bims-proteo Biomed News
on Proteostasis
Issue of 2025–11–02
57 papers selected by
Eric Chevet, INSERM
  1. Mechanisms and regulation of the Hsp70 chaperone network.
  2. Oligosaccharyltransferase Is Involved in Targeting to ER-Associated Degradation.
  3. Pharmacological targeting of RIG-I can selectively activate the integrated stress response.
  4. Mode of action of a DCAF16-recruiting targeted glue that can selectively degrade BRD9.
  5. Legionella pneumophila Effector Protein LegU1 Mediates the Ubiquitination of Endoplasmic Reticulum Chaperone BiP.
  6. Beyond Fructolysis: Ketohexokinase Orchestrates ER Proteostasis in Nutrient-Stressed Hepatocytes.
  7. Close-up of vesicular ER exit sites by volume electron imaging using FIB-SEM.
  8. Stage-specific TRIM10 expression regulates erythroid maturation.
  9. DDX3X acts as a selective dual switch regulator of mRNA translation in acute ER stress.
  10. Mechanism of autophagy initiation by transmembrane selective autophagy receptors.
  11. Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response.
  12. Leucine inhibits degradation of outer mitochondrial membrane proteins to adapt mitochondrial respiration.
  13. Drug ubiquitination: an unwelcome mode of action or a novel modality.
  14. Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks.
  15. Proteomic and Ubiquitinated Proteome Insights Into ER Stress Responses in Chinese Hamster Ovary Cells Under Mild Hypothermic Conditions.
  16. Circadian clock control of ribosome composition promotes rhythmic translation and termination fidelity.
  17. Navigating lipid droplet proteins - part I: ER targeting routes of lipid droplet-destined membrane proteins.
  18. Real-time tracking of mRNP complex assembly reveals various mechanisms that synergistically enhance translation repression.
  19. FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis.
  20. In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70.
  21. Chaperone dependency during biogenesis does not correlate with chaperone dependency during refolding.
  22. Auxin-Induced Depletion of Human CCR4-NOT Subunits Reveals Opposing Functions of CNOT1 and CNOT4 in mRNA Metabolism.
  23. Inhibition of Histone Deacetylases Induces Cancer Cell Apoptosis Through the PERK Pathway of ER Stress Response.
  24. SND3 is the membrane insertase within a distinct SEC61 translocon complex.
  25. Age-related decline in protein turnover highlights cystatin C dysfunction and links proteostasis collapse to neuroinflammation in the murine cortex.
  26. Molecular glue degrader function of SPOP enhances STING-dependent immunotherapy efficacy in melanoma models.
  27. Application of HIV-1 viral protein R-derived-peptides as new E3 ligase-binding components of BRD4 degraders.
  28. Lysosome heterogeneity and diversity mapped through its distinct cellular functions.
  29. PRosettaC outperforms AlphaFold3 for modeling PROTAC ternary complexes.
  30. Induced proximity-based therapeutic modalities.
  31. Selective silencing of antibiotic-tethered ribosomes as a resistance mechanism against aminoglycosides.
  32. Identification of a Highly Cooperative PROTAC Degrader Targeting GTP-Loaded KRAS(On) Alleles.
  33. Mucin 2 and unfolded protein response reshape the mucus barrier in inflammatory bowel disease (Review).
  34. Structural dissection of αβ-tubulin heterodimer assembly and disassembly by human tubulin-specific chaperones.
  35. Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model.
  36. Leukocyte-intrinsic ER stress responses contribute to chemotherapy-induced peripheral neuropathy.
  37. GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions.
  38. The GCN-2/eIF-2α/ATF-4 signaling pathway is involved in C. elegans defense against Salmonella enterica infection.
  39. PLM-interact: extending protein language models to predict protein-protein interactions.
  40. The unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence.
  41. Longevity-promoting mitochondrial unfolded protein response activation requires elements of the PeBoW complex.
  42. Impairment of lysosomal quality control in Huntington disease.
  43. NRBF2 homodimerization by its coiled-coil domain strengthens association with the PtdIns3K complex mediated by the mit domain to promote autophagy.
  44. Activation of IRE1 Endonuclease Activity Regulates Zika Virus Replication and Antiviral Response During Infection in Human Microglia.
  45. TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin-binding proteins.
  46. The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells.
  47. Helicase-mediated mechanism of SSU processome maturation and disassembly.
  48. Non-equilibrium strategies enabling ligand specificity by signaling receptors.
  49. CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival.
  50. Neuralized-like proteins differentially activate Notch ligand.
  51. The E3 ligase RNF32 controls the IκB kinase complex and NF-κB signaling in intestinal stem cells.
  52. Ins and outs of IRES elements: function and significance.
  53. Navigating lipid droplet proteins - part II: molecular mechanisms underlying ER-to-lipid droplet protein partitioning.
  54. MGTbind: a comprehensive database of molecular glue ternary interactome.
  55. The stringent response does not influence ribosome pausing in Bacillus subtilis.
  56. Substrate recognition by the human mitochondrial processing peptidase and its processing of PINK1.
  57. GPX4 Inhibition Enhances the Pro-Oxidant and ER Stress Effects of Tempol in Colon and Gastric Cancer Cell Lines.
  1. Nat Rev Mol Cell Biol. 2025 Oct 27.
    Mechanisms and regulation of the Hsp70 chaperone network.
    Anne Wentink, Rina Rosenzweig, Harm Kampinga, Bernd Bukau.
      The 70-kDa heat shock protein (Hsp70) chaperone is essential to maintain cellular protein homeostasis, facilitating the folding, assembly, membrane translocation and quality control of proteins. Hsp70s achieve their functions through 'selective promiscuity', interacting with a wide range of substrate proteins while minimizing undesired interactions. J-domain proteins (JDPs) and nucleotide exchange factors (NEFs) are key to substrate recognition, remodelling and release from chaperone complexes. JDPs either target Hsp70s to specific subcellular sites where substrates reside (recruiters) or bind substrates directly by using highly specific (specialists) or multiple, versatile (generalists) binding sites. Through diverse substrate-binding modes and regulatory mechanisms, the 50 human JDPs confer remarkable client specificity to Hsp70s, a function that is comparable to that achieved by close to 600 E3 ubiquitin ligases in targeting proteins for degradation. Moreover, JDPs, together with NEFs, dictate the fate of Hsp70 clients by directing them to distinct protein quality control pathways, resulting in their folding or degradation. These recent mechanistic insights into Hsp70 regulation not only highlight the versatility and complexity of the Hsp70 network but also offer new avenues for more specific interventions in ageing-related and other protein folding diseases.
    DOI:  https://doi.org/10.1038/s41580-025-00890-9
  2. Cells. 2025 Oct 14. pii: 1593. [Epub ahead of print]14(20):
    Oligosaccharyltransferase Is Involved in Targeting to ER-Associated Degradation.
    Marina Shenkman, Navit Ogen-Shtern, Chaitanya Patel, Haddas Saad, Bella Groisman, Metsada Pasmanik-Chor, Sonya M Schermann, Roman Körner, Gerardo Z Lederkremer.
      Most membrane and secretory proteins undergo N-glycosylation, catalyzed by oligosaccharyltransferase (OST), a membrane-bound complex in the endoplasmic reticulum (ER). Proteins failing quality control are degraded via ER-associated degradation (ERAD), involving retrotranslocation to cytosolic proteasomes, or relegated to ER subdomains and eliminated via ER-phagy. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics, we identified OST subunits as differential key interactors with a misfolded ER protein bait upon proteasomal inhibition, suggesting unexpected involvement in ERAD. Previous reports implied additional roles for OST subunits beyond N-glycosylation, such as quality control by ribophorin I. We tested OST engagement in glycoprotein and non-glycosylated protein ERAD; overexpression or partial knockdown of OST subunits interfered with ERAD in conditions that did not affect glycosylation. We studied the effects on model misfolded type I and II membrane-bound proteins, BACE476 and asialoglycoprotein receptor H2a, respectively, and on a soluble luminal misfolded glycoprotein, α1-antitrypsin NHK variant. OST subunits appear to participate in late ERAD stages, interacting with the E3 ligase HRD1 and facilitating retrotranslocation. Molecular dynamics simulations suggest membrane thinning by OST transmembrane domains, possibly assisting retrotranslocation via membrane distortion.
    Keywords:  ERAD; HRD1; OST; endoplasmic reticulum; retrotranslocation
    DOI:  https://doi.org/10.3390/cells14201593
  3. Sci Adv. 2025 Oct 31. 11(44): eadt3014
    Pharmacological targeting of RIG-I can selectively activate the integrated stress response.
    Caroline A Cuoco, Wen Ren, Kelsey R Baron, Sakina Gologo, Valerie Perea, Prakhyat Mathur, Prerona Bora, Amina Ta, Alan Chu, Rama Aldakhlallah, Derek Rhoades, Christian M Cole, Ee Phie Tan, William C Hou, Leonard Yoon, Xiaoyan Guo, Jessica D Rosarda, Phil S Baran, Martin Kampmann, Lakeisha Tillery, Kristen A Johnson, Evan T Powers, R Luke Wiseman, Jeffery W Kelly.
      The integrated stress response (ISR) is a eukaryotic stress-responsive signaling pathway that attenuates global protein synthesis while allowing selective translation of specific mRNAs, which together can reestablish homeostasis following acute stress. Diverse pathologic insults activate one or more of the four ISR kinases, which selectively phosphorylate eIF2α to mediate ISR functions. Recent results suggest that enhancing ISR kinase activity could ameliorate pathologies linked to numerous diseases, including many neurodegenerative disorders. However, few pharmacological strategies exist to selectively activate ISR kinases and downstream adaptive signaling. Here, we report that compound A8 can preferentially activate the ISR through the binding of the cytosolic pattern recognition receptor RIG-I, which subsequently activates the heme-regulated inhibitor (HRI) ISR kinase independent of an interferon response. The establishment of A8 and its active metabolite CC81 provides opportunities to probe the biological and therapeutic relationship between innate immune signaling and ISR activation in health and disease.
    DOI:  https://doi.org/10.1126/sciadv.adt3014
  4. Nat Commun. 2025 Oct 27. 16(1): 8516
    Mode of action of a DCAF16-recruiting targeted glue that can selectively degrade BRD9.
    Scott J Hughes, Wojciech J Stec, Colin T R Davies, David McGarry, Alicia Williams, Marta Carrara, Ivan Del Barco Barrantes, Rebecca Harris, Anna Tasegian, Dominic D G Owens, Alexander Fawcett, John Hellicar, Aleksandra Azevedo, Gregor P Meier, Andrew C Runcie, Liliana Greger, Martin O'Rourke, Ian Churcher, Martin Pass, Giles A Brown, Alessio Ciulli, Louise K Modis, Andrea Testa.
      Molecular glue degraders for therapeutic target proteins are emerging as a strategy in drug discovery. Here, we modify a BRD9 ligand with specific chemical fragments to create degrader compounds that we call Targeted Glues. When bound to the target protein, these create an altered protein-ligand interface that is recognised by a ligase. This interaction between the target and the E3 ligase leads to protein degradation and is stabilised by a reversible covalent interaction between our molecule and a specific cysteine in the ligase. By screening a library of BRD9 targeted compounds we discover AMPTX1, a potent selective and reversibly covalent BRD9 degrader. In cells, AMPTX-1 selectively recruits the E3 ligase, DCAF16, to BRD9 and drives BRD9 degradation, as demonstrated by co-immunoprecipitation-mass spectrometry. BRD9 degradation is primarily dependent on the engagement of the surface Cys58 of DCAF16; the formation of a covalent adduct to DCAF16 is facilitated by ternary complex formation with BRD9. BRD9 degradation is also achieved in vivo with AMPTX-1 in a mouse xenograft model after oral dosing due to the drug-like, orally bioavailable properties of the compound. This supports the concept that covalent recruitment of DCAF16 is a viable approach in the development of therapeutic degraders.
    DOI:  https://doi.org/10.1038/s41467-025-63594-w
  5. J Microbiol Biotechnol. 2025 Oct 28. 35 e2507056
    Legionella pneumophila Effector Protein LegU1 Mediates the Ubiquitination of Endoplasmic Reticulum Chaperone BiP.
    Beibei Zhao, Shiyu He, Minjun Huang, Yongjun Lu, Zhenhuang Ge.
