bims-proteo Biomed News
on Proteostasis
Issue of 2025–01–12
fifty papers selected by
Eric Chevet, INSERM
  1. Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy.
  2. The integrated stress response drives MET oncogene overexpression in cancers.
  3. Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae.
  4. Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.
  5. The efflux pump ABCC1/MRP1 constitutively restricts PROTAC sensitivity in cancer cells.
  6. Nonreceptor tyrosine kinase ABL1 regulates lysosomal acidification by phosphorylating the ATP6V1B2 subunit of the vacuolar-type H+-ATPase.
  7. Molecular basis for the stepwise and faithful maturation of the 20S proteasome.
  8. PERK Signaling Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation.
  9. VCP regulates early tau seed amplification via specific cofactors.
  10. Stress responses induced by perturbation of the ubiquitin-proteasome system.
  11. Protein quality control machinery: regulators of condensate architecture and functionality.
  12. Rickettsia parkeri forms extensive, stable contacts with the rough endoplasmic reticulum.
  13. The interplay between gut microbiota and the unfolded protein response: Implications for intestinal homeostasis preservation and dysbiosis-related diseases.
  14. Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis.
  15. Two FAM134B isoforms differentially regulate ER dynamics during myogenesis.
  16. Ribosome customization and functional diversification among P-stalk proteins regulate late poxvirus protein synthesis.
  17. Transcriptomics Unveil Canonical and Non-Canonical Heat Shock-Induced Pathways in Human Cell Lines.
  18. Genesis and regulation of C-terminal cyclic imides from protein damage.
  19. RACK1 MARylation regulates translation and stress granules in ovarian cancer cells.
  20. Transmission of unfolded protein response-a regulator of disease progression, severity, and spread in virus infections.
  21. Targeting N-Myc in neuroblastoma with selective Aurora kinase A degraders.
  22. Mitochondria- and ER-associated actin are required for mitochondrial fusion.
  23. Site-saturation mutagenesis of 500 human protein domains.
  24. E2-Ub-R74G strategy reveals E2-specific ubiquitin conjugation profiles in live cells.
  25. Chemically engineered antibodies for autophagy-based receptor degradation.
  26. Exquisite sensitivity of Polycystin-1 to H2O2 concentration in the endoplasmic reticulum.
  27. VCP controls KCC2 degradation through FAF1 recruitment and accelerates emergence from anesthesia.
  28. Regulation of physiological and pathological condensates by molecular chaperones.
  29. A deep learning method for predicting interactions for intrinsically disordered regions of proteins.
  30. Journey of PROTAC: From Bench to Clinical Trial and Beyond.
  31. Condensates act as translation hubs to coordinate multinucleate cell growth.
  32. Mitochondrial YME1L1 governs unoccupied protein translocase channels.
  33. Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function.
  34. Mutant Calreticulin in MPN: Mechanistic Insights and Therapeutic Implications.
  35. IRE1α-XBP1 safeguards hematopoietic stem and progenitor cells by restricting pro-leukemogenic gene programs.
  36. Phosphorylation of a nuclear condensate regulates cohesion and mRNA retention.
  37. TDP43 autoregulation gives rise to dominant negative isoforms that are tightly controlled by transcriptional and post-translational mechanisms.
  38. NXP800 activates the unfolded protein response, altering AR and E2F function to impact castration-resistant prostate cancer growth.
  39. Sexually dimorphic ATF4 expression in the fat confers female stress tolerance in Drosophila melanogaster.
  40. Human TRMT1 and TRMT1L paralogs ensure the proper modification state, stability, and function of tRNAs.
  41. BRAF V600 and ErbB inhibitors directly activate GCN2 in an off-target manner to limit cancer cell proliferation.
  42. Bile acid synthesis impedes tumor-specific T cell responses during liver cancer.
  43. TRMT1L-catalyzed m22G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.
  44. DNAJC13 localization to endosomes is opposed by its J domain and its disordered C- terminal tail.
  45. RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis.
  46. PPI-CoAttNet: A Web Server for Protein-Protein Interaction Tasks Using a Coattention Model.
  47. Valosin-containing protein p97 extracts capping protein CP110 from the mother centriole to promote ciliogenesis.
  48. Crypt density and recruited enhancers underlie intestinal tumour initiation.
  49. Disrupting the transmembrane domain interface between PMP22 and MPZ causes peripheral neuropathy.
  50. A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms.
  1. Nat Cell Biol. 2025 Jan 07.
    Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy.
    Mariya Licheva, Jeremy Pflaum, Riccardo Babic, Hector Mancilla, Jana Elsässer, Emily Boyle, David M Hollenstein, Jorge Jimenez-Niebla, Jonas Pleyer, Mio Heinrich, Franz-Georg Wieland, Joachim Brenneisen, Christopher Eickhorst, Johann Brenner, Shan Jiang, Markus Hartl, Sonja Welsch, Carola Hunte, Jens Timmer, Florian Wilfling, Claudine Kraft.
      Autophagy is a key cellular quality control mechanism. Nutrient stress triggers bulk autophagy, which nonselectively degrades cytoplasmic material upon formation and liquid-liquid phase separation of the autophagy-related gene 1 (Atg1) complex. In contrast, selective autophagy eliminates protein aggregates, damaged organelles and other cargoes that are targeted by an autophagy receptor. Phase separation of cargo has been observed, but its regulation and impact on selective autophagy are poorly understood. Here, we find that key autophagy biogenesis factors phase separate into initiation hubs at cargo surfaces in yeast, subsequently maturing into sites that drive phagophore nucleation. This phase separation is dependent on multivalent, low-affinity interactions between autophagy receptors and cargo, creating a dynamic cargo surface. Notably, high-affinity interactions between autophagy receptors and cargo complexes block initiation hub formation and autophagy progression. Using these principles, we converted the mammalian reovirus nonstructural protein µNS, which accumulates as particles in the yeast cytoplasm that are not degraded, into a neo-cargo that is degraded by selective autophagy. We show that initiation hubs also form on the surface of different cargoes in human cells and are key to establish the connection to the endoplasmic reticulum, where the phagophore assembly site is formed to initiate phagophore biogenesis. Overall, our findings suggest that regulated phase separation underscores the initiation of both bulk and selective autophagy in evolutionarily diverse organisms.
    DOI:  https://doi.org/10.1038/s41556-024-01572-y
  2. EMBO J. 2025 Jan 07.
    The integrated stress response drives MET oncogene overexpression in cancers.
    Marina Cerqua, Marco Foiani, Carla Boccaccio, Paolo M Comoglio, Dogus M Altintas.
      Cancer cells rely on invasive growth to survive in a hostile microenvironment; this growth is characterised by interconnected processes such as epithelial-to-mesenchymal transition and migration. A master regulator of these events is the MET oncogene, which is overexpressed in the majority of cancers; however, since mutations in the MET oncogene are seen only rarely in cancers and are relatively infrequent, the mechanisms that cause this widespread MET overexpression remain obscure. Here, we show that the 5' untranslated region (5'UTR) of MET mRNA harbours two functional stress-responsive elements, conferring translational regulation by the integrated stress response (ISR), regulated by phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) at serine 52. ISR activation by serum starvation, leucine deprivation, hypoxia, irradiation, thapsigargin or gemcitabine is followed by MET protein overexpression. We mechanistically link MET translation to the ISR by (i) mutation of the two uORFs within the MET 5'UTR, (ii) CRISPR/Cas9-mediated mutation of eIF2α (S52A), or (iii) the application of ISR pathway inhibitors. All of these interventions reduce stress-induced MET overexpression. Finally, we show that blocking stress-induced MET translation blunts MET-dependent invasive growth. These findings indicate that upregulation of the MET oncogene is a functional requirement linking integrated stress response to cancer progression.
    Keywords:  Integrated Stress Response; Invasive Growth; MET Oncogene
    DOI:  https://doi.org/10.1038/s44318-024-00338-4
  3. bioRxiv. 2024 Dec 22. pii: 2024.12.19.629525. [Epub ahead of print]
    Mapping the Genetic Architecture of the Adaptive Integrated Stress Response in S. cerevisiae.
    Rachel Baum, Jinyoung Kim, Ryan Y Muller, Nicholas Ingolia.
      The integrated stress response (ISR) is a conserved eukaryotic signaling pathway that responds to diverse stress stimuli to restore proteostasis. The strength and speed of ISR activation must be tuned properly to allow protein synthesis while maintaining proteostasis. Here, we describe how genetic perturbations change the dynamics of the ISR in budding yeast. We treated ISR dynamics, comprising timecourses of ISR activity across different levels of stress, as a holistic phenotype. We profiled changes in ISR dynamics across thousands of genetic perturbations in parallel using CRISPR interference with barcoded expression reporter sequencing (CiBER-seq). We treated cells with sulfometuron methyl, a titratable inhibitor of branched-amino acid synthesis, and measured expression of an ISR reporter. Perturbations to translation such as depletion of aminoacyl-tRNA synthetases or tRNA biogenesis factors reduced cell growth and caused a strikingly proportionate activation of the ISR activation. In contrast, impaired ribosome biogenesis reduced basal ISR activity and weakened ISR dynamics. Reduced ribosome capacity may lower the demand for amino acids and thereby explain these changes. Our work illustrates how CiBER-seq enables high-throughput measurements of complex and dynamic phenotypes that shed light on adaptive and homeostatic mechanisms.
    DOI:  https://doi.org/10.1101/2024.12.19.629525
  4. bioRxiv. 2024 Dec 18. pii: 2024.12.17.628947. [Epub ahead of print]
    Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.
    Hao-Yang Liu, Grant Ashby, Feng Yuan, Susovan Sarkar, Carl C Hayden, Jon M Huibregtse, Jeanne C Stachowiak.
      Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation.
    Significance: Ubiquitylation is essential for maintaining cellular homeostasis by regulating transmembrane protein trafficking, degradation, and signaling. Traditionally, poly-ubiquitylation has been viewed primarily as a signal for protein degradation, while mono-ubiquitylation is considered sufficient to trigger endocytosis. However, our work reveals a previously unrecognized role for poly-ubiquitylation in clathrin-mediated endocytosis, demonstrating that poly-ubiquitylated transmembrane proteins outcompete their non-ubiquitylated counterparts for incorporation into clathrin-coated vesicles, thereby establishing a competitive framework for endocytic cargo sorting. This mechanism reveals a selective sorting mechanism driven by the extent of ubiquitylation, which could regulate the removal of damaged proteins while protecting functional proteins at the plasma membrane.
