bims-proteo Biomed News
on Proteostasis
Issue of 2025–02–23
fifty papers selected by
Eric Chevet, INSERM
  1. Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress.
  2. TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the endoplasmic reticulum and into the Golgi.
  3. Native Fold Delay and its implications for co-translational chaperone binding and protein aggregation.
  4. Identification of actionable targeted protein degradation effector sites through Site-specific Ligand Incorporation-induced Proximity (SLIP).
  5. COMET enables direct screening for interactions between E3 ubiquitin ligases and their proteolytic target proteins.
  6. Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.
  7. The integrated stress response engages a cell-autonomous, ligand-independent, DR5-driven apoptosis switch.
  8. The ribosome as a platform to coordinate mRNA decay.
  9. Yeast TIA1 coordinates with Npl3 to promote ATG1 translation during starvation.
  10. Calcium release from damaged lysosomes triggers stress granule formation for cell survival.
  11. 7SL RNA and signal recognition particle orchestrate a global cellular response to acute thermal stress.
  12. Mapping the GDF15 Arm of the Integrated Stress Response in Human Cells and Tissues.
  13. Lysosomal Quality Control.
  14. Physiological Insights into ESCRT-Mediated Phagophore Closure: Potential Cytoprotective Roles for ATG8ylated Membranes.
  15. Small heat shock protein HSPB8 interacts with a pre-fibrillar TDP43 low complexity domain species to delay fibril formation.
  16. A general framework to analyze potential roles of tDRs in mammalian protein synthesis.
  17. Proteasome Inhibition Enhances Lysosome-mediated Targeted Protein Degradation.
  18. Cullin-RING ligase BioE3 reveals molecular-glue-induced neosubstrates and rewiring of the endogenous Cereblon ubiquitome.
  19. EndoMAP.v1, a Structural Protein Complex Landscape of Human Endosomes.
  20. A distant TANGO1 family member promotes vitellogenin export from the ER in C. elegans.
  21. Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants.
  22. Trafficking of K63-polyubiquitin modified membrane proteins in a macroautophagy-independent pathway is linked to ATG9A.
  23. SUMO2/3 conjugation of TDP-43 protects against aggregation.
  24. SecY translocon chaperones protein folding during membrane protein insertion.
  25. The core morning clock component CCA1 positively regulates the expression of UPR target genes for ER stress recovery.
  26. STING-induced noncanonical autophagy regulates endolysosomal homeostasis.
  27. PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection.
  28. Cell enlargement modulated by GATA4 and YAP instructs the senescence-associated secretory phenotype.
  29. Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase.
  30. Molecular mechanisms and pathological implications of unconventional protein secretion in human disease: from cellular stress to therapeutic targeting.
  31. A KIF1C-CNBP motor-adaptor complex for trafficking mRNAs to cell protrusions.
  32. AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack.
  33. Ribosome-associated tDRs in yeast.
  34. Molecular determinants of RNase MRP specificity and function.
  35. A privileged ER compartment for post-translational heteromeric assembly of an ion channel.
  36. Internal cap-initiated translation for efficient protein production from circular mRNA.
  37. Stress granules as transient reservoirs for autophagy proteins: a key mechanism for plant recovery from heat stress.
  38. The DYT6 dystonia causative protein THAP1 is responsible for proteasome activity via PSMB5 transcriptional regulation.
  39. Short internal open reading frames repress the translation of N-terminally truncated proteoforms.
  40. Proximal partners of the organellar N-terminal acetyltransferase NAA60: insights into Golgi structure and transmembrane protein topology.
  41. Degradation of circular RNA by the ribonuclease DIS3.
  42. RAB-10 cooperates with EHBP-1 to capture vesicular carriers during post-Golgi exocytic trafficking.
  43. Tracking proteasome degradation: A cross-organ analysis via intact degradomics mass spectrometry.
  44. De novo design of transmembrane fluorescence-activating proteins.
  45. Altered relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise.
  46. Deep-tissue transcriptomics and subcellular imaging at high spatial resolution.
  47. A structural atlas of death domain fold proteins reveals their versatile roles in biology and function.
  48. Clozapine as an E3 Ligand for PROTAC Technology.
  49. Organelle homeostasis requires ESCRTs.
  50. Nucleic-acid-induced ZCCHC3 condensation promotes broad innate immune responses.
  1. bioRxiv. 2025 Feb 05. pii: 2024.05.02.592277. [Epub ahead of print]
    Context specific ubiquitin modification of ribosomes regulates translation under oxidative stress.
    Shannon E Dougherty, Géssica C Barros, Matthew W Foster, Guoshou Teo, Hyungwon Choi, Gustavo M Silva.
      Cellular exposure to stress is known to activate several translational control pathways through ribosome ubiquitination. However, how unique patterns of ribosome ubiquitination act at the site-specific level to drive distinct modes of translation regulation remains unclear. To further understand the complexity of these ubiquitin signals, we developed a new targeted proteomics approach to quantify site-specific ubiquitin modification across the ribosome. This method increased the sensitivity and throughput of current approaches and allowed us to systematically measure the ubiquitin status of 78 ribosome peptides and ubiquitin linkages in response to stress. Using this method, we were able to detect the ubiquitination of several ribosome sites even in steady-state conditions, and to show that their modification increases non-stoichiometrically in a dynamic range of >4 orders of magnitude in response to hydrogen peroxide. Besides demonstrating new patterns of global ribosome ubiquitination, our study also revealed an unexpected increase of ubiquitination of ribosomal protein uS10/Rps20 and uS3/Rps3 independent of the canonical E3 ubiquitin ligase Hel2. Furthermore, we show that unique and mixed patterns of ribosome ubiquitination occur in a stress specific manner, depending on the nature of stressor and the enzymes involved. Finally, we showed that while deletion of HEL2 further induces the integrated stress response in response to the nucleotide alkylating agent 4-NQO, deletion of the E2 conjugase RAD6 leads to sustained translation only in response to H 2 O 2 . Our findings contribute to deciphering the complexity of the stress response at the translational level, revealing the induction of dynamic and selective ubiquitin codes, which shed light on the integration of important quality control pathways during cellular response to stress.
    DOI:  https://doi.org/10.1101/2024.05.02.592277
  2. bioRxiv. 2025 Feb 04. pii: 2024.09.27.615420. [Epub ahead of print]
    TMED9 coordinates the clearance of misfolded GPI-anchored proteins out of the endoplasmic reticulum and into the Golgi.
    Elsa Ronzier, Prasanna Satpute-Krishnan.
      The p24-family member, TMED9, has recently emerged as a player in secretory pathway protein quality control (PQC) that influences the trafficking and degradation of misfolded proteins. Here we show that TMED9 plays a central role in the PQC of GPI-anchored proteins (GPI-APs). Typically, upon release from the endoplasmic reticulum (ER)-resident chaperone calnexin, misfolded GPI-APs traffic to the Golgi by an ER-export pathway called Rapid ER stress-induced Export (RESET). From the Golgi, they access the plasma membrane where they are rapidly internalized for lysosomal degradation. We used biochemical and imaging approaches in cultured cells to demonstrate that at steady-state, the majority of misfolded GPI-APs reside in the ER in association with calnexin and TMED9. During RESET, they dissociate from calnexin and increase their association with TMED9. Inhibition of TMED9's function through siRNA-induced depletion or chemical inhibitor, BRD4780, blocked ER-export of misfolded GPI-APs. By contrast, TMED9-inhibition did not prevent ER-export of wild type GPI-APs, indicating a specific role for TMED9 in GPI-AP PQC. Intriguingly, we discovered that acute treatment with BRD4780 induced a shift in TMED9 localization away from the ER to the downstream Golgi cisternae and blocked the RESET pathway. Upon removal of BRD4780 following acute treatment, TMED9 regained access to the ER where TMED9 was able to associate with the RESET substrate and restore the RESET pathway. These results suggest that TMED9 plays a requisite role in RESET by capturing misfolded GPI-APs that are released by calnexin within the ER and conveying them to the Golgi.
    DOI:  https://doi.org/10.1101/2024.09.27.615420
  3. Nat Commun. 2025 Feb 15. 16(1): 1673
    Native Fold Delay and its implications for co-translational chaperone binding and protein aggregation.
    Ramon Duran-Romaña, Bert Houben, Paula Fernández Migens, Ying Zhang, Frederic Rousseau, Joost Schymkowitz.
      Because of vectorial protein translation, residues that interact in the native protein structure but are distantly separated in the primary sequence are unavailable simultaneously. Instead, there is a temporal delay during which the N-terminal interaction partner is unsatisfied and potentially vulnerable to non-native interactions. We introduce "Native Fold Delay" (NFD), a metric that integrates protein topology with translation kinetics to quantify such delays. We found that many proteins exhibit residues with NFDs in the range of tens of seconds. These residues, predominantly in well-structured, buried regions, often coincide with aggregation-prone regions. NFD correlates with co-translational engagement by the yeast Hsp70 chaperone Ssb, suggesting that native fold-delayed regions have a propensity to misfold. Supporting this, we show that proteins with long NFDs are more frequently co-translationally ubiquitinated and prone to aggregate upon Ssb deletion.
    DOI:  https://doi.org/10.1038/s41467-025-57033-z
  4. bioRxiv. 2025 Feb 04. pii: 2025.02.04.636303. [Epub ahead of print]
    Identification of actionable targeted protein degradation effector sites through Site-specific Ligand Incorporation-induced Proximity (SLIP).
    Zhangping Xiao, Efthymios Spyridon Gavriil, Fangyuan Cao, Xinyue Zhang, Stan Xiaogang Li, Sergei Kotelnikov, Patrycja Michalska, Friederike Marte, Chloe Huang, Yudi Lu, Yunxuan Zhang, Erika Bernardini, Dima Kozakov, Edward W Tate.
