bims-proteo Biomed News
on Proteostasis
Issue of 2024–12–15
forty-six papers selected by
Eric Chevet, INSERM
  1. Ubiquitin-dependent translation control mechanisms: Degradation and beyond.
  2. UGGT1-mediated reglucosylation of N-glycan competes with ER-associated degradation of unstable and misfolded glycoproteins.
  3. Loss of FIC-1-mediated AMPylation activates the UPR ER and upregulates cytosolic HSP70 chaperones to suppress polyglutamine toxicity.
  4. STIMulating IRE1: How store-operated Ca2+ entry intersects with ER proteostasis.
  5. TUDCA modulates drug bioavailability to regulate resistance to acute ER stress in Saccharomyces cerevisiae.
  6. Lysosomal Trafficking and Degradation of Extracellular Proteins via Multivalent Small Molecule Ligand Display on Dextran Scaffolds.
  7. Legionella uses host Rab GTPases and BAP31 to create a unique ER niche.
  8. Ubiquitin E3 ligases in the plant Arg/N-degron pathway.
  9. Reactive oxygen species control protein degradation at the mitochondrial import gate.
  10. The role of ER exit sites in maintaining P-body organization and integrity during Drosophila melanogaster oogenesis.
  11. Targeted protein degradation of PDE4 shortforms by a novel proteolysis targeting chimera.
  12. Phosphorylation of Optineurin by protein kinase D regulates Parkin-dependent mitophagy.
  13. Targeting DTX2/UFD1-mediated FTO degradation to regulate antitumor immunity.
  14. Circadian Control of Protein Synthesis.
  15. Hsp90 and cochaperones have two genetically distinct roles in regulating eEF2 function.
  16. Fluid mechanics of luminal transport in actively contracting endoplasmic reticulum.
  17. The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.
  18. CCDC134 controls TLR biogenesis through the ER chaperone Gp96.
  19. Amino acid sequence encodes protein abundance shaped by protein stability at reduced synthesis cost.
  20. Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs.
  21. Ordered ATP hydrolysis in the Hsp90 chaperone is regulated by Aha1 and a conserved post-translational modification.
  22. The ATAC complex represses the transcriptional program of the autophagy-lysosome pathway via its E3 ubiquitin ligase activity.
  23. Design principles to tailor Hsp104 therapeutics.
  24. The mechanism of mRNA cap recognition.
  25. GABRA1 frameshift variants impair GABAA receptor proteostasis.
  26. The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity.
  27. Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12.
  28. qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.
  29. Redesigning error control in cross-linking mass spectrometry enables more robust and sensitive protein-protein interaction studies.
  30. Rubicon regulates exosome secretion via the non-autophagic pathway.
  31. Crosslinking intermodular condensation in non-ribosomal peptide biosynthesis.
  32. Single-nucleus transcriptomic analysis reveals the regulatory circuitry of myofiber XBP1 during regenerative myogenesis.
  33. Dual BACH1 regulation by complementary SCF-type E3 ligases.
  34. Design of high specificity binders for peptide-MHC-I complexes.
  35. Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function.
  36. Acetylation of the yeast Hsp40 chaperone protein Ydj1 fine-tunes proteostasis and translational fidelity.
  37. Inhibition of ERO1L induces autophagy and apoptosis via endoplasmic reticulum stress in colorectal cancer.
  38. Kinetic principles of chemical cross-link formation for protein-protein interactions.
  39. CRL3ARMC5 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of RNA Pol II transcription.
  40. RudLOV is an optically synchronized cargo transport method revealing unexpected effects of dynasore.
  41. Janus-like behavior of intrinsically disordered regions in reticulophagy.
  42. Alpha-1 Antitrypsin Inclusions Sequester GRP78 in a Bile Acid-Inducible Manner.
  43. Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors.
  44. Disagreement on foundational principles of biological aging.
  45. A split intein and split luciferase-coupled system for detecting protein-protein interactions.
  46. Epitranscriptomic rRNA fingerprinting reveals tissue-of-origin and tumor-specific signatures.
  1. Cell Rep. 2024 Dec 10. pii: S2211-1247(24)01401-3. [Epub ahead of print]43(12): 115050
    Ubiquitin-dependent translation control mechanisms: Degradation and beyond.