    DOI:  https://doi.org/10.1101/2024.12.17.628947
  5. Cell Chem Biol. 2024 Dec 24. pii: S2451-9456(24)00489-6. [Epub ahead of print]
    The efflux pump ABCC1/MRP1 constitutively restricts PROTAC sensitivity in cancer cells.
    Gernot Wolf, Conner Craigon, Shao Thing Teoh, Patrick Essletzbichler, Svenja Onstein, Diane Cassidy, Esther C H Uijttewaal, Vojtech Dvorak, Yuting Cao, Ariel Bensimon, Ulrich Elling, Alessio Ciulli, Giulio Superti-Furga.
      Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that induce selective protein degradation by linking an E3 ubiquitin ligase enzyme to a target protein. This approach allows scope for targeting "undruggable" proteins, and several PROTACs have reached the stage of clinical candidates. However, the roles of cellular transmembrane transporters in PROTAC uptake and efflux remain underexplored. Here, we utilized transporter-focused genetic screens to identify the ATP-binding cassette transporter ABCC1/MRP1 as a key PROTAC resistance factor. Unlike the previously identified inducible PROTAC exporter ABCB1/MDR1, ABCC1 is highly expressed among cancers of various origins and constitutively restricts PROTAC bioavailability. Moreover, in a genome-wide PROTAC resistance screen, we identified candidates involved in processes such as ubiquitination, mTOR signaling, and apoptosis as genetic factors involved in PROTAC resistance. In summary, our findings reveal ABCC1 as a crucial constitutively active efflux pump limiting PROTAC efficacy in various cancer cells, offering insights for overcoming drug resistance.
    Keywords:  ABCC1; CRISPR/Cas9; MRP1; PROTACs; drug transport; genetic screening; solute carriers (SLCs)
    DOI:  https://doi.org/10.1016/j.chembiol.2024.11.009
  6. Autophagy. 2025 Jan 11. 1-20
    Nonreceptor tyrosine kinase ABL1 regulates lysosomal acidification by phosphorylating the ATP6V1B2 subunit of the vacuolar-type H+-ATPase.
    Caiwei Song, Qincai Dong, Yi Yao, Yan Cui, Chunmei Zhang, Lijun Lin, Lin Zhu, Yong Hu, Hainan Liu, Yanwen Jin, Ping Li, Xuan Liu, Cheng Cao.
      The vacuolar-type H+-ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. Its activity and assembly are tightly controlled by multiple pathways, of which phosphorylation-mediated regulation is poorly understood. In this report, we show that in response to starvation stimuli, the nonreceptor tyrosine kinase ABL1 directly interacts with ATP6V1B2, a subunit of the V1 domain of the V-ATPase, and phosphorylates ATP6V1B2 at Y68. Y68 phosphorylation in ATP6V1B2 facilitates the recruitment of the ATP6V1D subunit into the V1 subcomplex of V-ATPase, therefore potentiating the assembly of the V1 subcomplex with the membrane-embedded V0 subcomplex to form the integrated functional V-ATPase. ABL1 inhibition or depletion impairs V-ATPase assembly and lysosomal acidification, resulting in an increased lysosomal pH, a decreased lysosomal hydrolase activity, and consequently, the suppressed degradation of lumenal cargo during macroautophagy/autophagy. Consistently, the efficient removal of damaged mitochondrial residues during mitophagy is also impeded by ABL1 deficiency. Our findings suggest that ABL1 is a crucial autophagy regulator that maintains the adequate lysosomal acidification required for both physiological conditions and stress responses.Abbreviation: ANOVA: analysis of variance; Baf A1: bafilomycin A1; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CRK: CRK proto-oncogene, adaptor protein; CTSD: cathepsin D; DMSO: dimethylsulfoxide; EBSS: Earle's balanced salt solution; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; GST: glutathione S-transferase; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PD: Parkinson disease; PLA: proximity ligation assay; RFP: red fluorescent protein; WT: wild-type.
    Keywords:  ABL1; V-ATPase; kinase; lysosome; phosphorylation
    DOI:  https://doi.org/10.1080/15548627.2024.2448913
  7. Sci Adv. 2025 Jan 10. 11(2): eadr7943
    Molecular basis for the stepwise and faithful maturation of the 20S proteasome.
    Yaoyao Han, Qian Han, Qianqian Tang, Yixiao Zhang, Kai Liu.
      The proteasome degrades most superfluous and damaged proteins, and its decline is associated with many diseases. As the proteolytic unit, the 20S proteasome is assembled from 28 subunits assisted by chaperones PAC1/2/3/4 and POMP; then, it undergoes the maturation process, in which the proteolytic sites are activated and the assembly chaperones are cleared. However, mechanisms governing the maturation remain elusive. Here, we captured endogenous maturation intermediates of human 20S proteasome, which are low abundance and highly dynamic, and determined their structures by cryo-electron microscopy. Through structure-based functional studies, we identified the key switches that remodel and activate the proteolytic sites. Our results also revealed that the POMP degradation is tightly controlled by a dual-checking mechanism, while the α5 subunit senses POMP degradation to induce PAC1/2 release, achieving the full maturation. These findings elucidate mechanisms directing and safeguarding the proteasome maturation and set basis for building proteasomes to counteract the decline of protein degradation in aging and disease.
    DOI:  https://doi.org/10.1126/sciadv.adr7943
  8. bioRxiv. 2024 Dec 17. pii: 2024.12.13.628451. [Epub ahead of print]
    PERK Signaling Maintains Hematopoietic Stem Cell Pool Integrity under Endoplasmic Reticulum Stress by Promoting Proliferation.
    Manxi Zheng, Qinlu Peng, Erin M Kropp, Zhejuan Shen, Suxuan Liu, Zhengyou Yin, Sho Matono, Takao Iwawaki, Xiang Wang, Ken Inoki, Yang Mei, Qing Li, Lu Liu.
      The integrity of the hematopoietic stem cell (HSC) pool relies on efficient long-term self-renewal and the timely removal of damaged or differentiation-prone HSCs. Previous studies have demonstrated the PERK branch of the unfolded protein response (UPR) drives specific programmed cell death programs to maintain HSC pool integrity in response to ER stress. However, the role of PERK in regulating HSC fate in vivo remains unclear. Here, we demonstrate that PERK is dispensable for normal hematopoiesis and HSC self-renewal under steady-state conditions. In contrast, PERK is activated to promote HSC proliferation and depletion in response to ER stress induced by the inactivation of ER-associated degradation (ERAD), via the knockout of key components of ERAD Sel1L or Hrd1. Inhibition of PERK, either through genetic knockout or knock-in of a point mutation that eliminates PERK kinase activity, significantly restores the HSC defects induced by Sel1L or Hrd1 knockout. Mechanistic studies reveal that ERAD deficiency does not lead to HSC death or ROS accumulation. Instead, PERK promotes the activation of mTOR signaling and drives abnormal proliferation of HSCs, impairing their self-renewal potential. This process removes stressed HSCs, thereby maintaining HSC pool integrity. Our study uncovers a PERK-centered strategy employed by HSCs to preserve their pool integrity independently of apoptosis.
    Key points: PERK is not required for steady-state hematopoiesis but preserves hematopoietic stem cell pool integrity in response to increased ER stress.Under ER stress induced by ERAD deficiency, PERK is activated to promote mTOR signaling and HSC hyper-proliferation, depleting damaged HSCs.
    DOI:  https://doi.org/10.1101/2024.12.13.628451
  9. Mol Neurodegener. 2025 Jan 07. 20(1): 2
    VCP regulates early tau seed amplification via specific cofactors.
    Sushobhna Batra, Jaime Vaquer-Alicea, Clarissa Valdez, Skyler P Taylor, Victor A Manon, Anthony R Vega, Omar M Kashmer, Sourav Kolay, Andrew Lemoff, Nigel J Cairns, Charles L White, Marc I Diamond.
       BACKGROUND: Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. Seeding into the complex cytoplasmic milieu happens within hours, implying the existence of unknown factors that regulate this process.
    METHODS: We used proximity labeling to identify proteins that control seed amplification within 5 h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5 h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and pharmacological manipulations of VCP.
    RESULTS: VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on seeding in HEK293T tau biosensor cells and human neurons: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors only functioned when administered within 8 h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding.
    CONCLUSIONS: Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a cytoplasmic processing complex centered on VCP that directs seeds acutely towards degradation vs. amplification.
    Keywords:  APEX2; Cofactors; Disaggregase; Seeding; Tau; VCP; p97
    DOI:  https://doi.org/10.1186/s13024-024-00783-z
  10. Trends Biochem Sci. 2025 Jan 07. pii: S0968-0004(24)00283-4. [Epub ahead of print]
    Stress responses induced by perturbation of the ubiquitin-proteasome system.
    Mamta Rai, Liam C Hunt, Fabio Demontis.
      The ubiquitin-proteasome system is key for proteostasis and its disruption can induce several cellular adaptations. Here, we summarize the range of cellular responses that are induced by perturbation of distinct components of the ubiquitin-proteasome system, and how proteasome stress in a tissue can induce systemic responses in distant tissues.
    Keywords:  E2 ubiquitin-conjugating enzymes; UBA1; ingrained stress response; proteasome; systemic stress response; ubiquitination
    DOI:  https://doi.org/10.1016/j.tibs.2024.12.011
  11. Trends Biochem Sci. 2025 Jan 03. pii: S0968-0004(24)00275-5. [Epub ahead of print]
    Protein quality control machinery: regulators of condensate architecture and functionality.
    Anitha Rajendran, Carlos A Castañeda.