      Targeted protein degradation (TPD) is a rapidly emerging and potentially transformative therapeutic modality. However, the large majority of >600 known ubiquitin ligases have yet to be exploited as TPD effectors by proteolysis-targeting chimeras (PROTACs) or molecular glue degraders (MGDs). We report here a chemical-genetic platform, Site-specific Ligand Incorporation-induced Proximity (SLIP), to identify actionable ("PROTACable") sites on any potential effector protein in intact cells. SLIP uses genetic code expansion (GCE) to encode copper-free "click" ligation at a specific effector site in intact cells, enabling in situ formation of a covalent PROTAC-effector conjugate against a target protein of interest (POI). Modification at actionable effector sites drives degradation of the targeted protein, establishing the potential of these sites for TPD. Using SLIP, we systematically screened dozens of sites across E3 ligases and E2 enzymes from diverse classes, identifying multiple novel potentially PROTACable effector sites which are competent for TPD. SLIP adds a powerful approach to the proximity-induced pharmacology (PIP) toolbox, enabling future effector ligand discovery to fully enable TPD, and other emerging PIP modalities.
    DOI:  https://doi.org/10.1101/2025.02.04.636303
  5. Mol Cell. 2025 Feb 20. pii: S1097-2765(25)00062-0. [Epub ahead of print]85(4): 671-673
    COMET enables direct screening for interactions between E3 ubiquitin ligases and their proteolytic target proteins.
    Kenneth Wu, Zhen-Qiang Pan.
      In this issue of Molecular Cell, Sulter et al.1 describe a high-throughput method named COMET (combinatorial mapping of E3 targets) that enables direct screening for interactions between E3 ubiquitin ligases and their proteolytic substrate proteins.
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.028
  6. Brain. 2025 Feb 21. pii: awae407. [Epub ahead of print]
    Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.
    Thierry Touvier, Francesca A Veneri, Anke Claessens, Cinzia Ferri, Rosa Mastrangelo, Noémie Sorgiati, Francesca Bianchi, Serena Valenzano, Ubaldo Del Carro, Cristina Rivellini, Phu Duong, Michael E Shy, Jeffery W Kelly, John Svaren, R Luke Wiseman, Maurizio D'Antonio.
      Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 (Xbp1s). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.
    Keywords:  Charcot-Marie-Tooth; Schwann cell; XBP1; demyelinating neuropathy; proteostasis; unfolded protein response
    DOI:  https://doi.org/10.1093/brain/awae407
  7. Cell Death Dis. 2025 Feb 15. 16(1): 101
    The integrated stress response engages a cell-autonomous, ligand-independent, DR5-driven apoptosis switch.
    Francesca Zappa, Nerea L Muniozguren, Julia E Conrad, Diego Acosta-Alvear.
      The integrated stress response (ISR) is a fundamental signaling network that leverages the cell's biosynthetic capacity against different stresses to restore homeostasis. However, when homeostasis is unattainable, the ISR switches to drive cell death and eliminate irreparably damaged cells. Previous work has shown that persistent activity of the ISR kinase PERK during unyielding endoplasmic reticulum (ER) stress induces apoptosis downstream of death receptor 5 (DR5) [1]. ER stress provides activating signals that engage the ectodomain (ED) of DR5 to drive its unconventional activation in the Golgi apparatus [1, 2]. Here, using chemical genetics to uncouple stress sensing from ISR activation, we found that DR5 signaling from the Golgi apparatus is integral to the ISR and not specific to ER stress. Furthermore, we show that DR5 activation can be driven solely by increased expression and does not require its ED. These findings indicate that a general ISR kill switch eliminates irreversibly injured cells.
    DOI:  https://doi.org/10.1038/s41419-025-07403-8
  8. Nucleic Acids Res. 2025 Feb 08. pii: gkaf049. [Epub ahead of print]53(4):
    The ribosome as a platform to coordinate mRNA decay.
    Martin B D Müller, Thomas Becker, Timo Denk, Satoshi Hashimoto, Toshifumi Inada, Roland Beckmann.
      Messenger RNA (mRNA) homeostasis is a critical aspect of cellular function, involving the dynamic interplay between transcription and decay processes. Recent advances have revealed that the ribosome plays a central role in coordinating mRNA decay, challenging the traditional view that free mRNA is the primary substrate for degradation. This review examines the mechanisms whereby ribosomes facilitate both the licensing and execution of mRNA decay. This involves factors such as the Ccr4-Not complex, small MutS-related domain endonucleases, and various quality control pathways. We discuss how translational fidelity, as well as the presence of nonoptimal codons and ribosome collisions, can trigger decay pathways such as nonstop decay and no-go decay. Furthermore, we highlight the direct association of canonical exonucleases, such as Xrn1 and the Ski-exosome system, with the ribosome, underscoring the ribosome's multifaceted role as a platform for regulatory processes governing mRNA stability. By integrating recent findings, this review offers a comprehensive overview of the structural basis of how ribosomes not only facilitate translation but also serve as critical hubs for mRNA decay coordination.
    DOI:  https://doi.org/10.1093/nar/gkaf049
  9. Cell Rep. 2025 Feb 14. pii: S2211-1247(25)00087-7. [Epub ahead of print]44(2): 115316
    Yeast TIA1 coordinates with Npl3 to promote ATG1 translation during starvation.
    Shree Padma Metur, Xinxin Song, Sophie Mehta, Dimitra Dialynaki, Dibyendu Bhattacharyya, Zhangyuan Yin, Daolin Tang, Daniel J Klionsky.
      Macroautophagy/autophagy is crucial for cell survival during nutrient starvation. Autophagy requires the coordinated function of several Atg proteins, including the Atg1 kinase, for efficient induction and execution. Recently, several RNA-binding proteins (RBPs) have been shown to post-transcriptionally regulate ATG1. However, a comprehensive understanding of autophagy regulation by RBPs via ATG1 is yet to be elucidated. Here, we utilize an in vitro approach to identify RBPs that specifically interact with ATG1 untranslated regions. We show that Npl3 and Pub1 interact with the ATG1 5' and 3' untranslated regions during nitrogen starvation. Furthermore, Npl3 and Pub1 coordinate to facilitate ATG1 mRNA export to the cytoplasm and its subsequent interaction with the translational machinery. Significantly, in non-small cell lung cancer cell lines, mammalian Pub1, TIA1, also positively regulates ULK1 protein expression and autophagy during serum starvation. Overall, our study highlights the regulatory landscape that fine-tunes Atg1 protein expression to sustain autophagy during nutrient starvation.
    Keywords:  CP: Cell biology; CP: Molecular biology; RBPs; autophagy; mRNA metabolism; macroautophagy; mammalian cells; post-transcriptional regulation; stress; translation; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2025.115316
  10. Autophagy. 2025 Feb 17.
    Calcium release from damaged lysosomes triggers stress granule formation for cell survival.
    Aravinth Kumar Jayabalan, Aanuoluwakiitan Ayeni, Jingyue Jia.
      Lysosomes are essential membrane-bound organelles that integrate intracellular needs and external signals through multiple functions, including autophagy-mediated degradation and MTORC1 signaling. The integrity of the lysosomal membrane is therefore crucial for maintaining cellular homeostasis. Various endogenous and exogenous factors can damage lysosomes, contributing to diseases such as infections, cancer, and neurodegeneration. In response, cells mount defensive mechanisms to cope with such stress, including the formation of stress granules (SGs) - membraneless organelles composed of RNAs and protein complexes. While SGs have emerged as key players in repairing damaged lysosomes, how lysosomal damage triggers their formation and influences cell fate remains unclear. Here we report that the calcium signal from damaged lysosomes mediates SG formation and protects cells from lysosomal damage-induced cell death. Mechanistically, calcium leakage from damaged lysosomes signals the recruitment of calcium-activating protein PDCD6IP/ALIX and its partner PDCD6/ALG2. This complex recruits protein kinase EIF2AK2/PKR and its activator PRKRA/PACT, which phosphorylates translation initiator factor EIF2S1, stalling global translation initiation. This translation arrest leads to the accumulation of inactive messenger ribonucleoprotein complexes (mRNPs), resulting in SG formation. Cells deficient in SG formation show increased cell death when exposed to lysosomal damage from disease-associated factors including SARS-CoV-2ORF3a, adenovirus, malarial pigment, proteopathic MAPT/tau, or environmental hazards. Collectively, this study reveals how damaged lysosomes signal through calcium to trigger SG assembly, promoting cell survival. This establishes a novel link between membrane-bound and membraneless organelles, with implications for diseases involving lysosomal damage and SG dysfunction.
    Keywords:  Calcium signaling; cell survival; lysosomal damage; stress granules
    DOI:  https://doi.org/10.1080/15548627.2025.2468910
  11. Nat Commun. 2025 Feb 14. 16(1): 1630
    7SL RNA and signal recognition particle orchestrate a global cellular response to acute thermal stress.
    Bojan Bujisic, Hun-Goo Lee, Lilei Xu, Uri Weissbein, Carlos Rivera, Ivan Topisirovic, Jeannie T Lee.
      Non-coding 7SL RNA is an ancestor to mammalian Alu and B1 SINE RNAs and is thought to function exclusively within the Signal Recognition Particle (SRP), aiding in the translocation of secretory proteins into the endoplasmic reticulum for export. Here, we discover a function of 7SL/SRP unrelated to protein secretion. Under acute heat shock, 7SL and SRP together selectively arrest cellular transcription and translation machineries during early response to stress. Under thermal stress, 7SL is upregulated, accumulates in the nucleus, and binds to target genes repressed by heat shock. Concurrently, in the cytosol, SRP binds to ribosomes and inhibits new protein synthesis. Translational suppression occurs independently of the signal peptide and is abrogated by depleting SRP. Translation inhibition extends to the mitochondria, as nuclear-encoded genes with mitochondrial functions are enriched among SRP targets. Thus, apart from its role in protein export, 7SL/SRP orchestrates a global response to acute stress that encompasses the nucleus, cytosol, and mitochondria across transcription and translation.
    DOI:  https://doi.org/10.1038/s41467-025-56351-6
  12. bioRxiv. 2025 Feb 01. pii: 2025.01.31.635929. [Epub ahead of print]
    Mapping the GDF15 Arm of the Integrated Stress Response in Human Cells and Tissues.
    Janell Lm Smith, Kamaryn Tanner, Jack Devine, Anna S Monzel, Alan A Cohen, Martin Picard.