      The facultative intracellular pathogen Legionella pneumophila, which causes Legionnaires' disease, translocates over 300 effector proteins into the host cell. By hijacking numerous host cellular signaling pathways, these effectors promote bacterial survival and growth. One of the effector proteins, LegU1, is a F-box containing protein that binds to the host cell protein Skp1 to form a Skp1-Cullin-F-box protein (SCF) complex conferred E3 ubiquitin ligase activity. However, the role of LegU1 during L. pneumophila infection is incompletely known. Here, we demonstrate that LegU1 participates in modulation of the host BiP, an important endoplasmic reticulum chaperone that functions as both a master regulator and target protein of the unfolded protein response (UPR) process, during L. pneumophila infection. Ectopically expressed LegU1 localizes to the endoplasmic reticulum (ER) through the region from the 88th to the 136th residue. Deletion of LegU1 increases the protein level of BiP during L. pneumophila infection. We finally indicate that LegU1 interacts with and mediates the ubiquitinational degradation of BiP. Altogether, our study identifies BiP as a new substrate of LegU1 and provides new insight into how L. pneumophila modulates the host UPR pathway during infection.
    Keywords:  BiP; LegU1; Legionella pneumophila; ubiquitination
    DOI:  https://doi.org/10.4014/jmb.2507.07056
  6. Am J Physiol Gastrointest Liver Physiol. 2025 Oct 25.
    Beyond Fructolysis: Ketohexokinase Orchestrates ER Proteostasis in Nutrient-Stressed Hepatocytes.
    Salaheldeen Elsaid, Xiangdong Wu, Junkai Hu, Nigus Ambaye, Sui Seng Tee.
      Excessive intake of fructose and fats disrupt hepatocyte function by overwhelming endoplasmic reticulum (ER) capacity, leading to unresolved protein stress and progression to metabolic dysfunction-associated steatohepatitis (MASH)1. Ketohexokinase (KHK), the primary enzyme for fructose metabolism, is increasingly recognized for non-metabolic roles2,3, but its function in regulating ER proteostasis under nutrient stress remains unclear. We show that steatogenic conditions synergistically induce lipid accumulation and robust KHK expression accompanied by activation of the IRE1α-XBP1 branch of the unfolded protein response (UPR). This adaptive axis was observed in HepG2 cells, primary hepatocytes from GAN diet-fed mice, and liver biopsies from MASLD patients, establishing a conserved KHK-IRE1α axis across species. Khk knockdown disrupted this balance causing accumulation of misfolded and ubiquitinated proteins, proteotoxic stress, and a shift toward PERK-CHOP-driven apoptosis. Similar signatures in Khk-deficient mouse livers further underscore KHK's role in sustaining ER homeostasis. Our findings identify KHK as a dual-function enzyme: a metabolic gatekeeper of fructose flux and a proteostatic regulator that safeguards hepatocyte survival. While KHK contributes to steatosis, its complete loss destabilizes ER proteostasis, suggesting that selective inhibition of KHK enzymatic activity may offer therapeutic benefit without compromising ER function.
    Keywords:  ER stress; Ketohexokinase; MASH; Proteostasis; UPR
    DOI:  https://doi.org/10.1152/ajpgi.00235.2025
  7. J Cell Biol. 2026 Jan 05. pii: e202504178. [Epub ahead of print]225(1):
    Close-up of vesicular ER exit sites by volume electron imaging using FIB-SEM.
    Athul Nair, Akhil Nair, Patrick Stock, Arkash Jain, Elliott Somerville, Anwesha Sanyal, Jose Inacio Costa-Filho, Tom Kirchhausen.
      The architecture of ER exit sites (ERES), the first sites of membrane remodeling in protein secretion, remains unclear, with descriptions ranging from vesicular clusters to extended tubular structures. We addressed this divergence by visualizing ERES in cells not overexpressing secretory cargo using large-scale volume-focused ion beam scanning EM (FIB-SEM) after high-pressure freeze substitution. Automated segmentation in EM (ASEM), our 3D U-Net pipeline trained with sparsely labeled 50-70-nm COPI vesicles near the Golgi, accurately detected them in HeLa, SVG-A, and iPSC-derived neurons. Using the same model, we identified abundant clusters of ∼5-40 larger vesicles (∼65-85 nm) confined within ∼250 nm3 regions adjacent to flattened ER domains, consistent with vesicular ERES. Similar assemblies also appeared alongside tubular networks and varicosities extending from enlarged ER domains, previously described as the sole ERES in HeLa cells. These findings reveal that vesicular ERES are widespread and morphologically diverse, resolving longstanding contradictions in early secretory pathway organization.
    DOI:  https://doi.org/10.1083/jcb.202504178
  8. EMBO Rep. 2025 Oct 30.
    Stage-specific TRIM10 expression regulates erythroid maturation.
    Heesoo Kim, Wonji Shin, Dongeun Lee, Byunghoon Jeon, Yongbo Kim, Donghyuk Shin, Hyobin Jeong, Jun Young Hong, Sungwook Lee, Boyoun Park.
      Mammalian erythroid cells undergo extensive organelle and protein remodeling during erythropoiesis. The transcriptome and proteome of ubiquitin E3 ligases change dynamically during erythroid differentiation, yet mechanisms beyond E3 activity remain unclear. Here, we identify that tripartite motif-containing protein 10α (TRIM10α), an erythroid- and stage-specific E3 ligase, as crucial for stepwise erythroid maturation. TRIM10α self-association to localize on erythroblast surfaces, binding extracellular complement C1q, which facilitates pyrenocyte encapsulation and macrophage recognition. Surface C1q interacts with EpoR to promote lysosomal degradation, and its depletion prolongs Epo signaling. Notably, cytosolic TRIM10α enhances hemoglobin (Hb) maturation and sequesters Hb aggregates under oxidative conditions. Ultimately, TRIM10α self-ubiquitination and its binding to p62 are anticipated to lead to TRIM10α degradation, promoting the removal of Hb aggregates via autophagy. In contrast to TRIM10α, an alternatively spliced TRIM10β, which is barely expressed in human tissues and cells, forms deleterious aggregates, suggesting that evolutionary suppression of TRIM10β supports erythroid homeostasis. Our findings propose that aberrant TRIM10 expression drives erythroid-related diseases and highlight TRIM10 as a potential biomarker or therapeutic target.
    Keywords:  C1q; Erythropoiesis; Hemoglobin Maturation; TRIM10
    DOI:  https://doi.org/10.1038/s44319-025-00616-0
  9. Cell Rep. 2025 Oct 28. pii: S2211-1247(25)01264-1. [Epub ahead of print]44(11): 116493
    DDX3X acts as a selective dual switch regulator of mRNA translation in acute ER stress.
    Abd-El Monsif A Shawky, Allison V Eckhardt, Josef B Mick, Mahmoud F Dondeti, Kenneth Avanzino, Constantine A Simintiras, Maria Hatzoglou, Anastasios Vourekas.
      Regulation of eukaryotic mRNA translation initiation greatly impacts gene expression and is critical for cellular stress response. DDX3X is a ubiquitous DEAD-box RNA helicase whose precise role in scanning and translation regulation in non-stressed and stressed cells remains incompletely understood. Here, we show that DDX3X associates with thousands of mRNAs as part of the eIF4F-mediated 48S scanning complex and exerts dual regulatory effects, promoting or repressing translation of select mRNAs under basal conditions and reversing this regulation during acute endoplasmic reticulum stress. Initiation profiling reveals mechanistically distinct modes of DDX3X action linked to its binding patterns across the 5' UTR and coding sequence. We further uncover that mRNAs selectively regulated by DDX3X exhibit specific patterns of cytidine N4-acetylation near start codons, with shared de-repression observed upon NAT10 knockdown. Together, our findings reveal DDX3X as a context-sensitive regulator that has a possible functional connection with epitranscriptomic features in translation control.
    Keywords:  CP: Molecular biology; DDX3X; RNA helicase; ac4C; eIF3; endoplasmic reticulum stress; post-transcriptional modifications; post-transcriptional regulation of gene expression; translation initiation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116493
  10. EMBO J. 2025 Oct 30.
    Mechanism of autophagy initiation by transmembrane selective autophagy receptors.
    Elias Adriaenssens, Sascha Martens.
      Selective autophagy ensures the targeted degradation of damaged or surplus cellular components, including organelles, thereby safeguarding cellular homeostasis. This process relies on selective autophagy receptors (SARs) that link specific cargo to the autophagy machinery. These receptors exist in two distinct forms: soluble SARs that are recruited to the cargo on demand, and transmembrane SARs that are stably embedded in the membranes of organelles they target. While both receptor types converge on the same autophagy core machinery, they differ in how they recognize cargo, are regulated, and recruit this machinery to the site of degradation. In this review, we explore the unique challenges and strategies associated with transmembrane SARs, including how their activity is suppressed under basal conditions and activated in response to stress. We compare their mode of action with that of soluble SARs, highlight key differences in kinase regulation, including the roles of TBK1, ULK1, CK2, and Src, and discuss emerging models of autophagy initiation. We further highlight fundamental principles of organelle-selective autophagy and identify open questions that will guide future research.
    Keywords:  Autophagosome; ER-phagy; Mitophagy; Quality Control; Selective Autophagy
    DOI:  https://doi.org/10.1038/s44318-025-00615-w
  11. Nat Commun. 2025 Oct 28. 16(1): 9502
    Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response.
    Peipei Jiang, Qian Bian.
      Cells rapidly and extensively remodel their transcriptome in response to stress to restore homeostasis, but the underlying mechanisms are not fully understood. Here, we characterize the dynamic changes in transcriptome, epigenetics, and 3D genome organization during the integrated stress response (ISR). ISR induction triggers widespread transcriptional changes within 6 h, coinciding with increased binding of ATF4, a key transcriptional effector. Notably, ATF4 binds to hundreds of genes even under non-stress conditions, priming them for stronger activation upon stress. The transcriptional changes at ATF4-bound sites during ISR do not rely on increased H3K27 acetylation, chromatin accessibility, or rewired enhancer-promoter looping. Instead, ATF4-mediated gene activation is linked to the redistribution of CEBPγ from non-ATF4 sites to a subset of ATF4-bound regions, likely by forming an ATF4/CEBPγ heterodimer. CEBPγ preferentially targets the sites pre-occupied by ATF4, as well as genomic regions exhibiting a unique higher-order chromatin structure signature. Thus, the transcriptional responses during ISR are largely pre-wired by intrinsic chromatin properties. These findings provide critical insights into transcriptional remodeling during ISR with broader implications for other stress responses.
    DOI:  https://doi.org/10.1038/s41467-025-64577-7
  12. Nat Cell Biol. 2025 Oct 31.
    Leucine inhibits degradation of outer mitochondrial membrane proteins to adapt mitochondrial respiration.
    Qiaochu Li, Konstantin Weiss, Fuateima Niwa, Jan Riemer, Thorsten Hoppe.
      The mitochondrial proteome is remodelled to meet metabolic demands, but how metabolic cues regulate mitochondrial protein turnover remains unclear. Here we identify a conserved, nutrient-responsive mechanism in which the amino acid leucine suppresses ubiquitin-dependent degradation of outer mitochondrial membrane (OMM) proteins, stabilizing key components of the protein import machinery and expanding the mitochondrial proteome to enhance metabolic respiration. Leucine inhibits the amino acid sensor GCN2, which selectively reduces the E3 ubiquitin ligase cofactor SEL1L at mitochondria. Depletion of SEL1L phenocopies the effect of leucine, elevating OMM protein abundance and mitochondrial respiration. Disease-associated defects in leucine catabolism and OMM protein turnover impair fertility in Caenorhabditis elegans and render human lung cancer cells resistant to inhibition of mitochondrial protein import. These findings define a leucine-GCN2-SEL1L axis that links nutrient sensing to mitochondrial proteostasis, with implications for metabolic disorders and cancer.
    DOI:  https://doi.org/10.1038/s41556-025-01799-3
  13. Trends Biochem Sci. 2025 Oct 29. pii: S0968-0004(25)00245-2. [Epub ahead of print]
    Drug ubiquitination: an unwelcome mode of action or a novel modality.
    Richard A de Heiden, Monique P C Mulder.
      Recent reports by Orth, Pohl, et al. and Li, Garcia-Rivera et al. show that ubiquitin can mark drug-like molecules in cells. This non-canonical ubiquitination, initially discovered with proposed small-molecule HUWE1 inhibitors and a synthesis library component, respectively, offers a versatile chemical tool for probing protein regulation and developing new therapeutics.
    Keywords:  E3 ubiquitin ligase; HUWE1; RNF19; non-canonical ubiquitination; small-molecule ubiquitination; substrate-competitive inhibition
    DOI:  https://doi.org/10.1016/j.tibs.2025.10.004
  14. Cell Rep. 2025 Oct 29. pii: S2211-1247(25)01265-3. [Epub ahead of print]44(11): 116494
    Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks.
    Julie Smeyers, Juan A Oses-Prieto, Lin Yadanar, Man Wang, Marci Iadarola, Serena Lu, Karen S Wang, Taylor H Watanabe, Jayanta Debnath, Alma L Burlingame, Daniel A Mordes.