      Protein quality control (PQC) mechanisms including the ubiquitin (Ub)-proteasome system (UPS), autophagy, and chaperone-mediated refolding are essential to maintain protein homeostasis in cells. Recent studies show that these PQC mechanisms are further modulated by biomolecular condensates that sequester PQC components and compartmentalize reactions. Accumulating evidence points towards the PQC machinery playing a pivotal role in regulating the assembly, disassembly, and viscoelastic properties of several condensates. Here, we discuss how the PQC machinery can form their own condensates and also be recruited to known condensates under physiological or stress-induced conditions. We present molecular insights into how the multivalent architecture of polyUb chains, Ub-binding adaptor proteins, and other PQC machinery contribute to condensate assembly, leading to the regulation of downstream PQC outcomes and therapeutic potential.
    Keywords:  chaperones; condensates; phase separation; polyphasic linkage; protein quality control; ubiquitination
    DOI:  https://doi.org/10.1016/j.tibs.2024.12.003
  12. J Cell Biol. 2025 Mar 03. pii: e202406122. [Epub ahead of print]224(3):
    Rickettsia parkeri forms extensive, stable contacts with the rough endoplasmic reticulum.
    Yamilex Acevedo-Sánchez, Patrick J Woida, Caroline Anderson, Stephan Kraemer, Rebecca L Lamason.
      Upon invasion into the host cell, a subset of bacterial pathogens resides exclusively in the cytosol. While previous research revealed how they reshape the plasma membrane during invasion, subvert the immune response, and hijack cytoskeletal dynamics to promote their motility, it was unclear if these pathogens also interacted with the organelles in this crowded intracellular space. Here, we examined if the obligate intracellular pathogen Rickettsia parkeri interacts with the endoplasmic reticulum (ER), a large and dynamic organelle spread throughout the cell. Using live-cell microscopy and transmission and focused-ion-beam scanning electron microscopy, we show that R. parkeri forms extensive contacts with the rough ER that are ∼55 nm apart and cover more than half the bacterial surface. Depletion of the ER-specific tethers VAPA and VAPB reduced rickettsia-ER contacts, and VAPA and VAPB were localized around intracellular rickettsiae. Overall, our findings illuminate an interkingdom ER contact uniquely mediated by rickettsiae that mimics some characteristics of traditional host membrane contact sites.
    DOI:  https://doi.org/10.1083/jcb.202406122
  13. Microb Pathog. 2025 Jan 04. pii: S0882-4010(25)00004-X. [Epub ahead of print]200 107279
    The interplay between gut microbiota and the unfolded protein response: Implications for intestinal homeostasis preservation and dysbiosis-related diseases.
    Miriam Di Mattia, Michele Sallese, Loris Riccardo Lopetuso.
      The unfolded protein response (UPR) is a complex intracellular signal transduction system that orchestrates the cellular response during Endoplasmic Reticulum (ER) stress conditions to reestablish cellular proteostasis. If, on one side, prolonged ER stress conditions can lead to programmed cell death and autophagy as a cytoprotective mechanism, on the other, unresolved ER stress and improper UPR activation represent a perilous condition able to trigger or exacerbate inflammatory responses. Notably, intestinal and immune cells experience ER stress physiologically due to their high protein secretory rate. Indeed, there is evidence of UPR's involvement in both physiological and pathological intestinal conditions, while less is known about its bidirectional interaction with gut microbiota. However, gut microbes and their metabolites can influence ER stress and UPR pathways, and, in turn, ER stress conditions can shape gut microbiota composition, with important implications for overall intestinal health. Thus, targeting UPR components is an intriguing strategy for treating ER stress-linked dysbiosis and diseases, particularly intestinal inflammation.
    Keywords:  Autophagy; Immunity; KDEL receptors; Microbiota; Proteostasis
    DOI:  https://doi.org/10.1016/j.micpath.2025.107279
  14. Elife. 2025 Jan 10. pii: e75393. [Epub ahead of print]14
    Oversized cells activate global proteasome-mediated protein degradation to maintain cell size homeostasis.
    Shixuan Liu, Ceryl Tan, Chloe Melo-Gavin, Miriam B Ginzberg, Ron Blutrich, Nish Patel, Michael Rape, Kevin G Mark, Ran Kafri.
      Proliferating animal cells maintain a stable size distribution over generations despite fluctuations in cell growth and division size. Previously, we showed that cell size control involves both cell size checkpoints, which delay cell cycle progression in small cells, and size-dependent regulation of mass accumulation rates (Ginzberg et al., 2018). While we previously identified the p38 MAPK pathway as a key regulator of the mammalian cell size checkpoint (S. Liu et al., 2018), the mechanism of size-dependent growth rate regulation has remained elusive. Here, we quantified global rates of protein synthesis and degradation in cells of varying sizes, both under unperturbed conditions and in response to perturbations that trigger size-dependent compensatory growth slowdown. We found that protein synthesis rates scale proportionally with cell size across cell cycle stages and experimental conditions. In contrast, oversized cells that undergo compensatory growth slowdown exhibit a superlinear increase in proteasome-mediated protein degradation, with accelerated protein turnover per unit mass, suggesting activation of the proteasomal degradation pathway. Both nascent and long-lived proteins contribute to the elevated protein degradation during compensatory growth slowdown, with long-lived proteins playing a crucial role at the G1/S transition. Notably, large G1/S cells exhibit particularly high efficiency in protein degradation, surpassing that of similarly sized or larger cells in S and G2, coinciding with the timing of the most stringent size control in animal cells. These results collectively suggest that oversized cells reduce their growth efficiency by activating global proteasome-mediated protein degradation to promote cell size homeostasis.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.75393
  15. EMBO J. 2025 Jan 06.
    Two FAM134B isoforms differentially regulate ER dynamics during myogenesis.
    Viviana Buonomo, Kateryna Lohachova, Alessio Reggio, Sara Cano-Franco, Michele Cillo, Lucia Santorelli, Rossella Venditti, Elena Polishchuk, Ivana Peluso, Lorene Brunello, Carmine Cirillo, Sara Petrosino, Malan Silva, Rossella De Cegli, Sabrina Di Bartolomeo, Cesare Gargioli, Paolo Swuec, Mirko Cortese, Alexandra Stolz, Ramachandra M Bhaskara, Paolo Grumati.
      Endoplasmic reticulum (ER) plasticity and ER-phagy are intertwined processes essential for maintaining ER dynamics. We investigated the interplay between two isoforms of the ER-phagy receptor FAM134B in regulating ER remodeling in differentiating myoblasts. During myogenesis, the canonical FAM134B1 is degraded, while its isoform FAM134B2 is transcriptionally upregulated. The switch, favoring FAM134B2, is an important regulator of ER morphology during myogenesis. FAM134B2 partial reticulon homology domain, with its rigid conformational characteristics, enables efficient ER reshaping. FAM134B2 action increases in the active phase of differentiation leading to ER restructuring via ER-phagy, which then reverts to physiological levels when myotubes are mature and the ER is reorganized. Knocking out both FAM134B isoforms in myotubes results in an aberrant proteome landscape and the formation of dilated ER structures, both of which are rescued by FAM134B2 re-expression. Our results underscore how the fine-tuning of FAM134B isoforms and ER-phagy orchestrate the ER dynamics during myogenesis providing insights into the molecular mechanisms governing ER homeostasis in muscle cells.
    Keywords:  Autophagy; Endoplasmic Reticulum; FAM134B; Myogenesis; Reticulophagy
    DOI:  https://doi.org/10.1038/s44318-024-00356-2
  16. Cell Rep. 2025 Jan 08. pii: S2211-1247(24)01470-0. [Epub ahead of print]44(1): 115119
    Ribosome customization and functional diversification among P-stalk proteins regulate late poxvirus protein synthesis.
    Natalia Khalatyan, Daphne Cornish, Aaron J Ferrell, Jeffrey N Savas, Peter S Shen, Judd F Hultquist, Derek Walsh.
      Growing evidence suggests that ribosomes selectively regulate translation of specific mRNA subsets. Here, quantitative proteomics and cryoelectron microscopy demonstrate that poxvirus infection does not alter ribosomal subunit protein (RP) composition but skews 40S rotation states and displaces the 40S head domain. Genetic knockout screens employing metabolic assays and a dual-reporter virus further identified two RPs that selectively regulate non-canonical translation of late poxvirus mRNAs, which contain unusual 5' poly(A) leaders: receptor of activated C kinase 1 (RACK1) and RPLP2. RACK1 is a component of the altered 40S head domain, while RPLP2 is a subunit of the P-stalk, wherein RPLP0 anchors two heterodimers of RPLP1 and RPLP2 to the large 60S subunit. RPLP0 was required for global translation, yet RPLP1 was dispensable, while RPLP2 was specifically required for non-canonical poxvirus protein synthesis. From these combined results, we demonstrate that poxviruses structurally customize ribosomes and become reliant upon traditionally non-essential RPs from both ribosomal subunits for efficient initiation on their late mRNAs.
    Keywords:  CP: Molecular biology; CRISPR screen; P-stalk; RACK1; RPLP2; cryo-EM; poxvirus; ribosome customization; translational control
    DOI:  https://doi.org/10.1016/j.celrep.2024.115119
  17. bioRxiv. 2024 Dec 23. pii: 2024.12.22.629972. [Epub ahead of print]
    Transcriptomics Unveil Canonical and Non-Canonical Heat Shock-Induced Pathways in Human Cell Lines.
    Andrew Reinschmidt, Luis Solano, Yonny Chavez, William Drew Hulsy, Nikolas Nikolaidis.
      The cellular stress response (CSR) is a conserved mechanism that protects cells from environmental and physiological stressors. The heat shock response (HSR), a critical component of the CSR, utilizes molecular chaperones to mitigate proteotoxic stress caused by elevated temperatures. We hypothesized that while the canonical HSR pathways are conserved across cell types, specific cell lines may exhibit unique transcriptional responses to heat shock. To test this, we compared the transcriptomic responses of HEK293, HepG2, and HeLa cells under control conditions immediately following heat shock and after an 8-hour recovery period. RNA sequencing revealed conserved activation of canonical HSR pathways, including the unfolded protein response, alongside enrichment of the non-canonical "Receptor Ligand Activity" pathway across all cell lines. Cell line-specific variations were also observed, with HepG2 cells displaying more uniquely expressed genes and elevated expression levels (fold changes) of shared genes under stress conditions. Validation by qPCR confirmed the activation of key genes within the "Receptor Ligand Activity" pathway across time points. These findings provide insights into conserved and context-specific aspects of the HSR, contributing to a more comprehensive understanding of stress response mechanisms across mammalian cells.