      Mitochondrial stress activates the integrated stress response (ISR) and triggers cell-cell communication through secretion of the metabokine growth differentiation factor 15 (GDF15). However, the gene network underlying the ISR remains poorly defined, particularly across metabolically diverse cellular states and tissues. Using RNAseq data from fibroblasts subjected to metabolic perturbations, we develop an ISR GDF15 index quantifying the GDF15 arm of the ISR activation in human cells. Validation of ISR GDF15 index across 44 postmortem human tissues illustrates how this index can be applied to investigate tissue-specific and age-related ISR activation.
    DOI:  https://doi.org/10.1101/2025.01.31.635929
  13. Autophagy. 2025 Feb 19.
    Lysosomal Quality Control.
    Danielle Henn, Xi Yang, Ming Li.
      Healthy cells need functional lysosomes to degrade cargo delivered by autophagy and endocytosis. Defective lysosomes can lead to severe conditions such as lysosomal storage diseases (LSDs) and neurodegeneration. To maintain lysosome integrity and functionality, cells have evolved multiple quality control pathways corresponding to different types of stress and damage. These can be divided into five levels: regulation, reformation, repair, removal, and replacement. The different levels of lysosome quality control often work together to maintain the integrity of the lysosomal network. This review summarizes the different quality control pathways and discusses the less-studied area of lysosome membrane protein regulation and degradation, highlighting key unanswered questions in the field.
    Keywords:  ESCRT; Lysophagy; lysosome membrane protein regulation; lysosome membrane repair; lysosome quality control
    DOI:  https://doi.org/10.1080/15548627.2025.2469206
  14. Autophagy. 2025 Feb 20.
    Physiological Insights into ESCRT-Mediated Phagophore Closure: Potential Cytoprotective Roles for ATG8ylated Membranes.
    Kouta Hamamoto, Xinwen Liang, David M Opozda, Hong-Gang Wang, Yoshinori Takahashi.
      The endosomal sorting complex required for transport (ESCRT) machinery is a membrane abscission system that mediates various intracellular membrane remodeling processes, including macroautophagy/autophagy. In our recent study, we established the unique requirement of the ubiquitin E2 variant-like (UEVL) domain of the ESCRT-I subunit VPS37A in phagophore closure, the final step in autophagosome biogenesis, and determined the physiological impact of systemically inhibiting closure by targeting this region in mice. While the mutant mice exhibited phenotypes similar to those reported in mice deficient in generating ATG8 (mammalian Atg8 homologs)-conjugated (ATG8ylated) phagophores, certain phenotypes, such as neonatal lethality and liver injury, were found to be notably milder. Further investigation revealed that ATG8ylated phagophores promote TBK1-dependent SQSTM1 phosphorylation and droplet formation, leading to the formation of large insoluble aggregates upon closure inhibition. These findings suggest potential roles for ATG8ylated membranes in mitigating proteotoxicity by efficiently concentrating and sequestering soluble, reactive microaggregates and converting them into less reactive, insoluble large aggregates. The study highlights VPS37A UEVL mutant mice as a model for investigating the physiological and pathological roles of phagophores that extend beyond degradation.
    Keywords:  ATG8ylation; ESCRT; SQSTM1; TBK1; VPS37A UEVL mutant mouse; phagophore closure
    DOI:  https://doi.org/10.1080/15548627.2025.2468907
  15. bioRxiv. 2025 Jan 30. pii: 2025.01.28.635368. [Epub ahead of print]
    Small heat shock protein HSPB8 interacts with a pre-fibrillar TDP43 low complexity domain species to delay fibril formation.
    Khaled M Jami, Daniel C Farb, Kayla M Osumi, Catelynn C Shafer, Sophie Criscione, Dylan T Murray.
      The loss of cellular proteostasis through aberrant stress granule formation is implicated in neurodegenerative diseases. Stress granules are formed by biomolecular condensation involving protein-protein and protein-RNA interactions. These assemblies are protective, but can rigidify, leading to amyloid-like fibril formation, a hallmark of the disease pathology. Key proteins dictating stress granule formation and disassembly, such as TDP43, contain low-complexity (LC) domains that drive fibril formation. HSPB8, a small heat shock protein, plays a critical role modulating stress granule fluidity, preventing aggregation and promoting degradation of misfolded proteins. We examined the interaction between HSPB8 and the TDP43 LC using thioflavin T (ThT) and fluorescence polarization (FP) aggregation assays, fluorescence microscopy and photobleaching experiments, and crosslinking mass spectrometry (XL-MS). Our results indicate that HSPB8 delays TDP43 LC aggregation through domain-specific interactions with fibril nucleating species, without affecting fibril elongation rates. These findings provide mechanistic insight into how ATP-independent chaperones mediate LC domain aggregation and provide a basis for investigating how the TDP43 LC subverts chaperone activity in neurodegenerative disease.
    Significance Statement: ATP-independent chaperones facilitate clearance of aggregated proteins through autophagy. This study provides insight into the molecular mechanism by which small heat shock proteins interact with the aggregation-prone low complexity protein domains of RNA-binding proteins linked to neurodegenerative disease pathologies. The results provide a foundation for designing improved chaperones as therapeutics and illustrate a methodology to identify regions in low complexity domains for targeted drug development in the context of neurodegenerative disease.
    DOI:  https://doi.org/10.1101/2025.01.28.635368
  16. Methods Enzymol. 2025 ;pii: S0076-6879(24)00585-8. [Epub ahead of print]711 29-46
    A general framework to analyze potential roles of tDRs in mammalian protein synthesis.
    Nupur Bhatter, Pavel Ivanov.
      tRNA-derived RNAs (tDRs) are a heterogeneous class of small non-coding RNAs that have been implicated in numerous biological processes including the regulation of mRNA translation. A subclass of tDRs called tRNA-derived stress-induced RNAs (tiRNAs) have been shown to participate in translational control under stress where specific tiRNAs repress protein synthesis. Here, we use a prototypical tiRNA (5'-tiRNAAla) that inhibits mRNA translation in vitro and in cells as a model to study potential roles of tDRs in translational control. Specifically, we propose to use commercially available and custom-made in vitro translation systems together with sensitive luciferase-based mRNA reporters as well as transfection studies to determine potential effects of a given tDR on various aspects of protein synthesis. We overview methods to probe the capacity of specific tDRs to target specific steps of mRNA translation initiation, the most regulated step in translational control. Using 5'-tiRNAAla as an example, we analyze its effects on the integrity of the m7GTP (cap)-bound eIF4F complex and phosphorylation of eIF2α, the key regulatory molecule of the Integrated Stress Response. Using transfection studies, we also monitor whether tDRs can promote formation of stress granules (SGs), RNA granules are often formed in response to global translation repression in live cells. This simple workflow offers fast, scalable, and reliable analyses of a potential involvement of specific tDRs in the modulation of protein synthesis and provides initial hints on molecular mechanisms that underline such mRNA translation regulation.
    Keywords:  EIF2α phosphorylation; Protein synthesis; Stress granules; TiRNAs; Transfer RNA; Transfer RNA-derived RNAs (tDRs); in vitro translation assay
    DOI:  https://doi.org/10.1016/bs.mie.2024.11.018
  17. bioRxiv. 2025 Feb 01. pii: 2025.01.31.634950. [Epub ahead of print]
    Proteasome Inhibition Enhances Lysosome-mediated Targeted Protein Degradation.
    Ahmed M Elshazly, Nayyerehalsadat Hosseini, Shanwei Shen, Victoria Neely, Hisashi Harada, Steven Grant, Senthil K Radhakrishnan.
      Proteasome inhibitor drugs are currently used in the clinic to treat multiple myeloma and mantle cell lymphoma. These inhibitors cause accumulation of undegraded proteins, thus inducing proteotoxic stress and consequent cell death. However, cancer cells counteract this effect by activating an adaptive response through the transcription factor Nuclear factor erythroid 2-related factor 1 (NRF1, also known as NFE2L1). NRF1 induces transcriptional upregulation of proteasome and autophagy/lysosomal genes, thereby reducing proteotoxic stress and diminishing the effectiveness of proteasome inhibition. While suppressing this protective autophagy response is one potential strategy, here we investigated whether this heightened autophagy could instead be leveraged therapeutically. To this end, we designed an autophagy-targeting chimera (AUTAC) compound to selectively degrade the anti-apoptotic protein Mcl1 via the lysosome. Our results show that this lysosome-mediated targeted degradation is significantly amplified in the presence of proteasome inhibition, in a NRF1-dependent manner. The combination of the proteasome inhibitor carfilzomib and Mcl1 AUTAC synergistically promoted cell death in both wild-type and proteasome inhibitor-resistant multiple myeloma and lung cancer cells. Thus, our work offers a novel strategy for enhancing proteasome inhibitor efficacy by exploiting the adaptive autophagy response. More broadly, our study establishes a framework for amplifying lysosome-mediated targeted protein degradation, with potential applications in cancer therapeutics and beyond.
    DOI:  https://doi.org/10.1101/2025.01.31.634950
  18. Cell Commun Signal. 2025 Feb 19. 23(1): 101
    Cullin-RING ligase BioE3 reveals molecular-glue-induced neosubstrates and rewiring of the endogenous Cereblon ubiquitome.
    Laura Merino-Cacho, Orhi Barroso-Gomila, Mónica Pozo-Rodríguez, Veronica Muratore, Claudia Guinea-Pérez, Álvaro Serrano, Coralia Pérez, Sandra Cano-López, Ainhoa Urcullu, Mikel Azkargorta, Ibon Iloro, Carles Galdeano, Jordi Juárez-Jiménez, Ugo Mayor, Felix Elortza, Rosa Barrio, James D Sutherland.
       BACKGROUND: The specificity of the ubiquitination process is mediated by the E3 ligases. Discriminating genuine substrates of E3s from mere interacting proteins is one of the major challenges in the field. We previously developed BioE3, a biotin-based approach that uses BirA-E3 fusions together with ubiquitin fused to a low-affinity AviTag to obtain a site-specific and proximity-dependent biotinylation of the substrates. We proved the suitability of BioE3 to identify targets of RING and HECT-type E3 ligases.