      Loss-of-function variants in TBK1, encoding a protein kinase, are strongly associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how haploinsufficiency for TBK1 leads to age-related neurodegeneration remains unresolved. Here, we utilize sets of isogenic induced pluripotent stem cells (iPSCs) with loss of TBK1 or loss of optineurin (OPTN) for quantitative global proteomics and phospho-proteomics in both stem cells and excitatory neurons. We found that TBK1 sustains the abundance and phosphorylation of its interacting adapter proteins, AZI2/NAP1, TANK, and TBKBP1/SINTBAD. Moreover, TBK1 regulates the phosphorylation of endo-lysosomal proteins, such as GABARAPL2, the late-endosome GTPase RAB7A, and selective autophagy cargo receptor proteins-including novel phospho-sites in p62/SQSTM1-in neurons. Finally, we provide a census of the phospho-proteome in nascent human neurons for further studies. Overall, TBK1 serves as a point of convergence in ALS/FTD-linked endo-lysosomal networks that act in a cell-autonomous manner to maintain protein homeostasis in neurons.
    Keywords:  ALS; CP: Molecular biology; CP: Neuroscience; OPTN; TBK1; autophagy; dementia; iPSC disease modeling; neurodegeneration; phospho-proteomics; proteomics
    DOI:  https://doi.org/10.1016/j.celrep.2025.116494
  15. Biotechnol Bioeng. 2025 Oct 28.
    Proteomic and Ubiquitinated Proteome Insights Into ER Stress Responses in Chinese Hamster Ovary Cells Under Mild Hypothermic Conditions.
    David Ryan, Christiana-Kondylo Sideri, Michael Henry, Selvaprakash Karuppuchamy, Esen Efeoglu, Paula Meleady.
      Chinese hamster ovary (CHO) cells, widely utilised in biopharmaceutical production, experience various stressors during cell culture that can affect protein expression and folding, particularly within the endoplasmic reticulum (ER). Mild hypothermia is widely employed in CHO cell bioproduction to improve recombinant protein yield and quality; however, its impact on ER-associated pathways, particularly those governing protein folding and stress responses, remains insufficiently characterised. Mass spectrometry-based proteomics allows for the identification and relative quantification of proteins, enabling detailed insights into protein expression, modifications, and functional networks. This study investigates the impact of mild hypothermic conditions (31°C) on the whole cell proteome and ubiquitinated proteome of CHO cells, with a specific focus on ER proteins and ER stress. Using high-resolution mass spectrometry, we conducted a comprehensive proteomic and ubiquitinated proteomic analysis to quantify changes in protein abundance and ubiquitinated peptides under mild hypothermia. The downregulation of several proteins involved in the glycosylation of nascent polypeptides at 31°C, including DDOST, P4HB, PRKSCH and LMAN1, in all cell lines studied suggests that mild hypothermic shock disrupts the cell's normal ability to fold new proteins, leading to ER stress as the misfolded proteins build up. When this is coupled with the maintained cell viability and increased productivity at 31°C, it indicates the ER stress response can mitigate the build-up of misfolded proteins. The differential regulation of the transcription factor eIF2α, downregulated in non-producer cells but upregulated in producer cells at 31°C, suggests that recombinant protein-producing CHO cells possess a more adaptive ER stress response, enabling more efficient function under hypothermic culture conditions. Enhanced ubiquitination of misfolded protein substrates highlights an increased reliance on ER-associated degradation (ERAD) pathways to alleviate proteotoxic stress, as well as the wide range of biological processes that are regulated by ubiquitination as part of the hypothermic stress response. These findings provide new insights into the cellular adaptation mechanisms of CHO cells to mild hypothermia, with implications for optimising bioproduction strategies to improve yield and quality of therapeutic proteins. Our study highlights the importance of understanding the more complex aspects of the proteome and how this additional layer of detail can open new avenues for CHO cell engineering.
    Keywords:  Chinese hamster ovary (CHO) cells; endoplasmic reticulum (ER) stress; endoplasmic reticulum associated degradation (ERAD); hypothermia; protein folding; ubiquitination; unfolded protein response (UPR)
    DOI:  https://doi.org/10.1002/bit.70081
  16. Cell Rep. 2025 Oct 28. pii: S2211-1247(25)01255-0. [Epub ahead of print]44(11): 116484
    Circadian clock control of ribosome composition promotes rhythmic translation and termination fidelity.
    Teresa M Lamb, Kathrina D Castillo, Rachel Porter, Cheng Wu, Samuel O Purvine, Griffin Best, Erika Zink, Ebimobowei O Preh, Lunda Shen, Matthew S Sachs, Deborah Bell-Pedersen.
      Ribosome composition is dynamic, shifting with cell state and stress, but whether it varies with circadian time is unknown. Here, we uncover circadian clock-driven changes in ribosome composition in Neurospora crassa. Mass spectrometry of ribosomes across circadian time identified six ribosomal proteins and one associated factor under clock control. Rhythms in eL31 abundance were validated in purified ribosomes, and deletion of el31 disrupted translation rhythms in nearly half of rhythmically translated mRNAs. N. crassa eL31 promotes circadian control of translation termination and impacts elongation fidelity while maintaining Mg homeostasis, a key determinant of translational accuracy. These findings reveal that the circadian clock reprograms ribosome composition to orchestrate rhythmic translation and fidelity, temporally expanding the proteome beyond the static genome to align cellular function with time of day.
    Keywords:  CP: Molecular biology; Zuotin; circadian clock; eL31; magnesium ion homeostasis; ribosome composition; translation; translation fidelity
    DOI:  https://doi.org/10.1016/j.celrep.2025.116484
  17. Biochem Soc Trans. 2025 Oct 29. 53(5): 1401-1415
    Navigating lipid droplet proteins - part I: ER targeting routes of lipid droplet-destined membrane proteins.
    Louisa Magdalena Krauß, Bianca Schrul.
      Lipid droplets (LDs) are cytosolic lipid storage organelles that derive from the endoplasmic reticulum (ER). Their biogenesis and function are essential for maintaining cellular lipid homeostasis and require a spatiotemporally co-ordinated recruitment of specific membrane proteins to the LD surface. Many LD-destined proteins are inserted into the ER phospholipid bilayer in a monotopic hairpin topology before they can partition to the LD monolayer. About a third of all cellular proteins enter the ER during their biogenesis, either as ER-resident or as secretory proteins. Decades of research have provided a solid understanding of which molecular machineries ensure ER targeting fidelity of transmembrane-spanning proteins. The molecular mechanisms underlying the biogenesis of LD-destined monotopic proteins, however, are only beginning to emerge. In this first part of the bipartite review 'Navigating lipid droplet proteins,' we provide an overview of the general principles underlying protein targeting to the ER. We highlight recent advances and current challenges regarding the specific mechanisms for LD-destined proteins and discuss their physiological implications. The molecular mechanisms underlying the subsequent ER-to-LD protein partitioning are at the heart of the second part of this bipartite review.
    Keywords:  cellular targeting; endoplasmic reticulum; lipid droplets; membrane proteins; membranes; organelles; transmembrane proteins
    DOI:  https://doi.org/10.1042/BST20253051
  18. Cell Rep. 2025 Oct 28. pii: S2211-1247(25)01263-X. [Epub ahead of print]44(11): 116492
    Real-time tracking of mRNP complex assembly reveals various mechanisms that synergistically enhance translation repression.
    Marco Payr, Julia Meyer, Eva-Maria Geissen, Janosch Hennig, Olivier Duss.
      Protein biosynthesis must be highly regulated to ensure proper spatiotemporal gene expression and thus cellular viability. Translation is often modulated at the initiation stage by RNA-binding proteins through either promotion or repression of ribosome recruitment to the mRNA. However, it largely remains unknown how the kinetics of mRNA ribonucleoprotein (mRNP) assembly on untranslated regions (UTRs) relate to its translation regulation activity. Using Sex-lethal (Sxl)-mediated translation repression of msl-2 in female fly dosage compensation as a model system, we show that different mechanisms in mRNP assembly synergistically achieve tight translation repression. Using multicolor single-molecule fluorescence microscopy, we show that Sxl targets its binding sites via facilitated diffusion and multivalent binding, Unr recruitment is accelerated over 500-fold by RNA-bound Sxl, and Hrp48 further stabilizes RNA-bound Sxl indirectly via ATP-independent RNA remodeling. Overall, we provide a framework to study how multiple RBPs dynamically cooperate with RNA to achieve function.
    Keywords:  CP: Molecular biology; NMR; RNA chaperone; RNP dynamics; dosage compensation; mRNP complex formation; protein-RNA interactions; single-molecule FRET; translation regulation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116492
  19. Nat Cell Biol. 2025 Oct 29.
    FSP1-mediated lipid droplet quality control prevents neutral lipid peroxidation and ferroptosis.
    Mike Lange, Michele Wölk, Vivian Wen Li, Cody E Doubravsky, Joseph M Hendricks, Shunji Kato, Yurika Otoki, Benjamin Styler, Sean L Johnson, Cynthia A Harris, Kiyotaka Nakagawa, Isabel F Snodgrass, Dohee Kim, John W Newman, Maria Fedorova, James A Olzmann.
      Lipid droplets (LDs) are organelles that store and supply lipids, based on cellular needs. Although mechanisms preventing oxidative damage to membrane phospholipids are established, the vulnerability of LD neutral lipids to peroxidation and protective mechanisms are unknown. Here we identify LD-localized ferroptosis suppressor protein 1 (FSP1) as a critical regulator that prevents neutral lipid peroxidation by recycling coenzyme Q10 (CoQ10) to its lipophilic antioxidant form. Lipidomics reveal that FSP1 loss leads to the accumulation of oxidized triacylglycerols and cholesteryl esters, and biochemical reconstitution of FSP1 with CoQ10 and NADH suppresses triacylglycerol peroxidation in vitro. Notably, inducing polyunsaturated fatty acid-rich LDs triggers triacylglycerol peroxidation and LD-initiated ferroptosis when FSP1 activity is impaired. These findings uncover the first LD lipid quality-control pathway, wherein LD-localized FSP1 maintains neutral lipid integrity to prevent the build-up of oxidized lipids and induction of ferroptosis.
    DOI:  https://doi.org/10.1038/s41556-025-01790-y
  20. Cell Mol Life Sci. 2025 Oct 28. 82(1): 366
    In long-lasting cellular stress phases of melanoma cells, stress granules are dissolved by HSP70.
    Sebastian Staebler, Katharina Pieger, Jacqueline Perl, David Stieglitz, Aranya Thongmao, Nadja Schneider, Anja Katrin Bosserhoff, Silke Kuphal.
      Cancer cells must adapt to harsh microenvironmental conditions such as nutrient deprivation, hypoxia and immune pressure to sustain their high proliferative potential. One adaptive mechanism involves the formation of stress granules (SGs), which promote cell survival under stress. Interestingly, melanoma cells appear to tolerate such stressors, including hypoxia and chemotherapeutic agents, with minimal dependence on SG formation. In this study, we identify heat shock protein 70 (HSP70) as a key mediator of stress resistance in melanoma cells and the participation of the kinase CK2 in this process. We demonstrate that melanoma cells express high endogenous levels of HSP70, which preserves protein homeostasis and inhibits apoptosis under both environmental and drug-induced stress. Silencing of HSP70 led to increased SG formation and sensitized melanoma cells to apoptosis, particularly in response to the BRAF inhibitor Vemurafenib. These findings suggest that HSP70 plays a central role in melanoma cell survival by compensating for the need for SGs and promoting resistance to therapeutic stress.
    Keywords:  Cellular stress; Melanoma; Stress granules; Vemurafenib-resistance
    DOI:  https://doi.org/10.1007/s00018-025-05939-8
  21. Mol Syst Biol. 2025 Oct 28.
    Chaperone dependency during biogenesis does not correlate with chaperone dependency during refolding.
    Divya Yadav, İdil I Demiralp, Mark Fakler, Stephen D Fried.
      Many proteins require molecular chaperones to fold into their functional native forms. However, the roles of chaperones during primary biogenesis in vivo can differ from the functions they play during in vitro refolding experiments. Here, we use limited proteolysis mass spectrometry (LiP-MS) to probe structural changes incurred by the E. coli proteome when two key chaperones, trigger factor and DnaKJ, are deleted. While knocking out DnaKJ induces pervasive structural perturbations across the soluble E. coli proteome, trigger factor deletion only impacts a small number of proteins' structures. Overall, proteins which cannot spontaneously refold (or require chaperones to refold in vitro) are not more likely to be dependent on chaperones to fold in vivo. We find that chaperone-nonrefolders (proteins that cannot refold even with chaperone assistance) do not generally require chaperones to fold in vivo, strengthening the view that chaperone-nonrefolders are obligate co-translational folders. Hence, for some E. coli proteins, the vectorial nature of co-translational folding is the most important "chaperone".