    Pierce W Ford, Mythreyi Narasimhan, Eric J Bennett.
      Translation control mechanisms connect the largely static genome to the highly dynamic proteome. At each step in the translation cycle, multiple layers of regulation enable efficient protein biogenesis under optimal conditions and mediate responses to acute environmental challenges. Recent research has demonstrated that individual ribosomal protein ubiquitylation events act as molecular signals to specify quality control pathway outcomes. Here, we synthesize current knowledge of ubiquitin-mediated translation control mechanisms and highlight key outstanding questions. We compare and contrast ubiquitin-dependent mechanisms that regulate ribosome-associated quality control pathways at several steps in the translation cycle. We also explore how distinct ribosome ubiquitylation events on specific ribosomal proteins impact translation activity and how defects in specific ubiquitin-mediated regulatory steps impact physiology and health.
    Keywords:  CP: Molecular biology; NRD; eukaryotic translation; mRNA decay; non-functional rRNA decay; proteostasis; ribosomal protein degradation; ribosomal ubiquitylation; ribosome biogenesis; ribosome-associated quality control; translation regulation; translation stress response; translation surveillance pathways
    DOI:  https://doi.org/10.1016/j.celrep.2024.115050
  2. Elife. 2024 Dec 10. pii: RP93117. [Epub ahead of print]12
    UGGT1-mediated reglucosylation of N-glycan competes with ER-associated degradation of unstable and misfolded glycoproteins.
    Satoshi Ninagawa, Masaki Matsuo, Deng Ying, Shuichiro Oshita, Shinya Aso, Kazutoshi Matsushita, Mai Taniguchi, Akane Fueki, Moe Yamashiro, Kaoru Sugasawa, Shunsuke Saito, Koshi Imami, Yasuhiko Kizuka, Tetsushi Sakuma, Takashi Yamamoto, Hirokazu Yagi, Koichi Kato, Kazutoshi Mori.
      How the fate (folding versus degradation) of glycoproteins is determined in the endoplasmic reticulum (ER) is an intriguing question. Monoglucosylated glycoproteins are recognized by lectin chaperones to facilitate their folding, whereas glycoproteins exposing well-trimmed mannoses are subjected to glycoprotein ER-associated degradation (gpERAD); we have elucidated how mannoses are sequentially trimmed by EDEM family members (George et al., 2020; 2021 eLife). Although reglucosylation by UGGT was previously reported to have no effect on substrate degradation, here we directly tested this notion using cells with genetically disrupted UGGT1/2. Strikingly, the results showed that UGGT1 delayed the degradation of misfolded substrates and unstable glycoproteins including ATF6α. An experiment with a point mutant of UGGT1 indicated that the glucosylation activity of UGGT1 was required for the inhibition of early glycoprotein degradation. These and overexpression-based competition experiments suggested that the fate of glycoproteins is determined by a tug-of-war between structure formation by UGGT1 and degradation by EDEMs. We further demonstrated the physiological importance of UGGT1, since ATF6α cannot function properly without UGGT1. Thus, our work strongly suggests that UGGT1 is a central factor in ER protein quality control via the regulation of both glycoprotein folding and degradation.
    Keywords:  EDEM; ER protein quality control; UGGT1; cell biology; human; protein degradation; protein folding
    DOI:  https://doi.org/10.7554/eLife.93117
  3. bioRxiv. 2024 Nov 28. pii: 2024.11.27.625751. [Epub ahead of print]
    Loss of FIC-1-mediated AMPylation activates the UPR ER and upregulates cytosolic HSP70 chaperones to suppress polyglutamine toxicity.
    Kate M Van Pelt, Matthias C Truttmann.
