bims-proteo Biomed News
on Proteostasis
Issue of 2025–04–06
forty-five papers selected by
Eric Chevet, INSERM
  1. tRNA synthetase activity is required for stress granule and P-body assembly.
  2. Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration.
  3. The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.
  4. Sequence rules for a long SPOP-binding degron required for protein ubiquitylation.
  5. SEL1L-HRD1 ER-Associated Degradation Facilitates Prohormone Convertase 2 Maturation and Glucagon Production in Islet α Cells.
  6. Homeostatic regulation of nucleoporins is a central driver of nuclear pore biogenesis.
  7. PYM1 limits non-canonical Exon Junction Complex occupancy in a gene architecture dependent manner to tune mRNA expression.
  8. PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.
  9. PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.
  10. RAF1 kinase contributes to autophagic lysosome reformation.
  11. Therapeutic potential of allosteric HECT E3 ligase inhibition.
  12. Bacterial ubiquitin ligase engineered for small molecule and protein target identification.
  13. Stepwise recruitment of chaperone Hsc70 by DNAJB1 produces ordered arrays primed for bursts of amyloid fibril disassembly.
  14. Stress-dependent activation of PQM-1 orchestrates a second-wave proteostasis response for organismal survival.
  15. The Abelson kinase and the Nedd4 family E3 ligases co-regulate Notch trafficking to limit signaling.
  16. Heterobifunctional proteomimetic polymers for targeted protein degradation.
  17. At the crossroads of calcium signaling, protein synthesis and neuropeptide release.
  18. Translation dysregulation in cancer as a source for targetable antigens.
  19. Retromer promotes the lysosomal turnover of mtDNA.
  20. Remodelling of Cellular Protein Homeostasis by Enhanced ER-Mitochondrial Tethering.
  21. p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes.
  22. First responder to starvation: microreticulophagy clears aberrant membrane proteins in quick bites.
  23. Probing the E3 Ligase Adapter Cereblon with Chemical Biology.
  24. The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro.
  25. Plant ubiquitin E2 enzymes UBC32, UBC33, and UBC34 are involved in ERAD and function in host stress tolerance.
  26. ER nests are specialized ER subdomains in Arabidopsis where peroxisomes and lipid droplets form.
  27. Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment.
  28. SMYD5 is a ribosomal methyltransferase that trimethylates RPL40 lysine 22 through recognition of a KXY motif.
  29. Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis.
  30. Atomic context-conditioned protein sequence design using LigandMPNN.
  31. HEATR3 recognizes membrane rupture and facilitates xenophagy in response to Salmonella invasion.
  32. Protein aggregation identified in olfactory neuronal cells is associated with cognitive impairments in a subset of living schizophrenia patients.
  33. Characterization of MEK1/2 Degraders Uncovers a Kinase-Independent Role for MEK1/2 in the Stabilization and Maturation of CRAF.
  34. Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogen Campylobacter jejuni.
  35. iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation.
  36. Insights into non-proteinaceous ubiquitination.
  37. Infection-relevant conditions dictate differential versus coordinate expression of Salmonella chaperones and cochaperones.
  38. Vps41 functions as a molecular ruler for HOPS tethering complex function.
  39. Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression.
  40. Cell membranes sustain phospholipid imbalance via cholesterol asymmetry.
  41. Exogenous RNA surveillance by proton-sensing TRIM25.
  42. Atovaquone-induced activation of the PERK/eIF2α signaling axis mitigates metabolic radiosensitisation.
  43. Redox regulation of lung endothelial PERK, unfolded protein response (UPR) and proliferation via NOX1: Targeted inhibition as a potential therapy for PAH.
  44. Protein editing using a coordinated transposition reaction.
  45. Endosomal chloride/proton exchangers need inhibitory TMEM9 β-subunits for regulation and prevention of disease-causing overactivity.
  1. bioRxiv. 2025 Mar 13. pii: 2025.03.10.642431. [Epub ahead of print]
    tRNA synthetase activity is required for stress granule and P-body assembly.
    Max Baymiller, Noah S Helton, Benjamin Dodd, Stephanie L Moon.
      In response to stress, translation initiation is suppressed and ribosome runoff via translation elongation drives mRNA assembly into ribonucleoprotein (RNP) granules including stress granules and P-bodies. Defects in translation elongation activate the integrated stress response. If and how stalled ribosomes are removed from mRNAs during translation elongation stress to drive RNP granule assembly is not clear. We demonstrate the integrated stress response is induced upon tRNA synthetase inhibition in part via ribosome collision sensing. However, saturating levels of tRNA synthetase inhibitors do not induce stress granules or P-bodies and prevent RNP granule assembly upon exogenous stress. The loss of tRNA synthetase activity causes persistent ribosome stalls that can be released with puromycin but are not rescued by ribosome-associated quality control pathways. Therefore, tRNA synthetase activity is required for ribosomes to run off mRNAs during stress to scaffold cytoplasmic RNP granules. Our findings suggest ribosome stalls can persist in human cells and uniquely uncouple ribonucleoprotein condensate assembly from the integrated stress response.
    DOI:  https://doi.org/10.1101/2025.03.10.642431
  2. Cell Rep. 2025 Apr 03. pii: S2211-1247(25)00273-6. [Epub ahead of print]44(4): 115502
    Spatial proteomics of ER tubules reveals CLMN, an ER-actin tether at focal adhesions that promotes cell migration.
    Holly Merta, Kaitlynn Gov, Tadamoto Isogai, Blessy Paul, Achinta Sannigrahi, Arun Radhakrishnan, Gaudenz Danuser, W Mike Henne.
      The endoplasmic reticulum (ER) is structurally and functionally diverse, yet how its functions are organized within morphological subdomains is incompletely understood. Utilizing TurboID-based proximity labeling and CRISPR knockin technologies, we map the proteomic landscape of the human ER network. Sub-organelle proteomics reveals enrichments of proteins into ER tubules, sheets, and the nuclear envelope. We uncover an ER-enriched actin-binding protein, calmin/CLMN, and define it as an ER-actin tether that localizes to focal adhesions adjacent to ER tubules. Mechanistically, we find that CLMN depletion perturbs adhesion disassembly, actin dynamics, and cell movement. CLMN-depleted cells display decreased polarization of ER-plasma membrane contacts and calcium signaling factor STIM1 and altered calcium signaling near ER-actin interfaces, suggesting that CLMN influences calcium signaling to facilitate F-actin/adhesion dynamics. Collectively, we map the sub-organelle proteome landscape of the ER, identify CLMN as an ER-actin tether, and describe a non-canonical mechanism by which ER tubules engage actin to regulate cell migration.
    Keywords:  CLMN; CP: Cell biology; ER; TurboID; adhesion; calmin; endoplasmic reticulum; migration
    DOI:  https://doi.org/10.1016/j.celrep.2025.115502
  3. Genes Dev. 2025 Apr 01.
    The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin.
    Irina S Abaeva, Alexander G Bulakhov, Christopher U T Hellen, Tatyana V Pestova.
      Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2∼Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48 specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (UbA) and imposes K48 specificity by orienting UbA such that its K48 is directly positioned to attack the thioester bond of the Ube2D1∼Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin, suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.
    Keywords:  Listerin; RING domain; TCF25; ribosome-associated quality control; ubiquitination
    DOI:  https://doi.org/10.1101/gad.352389.124
  4. Biochem J. 2025 Mar 27. pii: BCJ20253041. [Epub ahead of print]
    Sequence rules for a long SPOP-binding degron required for protein ubiquitylation.
    Linda Makhlouf, Mukul Mishra, Hannah Makhlouf, Iain Manfield, Luca Busino, Elton Zeqiraj.
      The adaptor protein, Speckle-type BTB/POZ protein (SPOP), recruits substrates to the cullin-3-subclass of E3 ligase for selective protein ubiquitylation. The Myddosome protein, Myeloid differentiation primary response 88 (MyD88), is ubiquitylated by the SPOP-based E3 ligase to negatively regulate immune signaling, however, the sequence rules for SPOP-mediated substrate engagement and degradation are not fully understood. Here, we show that MyD88 interacts with SPOP through a long degron that contains the established SPOP-binding consensus and an N-terminal site that we name the Q-motif. Based on sequence similarity to MyD88, we show that additional substrates, including Steroid receptor coactivator-3 (SRC-3), SET domain-containing protein 2 (SETD2) and Caprin1, engage SPOP in this manner. We show that the Q-motif is a critical determinant of these interactions in mammalian cells and determine X-ray crystal structures that show the molecular basis of SPOP associations with these proteins. These studies reveal a new consensus sequence for substrate-binding to SPOP that is necessary for substrate ubiquitylation, thus expanding the sequence rules required for SPOP-mediated E3 ligase substrate recognition.
    Keywords:  Adaptor proteins; MyD88; SPOP; Ubiquitin ligases; Ubiquitin signalling
    DOI:  https://doi.org/10.1042/BCJ20253041
  5. bioRxiv. 2025 Mar 20. pii: 2025.03.20.644437. [Epub ahead of print]
    SEL1L-HRD1 ER-Associated Degradation Facilitates Prohormone Convertase 2 Maturation and Glucagon Production in Islet α Cells.
    Wenzhen Zhu, Linxiu Pan, Xianwei Cui, Anna Chiara Russo, Rohit Ray, Brent Pederson, Xiaoqiong Wei, Liangguang Leo Lin, Hannah Hafner, Brigid Gregg, Neha Shrestha, Chengyang Liu, Ali Naji, Peter Arvan, Darleen A Sandoval, Iris Lindberg, Ling Qi, Rachel B Reinert.