    Keywords:  Chaperones; Co-translational Folding; DnaK; Structural Proteomics; Trigger Factor
    DOI:  https://doi.org/10.1038/s44320-025-00166-6
  22. J Biol Chem. 2025 Oct 27. pii: S0021-9258(25)02714-0. [Epub ahead of print] 110862
    Auxin-Induced Depletion of Human CCR4-NOT Subunits Reveals Opposing Functions of CNOT1 and CNOT4 in mRNA Metabolism.
    Shardul Kulkarni, Courtney Smith, Oluwasegun T Akinniyi, Belinda M Giardine, Cheryl A Keller, Alexei Arnaoutov, Joseph C Reese.
      CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. Originally discovered in yeast, this complex is highly conserved across eukaryotes, although its composition and functions differ between mammals and yeast. For example, unlike yeast Not4, human CNOT4 (E3 Ubiquitin Ligase) does not form a stable complex with CCR4-NOT, and its functions are less clear compared to its yeast counterpart. To investigate the roles of CNOT1 (the central scaffold subunit) and CNOT4, we developed a rapid auxin-induced degron cell culture system that allows for the acute depletion of these proteins. We studied the effects of their absence on complex integrity, cell growth, and mRNA expression and turnover. Our transcriptome-wide analysis revealed that depleting CNOT1 altered the expression of thousands of transcripts, with the majority showing increased abundance and a general decrease in mRNA decay. Although CNOT4 does not associate with the complex through standard biochemical methods, BioID proximity labelling confirmed its association with the complex in cells. However, depleting CNOT4 did not affect the integrity of CCR4-NOT. In contrast to the effects observed with CNOT1 depletion, reducing CNOT4 levels led to only modest changes in RNA steady-state levels and unexpectedly accelerated global mRNA decay. Finally, we show that the changes in mRNA stability in CCR4-NOT-depleted cells correlated with the codon optimality of the transcripts. Our data suggest that CNOT4 exerts opposite effects on mRNA metabolism compared to CNOT1, and that CNOT4 may have functions distinct from those of complex subunits that promote mRNA degradation.
    Keywords:  CCR4-NOT; CNOT1; CNOT4; mRNA decay
    DOI:  https://doi.org/10.1016/j.jbc.2025.110862
  23. J Cell Mol Med. 2025 Nov;29(21): e70928
    Inhibition of Histone Deacetylases Induces Cancer Cell Apoptosis Through the PERK Pathway of ER Stress Response.
    Xuan Wang, Caiyun Yang, Yili Yang, Feng Xu, Donglan Yuan, Yuexi Gu.
      Activation of the endoplasmic reticulum (ER) stress is an adaptive response to disturbed ER homeostasis caused by the accumulation of misfolded or unfolded proteins, or an acute increase in the entry of newly synthesised or mutated proteins into the ER lumen. Overwhelmed or prolonged ER stress causes apoptotic cell death or a maladaptive state of the cell, resulting in various pathological diseases including cancer, inflammation and aging. With a screening of a chemical compound library, here we show that inhibition of histone deacetylases (HDACs) induces ER stress, along with increased retro-translocation of misfolded proteins from the ER lumen to the cytosol for proteasomal degradation. HDAC inhibitors (HDACi) activate the PERK-eIF2α subbranch of the unfolded protein response (UPR), whereas the IRE1α and ATF6 pathways are not affected. Inhibition of the PERK subbranch with specific siRNA or a small molecule inhibitor ameliorates HDACi-induced apoptotic cell death. In addition, a non-phosphorylatable mutant of eIF2α, a critical substrate that transduces the PERK-mediated ER stress response, abolishes apoptosis induced by HDACi, but not by the DNA damage reagent doxorubicin. HDACi reduce the sizes of tumours formed from wildtype but not eIF2αS51A-mutant cells in a xenograft model, further demonstrating the involvement of the PERK subbranch in HDACi-induced ER stress and cell death. Our study reveals novel effects of the well-studied family of HDAC inhibitors, which can be explored further in clinics to treat certain types of cancer manifested with abnormal ER stress conditions.
    Keywords:  ER stress; HDAC inhibitors; PERK pathway; apoptosis; quisinostat; unfolded protein response
    DOI:  https://doi.org/10.1111/jcmm.70928
  24. Nat Commun. 2025 Oct 29. 16(1): 9566
    SND3 is the membrane insertase within a distinct SEC61 translocon complex.
    Tzu-Jing Yang, Saumyak Mukherjee, Julian D Langer, Gerhard Hummer, Melanie A McDowell.
      During the biogenesis of most eukaryotic integral membrane proteins (IMPs), transmembrane domains are inserted into the endoplasmic reticulum membrane by a dedicated insertase or the SEC61 translocon. The SRP-independent (SND) pathway is the least understood route into the membrane, despite catering for a broad range of IMP types. Here, we show that Chaetomium thermophilum SND3 is a membrane insertase with an atypical fold. We further present a cryo-electron microscopy structure of a ribosome-associated SND3 translocon complex involved in co-translational IMP insertion. The structure reveals that the SND3 translocon additionally comprises the complete SEC61 translocon, CCDC47 and TRAPɑ. Here, the SEC61β N-terminus works together with CCDC47 to prevent substrate access to the translocon. Instead, molecular dynamics simulations show that SND3 disrupts the lipid bilayer to promote IMP insertion via its membrane-embedded hydrophilic groove. Structural and sequence comparisons indicate that the SND3 translocon is a distinct multipass translocon in fungi, euglenozoan parasites and other eukaryotic taxa.
    DOI:  https://doi.org/10.1038/s41467-025-65357-z
  25. Biogerontology. 2025 Oct 31. 26(6): 199
    Age-related decline in protein turnover highlights cystatin C dysfunction and links proteostasis collapse to neuroinflammation in the murine cortex.
    Daiana Burdusel, Sina Ghaemmaghami, Diana-Larisa Ancuta, Coman Cristin, Dirk Hermann, Thorsten Doeppner, Denisa F V Pirscoveanu, Aurel Popa-Wagner.
      One of the key hallmarks of aging is the breakdown of proteostasis-the finely tuned balance of protein synthesis, folding, trafficking, and degradation that maintains proteome integrity and cellular function. In this study, we employed 15N metabolic labeling to assess protein turnover in young and aged mice. Among the proteins examined, cystatin C exhibited the largest age-related reduction in turnover, alongside decreases in other proteins involved in neuroprotection, structural stability, and neurotransmission, including transthyretin, proteolipid protein 1, and the astrocytic glutamate transporter SLC1A3. Reduced proteostatic capacity is likely to increase neuronal susceptibility to proteotoxic stress, protein aggregation, and excitotoxic injury. Immunohistochemical analysis revealed a punctate accumulation of cystatin C in cortical layer IV, a region particularly vulnerable to age-related pathology. Moreover, gene expression profiling showed region-specific upregulation of inflammatory markers (Cd11b, Fcgr1, and Cr3), suggesting enhanced degradation of brain structures through phagocytic activity. Together, these findings demonstrate that aging disrupts proteostasis in a protein- and region-specific manner, with cystatin C emerging as a central mediator linking impaired clearance to neuroinflammation and cortical vulnerability. Interventions aimed at enhancing autophagy, proteasome function, or chaperone activity may represent promising strategies to counteract proteostasis collapse and mitigate neurodegeneration in the aging brain.
    Keywords:  Ageing; Brain; Cystatin C; Isotopic labelling; Neurodegeneration; Neuroinflammation; Proteostasis
    DOI:  https://doi.org/10.1007/s10522-025-10339-3
  26. J Clin Invest. 2025 Oct 28. pii: e191772. [Epub ahead of print]
    Molecular glue degrader function of SPOP enhances STING-dependent immunotherapy efficacy in melanoma models.
    Zhichuan Zhu, Xin Zhou, Max Xu, Jianfeng Chen, Kevin C Robertson, Gatphan N Atassi, Mark G Woodcock, Allie C Mills, Laura E Herring, Gianpietro Dotti, Pengda Liu.
      The E3 ligase SPOP plays a context-dependent role in cancer by targeting specific cellular proteins for degradation, thereby influencing cell behavior. However, its role in tumor immunity remains largely unexplored. In this study, we revealed that SPOP targeted the innate immune sensor STING for degradation in a CK1γ phosphorylation-dependent manner to promote melanoma growth. Stabilization of STING by escaping SPOP-mediated degradation enhanced anti-tumor immunity by increasing IFNβ production and ISG expression. Notably, small-molecule SPOP inhibitors not only blocked STING recognition by SPOP, but also acted as molecular glues, redirecting SPOP to target neo-substrates such as CBX4 for degradation. This CBX4 degradation led to increased DNA damage, which in turn activated STING and amplified innate immune responses. In a xenografted melanoma B16 tumor model, single-cell RNA-seq analysis demonstrated that SPOP inhibition induced the infiltration of immune cells associated with anti-PD1 responses. Consequently, SPOP inhibitors synergized with immune checkpoint blockade to suppress B16 tumor growth in syngeneic murine models and enhanced the efficacy of CD19-CAR-T therapy. Our findings highlight a molecular glue degrader property of SPOP inhibitors, with potential implications for other E3 ligase-targeting small molecules designed to disrupt protein-protein interactions.
    Keywords:  Cell biology; Cellular immune response; Oncology; Skin cancer; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI191772
  27. RSC Chem Biol. 2025 Oct 29.
    Application of HIV-1 viral protein R-derived-peptides as new E3 ligase-binding components of BRD4 degraders.
    Kohei Tsuji, Xueyuan Huang, Maho Miyamoto, Sayaka Sukegawa, Hidetomo Yokoo, Hiroaki Takeuchi, Yosuke Demizu, Hirokazu Tamamura.
      Proteolysis targeting chimeras (PROTACs) have become a new modality for drug development of particular importance for cancer chemotherapy. PROTACs are composed of a ligand that binds to the protein of interest (POI) tethered by a linker to a ubiquitin E3 ligase-binding motif. These molecules can degrade the POI by ubiquitination and subsequent digestion using the ubiquitin-proteasome system (UPS). Although more than six hundred E3 ligases are encoded in human genome, only a small number are currently utilized by PROTACs. Because the expression levels and activities of E3 ligases vary among the cell lines, it can be advantageous to develop PROTACs that utilize new E3 ligase-binding components. In our current work we report new E3 ligase-binding ligands that employ viral protein R (Vpr), an accessory protein of the human immunodeficiency virus type-1 (HIV-1). Vpr can bind to both the E3 ligase complex, Cul4A-DDB1-DCAF1 and host proteins, such as UNG2 and facilitate host protein degradation via the UPS. We envisioned that Vpr fragments may function in PROTACs as new E3 ligase-binding ligands. Herein, we designed, synthesized and evaluated bromodomain 4 (BRD4)-targeting PROTACs (BRD4-PROTACs) that employ a well-known BRD4 inhibitor (JQ1) as a warhead and Vpr-derived peptides as the E3 ligase-binding ligands. We successfully demonstrate that the Vpr-derived peptides can function as E3 ligase-targeting ligands for PROTAC development. We also evaluated PROTACs based on the HIV-1 latency-reversing activity of JQ-1. The chemical degraders are less effective than the parent inhibitor as a latency-reversing agent (LRA). However, the low cytotoxicity of the new peptidic PROTACs allowed the compounds to be tolerated at high does, leading to potent LRA activity.
    DOI:  https://doi.org/10.1039/d5cb00125k
  28. Cell Mol Life Sci. 2025 Oct 30. 82(1): 380
    Lysosome heterogeneity and diversity mapped through its distinct cellular functions.
    Indira Chavan, Arindam Bhattacharjee.
      Lysosomes respond to cellular nutrient availability and diverse oncoming vesicle traffic such as endocytosis and autophagy by switching between anabolic signaling or catabolic hydrolase activity, which coincides with a drastic shift in their cellular distribution, organelle contacts, ion homeostasis, membrane proteome and lipidome. Emerging evidence now reveals a dynamic remodeling of lysosomal membrane to counter membrane damage, acting via extensive lipid transfer from the endoplasmic reticulum or by localized membrane repair. Functionally, lysosomes play a key role in lipid metabolism and intracellular calcium signaling. Unsurprisingly, disease-associated lysosomes are either often hyperactive- thus promoting abnormal tissue growth, or hypoactive, promoting storage. Taken together, this presents an incredible functional diversity among the cellular population of lysosomes. Here, we discuss this intracellular heterogeneity and intercellular diversity in context of lysosomal function in health and disease.
    Keywords:  Lipid storage disorders; Lysosome plasticity; Lysosome quality control; Lysosome subpopulations; Phosphoinositides
    DOI:  https://doi.org/10.1007/s00018-025-05883-7
  29. Sci Rep. 2025 Oct 28. 15(1): 37620
    PRosettaC outperforms AlphaFold3 for modeling PROTAC ternary complexes.
