bims-proteo Biomed News
on Proteostasis
Issue of 2025–12–14
fifty-nine papers selected by
Eric Chevet, INSERM
  1. Induced ubiquitination bypasses canonical ERAD to drive ER protein degradation.
  2. GCN2 monitors mRNA translation termination.
  3. Phosphatidylcholine coordinates ER-autonomous and ER-nonautonomous adaptations to unfolded protein response dysfunction.
  4. Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling.
  5. Collision-induced ribosome degradation driven by ribosome competition and translational perturbations.
  6. TMEDs mediate versatile cargo transport in vesicle-dependent unconventional secretion.
  7. Linked-domain inhibitors designed to block UBE2D induce the unfolded protein response.
  8. Functionally diversified Caenorhabditis elegans BiP orthologs control body growth, reproduction, stress resistance, aging, and autophagy.
  9. The ribosome-associated complex regulates cytosolic translation upon mitoprotein-induced stress.
  10. A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy.
  11. Endoplasmic Reticulum Redoxome: Protein Folding and Beyond.
  12. Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.
  13. Chemically-induced degradation of the endoplasmic-reticulum stress sensor IRE1 by a VHL-recruiting chimera.
  14. Synthesis of Immunoproteasome N-Degron Prodrugs for Selective Protein Degradation.
  15. The RNA-binding protein PRRC2B preserves 5' TOP mRNA during starvation to maintain ribosome biogenesis during nutrient recovery.
  16. Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant UBB+1.
  17. A widespread protein misfolding mechanism is differentially rescued in vitro by chaperones based on gene essentiality.
  18. Endoplasmic reticulum disruption stimulates nuclear membrane mechanotransduction.
  19. AGPAT2 acts at the crossroads of lipid biosynthesis and DRP1-mediated ER morphogenesis.
  20. Cysteine-enabled cleavability to advance cross-linking mass spectrometry for global analysis of endogenous protein-protein interactions.
  21. HYPK promotes N-terminal protein acetylation through rapid ribosome exchange of NatA.
  22. Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.
  23. Context Matters: E3 Ligase-Ligand Pairing Strategies for Optimized PROTAC Performance.
  24. Diet-induced RKIP downregulation disrupts PC/PE-ER homeostasis to drive MASLD.
  25. Trans-Golgi network-associated noncanonical autophagy depends on the V-ATPase-ATG16L1 axis and mediates IL1B secretion.
  26. The deubiquitinating enzyme Cezanne stabilizes BRCA1 by counteracting APC/C and Ube2S-dependent Lys11-linked ubiquitination.
  27. Regulation via compartmentation: adaptive localization of the proteasome under stress.
  28. TAPT1 interacts with SUCO to maintain the homeostasis of newly synthesized proteins and brain development in mice.
  29. LRRC59 cooperates with nuclear transporters to restrain the nuclear envelope repair machinery and safeguard genome integrity.
  30. Endoplasmic reticulum-mitochondrion disconnection promotes metabolic reprogramming and cystogenesis in polycystic kidney disease.
  31. A reconstruction of the mammalian secretory pathway identifies mechanisms regulating antibody production.
  32. Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency.
  33. The degradation of extended protein isoforms points to a misfiring translation initiation process.
  34. Targeted protein O-GlcNAc reveals transcriptional functions for O-GlcNAc.
  35. Human RBM3 protein is prone to form neuronal aggregates opposed by the proteasome.
  36. Chronic enteritis triggered by diet westernization is driven by epithelial ATG16L1-mediated autophagy.
  37. Combinatorial use of VHL and KEAP1 PROTACs reveals unexpected synergy and hook effect relief.
  38. Dual mechanism of autophagy gene repression by PHF23 and therapeutic potential of its inhibition in protein aggregation disorders.
  39. eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules.
  40. Implementing N-terminomics and machine learning to probe Nt-arginylation.
  41. Protocol for identification of cell-surface proteins with horseradish peroxidase.
  42. Chaperone-mediated autophagy as a sex-specific modulator of synaptic proteostasis and neural function.
  43. Integrating rna structure and protein interactions to uncover the mechanisms of viral and cellular ires function.
  44. Communication between chloroplasts and the endoplasmic reticulum in plants under abiotic stress.
  45. A C-terminal cytoplasmic retention motif and nuclear localization signal regulates nuclear import of TP53INP2/DOR.
  46. Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag.
  47. ASNA1 is essential for cardiac development and function by regulating tail-anchored protein stability and vesicular transport in cardiomyocytes.
  48. circATF6 triggers calcium overload to synergize with EGFR-TKI in non-small cell lung cancer via modulation of proteostasis.
  49. Hijacking the unfolded protein response (UPR) pathway: Balancing viral infection and host cell survival.
  50. HSP70 Interactome-Mediated Proteolysis Targeting Chimera (HSP70-PROTAC) for Ferroptosis-Driven Cancer Treatment.
  51. UBA5 missense variants disrupt UFM1 activation: Structural, dynamic, and functional dissection.
  52. Beyond RNA modification: a novel role for tRNA modifying enzyme in oxidative stress response and metabolism.
  53. General trends in the calnexin-dependent expression and pharmacological rescue of clinical CFTR variants.
  54. Protocol for single-molecule analysis of synaptic protein complex-mediated vesicle recruitment.
  55. Leveraging Targeted Protein Degradation for G Protein-Coupled Receptors: The Development of CCR2 Molecular Degraders.
  56. Mutations in mitochondrial ferredoxin FDX2 suppress frataxin deficiency.
  57. CAPN15 is a non-proteasomal, ubiquitin-directed calpain protease that regulates cell adhesion by cleaving E-cadherin.
  58. Targeting the Protein-Protein Interaction Between the CDC37 Co-Chaperone and Client Kinases by an Allosteric RAF Dimer Breaker.
  59. Integrating endogenous TurboID and data-independent acquisition mass spectrometry for in vivo proximity labeling.
  1. bioRxiv. 2025 Dec 01. pii: 2025.11.28.691080. [Epub ahead of print]
    Induced ubiquitination bypasses canonical ERAD to drive ER protein degradation.
    Sydney J Tomlinson, Sean L Johnson, Anna H Kroskrity, Yun Hu, Kirandeep K Deol, Cynthia Y Zhang, Cynthia A Harris, Daniel K Nomura, James A Olzmann.
      Heterobifunctional proteolysis-targeting chimeras (PROTACs) have emerged as a powerful strategy to degrade disease-relevant proteins, enabling targeting of previously "undruggable" proteins. Current degrader molecules primarily target cytosolic substrates, yet nearly one-third of the proteome resides in or transits the endoplasmic reticulum (ER), including receptors, secreted factors, and biosynthetic enzymes with high therapeutic relevance. Whether ER-localized proteins can be broadly targeted for induced degradation remains an open question. To address this gap, we employed a panel of fluorescent reporter cell lines and used the dTAG chemical-genetic system to recruit cytosolic E3 ligases. While lumenal substrates segregated from the cytosol were resistant to degradation, recruitment of cytosolic ligases effectively degraded ER membrane proteins across multiple topologies and with post-translational modifications. CRISPR genetic screens revealed that the induced degradation required the expected cullin RING ligase complexes but surprisingly bypassed ER-associated degradation (ERAD) machinery, with the exception of the AAA ATPase VCP. Mechanistic studies demonstrated that substrate ubiquitination was essential for VCP binding, and cleavage of ubiquitin chains released VCP, suggesting a model in which VCP directly extracts substrates independent of a dislocation apparatus. Extending this strategy to an endogenous substrate, we synthesized an HMGCR ERAD-TAC by linking atorvastatin to a cereblon E3 ligase recruiter and found that HMGCR degradation was likewise VCP-dependent. Together, these findings demonstrate that ER membrane proteins are generally susceptible to induced degradation via cytosolic ligase recruitment, uncovering a VCP-centered mechanism that operates independently of membrane-embedded ERAD machinery. This work establishes foundational principles for extending targeted protein degradation to the early secretory pathway.
    SIGNIFICANCE STATEMENT: Targeted protein degradation has transformed drug discovery. Nearly one-third of the proteome reside in or transit the endoplasmic reticulum (ER), a compartment rich in therapeutically relevant but structurally complex targets. Whether these ER proteins can be broadly degraded using PROTACs has remained unknown. Here, we define the minimal requirements for degrading ER membrane proteins by recruiting cytosolic E3 ligases. Using chemical-genetic tools, genetic screens, and a statin-based degrader, we show that ubiquitination engages the VCP extraction machinery, enabling degradation of diverse ER membrane proteins independent of canonical ER-associated degradation components. These findings reveal a ubiquitin-driven route for membrane protein turnover, expand the landscape of druggable ER proteins, and establish principles for designing degraders operating in the early secretory pathway.
    DOI:  https://doi.org/10.1101/2025.11.28.691080
  2. Mol Cell. 2025 Dec 09. pii: S1097-2765(25)00932-3. [Epub ahead of print]
    GCN2 monitors mRNA translation termination.
    Kailey Worner, Katharine R Maschhoff, Gabrielle M Schuh, Wenqian Hu.
      Controlling mRNA translation is critical for proper protein production. Although translation initiation and elongation regulations are becoming increasingly clear, whether and how translation termination is monitored remains poorly understood. Using an acute protein degradation system coupled with phenotypic rescue via ectopic expression, here we show that the impaired translation termination reaction leads to the rapid activation of GCN2, resulting in eIF2α phosphorylation and inhibition of translation initiation, which occurs prior to ribosome collisions. Ribosome profiling analyses reveal that GCN2 monitors terminating ribosomes and prevents ribosome collisions and translation readthrough when translation termination is compromised. This rapid activation of GCN2 by compromised translation termination occurs in both stem and somatic cells and in mouse and human cells. These results suggest a conserved surveillance mechanism for translation termination.
    Keywords:  GCN2; dTAG; eIF2α phosphorylation; eRF1; terminating ribosome; translation termination
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.015
  3. J Biol Chem. 2025 Dec 06. pii: S0021-9258(25)02878-9. [Epub ahead of print] 111026
    Phosphatidylcholine coordinates ER-autonomous and ER-nonautonomous adaptations to unfolded protein response dysfunction.
    Haixiang Tong, Wei Li, Pangui Yuan, Xinyu Wang, Shanshan Pang, Haiqing Tang.
      The ER UPR plays a crucial role in maintaining proteostasis, with its dysfunction closely associated with aging and various diseases. However, how cells cope with ER UPR dysfunction remains largely unexplored. Here, we report that both ER-autonomous and ER-nonautonomous adaptive responses are activated by defects in the IRE-1/XBP-1 UPR branch in C. elegans. IRE-1/XBP-1 dysfunction not only triggers the activation of the PEK-1 UPR branch but also induces a lysosome-dependent cytosolic proteostatic response. Mechanistically, IRE-1/XBP-1 dysfunction downregulates phosphatidylcholine (PC) metabolism, reducing levels of membrane lipid PC. This PC deficiency drives BORC complex recruitment to lysosomes, triggering lysosomal activation. Furthermore, suppression of phosphatidylcholine metabolism alone sufficiently activates both the ER UPR and lysosomal pathways, thereby enhancing resilience to proteostatic stress and contributing to longevity. These findings provide insights into how cells integrate distinct adaptive responses to maintain systemic proteostasis when the ER UPR is compromised and identify phosphatidylcholine as a potent regulator of proteostasis and aging.
    Keywords:  UPR; aging; lysosome; phosphatidylcholine; proteostasis
    DOI:  https://doi.org/10.1016/j.jbc.2025.111026
  4. Nat Commun. 2025 Dec 10.
    Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling.
    Lianfeng Zhao, Zinan He, Yao Sun, Mengqi Sun, Zheyi Liu, Zhangsen Zhou, Ling Qi, Yuan Luo, Yewei Ji, Tingbo Liang.
      Toll-like receptor 3 (TLR3), an innate immune sensor for double-stranded RNA (dsRNA), traffics from the endoplasmic reticulum (ER) after synthesis to endolysosomes for proteolytic cleavage and activation. However, the molecular mechanisms governing TLR3 trafficking remain largely unclear. Here, we identify the ER-resident E3 ligase HMG-CoA reductase degradation protein 1 (HRD1), a core component of ER-associated degradation (ERAD), as a key regulator that promotes TLR3 trafficking and downstream signaling. HRD1 deficiency in macrophages significantly impairs poly(I:C)-induced TLR3 signaling and inflammatory responses in vitro and in vivo, caused by a marked reduction in TLR3 transport into endolysosomes and subsequent proteolytic processing. Mechanistically, HRD1 mediates ubiquitination of ER-localized TLR3 at lysine 813, which is required for its recognition and sorting by the endosomal sorting complex required for transport (ESCRT) machinery. This HRD1 function is decoupled from its canonical ERAD activity and the ER stress sensor inositol-requiring enzyme 1 alpha (IRE1α). Hence, our study identifies a previously unrecognized mechanism controlling TLR3 signaling and links HRD1-mediated ubiquitination to immune sensor trafficking during innate immune responses.
    DOI:  https://doi.org/10.1038/s41467-025-67219-0
  5. Nat Commun. 2025 Dec 12. 16(1): 11087
    Collision-induced ribosome degradation driven by ribosome competition and translational perturbations.
    Sihan Li, Okuto Shounai, Misaki Kato, Ken Ikeuchi, Toshifumi Inada.