      Targeted regulation of cellular proteostasis machinery represents a promising strategy for the attenuation of pathological protein aggregation. Recent work suggests that the unfolded protein response in the endoplasmic reticulum (UPR ER ) directly regulates the aggregation and toxicity of expanded polyglutamine (polyQ) proteins. However, the mechanisms underlying this phenomenon remain poorly understood. In this study, we report that perturbing ER homeostasis in Caenorhabditis elegans through the depletion of either BiP ortholog, hsp-3 or hsp-4, causes developmental arrest in worms expressing aggregation-prone polyQ proteins. This phenotype is rescued by the genetic deletion of the conserved UPR ER regulator, FIC-1. We demonstrate that the beneficial effects of fic-1 knock-out (KO) extend into adulthood, where the loss of FIC-1-mediated protein AMPylation in polyQ-expressing animals is sufficient to prevent declines in fitness and lifespan. We further show that loss of hsp-3 and hsp-4 leads to distinct, but complementary transcriptomic responses to ER stress involving all three UPR ER stress sensors (IRE-1, PEK-1, and ATF-6). We identify the cytosolic HSP70 family chaperone F44E5.4 , whose expression is increased in fic-1 -deficient animals upon ER dysregulation, as a key effector suppressing polyQ toxicity. Over-expression of F44E5.4 , but not other HSP70 family chaperones, is sufficient to rescue developmental arrest in polyQ-expressing embryos upon hsp-3 knock-down. Finally, we show that knock-down of ire-1 , pek-1 , or atf-6 blocks the upregulation of F44E5.4 in fic-1 -deficient worms. Taken together, our findings support a model in which the loss of FIC-1-mediated AMPylation engages UPR ER signaling to upregulate cytosolic chaperone activity in response to polyQ toxicity.
    DOI:  https://doi.org/10.1101/2024.11.27.625751
  4. Cell Calcium. 2024 Nov 29. pii: S0143-4160(24)00138-6. [Epub ahead of print]125 102980
    STIMulating IRE1: How store-operated Ca2+ entry intersects with ER proteostasis.
    Maria Livia Sassano, Robbe Van Gorp, Geert Bultynck, Patrizia Agostinis.
      The endoplasmic reticulum (ER) controls intracellular Ca2+ dynamics. Depletion of ER Ca2+ stores results in short-term activation of store-operated Ca2+ entry (SOCE) via STIM1/Orai1 at ER-plasma membrane (ER-PM) contact sites (MCSs) and the long-term activation of the unfolded protein response (UPR), securing ER proteostasis. Recent work by Carreras-Sureda and colleagues describes a bidirectional control between IRE1 and STIM1 within the ER lumen that regulates ER-PM contact assembly and SOCE to sustain T-cell activation and myoblast differentiation.
    Keywords:  IP3 receptors; IRE1; STIM1; Store-operated calcium entry; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.ceca.2024.102980
  5. Mol Biol Cell. 2024 Dec 11. mbcE24040147
    TUDCA modulates drug bioavailability to regulate resistance to acute ER stress in Saccharomyces cerevisiae.
    Sarah R Chadwick, Samuel Stack-Couture, Matthew D Berg, Sonja Di Gregorio, Bryan Lung, Julie Genereaux, Robyn D Moir, Christopher J Brandl, Ian M Willis, Erik L Snapp, Patrick Lajoie.
      Cells counter accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) through activation of the Unfolded Protein Response (UPR). Small molecules termed chemical chaperones can promote protein folding to alleviate ER stress. The bile acid tauroursodeoxycholic acid (TUDCA), has been described as a chemical chaperone. While promising in models of protein folding diseases, TUDCA's mechanism of action remains unclear. Here, we found TUDCA can rescue growth of yeast treated with the ER stressor tunicamycin (Tm), even in the absence of a functional UPR. In contrast, TUDCA failed to rescue growth on other ER stressors. Nor could TUDCA attenuate chronic UPR associated with specific gene deletions or over-expression of a misfolded mutant secretory protein. Neither pretreatment with or delayed addition of TUDCA conferred protection against Tm. Importantly, attenuation of Tm-induced toxicity required TUDCA's critical micelle forming concentration, suggesting a mechanism where TUDCA directly sequesters drugs. Indeed, in several assays, TUDCA treated cells closely resembled cells treated with lower doses of Tm. In addition, we found TUDCA can inhibit dyes from labeling intracellular compartments. Thus, our study challenges the model of TUDCA as a chemical chaperone and suggests that TUDCA decreases drug bioavailability, allowing cells to adapt to ER stress.