    Joseph M Schulz, Sarah I Schürer, Robert C Reynolds, Stephan C Schürer.
      Targeted protein degradation via PROTACs offers a promising therapeutic strategy, yet accurate modeling of ternary complexes remains a critical challenge in degrader design. In this study, we systematically benchmark two leading structure prediction tools, AlphaFold-3 and PRosettaC, against a curated dataset of 36 crystallographically resolved ternary complexes. Using DockQ as a quantitative interface scoring metric, we assess the structural fidelity of predicted complexes under both scaffold-inclusive and stripped configurations. Our results demonstrate that AlphaFold-3's performance is often inflated by accessory proteins such as Elongin B/C or DDB1, which contribute to overall interface area but not degrader-specific binding. PRosettaC, on the other hand, leverages chemically defined anchor points to yield more geometrically accurate models in select systems, though it frequently fails when linker sampling is insufficient or misaligned. To overcome the limitations of static benchmarking, we introduce a dynamic evaluation strategy using molecular dynamics simulations of the crystal structures. This frame-resolved analysis reveals that several PRosettaC models, while poorly aligned to the static crystal conformation, transiently achieve high DockQ alignment with specific frames along the MD trajectory. These findings underscore the importance of incorporating protein flexibility into benchmarking workflows and suggest that transient conformational compatibility may be overlooked in conventional evaluations. By combining constraint-based modeling with dynamic frame matching, this study provides a more nuanced framework for assessing ternary complex predictions and informs the selection of in silico tools for rational PROTAC development.
    Keywords:  AlphaFold; Computational modeling; PROTACs; PRosettaC; Protein modeling; Structure-based-drug-design
    DOI:  https://doi.org/10.1038/s41598-025-21502-8
  30. Nat Rev Drug Discov. 2025 Oct 31.
    Induced proximity-based therapeutic modalities.
    Elizabeth A King, Margot Meyers, Daniel K Nomura.
      Proximity is a key component of nearly all regulatory pathways within biological systems. Over the past few decades, the rapid development of induced proximity modalities has allowed for therapeutic intervention beyond classical occupancy-driven pharmacology. These modalities comprise multispecific small molecules or biologic agents that co-opt native biological pathways by inducing an interaction between biomolecules. Small-molecule 'molecular glues' modify protein surfaces to induce non-native interactions or to stabilize existing protein-protein interactions. They have been in the clinic since the 1980s but have more recently been shown to enable targeted protein degradation or inhibition and have been rationally designed to achieve this. Early discoveries on molecular glues spearheaded the development of next-generation heterobifunctional modalities for targeted protein degradation, such as proteolysis-targeting chimeras, which are seeing early-stage clinical success. Here, we aim to survey the field of induced proximity with a focus on potential therapeutic applications. We discuss the emergence of novel approaches to control cellular processes beyond protein degradation, including post-translational modifications, cellular localization and transcriptional activation. Some of these approaches are showing preclinical efficacy in various disease models.
    DOI:  https://doi.org/10.1038/s41573-025-01316-z
  31. Nat Commun. 2025 Oct 29. 16(1): 9568
    Selective silencing of antibiotic-tethered ribosomes as a resistance mechanism against aminoglycosides.
    Nilanjan Ghosh Dastidar, Nicola S Freyer, Valentyn Petrychenko, Ana C de A P Schwarzer, Bee-Zen Peng, Ekaterina Samatova, Christina Kothe, Marlen Schmidt, Frank Peske, Antonio Z Politi, Henning Urlaub, Niels Fischer, Marina V Rodnina, Ingo Wohlgemuth.
      Antibiotic resistance is a growing threat, underscoring the need to understand the underlying mechanisms. Aminoglycosides kill bacteria by disrupting translation fidelity, leading to the synthesis of aberrant proteins. Surprisingly, mutations in fusA, a gene encoding translation elongation factor G (EF-G), frequently confer resistance, even though EF-G neither participates in mRNA decoding nor blocks aminoglycoside binding. Here, we show that EF-G resistance variants selectively slow ribosome movement along mRNA when aminoglycosides are bound. This delay increases the chance that the drug dissociates before misreading occurs. Over several elongation cycles, this selective silencing of drug-bound ribosomes prevents error cluster formation, preserving proteome and membrane integrity. As a result, fusA mutations confer resistance early in treatment by preventing self-promoted aminoglycoside uptake. Translation on drug-free ribosomes remains sufficiently rapid to sustain near-normal bacterial growth. The mechanism of selective silencing of corrupted targets reveals a previously unrecognized antibiotic resistance strategy with potential therapeutic implications.
    DOI:  https://doi.org/10.1038/s41467-025-65298-7
  32. J Am Chem Soc. 2025 Oct 30.
    Identification of a Highly Cooperative PROTAC Degrader Targeting GTP-Loaded KRAS(On) Alleles.
    Vesna Vetma, Ilaria Puoti, Natalia K Karolak, Sohini Chakraborti, Emelyne Diers, Enrico Girardi, Shakil Khan, Giorgia Kidd, Katrin G Kropatsch, Ross Mclennan, Suzanne O'Connor, Matthias Samwer, Nicole Trainor, Claire Whitworth, Andre J Wijaya, Jeff Y F Wong, David Zollman, William Farnaby, Johannes Popow, Alessio Ciulli, Peter Ettmayer, Kirsten McAulay.
      Kirsten rat sarcoma viral oncogene homologue (KRAS) is a frequently mutated oncogene in multiple types of cancer and is a high priority target for oncology drug development. There are many different KRAS mutations, including mutations that favor the GTP-loaded hydrolysis-incompetent "active" state of KRAS, KRAS(on), that can lead to tumorigenesis. However, small molecule interventions thus far have predominantly targeted single mutations of "inactive" GDP-loaded KRAS, KRAS(off), such as KRASG12C. Here, we address this gap through the development of heterobifunctional VHL-based PROTACs capable of engaging and degrading KRAS(on), thus addressing a wider range of KRAS mutations. By studying ternary complex affinity, stability, and binding modes using SPR and X-ray cocrystal structures, we identified PROTACs that exhibit high positive cooperativity in forming ternary complexes with VHL and GCP-loaded KRAS as representative of KRAS(on) variants. Degrader activity profiling in relevant cancer cells supported the discovery of ACBI4, a PROTAC which forms a highly stable and cooperative ternary complex between VHL and GTP-bound KRAS and which potently degrades KRASG12R, leading to antiproliferative effect in KRAS mutant-driven cancer cells. ACBI4 provides a new chemical tool for studying the impact of degrading KRAS(on) mutants, which is not possible with current pan-KRAS inhibitors or degraders.
    DOI:  https://doi.org/10.1021/jacs.5c10354
  33. Mol Med Rep. 2026 Jan;pii: 18. [Epub ahead of print]33(1):
    Mucin 2 and unfolded protein response reshape the mucus barrier in inflammatory bowel disease (Review).
    Zhiwei Yuan, Zheng Xia, Liyun Ling, Jingzhe Xie, Feng Zhang.
      Mucin 2 (MUC2) is the primary structural component of the intestinal mucus layer and is essential for maintaining the integrity of the mucus barrier and influencing the development of inflammatory bowel disease (IBD). Disruption of MUC2 production or secretion compromises barrier function, increasing susceptibility to the chronic mucosal inflammation characteristic of IBD. Given their large size and complex folding requirements, immature MUC2 precursors easily accumulate in the endoplasmic reticulum (ER) and cause ER stress, leading to activation of the unfolded protein response (UPR). The UPR restores ER homeostasis by reducing protein synthesis, enhancing folding, and degrading misfolded proteins. The mammalian UPR has three known signaling branches: Pancreatic ER kinase, ER transmembrane inositol‑requiring enzymes 1α and β (IRE1α and IRE1β) and activating transcription factor 6. Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the ER. Importantly, IRE1β‑AGR2 signaling potentially serves as a superior regulatory mechanism for controlling UPR activation caused by the misfolding of MUC2 in goblet cells. The present review highlights the critical role of MUC2 dysfunction and UPR imbalance in IBD pathogenesis. Targeting the association between novel UPR signaling pathways and restoring MUC2 protein function may provide new insights into IBD research and treatment.
    Keywords:  anterior gradient 2; inflammatory bowel disease; inositol‑requiring enzymes 1β; mucin 2; unfolded protein response
    DOI:  https://doi.org/10.3892/mmr.2025.13728
  34. Science. 2025 Oct 30. 390(6772): eady2708
    Structural dissection of αβ-tubulin heterodimer assembly and disassembly by human tubulin-specific chaperones.
    Yeonjae Seong, Hyunmin Kim, Kyumi Byun, Yeon-Woo Park, Soung-Hun Roh.
      Microtubule assembly requires a set of chaperones known as tubulin-binding cofactors (TBCs). We used cryo-electron microscopy to visualize how human TBCD, TBCE, TBCC, and guanosine triphosphatase (GTPase) Arl2 mediate αβ-tubulin assembly and disassembly. We captured multiple conformational states, revealing how TBCs orchestrate tubulin heterodimer biogenesis. TBCD stabilizes monomeric β-tubulin and scaffolds the other cofactors. Guanosine triphosphate (GTP) binding to Arl2 induces conformational changes that toggle the complex between assembly and disassembly. TBCD and TBCE guide α- and β-tubulin into a partially assembled interface, and TBCC, acting as a molecular clamp, completes the heterodimer. TBCD also functions as a GTPase activating protein for β-tubulin. β-tubulin GTP hydrolysis is coupled to Arl2's GTPase activity, establishing a checkpoint that ensures that only fully matured heterodimers proceed. These findings provide a structural framework for tubulin heterodimer biogenesis and recycling, supporting cytoskeletal proteostasis.
    DOI:  https://doi.org/10.1126/science.ady2708
  35. Nat Commun. 2025 Oct 27. 16(1): 9213
    Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model.
    Kazuhide Asakawa, Takuya Tomita, Shinobu Shioya, Hiroshi Handa, Yasushi Saeki, Koichi Kawakami.
      Selective neuronal vulnerability is a defining feature of neurodegenerative disorders, exemplified by motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The nature of motor neurons underlying this selectivity remains unresolved. Here, by monitoring autophagy at single-cell resolution across the translucent zebrafish spinal cord, we identify motor neurons as the cell population with the highest autophagic flux. Large spinal motor neurons (SMNs), most susceptible to ALS, exhibit higher flux compared to smaller SMNs and ALS-resistant ocular motor neurons. Notably, large SMNs accelerates both autophagy and proteasome-mediated degradation, which are further augmented by TDP-43 loss. Additionally, acceleration of multiple unfolded protein response pathways indicates their innate tendency to accumulate misfolded proteins. Enhanced cellular degradation in large SMNs is neuroprotective as its inhibition halts axon outgrowth. These findings propose that cell size-associated degradation load underlies selective neuronal vulnerability in ALS, highlighting the alleviation of catabolic stress as a target of therapy and prevention.
    DOI:  https://doi.org/10.1038/s41467-025-65097-0
  36. Sci Transl Med. 2025 Oct 29. 17(822): eady5288
    Leukocyte-intrinsic ER stress responses contribute to chemotherapy-induced peripheral neuropathy.
    Miriam M Fonseca, Oriana Gelblung, Sarah D Pennypacker, Taylor Brooks, Michael Limia, James W Morgan, Xuewei Zhu, Luis C Tovias-Sanchez, Alejandro Pluma-Pluma, Ruth Elena Martinez, Mathew R Eber, Sun H Park, Cristina M Furdui, Deepika Awasthi, Alexander Emmanuelli, Byuri A Cho, Chen Tan, David I Shalowitz, Samuel S Lentz, Michael Kelly, Anderson O'Brien Cox, Lindsay Macnamara, Fang-Chi Hsu, Yusuke Shiozawa, Wesley Hsu, Takao Iwawaki, Lance D Miller, Glenn J Lesser, Roy Strowd, Juan R Cubillos-Ruiz, E Alfonso Romero-Sandoval.
      Chemotherapy-induced peripheral neuropathy (CIPN) is the most prevalent and limiting side effect of paclitaxel treatment in patients with cancer. CIPN affects sensory neurons through neuroinflammatory mechanisms, but how immune cells sense and interpret systemic paclitaxel exposure during treatment is unclear. Here, we found that paclitaxel administration activated the endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) in circulating and dorsal root ganglion-resident myeloid cells, engendering an inflammatory milieu that promotes CIPN. Mechanistically, paclitaxel induced the overproduction of mitochondria-derived reactive oxygen species (ROS) that provoked ER stress and IRE1α hyperactivation in macrophages. This process reprogrammed macrophages toward an inflammatory state characterized by IRE1α-dependent production of TNF-α, IL-1β, PGE2, IL-6, IL-5, GM-CSF, MCP-1, and MIP-2. Ablation of IRE1α in leukocytes, or treatment with a selective IRE1α pharmacological inhibitor, prevented dorsal root ganglion neuroinflammation and CIPN-related pain behaviors in mice. Furthermore, the development and severity of CIPN in patients with gynecological cancer were associated with the status of IRE1α activation in their circulating leukocytes. Our study uncovers leukocyte-intrinsic IRE1α as a key mediator of CIPN and suggests that targeting its dysregulated activation could help mitigate CIPN in patients with cancer who are receiving paclitaxel.