      Individual stalling of catalytically inactive ribosomes at the start codon triggers ubiquitination of ribosomal protein uS3 and subsequent 18S rRNA decay. While collisions between ribosomes during translation elongation represent a more widespread form of translation perturbation, their impact on ribosome stability remains unknown. Here, we clarify a bifurcation in ubiquitination-mediated ribosome turnover, identifying a collision-induced branch of uS3 ubiquitination and small subunit destabilization in yeast. This pathway eliminates not only non-functional ribosomes but also translationally active ones with a prokaryotic-like decoding center, driven by competition with wild-type ribosomes due to differing translation rates. We further show that endogenous ribosomal subunit stoichiometry shifts toward a small-subunit-shortage state via ubiquitination upon perturbed translation triggered by the anti-cancer drug cisplatin and the growth phase transition. These findings reveal a mechanism by which ribosome dynamics generally affects ribosome stability, implicating ribosome dysfunction, heterogeneity, and stress-related translational disturbances in small subunit degradation.
    DOI:  https://doi.org/10.1038/s41467-025-66026-x
  6. J Cell Biol. 2026 Jan 05. pii: e202503075. [Epub ahead of print]225(1):
    TMEDs mediate versatile cargo transport in vesicle-dependent unconventional secretion.
    Jianfei Zheng, Haodong Wang, Yuxin Sun, Pei Chang, Xing Deng, Lijingyao Zhang, Lin Zhu, Kangling Zhu, Dong Peng, Haiteng Deng, Min Zhang, Liang Ge.
      Unconventional protein secretion (UcPS) exports diverse signal peptide-lacking cargoes, yet its cargo selectivity remains poorly understood. Here, we identify TMED proteins as key regulators of vesicle-dependent UcPS, mediating selective cargo release via translocation into secretory carriers. TMED proteins act as translocators, facilitating cargo passage across lipid bilayers with assistance from HSP90 chaperones and partial cargo unfolding. Selectivity arises during translocation, where TMED cytoplasmic tails bind specific cargoes. The ER-Golgi intermediate compartment (ERGIC) is essential for TMED-mediated translocation and release. TMED homo-oligomerization enhances translocation, while hetero-tetramerization inhibits it. ERGIC localization promotes homo-oligomerization, which is further stabilized by cargo binding, forming a feed-forward mechanism to enhance translocation. These findings establish TMED proteins as central regulators of cargo diversity in UcPS, with their oligomerization and subcellular localization modulating translocation efficiency.
    DOI:  https://doi.org/10.1083/jcb.202503075
  7. Cell Chem Biol. 2025 Dec 05. pii: S2451-9456(25)00377-0. [Epub ahead of print]
    Linked-domain inhibitors designed to block UBE2D induce the unfolded protein response.
    Zara Bukhari, Li Gu, Anneroos E Nederstigt, Logan J Cope, Derek L Bolhuis, Kim Harvey, Tristan Allen, Spencer Hill, Yujie Yang, Guy Lawson, Cai Lu, Tommy Tran, Leah Pineda, Leanne Low, Andrew Chiang, Jason Song, Michelle V Fong, Vanessa M Rangel, William K Chan, Gary Kleiger, Dennis Goldfarb, Craig A Vierra, Nicholas G Brown, Joseph S Harrison.
      Ubiquitin (Ub) is a protein post-translational modifier that controls proteostasis through mechanisms spanning transcription, translation, and protein degradation. Ub conjugation occurs through a cascade of three enzyme classes (E1, E2, and E3s) involving >1,000 proteins that regulate the ubiquitination of cellular proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized. Here, we develop highly selective and potent pan-UBE2D/UBCH5 inhibitors by targeting the RING- and backside-binding sites with engineered linked-domain proteins. In HeLa cells, these inhibitors phenocopy the knockdown of UBE2D by enhancing chemosensitivity to cisplatin. Whole-cell proteomics reveals that ∼20% of the identified proteins are more abundant, and most do not have altered mRNA levels, suggesting that their protein turnover is regulated by UBE2D. Enrichment analysis of the altered mRNAs indicates that the linked-domain proteins trigger the unfolded protein response. These precision tools will enable new studies probing UBE2D's cellular roles and help to deconvolute complex Ub regulatory networks.
    Keywords:  E2; E2-ubiquitin conjugating enzymes; UBE2D; linked-domain proteins; proteostasis; proteostress; ubch5; ubiquitin; ubiquitin-like domains; unfolded protein response
    DOI:  https://doi.org/10.1016/j.chembiol.2025.11.007
  8. Nat Commun. 2025 Dec 12. 16(1): 11094
    Functionally diversified Caenorhabditis elegans BiP orthologs control body growth, reproduction, stress resistance, aging, and autophagy.
    Nicholas D Urban, Shannon M Lacy, Kate M Van Pelt, Benedict Abdon, Zachary Mattiola, Adam Klaiss, Sarah Tabler, Eric K F Donahue, Kristopher Burkewitz, Matthias C Truttmann.
      Cellular systems governing protein folding depend on functional redundancy and diversification to maintain proteostasis. Here, using Caenorhabditis elegans, we show two homologous ER-resident HSP70 chaperones, HSP-3 and HSP-4, have overlapping and distinct roles in ER proteostasis and organismal physiology. Their expression and function vary by tissue, age, and stress, impacting ER stress resistance, reproduction, body size, and lifespan. We also find HSP-3 and HSP-4 uniquely regulate dietary restriction and reduced insulin signaling-mediated longevity in C. elegans. Notably, knockdown of hsp-4, but not hsp-3, induces autophagy and enhances tolerance to protein aggregation stress; this process requires the ortholog of ER-Phagy receptor Sec-62 (C18E9.2) and IRE-1. Finally, human cell data suggests that the dissociation of chaperone Binding Immunoglobulin Protein (BiP) from IRE-1 during times of ER stress promotes autophagy by enhancing the interaction of IRE-1 and Sec-62. These findings reveal how ER chaperone diversification maximizes stress resilience and suggest a BiP-dependent regulation of autophagy.
    DOI:  https://doi.org/10.1038/s41467-025-65998-0
  9. FEBS J. 2025 Dec 07.
    The ribosome-associated complex regulates cytosolic translation upon mitoprotein-induced stress.
    Jiaxin Qian, Annika Nutz, Katja Hansen, Lea Bertgen, Mirita Franz-Wachtel, Boris Macek, Johannes M Herrmann, Doron Rapaport.
      The biogenesis of mitochondria relies on the import of newly synthesized precursor proteins from the cytosol. Tom70 is a mitochondrial surface receptor which recognizes precursors and serves as an interface between mitochondrial protein import and the cytosolic proteostasis network. Mitochondrial import defects trigger a complex stress response, in which compromised protein synthesis rates are a characteristic element. The molecular interplay that connects mitochondrial (dys)function to cytosolic translation rates in yeast cells is however poorly understood. Here, we show that the deletion of the two Tom70 paralogs of yeast (TOM70 and TOM71) leads to defects in mitochondrial biogenesis and slow cell growth. Surprisingly, upon heat stress, the deletion of ZUO1, a chaperone of the ribosome-associated complex (RAC), largely prevented the slow growth and the reduced translation rates in the tom70Δ/tom71Δ double deletion mutant. In contrast, the mitochondrial defects were not cured but even enhanced by ZUO1 deletion. Our study shows that Zuo1 is a critical component in the signaling pathway that mutes protein synthesis upon mitochondrial dysfunction. We propose a novel paradigm according to which RAC serves as a stress-controlled regulatory element of the cytosolic translation machinery.
    Keywords:  Tom70; mitochondria; protein import; proteostasis; ribosome‐associated complex
    DOI:  https://doi.org/10.1111/febs.70356
  10. bioRxiv. 2025 Nov 26. pii: 2025.11.24.690135. [Epub ahead of print]
    A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy.
    Vishnu Suresh Babu, Sayan Ghosh, Sridhar Bammidi, Ryu Jiwon, Ruchi Sharma, Dominique Meyer, Stacey Hose, Padmanabhan P Pattabiraman, José-Alain Sahel, Nathan J Kidley, Natércia F Braz, Martin J Slater, Stuart Lang, Arkasubhra Ghosh, Ji Yi, Srinivasa Sripathi, Kannan Rangaramanujam, Kapil Bharti, Debasish Sinha.
      Protein kinases canonically suppress autophagy, yet how cells activate autophagy during stress remains unclear. Here we reveal that AKT1 kinase promotes autophagy through a non-canonical pathway. AKT2 loss triggers compensatory AKT1 activation, which phosphorylates telomerase reverse transcriptase (TERT) at Serine 824, driving nuclear translocation. Nuclear TERT assembles with FOXO3 and c-MYC into a transcriptional complex that activates PERK, initiating a feed-forward loop. PERK-ATF4 signaling amplifies autophagy gene transcription while inducing selective ERphagy through receptors TEX264 and CCPG1. Using C. elegans , mouse models, and human iPSCs, we demonstrate this AKT1-TERT-c-MYC-FOXO3 axis is evolutionarily conserved and essential for proteostasis in post-mitotic cells. We developed a first-in-class allosteric AKT2 inhibitor through structure-guided design that selectively triggers beneficial AKT1 compensation, restoring autophagy in diseased cells. These findings reveal a transcriptional mechanism linking AKT1 activation to autophagy and provide a therapeutic strategy for diseases with defective ER quality control.
    DOI:  https://doi.org/10.1101/2025.11.24.690135
  11. Biochemistry. 2025 Dec 12.
    Endoplasmic Reticulum Redoxome: Protein Folding and Beyond.
    Percillia V S Oliveira, Tiphany C De Bessa, Francisco R M Laurindo.
      The endoplasmic reticulum (ER), the largest cellular organelle, is crucially dependent on its redox organization. First, to optimize disulfide bond formation in nascent proteins, it maintains a relatively oxidizing environment, reminiscent of the extracellular space. Second, it harbors several oxidoreductases from the protein disulfide isomerase (PDI) family, together with Ero1α oxidase and chaperones, which compose interplaying oxidative, reductive, and chaperone pathways to optimize protein processing. Third, disulfide formation and reshuffling in client proteins, involving thiol oxidation and disulfide exchange reactions, connect proteostasis to ER/cellular redox homeostasis. ER redox folding involves Ca2+-dependent liquid phase separation of PDI complexes. Calcium fluxes heavily interplay with dynamic redox regulation. ER stress disrupts the ER redox state and, in turn, is also regulated by cellular redox processes. Moreover, the ER makes membrane contacts with many other organelles such as plasma membrane, peroxisomes, and mitochondria, which are hubs for mutually dependent oxidant and calcium-linked effects. Furthermore, the ER redoxome extends to other subcellular and extracellular locations, a process we termed the "ER-dependent outreach redoxome (ERDOR)". ERDOR can occur by overflow of ER products such as H2O2, mobility of ER-associated domains or, mainly, via ER oxidoreductase translocation. The ER establishes a particular communication with the extracellular milieu via translocation of PDIs. Despite the low levels of extracellularly located ER oxidoreductases, they redox-regulate several molecular targets and may compose a peri/epicellular redox network. This article provides a comprehensive overview of the ER redoxome as an important emerging frontier to understand not only redox proteostasis but also intra- and intercellular redox communication.
    Keywords:  NADPH oxidase; NOX; oxidants; phenotype; protein disulfide isomerase; vascular smooth muscle cells
    DOI:  https://doi.org/10.1021/acs.biochem.5c00527
  12. J Biol Chem. 2025 Dec 09. pii: S0021-9258(25)02882-0. [Epub ahead of print] 111030
    Small heat shock protein HSPB5 uses disorder to bind zinc with high affinity.
    Maria K Janowska, Vanesa Racigh, Christoper N Woods, María Silvina Fornasari, Rachel E Klevit.
      Zinc is an essential metal that supports diverse cellular functions. Zinc exerts its biological activity through protein binding, serving as catalytic cofactors and structural stabilizers of many enzymes, transcription factors, and ubiquitin E3 ligases, among others. Despite total cellular zinc concentrations reaching hundreds of micromolar, free zinc levels are tightly buffered. Elevated free zinc promotes protein mismetalation and aggregation. While zinc is redox-inert, its cysteine-based protein ligands are readily oxidized. Oxidative modification of cysteines leads to zinc dissociation and a rapid increase in free zinc. With ∼3000 proteins in the human zinc proteome, uncontrolled zinc release could be highly deleterious. Metallothioneins buffer zinc under basal conditions, but their re-synthesis following oxidative inactivation occurs on the timescale of hours, raising the question of how free zinc is managed in the interim. Histidine, the second most prevalent zinc-coordinating residue, is resistant to oxidative modification. We characterized zinc binding by the small heat shock protein HSPB5 (αB-crystallin), a cysteine-free, histidine-rich protein chaperone that responds to cellular stress and found: (1) HSPB5 binds zinc with high affinity and rapid reversibility; (2) zinc binding requires the disordered HSPB5 N-terminal region; (3) zinc binding increases HSPB5 disorder; and (4) prolonged zinc exposure promotes formation of assemblies of oligomers cross-bridged by zinc. We propose that HSPB5 has evolved specialized zinc-dependent properties distinct among human sHSPs, enabling it to function not only as a protein chaperone but also as a conditional zinc reservoir under oxidative stress.
    Keywords:  HSPB5; alphaB crystallin; chaperone; intrinsic disorder; metalloproteins; small heat shock proteins; zinc
    DOI:  https://doi.org/10.1016/j.jbc.2025.111030
  13. Nat Commun. 2025 Dec 11.
    Chemically-induced degradation of the endoplasmic-reticulum stress sensor IRE1 by a VHL-recruiting chimera.