    METHODS: BioE3 experiments were performed in HEK293FT and U2OS stable cell lines expressing TRIPZ-bioGEFUb transiently transfected with BirA-cereblon (CRBN). Cells were seeded using biotin-free media, followed later by a short-biotin pulse. We evaluated the applicability of the BioE3 system to CRBN and molecular glues by Western blot and confocal microscopy, blocking the proteasome with bortezomib, inhibiting NEDDylation with MLN4924 and treating the cells with pomalidomide. For the identification of endogenous substrates and neosubstrates we analyzed the eluates of streptavidin pull-downs of BioE3 experiments by LC-MS/MS. Analysis of targets for which ubiquitination changes significantly upon treatment was done using two-sided Student's t-test. Orthogonal validations were performed by histidine pull-down, GFP-trap and computational modelling.
    RESULTS: Here we demonstrate that BioE3 is suitable for the multi-protein complex Cullin-RING E3s ligases (CRLs), the most utilized E3-type for targeted protein degradation (TPD) strategies. Using CRBN as proof of concept, one of the substrate receptors of CRL4 E3 ligase, we identified both endogenous substrates and novel neosubstrates upon pomalidomide treatment, including CSDE1 which contains a G-loop motif potentially involved in the binding to CRBN in presence of pomalidomide. Importantly, we observed a major rearrangement of the endogenous ubiquitination landscape upon treatment with this molecular glue.
    CONCLUSIONS: The ability of BioE3 to detect and compare both substrates and neosubstrates, as well as how substrates change in response to treatments, will facilitate both on-target and off-target identifications and offer a broader characterization and validation of TPD compounds, like molecular glues and PROTACs.
    Keywords:  E3 ligases; Immunomodulatory drugs; Molecular Glue; Targeted Protein Degradation; Ubiquitin
    DOI:  https://doi.org/10.1186/s12964-025-02091-5
  19. bioRxiv. 2025 Feb 09. pii: 2025.02.07.636106. [Epub ahead of print]
    EndoMAP.v1, a Structural Protein Complex Landscape of Human Endosomes.
    Miguel A Gonzalez-Lozano, Ernst W Schmid, Enya Miguel Whelan, Yizhi Jiang, Joao A Paulo, Johannes C Walter, J Wade Harper.
      Early/sorting endosomes are dynamic organelles that play key roles in proteome control by triaging plasma membrane proteins for either recycling or degradation in the lysosome 1,2,3 . These events are coordinated by numerous transiently-associated regulatory complexes and integral membrane components that contribute to organelle identity during endosome maturation 4 . While a subset of the several hundred protein components and cargoes known to associate with endosomes have been studied at the biochemical and/or structural level, interaction partners and higher order molecular assemblies for many endosomal components remain unknown. Here, we combine cross-linking and native gel mass spectrometry 5-8 of purified early endosomes with AlphaFold 9,10 and computational analysis to create a systematic human endosomal structural interactome. We present dozens of structural models for endosomal protein pairs and higher order assemblies supported by experimental cross-links from their native subcellular context, suggesting structural mechanisms for previously reported regulatory processes. Using induced neurons, we validate two candidate complexes whose interactions are supported by crosslinks and structural predictions: TMEM230 as a subunit of ATP8/11 lipid flippases 11 and TMEM9/9B as subunits of CLCN3/4/5 chloride-proton antiporters 12 . This resource and its accompanying structural network viewer provide an experimental framework for understanding organellar structural interactomes and large-scale validation of structural predictions.
    DOI:  https://doi.org/10.1101/2025.02.07.636106
  20. iScience. 2025 Feb 21. 28(2): 111860
    A distant TANGO1 family member promotes vitellogenin export from the ER in C. elegans.
    Jimmy H Mo, Chao Zhai, Kwangsek Jung, Yan Li, Yonghong Yan, Meng-Qiu Dong, Ho Yi Mak.
      Vitellogenin is thought to share a common ancestor with human apolipoprotein B (ApoB) for systemic lipid transport. In Caenorhabditis elegans, although a general route for inter-tissue vitellogenin transport has been described, the full mechanism that underlies its intracellular trafficking within the intestine remains obscure. In humans, the TANGO1 family of proteins generates membrane carriers to accommodate bulky ApoB-containing lipoprotein particles for their endoplasmic reticulum (ER) export. TANGO1 orthologs have hitherto been discovered in most metazoans, except nematodes. Here, we report the C. elegans TNGL-1 as a mediator of vitellogenin export from the ER. Depletion of TNGL-1 causes the retention of vitellogenin in the ER lumen. Furthermore, the TNGL-1 C-terminal unstructured domain and its luminal globular domain are required for its proper localization and cargo engagement, respectively. Our findings support TNGL-1 as a distant TANGO1 family member and point to the universal requirement of TANGO1-based mechanisms for the secretion of specific metazoan proteins.
    Keywords:  cell biology; functional aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2025.111860
  21. Cell. 2025 Feb 20. pii: S0092-8674(24)01376-X. [Epub ahead of print]188(4): 958-977.e23
    Plasmodesmata act as unconventional membrane contact sites regulating intercellular molecular exchange in plants.
    Jessica Pérez-Sancho, Marija Smokvarska, Gwennogan Dubois, Marie Glavier, Sujith Sritharan, Tatiana S Moraes, Hortense Moreau, Victor Dietrich, Matthieu P Platre, Andrea Paterlini, Ziqiang P Li, Laetitia Fouillen, Magali S Grison, Pepe Cana-Quijada, Françoise Immel, Valerie Wattelet, Mathieu Ducros, Lysiane Brocard, Clément Chambaud, Yongming Luo, Priya Ramakrishna, Vincent Bayle, Linnka Lefebvre-Legendre, Stéphane Claverol, Matej Zabrady, Pascal G P Martin, Wolfgang Busch, Marie Barberon, Jens Tilsner, Yrjö Helariutta, Eugenia Russinova, Antoine Taly, Yvon Jaillais, Emmanuelle M Bayer.
      Membrane contact sites (MCSs) are fundamental for intracellular communication, but their role in intercellular communication remains unexplored. We show that in plants, plasmodesmata communication bridges function as atypical endoplasmic reticulum (ER)-plasma membrane (PM) tubular MCSs, operating at cell-cell interfaces. Similar to other MCSs, ER-PM apposition is controlled by a protein-lipid tethering complex, but uniquely, this serves intercellular communication. Combining high-resolution microscopy, molecular dynamics, and pharmacological and genetic approaches, we show that cell-cell trafficking is modulated through the combined action of multiple C2 domains transmembrane domain proteins (MCTPs) 3, 4, and 6 ER-PM tethers and phosphatidylinositol-4-phosphate (PI4P) lipid. Graded PI4P amounts regulate MCTP docking to the PM, their plasmodesmata localization, and cell-cell permeability. SAC7, an ER-localized PI4P-phosphatase, regulates MCTP4 accumulation at plasmodesmata and modulates cell-cell trafficking capacity in a cell-type-specific manner. Our findings expand MCS functions in information transmission from intracellular to intercellular cellular activities.
    Keywords:  MCTP; endoplasmic reticulum plasma membrane; intercellular communication; membrane contact sites; phosphoinositide; plant biology; plasmodesmata
    DOI:  https://doi.org/10.1016/j.cell.2024.11.034
  22. Mol Biol Cell. 2025 Feb 19. mbcE24120535
    Trafficking of K63-polyubiquitin modified membrane proteins in a macroautophagy-independent pathway is linked to ATG9A.
    Francesco Scavone, Sharon Lian, Eeva-Liisa Eskelinen, Robert E Cohen, Tingting Yao.
      Cytoplasmic K63-linked polyubiquitin signals have well-established roles in endocytosis and selective autophagy. However, how these signals help to direct different cargos to different intracellular trafficking routes is unclear. Here we report that, when the K63-polyubiquitin signal is blocked by intracellular expression of a high-affinity sensor (named Vx3), many proteins originating from the plasma membrane are found trapped in clusters of small vesicles that co-localize with ATG9A, a transmembrane protein that plays an essential role in autophagy. Importantly, whereas ATG9A is required for cluster formation, other core autophagy machinery as well as selective autophagy cargo receptors are not required. Although the cargos are sequestered in the vesicular clusters in an ATG9-dependent manner, additional signals are needed to induce LC3 conjugation. Upon removal of the Vx3 block, K63-polyubiquitylated cargos are rapidly delivered to lysosomes. These observations suggest that ATG9A plays an unexpected role in the trafficking of K63-polyubiquitin modified membrane proteins. [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E24-12-0535
  23. Sci Adv. 2025 Feb 21. 11(8): eadq2475
    SUMO2/3 conjugation of TDP-43 protects against aggregation.
    Enza Maria Verde, Francesco Antoniani, Laura Mediani, Valentina Secco, Samuele Crotti, Maria Celidea Ferrara, Jonathan Vinet, Aleksandra Sergeeva, Xiao Yan, Carsten Hoege, Cristiana Stuani, Francesca Paron, Tzu-Ting Kao, Rohit Shrivastava, Jolanta Polanowska, Aymeric Bailly, Alessandro Rosa, Eleonora Aronica, Anand Goswami, Neil Shneider, Anthony A Hyman, Emanuele Buratti, Dimitris Xirodimas, Titus M Franzmann, Simon Alberti, Serena Carra.
      Cytosolic aggregation of the RNA binding protein TDP-43 (transactive response DNA-binding protein 43) is a hallmark of amyotrophic lateral sclerosis and frontotemporal dementia. Here, we report that during oxidative stress, TDP-43 becomes SUMO2/3-ylated by the SUMO E3 ligase protein PIAS4 (protein inhibitor of activated STAT 4) and enriches in cytoplasmic stress granules (SGs). Upon pharmacological inhibition of TDP-43 SUMO2/3-ylation or PIAS4 depletion, TDP-43 enrichment in SGs is accompanied by irreversible aggregation. In cells that are unable to assemble SGs, SUMO2/3-ylation of TDP-43 is strongly impaired, supporting the notion that SGs are compartments that promote TDP-43 SUMO2/3-ylation during oxidative stress. Binding of TDP-43 to UG-rich RNA antagonizes PIAS4-mediated SUMO2/3-ylation, while RNA dissociation promotes TDP-43 SUMO2/3-ylation. We conclude that SUMO2/3 protein conjugation is a cellular mechanism to stabilize cytosolic RNA-free TDP-43 against aggregation.