    DOI:  https://doi.org/10.1126/scitranslmed.ady5288
  37. EMBO Rep. 2025 Oct 31.
    GABARAP proteins regulate the packaging of HIV-1 genomic RNA into virions.
    Marjory Palaric, Margaux Versapuech, Delphine Judith, Corentin Aubé, Marjorie Leduc, Jacques Dutrieux, Emilie-Fleur Gautier, Jean-Christophe Paillart, Sarah Gallois-Montbrun, Clarisse Berlioz-Torrent.
      In addition to their role in canonical autophagy, autophagy proteins (ATG) contribute to various cellular processes, including phagocytosis, membrane remodeling, and vesicle secretion. Several viruses also exploit components of the autophagy pathway for their own replication. Here, we explore the role of ATG proteins in HIV-1 assembly. Postulating that host proteins crucial for virion assembly are present at the assembly site and can be incorporated within virions, we analyze the proteome of HIV-1 preparations using mass spectrometry. We identify an enrichment of macroautophagy-related terms, notably 3 of the 6 ATG8 (LC3/GABARAP) proteins. Functional studies reveal that GABARAP proteins are critical for the production of infectious virions. Knockout of GABARAP proteins reduces the packaging of viral genomic RNA (gRNA) into particles, impairing virion infectivity. GABARAPL1 associates with gRNA and interacts with Gag in an RNA-dependent manner. Additionally, GABARAP knockout increases cellular Gag:gRNA complexes and decreases gRNA association with membranes, suggesting that GABARAP proteins regulate gRNA fate during HIV-1 assembly by facilitating its packaging. This study uncovers a novel role for GABARAP proteins in HIV-1 genome packaging.
    Keywords:  Autophagy Protein; GABARAP; Genome Packaging; HIV-1; RNA
    DOI:  https://doi.org/10.1038/s44319-025-00607-1
  38. Virulence. 2025 Dec;16(1): 2580132
    The GCN-2/eIF-2α/ATF-4 signaling pathway is involved in C. elegans defense against Salmonella enterica infection.
    Shawndra Wibisono, Phillip Wibisono, Yiyong Liu, Jingru Sun.
      In response to external or endogenous stress, eukaryotic cells can activate a common adaptive pathway called the integrated stress response (ISR). The ISR reduces global protein translation but upregulates the expression of stress response proteins to either restore cellular homeostasis or, in case of severe or prolonged stress, promote cell death. The bZIP transcription factor ATF4 plays a deciding role in cellular fate upon ISR activation, but the precise mechanisms underlying such decision-making remain unclear. Although bacterial infection has previously been observed to induce the ISR, the effects of this pathway on bacterial pathogenesis and host defense are not well understood. The functions of ATF4 in this process remain even more elusive. Using the Caenorhabditis elegans model to explore the bacterial infection-induced ISR, we found that infection with Salmonella enterica induced the GCN-2/eIF-2α/ATF-4 signaling pathway to modulate host defense against the infection. More specifically, ATF-4 suppressed the expression of ribosomal protein genes in response to S. enterica exposure, reducing worm survival against the pathogen. Because ribosomal proteins are directly involved in protein translation, our data revealed an important, novel mechanism by which ATF-4 mediates the reduction of global translation under stress by inhibiting the expression of ribosomal proteins. ATF-4 also regulates components of mitochondrial electron transport and collagen genes in response to S. enterica infection; both regulations are linked to stress resistance and longevity. Overall, we have identified specific molecular mechanisms through which ATF-4 determines cell fate upon ISR activation, revealing how this pathway influences host outcomes during bacterial infection.
    Keywords:  ATF-4; C. elegans; GCN-2/eIF-2α/ATF-4 signaling pathway; Host-pathogen interactions; Salmonella enterica; host response to infection; integrated stress response; ribosomal proteins; stress resistance; stress response
    DOI:  https://doi.org/10.1080/21505594.2025.2580132
  39. Nat Commun. 2025 Oct 27. 16(1): 9012
    PLM-interact: extending protein language models to predict protein-protein interactions.
    Dan Liu, Francesca Young, Kieran D Lamb, Adalberto Claudio Quiros, Alexandrina Pancheva, Crispin J Miller, Craig Macdonald, David L Robertson, Ke Yuan.
      Computational prediction of protein structure from amino acid sequence alone has been achieved with unprecedented accuracy, yet the prediction of protein-protein interactions remains a challenge. Here, we assess the ability of protein language models (PLMs), routinely applied to protein folding, to be retrained for protein-protein interaction prediction. Existing models that exploit PLMs use a pre-trained PLM feature set, ignoring that the proteins are physically interacting. We propose PLM-interact, which goes beyond single proteins by jointly encoding protein pairs to learn their relationships, analogous to the next-sentence prediction task from natural language processing. This approach achieves state-of-the-art performance in a widely adopted cross-species protein-protein interaction prediction benchmark: trained on human data and tested on mouse, fly, worm, E. coli and yeast. In addition, we develop a fine-tuning method for PLM-interact to detect mutation effects on interactions. Finally, we report that the model outperforms existing approaches in predicting virus-host interaction at the protein level. Our work demonstrates that large language models can be extended to learn the intricate relationships among biomolecules from their sequences alone.
    DOI:  https://doi.org/10.1038/s41467-025-64512-w
  40. Microbes Infect. 2025 Oct 28. pii: S1286-4579(25)00114-5. [Epub ahead of print] 105582
    The unfolded protein response is a critical mediator in Campylobacter jejuni pathogenesis and host defence.
    Geunhye Hong, Zahra Omole, Cadi Davies, Janie Liaw, Anna D Grabowska, Barbara Canonico, Nicolae Corcionivoschi, Brendan W Wren, Ezra Aksoy, Nick Dorrell, Abdi Elmi, Ozan Gundogdu.
      Campylobacter jejuni is the major bacterial cause of foodborne gastroenteritis worldwide. How this pathogen interacts with the host defence machinery of human intestinal epithelial cells (IECs) and is involved in pathogenesis remains elusive. Bacterial pathogens utilise strategies to gain access to the eukaryotic cell machinery that can involve subversion of biological processes in host. Unfolded protein response (UPR) is a highly conserved host cell stress response to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) and is a conserved evolutionary response against invading pathogens. Several bacterial pathogens can induce the UPR for their own survival and thus design a dual scenario where UPR can both protect and facilitate pathogen evasion. Herein, we investigated whether UPR represents a virulence mechanism exploited by C. jejuni during bacterial invasion in human IECs. Our data show that following C. jejuni infection, we observe consistent upregulation of protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1α and (IRE1α), with activating transcription factor 6 (ATF6) activation occurring in a strain- and cell line-dependent manner. Chemical induction of UPR by thapsigargin in host cells reduced intracellular survival of C. jejuni while conversely pretreatment with UPR inhibitors increased intracellular survival of C. jejuni and attenuated IL-8 release. Finally, we show using C. jejuni mutants that the capsular polysaccharide and flagella contribute to UPR activation in IECs. Collectively, these findings provide observational insights into UPR activation during infections and how C. jejuni infection leads to UPR activation and inflammation, potentially contributing to downstream C. jejuni-mediated damage.
    Keywords:  Campylobacter jejuni; ER stress; Inflammation; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.micinf.2025.105582
  41. Genes Dev. 2025 Oct 29.
    Longevity-promoting mitochondrial unfolded protein response activation requires elements of the PeBoW complex.
    Adebanjo Adedoja, Niclole Stuhr, Yifei Zhou, Yuyao Zhang, Armen Yerevanian, Alexander A Soukas.
      Mitochondria play a crucial role in cellular energy metabolism and homeostasis and are strongly implicated in aging and age-related diseases. The outer mitochondrial membrane protein voltage-dependent anion channel (VDAC) plays multiple roles in mitochondrial homeostasis, including transport of metabolites, ATP, and Ca2+ Dysregulation of VDAC levels has been associated with cancer, neurodegeneration, metabolic disorders, and aging. Previously, we demonstrated that elevated VDAC-1 levels in Caenorhabditis elegans lead to increased mitochondrial permeability and reduced life span. Here we demonstrate that reduced VDAC-1 function extends life span through the activation of the mitochondrial unfolded protein response (UPRmt), a conserved stress response that maintains mitochondrial proteostasis and is linked to life span extension in multiple species. Leveraging unbiased genomic discovery, we identified genes encoding several proteins in the PeBoW complex as a critical mediator of UPRmt activation following VDAC-1 loss. More broadly, we demonstrated a universal requirement for several PeBoW component genes across diverse mitochondrial stressors in order to fully animate the UPRmt Our findings reveal a heretofore unappreciated role for PeBoW components in UPRmt induction and life span extension in response to mitochondrial stress, highlighting its essential function in mitochondrial quality control and longevity pathways.
    Keywords:  PeBoW; mito-stress; mitoUPR; mitochondria
    DOI:  https://doi.org/10.1101/gad.352979.125
  42. Cell Death Dis. 2025 Oct 27. 16(1): 762
    Impairment of lysosomal quality control in Huntington disease.
    Paola Rusmini, Francesco Mina, Marta Valenza, Martina Vitali, Veronica Ferrari, Barbara Tedesco, Elena Casarotto, Marta Cozzi, Marta Chierichetti, Ali Mohamed, Paola Pramaggiore, Laura Cornaggia, Carmelo Milioto, Maria Brodnanova, Rocio Magdalena, Prashant Koshal, Margherita Piccolella, Riccardo Cristofani, Mariarita Galbiati, Valeria Crippa, Angelo Poletti.
      Huntington disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion (polyQ) in the Huntingtin protein (muHTT), which makes it prone to misfolding and aggregation. muHTT aggregates sequester a wide variety of proteins essential for cell homeostasis, including chaperones and transcription factors, and their depletion may contribute to HD pathogenesis. Lysosomes are the main hubs for degradative and signaling activities in cells, and their functionality is crucial for cell homeostasis, especially for neurons. Different forms of cellular stresses, including proteotoxic stresses, can alter lysosome integrity and induce lysosomal membrane permeabilization (LMP). Damaged lysosomes are recognized by galectins, in particular galectin-3 (LGALS3) with activation of the lysosome quality control (LQC) system responsible for repairing, degrading, or replacing leaky lysosomes. The system is transcriptionally regulated by the transcription factors EB and E3 (TFEB and TFE3, respectively). Using HD mouse and cell models, we demonstrated that TFEB and TFE3 are sequestered in muHTT aggregates, and muHTT proteins associates with LMP triggering the translocation of LGALS3 to the lumen of lysosomes, with a close relation between polyQ size and severity of these events. Moreover, we demonstrated that TFEB and TFE3 silencing or overexpression modulate muHTT aggregation. TFEB and TFE3 knockdown worsens muHTT aggregation, while their overexpression reduces muHTT inclusions and concurrently reduces LGALS3 accumulation via lysophagy and lysosome replacement. Our findings suggest that both TFEB and TFE3 are implicated in HD, and their sequestration in muHTT inclusions increase the vulnerability of neurons to lysosome injury, altering LQC and contributing to disease pathogenesis. In physiologial conditions, lysosome membrane permeabilization occurs and activates TFEB and TFE3 triggering a response to induce lysophagy and lysosome biogenesis. In HD, muHTT sequesters TFEB and TFE3 into inclusions and the reduced TFEB/TFE3 bioavailability prevents the activation of lysophagy and leading to the accumulation of damaged lysosomes. Created in BioRender.
    DOI:  https://doi.org/10.1038/s41419-025-08103-z
  43. Autophagy. 2025 Oct 29.
    NRBF2 homodimerization by its coiled-coil domain strengthens association with the PtdIns3K complex mediated by the mit domain to promote autophagy.
    Na Li, Xiaohua Li, Xianxiu Qiu, Xuehua Pan, Shuai Wu, Jingyi Chen, Rong Liu, Jiahong Lu, Zhenyu Yue, Yanxiang Zhao.