      Proteolytic cleavage of proglucagon by prohormone convertase 2 (PC2) is required for islet α cells to generate glucagon. However, the regulatory mechanisms underlying this process remain largely unclear. Here, we report that SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD), a highly conserved protein quality control system responsible for clearing misfolded proteins from the ER, plays a key role in glucagon production by regulating turnover of the nascent proform of the PC2 enzyme (proPC2). Using a mouse model with SEL1L deletion in proglucagon-expressing cells, we observed a progressive decline in stimulated glucagon secretion and a reduction in pancreatic glucagon content. Mechanistically, we found that endogenous proPC2 is a substrate of SEL1L-HRD1 ERAD, and that degradation of misfolded proPC2 ensures the maturation of activation-competent proPC2 protein. These findings identify ERAD as a novel regulator of PC2 biology and an essential mechanism for maintaining α cell function.
    DOI:  https://doi.org/10.1101/2025.03.20.644437
  6. Cell Rep. 2025 Mar 26. pii: S2211-1247(25)00239-6. [Epub ahead of print] 115468
    Homeostatic regulation of nucleoporins is a central driver of nuclear pore biogenesis.
    Stephen Sakuma, Marcela Raices, Ethan Y S Zhu, Dana Mamriev, Charles I Fisher, Susanne Heynen-Genel, Maximiliano A D'Angelo.
      Nuclear pore complexes (NPCs) are channels that control access to the genome. The number of NPCs that cells assemble varies between different cell types and in disease. However, the mechanisms regulating NPC formation in mammalian cells remain unclear. Using a genome-wide small interfering RNA (siRNA) screen, we identify translation-related factors, proteasome components, and the CCR4-NOT complex as top regulators of NPC assembly and numbers. While inhibition of ribosomal function and protein translation reduces NPC formation, blocking protein degradation or CCR4-NOT function increases NPC numbers. We demonstrate that CCR4-NOT inhibition raises global mRNA levels, increasing the pool of nucleoporin mRNAs available for translation. Upregulation of nucleoporin complexes in CCR4-NOT-inhibited cells allows for higher NPC formation, increasing total NPC numbers in normal and cancer cells. Our findings uncover that nucleoporin mRNA stability and protein homeostasis are major determinants of NPC formation and highlight a role for the CCR4-NOT complex in negatively regulating NPC assembly.
    Keywords:  CCR4-CNOT; CP: Cell biology; CP: Molecular biology; degradation; nuclear pore complexes; nucleoporin; proteostasis; translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115468
  7. bioRxiv. 2025 Mar 15. pii: 2025.03.13.643037. [Epub ahead of print]
    PYM1 limits non-canonical Exon Junction Complex occupancy in a gene architecture dependent manner to tune mRNA expression.
    Manu Sanjeev, Lauren A Woodward, Michael L Schiff, Robert D Patton, Sean Myers, Debadrita Paul, Ralf Bundschuh, Guramrit Singh.
      The Exon Junction Complex (EJC) deposited upstream of exon-exon junctions during pre-mRNA splicing in the nucleus remains stably bound to RNA to modulate mRNA fate at multiple post-transcriptional steps until its disassembly during translation. Here, we investigated two EJC disassembly mechanisms in human embryonic kidney 293 (HEK293) cells, one mediated by PYM1, a factor that can bind both the ribosome and the RBM8A/MAGOH heterodimer of the EJC core, and another by the elongating ribosome itself. We find that EJCs lacking PYM1 interaction show no defect in translation-dependent disassembly but is required for translation-independent EJC destabilization. Surprisingly, PYM1 interaction deficient EJCs are enriched on sites away from the canonical EJC binding position including on transcripts without introns or with fewer and longer exons. Acute reduction of PYM1 levels in HEK293 cells results in a modest inhibition of nonsense-mediated mRNA decay and stabilization of mRNAs that localize to endoplasmic reticulum associated TIS-granules and are characterized by fewer and longer exons. We confirmed the previously reported PYM1-flavivirus capsid protein interaction and found that human cells expressing the capsid protein or infected with flaviviruses show similar changes in gene expression as upon PYM1 depletion. Thus, PYM1 acts as an EJC specificity factor that is hijacked by flaviviruses to alter global EJC occupancy and reshape host cell mRNA regulation.
    DOI:  https://doi.org/10.1101/2025.03.13.643037
  8. J Biol Chem. 2025 Mar 28. pii: S0021-9258(25)00316-3. [Epub ahead of print] 108467
    PEX1G843D remains functional in peroxisome biogenesis but is rapidly degraded by the proteasome.
    Connor J Sheedy, Soham P Chowdhury, Bashir A Ali, Julia Miyamoto, Eric Z Pang, Julien Bacal, Katherine U Tavasoli, Chris D Richardson, Brooke M Gardner.
      The PEX1/PEX6 AAA-ATPase is required for the biogenesis and maintenance of peroxisomes. Mutations in HsPEX1 and HsPEX6 disrupt peroxisomal matrix protein import and are the leading cause of Peroxisome Biogenesis Disorders (PBDs). The most common disease-causing mutation in PEX1 is the HsPEX1G843D allele, which results in a reduction of peroxisomal protein import. Here we demonstrate that in vitro the homologous yeast mutant, ScPex1G700D, reduces the stability of Pex1's active D2 ATPase domain and impairs assembly with Pex6, but can still form an active AAA-ATPase motor. In vivo, ScPex1G700D exhibits only a slight defect in peroxisome import. We generated model human HsPEX1G843D cell lines and show that PEX1G843D is rapidly degraded by the proteasome, but that induced overexpression of PEX1G843D can restore peroxisome import. Additionally, we found that the G843D mutation reduces PEX1's affinity for PEX6, and that impaired assembly is sufficient to induce degradation of PEX1WT. Lastly, we found that fusing a deubiquitinase to PEX1G843D significantly hinders its degradation in mammalian cells. Altogether, our findings suggest a novel regulatory mechanism for PEX1/PEX6 hexamer assembly and highlight the potential of protein stabilization as a therapeutic strategy for PBDs arising from the G843D mutation and other PEX1 hypomorphs.
    Keywords:  ATPases associated with diverse cellular activities (AAA); Peroxisome; neurodegenerative disease; proteasome; protein assembly; protein degradation; protein stability; ubiquitin thioesterase (OTUB1)
    DOI:  https://doi.org/10.1016/j.jbc.2025.108467
  9. Cell Rep. 2025 Mar 28. pii: S2211-1247(25)00254-2. [Epub ahead of print]44(4): 115483
    PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.
    Rajan S Thakur, Kate M O'Connor-Giles.
      Building synaptic connections requires coordinating a host of cellular activities from cell signaling to protein turnover, placing a high demand on intracellular communication. Membrane contact sites (MCSs) formed between organelles have emerged as key signaling hubs for coordinating diverse cellular activities, yet their roles in the developing nervous system remain obscure. We investigate the in vivo function of the endoplasmic reticulum (ER) MCS tethering and lipid-transfer protein PDZD8, which was recently linked to intellectual disability, in the nervous system. We find that PDZD8 is required for activity-dependent synaptic bouton formation in multiple paradigms. PDZD8 is sufficient to drive excess synaptic bouton formation through an autophagy-dependent mechanism and required for synapse development when autophagy is limited. PDZD8 accelerates autophagic flux by promoting lysosome maturation at ER-late endosome/lysosome MCSs. We propose that PDZD8 functions in the nervous system to increase autophagy during periods of high demand, including activity-dependent synaptic growth.
    Keywords:  CP: Cell biology; CP: Neuroscience; Drosophila; autophagy; lipid transfer protein; lysosomes; membrane contact sites; neurodevelopment; synapse
    DOI:  https://doi.org/10.1016/j.celrep.2025.115483
  10. Cell Rep. 2025 Apr 03. pii: S2211-1247(25)00261-X. [Epub ahead of print]44(4): 115490
    RAF1 kinase contributes to autophagic lysosome reformation.
    Stefanie Toifl, Sebastian Didusch, Karin Ehrenreiter, Enrico Desideri, Coralie Dorard, Manuela Baccarini.
      Autophagic lysosome reformation (ALR) is crucial for lysosomal homeostasis and therefore for different autophagic processes. Despite recent advances, the signaling mechanisms regulating ALR are incompletely understood. We show that RAF1, a member of the RAS/RAF/MEK/ERK pathway initiated by growth factors, has an essential, kinase-dependent role in lysosomal biology. RAF1 ablation impairs autophagy, and a proxisome screen identifies several proteins involved in autophagic and lysosomal pathways in the RAF1 molecular space. Two of these, SPG11 and the lipid phosphatase MTMR4, are RAF1 substrates. RAF1 ablation causes the appearance of enlarged autolysosomes and alters the phosphoinositide composition of autolysosomes. RAF1 and MTMR4 colocalize on autolysosomes, and overexpression of a MTMR4 mutant mimicking phosphorylation of the RAF1-dependent site rescues the lysosomal phenotypes induced by RAF1 ablation. Our data identify an RAF1 function in lysosomal homeostasis and a substrate through which the kinase regulates phospholipid metabolism at the lysosome, ALR, and autophagy.
    Keywords:  CP: Cell biology; RAF1 interactome; RAF1 substrates; autophagic lysosome reformation; autophagy; lysosomal homeostasis
    DOI:  https://doi.org/10.1016/j.celrep.2025.115490
  11. Cell. 2025 Apr 02. pii: S0092-8674(25)00274-0. [Epub ahead of print]
    Therapeutic potential of allosteric HECT E3 ligase inhibition.
    Alexander M K Rothman, Amir Florentin, Florence Zink, Catherine Quigley, Olivier Bonneau, Rene Hemmig, Amanda Hachey, Tomas Rejtar, Maulik Thaker, Rishi Jain, Shih-Min Huang, Daniel Sutton, Jan Roger, Ji-Hu Zhang, Sven Weiler, Simona Cotesta, Johannes Ottl, Salil Srivastava, Alina Kordonsky, Reut Avishid, Elon Yariv, Ritu Rathi, Oshrit Khvalevsky, Thomas Troxler, Sarah K Binmahfooz, Oded Kleifeld, Nicholas W Morrell, Marc Humbert, Matthew J Thomas, Gabor Jarai, Rohan E J Beckwith, Jennifer S Cobb, Nichola Smith, Nils Ostermann, John Tallarico, Duncan Shaw, Sabine Guth-Gundel, Gali Prag, David J Rowlands.
      Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.
    Keywords:  E6AP; HECT; SMURF1; allosteric inhibition; drug discovery; glycine hinge; pulmonary arterial hypertension; small molecule; ubiquitin E3 ligase; vascular remodeling
    DOI:  https://doi.org/10.1016/j.cell.2025.03.001
  12. bioRxiv. 2025 Mar 22. pii: 2025.03.20.644192. [Epub ahead of print]
    Bacterial ubiquitin ligase engineered for small molecule and protein target identification.
    James S Ye, Abir Majumdar, Brenden C Park, Miles H Black, Ting-Sung Hsieh, Adam Osinski, Kelly A Servage, Kartik Kulkarni, Jacinth Naidoo, Neal M Alto, Margaret M Stratton, Dominique Alfandari, Joseph M Ready, Krzysztof Pawłowski, Diana R Tomchick, Vincent S Tagliabracci.
      The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.
    DOI:  https://doi.org/10.1101/2025.03.20.644192
  13. Commun Biol. 2025 Mar 30. 8(1): 522
    Stepwise recruitment of chaperone Hsc70 by DNAJB1 produces ordered arrays primed for bursts of amyloid fibril disassembly.
    Jim Monistrol, Joseph G Beton, Erin C Johnston, Thi Lieu Dang, Bernd Bukau, Helen R Saibil.
      The Hsp70 chaperone system is capable of disassembling pathological aggregates such as amyloid fibres associated with serious degenerative diseases. Here we examine the role of the J-domain protein co-factor in amyloid disaggregation by the Hsc70 system. We used cryo-EM and tomography to compare the assemblies with wild-type DNAJB1 or inactive mutants. We show that DNAJB1 binds regularly along α-synuclein amyloid fibrils and acts in a 2-step recruitment of Hsc70, releasing DNAJB1 auto-inhibition before activating Hsc70 ATPase. The wild-type DNAJB1:Hsc70:Apg2 complex forms dense arrays of chaperones on the fibrils, with Hsc70 on the outer surface. When the auto-inhibition is removed by mutating DNAJB1 (ΔH5 DNAJB1), Hsc70 is recruited to the fibrils at a similar level, but the ΔH5 DNAJB1:Ηsc70:Apg2 complex is inactive, binds less regularly to the fibrils and lacks the ordered clusters. Therefore, we propose that 2-step activation of DNAJB1 regulates the ordered assembly of Hsc70 on the fibril. The localised, dense packing of chaperones could trigger a cascade of recruitment and activation to give coordinated, sequential binding and disaggregation from an exposed fibril end, as previously observed in AFM videos. This mechanism is likely to be important in maintaining a healthy cellular proteome into old age.
    DOI:  https://doi.org/10.1038/s42003-025-07906-2
  14. bioRxiv. 2025 Mar 14. pii: 2025.03.11.642454. [Epub ahead of print]
    Stress-dependent activation of PQM-1 orchestrates a second-wave proteostasis response for organismal survival.
    Laura Jones, Valeria Uvarova, Daniel O'Brien, Holly McIntyre, Natalie R Cohen, Robert H Dowen, Patricija van Oosten-Hawle.
      Stress responses are controlled by specialized stress-responsive proteostasis transcription factors that rapidly upregulate protein quality components to re-establish protein homeostasis and safeguard survival. Here we show that the zinc finger transcription factor PQM-1 is crucial for stress survival in response to thermal and oxidative challenges. We provide mechanistic insight into the regulation of PQM-1 during stress that depends on ILS-DAF-16 signaling, as well as phosphorylation on threonine residue 268 that is located within a conserved AKT motif. Our data show that in reproductively mature adults and during well-fed conditions, PQM-1 induction requires DAF-16 and occurs during the recovery period post heat shock. Moreover, PQM-1 co-localizes with DAF-16 in the nucleus during the stress recovery phase. This regulatory dependency on DAF-16 is bypassed under dietary restriction, allowing PQM-1 to promote stress resilience independent of the ILS pathway. During both conditions, PQM-1 is crucial for the upregulation of cytosolic and endoplasmic reticulum stress response genes required for organismal recovery and stress resilience. Our transcriptional and bioinformatic analysis reveals that PQM-1 regulates a distinct set of target genes during the stress recovery phase, suggesting that PQM-1 may be involved in vital secondary wave stress response. Thus, our findings uncover a previously unrecognized mechanism of stress-dependent PQM-1 activation that integrates multiple environmental cues to ensure proteostasis and organismal survival.
    DOI:  https://doi.org/10.1101/2025.03.11.642454
  15. J Cell Biol. 2025 Jun 02. pii: e202407066. [Epub ahead of print]224(6):
    The Abelson kinase and the Nedd4 family E3 ligases co-regulate Notch trafficking to limit signaling.
    Julio Miranda-Alban, Nicelio Sanchez-Luege, Fernando M Valbuena, Chyan Rangel, Ilaria Rebay.
      Precise output from the conserved Notch signaling pathway governs a plethora of cellular processes and developmental transitions. Unlike other pathways that use a cytoplasmic relay, the Notch cell surface receptor transduces signaling directly to the nucleus, with endocytic trafficking providing critical regulatory nodes. Here we report that the cytoplasmic tyrosine kinase Abelson (Abl) facilitates Notch internalization into late endosomes/multivesicular bodies (LEs), thereby limiting signaling output in both ligand-dependent and -independent contexts. Abl phosphorylates the PPxY motif within Notch, a molecular target for its degradation via Nedd4 family ubiquitin ligases. We show that Su(dx), a family member, mediates the Abl-directed LE regulation of Notch via the PPxY, while another family member, Nedd4Lo, contributes to Notch internalization into LEs through both PPxY-dependent and -independent mechanisms. Our findings demonstrate how a network of posttranslational modifiers converging at LEs cooperatively modulates Notch signaling to ensure the precision and robustness of its cellular and developmental functions.
    DOI:  https://doi.org/10.1083/jcb.202407066
  16. bioRxiv. 2025 Mar 12. pii: 2025.03.07.641543. [Epub ahead of print]
    Heterobifunctional proteomimetic polymers for targeted protein degradation.
    Max M Wang, Mihai I Truica, Brayley S Gattis, Julia Oktawiec, Vinay Sagar, Ananya A Basu, Paul A Bertin, Xiaoyu Zhang, Sarki A Abdulkadir, Nathan C Gianneschi.
      The burgeoning field of targeted protein degradation (TPD) has opened new avenues for modulating the activity of previously undruggable proteins of interest. To date, TPD has been dominated by small molecules containing separate linked domains for protein engagement and recruitment of cellular degradation machinery. The process of identifying active compounds has required tedious optimization and has been successful largely against a limited set of targets with well-defined, suitable docking pockets. Here we present a polymer chemistry approach termed the HYbrid DegRAding Copolymer (HYDRAC) to overcome standing challenges associated with the development of TPD. These copolymers densely display either peptide-based or small molecule-derived degradation inducers and target-binding peptide sequences for the selective degradation of disease-associated proteins. HYDRACs are synthesized in a facile manner, are modular in design, and are highly selective. Using the intrinsically disordered transcription factor MYC as an initial proof-of-concept, difficult to drug protein target, HYDRACs containing a MYC-inhibitory peptide copolymerized with a validated degron, showed robust and selective degradation of the target protein. Treatment of tumor-bearing mice with MYC-targeted HYDRACs showed decreased cell proliferation and increased tumor apoptosis, leading to significantly suppressed tumor growth in vivo . The versatility of the platform was demonstrated by substituting the degron for recruiters of three different E3 ligases (VHL, KEAP1, and CRBN), which all maintained MYC degradation. To demonstrate generalizability, HYDRACs were further designed against a second elusive target of clinical interest, KRAS, by employing a consensus RAS binding motif. RAS-targeted HYDRACs showed degradation in two cell lines harboring separate KRAS alleles, suggesting potential pan-KRAS activity. We envision the HYDRAC platform as a generalizable approach to developing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for TPD.
    DOI:  https://doi.org/10.1101/2025.03.07.641543
  17. Elife. 2025 Apr 02. pii: e106553. [Epub ahead of print]14
    At the crossroads of calcium signaling, protein synthesis and neuropeptide release.
    Jakob Rupert, Dragomir Milovanovic.
      By influencing calcium homeostasis, local protein synthesis and the endoplasmic reticulum, a small protein called Rab10 emerges as a crucial cytoplasmic regulator of neuropeptide secretion.
    Keywords:  Rab10; endoplasmic reticulum; exocytosis; mouse; neuropeptide; neuroscience; protein synthesis; synaptic transmission
    DOI:  https://doi.org/10.7554/eLife.106553
  18. Cancer Cell. 2025 Mar 21. pii: S1535-6108(25)00082-0. [Epub ahead of print]
    Translation dysregulation in cancer as a source for targetable antigens.
    Chen Weller, Osnat Bartok, Christopher S McGinnis, Heyilimu Palashati, Tian-Gen Chang, Dmitry Malko, Merav D Shmueli, Asuteka Nagao, Deborah Hayoun, Ayaka Murayama, Yuriko Sakaguchi, Panagiotis Poulis, Aseel Khatib, Bracha Erlanger Avigdor, Sagi Gordon, Sapir Cohen Shvefel, Marie J Zemanek, Morten M Nielsen, Sigalit Boura-Halfon, Shira Sagie, Nofar Gumpert, Weiwen Yang, Dmitry Alexeev, Pelgia Kyriakidou, Winnie Yao, Mirie Zerbib, Polina Greenberg, Gil Benedek, Kevin Litchfield, Ekaterina Petrovich-Kopitman, Adi Nagler, Roni Oren, Shifra Ben-Dor, Yishai Levin, Yitzhak Pilpel, Marina Rodnina, Jürgen Cox, Yifat Merbl, Ansuman T Satpathy, Yaron Carmi, Florian Erhard, Tsutomu Suzuki, Allen R Buskirk, Johanna Olweus, Eytan Ruppin, Andreas Schlosser, Yardena Samuels.