    DOI:  https://doi.org/10.1126/sciadv.adq2475
  24. Cell. 2025 Feb 12. pii: S0092-8674(25)00106-0. [Epub ahead of print]
    SecY translocon chaperones protein folding during membrane protein insertion.
    Xiaomin Ou, Chengying Ma, Dongjie Sun, Jinkun Xu, Yang Wang, Xiaofei Wu, Dali Wang, Song Yang, Ning Gao, Chen Song, Long Li.
      The Sec translocon is vital for guiding membrane protein insertion into lipid bilayers. The insertion and folding processes of membrane proteins are poorly understood. Here, we report cryo-electron microscopy structures of multi-spanning membrane proteins inserting through the SecY channel, the Sec translocon in prokaryotes. The high-resolution structures illustrate how bulky amino acids pass the narrow channel restriction. Comparison of different translocation states reveals that the cytoplasmic and extracellular cavities of the channel create distinct environments for promoting the unfolding and folding of transmembrane segments (TMs), respectively. Released substrate TMs are either flexible or stabilized by an unexpected hydrophilic groove between TM3 and TM4 of SecY. Disruption of the groove causes global defects in the folding of the membrane proteome. These findings demonstrate that beyond its role as a passive protein-conducting channel, the SecY translocon actively serves as a chaperone, employing multiple mechanisms to promote membrane protein insertion and folding.
    Keywords:  SecY channel; chaperone; membrane protein insertion; protein translocation
    DOI:  https://doi.org/10.1016/j.cell.2025.01.037
  25. Plant Commun. 2025 Feb 13. pii: S2590-3462(25)00046-X. [Epub ahead of print] 101284
    The core morning clock component CCA1 positively regulates the expression of UPR target genes for ER stress recovery.
    Gyeongik Ahn, In Jung Jung, Gyeong-Im Shin, Song Yi Jeong, Myung Geun Ji, Jin-Sung Huh, Ji-Won Hwang, Jeongsik Kim, Joon-Yung Cha, Sang Yeol Lee, Min Gab Kim, Woe-Yeon Kim.
      The endoplasmic reticulum (ER) is a cellular organelle responsible for protein synthesis and folding. When the protein folding capacity is exceeded, unfolded or misfolded proteins accumulate, causing ER stress and triggering the unfolded protein response (UPR) to restore ER proteostasis. Although UPR genes in plants are expressed in a diel cycle, the mechanisms by which the circadian clock regulates these genes are not well understood. Here, we demonstrate that ER stress sensitivity in root growth exhibits time-of-day phases and that the circadian clock regulates the expression of UPR target genes during ER stress. Notably, mutations in the core morning clock component CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) impair ER stress recovery. CCA1 forms a complex with the UPR modulator bZIP28 and acts as upstream regulator in ER stress recovery. Upon ER stress, CCA1 is stabilized and associates with bZIP28 at the ER stress response element of the BiP3 promoter, enhancing the ER stress response. Thus, CCA1 coordinates a time-dependent adaptive response to ER stress with bZIP28 to maintain ER proteostasis. Our results suggest that the circadian clock primes the timing and levels of ER chaperone expression to enhance ER stress tolerance.
    Keywords:  CCA1; Circadian clock; ER chaperone; ER stress; bZIP28
    DOI:  https://doi.org/10.1016/j.xplc.2025.101284
  26. Proc Natl Acad Sci U S A. 2025 Feb 25. 122(8): e2415422122
    STING-induced noncanonical autophagy regulates endolysosomal homeostasis.
    Tuozhi Huang, Chenglong Sun, Fenghe Du, Zhijian J Chen.
      The cGAS-STING pathway mediates innate immune responses to cytosolic DNA. In addition to its well-established role in inducing inflammatory cytokines, activation of the cGAS-STING pathway also induces noncanonical autophagy, a process involving the conjugation of the ATG8 family of ubiquitin-like proteins to membranes of the endolysosomal system. The mechanisms and functions of STING-induced autophagy remain poorly understood. In this study, we demonstrated that STING activation induced formation of pH-elevated Golgi-derived vesicles that led to ATG16L1 and V-ATPase-dependent noncanonical autophagy. We showed that STING-induced noncanonical autophagy resulted in activation of the MiT/TFE family of transcription factors (TFEB, TFE3, and MITF), which regulate lysosome biogenesis. We found that lipidation of the ATG8 proteins, particularly GABARAPs, inhibited phosphorylation of MiT/TFE transcription factors by mTORC1. The lipidated GABARAPs bound to the Folliculin-interacting proteins (FNIPs), thereby sequestering the FNIP-folliculin protein complexes from activating mTORC1, resulting in dephosphorylation and nuclear translocation of MiT/TFE transcription factors. Furthermore, we found that STING-induced autophagy activated Leucine-rich repeat kinase 2 (LRRK2), a protein implicated in Parkinson's disease, through GABARAPs lipidation. We further showed that STING-induced autophagy induced ALIX-mediated ESCRT machinery recruitment to mitigate endolysosomal perturbation. These results reveal the multifaceted functions of STING-induced noncanonical autophagy in regulating endolysosomal homeostasis.
    Keywords:  ESCRT; STING; TFEB; autophagy; cGAS
    DOI:  https://doi.org/10.1073/pnas.2415422122
  27. Nat Commun. 2025 Feb 21. 16(1): 1864
    PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection.
    Shuyuan Chen, Rachel Putnik, Xi Li, Alka Diwaker, Marina Vasconcelos, Shuzhen Liu, Sudershan Gondi, Junhui Zhou, Lei Guo, Lin Xu, Sebastian Temme, Klare Bersch, Stephen Hyland, Jianyi Yin, Ezra Burstein, Brian J Bahnson, Jeffrey C Gildersleeve, Catherine Leimkuhler Grimes, Hans-Christian Reinecker.
      Peptidoglycan recognition proteins (PGLYRPs) are implicated in the control of the intestinal microbiota; however, molecular requirements for peptidoglycan (PGN) binding and receptor signaling mechanisms remain poorly understood. Here we show that PGLYRP1 is a receptor for the disaccharide motif of lysine N-acetylglucosamine N-acetylmuramic tripeptide (GMTriP-K). PGLYRP1 is required for innate immune activation by GMTriP-K but not muramyl dipeptide (MDP). In macrophages, intracellular PGLYRP1 complexes with NOD2 and GEF-H1, both of which are required for GMTriP-K-regulated gene expression. PGLYRP1 localizes to the endoplasmic reticulum and interacts at the Golgi with NOD2 upon GMTriP-K stimulation. PGLYRP1 and dependent gene expression signatures are induced in both mouse intestinal inflammation and human ulcerative colitis. Importantly, PGLYRP1 activation by GMTriP-K can result in the protection of mice from TNBS-induced colitis. Mammalian PGLYRPs can function as intracellular pattern recognition receptors for the control of host defense responses in the intestine.
    DOI:  https://doi.org/10.1038/s41467-025-57126-9
  28. Nat Commun. 2025 Feb 17. 16(1): 1696
    Cell enlargement modulated by GATA4 and YAP instructs the senescence-associated secretory phenotype.
    Joae Joung, Yekang Heo, Yeonju Kim, Jaejin Kim, Haebeen Choi, Taerang Jeon, Yeji Jang, Eun-Jung Kim, Sang Heon Lee, Jae Myoung Suh, Stephen J Elledge, Mi-Sung Kim, Chanhee Kang.
      Dynamic changes in cell size are associated with development and pathological conditions, including aging. Although cell enlargement is a prominent morphological feature of cellular senescence, its functional implications are unknown; moreover, how senescent cells maintain their enlargement state is less understood. Here we show that an extensive remodeling of actin cytoskeleton is necessary for establishing senescence-associated cell enlargement and pro-inflammatory senescence-associated secretory phenotype (SASP). This remodeling is attributed to a balancing act between the SASP regulator GATA4 and the mechanosensor YAP on the expression of the Rho family of GTPase RHOU. Genetic or pharmacological interventions that reduce cell enlargement attenuate SASP with minimal effect on senescence growth arrest. Mechanistically, actin cytoskeleton remodeling couples cell enlargement to the nuclear localization of GATA4 and NF-κB via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. RhoU protein accumulates in mouse adipose tissue under senescence-inducing conditions. Furthermore, RHOU expression correlates with SASP expression in adipose tissue during human aging. Thus, our study highlights an unexpected instructive role of cell enlargement in modulating the SASP and reveals a mechanical branch in the senescence regulatory network.
    DOI:  https://doi.org/10.1038/s41467-025-56929-0
  29. EMBO Rep. 2025 Feb 20.
    Muskelin is a substrate adaptor of the highly regulated Drosophila embryonic CTLH E3 ligase.
    Chloe A Briney, Jesslyn C Henriksen, Chenwei Lin, Lisa A Jones, Leif Benner, Addison B Rains, Roxana Gutierrez, Philip R Gafken, Olivia S Rissland.
      The maternal-to-zygotic transition (MZT) is a conserved developmental process where the maternally-derived protein and mRNA cache is replaced with newly made zygotic gene products. We have previously shown that in Drosophila the deposited RNA-binding proteins ME31B, Cup, and Trailer Hitch are ubiquitylated by the CTLH E3 ligase and cleared. However, the organization and regulation of the CTLH complex remain poorly understood in flies because Drosophila lacks an identifiable substrate adaptor, and the mechanisms restricting the degradation of ME31B and its cofactors to the MZT are unknown. Here, we show that the developmental regulation of the CTLH complex is multi-pronged, including transcriptional control by OVO and autoinhibition of the E3 ligase. One major regulatory target is the subunit Muskelin, which we demonstrate is a substrate adaptor for the Drosophila CTLH complex. Finally, we find that Muskelin has few targets beyond the three known RNA-binding proteins, showing exquisite target specificity. Thus, multiple levels of integrated regulation restrict the activity of the embryonic CTLH complex to early embryogenesis, during which time it regulates three important RNA-binding proteins.
    Keywords:  E3 Ligase; Maternal-to-zygotic Transition; Protein Degradation
    DOI:  https://doi.org/10.1038/s44319-025-00397-6
  30. Mol Biol Rep. 2025 Feb 15. 52(1): 236
    Molecular mechanisms and pathological implications of unconventional protein secretion in human disease: from cellular stress to therapeutic targeting.
