (PKR) pathway. Here, we examine the process of PKR activation in response to an immunostimulatory MERS-CoV mutant encoding an inactive endoribonuclease U and a deletion of accessory protein NS4a. We show that PKR condenses and activates on viral dsRNA proximal to viral double-membrane vesicles (DMVs). Condensates composed of activated PKR disassociate from dsRNA and dissolve, releasing activated PKR molecules into the cytosol where they phosphorylate eIF2α to initiate the integrated stress response. MERS-CoV NS4a protein prevents PKR activation by condensing on dsRNA and occluding PKR binding. Lastly, PKR condensation coincided with its activation in response to Zika virus. These findings establish a comprehensive model for PKR activation in response to positive-strand RNA viruses that replicate within membrane-associated complexes and elucidate how MERS-CoV antagonizes this crucial antiviral pathway.
    GRAPHICAL ABSTRACT:
    HIGHLIGHTS: PKR condenses on viral dsRNA exposed at membrane-associated replication complexesPKR condensation initiates autophosphorylation of PKRActivated p-PKR molecules disassociate from dsRNA and re-localize to the cytosolMERS-CoV NS4a inhibits PKR activation by occluding PKR condensation on dsRNA.
    DOI:  https://doi.org/10.1101/2025.08.18.670656
  50. Cell Rep. 2025 Sep 01. pii: S2211-1247(25)00975-1. [Epub ahead of print]44(9): 116204
    Polysomes and mRNA control the biophysical properties of the eukaryotic cytoplasm.
    Vamshidhar R Gade, Stephanie Heinrich, Matteo Paloni, Pablo A Gómez-García, Ajla Dzanko, Alexandra Oswald, Désirée Marchand, Sarah Khawaja, Alessandro Barducci, Karsten Weis.
      The organization and biophysical properties of the cytoplasm influence all cellular reactions, including molecular interactions and the mobility of biomolecules. The cytoplasm does not behave like a simple fluid but is a densely crowded and highly organized environment. However, its detailed properties, the molecular mechanisms that control them, and how they influence the cellular biochemistry remain poorly understood. Here, we investigate the diffusive properties of the cytoplasm in silico and in vivo, employing mRNPs (messenger ribonucleoproteins) and GEM (genetically encoded multimeric) particles as rheological probes. Cytoplasmic diffusivity increases upon polysome disassembly or a reduction in mRNA levels. Reducing ribosome concentration by up to 20%-25% without altering polysome levels has no effect in vivo. Furthermore, mRNA condensation into P-bodies upon polysome disassembly does not affect cytosolic diffusion in budding yeast. Our results demonstrate that mRNAs and their organization into polysomes regulate the biophysical properties of the eukaryotic cytoplasm.
    Keywords:  CP: Molecular biology; GEMs; biophysical properties; cytosolic diffusion; eukarytoic cytoplasm; mRNA; mRNPs; polysomes; translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116204
  51. Sci Adv. 2025 Aug 29. 11(35): eadv9401
    DKC1-mediated pseudouridylation of rRNA targets hnRNP A1 to sustain IRES-dependent translation and ATF4-driven metabolic adaptation.
    Anamika Gupta, Mohit Bansal, Jane Ding, Suman Karki, Madhuparna Pandit, Sunil Sudarshan, Han-Fei Ding.
      The pseudouridine synthase DKC1 regulates internal ribosome entry site (IRES)-dependent translation and is up-regulated in cancers by the MYC family of oncogenes. The functional significance of DKC1 up-regulation and the mechanistic connection between pseudouridylation and IRES-mediated translation remain poorly understood. Here, we report that DKC1 drives an ATF4-mediated transcriptional program that supports amino acid metabolism and stress adaptation. We identify hnRNP A1, an IRES trans-acting factor, as a critical downstream mediator of DKC1 in sustaining ATF4 expression and IRES-dependent translation. Mechanistically, DKC1-mediated pseudouridylation at two specific 28S ribosomal RNA sites is crucial for maintaining hnRNP A1 protein expression. In turn, hnRNP A1 binds and stabilizes ATF4 messenger RNA, preferentially promoting IRES-dependent translation of ATF4 variant 1. Furthermore, cellular stress induces hnRNP A1, which is necessary for stress-induced ATF4 protein expression. Collectively, our findings uncover an MYC-driven DKC1-hnRNP A1 axis that links IRES-dependent translation and ATF4-mediated metabolic adaptation, thereby supporting cancer cell survival under metabolic stress during tumor progression.