      Aberrant peptides presented by major histocompatibility complex (MHC) molecules are targets for tumor eradication, as these peptides can be recognized as foreign by T cells. Protein synthesis in malignant cells is dysregulated, which may result in the generation and presentation of aberrant peptides that can be exploited for T cell-based therapies. To investigate the role of translational dysregulation in immunological tumor control, we disrupt translation fidelity by deleting tRNA wybutosine (yW)-synthesizing protein 2 (TYW2) in tumor cells and characterize the downstream impact on translation fidelity and immunogenicity using immunopeptidomics, genomics, and functional assays. These analyses reveal that TYW2 knockout (KO) cells generate immunogenic out-of-frame peptides. Furthermore, Tyw2 loss increases tumor immunogenicity and leads to anti-programmed cell death 1 (PD-1) checkpoint blockade sensitivity in vivo. Importantly, reduced TYW2 expression is associated with increased response to checkpoint blockade in patients. Together, we demonstrate that defects in translation fidelity drive tumor immunogenicity and may be leveraged for cancer immunotherapy.
    Keywords:  cancer immunopeptidome; immune checkpoint blockade; neoantigens; non-canonical peptides; translation fidelity
    DOI:  https://doi.org/10.1016/j.ccell.2025.03.003
  19. Sci Adv. 2025 Apr 04. 11(14): eadr6415
    Retromer promotes the lysosomal turnover of mtDNA.
    Parisa Kakanj, Mari Bonse, Arya Kshirsagar, Aylin Gökmen, Felix Gaedke, Ayesha Sen, Belén Mollá, Elisabeth Vogelsang, Astrid Schauss, Andreas Wodarz, David Pla-Martín.
      Mitochondrial DNA (mtDNA) is exposed to multiple insults produced by normal cellular function. Upon mtDNA replication stress, the mitochondrial genome transfers to endosomes for degradation. Using proximity biotinylation, we found that mtDNA stress leads to the rewiring of the mitochondrial proximity proteome, increasing mitochondria's association with lysosomal and vesicle-related proteins. Among these, the retromer complex, particularly VPS35, plays a pivotal role by extracting mitochondrial components. The retromer promotes the formation of mitochondrial-derived vesicles shuttled to lysosomes. The mtDNA, however, directly shuttles to a recycling organelle in a BAX-dependent manner. Moreover, using a Drosophila model carrying a long deletion on the mtDNA (ΔmtDNA), we found that ΔmtDNA activates a specific transcriptome profile to counteract mitochondrial damage. Here, Vps35 expression restores mtDNA homoplasmy and alleviates associated defects. Hence, we demonstrate the existence of a previously unknown quality control mechanism for the mitochondrial matrix and the essential role of lysosomes in mtDNA turnover to relieve mtDNA damage.
    DOI:  https://doi.org/10.1126/sciadv.adr6415
  20. Contact (Thousand Oaks). 2025 Jan-Dec;8:8 25152564251329704
    Remodelling of Cellular Protein Homeostasis by Enhanced ER-Mitochondrial Tethering.
    Elisa Tonelli, Justyna Malecka, Elettra Barberis, Camilla Romano, Emanuela Pessolano, Maria Talmon, Armando A Genazzani, Claudio Casali, Marco Biggiogera, Marcello Manfredi, Laura Tapella, Dmitry Lim, Giulia Dematteis.
      Alterations of endoplasmic reticulum (ER)-mitochondrial interaction have been associated with different pathological conditions, including neurodegenerative diseases, characterized by dysregulation of protein homeostasis. However, little is known about how enhanced ER-mitochondrial tethering affects cellular proteostatic machinery. Here, we transiently overexpressed synthetic ER-mitochondrial linkers (EMLs), stabilizing the ER-mitochondrial distance at ≤5 nm (denominated as 5 nm-EML) and ∼10 nm (10 nm-EML), in HeLa cells. No alterations were found in cell growth, although metabolic activity and total ATP were significantly reduced. In EML-expressing cells, global protein synthesis was significantly reduced, accompanied by a reduction of total PERK and eIF2α protein levels, but increased p-eIF2α. Unfolded protein response (UPR) markers ATF4 and ATF6 were upregulated, suggesting that enhanced ER-mitochondrial tethering deranges protein synthesis and induces a low-grade ER stress/UPR. To further investigate ER-mitochondrial tethering-induced protein dyshomeostasis, we performed shotgun mass spectrometry proteomics followed by bioinformatic analysis. Analysis of highly changed proteins and the most significantly overrepresented gene ontology (GO) terms revealed that ≤5 nm tethering preferentially affected the expression of proteins involved in RNA processing and splicing and proteasomal protein degradation, while ∼10 nm tethering preferentially affected protein translation. Both EMLs affected expression of proteins involved in mitochondrial bioenergetics and metabolism, defense against oxidative stress, ER protein homeostasis, signaling and secretion. Finally, lipidomic analysis suggests that 5 nm-EML and 10 nm-EML differentially affect lipid homeostasis. Altogether, our results suggest that enhanced ER-mitochondrial tethering leads to a profound remodeling of cellular protein homeostasis, which may play a key role in pathogenesis of Alzheimer's and other neurodegenerative diseases.
    Keywords:  MAMs; MERCS; mitochondria-ER contact sites; proteostasis
    DOI:  https://doi.org/10.1177/25152564251329704
  21. bioRxiv. 2025 Mar 20. pii: 2025.03.20.644464. [Epub ahead of print]
    p62 sorts Lupus La and selected microRNAs into breast cancer-derived exosomes.
    Jordan Matthew Ngo, Justin Krish Williams, Morayma Mercedes Temoche-Diaz, Abinayaa Murugupandiyan, Randy Schekman.
      Exosomes are multivesicular body-derived extracellular vesicles that are secreted by metazoan cells. Exosomes have utility as disease biomarkers, and exosome-mediated miRNA secretion has been proposed to facilitate tumor growth and metastasis. Previously, we demonstrated that the Lupus La protein (La) mediates the selective incorporation of miR-122 into metastatic breast cancer-derived exosomes; however, the mechanism by which La itself is sorted into exosomes remains unknown. Using unbiased proximity labeling proteomics, biochemical fractionation, superresolution microscopy and genetic tools, we establish that the selective autophagy receptor p62 sorts La and miR-122 into exosomes. We then performed small RNA sequencing and found that p62 depletion reduces the exosomal secretion of tumor suppressor miRNAs and results in their accumulation within cells. Our data indicate that p62 is a quality control factor that modulates the miRNA composition of exosomes. Cancer cells may exploit p62-dependent exosome cargo sorting to eliminate tumor suppressor miRNAs and thus to promote cell proliferation.
    DOI:  https://doi.org/10.1101/2025.03.20.644464
  22. Autophagy. 2025 Apr 02.
    First responder to starvation: microreticulophagy clears aberrant membrane proteins in quick bites.
    Yaneris Alvarado Cartagena, Valeriya Gyurkovska, Nava Segev.
      Cells can use two different pathways for recycling their non-essential components in the lysosome during nutritional stress: macroautophagy and microautophagy. While the well-established macroautophagy pathway requires de novo formation of the double-membrane autophagosome, microautophagy involves direct engulfment of cargo by the lysosomal membrane. Recently, using a yeast model, we identified a novel microreticulophagy pathway induced by nutritional stress that selectively clears aberrant membrane proteins that accumulate during normal growth. This effective clearance occurs rapidly and precedes the degradation of normal ER- or mitochondrial-membrane proteins by macroautophagy. We showed that the nutritional-stress induced selective microreticulophagy pathway requires the ubiquitin-ligase Rsp5, its adaptor Ssh4, and the ESCRT complex. Moreover, live-cell fluorescence microscopy with high temporal and special resolution demonstrated that individual microautophagy events occur within seconds. Thus, cells use the effective microreticulophagy pathway to dispose of misfolded or excess membrane proteins as a first response to starvation. If the stress persists, the more costly macroautophagy pathway is activated for degrading normal cellular components. These findings point to an intricate interplay between microautophagy and macroautophagy during nutritional stress, which optimizes stress responses and could have significant implications for understanding how cells maintain homeostasis or progress to disease states.
    Keywords:  Aberrant membrane proteins; ER-phagy; ERAD; macroautophagy; microautophagy; nutritional stress
    DOI:  https://doi.org/10.1080/15548627.2025.2487675
  23. Acc Chem Res. 2025 Apr 01.
    Probing the E3 Ligase Adapter Cereblon with Chemical Biology.
    Wenqing Xu, Christina M Woo.