    Yukun Liu, Haolin Zhang, Xianghua Li, Tianlong He, Wenting Zhang, Cuicui Ji, Juan Wang.
      Unconventional protein secretion (UcPS) encompasses diverse non-canonical cellular export mechanisms that operate independently of the classical secretory pathway, representing a crucial cellular response to various physiological and pathological conditions. This comprehensive review synthesizes current understanding of UcPS mechanisms, particularly focusing on their roles in disease pathogenesis and progression. Recent advances in proteomics and cellular biology have revealed that UcPS facilitates the secretion of various biomedically significant proteins, including inflammatory mediators, growth factors, and disease-associated proteins, through multiple pathways such as membrane translocation, secretory lysosomes, and membrane-bound organelles. Notably, dysregulation of UcPS mechanisms has been implicated in various pathological conditions, including chronic inflammation, neurodegenerative disorders, and malignant transformation. We critically evaluate the molecular machinery governing UcPS, its regulation under cellular stress, and its contribution to disease mechanisms. Furthermore, we examine emerging therapeutic strategies targeting UcPS pathways, highlighting both opportunities and challenges in developing novel interventional approaches.
    Keywords:  Cancer; Inflammation; Neurodegenerative diseases; Unconventional protein secretion
    DOI:  https://doi.org/10.1007/s11033-025-10316-6
  31. Cell Rep. 2025 Feb 20. pii: S2211-1247(25)00117-2. [Epub ahead of print]44(3): 115346
    A KIF1C-CNBP motor-adaptor complex for trafficking mRNAs to cell protrusions.
    Konstadinos Moissoglu, Tianhong Wang, Alexander N Gasparski, Michael Stueland, Elliott L Paine, Lisa M Jenkins, Stavroula Mili.
      mRNA localization to subcellular compartments is a widely used mechanism that functionally contributes to numerous processes. mRNA targeting can be achieved upon recognition of RNA cargo by molecular motors. However, our molecular understanding of how this is accomplished is limited, especially in higher organisms. We focus on a pathway that targets mRNAs to peripheral protrusions of mammalian cells and which is important for cell migration. Trafficking occurs through active transport on microtubules, mediated by the KIF1C kinesin. Here, we identify the RNA-binding protein CNBP as a factor required for mRNA localization to protrusions. CNBP binds directly to GA-rich sequences in the 3' UTR of protrusion-targeted mRNAs. CNBP also interacts with KIF1C and is required for KIF1C recruitment to mRNAs and their trafficking on microtubules to the periphery. This work provides a molecular mechanism for KIF1C recruitment to mRNA cargo and reveals a motor-adaptor complex for mRNA transport to cell protrusions.
    Keywords:  CNBP; CP: Cell biology; CP: Molecular biology; GA-rich element; KIF1C; RNA transport; kinesin adaptor; localization; microtubule; protrusion-localized mRNA
    DOI:  https://doi.org/10.1016/j.celrep.2025.115346
  32. Mol Cell. 2025 Feb 14. pii: S1097-2765(25)00054-1. [Epub ahead of print]
    AcrVIB1 inhibits CRISPR-Cas13b immunity by promoting unproductive crRNA binding accessible to RNase attack.
    Katharina G Wandera, Stefan Schmelz, Angela Migur, Anuja Kibe, Peer Lukat, Tatjana Achmedov, Neva Caliskan, Wulf Blankenfeldt, Chase L Beisel.
      Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.
    Keywords:  AcrVIB1; Cas13b; anti-CRISPR protein; cryo-EM; cryoelectron microscopy; immune defense; phage
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.020
  33. Methods Enzymol. 2025 ;pii: S0076-6879(24)00573-1. [Epub ahead of print]711 85-101
    Ribosome-associated tDRs in yeast.
    Alessia Rosina, Norbert Polacek, Robert Rauscher.
      The regulation of gene expression in response to environmental stress is a key process that ensures cellular survival across all three domains of life. The adjustment of protein synthesis appears to be one of the initial steps toward the response and adaptation to stress. Ribosome-associated non-coding RNAs (rancRNAs) efficiently regulate translation as an immediate response to stress by directly targeting the ribosome and fine-tuning translation. tRNA-derived RNAs (tDRs) are part of the RNA species that constitute the functionally diverse class of rancRNAs. Here we report a new experimental approach for creating deep sequencing libraries of ribosome-associated small RNAs in yeast utilizing state-of-the-art technologies. Our new strategy is supported by validating previously identified rancRNAs and discovering novel tDRs interacting with the Saccharomyces cerevisiae ribosome.
    Keywords:  RancRNA; Stress response; Translation regulation; Yeast tRNA-derived RNA
    DOI:  https://doi.org/10.1016/bs.mie.2024.11.006
  34. bioRxiv. 2025 Jan 28. pii: 2025.01.28.635360. [Epub ahead of print]
    Molecular determinants of RNase MRP specificity and function.
    Eric M Smith, Jimmy Ly, Sofia Haug, Iain M Cheeseman.
      RNase MRP and RNase P are evolutionarily related complexes that facilitate rRNA and tRNA biogenesis, respectively. The two enzymes share nearly all protein subunits and have evolutionarily related catalytic RNAs. Notably, RNase P includes a unique subunit, Rpp21, whereas no RNase MRP-specific proteins have been found in humans, limiting molecular analyses of RNase MRP function. Here, we identify the RNase MRP-specific protein, C18orf21/RMRPP1. RMRPP1 and Rpp21 display significant structural homology, but we identify specific regions that drive interactions with their respective complexes. Additionally, we reveal that RNase MRP is required for 40S, but not 60S, ribosome biogenesis uncovering an alternative pathway for ribosome assembly. Finally, we identify Nepro as an essential rRNA processing factor that associates with the RNase MRP complex. Together, our findings elucidate the molecular determinants of RNase MRP function and underscore its critical role in ribosome biogenesis.
    DOI:  https://doi.org/10.1101/2025.01.28.635360
  35. bioRxiv. 2025 Jan 31. pii: 2025.01.30.635714. [Epub ahead of print]
    A privileged ER compartment for post-translational heteromeric assembly of an ion channel.
    Sudharsan Kannan, William Kasberg, Liliana R Ernandez, Anjon Audhya, Gail A Robertson.
      Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric assemblies are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-à-go-go related gene ( hERG ) is essential for the normal function of cardiac I Kr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome. While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, driven by an N-terminal "RXR" ER retention signal and phase separation, are distinct from other membranous compartments and proteasomal degradation pathways. The puncta represent a privileged ER sub-compartment distinct from that of ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurs by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly.
    DOI:  https://doi.org/10.1101/2025.01.30.635714
  36. Nat Biotechnol. 2025 Feb 19.
    Internal cap-initiated translation for efficient protein production from circular mRNA.
    Kosuke Fukuchi, Yuko Nakashima, Naoko Abe, Seigo Kimura, Fumitaka Hashiya, Yuichi Shichino, Yiwei Liu, Ryoko Ogisu, Satomi Sugiyama, Daisuke Kawaguchi, Masahito Inagaki, Zheyu Meng, Shiryu Kajihara, Mizuki Tada, Satoshi Uchida, Ting-Ting Li, Ramkrishna Maity, Tairin Kawasaki, Yasuaki Kimura, Shintaro Iwasaki, Hiroshi Abe.
      Circular mRNA faces challenges in enhancing its translation potential as an RNA therapeutic. Here we introduce two molecular designs that bolster circular mRNA translation through an internal cap-initiated mechanism. The first consists of a circular mRNA with a covalently attached N7-methylguanosine (m7G) cap through a branching structure (cap-circ mRNA). This modification allows circular mRNA to recruit translation machinery and produce proteins more efficiently than internal ribosome entry site (IRES)-containing circular mRNAs. Combining with an N1-methylpseudouridine (m1Ψ) modification, cap-circ mRNA exhibits a lower acute immunostimulatory effect, maintaining high translation in mice. The second design features the non-covalent attachment of an m7G cap to a circular mRNA through hybridization with an m7G cap-containing oligonucleotide, enhancing translation by more than 50-fold. This setup allows circular mRNAs to synthesize reporter proteins upon hybridizing with capped mRNAs or long non-coding RNAs and to undergo rolling circle-type translation. These advancements broaden the therapeutic applications of circular mRNAs by minimizing their molecular size, elevating translation efficiency and facilitating cell-type-selective translation.
    DOI:  https://doi.org/10.1038/s41587-025-02561-8
  37. Autophagy. 2025 Feb 19. 1-3
    Stress granules as transient reservoirs for autophagy proteins: a key mechanism for plant recovery from heat stress.
    Lei Feng, Xibao Li, Wenjin Shen, Caiji Gao.
      Stress granules (SGs) are transient, non-membrane-bound cytoplasmic condensates that form in response to environmental stresses, serving as protective reservoirs for mRNAs and proteins. In plants, SGs play a crucial role in stress adaptation, but their relationship with macroautophagy/autophagy, a key process for degrading damaged organelles and misfolded proteins, remains poorly understood. In a recent study, we revealed that key autophagy proteins, including components of the ATG1-ATG13 kinase complex, the class III phosphatidylinositol 3-kinase (PtdIns3K) complex, and the ATG8-PE system, translocate to SGs during heat stress (HS) in Arabidopsis thaliana. Using biochemical, cell biological and genetic approaches, we demonstrated that ATG proteins accumulate on HS-induced SGs and are released to the cytosol upon SG disassembly during the post-HS recovery stage. This process facilitates rapid autophagy activation. Notably, a SG-deficient mutant (ubp1abc) exhibits delayed autophagy activation and impaired clearance of ubiquitinated protein aggregates, highlighting the importance of SGs in regulating autophagy. Our findings uncover a novel mechanism by which SGs sequester autophagy proteins during stress, ensuring their rapid availability for stress recovery, and provide new insights into the interplay between SGs and autophagy in plant stress responses.Abbreviation: ATG, autophagy related; HS, heat stress; PtdIns3K, phosphatidylinositol 3-kinase; RBP47B, RNA-binding protein 47B; SG, stress granule; UBP1, ubiquitin-specific protease 1.