    Jin Du, Elisia Villemure, Matthew Johnson, Caleigh Azumaya, Catarina J Gaspar, Scot Marsters, David Lawrence, Scott Foster, Alexis Rohou, Tommy K Cheung, Christopher M Rose, Thomas Garner, Soo Ro, Kevin Clark, Maureen H Beresini, Marie-Gabrielle Braun, Joachim Rudolph, Peter Hsu, Avi Ashkenazi.
      The endoplasmic-reticulum (ER) transmembrane protein IRE1 mitigates ER stress through kinase-endoribonuclease and scaffolding activities. Cancer cells often co-opt IRE1 to facilitate growth. An IRE1-RNase inhibitor has entered clinical trials; however, recent work uncovered a significant nonenzymatic IRE1 dependency in cancer. To fully disrupt IRE1, we describe a proteolysis-targeting chimera (G6374) that couples an IRE1-kinase ligand to a compound that binds the ubiquitin Cullin-RING Ligase (CRL) substrate receptor, VHL. G6374 induces a stable, cooperative interaction between IRE1 and VHL, driving K48-linked ubiquitination on two principal lysine residues in the IRE1-kinase domain and inducing proteasomal IRE1 degradation. Cryogenic electron microscopy and mutagenesis studies reveal a 2:2 IRE1:VHL ternary-complex topology and critical interactional features, informing future designs. G6374 blocks growth of IRE1-dependent cancer cells irrespective of their dependency mode, while sparing IRE1-independent cells. We provide a proof-of-concept for VHL-based degradation of an ER-transmembrane protein, advancing strategies to fully disrupt IRE1.
    DOI:  https://doi.org/10.1038/s41467-025-66382-8
  14. Curr Protoc. 2025 Dec;5(12): e70278
    Synthesis of Immunoproteasome N-Degron Prodrugs for Selective Protein Degradation.
    Cody A Loy, Timothy J Harris, Darci J Trader.
      Targeted protein degradation using bifunctional molecules like proteolysis targeting chimeras (PROTACs) has revolutionized drug discovery but remains limited by E3 ligase availability and lack of cell selectivity. N-degron degraders provide a smaller, more drug-like alternative that harnesses endogenous N-terminal degradation signals independent of recruited ligases. However, they are prone to proteolysis and lack mechanisms for tissue-specific activation. To overcome these challenges, we developed a "caged" N-degron degrader selectively activated by the immunoproteasome (iCP), a proteasome isoform induced in cancer and inflammatory settings. By appending an iCP-recognition tetrapeptide (Ala-Thr-Met-Trp) capped with a morpholine group to the N-terminus, we shielded the degron from nonspecific cleavage and enabled selective activation in iCP-expressing cells. This design improved stability, enhanced degradation potency, and minimized activity in healthy cells. Our findings establish a generalizable strategy to spatially control N-degron activity through disease-specific proteolytic environments, advancing safer and more selective protein degradation therapeutics. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Synthesis and characterization of an N-degron degrader for Abl Basic Protocol 2: Synthesis and characterization of an immunoproteasome N-degron prodrug Basic Protocol 3: Assessing protein degradation in cells.
    Keywords:  N‐degron; immunoproteasome; prodrug; proteostasis; targeted protein degradation
    DOI:  https://doi.org/10.1002/cpz1.70278
  15. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1334. [Epub ahead of print]53(22):
    The RNA-binding protein PRRC2B preserves 5' TOP mRNA during starvation to maintain ribosome biogenesis during nutrient recovery.
    Nadav Goldberg, Doron Bril, Miriam Eisenstein, Tsviya Olender, Alon Savidor, Shani Bialik, Shmuel Pietrokovski, Adi Kimchi.
      PRRC2B is an intrinsically disordered RNA-binding protein that is part of the cell's translation machinery. Here, we show that PRRC2B has two alternatively spliced mRNA transcripts producing major long and minor short isoforms. Mass spectrometry-based interaction studies indicated that both isoforms associate with the 40S ribosomal subunit and translation initiation factors. Importantly, the long isoform also interacted with additional RNA-binding proteins through its unique Arg/Gly-rich region. Among these is LARP1, a regulator of 5' terminal oligopyrimidine (TOP) mRNAs under conditions of mTOR inhibition. We discovered that, like LARP1, PRRC2B-long isoform binds to 5' TOP mRNAs. Moreover, it is necessary for the post-transcriptional preservation of their mRNA levels, particularly those encoding ribosomal proteins, during amino acid starvation. In its absence, the rapid de novo translation of ribosomal proteins that takes place upon nutrient recovery is impeded. Overall, our study elucidates a newly discovered function for PRRC2B as an RNA-binding protein that regulates ribosomal biogenesis upon metabolic shift, in addition to its established function in initiating translation of specific mRNA targets.
    DOI:  https://doi.org/10.1093/nar/gkaf1334
  16. Proc Natl Acad Sci U S A. 2025 Dec 16. 122(50): e2504528122
    Molecular mechanisms underlying p62-dependent secretion of the Alzheimer-associated ubiquitin variant UBB+1.
    Ajay R Wagh, Michael H Glickman.
      UBB+1, a ubiquitin variant protein resulting from a frameshift in the ubiquitin-B gene, is a pathological hallmark of Alzheimer disease (AD). At the cellular level, UBB+1 disrupts the ubiquitin-proteasome system while inducing autophagy. Notably, UBB+1 itself is secreted via autophagosome-like vesicles. Here, we demonstrate that UBB+1 can be removed from the cell by degradative and secretory autophagy. Sequestosome 1 (SQSTM1)/p62 functions as a pivotal ubiquitin receptor for UBB+1, recognizing its ubiquitin domain and facilitating loading into autophagosomes. Oligomerization of SQSTM1/p62 was critical to isolate UBB+1 in bodies preventing its aggregation. Intriguingly, both gain- and loss-of-function SQSTM1/p62 suppressed UBB+1 secretion, causing intracellular retention: SQSTM1/p62 knockout led to UBB+1 accumulation in insoluble aggregates, while its overexpression promoted the formation of p62-UBB+1 bodies. We further identified distinct roles for SNARE-mediated membrane fusion in secretory autophagy of UBB+1. Specifically, the R-SNARE SEC22B and the Q-SNAREs Syntaxin-4 and SNAP23 participated in UBB+1 exocytosis. Disruption of SEC22B impaired the fusion of UBB+1-containing autophagosomes with the plasma membrane, reducing UBB+1 secretion without affecting its intracellular turnover. Inhibition of lysosomes partially stabilized UBB+1 indicating that degradation and secretion are complementary processes that determine the fate of UBB+1. This study elucidates the dual roles of autophagy in managing neurotoxic proteins, highlighting SQSTM1/p62 as a key mediator of UBB+1 trafficking and secretion. Although ubiquitin typically acts as a degradation signal, our findings reveal a rare instance of a ubiquitin-related protein driving secretory autophagy. These findings advance our understanding of cellular mechanisms underlying the clearance of misfolded proteins in neurodegenerative diseases.
    Keywords:  Alzheimer’s disease; autophagy; p62; trafficking; ubiquitin
    DOI:  https://doi.org/10.1073/pnas.2504528122
  17. Nat Commun. 2025 Dec 12. 16(1): 10870
    A widespread protein misfolding mechanism is differentially rescued in vitro by chaperones based on gene essentiality.
    Ian Sitarik, Quyen V Vu, Justin Petucci, Paulina Frutos, Hyebin Song, Edward P O'Brien.
      Protein misfolding involving changes in non-covalent lasso entanglement (NCLE) status has been proposed based on simulations and biochemical assays of a small number of proteins. Here, we detect hallmarks of these misfolded states across hundreds of proteins by integrating E. coli proteome-wide limited-proteolysis mass spectrometry data with structural datasets of protein native structures. Proteins containing native NCLEs are twice as likely to misfold, predominantly in regions where these NCLEs naturally occur. Surprisingly, the chaperones DnaK and GroEL do not typically correct this misfolding, except in the case of essential proteins. Statistical analysis links this differential rescue activity to weaker loop-closing contacts in the NCLEs of essential proteins, suggesting misfolding involving these loops is easier to rectify by chaperones. Molecular simulations indicate a mechanism where premature NCLE loop closure, prior to proper placement of the threading segment, leads to persistent misfolded states. This mechanism can explain why, in this mass spectrometry dataset, proteins with NCLEs are more likely to misfold and misfold in NCLE regions. These results suggest the potential for widespread NCLE misfolding, that such misfolded states in non-essential proteins could bypass the refolding action of chaperones, and that some protein sequences may have evolved to allow chaperone rescue from this class of misfolding.
    DOI:  https://doi.org/10.1038/s41467-025-66236-3
  18. Nat Cell Biol. 2025 Dec 09.
    Endoplasmic reticulum disruption stimulates nuclear membrane mechanotransduction.
    Zhouyang Shen, Zaza Gelashvili, Philipp Niethammer.
      Cytosolic phospholipase A2 (cPLA2) controls some of the most powerful inflammatory lipids in vertebrates by releasing their metabolic precursor, arachidonic acid, from the inner nuclear membrane (INM). Ca2+ and INM tension (TINM) are thought to govern the interactions and activity of cPLA2 at the INM. However, as compensatory membrane flow from the contiguous endoplasmic reticulum (ER) may prevent TINM, the conditions permitting nuclear membrane mechanotransduction by cPLA2 or other mediators remain unclear. To test whether the ER buffers TINM, we created the genetically encoded, Ca²⁺-insensitive TINM biosensor amphipathic lipid-packing domain inside the nucleus (ALPIN). Confocal time-lapse imaging of ALPIN- or cPLA2-INM interactions, along with ER morphology, nuclear shape/volume and cell lysis revealed a link between TINM and disrupted ER-nuclear membrane contiguity in osmotically or ferroptotically stressed mammalian cells and at zebrafish wound margins in vivo. By combining ALPIN imaging with Ca2+-induced ER disruption, we reveal the causality of this correlation, which suggests that compensatory membrane flow from the ER buffers TINM without preventing it. Besides consolidating the biomechanical basis of cPLA2 activation by nuclear deformation, our results identify cell stress- and cell death-induced ER disruption as an additional nuclear membrane mechanotransduction trigger.
    DOI:  https://doi.org/10.1038/s41556-025-01820-9
  19. Nat Commun. 2025 Dec 12.
    AGPAT2 acts at the crossroads of lipid biosynthesis and DRP1-mediated ER morphogenesis.
    Yoshihiro Adachi, Mauricio Torres, Allen H Hunter, Woo Jung Cho, Meixia Pan, Xianlin Han, Guangwei Du, Ling Qi, Miho Iijima, Hiromi Sesaki.
      The morphology of the endoplasmic reticulum (ER), characterized by central sheets and peripheral tubules, is controlled by membrane-shaping proteins. However, the role of lipids in ER morphogenesis remains elusive, despite the ER being the major site for lipid synthesis. Here, by examining the role of eighteen phosphatidic acid (PA)-generating enzymes in ER morphology, we identify lysophosphatidic acid acyltransferase 2 (AGPAT2) as a critical factor in mouse and human cells. AGPAT2 produces PA in the glycerophospholipid/triacylglycerol biosynthesis pathway, and its mutations cause congenital generalized lipodystrophy. We find that AGPAT2-generated PA drives ER tubulation through gene knockout, 3D structural analysis by FIB-SEM, super-resolution microscopy, lipidomics, AlphaFold, and in vitro reconstitutions of ER tubulation and AGPAT2 activity. AGPAT2 interacts with and supplies PA to the PA-binding, dynamin-related GTPase, DRP1, which subsequently tubulates the ER in a manner independent of GTP hydrolysis and oligomerization, distinct from its function in mitochondrial division. Consistently, the reduction of PA levels by ectopic expression of a PA phosphatase, LIPIN1, transforms ER tubules into sheets. Our results reveal an unforeseen interplay between lipid biosynthesis and membrane organization in the ER.
    DOI:  https://doi.org/10.1038/s41467-025-66474-5
  20. Nat Commun. 2025 Dec 12. 16(1): 11093
    Cysteine-enabled cleavability to advance cross-linking mass spectrometry for global analysis of endogenous protein-protein interactions.
    Fenglong Jiao, Merav Braitbard, Clinton Yu, Ben Shor, Bjorn-Erik Wulff, Dina Schneidman-Duhovny, Lan Huang.
      Cross-linking mass spectrometry (XL-MS) is a powerful technology for probing protein-protein interactions (PPIs) and elucidating architectures of protein complexes at the systems level. While successful, the proteome coverage remains limited. To expand the scope of global PPI profiling, we introduce an innovative cysteine-based cleavable XL-MS platform using non-cleavable heterobifunctional lysine-cysteine (K-C) cross-linkers. The oxidation-induced transformation of cysteine cleavability enables unambiguous identification of cross-linked peptides. This strategy has been successfully applied to proteome-wide XL-MS analysis of intact cells, and its broad applicability has been demonstrated using three heterobifunctional cross-linkers. A total of 25,401 unique linkages from 2007 proteins have been identified, significantly expanding the existing XL-PPI map and increasing the interconnectivity of the human interactome. The XL-data generated here has been coupled with AlphaFold-based predictions and integrative modeling to reveal the structural characteristics of cellular networks, offering insights into the organization of native protein complexes in cells including the SERBP1-ribosome and eEF1A1-eEF1B complexes. Due to the effectiveness in modulating cysteine cleavability, our work presents an avenue for developing bifunctional/multifunctional cross-linking reagents to further advance XL-MS technologies. Additionally, the same strategy can be easily adapted to facilitate the characterization of cysteine modifications, reactivity and interactions to benefit chemical proteomics.