      ConspectusThe E3 ligase substrate adapter cereblon (CRBN) has garnered widespread interest from the research laboratory to the clinic. CRBN was first discovered for its association with neurological development and subsequently identified as the target of thalidomide and lenalidomide, therapeutic agents used in the treatment of hematopoietic malignancies. Both thalidomide and lenalidomide have been repurposed as ligands for targeted protein degradation therapeutic modalities. These agents were proposed to mimic a naturally occurring ligand, although the native substrate recognition mechanism of CRBN remained elusive. Chemical biology, which involves the use of chemical tools to modulate and probe biological systems, can provide unique insights into the molecular mechanisms and interactions of proteins with their cognate ligands. Here we describe our use of chemical biology approaches, including photoaffinity labeling, chemical proteomics, and targeted protein degradation, to interrogate the biological activities of CRBN in the presence or absence of its ligands. Our development of a photoaffinity labeling probe derived from lenalidomide, termed photolenalidomide, enabled mapping of the binding site on CRBN and identification of a new target recruited to CRBN by lenalidomide through chemical proteomics. Further derivatization of the lenalidomide scaffold afforded DEG-77, a potent degrader with therapeutic efficacy against acute myeloid leukemia. Our parallel development of chemically defined probes that are inspired by heterobifunctional targeted protein degradation agents and functionally engage CRBN in cells revealed that thalidomide is a peptidomimetic of an underappreciated protein modification termed the C-terminal cyclic imide, which arises from intramolecular cyclization of asparagine or glutamine residues and represents a degron endogenously recognized by CRBN. Protein engineering and proteomic efforts validated the CRBN-dependent regulation of proteins bearing the C-terminal cyclic imide modification in vitro and in cells and the prevalence of the C-terminal cyclic imide in the biological system. Application of C-terminal cyclic imides as a class of cyclimid ligands for targeted protein degradation led to the development of a variety of heterobifunctional degraders with distinct efficacy and target selectivity, whereas examination of the occurrence of C-terminal cyclic imides as a form of protein damage uncovered the intrinsic and extrinsic factors that predispose peptides and proteins to C-terminal cyclic imide formation and the role of CRBN in mitigating the accumulation of damaged proteins with a propensity for aggregation. Future investigation of C-terminal cyclic imides, synthetic ligands, and their relationship to CRBN biology will illuminate regulatory mechanisms that are controlled by CRBN and drive the pursuit of additional functional chemistries on proteins and the biological pathways that intercept them.
    DOI:  https://doi.org/10.1021/acs.accounts.5c00059
  24. Life Sci Alliance. 2025 Jun;pii: e202503243. [Epub ahead of print]8(6):
    The RBR E3 ubiquitin ligase HOIL-1 can ubiquitinate diverse non-protein substrates in vitro.
    Xiangyi S Wang, Jenny Jiou, Anthony Cerra, Simon A Cobbold, Marco Jochem, Ka Hin Toby Mak, Leo Corcilius, John Silke, Richard J Payne, Ethan D Goddard-Borger, David Komander, Bernhard C Lechtenberg.
      HOIL-1 is a RING-between-RING-family E3 ubiquitin ligase and a component of the linear ubiquitin chain assembly complex. Although most E3 ubiquitin ligases conjugate ubiquitin to protein lysine sidechains, HOIL-1 has also been reported to ubiquitinate hydroxyl groups in protein serine and threonine sidechains and glucosaccharides, such as glycogen and its building block maltose, in vitro. However, HOIL-1 substrate specificity is currently poorly defined. Here, we show that HOIL-1 is unable to ubiquitinate lysine but can efficiently ubiquitinate serine and a variety of model and physiologically relevant di- and monosaccharides in vitro. We identify a critical catalytic histidine residue, His510, in the flexible catalytic site of HOIL-1 that enables this O-linked ubiquitination and prohibits ubiquitin discharge onto lysine sidechains. We use HOIL-1's in vitro non-proteinaceous ubiquitination activity to produce preparative amounts of different ubiquitinated saccharides that can be used as tool compounds and standards in the rapidly emerging field of non-proteinaceous ubiquitination. Finally, we report an engineered, constitutively active HOIL-1 variant that simplifies in vitro generation of ubiquitinated saccharides.
    DOI:  https://doi.org/10.26508/lsa.202503243
  25. BMC Plant Biol. 2025 Apr 02. 25(1): 412
    Plant ubiquitin E2 enzymes UBC32, UBC33, and UBC34 are involved in ERAD and function in host stress tolerance.
    Chaofeng Wang, Bangjun Zhou, Yi Zhang, Lirong Zeng.
       BACKGROUND: Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a critical component of the ER-mediated protein quality control (ERQC) system and plays a vital role in plant stress responses. However, the ubiquitination machinery underlying plant ERAD-particularly the ubiquitin-conjugating enzymes (E2s)-and their contributions to stress tolerance remain poorly understood.
    RESULTS: In this study, we identified UBC32, UBC33, and UBC34 as ER-localized ubiquitin E2 enzymes involved in ERAD and demonstrated their roles in biotic and abiotic stress tolerance in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). In response to biotic stress, UBC33 and UBC34 collectively contribute more substantially than UBC32 to plant immunity against Pseudomonas syringae pv. tomato (Pst). Under abiotic stress and ER stress induced by tunicamycin (TM), all three E2s play important roles. Notably, mutation of UBC32 enhances tolerance to TM-induced ER stress, whereas the loss of function in UBC33 or UBC34 suppresses this response. Additionally, UBC32, UBC33, and UBC34 act synergistically in Arabidopsis seed germination under salt stress and abscisic acid (ABA) treatment. While the single mutants atubc32, atubc33, and atubc34 exhibit germination rates comparable to Col-0 under salt stress or ABA treatment, the double mutants atubc32/33, atubc32/34, and atubc33/34 show a significantly greater reduction in germination rate. Interestingly, the atubc32/33/34 triple mutant exhibits a seed germination rate under salt stress and ABA treatment, as well as a level of host immunity to Pst, comparable to that of the atubc33/34 and atubc32/34 double mutants.
    CONCLUSIONS: Our findings establish UBC32, UBC33, and UBC34 as key components of the plant ERAD machinery, contributing to plant tolerance to both abiotic and biotic stress. Despite their close phylogenetic relationship, these E2 enzymes exhibit redundant, synergistic, or antagonistic roles depending on the specific stress response pathway, underscoring the complexity of their functional interactions.
    Keywords:   Pseudomonas syringae Pv. tomato ; Abiotic stress; Biotic stress; ER stress; Endoplasmic reticulum (ER)-associated protein degradation (ERAD); Ubiquitin-conjugating enzyme (E2)
    DOI:  https://doi.org/10.1186/s12870-025-06419-8
  26. Dev Cell. 2025 Mar 25. pii: S1534-5807(25)00152-2. [Epub ahead of print]
    ER nests are specialized ER subdomains in Arabidopsis where peroxisomes and lipid droplets form.
    Zachary J Wright, Nathan E Tharp, Bonnie Bartel.
      Organelles are defining features of eukaryotic cells, yet much remains to be learned about organelle biogenesis. Lipid droplets and peroxisomes, which play opposing roles in storing and catabolizing fats, form from a mysterious domain in the endoplasmic reticulum (ER). We used live-cell fluorescence microscopy to visualize peroxisome and lipid droplet biogenesis in young Arabidopsis seedlings, where lipid catabolism is active, and peroxisomes can be unusually large. We found that the ER domains where these organelles are born, which we term ER nests, are complex, dynamic structures that exclude general ER proteins but accumulate other proteins, including lipid biosynthetic enzymes and the COPII component SAR1. Furthermore, ER nests appear to define peroxisome-lipid droplet contact sites. Our findings provide a framework for understanding how these domains form and sort their protein components, illuminate eukaryotic lipid biosynthesis, and elucidate how distinct organelles arise from the ER.
    Keywords:  Arabidopsis cell biology; RHD3/atlastin; SAR1; endomembrane trafficking; lipid droplet; organelle biogenesis; peroxisome
    DOI:  https://doi.org/10.1016/j.devcel.2025.03.005
  27. Nat Commun. 2025 Apr 02. 16(1): 3144
    Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment.
    Zhuoyao Chen, Gamma Chi, Timea Balo, Xiangrong Chen, Beatriz Ralsi Montes, Steven C Clifford, Vincenzo D'Angiolella, Timea Szabo, Arpad Kiss, Tibor Novak, András Herner, András Kotschy, Alex N Bullock.
      Neomorphic mutations and drugs can elicit unanticipated effects that require mechanistic understanding to inform clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. Here, we identify that these neomorphic interactions are mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 capture 2:1 and 2:2 KBTBD4-HDAC2 complexes, as well as a 2:1:1 KBTBD4-HDAC2-CoREST1 complex, at resolutions spanning 2.7 to 3.3 Å. The mutant and drug-induced complexes adopt similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 is identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshapes the same surface of KBTBD4 providing an example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4, while structure-activity relationship (SAR) analysis of UM171 reveals analog S234984 as a more potent molecular glue for future studies.
    DOI:  https://doi.org/10.1038/s41467-025-58350-z
  28. Cell Rep. 2025 Apr 03. pii: S2211-1247(25)00289-X. [Epub ahead of print]44(4): 115518
    SMYD5 is a ribosomal methyltransferase that trimethylates RPL40 lysine 22 through recognition of a KXY motif.
    Joshua J Hamey, Manan Shah, John D Wade, Tara K Bartolec, Richard E H Wettenhall, Kate G R Quinlan, Nicholas A Williamson, Marc R Wilkins.
      The eukaryotic ribosome is highly modified by protein methylation, yet many of the responsible methyltransferases remain unknown. Here, we identify SET and MYND domain-containing protein 5 (SMYD5) as a ribosomal protein methyltransferase that catalyzes trimethylation of RPL40/eL40 at lysine 22. Through a systematic mass spectrometry-based approach, we identify 12 primary sites of protein methylation in ribosomes from K562 cells, including at RPL40 K22. Through in vitro methylation of synthetic RPL40 using fractionated lysate, we then identify SMYD5 as a candidate RPL40 K22 methyltransferase. We show that recombinant SMYD5 has robust activity toward RPL40 K22 in vitro and that active site mutations ablate this activity. Knockouts of SMYD5 in K562 cells show a complete loss of RPL40 K22 methylation and decreased polysome levels. We show that SMYD5 does not methylate histones in vitro, and by systematic analysis of its recognition motif, we find that SMYD5 requires a KXY motif for methylation, explaining its lack of activity toward histones.
    Keywords:  CP: Molecular biology; lysine methylation; methyltransferase; ribosome; substrate specificity; translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.115518
  29. Elife. 2025 Apr 02. pii: RP94930. [Epub ahead of print]13
    Rab10 regulates neuropeptide release by maintaining Ca2+ homeostasis and protein synthesis.