    DOI:  https://doi.org/10.1101/2024.12.22.629972
  18. Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2415976121
    Genesis and regulation of C-terminal cyclic imides from protein damage.
    Wenqing Xu, Zhenguang Zhao, Matthew Su, Atul D Jain, Hannah C Lloyd, Ethan Yang Feng, Nick Cox, Christina M Woo.
      C-Terminal cyclic imides are posttranslational modifications that can arise from spontaneous intramolecular cleavage of asparagine or glutamine residues resulting in a form of irreversible protein damage. These protein damage events are recognized and removed by the E3 ligase substrate adapter cereblon (CRBN), indicating that these aging-related modifications may require cellular quality control mechanisms to prevent deleterious effects. However, the factors that determine protein or peptide susceptibility to C-terminal cyclic imide formation or their effect on protein stability have not been explored in detail. Here, we characterize the primary and secondary structures of peptides and proteins that promote intrinsic formation of C-terminal cyclic imides in comparison to deamidation, a related form of protein damage. Extrinsic effects from solution properties and stressors on the cellular proteome additionally promote C-terminal cyclic imide formation on proteins like glutathione synthetase that are susceptible to aggregation if the protein damage products are not removed by CRBN. This systematic investigation provides insight into the regions of the proteome that are prone to these unexpectedly frequent modifications, the effects of this form of protein damage on protein stability, and the biological role of CRBN.
    Keywords:  cereblon; deamidation; post-translational modification; protein aggregation; protein damage
    DOI:  https://doi.org/10.1073/pnas.2415976121
  19. J Cell Biol. 2025 Feb 03. pii: e202401101. [Epub ahead of print]224(2):
    RACK1 MARylation regulates translation and stress granules in ovarian cancer cells.
    Sridevi Challa, Tulip Nandu, Hyung Bum Kim, Xuan Gong, Charles W Renshaw, Wan-Chen Li, Xinrui Tan, Marwa W Aljardali, Cristel V Camacho, Jin Chen, W Lee Kraus.
      Mono(ADP-ribosyl)ation (MARylation) is emerging as a critical regulator of ribosome function and translation. Herein, we demonstrate that RACK1, an integral component of the ribosome, is MARylated by the mono(ADP-ribosyl) transferase (MART) PARP14 in ovarian cancer cells. MARylation of RACK1 is required for stress granule formation and promotes the colocalization of RACK1 in stress granules with G3BP1, eIF3η, and 40S ribosomal proteins. In parallel, we observed reduced translation of a subset of mRNAs, including those encoding key cancer regulators (e.g., AKT). Treatment with a PARP14 inhibitor or mutation of the sites of MARylation on RACK1 blocks these outcomes, as well as the growth of ovarian cancer cells in culture and in vivo. To reset the system after prolonged stress and recovery, the ADP-ribosyl hydrolase TARG1 deMARylates RACK1, leading to the dissociation of the stress granules and the restoration of translation. Collectively, our results demonstrate a therapeutically targetable pathway that controls polysome assembly, translation, and stress granule dynamics in ovarian cancer cells.
    DOI:  https://doi.org/10.1083/jcb.202401101
  20. mBio. 2025 Jan 08. e0352224
    Transmission of unfolded protein response-a regulator of disease progression, severity, and spread in virus infections.
    Vibhu Prasad.
      The unfolded protein response (UPR) is a cell-autonomous stress response aimed at restoring homeostasis due to the accumulation of misfolded proteins in the endoplasmic reticulum (ER). Viruses often hijack the host cell machinery, leading to an accumulation of misfolded proteins in the ER. The cell-autonomous UPR is the immediate response of an infected cell to this stress, aiming to restore normal function by halting protein translation, degrading misfolded proteins, and activating signaling pathways that increase the production of molecular chaperones. The cell-non-autonomous UPR involves the spreading of UPR signals from initially stressed cells to neighboring unstressed cells that lack the stressor. Though viruses are known modulators of cell-autonomous UPR, recent advancements have highlighted that cell-non-autonomous UPR plays a critical role in elucidating how local infections cause systemic effects, thereby contributing to disease symptoms and progression. Additionally, by utilizing cell-non-autonomous UPR, viruses have devised novel strategies to establish a pro-viral state, promoting virus spread. This review discusses examples that have broadened the understanding of the role of UPR in virus infections and disease progression by looking beyond cell-autonomous to non-autonomous processes and mechanistic details of the inducers, spreaders, and receivers of UPR signals.
    Keywords:  cell-non-autonomous UPR; disease progression; pro-viral state; unfolded protein response (UPR); virus infections
    DOI:  https://doi.org/10.1128/mbio.03522-24
  21. Cell Chem Biol. 2025 Jan 06. pii: S2451-9456(24)00516-6. [Epub ahead of print]
    Targeting N-Myc in neuroblastoma with selective Aurora kinase A degraders.
    Jian Tang, Ramkumar Moorthy, Laura E Hirsch, Özlem Demir, Zachary D Baker, Jordan A Naumann, Katherine F M Jones, Michael J Grillo, Ella S Haefner, Ke Shi, Michaella J Levy, Harshita B Gupta, Hideki Aihara, Reuben S Harris, Rommie E Amaro, Nicholas M Levinson, Daniel A Harki.
      The N-Myc transcription factor, encoded by MYCN, is a mechanistically validated, yet challenging, target for neuroblastoma (NB) therapy development. In normal neuronal progenitors, N-Myc undergoes rapid degradation, while, in MYCN-amplified NB cells, Aurora kinase A (Aurora-A) binds to and stabilizes N-Myc, resulting in elevated protein levels. Here, we demonstrate that targeted protein degradation of Aurora-A decreases N-Myc levels. A potent Aurora-A degrader, HLB-0532259 (compound 4), was developed from an Aurora-A-binding ligand that engages the Aurora-A/N-Myc complex. HLB-0532259 promotes the degradation of Aurora-A, which elicits concomitant N-Myc degradation, with nanomolar potency and excellent selectivity. HLB-0532259 surpasses the cellular efficacy of established allosteric Aurora-A inhibitors, exhibits favorable pharmacokinetic properties, and elicits tumor reduction in a murine xenograft NB model. This study broadly delineates a strategy for targeting "undruggable" proteins that are reliant on accessory proteins for cellular stabilization.
    Keywords:  Aurora kinase A; MYCN; N-Myc; PROTAC; TPD; neuroblastoma; targeted protein degradation; transcription factor
    DOI:  https://doi.org/10.1016/j.chembiol.2024.12.006
  22. Nat Commun. 2025 Jan 07. 16(1): 451
    Mitochondria- and ER-associated actin are required for mitochondrial fusion.
    Priya Gatti, Cara Schiavon, Julien Cicero, Uri Manor, Marc Germain.
      Mitochondria are crucial for cellular metabolism and signalling. Mitochondrial activity is modulated by mitochondrial fission and fusion, which are required to properly balance metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at mitochondria-endoplasmic reticulum (ER) contact sites, and requires the formation of actin filaments that drive mitochondrial constriction and the recruitment of the fission protein DRP1. The role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing actin polymerisation on either mitochondria or the ER disrupts both fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion require INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the fusion protein MFN2. Together, our work introduces a method for perturbing organelle-associated actin and demonstrates a previously unknown role for actin in mitochondrial fusion.
    DOI:  https://doi.org/10.1038/s41467-024-55758-x
  23. Nature. 2025 Jan 08.
    Site-saturation mutagenesis of 500 human protein domains.
    Antoni Beltran, Xiang'er Jiang, Yue Shen, Ben Lehner.
      Missense variants that change the amino acid sequences of proteins cause one-third of human genetic diseases1. Tens of millions of missense variants exist in the current human population, and the vast majority of these have unknown functional consequences. Here we present a large-scale experimental analysis of human missense variants across many different proteins. Using DNA synthesis and cellular selection experiments we quantify the effect of more than 500,000 variants on the abundance of more than 500 human protein domains. This dataset reveals that 60% of pathogenic missense variants reduce protein stability. The contribution of stability to protein fitness varies across proteins and diseases and is particularly important in recessive disorders. We combine stability measurements with protein language models to annotate functional sites across proteins. Mutational effects on stability are largely conserved in homologous domains, enabling accurate stability prediction across entire protein families using energy models. Our data demonstrate the feasibility of assaying human protein variants at scale and provides a large consistent reference dataset for clinical variant interpretation and training and benchmarking of computational methods.
    DOI:  https://doi.org/10.1038/s41586-024-08370-4
  24. Nat Chem Biol. 2025 Jan 06.
    E2-Ub-R74G strategy reveals E2-specific ubiquitin conjugation profiles in live cells.
    Siqi Shen, Hang Yin.
      The E2 ubiquitin (Ub)-conjugating enzyme primarily determines Ub conjugation as Ub-isopeptide (lysine), Ub-oxyester (serine/threonine) or Ub-thioester (cysteine). However, E2-specific Ub conjugation profiles within cells remain elusive. Here we developed the fusion E2-Ub-R74G profiling (FUSEP) strategy to access E2-specific Ub conjugation profiles in cells with amino acid resolution. The probe-specific leucine-glycine-glycine-glycine-modified Ub remnant enables systematic studies of non-lysine Ub conjugation and provides site-specific information. Multiple E2 enzymes were found to be involved in non-lysine ubiquitination. Profiling with UBE2D3-Ub-R74G probes identified a post-translational modification, tyrosine ubiquitination, in human Cullin-1, a scaffold protein for Cullin-RING E3 Ub ligases. This modification is distinct from lysine ubiquitination. A single-pass membrane-bound E3 ligase, RNF149, was identified to pair with UBE2D3 to regulate pyroptosis by ubiquitinating apoptosis-associated speck-like protein ASC. The availability of this toolbox paves the way for uncovering E2-specific Ub conjugation profiles and identifying previously unknown E3 Ub ligases for potential therapeutic applications.
    DOI:  https://doi.org/10.1038/s41589-024-01809-9
  25. Nat Chem Biol. 2025 Jan 09.
    Chemically engineered antibodies for autophagy-based receptor degradation.