    DOI:  https://doi.org/10.1091/mbc.E24-04-0147
  6. Biomacromolecules. 2024 Dec 12.
    Lysosomal Trafficking and Degradation of Extracellular Proteins via Multivalent Small Molecule Ligand Display on Dextran Scaffolds.
    Benoit Louage, Demi Defreyne, Heleen Lauwers, Jamie De Baere, Annemiek Uvyn, Haixia Peng, Yong Chen, Bruno G De Geest.
      Targeted protein degradation (TPD) marks a shift in drug development from conventional inhibition to the complete removal of pathological proteins. Traditional TPD technologies target intracellular proteins of interest (POIs) for degradation but are ineffective against extracellular cell surface and soluble proteins, a significant portion of the human proteome. Recent advances involve the formation of ternary complexes between a POI and a cell surface lysosomal trafficking receptor, directing POIs to lysosomes for degradation. We report on DEXtran TRAfficking Chimeras (DEXTRACs) comprising multiple copies of synthetic small molecule ligands for a model POI and the cation-independent mannose-6-phosphate receptor (CI-M6PR) lysosomal trafficking receptor. These ligands are arranged along the dextran backbones. We demonstrate that DEXTRACs leverage multivalency with their efficacy dependent on the dextran chain length and ligand density to form high-avidity ternary complexes. Our in vitro studies confirmed that DEXTRACs traffic the target POI to lysosomes and facilitate its degradation.
    DOI:  https://doi.org/10.1021/acs.biomac.4c01603
  7. Cell Rep. 2024 Dec 10. pii: S2211-1247(24)01404-9. [Epub ahead of print]43(12): 115053
    Legionella uses host Rab GTPases and BAP31 to create a unique ER niche.
    Attinder Chadha, Yu Yanai, Hiromu Oide, Yuichi Wakana, Hiroki Inoue, Saradindu Saha, Manish Paul, Mitsuo Tagaya, Kohei Arasaki, Shaeri Mukherjee.
      The bacterium Legionella pneumophila secretes numerous effector proteins that manipulate endoplasmic reticulum (ER)-derived vesicles to form the Legionella-containing vacuole (LCV). Despite extensive studies, whether the LCV membrane is separate from or connected to the host ER network remains unclear. Here, we show that the smooth ER (sER) is closely associated with the LCV early in infection. Remarkably, Legionella forms a distinct rough ER (rER) niche at later stages, disconnected from the host ER network. We discover that host small GTPases Rab10 and Rab4 and an ER protein, BAP31, play crucial roles in transitioning the LCV from an sER to an rER. Additionally, we have identified a Legionella effector, Lpg1152, that binds to BAP31. Interestingly, the optimal growth of Legionella is dependent on both BAP31 and Lpg1152. These findings detail the complex interplay between host and pathogen in transforming the LCV membrane from a host-associated sER to a distinct rER.
    Keywords:  CP: Cell biology; CP: Microbiology; Legionella; Rab GTPase; bacteria; endoplasmic reticulum; pathogen
    DOI:  https://doi.org/10.1016/j.celrep.2024.115053
  8. Biochem J. 2024 Dec 18. 481(24): 1949-1965
    Ubiquitin E3 ligases in the plant Arg/N-degron pathway.
    Keely E A Oldham, Peter D Mabbitt.