    Jian Dong, Mian Chen, Jan R T van Weering, Ka Wan Li, August B Smit, Ruud F Toonen, Matthijs Verhage.
      Dense core vesicles (DCVs) transport and release various neuropeptides and neurotrophins that control diverse brain functions, but the DCV secretory pathway remains poorly understood. Here, we tested a prediction emerging from invertebrate studies about the crucial role of the intracellular trafficking GTPase Rab10, by assessing DCV exocytosis at single-cell resolution upon acute Rab10 depletion in mature mouse hippocampal neurons, to circumvent potential confounding effects of Rab10's established role in neurite outgrowth. We observed a significant inhibition of DCV exocytosis in Rab10-depleted neurons, whereas synaptic vesicle exocytosis was unaffected. However, rather than a direct involvement in DCV trafficking, this effect was attributed to two ER-dependent processes, ER-regulated intracellular Ca2+ dynamics, and protein synthesis. Gene Ontology analysis of differentially expressed proteins upon Rab10 depletion identified substantial alterations in synaptic and ER/ribosomal proteins, including the Ca2+ pump SERCA2. In addition, ER morphology and dynamics were altered, ER Ca2+ levels were depleted, and Ca2+ homeostasis was impaired in Rab10-depleted neurons. However, Ca2+ entry using a Ca2+ ionophore still triggered less DCV exocytosis. Instead, leucine supplementation, which enhances protein synthesis, largely rescued DCV exocytosis deficiency. We conclude that Rab10 is required for neuropeptide release by maintaining Ca2+ dynamics and regulating protein synthesis. Furthermore, DCV exocytosis appeared more dependent on (acute) protein synthesis than synaptic vesicle exocytosis.
    Keywords:  ER; Rab10; exocytosis; mouse; neuropeptide; neuroscience; protein synthesis; synaptic transmission
    DOI:  https://doi.org/10.7554/eLife.94930
  30. Nat Methods. 2025 Mar 28.
    Atomic context-conditioned protein sequence design using LigandMPNN.
    Justas Dauparas, Gyu Rie Lee, Robert Pecoraro, Linna An, Ivan Anishchenko, Cameron Glasscock, David Baker.
      Protein sequence design in the context of small molecules, nucleotides and metals is critical to enzyme and small-molecule binder and sensor design, but current state-of-the-art deep-learning-based sequence design methods are unable to model nonprotein atoms and molecules. Here we describe a deep-learning-based protein sequence design method called LigandMPNN that explicitly models all nonprotein components of biomolecular systems. LigandMPNN significantly outperforms Rosetta and ProteinMPNN on native backbone sequence recovery for residues interacting with small molecules (63.3% versus 50.4% and 50.5%), nucleotides (50.5% versus 35.2% and 34.0%) and metals (77.5% versus 36.0% and 40.6%). LigandMPNN generates not only sequences but also sidechain conformations to allow detailed evaluation of binding interactions. LigandMPNN has been used to design over 100 experimentally validated small-molecule and DNA-binding proteins with high affinity and high structural accuracy (as indicated by four X-ray crystal structures), and redesign of Rosetta small-molecule binder designs has increased binding affinity by as much as 100-fold. We anticipate that LigandMPNN will be widely useful for designing new binding proteins, sensors and enzymes.
    DOI:  https://doi.org/10.1038/s41592-025-02626-1
  31. Proc Natl Acad Sci U S A. 2025 Apr 08. 122(14): e2420544122
    HEATR3 recognizes membrane rupture and facilitates xenophagy in response to Salmonella invasion.
    Masashi Arakawa, Keiya Uriu, Koki Saito, Mai Hirose, Kaoru Katoh, Krisana Asano, Akio Nakane, Tatsuya Saitoh, Tamotsu Yoshimori, Eiji Morita.
      Bacterial invasion into the cytoplasm of epithelial cells triggers the activation of the cellular autophagic machinery as a defense mechanism, a process known as xenophagy. In this study, we identified HEATR3, an LC3-interacting region (LIR)-containing protein, as a factor involved in this defense mechanism using quantitative mass spectrometry analysis. HEATR3 localizes intracellularly invading Salmonella, and HEATR3 deficiency promotes Salmonella proliferation in the cytoplasm. HEATR3 also localizes to lysosomes damaged by chemical treatment, suggesting that Salmonella recognition is facilitated by damage to the host cell membrane. HEATR3 deficiency impairs LC3 recruitment to damaged membranes and blocks the delivery of the target to the lysosome. These phenotypes were rescued by exogenous expression of wild-type HEATR3 but not by the LIR mutant, indicating the crucial role of the HEATR3-LC3 interaction in the receptor for selective autophagy. HEATR3 is delivered to lysosomes in an autophagy-dependent manner. Although HEATR3 recruitment to the damaged membrane was unaffected by ATG5 or FIP200 deficiency, it was markedly impaired by treatment with a calcium chelator, suggesting involvement upstream of the autophagic pathway. These findings suggest that HEATR3 serves as a receptor for selective autophagy and is able to identify damaged membranes, facilitate the removal of damaged lysosomes, and target invading bacteria within cells.
    Keywords:  HEATR3; NOD2 signaling; autophagy; salmonella infection; xenophagy receptor
    DOI:  https://doi.org/10.1073/pnas.2420544122
  32. Mol Psychiatry. 2025 Apr 03.
    Protein aggregation identified in olfactory neuronal cells is associated with cognitive impairments in a subset of living schizophrenia patients.
    Leslie G Nucifora, Koko Ishizuka, Nagat El Demerdash, Brian J Lee, Michael T Imai, Carlos Ayala-Grosso, Gayane Yenokyan, Nicola G Cascella, Sandra Lin, David J Schretlen, Philip D Harvey, Russell L Margolis, Christopher A Ross, Akira Sawa, Frederick C Nucifora.
      Schizophrenia is a heterogeneous disorder, and likely results from multiple pathophysiological mechanisms. Protein aggregation, resulting from disruption of protein homeostasis (proteostasis), has been implicated in many diseases, including cancer, cardiac and pulmonary diseases, muscle diseases, and neurodegenerative disorders, but is a relatively new pathophysiological hypothesis for schizophrenia. Genetic findings implicate proteostasis in schizophrenia, and individual proteins associated with the disorder may undergo aggregation. While there is some evidence of associations between genetic variants and protein aggregation, the extent to which genetic variations influence protein aggregation remains unknown. We have previously reported increased protein insolubility and increased ubiquitination of the insoluble protein fraction, two markers of protein aggregation, in human postmortem brains from a subset of patients with schizophrenia. In the present study, we investigate whether protein aggregation is observed in an independent model system, olfactory neuronal cells derived from living patients with schizophrenia, and examine the relationship between aggregation and patient clinical and cognitive status. We demonstrate that, as in postmortem brain, olfactory neurons from a subset of patients with schizophrenia exhibit protein aggregation, identified by increased protein insolubility and ubiquitination of the insoluble protein fraction, and by ubiquitin positive protein aggregates. Patients with protein aggregation exhibit more severe cognitive deficits than those without aggregation, as revealed by between-group comparisons and correlational analyses. Understanding the mechanisms of the aggregation process, the factors that differentiate individuals who develop aggregates from those who do not, and the relationship between aggregation and cell function, has important implications for the pathophysiology of schizophrenia, and may provide insight into disease heterogeneity and novel therapeutic targets.
    DOI:  https://doi.org/10.1038/s41380-025-02956-8
  33. bioRxiv. 2025 Mar 14. pii: 2025.03.11.642495. [Epub ahead of print]
    Characterization of MEK1/2 Degraders Uncovers a Kinase-Independent Role for MEK1/2 in the Stabilization and Maturation of CRAF.
    Jason S Wasserman, Alison M Kurimchak, Carlos Herrera-Montávez, Glenn A Doyle, Brandon D Fox, Ishadi K M Kodikara, Xiaoping Hu, Jianping Hu, Jian Jin, James S Duncan.
      Altered MAPK signaling frequently occurs in human disease. MEK1 and MEK2 (MEK1/2) are central protein kinases in the MAPK signaling cascade that phosphorylate ERK1/2 promoting cell growth. MEK1/2 degraders offer a strategy to characterize both kinase-dependent and independent functions of MEK1/2. Here, we discovered that MEK1/2 degradation, but not kinase inhibition, caused the subsequent degradation of upstream kinase CRAF via a cell-intrinsic mechanism. MEK1/2 binding to CRAF, but not MEK1/2 catalytic activity, was required for CRAF protein stability and maturation to a functional kinase. In the absence of MEK1/2, a minor pool of newly synthesized immature CRAF that had anti-apoptotic functions remained. Finally, we showed that a stable primed CRAF-MEK1/2 signaling complex existed in cells that required RAS binding to potentiate MEK-ERK phosphorylation. Together, we've discovered a previously unrecognized kinase-independent function of MEK1/2, while contextualizing MEK1/2 as an integral component of the CRAF activation cycle beyond the conventional CRAF-MEK kinase- substrate paradigm.
    DOI:  https://doi.org/10.1101/2025.03.11.642495
  34. Nat Commun. 2025 Mar 30. 16(1): 3078
    Complementary Ribo-seq approaches map the translatome and provide a small protein census in the foodborne pathogen Campylobacter jejuni.
    Kathrin Froschauer, Sarah L Svensson, Rick Gelhausen, Elisabetta Fiore, Philipp Kible, Alicia Klaude, Martin Kucklick, Stephan Fuchs, Florian Eggenhofer, Chao Yang, Daniel Falush, Susanne Engelmann, Rolf Backofen, Cynthia M Sharma.