    DOI:  https://doi.org/10.1038/s41467-025-66023-0
  21. Mol Cell. 2025 Dec 10. pii: S1097-2765(25)00934-7. [Epub ahead of print]
    HYPK promotes N-terminal protein acetylation through rapid ribosome exchange of NatA.
    Alfred M Lentzsch, Ziyi Fan, Inayat U Irshad, Edward P O'Brien, Ajeet K Sharma, Rachel Green, Shu-Ou Shan.
      Numerous protein biogenesis factors cotranslationally facilitate the maturation of nascent proteins. Among them, N-terminal acetyltransferase A (NatA) acetylates the N terminus of ∼40% of the eukaryotic proteome. NatA is bound to Huntingtin-interacting protein K (HYPK), which inhibits NatA activity in vitro but enhances function in vivo. Here, kinetic and in-cell measurements resolve this paradox, showing that HYPK acts as a ribosome exchange factor for NatA. Without HYPK, hyper-tight ribosome binding prevents NatA from accessing additional ribosomes following each round of acetylation. HYPK accelerates NatA dissociation from the ribosome to license multiple turnovers, allowing a sub-stoichiometric level of this enzyme to globally acetylate the nascent proteome. Our results uncover a previously unidentified function of HYPK and demonstrate that a "Goldilocks" zone of ribosome interaction kinetics is required for cotranslational protein biogenesis machineries to act on all translating ribosomes in the cell.
    Keywords:  HYPK; N-acetyltransferase; cotranslational protein modification; exchange factor; ribosome
    DOI:  https://doi.org/10.1016/j.molcel.2025.11.017
  22. Nat Commun. 2025 Dec 06.
    Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.
    Arden Darko-Boateng, Emmanuel Afriyie, Travis J Morgenstern, Sri Karthika Shanmugam, Xinle Zou, Yianni D Laloudakis, Papiya Choudhury, Meera Desai, Robert S Kass, Francesca Vallese, Oliver B Clarke, Henry M Colecraft.
      Targeted protein degradation/downregulation (TPD/TPDR) is a disruptive paradigm for developing therapeutics. <2% of ~600 E3 ligases have been exploited for this modality, and efficacy for multi-subunit ion channels has not been demonstrated. NEDD4-2 E3 ligase regulates myriad ion channels, but its utility for TPD/TPDR is uncertain due to complex regulatory mechanisms. Here, we identify a nanobody that binds NEDD4-2 HECT domain without disrupting catalysis sites as revealed by cryo-electron microscopy and in vitro ubiquitination assays. Recruiting NEDD4-2 to diverse ion channels (CaV2.2; KCNQ1; and epithelial Na+ channel, ENaC, with a Liddle syndrome mutation) using divalent nanobodies (DiVas) strongly suppresses their surface density and function. Global proteomics indicates DiVa recruitment of endogenous NEDD4-2 to KCNQ1-YFP yields dramatically lower off-target effects compared to NEDD4-2 overexpression. The results establish utility of NEDD4-2 recruitment for TPD/TPDR, validate ion channels as susceptible to this modality, and introduce a general method to generate ion channel inhibitors.
    DOI:  https://doi.org/10.1038/s41467-025-67068-x
  23. Protein Cell. 2025 Dec 10. pii: pwaf107. [Epub ahead of print]
    Context Matters: E3 Ligase-Ligand Pairing Strategies for Optimized PROTAC Performance.
    Luyao Yin, Pengcheng Shu, Xiaozhong Peng.
      PROTACs (proteolysis targeting chimeras) offer a revolutionary strategy to degrade proteins previously considered "undruggable." While the importance of the target protein ligand and linker is well-established, the strategic selection of an E3 ubiquitin ligase and its corresponding ligand is an equally critical but underexplored determinant of PROTAC efficacy and selectivity. This perspective systematically analyzes how E3 ligase-ligand pairing dictates degradation outcomes across diverse biological contexts. Our analysis, incorporating head-to-head comparisons, demonstrates that no single E3 ligand is universally superior. Instead, degradation efficiency is profoundly modulated by ternary complex cooperativity, cell-type specificity, and tissue distribution. CRBN-based degraders frequently excel in hematologic malignancies, while VHL-based PROTACs show advantages in certain solid tumors. We further highlight emerging E3 ligands (e.g., from IAP, DCAF families) as promising tools to overcome resistance and expand the degradable proteome. The perspective also explores innovative frontiers, including the potential for targeting non-protein substrates and the application of PROTACs as versatile chemical knockdown tools in research. Ultimately, this paper underscores the central paradigm that "context dictates strategy" in E3 ligase selection, providing a critical framework for optimizing PROTAC design and broadening their therapeutic and research applications.
    Keywords:  CRBN; E3 Ligand; E3 Ligase; PROTAC; VHL
    DOI:  https://doi.org/10.1093/procel/pwaf107
  24. Nat Commun. 2025 Dec 12. 16(1): 11092
    Diet-induced RKIP downregulation disrupts PC/PE-ER homeostasis to drive MASLD.
    Mengjie Li, Qian Ou, Qiang Qin, Jiaxi Chen, Sen Yang, Jie Zhao, Hua Meng, Xu Li, Pinglong Xu, Cunqi Ye, Xiaojian Wang.
      High-fat diet (HFD) is a risk factor for metabolic dysfunction-associated steatotic liver disease (MASLD), yet the molecular pathways that connect dietary fats to liver dysfunction remain unclear. Here, we discover that hepatic downregulation of Raf kinase inhibitory protein (RKIP) in MASLD patients and male mice is linked to fatty acid uptake, which causes endoplasmic reticulum (ER)-associated degradation of RKIP by inhibiting its S-palmitoylation. Via facilitating the m6A-modified RNA binding of YTHDF1, RKIP is required for the efficient translation of PEMT, an essential enzyme in maintaining phosphatidylcholine (PC) / phosphatidylethanolamine (PE) ratio and ER homeostasis. Hepatocyte-specific RKIP depletion in male mice exacerbates the PC/PE imbalance and ER stress, resulting in lipid droplets accumulation and MASLD progression. Notably, RKIP correlates positively with PEMT protein but inversely with MASLD development. These findings uncover a cellular mechanism of HFD-RKIP-PEMT that underlies diet-induced liver metabolic disease and propose RKIP as a target for MASLD prevention.
    DOI:  https://doi.org/10.1038/s41467-025-65982-8
  25. Autophagy. 2025 Dec 08.
    Trans-Golgi network-associated noncanonical autophagy depends on the V-ATPase-ATG16L1 axis and mediates IL1B secretion.
    Jiahui Long, Huazhong Xie, Hao Shan, Ruoqing Chen, Jialing Zhong, Jiuge Tang, Dongmei Fang, Yao Wang, Peiqing Liu, Xiao-Ming Yin, Min Li.
      Formation of MAP1LC3/LC3 (microtubule associated protein 1 light chain 3)-positive structures that does not require all of the core ATG (autophagy related) proteins is emerging in the process of noncanonical autophagy (NCA). While LC3 lipidation on endolysosomal membranes has been well characterized, the involvement of other membrane sources and the regulatory mechanisms governing LC3 lipidation in alternative forms of NCA remain poorly understood. Here, we demonstrate the occurrence of LC3 lipidation on the trans-Golgi network (TGN) platform. Different from canonical autophagosomes, these LC3-positive structures do not fuse with lysosomes, and fail to degrade long-lived proteins. In addition, the functional vacuolar-type H+-translocating ATPase (V-ATPase)-ATG16L1 axis is found to be essential for TGN-associated NCA. Notably, in this process, the cytosolic but not lysosomal V1 complex of the V-ATPase assembles at the TGN and plays a pivotal role in further induction of NCA. Eventually, IL1B/IL-1β (interleukin 1 beta) secretion is found to be efficiently enhanced by such TGN-associated NCA, independently of GSDM (gasdermin)-mediated pore formation. Thus, besides the known endolysosome-related NCA, we identify a distinct form of TGN-associated NCA mediated by the V-ATPase-ATG16L1 axis. Such NCA might work as a protein-transport route for the extracellular secretion of IL1B, revealing a mechanism linking Golgi-derived NCA to inflammatory cytokines release.
    Keywords:  ATG16L1; CASM; TGN; V-ATPase; membrane atg8ylation; unconventional protein secretion
    DOI:  https://doi.org/10.1080/15548627.2025.2601896
  26. PLoS Biol. 2025 Dec;23(12): e3003545
    The deubiquitinating enzyme Cezanne stabilizes BRCA1 by counteracting APC/C and Ube2S-dependent Lys11-linked ubiquitination.
    Longqiang Wang, Xiao Wu, Atanu Paul, Jun Yao, Bin Wang.
      The breast and ovarian tumor suppressor BRCA1 is a cell cycle-regulated protein and tumors with reduced BRCA1 protein level may share molecular features of BRCA1-mutant tumor and respond to PARPi therapy. Here, we identify that BRCA1 protein stability is controlled through ubiquitin lysine 11 (K11)-linkage modification under the regulation of Cezanne deubiquitinating enzyme, APC/C E3 ligase, and Ube2S E2 conjugating enzyme in a cell cycle-dependent manner. Cezanne-deficiency leads to increased BRCA1 K11-ubiquitination, decreased BRCA1 protein level, and increased cellular sensitivity to PARPi. The BRCA1 K11-linked ubiquitination is carried out through a degron on BRCA1 that is recognized by APC/C cofactor Cdh1. Tumor expression and mutational analyses indicate that Cezanne low or Ube2S high expression is associated with "BRCAness" and correlated with poor prognosis in breast cancer patients. Thus, our study has demonstrated a ubiquitin K11-linked ubiquitination pathway that regulates BRCA1 protein stability, dysregulation of which predicts BRCA1-deficiency that may be effectively targeted with PARPi therapy.
    DOI:  https://doi.org/10.1371/journal.pbio.3003545
  27. Biochem Soc Trans. 2025 Dec 11. pii: BST20253082. [Epub ahead of print]
    Regulation via compartmentation: adaptive localization of the proteasome under stress.
    Aaron Ciechanover, Ido Livneh.
      Protein degradation by the ubiquitin-proteasome system and autophagy are essential mechanisms that are involved in virtually all cellular activities, and their inadequate function was shown to underlie the pathogenesis of various medical conditions. Much of the study into these proteolytic systems has been focused on the components that facilitate the selective substrate identification and targeting for degradation. Given that most of the specific breakdown of proteins is mediated via their modification by ubiquitin, much research was dedicated to the enzymes which are responsible for substrate recognition and ubiquitination-E3 ubiquitin ligases. In addition to the complexity of substrate recognition and targeting for degradation, the mechanisms governing proteasome function were found to be tightly regulated, including the assembly of the different proteasomal sub-complexes, its different compositions and specialized subtypes such as the immunoproteasome, posttranslational modification of proteasomal subunits, and adaptations in its activity in face of different cellular states and stress conditions. Studies from recent years have highlighted an as-yet unexplored tier of proteasome regulation, namely its subcellular compartmentation and trafficking. Intracellular proteasome shuttling was shown to serve as an essential stress-coping mechanism in tumor cells and is emerging as a potential target for therapeutic interventions.
    Keywords:  proteasome dynamics; proteasomes; proteostasis; stress response; ubiquitin proteasome system; ubiquitin signaling
    DOI:  https://doi.org/10.1042/BST20253082
  28. Proc Natl Acad Sci U S A. 2025 Dec 16. 122(50): e2501361122
    TAPT1 interacts with SUCO to maintain the homeostasis of newly synthesized proteins and brain development in mice.
    Jiawei Gao, Fuqiang Yang, Yisheng Jiang, Yu Zheng, Lu Cai, Xiahe Huang, Li Yuan, Yingchun Wang, Yaqing Wang, Zhiheng Xu.
      Genetic mutations in Tapt1 cause complex skeletal dysplasia and structural brain abnormalities. Although the pathogenesis underlying skeletal dysplasia has been explored, the functions and potential mechanisms of transmembrane anterior-posterior transition 1 (TAPT1) during brain development have not been reported. Here, we show that the brains of Tapt1 conditional knockout mice exhibit severe neurodevelopmental defects, including impaired proliferation and differentiation of neural progenitor cells and defects in dendritic and synaptic development, leading to severe microcephaly, motor dysfunction, and early death. Mechanically, we reveal that TAPT1 interacts with SUCO in the endoplasmic reticulum to maintain newly synthesized proteins, including those important for brain development. The TAPT1-SUCO complex plays an essential role in the homeostasis of newly synthesized proteins, and its loss causes overactivated protein degradation, as well as impaired endoplasmic reticulum-to-Golgi trafficking and organelle structures. Our results thus provide insights into the pathogenesis of TAPT1 and SUCO mutation-associated diseases that share similar pathologies.
    Keywords:  ER; SUCO; TAPT1; brain development; newly synthesized proteins
    DOI:  https://doi.org/10.1073/pnas.2501361122
  29. Nat Commun. 2025 Dec 12.
    LRRC59 cooperates with nuclear transporters to restrain the nuclear envelope repair machinery and safeguard genome integrity.
    Romy Timmer, Aurélie Bellanger, Sarah Peeters, Hera Kim, Laura Rodriguez de la Ballina, Sissel Eikvar, Annemijn J Arns, Esmée Oortgijs, Nikolina Sekulić, Winnok H De Vos, Coen Campsteijn.