      Regulation of protein longevity via the ubiquitin (Ub) - proteasome pathway is fundamental to eukaryotic biology. Ubiquitin E3 ligases (E3s) interact with substrate proteins and provide specificity to the pathway. A small subset of E3s bind to specific exposed N-termini (N-degrons) and promote the ubiquitination of the bound protein. Collectively these E3s, and other N-degron binding proteins, are known as N-recognins. There is considerable functional divergence between fungi, animal, and plant N-recognins. In plants, at least three proteins (PRT1, PRT6, and BIG) participate in the Arg/N-degron pathway. PRT1 has demonstrated E3 ligase activity, whereas PRT6 and BIG are candidate E3s. The Arg/N-degron pathway plays a central role in plant development, germination, and submersion tolerance. The pathway has been manipulated both to improve crop performance and for conditional protein degradation. A more detailed structural and biochemical understanding of the Arg/N-recognins and their substrates is required to fully realise the biotechnological potential of the pathway. This perspective focuses on the structural and molecular details of substrate recognition and ubiquitination in the plant Arg/N-degron pathway. While PRT1 appears to be plant specific, the PRT6 and BIG proteins are similar to UBR1 and UBR4, respectively. Analysis of the cryo-EM structures of Saccharomyces UBR1 suggests that the mode of ubiquitin conjugating enzyme (E2) and substrate recruitment is conserved in PRT6, but regulation of the two N-recognins may be significantly different. The structurally characterised domains from human UBR4 are also likely to be conserved in BIG, however, there are sizeable gaps in our understanding of both proteins.
    Keywords:  plant signal transduction; protein turnover; structural biology; ubiquitin signalling
    DOI:  https://doi.org/10.1042/BCJ20240132
  9. Mol Cell. 2024 Dec 05. pii: S1097-2765(24)00909-2. [Epub ahead of print]84(23): 4612-4628.e13
    Reactive oxygen species control protein degradation at the mitochondrial import gate.
    Rachael McMinimy, Andrew G Manford, Christine L Gee, Srividya Chandrasekhar, Gergey Alzaem Mousa, Joelle Chuang, Lilian Phu, Karen Y Shih, Christopher M Rose, John Kuriyan, Baris Bingol, Michael Rapé.
      While reactive oxygen species (ROS) have long been known to drive aging and neurodegeneration, their persistent depletion below basal levels also disrupts organismal function. Cells counteract loss of basal ROS via the reductive stress response, but the identity and biochemical activity of ROS sensed by this pathway remain unknown. Here, we show that the central enzyme of the reductive stress response, the E3 ligase Cullin 2-FEM1 homolog B (CUL2FEM1B), specifically acts at mitochondrial TOM complexes, where it senses ROS produced by complex III of the electron transport chain (ETC). ROS depletion during times of low ETC activity triggers the localized degradation of CUL2FEM1B substrates, which sustains mitochondrial import and ensures the biogenesis of the rate-limiting ETC complex IV. As complex III yields most ROS when the ETC outpaces metabolic demands or oxygen availability, basal ROS are sentinels of mitochondrial activity that help cells adjust their ETC to changing environments, as required for cell differentiation and survival.
    Keywords:  FEM1B; TOM complex; electron transport chain; mitochondria; proteasome; reductive stress response; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.004
  10. EMBO Rep. 2024 Dec 09.
    The role of ER exit sites in maintaining P-body organization and integrity during Drosophila melanogaster oogenesis.
    Samantha N Milano, Livia V Bayer, Julie J Ko, Caroline E Casella, Diana P Bratu.
      Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro, a nuanced understanding of how their assembly is regulated in vivo remains elusive. Here, we investigate the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, we find that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies. Moreover, we demonstrate that altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch), suggesting a potential role for ER exit sites in P-body organization. Furthermore, we show that in the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar, are reduced. Together, our data highlights the crucial role of ER exit sites in governing P-body organization.
    Keywords:  Biological Condensates; ER Exit Sites; Oogenesis; P-bodies; Post-transcriptional Regulation
    DOI:  https://doi.org/10.1038/s44319-024-00344-x
  11. FEBS J. 2024 Dec 13.
    Targeted protein degradation of PDE4 shortforms by a novel proteolysis targeting chimera.
    Yuan Yan Sin, Aoife Giblin, Aleksandra Judina, Punchita Rujirachaivej, Laura G Corral, Eliza Glennon, Zhi Xian Tai, Tian Feng, Eduardo Torres, Alina Zorn, Julia Gorelik, Elka Kyurkchieva, Pa Thai Yenchitsomanus, Cathy Swindlehurst, Kyle Chan, David Stirling, George S Baillie.