      The mammalian class III phosphatidylinositol-3-kinase complex (PtdIns3K) forms two biochemically and functionally distinct subcomplexes including the ATG14-containing complex I (PtdIns3K-C1) and the UVRAG-containing complex II (PtdIns3K-C2). Both subcomplexes adopt a V-shaped architecture with a BECN1-ATG14 or UVRAG adaptor arm and a PIK3R4/VPS15-PIK3C3/VPS34 catalytic arm. NRBF2 is a pro-autophagic modulator that specifically associates with PtdIns3K-C1 to enhance its kinase activity and promotes macroautophagy/autophagy. How NRBF2 exerts such a positive effect is not fully understood. Here we report that NRBF2 binds to PIK3R4/VPS15 with moderate affinity through a conserved site on its N-terminal MIT domain. The NRBF2-PIK3R4/VPS15 interaction is incompatible with the UVRAG-containing PtdIns3K-C2 because the C2 domain of UVRAG outcompetes NRBF2 for PIK3R4/VPS15 binding. Our crystal structure of the NRBF2 coiled-coil (CC) domain reveals a symmetric homodimer with multiple hydrophobic pairings at the CC interface, which is in distinct contrast to the asymmetric dimer observed in the yeast ortholog Atg38. Mutations in the CC domain that rendered NRBF2 monomeric led to weakened binding to PIK3R4/VPS15 and only partial rescue of autophagy deficiency in nrbf2 knockout cells. In comparison, NRBF2 with its CC domain replaced by a dimeric Gcn4 module showed proautophagic activity comparable to wild type while NRBF2 carrying a tetrameric Gcn4 module showed further enhanced activity. We propose that the oligomeric state of NRBF2 mediated by its CC domain is critical for strengthening the moderate NRBF2-PIK3R4/VPS15 interaction mediated by its MIT domain to fully activate PtdIns3K-C1 and promote autophagy.
    Keywords:  Autophagy; MIT; NRBF2; PIK3R4/VPS15; PtdIns3K; coiled-coil
    DOI:  https://doi.org/10.1080/15548627.2025.2580438
  44. Viruses. 2025 Sep 24. pii: 1291. [Epub ahead of print]17(10):
    Activation of IRE1 Endonuclease Activity Regulates Zika Virus Replication and Antiviral Response During Infection in Human Microglia.
    Tomás Hernández-Díaz, Aarón Oyarzún-Arrau, Aracelly Gaete-Argel, Delia López-Palma, Javier López-Schettini, Dominique Fernández, Fernando Valiente-Echeverría, Fabiola Osorio, Ricardo Soto-Rifo.
      Zika virus (ZIKV) can infect and replicate in the endoplasmic reticulum (ER) of different human cell types, including neural progenitor cells, radial glial cells, astrocytes, and microglia in the brain. ZIKV infection of microglia is expected to trigger both ER stress and the induction of an antiviral response through production of type-I interferons and pro-inflammatory cytokines, contributing to neuroinflammation during infection. Despite their critical role in ZIKV pathogenesis, the interplay between ER stress and the antiviral response during infection has not been fully characterized in human microglia. In this work, we show that infection of a human microglia cell line with ZIKV triggers the induction of an antiviral response and the activation of the endonuclease activity of the unfolded protein response sensor IRE1. Interestingly, we observed that both IRE1 and XBP1 were sequestered to the viral replication sites during infection. Moreover, pharmacological inhibition or hyperactivation of the endonuclease activity of IRE1 resulted in reduced viral titers. As such, while inhibition of IRE1 resulted in an increased type-I interferon response, hyperactivation led to a decrease in ZIKV RNA levels and the appearance of ER-derived cytoplasmic structures containing NS3, IRE1, and XBP1. Together, our data indicate that regulation of the endonuclease activity of IRE1 is critical for both ZIKV replication and immune activation, highlighting the potential of the ER stress sensor as a target for the development of antivirals to treat ZIKV infections.
    Keywords:  ER stress; IRE1; Zika virus; innate immune response; microglia
    DOI:  https://doi.org/10.3390/v17101291
  45. Nat Cell Biol. 2025 Oct 31.
    TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin-binding proteins.
    Shan Lu, Sitao Zhang, Spencer Oung, Jolene K Diedrich, Peng Han, Olatz Arnold-Garcia, Takuya Ohkubo, Olubankole Aladesuyi Arogundade, Sonia Vazquez-Sanchez, Ke Zhang, John Ravits, John R Yates Iii, Don W Cleveland.
      In multiple neurodegenerative diseases, the RNA-binding protein TDP-43 forms cytoplasmic aggregates of distinct morphologies, including skein-like, small rounded granular and large spherical inclusions. Here, whereas the N-terminal self-oligomerization domain regulates TDP-43 demixing into cytoplasmic droplets, inhibition of N-terminal self-oligomerization domain-mediated oligomerization is shown to promote the formation of skein-like inclusions. Utilizing proximity labelling-mass spectrometry, cellular stresses are shown to induce TDP-43 association with actin-binding proteins that include filamins and α-actinin. Small interfering RNA-mediated reduction of filamin in Drosophila ameliorates cell loss from cytoplasmic TDP-43, consistent with the filamin-TDP-43 interaction enhancing cytotoxicity. TDP-43's association with actin-binding proteins is mediated by BAG3, a HSP70 family nucleotide exchange factor that regulates the proteostasis of actin-binding proteins. BAG2, another HSP70 nucleotide exchange factor, facilitates the formation of small, rounded TDP-43 inclusions. We demonstrate that both TDP-43 self-oligomerization and its binding partners, including HSP70 and cochaperones BAG2 and BAG3, drive the formation of the different types of TDP-43 inclusion.
    DOI:  https://doi.org/10.1038/s41556-025-01789-5
  46. Adv Sci (Weinh). 2025 Oct 29. e12652
    The Crosstalk Between CRL5 and APC/C E3 Ligases Regulates Metastasis and Chemosensitivity of Cancer Cells.
    Danrui Cui, Ruirui Qu, Tianqi Li, Linchen Wang, Xiaoyu Chen, Shengpeng Shao, Xue Liang, Jun Xu, Yi Sun, Xiufang Xiong, Yongchao Zhao.
      Cullin-RING ligases (CRLs) and the anaphase-promoting complex/cyclosome (APC/C) are two major multi-subunit ubiquitin ligases essential for protein homeostasis. The underlying mechanism and biological consequence of their crosstalk remain elusive. Here, tandem affinity purification followed by LC-MS/MS is employed, and identified APC11-the RING subunit of APC/C-as a bona fide binding partner of CUL5, the scaffold of CRL5. On one hand, APC11 interacts with CUL5 and inhibits its neddylation. Consequently, APC11 knockdown enhances CUL5 neddylation by promoting its interaction with the neddylation E2 UBE2F. This leads to the degradation of SOCS3, a substrate receptor of CRL5, and the subsequent accumulation of its substrate, integrin β1, ultimately promoting cancer metastasis. On the other hand, CUL5-APC11 binding stabilizes APC11 by facilitating its atypical K27/K29/K33-linked polyubiquitylation at Lys83, a process catalyzed by ITCH E3 ligase. CUL5 loss delays mitotic exit, induces aneuploidy, and sensitizes cancer cells to the microtubule-targeting drug paclitaxel by destabilizing APC11. Collectively, the study revealed a new crosstalk between CUL5 and APC11 of two major E3 ligases, and targeting this crosstalk can provide a new strategy for blocking metastasis and triggering chemosensitization.
    Keywords:  APC11; CUL5; chemosensitivity; metastasis; neddylation
    DOI:  https://doi.org/10.1002/advs.202512652
  47. Nature. 2025 Oct 29.
    Helicase-mediated mechanism of SSU processome maturation and disassembly.
    Olga Buzovetsky, Sebastian Klinge.
      Eukaryotic ribosomal small subunit (SSU) assembly requires the SSU processome, a nucleolar precursor containing the RNA chaperone U3 small nucleolar RNA (snoRNA). The underlying molecular mechanisms of SSU processome maturation, remodelling, disassembly and RNA quality control, and the transitions between states remain unknown owing to a paucity of intermediates1-3. Here we report 16 native SSU processome structures alongside genetic data, revealing how two helicases, the Mtr4-exosome and Dhr1, are controlled for accurate and unidirectional ribosome biogenesis. Our data show how irreversible pre-ribosomal RNA degradation by the redundantly tethered RNA exosome couples the transformation of the SSU processome into a pre-40S particle, during which Utp14 can probe evolving surfaces, ultimately positioning and activating Dhr1 to unwind the U3 snoRNA and initiate nucleolar pre-40S release. This study highlights a paradigm for large dynamic RNA-protein complexes in which irreversible RNA degradation drives compositional changes and communicates these changes to govern enzyme activity while maintaining overall quality control.
    DOI:  https://doi.org/10.1038/s41586-025-09688-3
  48. Elife. 2025 Oct 29. pii: RP107524. [Epub ahead of print]14
    Non-equilibrium strategies enabling ligand specificity by signaling receptors.
    Andrew Goetz, Jeremy Barrios, Ralitsa Radostinova Madsen, Purushottam D Dixit.
      Signaling receptors often encounter multiple ligands and have been shown to respond selectively to generate appropriate, context-specific outcomes. At thermal equilibrium, ligand specificity is limited by the relative affinities of ligands for their receptors. Here, we present a non-equilibrium model in which receptors overcome thermodynamic constraints to preferentially signal from specific ligands while suppressing others. In our model, multi-site phosphorylation and active receptor degradation act in concert to regulate ligand specificity, with receptor degradation, a common motif in eukaryotes, providing a previously under-appreciated layer of control. Here, ligand-bound receptors undergo sequential phosphorylation, with progression restarted by ligand unbinding or receptor turnover. High-affinity complexes are kinetically sorted toward degradation-prone states, while low-affinity complexes are sorted toward inactivated states, both limiting signaling. As a result, network activity is maximized for ligands with intermediate affinities. This mechanism explains paradoxical experimental observations in receptor tyrosine kinase signaling, including non-monotonic dependence of signaling output on ligand affinity and kinase activity. Given the ubiquity of multi-site phosphorylation and ligand-induced degradation across signaling receptors, we propose that kinetic sorting may be a general non-equilibrium ligand-discrimination strategy used by multiple signaling receptors.
    Keywords:  computational biology; human; physics of living systems; proofreading; signaling networks; specificity; systems biology
    DOI:  https://doi.org/10.7554/eLife.107524
  49. Cell Death Dis. 2025 Oct 31. 16(1): 772
    CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival.
    Aseel Sharaireh, Marta Guevara-Ferrer, Anna M Ludlaim, Jonathan D Humphries, Alexander M Phillips, Andrew W Dowsey, Zehan Zhang, John R Counsell, Richard D Unwin, Sara E Mole, Ahad A Rahim, Tristan R McKay.
      Inherited biallelic mutations in the CLN7 gene result in the variant late infantile onset neuronal ceroid lipofuscinosis, a subtype of Batten disease (BD), a severe and fatal childhood neurodegenerative disease. Intriguingly, CLN7 genetic variants have also been associated with retinopathies, amyotrophic lateral sclerosis, and frontotemporal dementia. CLN7 encodes a transmembrane protein localizing to endolysosomal membranes with outward-facing chloride channel activity. Loss of CLN7 function results in cortical neurons accumulating swollen lipofuscin-containing lysosomes, leading to neuroinflammation and neurodegeneration. The molecular mechanisms underlying CLN7 BD neuropathology are not completely understood. We have generated iPSC lines from two CLN7 BD patients and age-matched unaffected controls to interrogate intracellular molecular phenotypes in iPSC-derived neural progenitor cells (iNPC). Taking a multi-omics approach we have identified disease-modified activities in endolysosomal transport in iNPCBD that lead to lysosomal dysfunction and decreased mitophagy, resulting in the accumulation of metabolically defective mitochondria. We further observe a breakdown in nuclear functions that centre on RNA processing and nuclear export, linking to CLN7 protein interactions at the stress granule. We have identified dual and distinct functions for CLN7, promoting cell survival during the cellular stress response. CLN7 loss of function in BD results in neuronal apoptosis.
    DOI:  https://doi.org/10.1038/s41419-025-08063-4
  50. EMBO Rep. 2025 Oct 31.
    Neuralized-like proteins differentially activate Notch ligand.
    Alina Airich, Oren Gozlan, Ekaterina Seib, Gittel Leah Shaingarten, Lena-Sophie Wilschrey, Liora Lindenboim, David Sprinzak, Thomas Klein.
      Notch signalling is a major signalling pathway coordinating cellular processes between neighbouring animal cells. In Drosophila, two E3 ubiquitin ligases, Neuralized (Neur) and Mindbomb1 (Mib1), regulate Notch ligand activation and are essential for development. However, the mammalian orthologs of Neur, Neuralized-like (NEURL) 1 and 1B, appear to be dispensable for development, as double knock-out mice show no overt developmental defects. Thus, it is unclear if and how NEURL proteins regulate the mammalian Notch ligands. To address this question, we examined NEURL proteins' ability to activate Notch ligands in a humanized Drosophila model and mammalian cell culture. We found that, unlike MIB1, NEURL proteins activate Notch only with a subset of mammalian ligands, which contain a Neuralized binding motif. This motif has the consensus sequence NxxN and is present only in Notch ligands DLL1 and JAG1, but not in DLL4 and JAG2. Thus, our results reveal a differential regulatory mechanism of Notch activation in mammals, which can potentially explain the limited role of NEURL proteins in mammalian development and homeostasis.