      Nuclear envelope (NE) rupture is a hallmark of cancer cells, and persistent NE damage drives genome instability and inflammation. NE repair relies on activation of the endosomal sorting complex required for transport (ESCRT)-III repair machinery by the LEMD2-CHMP7 compartmentalization sensor, but little is known beyond these core factors. Here, we use convergent proximity proteomics to inventorise proteins mobilized to the NE upon assembly of LEMD2-CHMP7 and activation of ESCRT-III. Within this NE repairome, we identify LRRC59 as a critical regulator of LEMD2 accumulation at NE ruptures. We find that LRRC59, together with the nuclear transporters KPNB1 and XPO1, restricts the assembly of LEMD2-CHMP7 complexes to the site of rupture. Disruption of this regulatory axis escalates LEMD2-CHMP7 spreading across the NE, driving torsional DNA damage in ruptured nuclei and micronuclei. Thus, our work identifies a central regulatory layer of NE repair centered on LRRC59 and KPNB1. We propose that altered LRRC59 levels and deregulated nuclear transport coordinately compromise NE repair, driving genome instability and cancer development.
    DOI:  https://doi.org/10.1038/s41467-025-65994-4
  30. bioRxiv. 2025 Nov 12. pii: 2025.10.31.685870. [Epub ahead of print]
    Endoplasmic reticulum-mitochondrion disconnection promotes metabolic reprogramming and cystogenesis in polycystic kidney disease.
    Biswajit Padhy, Jian Xie, Danish Idrees, Chih-Jen Cheng, Chou-Long Huang.
      Mutations in PKD1 and PKD2 cause autosomal-dominant polycystic kidney disease (ADPKD), characterized by fluid-filled cysts, aberrant cell proliferation, and widespread genetic and epigenetic remodeling. While mitochondrial dysfunction and metabolic shifts are central to disease progression, the mechanisms linking PKD mutations to these changes remain unclear. Here, we demonstrate that ER-mitochondria connectivity was disrupted in Pkd1- and Pkd2- deleted mice, preceding cyst formation. This disconnection induces mitochondrial stress, triggering epigenetic remodeling and transcriptional activation of pathways driving proliferation and metabolic reprogramming. Remarkably, restoring PKD function in the ER or pharmacologically enhancing ER-mitochondria connection ameliorates mitochondrial dysfunction, epigenetic shifts, and cystogenesis. These findings reveal a critical role for ER-localized PKD in maintaining mitochondrial integrity and transcriptional homeostasis. Mitochondrial dysfunction resulting from ER-mitochondria uncoupling emerges as a key driver of cystogenesis in ADPKD, and correcting this defect may offer a promising therapeutic strategy.
    Significance: Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent monogenic cause of kidney failure, marked by fluid-filled cysts, aberrant cell proliferation, metabolic reprogramming, and extensive genetic and epigenetic alterations. The mechanisms by which loss-of-function mutations in PKD1 and PKD2 drive disease progression remain poorly understood. Here, we demonstrate that ER-mitochondria contacts are disrupted in Pkd -mutant mice prior to cyst formation. This disconnection induces mitochondrial dysfunction and epigenetic remodeling, which in turn promote metabolic reprogramming and cystogenesis. Restoration of PKD function in the ER or pharmacological enhancement of ER-mitochondria coupling mitigates these pathological changes. Our findings uncover a critical role for ER-mitochondria crosstalk in suppressing cystogenesis and identify a promising therapeutic target for ADPKD.
    DOI:  https://doi.org/10.1101/2025.10.31.685870
  31. Cell Syst. 2025 Dec 10. pii: S2405-4712(25)00286-8. [Epub ahead of print] 101453
    A reconstruction of the mammalian secretory pathway identifies mechanisms regulating antibody production.
    Helen Masson, Jasmine Tat, Pablo Di Giusto, Athanasios Antonakoudis, Isaac Shamie, Hratch Baghdassarian, Mojtaba Samoudi, Caressa M Robinson, Chih-Chung Kuo, Natalia Koga, Sonia Singh, Angel Gezalyan, Zerong Li, Alexia Movsessian, Anne Richelle, Nathan E Lewis.
      The secretory pathway processes >30% of mammalian proteins, orchestrating their synthesis, modification, trafficking, and quality control across multiple organelles via coordinated interactions, making its regulation difficult to decipher. To advance such research, we present secRecon, a reconstruction of the mammalian secretory pathway, comprising 1,127 manually curated genes organized within 77 secretory process terms, annotated with functional roles, subcellular localization, protein interactions, and complexes. Applying secRecon to omics data revealed distinct secretory topologies in antibody-producing plasma cells versus Chinese hamster ovary (CHO) cells, with CHO-specific deficiencies in proteostasis, translocation, and N-glycosylation genes, highlighting targets to enhance secretion. Analysis of single-cell SEC-seq data uncovered diversity in IgG-secreting plasma cells that is shaped by the unfolded protein response, endoplasmic reticulum (ER)-associated degradation, and vesicle trafficking and identified distinct secretory machinery genes as markers of plasma cell differentiation. These results show that secRecon enables the discovery of mechanisms controlling protein secretion and supports applications in both biomedical research and biotechnology. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  CHO cells; SEC-seq; multi-omics; plasma cells; secRecon; secretory pathway reconstruction
    DOI:  https://doi.org/10.1016/j.cels.2025.101453
  32. Redox Biol. 2025 Dec 09. pii: S2213-2317(25)00479-3. [Epub ahead of print]89 103966
    Activation of the integrated stress response contributes to developmental delay and seizures caused by mitochondrial prolyl-tRNA synthetase (PARS2) deficiency.
    Man Xu, Yuxin Liu, Runhua Ma, Wenlu Fan, Jingwei Duan, Huan Deng, Yanbin Ma, Wanzhong Ge.
      Mutations in mitochondrial aminoacyl-tRNA synthetases (mtARSs) causes mitochondrial defects and serious, progressive and usually lethal diseases with exceptional heterogeneous and tissue-specific clinical manifestations. However, the pathogenic mechanisms for specific mtARS related diseases are largely unknown and currently there is no highly effective treatment or cure for these diseases. In the present study, we generate Drosophila models with human mitochondrial prolyl-tRNA synthetase (PARS2) deficiency by knocking out or knocking down dPARS2, the Drosophila ortholog of human PARS2, and further characterize the disease-associated defects and explore the molecular basis of these phenotypes. Inactivation of dPARS2 in Drosophila causes developmental delay and seizure, two main clinical features in human PARS2 deficiency-associated patients. Biochemical analysis demonstrates that loss of dPARS2 activity results in reduced mitochondrial tRNAPro aminoacylation, decreased levels of OXPHOS complex proteins, defective assembly and altered enzyme activities of OXPHOS complexes. Interestingly, we discover that dPARS2 deficiency activates the integrated stress response (ISR), which reduces global protein translation and increases activity of ATF4 in our neuronal dPARS2 knockdown model. Importantly, blockade of ISR activation by genetic suppression of GCN2 kinase prevents developmental delay and seizure phenotypes in dPARS2-deficient flies. Furthermore, the genetic suppression of ATF4, the ISR key effector, also reverses these developmental and behavioral abnormalities associated with dPARS2 deficiency. Furthermore, a disease-associated PARS2 V95I variant causes mitochondrial dysfunction and ISR activation in human cells, verifying the findings in the Drosophila models. Together, these results not only provide evidence for PARS2 deficiency associated mitochondrial dysfunction, but also reveal a novel pathogenic mechanism involved in ISR activation in the PARS2 deficiency related disease, indicating a novel disease treatment approach by targeting ISR.
    Keywords:  Developmental delay; Integrated stress response; Mitochondrial aminoacyl-tRNA synthetases; PARS2; Seizures
    DOI:  https://doi.org/10.1016/j.redox.2025.103966
  33. Mol Genet Genomics. 2025 Dec 12. 301(1): 3
    The degradation of extended protein isoforms points to a misfiring translation initiation process.
    Michael L Tress.
      There is ever increasing evidence for significant amounts of translation upstream of known AUG start codons in protein coding genes. Some of this translation is from upstream open reading frames (ORFs) that are unconnected to the main coding exons, but upstream initiation codons that are in-frame with coding exons can produce N-terminally extended protein isoforms. N-terminal extensions have much more proteomics support than the shorter proteins predicted to be produced from upstream ORFs. The upstream regions that produce N-terminal extensions have certain characteristics in common. They are highly GC-rich, most of the predicted start codons are non-AUG, and most do not conserve their reading frames beyond simians. The extended isoforms themselves are found significantly more frequently in dysregulated cells than in normal tissues. Approximately one in seven of these N-terminal extensions are upstream of signal peptides and would almost certainly block their recognition by the signal recognition particle. As a result, N-terminally extended isoforms containing exposed, hydrophobic signal peptides would be expected to accumulate in the cytoplasm. However, this analysis finds that those N-terminal extensions that would block signal recognition are practically not detected at the protein level even though the transcripts that would produce these extensions are found as expected in ribosome profiling experiments. This is a clear indication that these mislocated proteins are degraded after translation. Theprobable degradation of these extended proteins strongly suggests that their translation is a side effect of inefficient translation initiation.
    Keywords:  Protein degradation; Proteomics; Ribosome profiling; Signal peptides; Translation initiation; Upstream translation
    DOI:  https://doi.org/10.1007/s00438-025-02324-9
  34. Cell Chem Biol. 2025 Dec 08. pii: S2451-9456(25)00353-8. [Epub ahead of print]
    Targeted protein O-GlcNAc reveals transcriptional functions for O-GlcNAc.
    Alison C Mody, Daniel H Ramirez, Christina M Woo.
      O-Linked β-N-acetylglucosamine (O-GlcNAc) is an essential nucleocytoplasmic post-translational modification (PTM) installed on many substrates by a single O-GlcNAc transferase (OGT), although functional outcomes for most of these modifications are unknown. Induced proximity methods to write and erase PTMs from desired targets can accelerate functional annotation and identify therapeutic opportunities for PTMs like O-GlcNAc. Here, we report an induced-proximity method with a destabilized nanobody-OGT fusion and demonstrate its general utility for targeted protein O-GlcNAc against 21 substrates followed by annotation of the direct effects of O-GlcNAc on transcription factors in cells. Deeper investigation of AP-1 transcriptional activation reveals an inhibitory nutrient-sensing event regulated by O-GlcNAc on transcription factors c-Fos and c-Jun. Collectively, these data illustrate the rapid investigation of O-GlcNAc functions in cells enabled by a generalizable induced proximity method for targeted protein O-GlcNAc.
    Keywords:  AP-1; O-GlcNAc; OGT; c-Fos; c-Jun; induced proximity; nutrient-sensing; transcription
    DOI:  https://doi.org/10.1016/j.chembiol.2025.11.003
  35. Biol Open. 2025 Dec 12. pii: bio.062179. [Epub ahead of print]
    Human RBM3 protein is prone to form neuronal aggregates opposed by the proteasome.
    Suman Kumar, Tina Kleven, Rafal Ciosk.
      Maintenance of proteostasis is critical for neuronal functions, as the accumulation of misfolded or damaged proteins leads to neurodegeneration. Cooling is generally neuroprotective and is used in various clinical settings. However, how it impacts neuronal proteostasis remains unclear. In rodents, the neuroprotective effects of cold have been largely attributed to the cold-inducible protein RBM3. Here, studying the human RBM3 in cultured neurons subjected to profound hypothermia, we observed its cold-induced aggregation. These RBM3 aggregates are distinct from stress granules, occur specifically in differentiated neurons, and form also at physiological temperature upon proteasomal inhibition. Thus, in humans, RBM3 aggregation may be normally counteracted by the proteasome to maintain neuronal health. Exploring the natural variation between RBM3 proteins in hibernating versus non-hibernating mammals, we discuss how the aggregation could be prevented in animals with fluctuating body temperature. These findings are important for the understanding of RBM3 functions and neuronal proteostasis and have implications for medical treatments involving incidental and induced hypothermia.
    Keywords:  Aggregation; Hibernation; Hypothermia; Neurodegeneration; Proteostasis; RBM3
    DOI:  https://doi.org/10.1242/bio.062179
  36. Autophagy. 2025 Dec 11.
    Chronic enteritis triggered by diet westernization is driven by epithelial ATG16L1-mediated autophagy.
    Lisa Mayr, Julian Schwärzler, Laura Scheffauer, Zhigang Rao, Dietmar Rieder, Felix Grabherr, Moritz Meyer, Jakob Scheler, Almina Jukic, Luis Zundel, Verena Wieser, Andreas Zollner, Anna Simonini, Stefanie Auer, Lisa Amann, Maureen Philipp, Johannes Leierer, Richard Hilbe, Günter Weiss, Patrizia Moser, Philip Rosenstiel, Qitao Ran, Richard S Blumberg, Arthur Kaser, Andreas Koeberle, Zlatko Trajanoski, Herbert Tilg, Timon E Adolph.
      Macroautophagy/autophagy exerts multilayered protective functions in intestinal epithelial cells (IECs) while a loss-of-function genetic variant in ATG16L1 (autophagy related 16 like 1) is associated with risk for developing Crohn disease (CD). Westernization of diet, partly characterized by excess of long-chain fatty acids, contributes to CD, and a metabolic control of intestinal inflammation is emerging. Here, we report an unexpected inflammatory function for ATG16L1-mediated autophagy in Crohn-like metabolic enteritis of mice induced by polyunsaturated fatty acid (PUFA) excess in a western diet. Dietary PUFAs induce ATG16L1-mediated conventional autophagy in IECs, which is required for PUFA-induced chemokine production and metabolic enteritis. By transcriptomic and lipidomic profiling of IECs, we demonstrate that ATG16L1 is required for PUFA-induced inflammatory stress signaling specifically mediated by TLR2 (toll-like receptor 2) and the production of arachidonic acid metabolites. Our study identifies ATG16L1-mediated autophagy in IECs as an inflammatory hub driving metabolic enteritis, which challenges the perception of protective autophagy in the context of diet westernization.Abbreviations: AA: arachidonic acid; ATG16L1: autophagy related 16 like 1; CD: Crohn disease; CXCL1: C-X-C motif chemokine ligand 1; ER: endoplasmic reticulum; GFP: green fluorescent protein; GPX4: glutathione peroxidase 4; IBD: inflammatory bowel disease; IECs: intestinal epithelial cells; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; PUFA: polyunsaturated fatty acid; SDA: stearidonic acid; TLR2: toll-like receptor 2; WT: wild-type.