      Cyclic AMP (cAMP) has a crucial role in many vital cellular processes and there has been much effort expended in the discovery of inhibitors against the enzyme superfamily that degrades this second messenger, namely phosphodiesterases (PDEs). The journey of competitive PDE inhibitors to the clinic has been hampered by side effects profiles that have resulted from a lack of selectivity for subfamilies and individual isoforms because of high conservation of catalytic site sequences and structures. Here we introduce a proteolysis targeting chimera (PROTAC) that can specifically target a small subset of isoforms from the PDE4 family to send the enzyme for degradation at the proteasome by recruiting a ubiquitin E3 ligase into proximity with the PDE. We constructed our PDE4 PROTAC (KTX207) using a previously characterized PDE4 inhibitor, and we show that evolution of the compound into a PROTAC improves selectivity, potency and enables a long-lasting effect even after the compound is removed from cells after a short treatment duration. Functionally, KTX207 is more effective at increasing cAMP, is 100 times more anti-inflammatory, and is significantly better at reducing the growth in cancer cell models than the PDE4 inhibitor alone. Our study highlights the advantages of targeted degradation versus active-site occupancy for PDE4 inhibition and discusses the potential of this novel pharmacological approach to improve the safety profile of PDE4 inhibition in the future.
    Keywords:  PDE4; PROTAC; phosphodiesterases; protein degradation
    DOI:  https://doi.org/10.1111/febs.17359
  12. iScience. 2024 Dec 20. 27(12): 111384
    Phosphorylation of Optineurin by protein kinase D regulates Parkin-dependent mitophagy.
    Robert Weil, Emmanuel Laplantine, Messaouda Attailia, Anne Oudin, Shannel Curic, Aya Yokota, Elie Banide, Pierre Génin.
      Degradation of damaged mitochondria, a process called mitophagy, plays a role in mitochondrial quality control and its dysfunction has been linked to neurodegenerative pathologies. The PINK1 kinase and the ubiquitin ligase Parkin-mediated mitophagy represents the most common pathway in which specific receptors, including Optineurin (Optn), target ubiquitin-labeled mitochondria to autophagosomes. Here, we show that Protein Kinases D (PKD) are activated and recruited to damaged mitochondria. Subsequently, PKD phosphorylate Optn to promote a complex with Parkin leading to enhancement of its ubiquitin ligase activity. Paradoxically, inhibiting PKD activity enhances the interaction between Optn and LC3, promotes the recruitment of Parkin to mitochondria, and increases the mitophagic function of Optn. This enhancement of mitophagy is characterized by increased production of mitochondrial ROS and a reduction in mitochondrial mass. The PKD kinases may therefore regulate Optn-dependent mitophagy by amplifying the Parkin-mediated degradation signals to improve the cell response against oxidative stress damage.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111384
  13. Proc Natl Acad Sci U S A. 2024 Dec 17. 121(51): e2407910121
    Targeting DTX2/UFD1-mediated FTO degradation to regulate antitumor immunity.
    Yan-Hong Cui, Jiangbo Wei, Hao Fan, Wenlong Li, Lijie Zhao, Emma Wilkinson, Jack Peterson, Lishi Xie, Zhongyu Zou, Seungwon Yang, Mark A Applebaum, Justin Kline, Jing Chen, Chuan He, Yu-Ying He.
      Here, we show that vitamin E succinate (VES) acts as a degrader for the m6A RNA demethylase fat mass and obesity-associated protein (FTO), thus suppressing tumor growth and resistance to immunotherapy. FTO is ubiquitinated by its E3 ligase DTX2, followed by UFD1 recruitment and subsequent degradation in the proteasome. VES binds to FTO and DTX2, leading to enhanced FTO-DTX2 interaction, FTO ubiquitination, and degradation in FTO-dependent tumor cells. VES suppressed tumor growth and enhanced antitumor immunity and response to immunotherapy in vivo in mouse models. Genetic FTO knockdown or VES treatment increased m6A methylation in the LIF (Leukemia Inhibitory Factor) gene and decreased LIF mRNA decay, and thus sensitized melanoma cells to T cell-mediated cytotoxicity. Taken together, our findings reveal the underlying molecular mechanism for FTO protein degradation and identify a dietary degrader for FTO that inhibits tumor growth and overcomes immunotherapy resistance.