    Binghua Cheng, Meiqing Li, Jiwei Zheng, Jiaming Liang, Yanyan Li, Ruijing Liang, Hui Tian, Zeyu Zhou, Li Ding, Jian Ren, Wenli Shi, Wenjie Zhou, Hailiang Hu, Long Meng, Ke Liu, Lintao Cai, Ximing Shao, Lijing Fang, Hongchang Li.
      Cell surface receptor-targeted protein degraders hold promise for drug discovery. However, their application is restricted because of the complexity of creating bifunctional degraders and the reliance on specific lysosome-shuttling receptors or E3 ubiquitin ligases. To address these limitations, we developed an autophagy-based plasma membrane protein degradation platform, which we term AUTABs (autophagy-inducing antibodies). Through covalent conjugation with polyethylenimine (PEI), the engineered antibodies acquire the capacity to degrade target receptors through autophagy. The degradation activities of AUTABs are self-sufficient, without necessitating the participation of lysosome-shuttling receptors or E3 ubiquitin ligases. The broad applicability of this platform was then illustrated by targeting various clinically important receptors. Notably, combining specific primary antibodies with a PEI-tagged secondary nanobody also demonstrated effective degradation of target receptors. Thus, our study outlines a strategy for directing plasma membrane proteins for autophagic degradation, which possesses desirable attributes such as ease of generation, independence from cell type and broad applicability.
    DOI:  https://doi.org/10.1038/s41589-024-01803-1
  26. Redox Biol. 2024 Dec 31. pii: S2213-2317(24)00464-6. [Epub ahead of print]80 103486
    Exquisite sensitivity of Polycystin-1 to H2O2 concentration in the endoplasmic reticulum.
    Elisa Speranza, Ilaria Sorrentino, Alessandra Boletta, Roberto Sitia.
      Aquaporin11 (AQP11) is an endoplasmic reticulum (ER) resident peroxiporin. It allows H2O2 transport from the lumen to the cytosol, guaranteeing redox homeostasis and signaling in and between the two organelles. Interestingly, Aqp11-/- mice develop a fatal, early onset polycystic kidney disease (PKD) similar to Autosomal Dominant PKD, a condition frequently associated with mutations of polycystin-1 (PC-1) in human patients. Here we investigated the molecular mechanisms of AQP11-associated PKD. Using different cell models, we show that transient downregulation of AQP11 selectively prevents the biogenesis of overexpressed PC-1. Expression of catalase in the ER lumen rescues the phenotype, demonstrating a direct role of (H2O2)ER in controlling the complex maturation of PC-1. Analysis of endogenous Pc-1 revealed an additional regulatory role at the pre-translational level. Taken together, our results show that AQP11 controls the complex biogenesis of PC-1 at multiple levels governing H2O2 intra and inter-organellar fluxes, with important implications in the pathogenesis and onset of PKD.
    Keywords:  Aquaporin 11; H(2)O(2); Polycystic kidney disease; Polycystin-1; Protein transport; Redox homeostasis
    DOI:  https://doi.org/10.1016/j.redox.2024.103486
  27. Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2414016122
    VCP controls KCC2 degradation through FAF1 recruitment and accelerates emergence from anesthesia.
    Peng Chen, Jiang-Jian Hu, Yuexin Liu, Boxu Cao, Xue-Jun Song.
      Ubiquitin-proteasomal degradation of K+/Cl- cotransporter 2 (KCC2) in the ventral posteromedial nucleus (VPM) has been demonstrated to serve as a common mechanism by which the brain emerges from anesthesia and regains consciousness. Ubiquitin-proteasomal degradation of KCC2 during anesthesia is driven by E3 ligase Fbxl4. However, the mechanism by which ubiquitinated KCC2 is targeted to the proteasome has not been elucidated. We report in cultured neuro-2a cells that the valosin-containing protein (VCP) transported ubiquitinated KCC2 to the proteasome and in mice in vivo experiments that inhibition of VCP restored KCC2 expression in the VPM and enhanced the effects of anesthesia. In cultured neuro-2a cells, propofol-induced degradation of KCC2 was inhibited by VCP inhibitor DBeQ and VCP knockout plasmid sgRNA(VCP). Propofol-induced enhanced interaction between VCP and KCC2 was inhibited by knockout of Fbxl4 or Fas-associated factor 1 (FAF1). In in vivo studies, pharmacological or genetic inhibition of VCP in the VPM significantly prevented KCC2 degradation and enhanced propofol anesthesia; these effects were abrogated by a KCC2 antagonist VU0463271. These results demonstrate that the VCP controls ubiquitin-proteasomal degradation of KCC2 dependent on FAF1 recruitment and serves as a mechanism for the ubiquitin-proteasomal degradation of KCC2, which is responsible for the subsequent emergence from anesthesia.
    Keywords:  FAF1; KCC2 degradation; VCP; emergence from anesthesia
    DOI:  https://doi.org/10.1073/pnas.2414016122
  28. FEBS J. 2025 Jan 05.
    Regulation of physiological and pathological condensates by molecular chaperones.
    Nadeen Akaree, Valentina Secco, Flonia Levy-Adam, Amal Younis, Serena Carra, Reut Shalgi.
      Biomolecular condensates are dynamic membraneless compartments that regulate a myriad of cellular functions. A particular type of physiological condensate called stress granules (SGs) has gained increasing interest due to its role in the cellular stress response and various diseases. SGs, composed of several hundred RNA-binding proteins, form transiently in response to stress to protect mRNAs from translation and disassemble when the stress subsides. Interestingly, SGs contain several aggregation-prone proteins, such as TDP-43, FUS, hnRNPA1, and others, which are typically found in pathological inclusions seen in autopsy tissues from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. Moreover, mutations in these genes lead to the familial form of ALS and FTD. This has led researchers to propose that pathological aggregation is seeded by aberrant SGs: SGs that fail to properly disassemble, lose their dynamic properties, and become pathological condensates which finally 'mature' into aggregates. Here, we discuss the evidence supporting this model for various ALS/FTD-associated proteins. We further continue to focus on molecular chaperone-mediated regulation of ALS/FTD-associated physiological condensates on one hand, and pathological condensates on the other. In addition to SGs, we review ALS/FTD-relevant nuclear condensates, namely paraspeckles, anisosomes, and nucleolar amyloid bodies, and discuss their emerging regulation by chaperones. As the majority of chaperoning mechanisms regulate physiological condensate disassembly, we highlight parallel themes of physiological and pathological condensation regulation across different chaperone families, underscoring the potential for early disease intervention.
    Keywords:  ALS; FTD; FUS; LLPS; TDP‐43; aggregation; chaperones; condensates; proteostasis; stress granules
    DOI:  https://doi.org/10.1111/febs.17390
  29. bioRxiv. 2024 Dec 20. pii: 2024.12.19.629373. [Epub ahead of print]
    A deep learning method for predicting interactions for intrinsically disordered regions of proteins.
    Kartik Majila, Varun Ullanat, Shruthi Viswanath.
      Intrinsically disordered proteins or regions (IDPs or IDRs) exist as ensembles of conformations in the monomeric state and can adopt diverse binding modes, making their experimental and computational characterization challenging. Here, we developed Disobind, a deep-learning method that predicts inter-protein contact maps and interface residues for an IDR and a partner protein, leveraging sequence embeddings from a protein language model. Several current methods, in contrast, provide partner-independent predictions, require the structure of either protein, and/or are limited by the MSA quality. Disobind performs better than AlphaFold-multimer and AlphaFold3. Combining the Disobind and AlphaFold-multimer predictions further improves the performance. However, Disobind is limited to binary IDP-partner complexes, where the two proteins are known to bind, and the input sequence fragments are less than one hundred residues long. The predictions can be used to localize IDRs in integrative structures of large assemblies, characterize protein-protein interactions involving IDRs, and modulate IDR-mediated interactions.
    DOI:  https://doi.org/10.1101/2024.12.19.629373
  30. Biochemistry. 2025 Jan 10.
    Journey of PROTAC: From Bench to Clinical Trial and Beyond.
    Kyli Berkley, Julian Zalejski, Nidhi Sharma, Ashutosh Sharma.
      Proteolysis-targeting chimeras (PROTACs) represent a transformative advancement in drug discovery, offering a method to degrade specific intracellular proteins. Unlike traditional inhibitors, PROTACs are bifunctional molecules that target proteins for elimination, enabling the potential treatment of previously "undruggable" proteins. This concept, pioneered by Crews and his team, introduced the use of small molecules to link a target protein to an E3 ubiquitin ligase, inducing ubiquitination and subsequent degradation of the target protein. By promoting protein degradation rather than merely inhibiting function, PROTACs present a novel therapeutic strategy with enhanced specificity and effectiveness, especially in areas such as cancer and neurodegenerative diseases. Since their initial discovery, the field of PROTAC research has rapidly expanded with numerous PROTACs now designed to target a wide range of disease-relevant proteins. The substantial research, investment, and collaboration across academia and the pharmaceutical industry reflect the growing interest in PROTACs. This Review discusses the journey of PROTACs from initial discovery to clinical trials, highlighting advancements and challenges. Additionally, recent developments in fluorescent and photogenic PROTACs, used for real-time tracking of protein degradation, are presented, showcasing the evolving potential of PROTACs in targeted therapy.
    Keywords:  Cancer; Clinical Trial; E3 Ubiquitin Ligase; Fluorescent PROTAC; Photogenic PROTAC; Protein Degradation; Real Time Tracking
    DOI:  https://doi.org/10.1021/acs.biochem.4c00577
  31. bioRxiv. 2024 Dec 17. pii: 2024.12.12.628219. [Epub ahead of print]
    Condensates act as translation hubs to coordinate multinucleate cell growth.
    Zachary M Geisterfer, Ameya P Jalihal, Sierra J Cole, Amy S Gladfelter.
      Coordination between growth and division is a fundamental feature of cells. In many syncytia, cell growth must couple with multiple nuclear divisions in one cytoplasm. In the fungus, Ashbya gossypii, cell-cycle progression and hyphal elongation require condensates formed by the protein Whi3 in complex with distinct mRNA species. We hypothesized the condensates may act through local translation regulation and find that Whi3 target mRNAs show distinct spatial biases in translation in vivo. Whi3-RNA condensates can both promote and repress RNA translation in an RNA- and condensate size-dependent manner in vitro. Interestingly, we observe a sub-condensate enrichment of translation that is tunable by RNA valency and protein phospho-state. Together, these data suggest that Whi3 condensates generate a continuum of translation states, resulting in asynchronous nuclear divisions coordinated with growth. This local regulation requires a minimal complement of molecular components at the nano scale to support global coordination at the cell scale.