    Keywords:  ATG16L1; Crohn disease; glutathione peroxidase 4; intestinal epithelial cells; intestinal inflammation; polyunsaturated fatty acids
    DOI:  https://doi.org/10.1080/15548627.2025.2600906
  37. Genes Dev. 2025 Dec 08.
    Combinatorial use of VHL and KEAP1 PROTACs reveals unexpected synergy and hook effect relief.
    Sehbanul Islam, Haihong Jin, Dong Liu, Dan Lu, Yunkai Zhang, Eric Christenson, Renxu Chang, Joel Austin, Anneliese Faustino, Thomas Beer, Hsin-Yao Tang, Lan Huang, James R Tonra, Luca Busino.
      Proteolysis-targeting chimeras (PROTACs) are bifunctional molecules bridging a protein with an E3 ubiquitin ligase, promoting its ubiquitylation and degradation. However, PROTACs are not without limitations, including suboptimal target degradation and the "hook effect," a phenomenon where high PROTAC concentrations reduce efficacy due to inactive binary complex formation. In this study, we introduce a novel dual-PROTAC strategy utilizing two distinct E3 ligases, such as KEAP1 and VHL, to synergistically degrade KRAS(G12D) and androgen receptor (AR) by promoting ubiquitin chain elongation and also mitigating the hook effect. In conclusion, a dual-E3 ligase approach represents a promising avenue for optimizing PROTAC-based therapeutics.
    Keywords:  KRAS; PROTAC; degradation
    DOI:  https://doi.org/10.1101/gad.352916.125
  38. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1317. [Epub ahead of print]53(22):
    Dual mechanism of autophagy gene repression by PHF23 and therapeutic potential of its inhibition in protein aggregation disorders.
    Seon Ah Choi, Jaebeom Kim, Young Suk Yu, Jaemin Kim, Se In Kim, Hamin Bae, Junhee Kwon, Keun Il Kim, Sung Hee Baek.
      Autophagy is a conserved self-digestion pathway essential for maintaining cellular homeostasis. While the transcriptional and epigenetic activation of autophagy under nutrient-deprived condition is well studied, the repression mechanisms of autophagy under basal conditions remain poorly understood. Here, we identify plant homeodomain finger protein 23 (PHF23) as an epigenetic repressor of autophagy through a CRISPR interference screen. Importantly, PHF23 inhibits autophagy gene expression via two distinct mechanisms: by recruiting the nucleosome remodeling and deacetylase (NuRD) complex to autophagy gene promoters, and by reducing chromatin accessibility at enhancers through downregulation of AP-1 and C/EBPβ transcription factors. This dual repression requires an intact plant homeodomain (PHD) and is relieved following PHF23 degradation under amino acid starvation or mTOR inhibition. Notably, genetic or pharmacological inhibition of PHF23 induces autophagy and promotes the autophagic clearance of pathological protein aggregates, including Tau and α1-antitrypsin Z (ATZ) variant, highlighting PHF23 as a potential therapeutic target in proteotoxic diseases.
    DOI:  https://doi.org/10.1093/nar/gkaf1317
  39. Cell Rep. 2025 Dec 08. pii: S2211-1247(25)01415-9. [Epub ahead of print]44(12): 116643
    eIF3d and eIF3e mediate selective translational control of hypoxia that can be inhibited by small molecules.
    Stephen C Purdy, Kate Matlin, Christopher Alderman, Amber Baldwin, Natasha Shrivastava, Goksu Sarioglu, Somnath Dutta, Kristofor J Webb, Arthur Wolin, Dillon P Boulton, Annika Gustafson, Jyoti Kapali, John D Landua, Michael T Lewis, M Cecilia Caino, James C Costello, William Old, Xiang Wang, Rui Zhao, Heide L Ford, Neelanjan Mukherjee.
      Exposure to hypoxia is linked to increased cellular plasticity and enhanced metastasis, effects that are primarily attributed to the transcriptional activation of large gene programs downstream of hypoxia-inducible factors (HIFs). However, translational effects in hypoxia, which likely precede transcriptional effects, have remained largely unexplored. Using ribosome profiling, we uncovered a selective translational response in acute hypoxia that is eukaryotic initiation factor (eIF)3d/eIF3e dependent and controls downstream hypoxic responses, including HIF1α accumulation and cellular invasion. We further demonstrated that eIF3e copy number and eIF3e and eIF3d expression signatures are associated with worsened outcomes for patients with breast cancer. Finally, we identified a class of novel small molecules that target eIF3e specifically, reducing the translational response to hypoxia and to endoplasmic reticulum (ER) stress, another stressor that is dependent on eIF3d-/eIF3e-mediated translation. Our data uncover critical functions for eIF3d/eIF3e in the hypoxic response and identify a potential means to inhibit stress-induced translation, and potentially plasticity and metastasis, mediated by eIF3e.
    Keywords:  CP: cancer; CP: molecular biology; breast cancer invasion; eIF3d; eIF3e; hypoxia; selective mRNA translation
    DOI:  https://doi.org/10.1016/j.celrep.2025.116643
  40. Nat Commun. 2025 Dec 09.
    Implementing N-terminomics and machine learning to probe Nt-arginylation.
    Shinyeong Ju, Laxman Nawale, Seonjeong Lee, Jung Gi Kim, Hankyul Lee, Narae Park, Dong Hyun Kim, Hyunjoo Cha-Molstad, Cheolju Lee.
      N-terminal arginylation (Nt-arginylation) is a multifunctional post-translational modification (PTM) with roles in protein quality control, organelle homeostasis and stress signaling, but its study has been limited by technical challenges. Here, we develop an integrated approach combining N-terminomics with machine learning-based filtering to identify in cellulo Nt-arginylation. Using Arg-starting missed cleavage peptides as proxies for ATE1-mediated arginylation, we train a transfer learning model to predict mass spectra and retention times. By applying the prediction models with an additional statistical filter, we identify 134 Nt-arginylation sites in thapsigargin-treated HeLa cells. Arginylation is enriched in proteins from various organelles, especially at caspase cleavage and signal peptide processing sites. Eight of twelve tested proteins are further validated for their interaction with p62 ZZ domain. Temporal profiling reveals that ATF4 increases early post-stress, followed by arginylation at caspase-3 substrates and ER signal-cleaved proteins. Our approach enables sensitive detection of rare N-terminal modifications, offering potential for biomarker and drug target discovery.
    DOI:  https://doi.org/10.1038/s41467-025-66883-6
  41. STAR Protoc. 2025 Dec 11. pii: S2666-1667(25)00661-6. [Epub ahead of print]6(4): 104255
    Protocol for identification of cell-surface proteins with horseradish peroxidase.
    Ling Wu, Vijaya Pandey, James A Wohlschlegel, Baljit S Khakh.
      Cell-surface proteins (CSPs) play a central role in intercellular communication. Here, we outline a protocol for the cell-type-specific proteomic profiling of CSPs in mice based on horseradish peroxidase (HRP). We describe steps for adeno-associated virus (AAV) injection, biotinylation reaction, tissue homogenization, streptavidin pull-down, and mass spectrometry characterization, followed by quality control and data interpretation. This approach enables systematic identification of CSPs underlying cell-cell interactions in health and disease. For complete details on the use and execution of this protocol, please refer to Wu et al.1.
    Keywords:  mass spectrometry; molecular biology; molecular/chemical probes; neuroscience; protein biochemistry; proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2025.104255
  42. Autophagy. 2025 Dec 08.
    Chaperone-mediated autophagy as a sex-specific modulator of synaptic proteostasis and neural function.
    Rongcan Luo.
      Chaperone-mediated autophagy (CMA), once considered a secondary or auxiliary degradation pathway, is now recognized as a central regulator of synaptic proteostasis. A recent study by Khawaja et al. (2025) in Nature Cell Biology provides compelling evidence that CMA actively remodels the synaptic proteome in a sex-specific manner. Using a conditional knockout strategy based on Lamp2a-floxed mice crossed with a Camk2a-Cre driver line to achieve excitatory neuron-specific deletion of Lamp2a in adult mice, the authors revealed sexually divergent synaptic phenotypes: females exhibit enhanced presynaptic neurotransmitter release and GRIN/NMDAR-mediated plasticity, while males show increased postsynaptic GRIA/AMPAR activity due to impaired receptor endocytosis. These changes are driven by sex-specific degradation of synaptic proteins such as SYN1 (synapsin I) in females and AP2A/α-Adaptin in males. Importantly, reactivation of CMA - either genetically or pharmacologically - rescues synaptic dysfunction, seizure susceptibility, and memory deficits in aged mice and Alzheimer disease models. This commentary contextualizes these findings within the broader framework of activity-dependent proteostasis, sex-specific autophagy modulation, and therapeutic potential of CMA in brain aging and neurodegeneration.
    Keywords:  Activity-dependent degradation; LAMP2A; chaperone-mediated autophagy; memory; neurodegenerative disorders; neuronal excitability; sex differences; synaptic proteome
    DOI:  https://doi.org/10.1080/15548627.2025.2601849
  43. Biol Direct. 2025 Dec 08.
    Integrating rna structure and protein interactions to uncover the mechanisms of viral and cellular ires function.
    Riccardo Delli Ponti, Andrea Vandelli, Laura Broglia, Gian Gaetano Tartaglia.
       BACKGROUND: RNAs fold into complex structures that critically influence gene expression. A prominent class of regulatory elements resides in the 5' untranslated region (5' UTR), where internal ribosome entry sites (IRESs) promote cap-independent translation by directly engaging the ribosome. First discovered in viral genomes, IRESs have been classified into four types according to their structural compactness and factor requirements. While viral IRESs are well studied, cellular IRESs remain poorly understood: they display limited sequence conservation, reduced structural compactness, and variable dependence on auxiliary RNA-binding proteins known as IRES trans-acting factors (ITAFs). Whether their activity relies mainly on RNA structure or protein assistance remains unresolved. Here, we present a computational framework that combines in silico mutagenesis and RNA-protein interaction profiling to investigate IRES mechanisms and guide the design of synthetic elements.
    RESULTS: Using the Hepatitis C Virus (HCV) IRES as a benchmark, we performed systematic single-nucleotide mutagenesis coupled with structural predictions. Mutations were classified as synonymous or non-synonymous based on their effect on the secondary structure. The HCV IRES showed overall robustness, but the domain interacting with eIF3 was particularly sensitive, consistent with its essential role in translation initiation. Extending this approach to other viral IRES families revealed distinct profiles of resilience: Aphthoviruses retained structural integrity despite extensive sequence variation, whereas Cripaviruses displayed higher variability. We then applied the same analysis to cellular IRESs, which proved to be more structurally sensitive, suggesting stronger reliance on cofactor support. To probe this connection, we used the catRAPID approach to predict interactions with translation-related proteins. The method distinguished IRESs with known ITAF binding, such as PTBP1, and highlighted stability-promoting mutations that increased the predicted affinity for translation factors.
    CONCLUSIONS: Our in silico analysis indicates that mutational tolerance mirrors IRES cofactor dependence: compact viral IRESs are structurally robust, whereas non-viral IRESs are more reliant on protein interactions. By linking structure prediction with interaction profiling, we identify variants that both stabilize IRESs and improve binding to ITAFs or translation factors. This framework provides mechanistic insight into sequence-structure-function relationships and supports the rational design of synthetic IRES elements for therapeutic and biotechnological applications.
    Keywords:  IRES trans-acting factor (ITAF); Internal ribosome entry sites (IRESs); Protein-RNA interactions; RNA secondary structure; RNA viruses; RNA-binding proteins (RBPs); Ribosome; Translation regulation
    DOI:  https://doi.org/10.1186/s13062-025-00706-y
  44. Trends Plant Sci. 2025 Dec 09. pii: S1360-1385(25)00331-0. [Epub ahead of print]
    Communication between chloroplasts and the endoplasmic reticulum in plants under abiotic stress.
    Thomas Depaepe, Sergi Munné-Bosch.
      Chloroplasts and the endoplasmic reticulum (ER) are vital organelles for plant cellular function, yet their communication remains relatively underexplored. Beyond photosynthesis and protein folding, both organelles serve as metabolic hubs and stress sensors, and their crosstalk represents a crucial missing link in plant stress biology. The discovery of membrane contact sites (MCSs) underscores this interdependence, revealing exchanges of biomolecules such as lipids that sustain cellular homeostasis. Evidence also points to stress metabolites, secondary messengers, and hormones as possible mediators in communication, particularly under adverse conditions. By discussing established and putative signals and pointing to emerging technologies, we show that ER-chloroplast communication is critical to understanding abiotic stress adaptation and may open new avenues for improving crop resilience in a changing climate.