    Keywords:  ATG8; Arabidopsis thaliana; UBP1; autophagy; heat stress; stress granules
    DOI:  https://doi.org/10.1080/15548627.2025.2465395
  38. Nat Commun. 2025 Feb 14. 16(1): 1600
    The DYT6 dystonia causative protein THAP1 is responsible for proteasome activity via PSMB5 transcriptional regulation.
    Yan Wang, Yi Wang, Tomohiro Iriki, Eiichi Hashimoto, Maki Inami, Sota Hashimoto, Ayako Watanabe, Hiroshi Takano, Ryo Motosugi, Shoshiro Hirayama, Hiroki Sugishita, Yukiko Gotoh, Ryoji Yao, Jun Hamazaki, Shigeo Murata.
      The proteasome plays a pivotal role in protein degradation, and its impairment is associated with various pathological conditions, including neurodegenerative diseases. It is well understood that Nrf1 coordinates the induction of all proteasome genes in response to proteasome dysfunction. However, the molecular mechanism regulating the basal expression of the proteasome remains unclear. Here we identify the transcription factor THAP1, the causative gene of DYT6 dystonia, as a regulator of proteasome activity through a genome-wide genetic screen. We demonstrated that THAP1 directly regulates the expression of the PSMB5 gene, which encodes the central protease subunit β5. Depletion of THAP1 disrupts proteasome assembly, leading to reduced proteasome activity and the accumulation of ubiquitinated proteins. These findings uncover a regulatory mechanism for the proteasome and suggest a potential role for proteasome dysfunction in the pathogenesis of dystonia.
    DOI:  https://doi.org/10.1038/s41467-025-56867-x
  39. EMBO Rep. 2025 Feb 17.
    Short internal open reading frames repress the translation of N-terminally truncated proteoforms.
    Raphael Fettig, Zita Gonda, Niklas Walter, Paul Sallmann, Christiane Thanisch, Markus Winter, Susanne Bauer, Lei Zhang, Greta Linden, Margarethe Litfin, Marina Khamanaeva, Sarah Storm, Christina Münzing, Christelle Etard, Olivier Armant, Olalla Vázquez, Olivier Kassel.
      Internal translation initiation sites, as revealed by ribosome profiling experiments can potentially drive the translation of many N-terminally truncated proteoforms. We report that internal short open reading frame (sORF) within coding sequences regulate their translation. nTRIP6 represents a short nuclear proteoform of the cytoplasmic protein TRIP6. We have previously reported that nTRIP6 regulates the dynamics of skeletal muscle progenitor differentiation. Here we show that nTRIP6 is generated by translation initiation at an internal AUG after leaky scanning at the canonical TRIP6 AUG. The translation of nTRIP6 is repressed by an internal sORF immediately upstream of the nTRIP6 AUG. Consistent with this representing a more general regulatory feature, we have identified other internal sORFs which repress the translation of N-terminally truncated proteoforms. In an in vitro model of myogenic differentiation, the expression of nTRIP6 is transiently upregulated through a mechanistic Target of Rapamycin Complex 1-dependent increase in translation initiation at the internal AUG. Thus, the translation of N-terminally truncated proteoforms can be regulated independently of the canonical ORF.
    Keywords:  Myogenesis; Proteoforms; Short ORFs; Translation; Trip6
    DOI:  https://doi.org/10.1038/s44319-025-00390-z
  40. Open Biol. 2025 Feb;15(2): 240225
    Proximal partners of the organellar N-terminal acetyltransferase NAA60: insights into Golgi structure and transmembrane protein topology.
    Sebastian Tanco, Veronique Jonckheere, Arun Kumar Tharkeshwar, Annelies Bogaert, Kris Gevaert, Wim Anneart, Petra Van Damme.
      Biotin identification (BioID) is an interactomics approach that utilizes proximity labelling to map the local interactome or proxeome of proteins within a cell. This study applies BioID to investigate proteins proximal to NAA60 (N-alpha-acetyltransferase 60), an N-terminal acetyltransferase (NAT) of pathological significance in human disease, characterized by its unique Golgi localization. NAA60 is known to N-terminally acetylate transmembrane proteins that present their N-terminus on the cytosolic face of the membrane, and its involvement in maintaining Golgi structure has previously been established. Using a stable cell-line expressing an NAA60-BirA* fusion protein, we isolated biotinylated proteins through streptavidin affinity purification. Mass spectrometry analysis revealed over 100 proximal partners of NAA60, enriched in proteins localized on the trans-side of the Golgi apparatus. High-confidence proximity interactors included golgins and GRASP proteins, essential for Golgi integrity. Considering the transmembrane nature of NAA60, the identification of biotinylated peptides inferred the topology of transmembrane protein interactors within the secretory pathway. Subsequent suborganellar localization analysis revealed a more prominent medial/trans-Golgi localization of NAA60. Our findings underscore the role of NAA60 and its interactors in maintaining Golgi structural integrity and highlight the effectiveness of BioID in generating critical protein topology data, invaluable for enhancing the prediction of protein topology within cellular compartments.
    Keywords:  Golgi fragmentation; N-terminal acetylation; NAA60; proxeome; proximity-dependent biotin identification (BioID); transmembrane protein topology
    DOI:  https://doi.org/10.1098/rsob.240225
  41. Mol Cell. 2025 Feb 10. pii: S1097-2765(25)00046-2. [Epub ahead of print]
    Degradation of circular RNA by the ribonuclease DIS3.
    Xiao Tao, Si-Nan Zhai, Chu-Xiao Liu, Youkui Huang, Jia Wei, Yi-Lin Guo, Xiao-Qi Liu, Xiang Li, Li Yang, Ling-Ling Chen.
      Features of circular RNAs (circRNAs) produced by back-splicing of eukaryotic exon(s) make them resistant to degradation by linear RNA decay machineries. Thus, a general circRNA degradation pathway under normal conditions has remained largely elusive. Here, we report that the endonucleolytic enzyme DIS3 is responsible for the degradation of circRNAs. Depletion of DIS3 leads to the upregulation of more than 60% of circRNAs with little effect on their linear cognates. Such DIS3-mediated circRNA degradation is conserved, occurs in the cytoplasm, and relies on DIS3's endonucleolytic activity but is independent of the RNA exosome complex. Sequence enrichment analyses suggest that DIS3 prefers to degrade circRNAs containing U-rich motifs. Correspondingly, synthesized RNA circles with or without U-rich motifs exhibit decreased or increased stabilities, respectively. Together, these findings suggest a general regulation of circRNA turnover by DIS3.
    Keywords:  DIS3; RNA decay; U-rich motifs; circRNAs; circular RNAs; degradation; endonuclease; turnover
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.012
  42. J Cell Biol. 2025 Apr 07. pii: e202410003. [Epub ahead of print]224(4):
    RAB-10 cooperates with EHBP-1 to capture vesicular carriers during post-Golgi exocytic trafficking.
    Shuai Liu, Jie Wei, Liangyujie Zhong, Sirao Hai, Shibo Song, Chaoyi Xie, Zeyu Huang, Zihang Cheng, Jing Zhang, Anna Du, Pei Zhang, Yanling Yan, Anbing Shi.
      Post-Golgi exocytic trafficking, fundamental for secretion and cell surface component integration, remains incompletely understood at the molecular level. Here, we investigated this process using Caenorhabditis elegans and mammalian cell models, revealing a novel exocytic carrier capturing mechanism involving the small GTPase RAB-10/Rab10 and its effector EHBP-1/EHBP1. EHBP-1, localized in recycling endosomes, selectively captures RAB-10-positive lipoprotein exocytic carriers through its interaction with active RAB-10, thereby promoting the delivery of exocytic cargo to recycling endosomes. A detailed mechanistic examination demonstrated the synergy between EHBP-1's RAB-10-binding coiled-coil domain and its PI(4,5)P2-binding C2 domain in the capturing process. Of note, we identified LST-6/DENND5 as a specialized guanine nucleotide exchange factor (GEF) for RAB-10 in this particular pathway, distinct from the GEF involved in basolateral recycling. Following the RAB-10-EHBP-1-mediated capture, the exocyst complex carries out its function. Taken together, this study suggests a potential tethering mechanism for basolateral post-Golgi exocytic carriers, highlighting the coordination among membrane compartments in regulating this trafficking route.
    DOI:  https://doi.org/10.1083/jcb.202410003
  43. Proc Natl Acad Sci U S A. 2025 Feb 25. 122(8): e2419607122
    Tracking proteasome degradation: A cross-organ analysis via intact degradomics mass spectrometry.
    Katharina I Zittlau, Daniel Zachor-Movshovitz, Yegor Leushkin, Roy Schimmel Brener, David Morgenstern, Gili Ben-Nissan, Michal Sharon.
      The proteasome is a multisubunit degradation machinery that is essential for maintaining protein homeostasis by breaking down unnecessary or damaged proteins into peptides. While most of these peptides are further processed into amino acids, a subset evades complete degradation and plays key roles in biological processes such as antigen presentation, signaling, and apoptosis. However, the variability in peptide lengths and the diverse composition of proteasomes make their comprehensive identification and characterization particularly challenging. Here, we present a method that enables real-time identification of generated peptides, as well as uncleaved and partially cleaved protein substrates, revealing the processive nature of protein proteasomal degradation. Our intact degradomics workflow is based on intact mass spectrometry measurements and treats the enzymatically produced peptides as if they were generated within the mass spectrometer, akin to top-down products. We applied this approach to determine the kinetic profile of proteasome degradation and compare the real-time activity of proteasomes isolated from different mouse organs, uncovering distinct functionalities of the complex. Overall, this method offers a valuable tool for studying peptide degradation products across various proteasome configurations, while also enabling the investigation of how interacting proteins, inhibitors, and activators influence proteasome activity. Furthermore, its adaptability makes it applicable to a wide range of other proteolytic complexes, broadening its potential impact in the field.
    Keywords:  mass spectrometry; proteasome; protein degradation; top-down MS
    DOI:  https://doi.org/10.1073/pnas.2419607122
  44. Nature. 2025 Feb 19.
    De novo design of transmembrane fluorescence-activating proteins.
    Jingyi Zhu, Mingfu Liang, Ke Sun, Yu Wei, Ruiying Guo, Lijing Zhang, Junhui Shi, Dan Ma, Qi Hu, Gaoxingyu Huang, Peilong Lu.