    DOI:  https://doi.org/10.1101/2024.12.12.628219
  32. Nat Cell Biol. 2025 Jan 07.
    Mitochondrial YME1L1 governs unoccupied protein translocase channels.
    Meng-Chieh Hsu, Hiroki Kinefuchi, Linlin Lei, Reika Kikuchi, Koji Yamano, Richard J Youle.
      Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase fused to the N terminus of the inner membrane protein MIC60. While stabilization of the dihydrofolate reductase domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, notably, plugging the channel of translocase of the outer membrane) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 seems to play a crucial role in mitochondrial quality control to counteract precursor stalling in the translocase of the outer membrane complex and unoccupied TIM23 channels.
    DOI:  https://doi.org/10.1038/s41556-024-01571-z
  33. Elife. 2025 Jan 07. pii: RP100928. [Epub ahead of print]13
    Noncanonical roles of ATG5 and membrane atg8ylation in retromer assembly and function.
    Masroor Ahmad Paddar, Fulong Wang, Einar S Trosdal, Emily Hendrix, Yi He, Michelle R Salemi, Michal Mudd, Jingyue Jia, Thabata Duque, Ruheena Javed, Brett S Phinney, Vojo Deretic.
      ATG5 is one of the core autophagy proteins with additional functions such as noncanonical membrane atg8ylation, which among a growing number of biological outputs includes control of tuberculosis in animal models. Here, we show that ATG5 associates with retromer's core components VPS26, VPS29, and VPS35 and modulates retromer function. Knockout of ATG5 blocked trafficking of a key glucose transporter sorted by the retromer, GLUT1, to the plasma membrane. Knockouts of other genes essential for membrane atg8ylation, of which ATG5 is a component, affected GLUT1 sorting, indicating that membrane atg8ylation as a process affects retromer function and endosomal sorting. The contribution of membrane atg8ylation to retromer function in GLUT1 sorting was independent of canonical autophagy. These findings expand the scope of membrane atg8ylation to specific sorting processes in the cell dependent on the retromer and its known interactors.
    Keywords:  active tuberculosis; atg8ylation; autophagy; cell biology; glucose transport; human; latent tuberculosis; membrane transport; mouse
    DOI:  https://doi.org/10.7554/eLife.100928
  34. Curr Hematol Malig Rep. 2025 Jan 08. 20(1): 4
    Mutant Calreticulin in MPN: Mechanistic Insights and Therapeutic Implications.
    Mifra Faiz, Merle Riedemann, Jonas S Jutzi, Ann Mullally.
       PURPOSE OF REVIEW: More than a decade following the discovery of Calreticulin (CALR) mutations as drivers of myeloproliferative neoplasms (MPN), advances in the understanding of CALR-mutant MPN continue to emerge. Here, we summarize recent advances in mehanistic understanding and in targeted therapies for CALR-mutant MPN.
    RECENT FINDINGS: Structural insights revealed that the mutant CALR-MPL complex is a tetramer and the mutant CALR C-terminus is exposed on the cell surface. Targeting mutant CALR utilizing antibodies is the leading therapeutic approach, while mutant CALR-directed vaccines are also in early clinical trials. Additionally, chimeric antigen receptor (CAR) T-cells directed against mutant CALR are under evaluation in preclinical models. Approaches addressing the cellular effects of mutant CALR beyond MPL-JAK-STAT activation, such as targeting the unfolded protein response, proteasome, and N-glycosylation pathways, have been tested in preclinical models. In CALR-mutant MPN, the path from discovery to mechanistic understanding to direct therapeutic targeting has advanced rapidly. The longer-term goal remains clonally-selective therapies that modify the disease course in patients.
    DOI:  https://doi.org/10.1007/s11899-024-00749-4
  35. Nat Immunol. 2025 Jan 09.
    IRE1α-XBP1 safeguards hematopoietic stem and progenitor cells by restricting pro-leukemogenic gene programs.
    Brendan M Barton, Francheska Son, Akanksha Verma, Saswat Kumar Bal, Qianzi Tang, Rui Wang, Katharine Umphred-Wilson, Rehan Khan, Josephine Trichka, Han Dong, Claudia Lentucci, Xi Chen, Yinghua Chen, Yuning Hong, Cihangir Duy, Olivier Elemento, Ari M Melnick, Jin Cao, Xi Chen, Laurie H Glimcher, Stanley Adoro.
      Hematopoietic stem cells must mitigate myriad stressors throughout their lifetime to ensure normal blood cell generation. Here, we uncover unfolded protein response stress sensor inositol-requiring enzyme-1α (IRE1α) signaling in hematopoietic stem and progenitor cells (HSPCs) as a safeguard against myeloid leukemogenesis. Activated in part by an NADPH oxidase-2 mechanism, IRE1α-induced X-box binding protein-1 (XBP1) mediated repression of pro-leukemogenic programs exemplified by the Wnt-β-catenin pathway. Transcriptome analysis and genome-wide mapping of XBP1 targets in HSPCs identified an '18-gene signature' of XBP1-repressed β-catenin targets that were highly expressed in acute myeloid leukemia (AML) cases with worse prognosis. Accordingly, IRE1α deficiency cooperated with a myeloproliferative oncogene in HSPCs to cause a lethal AML in mice, while genetic induction of XBP1 suppressed the leukemia stem cell program and activity of patient-derived AML cells. Thus, IRE1α-XBP1 signaling safeguards the integrity of the blood system by restricting pro-leukemogenic programs in HSPCs.
    DOI:  https://doi.org/10.1038/s41590-024-02063-w
  36. Nat Commun. 2025 Jan 04. 16(1): 390
    Phosphorylation of a nuclear condensate regulates cohesion and mRNA retention.
    Alexa B R McIntyre, Adrian Beat Tschan, Katrina Meyer, Severin Walser, Arpan Kumar Rai, Keisuke Fujita, Lucas Pelkmans.
      Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During the cell cycle, nuclear speckles dissolve following phosphorylation of their protein components. Here, we identify the PP1 family as the phosphatases that counteract kinase-mediated dissolution. PP1 overexpression increases speckle cohesion and leads to retention of mRNA within speckles and the nucleus. Using APEX2 proximity labeling combined with RNA-sequencing, we characterize the recruitment of specific RNAs. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm, particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increases with nuclear speckle cohesion, the ratios of most mRNA species to each other are constant, indicating non-selective retention. We further find that cellular responses to heat shock, oxidative stress, and hypoxia include changes to the phosphorylation and cohesion of nuclear speckles and to mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization.
    DOI:  https://doi.org/10.1038/s41467-024-55469-3
  37. Cell Rep. 2025 Jan 08. pii: S2211-1247(24)01464-5. [Epub ahead of print]44(1): 115113
    TDP43 autoregulation gives rise to dominant negative isoforms that are tightly controlled by transcriptional and post-translational mechanisms.
    Megan M Dykstra, Kaitlin Weskamp, Nicolás B Gómez, Jacob Waksmacki, Elizabeth Tank, M Rebecca Glineburg, Allison Snyder, Emile Pinarbasi, Michael Bekier, Xingli Li, Morgan R Miller, Jen Bai, Shameena Shahzad, Neha Nedumaran, Clare Wieland, Corey Stewart, Sydney Willey, Nikolas Grotewold, Jonathon McBride, John J Moran, Aditya V Suryakumar, Michael Lucas, Peter M Tessier, Michael Ward, Peter K Todd, Sami J Barmada.
      The nuclear RNA-binding protein TDP43 is integrally involved in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Previous studies uncovered N-terminal TDP43 isoforms that are predominantly cytosolic in localization, prone to aggregation, and enriched in susceptible spinal motor neurons. In healthy cells, however, these shortened (s)TDP43 isoforms are difficult to detect in comparison to full-length (fl)TDP43, raising questions regarding their origin and selective regulation. Here, we show that sTDP43 is created as a by-product of TDP43 autoregulation and cleared by nonsense-mediated RNA decay (NMD). sTDP43-encoding transcripts that escape NMD are rapidly degraded post-translationally via the proteasome and macroautophagy. Circumventing these regulatory mechanisms by overexpressing sTDP43 results in neurodegeneration via N-terminal oligomerization and impairment of flTDP43 splicing activity, in addition to RNA-binding-dependent gain-of-function toxicity. Collectively, these studies highlight endogenous mechanisms that tightly regulate sTDP43 expression and underscore the consequences of aberrant sTDP43 accumulation in disease.
    Keywords:  ALS; CP: Molecular biology; CP: Neuroscience; FTLD-TDP; TDP43; cryptic splicing; neurodegeneration; nonsense-mediated RNA decay
    DOI:  https://doi.org/10.1016/j.celrep.2024.115113
  38. Clin Cancer Res. 2025 Jan 09.
    NXP800 activates the unfolded protein response, altering AR and E2F function to impact castration-resistant prostate cancer growth.
    Jonathan Welti, Denisa Bogdan, Ines Figueiredo, Ilsa Coleman, Juan Jiménez Vacas, Kate Liodaki, Franziska Weigl, Lorenzo Buroni, Wanting Zeng, Ilona Bernett, Claudia Bertan, Theodoros I Roumeliotis, Amandeep Bhamra, Jan Rekowski, Bora Gurel, Antje J Neeb, Jian Ning, Dapei Li, Veronica S Gil, Ruth Riisnaes, Susana Miranda, Mateus Crespo, Ana Ferreira, Nina Tunariu, Elisa Pasqua, Nicola Chessum, Matthew Cheeseman, Robert Te Poele, Marissa Powers, Suzanne Carreira, Jyoti Choudhary, Paul Clarke, Udai Banerji, Amanda Swain, Keith Jones, Wei Yuan, Paul Workman, Peter S Nelson, Johann S de Bono, Adam Sharp.