    Keywords:  ER–chloroplast signaling; lipid-derived signaling; membrane contact sites (MCSs); organelle communication; retrograde signaling; stress tolerance
    DOI:  https://doi.org/10.1016/j.tplants.2025.11.009
  45. J Cell Sci. 2025 Dec 10. pii: jcs.264267. [Epub ahead of print]
    A C-terminal cytoplasmic retention motif and nuclear localization signal regulates nuclear import of TP53INP2/DOR.
    Birendra Kumar Shrestha, Eva Sjøttem, Hallvard Lauritz Olsvik, Isaac Odonkor, Aud Øvervatn, Hanne Britt Brenne, Jack-Ansgar Bruun, Trond Lamark, Terje Johansen.
      Tumor protein p53 inducible nuclear protein 2 (TP53INP2) is a multifunctional protein involved in transcriptional coactivation, ribosomal RNA synthesis, and autophagy, regulated by subcellular localization. Using CRISPR/Cas9-generated TP53INP2 knockout HeLa cells reconstituted with EGFP-TP53INP2, we show that TP53INP2 is predominantly degraded by nuclear proteasomes under basal conditions. Under stress, including starvation and various chemical stress inducers, TP53INP2 accumulates in the cytoplasm independently of ATG5, CRM1-mediated export, phosphorylation, ubiquitination, or acetylation. We identify a nuclear localization signal (NLS) overlapping a nucleolar localization signal (NoLS) in the C-terminus, which mediates nuclear import and nucleolar enrichment. Deletion of this region redirects TP53INP2 to LC3B-positive puncta. A conserved nine-amino acid cytoplasmic retention motif (CRM) in the C-terminus prevents nuclear re-entry under stress. This motif and regulation of subcellular localization is conserved in the related TP53INP1 protein. Fluorescence recovery after photobleaching (FRAP) and importin-binding assays show that nutrient starvation disrupts nuclear import of TP53INP2. Finally, starvation enhances TP53INP2 translation via the m6A demethylase FTO, without altering mRNA stability. These findings uncover coordinated regulation of TP53INP2 localization and turnover by cellular stress.
    Keywords:  Autophagy; Cytoplasmic retention; DOR; LC3B; Nuclear localization; Proteasome; TP53INP1; TP53INP2
    DOI:  https://doi.org/10.1242/jcs.264267
  46. J Am Chem Soc. 2025 Dec 08.
    Multiplexed Analysis of Multicomponent Biomolecular Condensates without Any Tag.
    Gyula Pálfy, Johannes Schmoll, Maria E Pérez, Fred F Damberger, Yaning Han, Leonidas Emmanouilidis, Frédéric H-T Allain, Mihajlo Novakovic.
      Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components, such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labeling of various droplet components, which may affect phase separation behavior and are thus not able to probe biomolecules in their chemically native state. Here, we introduce a noninvasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both the dilute and condensed phases. We demonstrate the robust performance of this 1D 1H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.
    DOI:  https://doi.org/10.1021/jacs.5c14476
  47. PLoS Genet. 2025 Dec;21(12): e1011964
    ASNA1 is essential for cardiac development and function by regulating tail-anchored protein stability and vesicular transport in cardiomyocytes.
    Wei Feng, Zengming Zhang, Zeyu Chen, Li Wang, Mao Ye, Yusu Gu, Titania Huang, Harrison Ngo, Ju Chen.
      Recent studies have linked compound heterozygous mutations in ASNA1 to progressive dilated cardiomyopathy and early infantile mortality in humans. However, the specific role of ASNA1 in cardiomyocytes and the molecular mechanisms underlying ASNA1-related cardiomyopathy remain poorly understood. Tail-anchored (TA) proteins, characterized by a single C-terminal transmembrane domain (TMD), require post-translational targeting to intracellular membranes, a process primarily mediated by the evolutionarily conserved Guided Entry of Tail-anchored proteins (GET) pathway in yeast and the Transmembrane Recognition Complex (TRC) pathway in mammals. ASNA1 (also known as TRC40 or GET3) serves as the central ATP-dependent chaperone delivering TA proteins to the endoplasmic reticulum (ER) membrane. To address ASNA1's role in the heart, we generated constitutive and inducible cardiomyocyte-specific Asna1 knockout mouse models. Constitutive Asna1 deletion during embryogenesis caused perinatal lethality with marked ventricular myocardial thinning by embryonic day 16.5, whereas inducible deletion in adult cardiomyocytes led to rapid ventricular dilation, impaired cardiac function, pathological remodeling, and early mortality. Mechanistically, ASNA1 deficiency destabilized the pre-targeting complex and reduced the expression of multiple TA protein substrates, impairing membrane trafficking and protein transport. Transcriptomic analyses revealed compensatory upregulation of genes involved in protein trafficking and Golgi-to-ER transport, reflecting maladaptive responses to disrupted vesicular transport. Collectively, our findings identify ASNA1 as a critical regulator of TA protein stability and vesicular trafficking in cardiomyocytes, whose loss disrupts cardiac proteostasis and contributes to the cardiomyopathy pathogenesis. Our work provides mechanistic insights into ASNA1-related cardiac disease and highlights potential therapeutic targets.
    DOI:  https://doi.org/10.1371/journal.pgen.1011964
  48. Cell Rep. 2025 Dec 05. pii: S2211-1247(25)01431-7. [Epub ahead of print]44(12): 116659
    circATF6 triggers calcium overload to synergize with EGFR-TKI in non-small cell lung cancer via modulation of proteostasis.
    Qianfan Hu, Yajing Wang, Yuxian Qian, Zi Wang, Hui Wang, Frank Qingyun Wang, Rutao Li, Yipeng Feng, Yijian Zhang, Hanlin Ding, Weiran Zhou, Wei Zhang, Qixing Mao, Wenjie Xia, Gaochao Dong, Feng Jiang.
      Drug-tolerant persister (DTP) cells are a reversible, refractory population that contributes to tumor relapse during epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy in non-small cell lung cancer (NSCLC). Targeting the vulnerabilities of DTP cells constitutes an effective strategy to enhance the long-term efficacy of EGFR-TKIs. Here, we identify the circular RNA circATF6, which is specifically downregulated in DTP cells due to regulation by the splicing factor ADAR1. Restoration of circATF6 expression significantly enhances osimertinib (Osi) efficacy both in vitro and in vivo. Mechanistically, circATF6 interacts with the endoplasmic reticulum (ER) chaperone BiP/GRP78, inhibiting the activated adaptive unfolded protein response (UPR). This inhibition disrupts proteostasis, leading to ER fragmentation and Ca2+ overload in the cytoplasm, ultimately triggering apoptosis in DTP cells. Notably, lipid nanoparticle (LNP)-encapsulated circATF6 enhanced the antitumor effects of EGFR-TKI without significant toxicity. Our study provides a therapeutic strategy targeting circATF6 to synergize with EGFR-TKI in NSCLC.
    Keywords:  CP: cancer; CP: molecular biology; EGFR-TKI; ER chaperone; calcium; drug-tolerant persister; proteostasis; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2025.116659
  49. J Biol Chem. 2025 Dec 10. pii: S0021-9258(25)02892-3. [Epub ahead of print] 111040
    Hijacking the unfolded protein response (UPR) pathway: Balancing viral infection and host cell survival.
    Haoyu Chen, Duxuan Liu, Jing Hua, Mingjie Wu, Yanhong Hua, Chenwei Feng, Zhen He, Peter Moffett, Kun Zhang.
      The endoplasmic reticulum (ER) unfolded protein response (UPR) is a conserved eukaryotic pathway crucial for restoring cellular homeostasis under ER stress. However, diverse viruses infecting mammalian or plant cells strategically hijack and manipulate the UPR pathways featuring sensor proteins IRE1, PERK, ATF6 and bZIP17/28 to promote viral replication . While the UPR is designed to provide cellular adaptive functions in response to stresses, viruses can instead exploit UPR components to instead enhance viral protein folding and remodel ER membranes for viral replication. Exploiting the UPR presents a critical dilemma: while mild UPR activation facilitates virus replication and survival, excessive or prolonged activation triggers host programmed cell death (PCD), prematurely terminating infection. Navigating this UPR tightrope is central for successful infection and replication of the virus. Viruses are not passive triggers of ER stress and the UPR. Viruses have evolved sophisticated "braking mechanisms" to actively modulate UPR signaling intensity. By fine-tuning the UPR, viruses harness the beneficial aspects of the UPR while crucially preventing the activation cascade from reaching the lethal threshold that initiates PCD. By carefully controlling the UPR balance, viruses ensure host cell survival for sufficient duration to maximize viral progeny production. This review details the intricate interactions between the cellular UPR and infecting viruses, including links to cellular clearance pathways like autophagy and ER-associated degradation (ERAD), across different viral families (flaviviruses, coronaviridae, and potyviruses) and hosts (plants and animals). Understanding the sophisticated viral manipulation of the UPR equilibrium reveals fundamental insights into host-pathogen co-evolution and highlights novel potential targets for antiviral strategies aimed at disrupting this delicate balance.
    Keywords:  Apoptosis; ER stress; Programed cell death (PCD); Unfold protein response (UPR); Virus
    DOI:  https://doi.org/10.1016/j.jbc.2025.111040
  50. Adv Sci (Weinh). 2025 Dec 12. e13655
    HSP70 Interactome-Mediated Proteolysis Targeting Chimera (HSP70-PROTAC) for Ferroptosis-Driven Cancer Treatment.
    Jinyun Dong, Yulong Li, Hui Liang, Zumei Wu, Shiqun Wang, Yichao Wang, Jieyu Xu, Yanning Lan, Maohua Cai, Guangzhao Pan, Haiyan Yang, Kai Miao, Zhe-Sheng Chen, Fangfang Tao, Xuelei Ma, Jiang-Jiang Qin.
      Targeted protein degradation (TPD) represents a transformative therapeutic paradigm that harnesses the cellular degradation machinery to pharmacologically eliminate disease-causing proteins with aberrant expression. This work here reports the first design of an HSP70 interactome-mediated proteolysis targeting chimera (HSP70-PROTAC) for the degradation of the intracellular therapeutically relevant proteins via dual processes of ubiquitin-proteasomal degradation (UPS) and chaperone-mediated autophagy (CMA). By hijacking the highly expressed heat shock cognate protein (Hsc70) isoform complex in tumor tissues to glutathione peroxidase 4 (GPX4) protein, this work successfully develops an HSP70-PROTAC molecule GDAz-3 that potently and rapidly eliminates GPX4 in HT1080 cells, thereby triggering ferroptosis with high selectivity. Correspondingly, GDAz-3 exhibits a remarkable tumor-inhibitory effect in the HT1080 xenograft tumor mouse model without obvious toxicity. In addition, this work demonstrates the versatility of HSP70-based PROTACs by effectively degrading additional endogenous bromodomain-containing protein 4 (BRD4) in cancer cells. More importantly, the degradation of GPX4 mediated by GDAz-3 occurs with comparable efficiency in CRBN/VHL-knockdown cells and 786-O cells intrinsically lacking VHL expression, which facilitates expanding the application scope and overcoming drug resistance of traditional PROTAC. These findings suggest that HSP70-PROTAC is a novel and feasible strategy for the future development of TPD technology.
    Keywords:  cancer; ferroptosis; glutathione peroxidase 4; heat shock protein 70; targeted protein degradation
    DOI:  https://doi.org/10.1002/advs.202513655
  51. Int J Biol Macromol. 2025 Dec 06. pii: S0141-8130(25)10097-4. [Epub ahead of print]337(Pt 2): 149540
    UBA5 missense variants disrupt UFM1 activation: Structural, dynamic, and functional dissection.
    Liqiang Ai, Wenbo Han, Shengwei Xiao, Yuanjiang Huang, Yingbao Zhu, Boquan Jia, Qiao Xiao, Wen Huang, Ranhui Duan.
      UFMylation is an evolutionarily conserved ubiquitin-like modification essential for cellular homeostasis. UBA5 acts as the sole E1 enzyme initiating this process, and biallelic UBA5 variants cause severe neurodevelopmental disorders. However, the specific mechanisms by which missense variants disrupt UFM1 activation remain unclear. Here, we applied AlphaFold2/3 modeling and molecular dynamics simulations to examine conformational states of UBA5 during activation, focusing on 11 clinically relevant missense variants within the adenylation domain. Structural analysis revealed that variants impair key mechanistic steps: monomer stabilization exemplified by p.Arg72Cys and p.Gly168Glu, dimerization disrupted by p.Val260Met, ATP coordination compromised in p.Arg55His, UFM1 recruitment weakened in p.Cys303Arg, and thioester bond formation impaired in p.Leu254Pro.Molecular dynamics simulations revealed variant-specific perturbations across activation stages: p.Arg72Cys and p.Gly168Glu increased flexibility and solvent exposure in the monomeric core; p.Val260Met and p.Leu254Pro altered dimer interface packing; p.Cys303Arg expanded and destabilized the UFM1-binding interface, while p.Arg55His reduced local flexibility near the ATP-binding pocket yet maintained global compactness. Biochemical and cellular assays, including thermal stability, ATP-binding, UFM1 charging, and substrate UFMylation, confirmed these defects and aligned with computational predictions. This study defines how individual UBA5 variants compromise structural transitions required for UFM1 activation and establishes a mechanistic framework linking variant-specific disruptions to disease pathogenesis.
    Keywords:  Missense variants; Molecular dynamics; UBA5
    DOI:  https://doi.org/10.1016/j.ijbiomac.2025.149540
  52. Nucleic Acids Res. 2025 Nov 26. pii: gkaf1276. [Epub ahead of print]53(22):
    Beyond RNA modification: a novel role for tRNA modifying enzyme in oxidative stress response and metabolism.