    Keywords:  DTX2; FTO; UFD1; m6A RNA methylation; ubiquitin-mediated proteasomal degradation
    DOI:  https://doi.org/10.1073/pnas.2407910121
  14. Bioessays. 2024 Dec 12. e202300158
    Circadian Control of Protein Synthesis.
    Nathan R James, John S O'Neill.
      Daily rhythms in the rate and specificity of protein synthesis occur in most mammalian cells through an interaction between cell-autonomous circadian regulation and daily cycles of systemic cues. However, the overall protein content of a typical cell changes little over 24 h. For most proteins, translation appears to be coordinated with protein degradation, producing phases of proteomic renewal that maximize energy efficiency while broadly maintaining proteostasis across the solar cycle. We propose that a major function of this temporal compartmentalization-and of circadian rhythmicity in general-is to optimize the energy efficiency of protein synthesis and associated processes such as complex assembly. We further propose that much of this temporal compartmentalization is achieved at the level of translational initiation, such that the translational machinery alternates between distinct translational mechanisms, each using a distinct toolkit of phosphoproteins to preferentially recognize and translate different classes of mRNA.
    Keywords:  biphasic model; circadian rhythms; protein synthesis; temporal compartmentalization; translational initiation
    DOI:  https://doi.org/10.1002/bies.202300158
  15. PLoS Genet. 2024 Dec 09. 20(12): e1011508
    Hsp90 and cochaperones have two genetically distinct roles in regulating eEF2 function.
    Melody D Fulton, Danielle J Yama, Ella Dahl, Jill L Johnson.
      Protein homeostasis relies on the accurate translation and folding of newly synthesized proteins. Eukaryotic elongation factor 2 (eEF2) promotes GTP-dependent translocation of the ribosome during translation. eEF2 folding was recently shown to be dependent on Hsp90 as well as the cochaperones Hgh1, Cns1, and Cpr7. We examined the requirement for Hsp90 and cochaperones more closely and found that Hsp90 and cochaperones have two distinct roles in regulating eEF2 function. Yeast expressing one group of Hsp90 mutations or one group of cochaperone mutations had reduced steady-state levels of eEF2. The growth of Hsp90 mutants that affected eEF2 accumulation was also negatively affected by deletion of the gene encoding Hgh1. Further, mutations in yeast eEF2 that mimic disease-associated mutations in human eEF2 were negatively impacted by loss of Hgh1 and growth of one mutant was partially rescued by overexpression of Hgh1. In contrast, yeast expressing different groups of Hsp90 mutations or a different cochaperone mutation had altered sensitivity to diphtheria toxin, which is dictated by a unique posttranslational modification on eEF2. Our results provide further evidence that Hsp90 contributes to proteostasis not just by assisting protein folding, but also by enabling accurate translation of newly synthesized proteins. In addition, these results provide further evidence that yeast Hsp90 mutants have distinct in vivo effects that correlate with defects in subsets of cochaperones.
    DOI:  https://doi.org/10.1371/journal.pgen.1011508
  16. Elife. 2024 Dec 13. pii: RP93518. [Epub ahead of print]13
    Fluid mechanics of luminal transport in actively contracting endoplasmic reticulum.
    Pyae Hein Htet, Edward Avezov, Eric Lauga.