       PURPOSE: Advanced prostate cancer (PCa) is invariably fatal with the androgen receptor (AR) being a major therapeutic target. AR signaling inhibitors have improved overall survival for men with advanced PCa, but treatment resistance is inevitable and includes reactivation of AR signaling. Novel therapeutic approaches targeting these mechanisms to block tumor growth is an urgent unmet clinical need. One attractive strategy is to target heat shock proteins critical to AR functional activity.
    EXPERIMENTAL DESIGN: We first did transcriptome analysis on multiple castration-resistant PCa (CRPC) cohorts to correlate the association between the GO Cellular Response to Heat gene expression signature and overall survival. Next, we analyzed the impact of targeting the heat shock factor 1 (HSF1) pathway, with an inhibitor in clinical development, namely NXP800, in models of treatment-resistant PCa. Finally, we confirmed our mechanistic and phenotypic findings using an NXP800-resistant model and an in-vivo model of CRPC.
    RESULTS: We report that in multiple CRPC transcriptome cohorts the GO Cellular Response to Heat gene expression signature associates with AR signaling and worse clinical outcome. We demonstrate the effects of targeting the HSF1 pathway, central to cellular stress, with an inhibitor in clinical development, namely NXP800 (formerly CCT361814), in PCa. Targeting the HSF1 pathway with the inhibitor NXP800 decreases HSP72 expression, activates the unfolded protein response, and inhibits AR- and E2F-mediated activity, inhibiting the growth of treatment-resistant PCa models.
    CONCLUSIONS: Overall, NXP800 has anti-tumor activity against treatment-resistant PCa models, including molecular subtypes with limited treatment options, supporting its consideration for PCa-specific clinical development.
    DOI:  https://doi.org/10.1158/1078-0432.CCR-24-2386
  39. bioRxiv. 2024 Dec 27. pii: 2024.12.27.630478. [Epub ahead of print]
    Sexually dimorphic ATF4 expression in the fat confers female stress tolerance in Drosophila melanogaster.
    Lydia Grmai, Melissa Mychalczuk, Aditya Arkalgud, Deepika Vasudevan.
      Metabolic differences between males and females have been well documented across many species. However, the molecular basis of these differences and how they impact tolerance to nutrient deprivation is still under investigation. In this work, we use Drosophila melanogaster to demonstrate that sex-specific differences in fat tissue metabolism are driven, in part, by dimorphic expression of the Integrated Stress Response (ISR) transcription factor, ATF4. We found that female fat tissues have higher ATF4 activity than their male counter parts under homeostatic conditions. This dimorphism was partly due to a female bias in transcript abundance of specific ATF4 splice isoforms. We found that the canonical sex determinants transformer (tra) and doublesex (dsx) drive such dimorphic ATF4 transcript abundance. These differences persist in a genetic model of nutrient deprivation, where female animals showed greater resistance to lethality than males in an ATF4-dependent manner. These results suggest that higher ATF4 activity confers higher tolerance to stress in females. Together, our data describe a previously unknown facet of ISR signaling wherein sexual identity of adipose tissue confers differential stress tolerance in males and females. Since energy storage mechanisms are known to be dimorphic and have been linked to ATF4 regulation, our studies provide a mechanistic starting point for understanding how sexual identity influences metabolic disease outcomes.
    Keywords:  ATF4; adipose; dimorphism; doublesex; methioninase; splicing; stress; transformer
    DOI:  https://doi.org/10.1101/2024.12.27.630478
  40. Cell Rep. 2025 Jan 08. pii: S2211-1247(24)01443-8. [Epub ahead of print]44(1): 115092
    Human TRMT1 and TRMT1L paralogs ensure the proper modification state, stability, and function of tRNAs.
    Kejia Zhang, Aidan C Manning, Jenna M Lentini, Jonathan Howard, Felix Dalwigk, Reza Maroofian, Stephanie Efthymiou, Patricia Chan, Sergei I Eliseev, Zi Yang, Hayley Chang, Ehsan Ghayoor Karimiani, Behnoosh Bakhshoodeh, Henry Houlden, Stefanie M Kaiser, Todd M Lowe, Dragony Fu.
      The tRNA methyltransferase 1 (TRMT1) enzyme catalyzes the N2,N2-dimethylguanosine (m2,2G) modification in tRNAs. Intriguingly, vertebrates encode an additional tRNA methyltransferase 1-like (TRMT1L) paralog. Here, we use a comprehensive tRNA sequencing approach to decipher targets of human TRMT1 and TRMT1L. We find that TRMT1 methylates all known tRNAs containing guanosine at position 26, while TRMT1L represents the elusive enzyme catalyzing m2,2G at position 27 in tyrosine tRNAs. Surprisingly, TRMT1L is also necessary for maintaining 3-(3-amino-3-carboxypropyl)uridine (acp3U) modifications in a subset of tRNAs through a process that can be uncoupled from methyltransferase activity. We also demonstrate that tyrosine and serine tRNAs are dependent upon m2,2G modifications for their stability and function in translation. Notably, human patient cells with disease-associated TRMT1 variants exhibit reduced levels of tyrosine and serine tRNAs. These findings uncover unexpected roles for TRMT1 paralogs, decipher functions for m2,2G modifications, and pinpoint tRNAs dysregulated in human disorders caused by tRNA modification deficiency.
    Keywords:  CP: Molecular biology; RNA modification; TRMT1; TRMT1L; Trm1; intellectual disability; m2,2G; paralog; tRNA; translation
    DOI:  https://doi.org/10.1016/j.celrep.2024.115092
  41. bioRxiv. 2024 Dec 20. pii: 2024.12.19.629301. [Epub ahead of print]
    BRAF V600 and ErbB inhibitors directly activate GCN2 in an off-target manner to limit cancer cell proliferation.
    C Ryland, Nasreen C Marikar, Vu Nguyen, Varuna Nangia, Alicia M Darnell, Matthew G Vander Heiden, Philip Reigan, Sabrina L Spencer.
      Targeted kinase inhibitors are well known for their promiscuity and off-target effects. Herein, we define an off-target effect in which several clinical BRAF V600 inhibitors, including the widely used dabrafenib and encorafenib, interact directly with GCN2 to activate the Integrated Stress Response and ATF4. Blocking this off-target effect by co-drugging with a GCN2 inhibitor in A375 melanoma cells causes enhancement rather than suppression of cancer cell outgrowth, suggesting that the off-target activation of GCN2 is detrimental to these cells. This result is mirrored in PC9 lung cancer cells treated with erlotinib, an EGFR inhibitor, that shares the same off-target activation of GCN2. Using an in silico kinase inhibitor screen, we identified dozens of FDA-approved drugs that appear to share this off-target activation of GCN2 and ATF4. Thus, GCN2 activation may modulate the therapeutic efficacy of some kinase inhibitors, depending on the cancer context.
    DOI:  https://doi.org/10.1101/2024.12.19.629301
  42. Science. 2025 Jan 10. 387(6730): 192-201
    Bile acid synthesis impedes tumor-specific T cell responses during liver cancer.
    Siva Karthik Varanasi, Dan Chen, Yingluo Liu, Melissa A Johnson, Cayla M Miller, Souradipta Ganguly, Kathryn Lande, Michael A LaPorta, Filipe Araujo Hoffmann, Thomas H Mann, Marcos G Teneche, Eduardo Casillas, Kailash C Mangalhara, Varsha Mathew, Ming Sun, Isaac J Jensen, Yagmur Farsakoglu, Timothy Chen, Bianca Parisi, Shaunak Deota, Aaron Havas, Jin Lee, H Kay Chung, Andrea Schietinger, Satchidananda Panda, April E Williams, Donna L Farber, Debanjan Dhar, Peter D Adams, Gen-Sheng Feng, Gerald S Shadel, Mark S Sundrud, Susan M Kaech.
      The metabolic landscape of cancer greatly influences antitumor immunity, yet it remains unclear how organ-specific metabolites in the tumor microenvironment influence immunosurveillance. We found that accumulation of primary conjugated and secondary bile acids (BAs) are metabolic features of human hepatocellular carcinoma and experimental liver cancer models. Inhibiting conjugated BA synthesis in hepatocytes through deletion of the BA-conjugating enzyme bile acid-CoA:amino acid N-acyltransferase (BAAT) enhanced tumor-specific T cell responses, reduced tumor growth, and sensitized tumors to anti-programmed cell death protein 1 (anti-PD-1) immunotherapy. Furthermore, different BAs regulated CD8+ T cells differently; primary BAs induced oxidative stress, whereas the secondary BA lithocholic acid inhibited T cell function through endoplasmic reticulum stress, which was countered by ursodeoxycholic acid. We demonstrate that modifying BA synthesis or dietary intake of ursodeoxycholic acid could improve tumor immunotherapy in liver cancer model systems.
    DOI:  https://doi.org/10.1126/science.adl4100
  43. Cell Rep. 2025 Jan 08. pii: S2211-1247(24)01518-3. [Epub ahead of print]44(1): 115167
    TRMT1L-catalyzed m22G27 on tyrosine tRNA is required for efficient mRNA translation and cell survival under oxidative stress.
    Sseu-Pei Hwang, Han Liao, Katherine Barondeau, Xinyi Han, Cassandra Herbert, Hunter McConie, Amirtha Shekar, Dimitri G Pestov, Patrick A Limbach, Jeffrey T Chang, Catherine Denicourt.
      tRNA modifications are critical for several aspects of their functions, including decoding, folding, and stability. Using a multifaceted approach encompassing eCLIP-seq and nanopore tRNA-seq, we show that the human tRNA methyltransferase TRMT1L interacts with the component of the Rix1 ribosome biogenesis complex and binds to the 28S rRNA as well as to a subset of tRNAs. Mechanistically, we demonstrate that TRMT1L is responsible for catalyzing N2,N2-dimethylguanosine (m22G) solely at position 27 of tRNA-Tyr-GUA. Surprisingly, TRMT1L depletion also impaired the deposition of 3-(3-amino-3-carboxypropyl) uridine (acp3U) and dihydrouridine on tRNA-Tyr-GUA, Cys-GCA, and Ala-CGC. TRMT1L knockout cells have a marked decrease in tRNA-Tyr-GUA levels, coinciding with a reduction in global translation rates and hypersensitivity to oxidative stress. Our results establish TRMT1L as the elusive methyltransferase catalyzing the m22G27 modification on tRNA Tyr, resolving a long-standing gap of knowledge and highlighting its potential role in a tRNA modification circuit crucial for translation regulation and stress response.