    Louna Fruchard, Claudia Sudol, Caroline Rouard, Aurore Treffkorn-Maurau, Léo Hardy, Julia Bos, Magalie Duchateau, Quentin Giai Gianetto, Mariette Matondo, Frédéric Bonhomme, Quentin Thuillier, Virginie Marchand, Yuri Motorin, Damien Bregeon, Didier Mazel, Djemel Hamdane, Zeynep Baharoglu.
      RNA modifications play a fundamental role in regulating essential cellular processes, including translation fidelity and stress adaptation. While these modifications are installed post-transcriptionally by specialized enzymes, their broader functional roles remain largely unexplored. Here, we uncover an unexpected function for the Vibrio cholerae tRNA dihydrouridine synthase B (VcDusB) beyond its canonical role in tRNA dihydrouridylation. We show that deletion of dusB severely compromises V. cholerae resistance to oxidative stress, not through the loss of tRNA modification, but via disruption of an intrinsic NADPH oxidase activity. Mutational analyses reveal that DusB redox function is essential for survival under oxidative stress. Proteomic and transposon insertion sequencing analysis further linked DusB to NADPH homeostasis and metabolic reprogramming during stress adaptation. These findings redefine DusB as a bifunctional enzyme coupling tRNA modification to redox regulation, expanding the functional repertoire of RNA-modifying enzymes in stress adaptation. More broadly, this work paves the way for exploring the evolutionary versatility of tRNA-modifying enzymes, suggesting that their functions extend far beyond RNA metabolism to direct integration of translational control with cellular redox state.
    DOI:  https://doi.org/10.1093/nar/gkaf1276
  53. Elife. 2025 Dec 11. pii: RP107180. [Epub ahead of print]14
    General trends in the calnexin-dependent expression and pharmacological rescue of clinical CFTR variants.
    Austin Tedman, John A Olson, Minsoo Kim, Catherine Foye, JaNise J Jackson, Eli F McDonald, Andrew G McKee, Karen Noguera, Charles P Kuntz, Jens Meiler, Kathryn E Oliver, Lars Plate, Jonathan P Schlebach.
      Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR). Though most people with CF have one or two copies of the ΔF508 mutation, there are hundreds of other distinct CF mutations that vary in their mechanistic effects and response to therapeutics. Endogenous chaperones are known to have divergent effects on the druggability of CF variants. Nevertheless, it remains unclear how this proteostatic modulation is related to the underlying mechanistic effects of distinct classes of CF mutations. Here, we survey the effects of a previously discovered effector (calnexin, CANX) on the expression and pharmacological rescue of 232 CF variants using deep mutational scanning. We find that CANX is generally required for robust plasma membrane expression of the CFTR protein, particularly for CF variants that perturb its second nucleotide-binding domain. CANX also appears to be critical for the pharmacological rescue of CF variants with poor basal expression. Though corrector selectivity is generally dictated by the properties of mutations, we find that CANX enhances the sensitivity of CF variants within a domain-swapped region of membranes spanning domain 2 to the type III corrector VX-445. Overall, mutagenic trends suggest CANX modulates the later stages of CFTR assembly and disproportionately affects variants bearing mutations within the C-terminal domains. Interestingly, we find that the loss of CANX results in widespread perturbations of CF variant interactomes and that the proteostatic effects of CANX are generally decoupled from changes in CFTR activity. Together, our findings reveal how the proteostasis machinery may shape the variant-specific effects of corrector molecules.
    Keywords:  CFTR; biochemistry; chemical biology; corrector; cystic fibrosis; human; membrane protein folding; protein misfolding; proteostasis
    DOI:  https://doi.org/10.7554/eLife.107180
  54. STAR Protoc. 2025 Dec 05. pii: S2666-1667(25)00655-0. [Epub ahead of print]6(4): 104249
    Protocol for single-molecule analysis of synaptic protein complex-mediated vesicle recruitment.
    Akshay Kapadia, Anne-Sophie Hafner.
      Single-molecule pull-down (SIM-Pull) combined with total internal reflection fluorescence (TIRF) microscopy enables direct visualization of proteins and multi-protein complexes. Here, we present an extended SIM-Pull protocol for analyzing protein interactions at the active zone and their ability to recruit isolated synaptic vesicles (SVs). We describe steps for visualizing and quantifying SV recruitment mediated by STX1A-SNARE and RIM1-Rab3a interactions. This technique allows the examination of subcellular vesicle-associated protein-protein interactions at a molecular level in a near-native cellular context.
    Keywords:  Cell Biology; Microscopy; Neuroscience
    DOI:  https://doi.org/10.1016/j.xpro.2025.104249
  55. J Med Chem. 2025 Dec 11.
    Leveraging Targeted Protein Degradation for G Protein-Coupled Receptors: The Development of CCR2 Molecular Degraders.
    Khaled Essa, Natalia V Ortiz Zacarías, Sascha Roth, Bo-Tao Xin, Sabine de Winter, Cas van der Horst, Bente Bleijs, Richard A de Heiden, Rodanthi Voulgaraki, Elsa Neubert, Huib Ovaa, Erik H J Danen, Monique P C Mulder, Kevin Moreau, Daan van der Es, Laura H Heitman.
      Targeted protein degradation (TPD) is one of the most prominent and rapidly advancing modalities in drug discovery. However, only few degraders have been reported for the membrane-bound G protein-coupled receptors (GPCRs). Therefore, using CC chemokine receptor 2 (CCR2) as a GPCR model, we synthesized potential CCR2 molecular degraders. The putative proteolysis-targeting chimeras (PROTACs) employed an allosteric intracellular CCR2 ligand tethered to commonly used E3 ligase ligands. Among these compounds, LUF7996 (8) demonstrated engagement of both CCR2 and the E3 ligase cereblon and displayed sustained and concentration-dependent degradation of CCR2 over 24 h. Mechanistic studies revealed the reliance of LUF7996 on the lysosomal pathway to induce CCR2 degradation. Finally, LUF7996 (8) efficiently inhibited monocyte migration in a transwell assay. Collectively, the developed assessment workflow led to identification of the first CCR2 molecular degraders and has the potential to expand the repertoire of degraders targeting the pharmacologically rich GPCRs.
    DOI:  https://doi.org/10.1021/acs.jmedchem.5c02920
  56. Nature. 2025 Dec 10.
    Mutations in mitochondrial ferredoxin FDX2 suppress frataxin deficiency.
    Joshua D Meisel, Pallavi R Joshi, Amy N Spelbring, Hong Wang, Sandra M Wellner, Presli P Wiesenthal, Maria Miranda, Jason G McCoy, David P Barondeau, Gary Ruvkun, Vamsi K Mootha.
      Frataxin is a key component of an ancient, mitochondrial iron-sulfur cluster biosynthetic machinery, serving as an allosteric activator of the cysteine desulfurase NFS1 (refs. 1-5). Loss of frataxin levels underlies Friedreich's ataxia6, the most common inherited ataxia. Yeast, Caenorhabditis elegans and human cells can tolerate loss of frataxin when grown in 'permissive' low oxygen tensions7. Here we conducted an unbiased, genome-scale forward genetic screen in C. elegans leveraging permissive and non-permissive oxygen tensions to discover suppressor mutations that bypass the need for frataxin. All mutations act dominantly and are in the ferredoxin FDX2/fdx-2 or in the cysteine desulfurase NFS1/nfs-1 genes, resulting in amino-acid substitutions at the FDX2-NFS1 binding interface. Our genetic and biochemical analyses show that the suppressor mutations boost iron-sulfur cluster levels in the absence of frataxin. We also demonstrate that an excess of FDX2 inhibits frataxin-stimulated NFS1 activity in vitro and blocks the synthesis of iron-sulfur clusters in mammalian cell culture. These findings are consistent with structural and biochemical evidence that frataxin and FDX2 compete for occupancy at the same site on NFS1 (refs. 8,9). We show that lowering levels of wild-type FDX2 through loss of one gene copy can ameliorate the growth of frataxin mutant C. elegans or the ataxia phenotype of a mouse model of Friedreich's ataxia under normoxic conditions. These genetic and biochemical studies indicate that restoring the stoichiometric balance of frataxin and FDX2 through partial knockdown of FDX2 may be a potential therapy for Friedreich's ataxia.
    DOI:  https://doi.org/10.1038/s41586-025-09821-2
  57. J Biol Chem. 2025 Dec 09. pii: S0021-9258(25)02886-8. [Epub ahead of print] 111034
    CAPN15 is a non-proteasomal, ubiquitin-directed calpain protease that regulates cell adhesion by cleaving E-cadherin.
    Aya Noguchi, Hikaru Tsuchiya, Hiroshi Shitara, Yasushi Saeki, Yasuko Ono, Shoji Hata.
      CAPN15, a member of the calpain protease family, contains a unique N-terminal region with five Zn2+-finger domains, including two ubiquitin-binding Npl4-type Zn2+-finger domains. However, the role of these domains in CAPN15 function remains unknown. In this study, we show that CAPN15 functions as a non-proteasomal, ubiquitin-directed protease, regulating cell surface E-cadherin. In cultured epithelial cells, CAPN15 knockout resulted in densely packed morphology accompanied by the accumulation of cell surface E-cadherin, suggesting an increased cell adhesion. This defect was rescued by expressing wild-type CAPN15, but not protease-inactive or ubiquitin-binding-deficient CAPN15. CAPN15 recognizes the ubiquitinated E-cadherin-catenin complex through its N-terminal Zn2+-finger region and cleaves E-cadherin near its transmembrane domain. Since the truncated form is directed for lysosomal degradation, CAPN15 stands as a negative regulator of E-cadherin. E-cadherin accumulation was also observed in the epithelial tissues of Capn15 knockout mice, further corroborating the in vivo relevance of CAPN15 to a mechanism controlling E-cadherin function. These findings reveal a previously unappreciated ubiquitin-dependent proteolytic pathway involving CAPN15 and provide important insight into the regulation of cell adhesion.
    Keywords:  cadherin; calpain; cell adhesion; ubiquitin; ubiquitin-dependent protease
    DOI:  https://doi.org/10.1016/j.jbc.2025.111034
  58. J Biol Chem. 2025 Dec 08. pii: S0021-9258(25)02870-4. [Epub ahead of print] 111018
    Targeting the Protein-Protein Interaction Between the CDC37 Co-Chaperone and Client Kinases by an Allosteric RAF Dimer Breaker.
    Alison Yu, Shrhea Banerjee, Sravani Malasani, Bamidele Towolawi, Zhiwei Liu, Zhihong Wang.
      CDC37, a selectivity co-chaperone in the HSP90 chaperone machinery, plays a crucial role in facilitating the recognition of client kinases and aiding the folding and maturation of these kinases. RAF kinases, central component of the MAPK signaling pathway, rely on their interaction with CDC37 for stability and function. The RAF dimer interface, a key determinant of RAF kinase activity, overlaps with the CDC37-kinase client recognition motif, known as the αC helix-β4 loop region. Here, we report that Braftide, a peptide originally designed as a potent allosteric RAF kinase dimer disruptor, also triggers proteasome-mediated protein degradation of RAF kinases with an unclear mechanism of action. This study elucidates the mechanism underlying Braftide's dual functionality and assesses the potential of targeting kinase-chaperone interaction in cancer cell lines. Using co-immunoprecipitation and NanoBiT assays, we confirmed Braftide's ability to selectively disrupt the CDC37-client kinase interaction while sparing HSP90. Through deuterium exchange mass spectrometry, molecular dynamic simulations, and in vitro crosslinking analyses, we mapped Braftide's binding region within the BRAF kinase domain, as well as the CDC37 region implicated in the association of CDC37-client kinase complex. Consequently, this disruption destabilizes RAF kinase clients, resulting in proteasomal degradation, reduced cellular proliferation, and increased apoptosis in cancer cell lines. Furthermore, Braftide exhibits synergy with HSP90 inhibitors, jointly destabilizing both the CDC37-RAF complex and HSP90. Our work identifies the αC helix-β4 loop as a novel allosteric site for targeting kinase-chaperone interactions and demonstrates the feasibility of disrupting the CDC37-client kinase interaction as an innovative therapeutic strategy.
    DOI:  https://doi.org/10.1016/j.jbc.2025.111018
  59. EMBO J. 2025 Dec 11.
    Integrating endogenous TurboID and data-independent acquisition mass spectrometry for in vivo proximity labeling.
    David S Fay, Boopathi Balasubramaniam, Sean M Harrington, Philip T Edeen.
      Proximity labeling has emerged as a powerful approach for identifying protein-protein interaction networks within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive revised proximity labeling workflow, integrating TurboID labeling of endogenously expressed fusion proteins and data-independent acquisition (DIA) mass spectrometry (MS). We benchmark this pipeline with a study of five conserved Caenorhabditis elegans proteins-NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4- that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Profiling of NEKL-MLT interactomes across 23 experiments validated our approach through the identification of known NEKL-MLT binding partners and conserved nekl-mlt genetic interactors, including the discovery of several novel functional interactors. Importantly, inclusion of methodological variations, stringent controls, and filtering strategies enhanced sensitivity and reproducibility, defining a set of intuitive quantitative metrics for routine assessment of experimental quality. We show that DIA-based interactome workflows produce physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our study underscores the utility of DIA mass spectrometry in proximity labeling applications and highlights the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors.
    Keywords:   C. elegans ; NIMA-related Kinases; Proximity Labeling; TurboID
    DOI:  https://doi.org/10.1038/s44318-025-00660-5
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