    DOI:  https://doi.org/10.1126/sciadv.adv9401
  52. Nat Metab. 2025 Sep 01.
    ATF6 activation alters colonic lipid metabolism causing tumour-associated microbial adaptation.
    Olivia I Coleman, Adam Sorbie, Alessandra Riva, Miriam von Stern, Stephanie Kuhls, Denise M Selegato, Luisa Siegert, Isabel Keidel, Nikolai Köhler, Jakob Wirbel, Tim Kacprowski, Andreas Dunkel, Josch K Pauling, Johannes Plagge, Diego Mediel-Cuadra, Sophia J Wagner, Ines Chadly, Sandra Bierwith, Tingying Peng, Thomas Metzler, Xin Li, Mathias Heikenwälder, Clemens Schafmayer, Sebastian Hinz, Christian Röder, Christoph Röcken, Michael Zimmermann, Philip Rosenstiel, Katja Steiger, Moritz Jesinghaus, Gerhard Liebisch, Josef Ecker, Christina Schmidt, Georg Zeller, Klaus-Peter Janssen, Dirk Haller.
      Endoplasmic reticulum unfolded protein responses contribute to cancer development, with activating transcription factor 6 (ATF6) involved in microbiota-dependent tumorigenesis. Here we show the clinical relevance of ATF6 in individuals with early-onset and late colorectal cancer, and link ATF6 signalling to changes in lipid metabolism and intestinal microbiota. Transcriptional analysis in intestinal epithelial cells of ATF6 transgenic mice (nATF6IEC) identifies bacteria-specific changes in cellular metabolism enriched for fatty acid biosynthesis. Untargeted metabolomics and isotype labelling confirm ATF6-related enrichment of long-chain fatty acids in colonic tissue of humans, mice and organoids. FASN inhibition and microbiota transfer in germ-free nATF6IEC mice confirm the causal involvement of ATF6-induced lipid alterations in tumorigenesis. The selective expansion of tumour-relevant microbial taxa, including Desulfovibrio fairfieldensis, is mechanistically linked to long-chain fatty acid exposure using bioorthogonal non-canonical amino acid tagging, and growth analysis of Desulfovibrio isolates. We postulate chronic ATF6 signalling to select for tumour-promoting microbiota by altering lipid metabolism.
    DOI:  https://doi.org/10.1038/s42255-025-01350-6
  53. Nat Aging. 2025 Sep 03.
    The aging factor EPS8 induces disease-related protein aggregation through RAC signaling hyperactivation.
    Seda Koyuncu, Yaiza Dominguez-Canterla, Rafael Alis, Nassima Salarzai, Dunja Petrovic, Nuria Flames, David Vilchez.
      Aging is a major risk factor for neurodegenerative diseases associated with protein aggregation, including Huntington's disease and amyotrophic lateral sclerosis (ALS). Although these diseases involve different aggregation-prone proteins, their common late onset suggests a link to converging changes resulting from aging. In this study, we found that age-associated hyperactivation of EPS8/RAC signaling in Caenorhabditis elegans promotes the pathological aggregation of Huntington's disease-related polyglutamine repeats and ALS-associated mutant FUS and TDP-43 variants. Conversely, knockdown of eps-8 or RAC orthologs prevents protein aggregation and subsequent deficits in neuronal function during aging. Similarly, inhibiting EPS8 signaling reduces protein aggregation and neurodegeneration in human cell models. We further identify the deubiquitinating enzyme USP4 as a regulator of EPS8 ubiquitination and degradation in both worms and human cells. Notably, reducing USP-4 upregulation during aging prevents EPS-8 accumulation, extends longevity and attenuates disease-related changes. Our findings suggest that targeting EPS8 and its regulatory mechanisms could provide therapeutic strategies for age-related diseases.
    DOI:  https://doi.org/10.1038/s43587-025-00943-w
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