    Keywords:  CP: Molecular biology; N2,N2-dimethylguanosine; RNA methyltransferases; TRMT1L; eCLIP-seq; m(2)(2)G; nanopore tRNA-seq; oxidative stress response; rRNA; tRNA; translation
    DOI:  https://doi.org/10.1016/j.celrep.2024.115167
  44. bioRxiv. 2024 Dec 20. pii: 2024.12.19.629517. [Epub ahead of print]
    DNAJC13 localization to endosomes is opposed by its J domain and its disordered C- terminal tail.
    Hayden Adoff, Brandon Novy, Emily Holland, Braden T Lobingier.
      Endosomes are a central sorting hub for membrane cargos. DNAJC13/RME-8 plays a critical role in endosomal trafficking by regulating the endosomal recycling or degradative pathways. DNAJC13 localizes to endosomes through its N-terminal Plekstrin Homology (PH)-like domain, which directly binds endosomal phosphoinositol-3-phosphate (PI(3)P). However, little is known about how DNAJC13 localization is regulated. Here, we show that two regions within DNAJC13, its J domain and disordered C-terminal tail, act as negative regulators of its PH-like domain. Using a structure-function approach combined with quantitative proteomics, we mapped these control points to a conserved YLT motif in the C-terminal tail as well as the catalytic HPD triad in its J domain. Mutation of either motif enhanced DNAJC13 endosomal localization in cells and increased binding to PI(3)P in vitro . Further, these effects required the N-terminal PH-like domain. We show that, similar to other PI(3)P binding domains, the N-terminal PH-like domain binds PI(3)P weakly in isolation and requires oligomerization for efficient PI(3)P binding and endosomal localization. Together, these results demonstrate that interaction between DNAJC13 and PI(3)P serves as a molecular control point for regulating DNAJC13 localization to endosomes.
    Significance Statement: DNAJC13 controls endosomal sorting by regulating proteins which mediate the endosomal recycling and degradative subdomains.Here we show that subcellular localization of DNAJC13 is regulated through the coordinated action of three of its domains: the PH-like domain which has low affinity for PI(3)P, the J domain, and a YLT motif in its disordered C-terminus.This study defines a novel mechanism by which DNAJC13 function is regulated.
    DOI:  https://doi.org/10.1101/2024.12.19.629517
  45. EMBO J. 2025 Jan 06.
    RHEB neddylation by the UBE2F-SAG axis enhances mTORC1 activity and aggravates liver tumorigenesis.
    Fengwu Zhang, Xiufang Xiong, Zhijian Li, Haibo Wang, Weilin Wang, Yongchao Zhao, Yi Sun.
      Small GTPase RHEB is a well-known mTORC1 activator, whereas neddylation modifies cullins and non-cullin substrates to regulate their activity, subcellular localization and stability. Whether and how RHEB is subjected to neddylation modification remains unknown. Here, we report that RHEB is a substrate of NEDD8-conjugating E2 enzyme UBE2F. In cell culture, UBE2F depletion inactivates mTORC1, inhibiting cell cycle progression, cell growth and inducing autophagy. Mechanistically, UBE2F cooperates with E3 ligase SAG in neddylation of RHEB at K169 to enhance its lysosome localization and GTP-binding affinity. Furthermore, liver-specific Ube2f knockout attenuates steatosis and tumorigenesis induced by Pten loss in an mTORC1-dependent manner, suggesting a causal role of UBE2F in liver tumorigenesis. Finally, UBE2F expression levels and mTORC1 activity correlate with patient survival in hepatocellular carcinoma. Collectively, our study identifies RHEB as neddylation substrate of the UBE2F-SAG axis, and highlights the UBE2F-SAG axis as a potential target for the treatment of non-alcoholic fatty liver disease and hepatocellular carcinoma.
    Keywords:  Liver Steatosis and Tumorigenesis; Neddylation; RHEB; UBE2F; mTORC1
    DOI:  https://doi.org/10.1038/s44318-024-00353-5
  46. J Chem Inf Model. 2025 Jan 06.
    PPI-CoAttNet: A Web Server for Protein-Protein Interaction Tasks Using a Coattention Model.
    Qingyu Bian, Zheyuan Shen, Jian Gao, Liteng Shen, Yang Lu, Qingnan Zhang, Roufen Chen, Donghang Xu, Tao Liu, Jinxin Che, Yan Lu, Xiaowu Dong.
      Predicting protein-protein interactions (PPIs) is crucial for advancing drug discovery. Despite the proposal of numerous advanced computational methods, these approaches often suffer from poor usability for biologists and lack generalization. In this study, we designed a deep learning model based on a coattention mechanism that was capable of both PPI and site prediction and used this model as the foundation for PPI-CoAttNet, a user-friendly, multifunctional web server for PPI prediction. This platform provides comprehensive services for online PPI model training, PPI and site prediction, and prediction of interactions with proteins associated with highly prevalent cancers. In our Homo sapiens test set for PPI prediction, PPI-CoAttNet achieved an AUC of 0.9841 and an F1 score of 0.9440, outperforming most state-of-the-art models. Additionally, these results are generated in real time, delivering outcomes within minutes. We also evaluated PPI-CoAttNet for downstream tasks, including novel E3 ligase scoring, demonstrating outstanding accuracy. We believe that this tool will empower researchers, especially those without computational expertise, to leverage AI for accelerating drug development.
    DOI:  https://doi.org/10.1021/acs.jcim.4c01365
  47. Mol Biol Cell. 2025 Jan 09. mbcE24100455
    Valosin-containing protein p97 extracts capping protein CP110 from the mother centriole to promote ciliogenesis.
    Rahit Dewanji, Naava Naslavsky, Steve Caplan.
      The primary cilium is a crucial signaling organelle that can be generated by most human cells, and impediments to primary ciliogenesis lead to a variety of developmental disorders known as ciliopathies. The removal of the capping protein, CP110, from the mother centriole is a crucial early step that promotes generation of the ciliary vesicle and ciliogenesis. Recent studies have demonstrated that CP110 undergoes polyubiquitination and degradation in the proteosome, but the mechanisms of unfolding and removal from the mother centriole remain unknown. Herein we demonstrate that p97/Valosin-containing protein (VCP or Cdc48), a member of the ATPase Associated with diverse Activities (AAA) protein family, is responsible for removal of CP110 from the mother centriole. We show that use of p97 knock-down or inhibition impairs ciliogenesis, in a mechanism dependent on CP110. Our findings demonstrate a novel role for p97 in the process of primary ciliogenesis, and support a mechanism by which ubiquitinated CP110 is degraded in a process that requires p97-mediated unfolding and removal from the mother centriole.
    DOI:  https://doi.org/10.1091/mbc.E24-10-0455
  48. Nature. 2025 Jan 08.
    Crypt density and recruited enhancers underlie intestinal tumour initiation.
    Liam Gaynor, Harshabad Singh, Guodong Tie, Krithika Badarinath, Shariq Madha, Andrew Mancini, Swarnabh Bhattacharya, Mikio Hoshino, Frederic J de Sauvage, Kazutaka Murata, Unmesh Jadhav, Ramesh A Shivdasani.
      Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence1,2. Mutation of Adenomatous polyposis coli (Apc), the most common initiating event in conventional adenomas3, activates Wnt signalling, hence conferring fitness on mutant intestinal stem cells (ISCs)4,5. Apc mutations may occur in ISCs that arose by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar6,7, it is unclear if both generate tumours equally well in uninjured intestines. Also unknown is whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here, we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs not associated with adenomas. These cis-elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumourigenesis.
    DOI:  https://doi.org/10.1038/s41586-024-08573-9
  49. iScience. 2024 Nov 15. 27(11): 110989
    Disrupting the transmembrane domain interface between PMP22 and MPZ causes peripheral neuropathy.
    Natalya Pashkova, Tabitha A Peterson, Christopher P Ptak, Stanley C Winistorfer, Debbie Guerrero-Given, Naomi Kamasawa, Christopher A Ahern, Michael E Shy, Robert C Piper.
      Peripheral Myelin Protein 22 (PMP22) and MPZ are abundant myelin membrane proteins in Schwann cells. The MPZ adhesion protein holds myelin wraps together across the intraperiod line. PMP22 is a tetraspan protein belonging to the Claudin superfamily. Loss of either MPZ or PMP22 causes severe demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy, and duplication of PMP22 causes the most common form of CMT, CMT1A. Yet, the molecular functions provided by PMP22 and how its alteration causes CMT are unknown. Here, we find MPZ and PMP22 form a specific complex through interfaces within their transmembrane domains. We also find that the PMP22 A67T patient variant that causes a loss-of-function (hereditary neuropathy with pressure palsies) phenotype maps to this interface, and blocks MPZ association without affecting localization to the plasma membrane or interactions with other proteins. These data define the molecular basis for the MPZ ∼ PMP22 interaction and indicate this complex fulfills an important function in myelinating cells.
    Keywords:  Biochemistry; Cell biology; Molecular biology; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2024.110989
  50. Elife. 2025 Jan 09. pii: RP93183. [Epub ahead of print]12
    A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms.
    David Trombley McSwiggen, Helen Liu, Ruensern Tan, Sebastia Agramunt Puig, Lakshmi B Akella, Russell Berman, Mason Bretan, Hanzhe Chen, Xavier Darzacq, Kelsey Ford, Ruth Godbey, Eric Gonzalez, Adi Hanuka, Alec Heckert, Jaclyn J Ho, Stephanie L Johnson, Reed Kelso, Aaron Klammer, Ruchira Krishnamurthy, Jifu Li, Kevin Lin, Brian Margolin, Patrick McNamara, Laurence Meyer, Sarah E Pierce, Akshay Sule, Connor Stashko, Yangzhong Tang, Daniel J Anderson, Hilary P Beck.
      The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging >106 cells/day and screening >104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.
    Keywords:  cell biology; drug discovery; estrogen receptor; high-throughput imaging; human; live-cell imaging; physics of living systems; protein motion; single-molecule imaging
    DOI:  https://doi.org/10.7554/eLife.93183
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