      In contrast to transcriptome maps, bacterial small protein (≤50-100 aa) coding landscapes, including overlapping genes, are poorly characterized. However, an emerging number of small proteins have crucial roles in bacterial physiology and virulence. Here, we present a Ribo-seq-based high-resolution translatome map for the major foodborne pathogen Campylobacter jejuni. Besides conventional Ribo-seq, we employed translation initiation site (TIS) profiling to map start codons and also developed a translation termination site (TTS) profiling approach, which revealed stop codons not apparent from the reference genome in virulence loci. Our integrated approach combined with independent validation expanded the small proteome by two-fold, including CioY, a new 34 aa component of the CioAB oxidase. Overall, our study generates a high-resolution annotation of the C. jejuni coding landscape, provided in an interactive browser, and showcases a strategy for applying integrated Ribo-seq to other species to enrich our understanding of small proteomes.
    DOI:  https://doi.org/10.1038/s41467-025-58329-w
  35. BMC Methods. 2025 ;2(1): 5
    iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation.
    Sarah Lecinski, Jamieson A L Howard, Chris MacDonald, Mark C Leake.
       Background: Cells employ myriad regulatory mechanisms to maintain protein homeostasis, termed proteostasis, to ensure correct cellular function. Dysregulation of proteostasis, which is often induced by physiological stress and ageing, often results in protein aggregation in cells. These aggregated structures can perturb normal physiological function, compromising cell integrity and viability, a prime example being early onset of several neurodegenerative diseases. Understanding aggregate dynamics in vivo is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are not fully understood.
    Methods: Here, we report an improved methodology for production of fluorescent aggregates in model budding yeast which can be detected, tracked and quantified using fluorescence microscopy in live cells. This new openly-available technology, iPAR (inducible Protein Aggregation Reporter), involves monomeric fluorescent protein reporters fused to a ∆ssCPY* aggregation biomarker, with expression controlled under the copper-regulated CUP1 promoter.
    Results: Monomeric tags overcome challenges associated with non-physiological reporter aggregation, whilst CUP1 provides more precise control of protein production. We show that iPAR and the associated bioimaging methodology enables quantitative study of cytoplasmic aggregate kinetics and inheritance features in vivo. We demonstrate that iPAR can be used with traditional epifluorescence and confocal microscopy as well as single-molecule precise Slimfield millisecond microscopy. Our results indicate that cytoplasmic aggregates are mobile and contain a broad range of number of iPAR molecules, from tens to several hundred per aggregate, whose mean value increases with extracellular hyperosmotic stress.
    Discussion: Time lapse imaging shows that although larger iPAR aggregates associate with nuclear and vacuolar compartments, we show directly, for the first time, that these proteotoxic accumulations are not inherited by daughter cells, unlike nuclei and vacuoles. If suitably adapted, iPAR offers new potential for studying diseases relating to protein oligomerization processes in other model cellular systems.
    Supplementary Information: The online version contains supplementary material available at 10.1186/s44330-025-00023-w.
    Keywords:  Cell ageing; Confocal microscopy; Inheritance; Protein aggregation; Saccharomyces cerevisiae; Single-molecule
    DOI:  https://doi.org/10.1186/s44330-025-00023-w
  36. Biochem Soc Trans. 2025 Apr 03. pii: BST20253019. [Epub ahead of print]53(2):
    Insights into non-proteinaceous ubiquitination.
    Emily L Dearlove, Danny T Huang.
      Ubiquitination plays a key role in the regulation of numerous diverse cellular functions. This process involves the covalent attachment of ubiquitin to protein substrates by a cascade of enzymes. In recent years, various non-proteinaceous substrates of ubiquitination have been discovered, expanding the potential for the functions of ubiquitination beyond its conventional role as a post-translational modification. Here, we profile the non-proteinaceous substrates of ubiquitination reported to date, the enzymes that regulate these activities, and the mechanistic details of substrate modification. The biological functions linked to these modifications are discussed, and finally, we highlight the challenges hindering further progress in the identification and functional characterization of non-proteinaceous substrates of ubiquitination within cellular contexts.
    Keywords:  adenosine diphosphate ribose; carbohydrate; lipid; nucleic acids; ubiquitin
    DOI:  https://doi.org/10.1042/BST20253029
  37. mBio. 2025 Mar 31. e0022725
    Infection-relevant conditions dictate differential versus coordinate expression of Salmonella chaperones and cochaperones.
    Carissa Chan, Keiichiro Mukai, Eduardo A Groisman.
      Molecular chaperones are critical for protein homeostasis. In bacteria, chaperone trigger factor (TF) folds proteins co-translationally, and chaperone DnaK requires a J-domain cochaperone and nucleotide exchange factor GrpE to fold proteins largely post-translationally. However, when the pathogen Salmonella enterica serovar Typhimurium faces the infection-relevant condition of cytoplasmic Mg2+ starvation, DnaK reduces protein synthesis independently. This raises the possibility that bacteria differentially express chaperones and cochaperones. We now report that S. Typhimurium responds to cytoplasmic Mg2+ starvation by increasing mRNA amounts of dnaK while decreasing those of the TF-encoding gene tig and J-domain cochaperone genes dnaJ and djlA. This differential strategy requires the master regulator of Mg2+ homeostasis and virulence PhoP, which increases dnaK mRNA amounts by lowering the ATP concentration, thereby hindering proteolysis of the alternative sigma factor RpoH responsible for dnaK transcription. We also establish that DnaK exerts negative feedback on the RpoH protein and RpoH-dependent transcripts independently of J-domain cochaperones. Thus, bacteria express chaperones and cochaperones coordinately or differentially depending on the specific stress perturbing protein homeostasis.IMPORTANCEMolecular chaperones typically require cochaperones to fold proteins and to prevent protein aggregation, and the corresponding genes are thus coordinately expressed. We have now identified an infection-relevant stress condition in which the genes specifying chaperone DnaK and cochaperone DnaJ are differentially expressed despite belonging to the same operon. This differential strategy requires the master regulator of Mg2+ homeostasis and virulence in the pathogen Salmonella enterica serovar Typhimurium. Moreover, it likely reflects that Salmonella requires dnaK, but not J-domain cochaperone-encoding genes, for survival against cytoplasmic Mg2+ starvation and expresses genes only when needed. Thus, the specific condition impacting protein homeostasis determines the coordinate versus differential expression of molecular chaperones and cochaperones.
    Keywords:  DnaK; J-domain cochaperones; PhoP; RpoH; magnesium
    DOI:  https://doi.org/10.1128/mbio.00227-25
  38. J Cell Sci. 2025 Mar 31. pii: jcs.263788. [Epub ahead of print]
    Vps41 functions as a molecular ruler for HOPS tethering complex function.
    Caroline König, Dmitry Shvarev, Jieqiong Gao, Eduard Haar, Nicole Susan, Kathrin Auffarth, Lars Langemeyer, Arne Moeller, Christian Ungermann.
      Fusion at the lysosome (or the yeast vacuole) requires the conserved hexameric HOPS tethering complex. HOPS binds to the vacuolar Rab7-like GTPase Ypt7 via its subunits Vps41 and Vps39 and supports fusion by promoting SNARE assembly. In contrast to its sister complex CORVET, the Ypt7-interacting domain of Vps41 in the HOPS complex is connected to the core by a long, extended α-solenoid domain. Here, we show that this solenoid acts as a molecular ruler to position the Ypt7-interaction part of Vps41 relative to the core of HOPS to support function. Mutant complexes with a shortened or extended α-solenoid part in Vps41 still tether membranes, but fail to efficiently support their fusion. In vivo, Vps41 mutants grow poorly, show defects in vacuolar morphology, endolysosomal sorting and autophagy. Importantly, if a length-compensating linker is inserted instead of the shortened α-solenoid, these defects are rescued. This suggests that the Rab-specific Vps41 subunit requires the exact length, but not the α-solenoid domain for functionality, implying a revised model how HOPS supports fusion.
    Keywords:  HOPS; Rab GTPase; Vacuole; Vps41; Ypt7; α-solenoid
    DOI:  https://doi.org/10.1242/jcs.263788
  39. J Clin Invest. 2025 Apr 01. pii: e189197. [Epub ahead of print]135(7):
    Lysyl hydroxylase 2 glucosylates collagen VI to drive lung cancer progression.
    Shike Wang, Houfu Guo, Reo Fukushima, Masahiko Terajima, Min Liu, Guan-Yu Xiao, Lenka Koudelková, Chao Wu, Xin Liu, Jiang Yu, Emma Burris, Jun Xu, Alvise Schiavinato, William K Russell, Mitsuo Yamauchi, Xiaochao Tan, Jonathan M Kurie.
      Lysyl hydroxylase 2 (LH2) is highly expressed in multiple tumor types and accelerates disease progression by hydroxylating lysine residues on fibrillar collagen telopeptides to generate stable collagen cross links in tumor stroma. Here, we show that a galactosylhydroxylysyl glucosyltransferase (GGT) domain on LH2-modified type-VI collagen (Col6) to promote lung adenocarcinoma (LUAD) growth and metastasis. In tumors generated by LUAD cells lacking LH2 GGT domain activity, stroma was less stiff, and stable types of collagen cross links were reduced. Mass spectrometric analysis of total and glycosylated peptides in parental and GGT-inactive tumor samples identified Col6 chain α3 (Col6a3), a component of the Col6 heterotrimeric molecule, as a candidate LH2 substrate. In gain- and loss-of-function studies, high Col6a3 levels increased tumor growth and metastatic activity and enhanced the proliferative, migratory, and invasive activities of LUAD cells. LH2 coimmunoprecipitated with Col6a3, and LH2 glucosylated Col6 in an in vitro reaction. Glucosylation increased the integrin-binding and promigratory activities of Col6 in LUAD cells. Col6a3 K2049 was deglucosylated in GGT-inactive tumor samples, and mutagenesis of Col6a3 K2049 phenocopied Col6a3 deficiency or LH2 GGT domain inactivation in LUAD cells. Thus, LH2 glucosylates Col6 to drive LUAD progression. These findings show that the GGT domain of LH2 is protumorigenic, identify Col6 as a candidate effector, and provide a rationale to develop pharmacological strategies that target LH2's GGT domain in cancer cells.