      The recognition of ligands by transmembrane proteins is essential for the exchange of materials, energy and information across biological membranes. Progress has been made in the de novo design of transmembrane proteins1-6, as well as in designing water-soluble proteins to bind small molecules7-12, but de novo design of transmembrane proteins that tightly and specifically bind to small molecules remains an outstanding challenge13. Here we present the accurate design of ligand-binding transmembrane proteins by integrating deep learning and energy-based methods. We designed pre-organized ligand-binding pockets in high-quality four-helix backbones for a fluorogenic ligand, and generated a transmembrane span using gradient-guided hallucination. The designer transmembrane proteins specifically activated fluorescence of the target fluorophore with mid-nanomolar affinity, exhibiting higher brightness and quantum yield compared to those of enhanced green fluorescent protein. These proteins were highly active in the membrane fraction of live bacterial and eukaryotic cells following expression. The crystal and cryogenic electron microscopy structures of the designer protein-ligand complexes were very close to the structures of the design models. We showed that the interactions between ligands and transmembrane proteins within the membrane can be accurately designed. Our work paves the way for the creation of new functional transmembrane proteins, with a wide range of applications including imaging, ligand sensing and membrane transport.
    DOI:  https://doi.org/10.1038/s41586-025-08598-8
  45. bioRxiv. 2025 Feb 05. pii: 2025.01.14.633043. [Epub ahead of print]
    Altered relaxation and Mitochondria-Endoplasmic Reticulum Contacts Precede Major (Mal)adaptations in Aging Skeletal Muscle and are Prevented by Exercise.
    Ryan J Allen, Ana Kronemberger, Qian Shi, Marshall Pope, Elizabeth Cuadra-Muñoz, Wangkuk Son, Long-Sheng Song, Ethan J Anderson, Renata O Pereira, Vitor A Lira.
      Sarcopenia, or age-related muscle dysfunction, contributes to morbidity and mortality. Besides decreases in muscle force, sarcopenia is associated with atrophy and fast-to-slow fiber type switching, which is typically secondary to denervation in humans and rodents. However, very little is known about cellular changes preceding these important (mal)adaptations. To this matter, mitochondria and the sarcoplasmic reticulum are critical for tension generation in myofibers. They physically interact at the boundaries of sarcomeres forming subcellular hubs called mitochondria-endo/sarcoplasmic reticulum contacts (MERCs). Yet, whether changes at MERCs ultrastructure and proteome occur early in aging is unknown. Here, studying young adult and older mice we reveal that aging slows muscle relaxation leading to longer excitation-contraction-relaxation (ECR) cycles before maximal force decreases and fast-to-slow fiber switching takes place. We reveal that muscle MERC ultrastructure and mitochondria-associated ER membrane (MAM) protein composition are also affected early in aging and are closely associated with rate of muscle relaxation. Additionally, we demonstrate that regular exercise preserves muscle relaxation rate and MERC ultrastructure in early aging. Finally, we profile a set of muscle MAM proteins involved in energy metabolism, protein quality control, Ca 2+ homeostasis, cytoskeleton integrity and redox balance that are inversely regulated early in aging and by exercise. These may represent new targets to preserve muscle function in aging individuals.
    DOI:  https://doi.org/10.1101/2025.01.14.633043
  46. Science. 2025 Feb 20. eadq2084
    Deep-tissue transcriptomics and subcellular imaging at high spatial resolution.
    Valentina Gandin, Jun Kim, Liang-Zhong Yang, Yumin Lian, Takashi Kawase, Amy Hu, Konrad Rokicki, Greg Fleishman, Paul Tillberg, Alejandro Aguilera Castrejon, Carsen Stringer, Stephan Preibisch, Zhe J Liu.
      Limited color channels in fluorescence microscopy have long constrained spatial analysis in biological specimens. Here, we introduce cycle Hybridization Chain Reaction (HCR), a method that integrates multicycle DNA barcoding with HCR to overcome this limitation. cycleHCR enables highly multiplexed imaging of RNA and proteins using a unified barcode system. Whole-embryo transcriptomics imaging achieved precise three-dimensional gene expression and cell fate mapping across a specimen depth of ~310 μm. When combined with expansion microscopy, cycleHCR revealed an intricate network of 10 subcellular structures in mouse embryonic fibroblasts. In mouse hippocampal slices, multiplex RNA and protein imaging uncovered complex gene expression gradients and cell-type-specific nuclear structural variations. cycleHCR provides a quantitative framework for elucidating spatial regulation in deep tissue contexts for research and potentially diagnostic applications.
    DOI:  https://doi.org/10.1126/science.adq2084
  47. Proc Natl Acad Sci U S A. 2025 Feb 25. 122(8): e2426986122
    A structural atlas of death domain fold proteins reveals their versatile roles in biology and function.
    Emily J Wu, Ankita T Kandalkar, Julian F Ehrmann, Alexander B Tong, Jing Zhang, Qian Cong, Hao Wu.
      Death domain fold (DDF) superfamily proteins are critically important players in pathways of cell death and inflammation. DDFs are often essential scaffolding domains in receptors, adaptors, or effectors of these pathways by mediating homo- and hetero-oligomerization including helical filament assembly. At the downstream ends of these pathways, effector oligomerization by DDFs brings the enzyme domains into proximity for their dimerization and activation. Hundreds of structures of these domains have been solved. However, a comprehensive understanding of DDFs is lacking. In this article, we report the curation of a DDF structural atlas as a public website (deathdomain.org) and deduce the common and distinct principles of DDF-mediated oligomerization among the four families (death domain or DD, death effector domain or DED, caspase recruitment domain or CARD, and pyrin domain or PYD). We further annotate DDFs genome-wide based on AlphaFold-predicted models and protein sequences. These studies reveal mechanistic rules for this widely distributed domain superfamily.
    Keywords:  DPAM; ECOD; cell death; death domain fold; inflammation
    DOI:  https://doi.org/10.1073/pnas.2426986122
  48. ACS Med Chem Lett. 2025 Feb 13. 16(2): 258-262
    Clozapine as an E3 Ligand for PROTAC Technology.
    Reina Takano, Nobumichi Ohoka, Takashi Kurohara, Noriaki Arakawa, Kenji Ohgane, Takao Inoue, Hidetomo Yokoo, Yosuke Demizu.
      New ubiquitin ligase (E3) ligands are crucial for developing proteolysis-targeting chimeras (PROTACs) to induce the degradation of a target protein. In this study, we developed a PROTAC using the antipsychotic drug clozapine as a new E3 ligand. First, a clozapine PROTAC targeting a model target HaloTag protein (Halo-PEG-Clozapine) was synthesized, and the PROTAC induced degradation of the HaloTag-fused protein in a cell culture system. Another clozapine PROTAC targeting the cancer therapeutic target estrogen receptor α (ERα) (Tamoxifen-PEG-Clozapine) was synthesized and induced degradation of the ERα protein in MCF-7 breast cancer cells. Experiments with inhibitors and siRNAs showed that Tamoxifen-PEG-Clozapine degraded ERα via a ubiquitin-proteasome system that uses the ubiquitin protein ligase E3 component N-recognin 5. These results indicate that clozapine is a promising E3 ligand that may expand the molecular design of PROTACs, contributing to the advancement of drug discovery by facilitating the degradation of disease-related proteins.
    DOI:  https://doi.org/10.1021/acsmedchemlett.4c00500
  49. Curr Opin Cell Biol. 2025 Feb 14. pii: S0955-0674(25)00019-5. [Epub ahead of print]93 102481
    Organelle homeostasis requires ESCRTs.
    Tsan-Wen Lu, Adam Frost, Frank R Moss.
      The endosomal sorting complexes required for transport (ESCRT) catalyze membrane shape transformations throughout the cell. Canonical functions of the ESCRTs include endosomal multivesicular body biogenesis, enveloped virus budding, and abscission of daughter cell plasma membranes. The ESCRT machinery is also required for membranous organelle homeostasis generally, including by facilitating lipid transport at membrane contact sites, repairing membrane damage, driving lysosomal catabolism, and maintaining nuclear envelope integrity, among other roles. Here, we review a subset of recent discoveries and highlight opportunities to better understand how ESCRT activities support cell health.
    DOI:  https://doi.org/10.1016/j.ceb.2025.102481
  50. Mol Cell. 2025 Feb 14. pii: S1097-2765(25)00061-9. [Epub ahead of print]
    Nucleic-acid-induced ZCCHC3 condensation promotes broad innate immune responses.
    Miao Shi, Tao Jiang, Mengfan Zhang, Quanjin Li, Kexin Liu, Ni Lin, Xinlu Wang, Amin Jiang, Yina Gao, Yong Wang, Songqing Liu, Liguo Zhang, Dong Li, Pu Gao.
      Retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) and cyclic GMP-AMP synthase (cGAS) recognize aberrant nucleic acids and initiate antiviral responses. Host factor zinc finger CCHC domain-containing protein 3 (ZCCHC3) positively regulates both RLRs- and cGAS-mediated signaling through unknown mechanisms. Here, we show that ZCCHC3 employs a broad and unified strategy to promote these pathways in human cell lines. Rather than developing strong protein-protein interactions, ZCCHC3 harbors multiple nucleic-acid-binding modules and undergoes robust liquid phase condensation with nucleic acids. RNA-induced ZCCHC3 condensates enrich and activate RLRs, which then facilitate the interaction of RLRs with the downstream adaptor mitochondrial antiviral-signaling (MAVS). Direct and high-resolution structure determination of liquid condensates confirms the assembly of active-form MAVS filaments. Furthermore, ZCCHC3 efficiently promotes the condensation and enrichment of DNA, cGAS, ATP, and GTP, thereby enhancing cGAS signaling. ZCCHC3 mutants defective in RNA/DNA-induced condensation lost their regulatory efficiency in both pathways. These results highlight unexpectedly broad connections between biomolecular condensation and innate immunity.
    Keywords:  MAVS; MDA5; RIG-I; STING; ZCCHC3; cGAMP; cGAS; nucleic-acid sensing; phase separation; structural biology
    DOI:  https://doi.org/10.1016/j.molcel.2025.01.027
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