    Keywords:  DSL ligands; E3 ubiquitin ligases; Neuralized; Notch signalling
    DOI:  https://doi.org/10.1038/s44319-025-00601-7
  51. Mol Cell. 2025 Oct 29. pii: S1097-2765(25)00822-6. [Epub ahead of print]
    The E3 ligase RNF32 controls the IκB kinase complex and NF-κB signaling in intestinal stem cells.
    Angela Lauriola, Juliana Haydeé Enriqué Steinberg, Motoharu Sarubo, Elena Maspero, Fabiana Alejandra Rossi, Yasuhiro Mouri, Marco Pedretti, Mohsen Hajisadeghian, Vincenzo Taibi, Andrea Vettori, Nicola Vitulo, Michael Assfalg, Mariapina D'Onofrio, Mario Rossi, Akihiro Yasue, Alessandra Astegno, Simona Polo, Spartaco Santi, Yasusei Kudo, Daniele Guardavaccaro.
      Nuclear factor κB (NF-κB) signaling is a central pathway regulating a plethora of cellular functions. Here, we find that RNF32, a RING E3 ubiquitin ligase whose expression is enriched in murine intestinal stem cells, regulates the activity of the IκB kinase (IKK) complex, the signal integration hub for NF-κB activation. The E3 ligase activity of RNF32 depends on calmodulin, the primary calcium sensor in eukaryotic cells. Increased levels of intracellular calcium ion (Ca2+) induce RNF32 binding to calmodulin, RNF32 activation, and autoubiquitylation. In turn, polyubiquitin chains conjugated to RNF32 recruit NEMO, the regulatory subunit of the IKK complex. Moreover, Ca2+ rise triggers RNF32 phase separation, which is required for the formation of NEMO condensates and IKK activation. Finally, we show that RNF32 is required for NF-κB activation triggered by bacterial lipopolysaccharides. Collectively, our findings uncover a mechanism controlling NF-κB signaling in the intestinal epithelium.
    DOI:  https://doi.org/10.1016/j.molcel.2025.10.005
  52. Biochem Soc Trans. 2025 Oct 29. 53(5): 1377-1386
    Ins and outs of IRES elements: function and significance.
    Encarnacion Martínez-Salas.
      RNA and proteins are key components of all organisms. Internal ribosome entry site (IRES) elements are a diverse type of RNA regulatory structural elements that mediate end-independent, internal translation initiation in viral mRNAs and certain cellular mRNAs translated under stress conditions. Notably, viral IRES elements regulate translation initiation via a dynamic, modular RNA structure organization, which serves as the anchoring site for the ribosome guided by RNA-RNA and/or RNA-protein interactions. The implementation of advanced transcriptomics, proteomics, and computational methodologies has facilitated the identification of novel RNAs potentially translated using cap-independent mechanisms, harboring RNA structural elements with distinctive features. Here, we present a summary of the current understanding of IRES elements, focusing on the molecular functions and the RNA-binding proteins regulating IRES activity.
    Keywords:  IRES; RNA; RNA-binding proteins; bioinformatics; translation initiation
    DOI:  https://doi.org/10.1042/BST20253097
  53. Biochem Soc Trans. 2025 Oct 29. 53(5): 1417-1429
    Navigating lipid droplet proteins - part II: molecular mechanisms underlying ER-to-lipid droplet protein partitioning.
    Louisa Magdalena Krauß, Bianca Schrul.
      Lipid droplets (LDs) originate from the endoplasmic reticulum (ER) and are unique among cellular organelles, as they consist of a hydrophobic core of neutral lipids that is surrounded by a phospholipid monolayer. Proteins and enzymes embedded into this monolayer are essential for regulating dynamic lipid storage and consumption and hence, for the cellular adaptation to metabolic changes. Their activity and abundance on the LD surface must therefore be well-controlled. Many of these proteins are first inserted into the phospholipid bilayer membrane of the ER before they partition to the LD monolayer. While a monotopic membrane topology is required for enabling the targeting of these ERTOLD proteins from the ER to LDs, the molecular mechanisms underlying this partitioning are only beginning to emerge. In this second part of the bipartite review 'Navigating lipid droplet proteins,' we discuss recent conceptual advances regarding ER-to-LD protein partitioning and focus on novel insights into the structural dynamics of LD-destined proteins, how their partitioning to LDs is temporally controlled, and the hierarchies involved in selective and competitive protein recruitment to LDs according to metabolic needs and functions.
    Keywords:  endoplasmic reticulum; intracellular transport; lipid droplets; lipid metabolism; membrane proteins; organelle biogenesis
    DOI:  https://doi.org/10.1042/BST20253052
  54. Nucleic Acids Res. 2025 Oct 29. pii: gkaf1075. [Epub ahead of print]
    MGTbind: a comprehensive database of molecular glue ternary interactome.
    Jintao Zhu, Yiyan Liao, Haoyu Lin, Juan Xie, Zhichao Deng, Jinyu Han, Zhen Zhang, Jinchuan Xiao, Zhiyao Wang, Shuaipeng Zhang, Luhua Lai, Jianfeng Pei.
      Molecular glues (MGs) are an emerging class of small molecules capable of inducing or enhancing protein-protein interactions, leading to reprograming of cellular events. In recent years, MG discovery has gained significant attention in drug discovery and synthetic biology studies. However, MG discovery remains exceptionally challenging as it continues to rely on serendipity and experimental screening efforts. In order to contribute data resources to facilitate the rational design of MGs, we developed the molecular glue and ternary binding (MGTbind) database, providing comprehensive resources about ternary structures and experimental data for the coverage of MG-engaged interactome. MGTbind database contains 3093 manually curated MGs with their chemical structures and physicochemical properties, along with 3924 ternary interactions with bioactivity measurements (e.g. degradation capacities for MG degraders, in vitro biochemical activities and cellular activities). Hierarchical binding affinity is also collected to enable exploring of cooperative binding mechanisms. Ternary complex structural data are systematically integrated, either from the Protein Data Bank or generated by AlphaFold 3. All of them can be intuitively analyzed and visualized along with various predicted metrics. A fully functional and user-friendly web interface that allows global search and smooth browsing is provided and is freely accessible at https://mgtbind.pkumdl.cn.
    DOI:  https://doi.org/10.1093/nar/gkaf1075
  55. Nucleic Acids Res. 2025 Oct 28. pii: gkaf1088. [Epub ahead of print]53(20):
    The stringent response does not influence ribosome pausing in Bacillus subtilis.
    Yaozu Han, Alberto Agnolin, Frans van der Kloet, Christopher Sauer, Paul Igor Costea, Max Fabian Felle, Mathis Appelbaum, Leendert W Hamoen.
      The stringent response represses translation and is activated when cells enter the stationary phase and intracellular amino acid levels drop. Bacillus subtilis, a well-known industrial enzyme production organism, secretes most enzymes during the stationary phase. However, translation pausing can affect protein folding and production. Here, we examined whether the stringent response affects ribosome pausing in B. subtilis. To investigate this, genome-wide ribosome profiling was performed using a stringent response mutant that overexpressed the α-amylase AmyM. Blocking the stringent response did increase overall protein synthesis, however AmyM production was reduced. Ribosome profiling revealed that this was not caused by a reduction in translation. In fact, absence of the stringent response did not markedly influence ribosome pausing. Late into the stationary phase an increased ribosome pausing at tryptophan codons was observed, suggesting a depletion of tryptophan. A strong suppression of tryptophan biosynthesis and acquisition genes, under control of the trp RNA-binding attenuation protein TRAP, likely accounts for this. Why this occurs is unclear since TRAP does not belong to the stringent response regulon of B. subtilis. Finally, the genome-wide ribosome profiles revealed several operon genes with unusually low translation activities, illustrating the importance of translation initiation as a regulatory element of expression.
    DOI:  https://doi.org/10.1093/nar/gkaf1088
  56. J Biol Chem. 2025 Oct 27. pii: S0021-9258(25)02712-7. [Epub ahead of print] 110860
    Substrate recognition by the human mitochondrial processing peptidase and its processing of PINK1.
    Andrew N Bayne, Danielle M Simons, Naoto Soya, Karl Grenier, Gergely L Lukacs, Edward A Fon, Jean-François Trempe.
      Nuclear-encoded mitochondrial proteins rely on N-terminal targeting sequences (N-MTS) for their import. Most N-MTSs are cleaved in the matrix by the mitochondrial processing peptidase (MPP), a heterodimeric metalloprotease composed of (α) and catalytic (β) subunits, essential for the maturation of imported proteins. Import and processing of PINK1, a kinase implicated in Parkinson's disease, govern its ability to sense mitochondrial damage. The current paradigm suggests PINK1 undergoes two sequential processing steps: first, MPP removes the PINK1 N-MTS in the matrix; second, the inner mitochondrial membrane protease PARL cleaves the PINK1 transmembrane domain, leading to PINK1 degradation. Upon depolarization, PINK1 escapes proteolysis and accumulates on mitochondria to initiate mitophagy. However, the MPP cleavage site on PINK1, the role of MPP in PINK1 signalling, and the mechanisms of substrate recognition by human MPP remain unclear. Here, we define the MPP cleavage site on PINK1 between Ala28-Tyr29 and show it is inefficiently processed compared to canonical N-MTSs. In cells, MPP cleavage is dispensable for both PARL processing and PINK1 function, decoupling PINK1 import and damage sensing from its N-MTS removal. However, in vitro, the PINK1 N-MTS binds potently to MPP, inhibits the cleavage of other substrates, and traps MPP in a slowly processing complex. Exploiting PINK1 as a mechanistic probe, we use hydrogen-deuterium exchange mass spectrometry to map the PINK1 binding site on MPPα. We identify a two-step mechanism involving MPPα lid rearrangement followed by active site engagement, providing key insight into PINK1's unique import pathway and fundamental MPP processing mechanisms.
    Keywords:  PTEN-induced putative kinase 1 (PINK1); Parkinson disease; hydrogen-deuterium exchange; mitochondria; mitochondrial processing peptidase (MPP); protein import; protein processing
    DOI:  https://doi.org/10.1016/j.jbc.2025.110860
  57. Curr Issues Mol Biol. 2025 Oct 16. pii: 856. [Epub ahead of print]47(10):
    GPX4 Inhibition Enhances the Pro-Oxidant and ER Stress Effects of Tempol in Colon and Gastric Cancer Cell Lines.
    Gorkem Ozdemir, Halil Mahir Kaplan.
      Tempol, a synthetic nitroxide, exhibits dual antioxidant and pro-oxidant activity, requiring millimolar concentrations to induce oxidative stress, which limits its therapeutic use. Glutathione Peroxidase 4 (GPX4) is a critical lipid peroxidase that prevents ferroptosis, and its inhibition has emerged as a strategy to sensitize cancer cells to oxidative stress. To enhance Tempol's efficacy, we investigated its interaction with ML210, a GPX4 inhibitor, in human colon (HT29) and gastric (CRL-1739) cancer cell lines. We quantified cell viability, oxidative stress markers (H2O2, Total Oxidant Status (TOS), and Total Antioxidant Status (TAS)) and endoplasmic reticulum (ER) stress proteins (ATF6, GRP78, and IRE1α) in in vitro assays. Synergy was assessed using Bliss independence analysis. The combination of Tempol (2 mM) and ML210 (0.05 μM) markedly reduced viability in both cell lines. Bliss analysis revealed slight/moderate synergy for cytotoxicity (Δ = +0.15 in HT29; Δ = +0.26 in CRL-1739) and strong synergy for H2O2 accumulation (Δ = +1.92-2.23 across replicates). In contrast, TOS showed moderate-to-strong antagonism across both cell lines, and TAS demonstrated slight synergistic or antagonistic effects. ER stress markers exhibited marker and cell line specific synergy: ATF6 showed strong synergy, IRE1α slight synergy in both lines, and GRP78 activation was highly variable, showing strong synergy in CRL-1739 cells but moderate antagonism in HT29 cells. These findings indicate that the cooperative action of Tempol and ML210 is ROS-pool-specific and pathway-selective in the ER. These findings demonstrate that ML210 potentiates Tempol's pro-oxidant pressure by targeting GPX4, selectively amplifying H2O2 accumulation and ER stress engagement without collapsing global redox balance. This study provides mechanistic rationale for redox-proteostasis co-targeting in gastric and colon cancers and establishes a foundation for in vivo validation.
    Keywords:  ATF6; ER stress; GPX4 inhibitor; GRP78; IRE1α; ML210; colon cancer; gastric cancer; tempol; unfolded protein response
    DOI:  https://doi.org/10.3390/cimb47100856
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