    Keywords:  Cell biology; Collagens; Integrins; Lung cancer; Oncology
    DOI:  https://doi.org/10.1172/JCI189197
  40. Cell. 2025 Mar 26. pii: S0092-8674(25)00270-3. [Epub ahead of print]
    Cell membranes sustain phospholipid imbalance via cholesterol asymmetry.
    Milka Doktorova, Jessica L Symons, Xiaoxuan Zhang, Hong-Yin Wang, Jan Schlegel, Joseph H Lorent, Frederick A Heberle, Erdinc Sezgin, Edward Lyman, Kandice R Levental, Ilya Levental.
      Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PMs). Using lipidomics, we report that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared with exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization.
    Keywords:  cholesterol; lipid asymmetry; lipid diffusion; membrane packing; membrane structure; peripheral protein; permeability; phospholipid; plasma membrane; protein-membrane interactions
    DOI:  https://doi.org/10.1016/j.cell.2025.02.034
  41. Science. 2025 Apr 04. 388(6742): eads4539
    Exogenous RNA surveillance by proton-sensing TRIM25.
    Myeonghwan Kim, Youngjoon Pyo, Seong-In Hyun, Minseok Jeong, Yeon Choi, V Narry Kim.
      Exogenous messenger RNAs (mRNAs) require cellular machinery for delivery and translation but also encounter inhibitory factors. To investigate their regulation, we performed genome-wide CRISPR screens with in vitro-transcribed mRNAs in lipid nanoparticles (LNPs). Heparan sulfate proteoglycans (HSPGs) and vacuolar adenosine triphosphatase (V-ATPase) were identified as mediators of LNP uptake and endosomal escape, respectively. TRIM25-an RNA binding E3 ubiquitin ligase-emerged as a key suppressor inducing turnover of both linear and circular mRNAs. The endoribonucleases N4BP1 and KHNYN, along with the antiviral protein ZAP, act redundantly in TRIM25-dependent surveillance. TRIM25 specifically targets mRNAs delivered by endosomes, and its RNA affinity increases at acidic pH, suggesting activation by protons released from ruptured endosomes. N1-methylpseudouridine modification reduces TRIM25's RNA binding, helping RNAs evade its suppressive effect. This study comprehensively maps cellular pathways regulating LNP-mRNAs, offering insights into RNA immunity and therapeutics.
    DOI:  https://doi.org/10.1126/science.ads4539
  42. Cell Commun Signal. 2025 Apr 02. 23(1): 164
    Atovaquone-induced activation of the PERK/eIF2α signaling axis mitigates metabolic radiosensitisation.
    Jie Feng, Varun Pathak, Niall M Byrne, Sarah Chambers, Tongchuan Wang, Rayhanul Islam, Reinhold J Medina, Jonathan A Coulter.
       BACKGROUND: Hypoxia, a key feature of most solid tumours, including head and neck cancer, reduces radiotherapy efficacy by promoting radiation resistance through micro-environmental and genomic alterations. Addressing these resistance mechanisms is crucial, as radiotherapy remains central to managing locally advanced disease. Atovaquone, a mitochondrial electron transport chain complex III inhibitor, is reported to reduce tumour hypoxia in preclinical models, however, this response does not consistently enhance radiation sensitivity. This work examines the potential of atovaquone to modify the hypoxic response in models of head and neck squamous cell carcinoma (HNSCC), uncovering an adaptive resistance mechanism driven by integrated stress response (ISR) signaling that limits the radiosensitising potential of this approach.
    METHODS: The bioenergetic response of HNSCC cells to atovaquone was assessed using the Seahorse XFe96 Analyzer with the XF Cell Mito Stress Test. Radiation dose modifying effects of atovaquone were tested by clonogenic survival assays, while ROS yields were analysed by flow cytometry. Western blotting and quantitative reverse transcription-PCR were employed to study activation of ISR signaling and the overall influence of atovaquone on the hypoxic response. Finally, the role of the ISR activation in modulating radiosensitivity was investigated using both siRNA and pharmacological inhibition of eIF2α, a central regulator of the ISR.
    RESULTS: Herein we report that atovaquone significantly disrupts mitochondrial respiration, triggering phosphorylation of eIF2α, a pivotal regulator of the ISR, and a master regulator of protein synthesis. Notably, atovaquone also increased the autophagic load under hypoxia, while autophagy inhibition significantly enhanced apoptosis, improving radiation sensitivity. Combined eIF2α inhibition and atovaquone promotes cell cycle redistribution and significantly enhances mitochondrial ROS production and compared to atovaquone alone, restoring atovaquone mediated radiosensitisation.
    CONCLUSIONS: Our data highlight dual counter opposing impacts of atovaquone, serving as a hypoxic radiosensitiser though oxidative phosphorylation (OXPHOS) inhibition, but also in promoting stress induced ISR signaling, conferring resistance to radiation treatment. Importantly, if ISR activation is impeded, the metabolic radiosensitising properties of atovaquone is restored. These data provide a new insight to a molecular response that could help counteract hypoxia-induced radioresistance.
    Keywords:  Autophagy; ER stress; Hypoxia; Radiosensitisation
    DOI:  https://doi.org/10.1186/s12964-025-02160-9
  43. Redox Biol. 2025 Feb 19. pii: S2213-2317(25)00067-9. [Epub ahead of print]82 103554
    Redox regulation of lung endothelial PERK, unfolded protein response (UPR) and proliferation via NOX1: Targeted inhibition as a potential therapy for PAH.
    Christian J Goossen, Alex Kufner, Christopher M Dustin, Imad Al Ghouleh, Shuai Yuan, Adam C Straub, John Sembrat, Jeffrey J Baust, Delphine Gomez, Damir Kračun, Patrick J Pagano.
       AIMS: Reactive oxygen species (ROS) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH) and NADPH oxidases (NOXs) as sources of ROS are implicated in the development of the disease. We previously showed that NOX isozyme 1 (NOX1)-derived ROS contributes to pulmonary vascular endothelial cell (EC) proliferation in response to PAH triggers in vitro. However, whether and how NOX1 is involved in PAH in vivo have not been explored nor has NOX1 been examined as a viable and effective therapeutic disease target.
    METHODS AND RESULTS: Herein, infusion of mice exposed to Sugen/hypoxia (10 % O2) with a specific NOX1 inhibitor, NOXA1ds, delivered via osmotic minipumps (i.p.), significantly suppressed pathological changes in hemodynamic parameters characteristic of PAH. Furthermore, lungs of human patients with idiopathic PAH (iPAH) and exploratory RNA-seq analysis of hypoxic human pulmonary ECs, in which NOX1 was suppressed, were probed. The findings showed a clear indication of NOX1 in the promotion of both protein disulfide isomerase (PDI) and the unfolded protein response (UPR; in particular, the PERK arm of the pathway including eIF2α and ATF4) leading to proliferation. In aggregate, these results are consistent with a causal role for NOX1 in the development of mouse and human PAH and reveal a novel and mechanistic pathway by which NOX1 activates the UPR response during EC proliferation.
    CONCLUSION: NOX1 promotes phenotypic changes in ECs that are pivotal to proliferation and PAH through activation of the UPR. Taken together, our results are consistent with selective inhibition of NOX1 as a novel modality for attenuating PAH.
    Keywords:  Hypoxia; NADPH oxidases; NOX inhibitors; NOXA1ds; Pulmonary arterial hypertension; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.redox.2025.103554
  44. Science. 2025 Apr 04. 388(6742): 68-74
    Protein editing using a coordinated transposition reaction.
    Yi Hua, Nicholas E S Tay, Xuanjia Ye, Jeremy A Owen, Hengyuan Liu, Robert E Thompson, Tom W Muir.
      Protein engineering through the ligation of polypeptide fragments has proven enormously powerful for studying biochemical processes. In general, this strategy necessitates a final protein-folding step, constraining the types of systems amenable to the approach. Here, we report a method that allows internal regions of target proteins to be replaced in a single operation. Conceptually, our system is analogous to a DNA transposition reaction but uses orthogonal pairs of engineered split inteins to mediate the editing process. This "protein transposition" reaction is applied to several systems, including folded protein complexes, allowing the efficient introduction of a variety of noncoded elements. By carrying out a molecular "cut and paste" under native protein-folding conditions, our approach substantially expands the scope of protein semisynthesis.
    DOI:  https://doi.org/10.1126/science.adq8540
  45. Nat Commun. 2025 Apr 01. 16(1): 3117
    Endosomal chloride/proton exchangers need inhibitory TMEM9 β-subunits for regulation and prevention of disease-causing overactivity.
    Rosa Planells-Cases, Viktoriia Vorobeva, Sumanta Kar, Franziska W Schmitt, Uwe Schulte, Marina Schrecker, Richard K Hite, Bernd Fakler, Thomas J Jentsch.
      The function of endosomes critically depends on their ion homeostasis. A crucial role of luminal Cl-, in addition to that of H+, is increasingly recognized. Both ions are transported by five distinct endolysosomal CLC chloride/proton exchangers. Dysfunction of each of these transporters entails severe disease. Here we identified TMEM9 and TMEM9B as obligatory β-subunits for endosomal ClC-3, ClC-4, and ClC-5. Mice lacking both β-subunits displayed severely reduced levels of all three CLCs and died embryonically or shortly after birth. TMEM9 proteins regulate trafficking of their partners. Surprisingly, they also strongly inhibit CLC ion transport. Tonic inhibition enables the regulation of CLCs and prevents toxic Cl- accumulation and swelling of endosomes. Inhibition requires a carboxy-terminal TMEM9 domain that interacts with CLCs at multiple sites. Disease-causing CLCN mutations that weaken inhibition by TMEM9 proteins cause a pathogenic gain of ion transport. Our work reveals the need to suppress, in a regulated manner, endolysosomal chloride/proton exchange. Several aspects of endosomal ion transport must be revised.
    DOI:  https://doi.org/10.1038/s41467-025-58546-3
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