      The endoplasmic reticulum (ER), the largest cellular compartment, harbours the machinery for the biogenesis of secretory proteins and lipids, calcium storage/mobilisation, and detoxification. It is shaped as layered membranous sheets interconnected with a network of tubules extending throughout the cell. Understanding the influence of the ER morphology dynamics on molecular transport may offer clues to rationalising neuro-pathologies caused by ER morphogen mutations. It remains unclear, however, how the ER facilitates its intra-luminal mobility and homogenises its content. It has been recently proposed that intra-luminal transport may be enabled by active contractions of ER tubules. To surmount the barriers to empirical studies of the minuscule spatial and temporal scales relevant to ER nanofluidics, here we exploit the principles of viscous fluid dynamics to generate a theoretical physical model emulating in silico the content motion in actively contracting nanoscopic tubular networks. The computational model reveals the luminal particle speeds, and their impact in facilitating active transport, of the active contractile behaviour of the different ER components along various time-space parameters. The results of the model indicate that reproducing transport with velocities similar to those reported experimentally in single-particle tracking would require unrealistically high values of tubule contraction site length and rate. Considering further nanofluidic scenarios, we show that width contractions of the ER's flat domains (perinuclear sheets) generate local flows with only a short-range effect on luminal transport. Only contractions of peripheral sheets can reproduce experimental measurements, provided they are able to contract fast enough.
    Keywords:  biological fluid mechanics; cell biology; endoplasmic reticulum; fluid dynamics; intracellular transport; membrane dynamics; networks; organelles; physical simulation; physics of living systems
    DOI:  https://doi.org/10.7554/eLife.93518
  17. bioRxiv. 2024 Nov 26. pii: 2024.11.26.625361. [Epub ahead of print]
    The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks.
    Wei Song, Yichen Zhao, Annamaria Ruggiano, Christina Redfield, Joseph A Newman, Xiaosheng Zhu, Abimael Cruz-Migoni, Rebecca Roddan, Peter McHugh, Paul Elliott, Kristijan Ramadan.
      DNA-protein crosslinks (DPCs) are endogenous and chemotherapy-induced genotoxic DNA lesions and, if not repaired, lead to embryonic lethality, neurodegeneration, premature ageing, and cancer. DPCs are heavily polyubiquitinated, and the SPRTN protease and 26S proteasome emerged as two central enzymes for DPC proteolysis. The proteasome recognises its substrates by their ubiquitination status. How SPRTN protease, an essential enzyme for DPC proteolysis, achieves specificity for DPCs still needs to be discovered. We found that the N-terminal SPRTN catalytic region (SprT) possesses a ubiquitin-binding domain named the U biquitin interface of S prT D omain (USD). Using multiple biochemical, biophysical, and structural approaches, we reveal that USD binds ubiquitin chains. SPRTN binding to ubiquitin chains via USD leads to ∼ 67-fold higher activation of SPRTN proteolysis towards polyubiquitinated DPCs than the unmodified DPCs. This study reveals the ubiquitination of DPCs is the key signal for SPRTN's substrate specificity and rapid proteolysis.
    DOI:  https://doi.org/10.1101/2024.11.26.625361
  18. J Exp Med. 2025 Mar 03. pii: e20240825. [Epub ahead of print]222(3):
    CCDC134 controls TLR biogenesis through the ER chaperone Gp96.
    Léa Bernaleau, Michaela Drobek, Fenja Blank, Philipp Walch, Maeva Delacrétaz, Ales Drobek, Marta Monguió-Tortajada, Petr Broz, Olivia Majer, Manuele Rebsamen.
      Toll-like receptors (TLRs) are central to initiate immune responses against invading pathogens. To ensure host defense while avoiding aberrant activation leading to pathogenic inflammation and autoimmune diseases, TLRs are tightly controlled by multilevel regulatory mechanisms. Through a loss-of-function genetic screen in a reporter cell line engineered to undergo cell death upon TLR7-induced IRF5 activation, we identified here CCDC134 as an essential factor for TLR responses. CCDC134 deficiency impaired endolysosomal TLR-induced NF-κB, MAPK, and IRF5 activation, as well as downstream production of proinflammatory cytokines and type I interferons. We further demonstrated that CCDC134 is an endoplasmic reticulum (ER)-resident interactor of Gp96 (HSP90B1/Grp94), an ER chaperone essential for folding and trafficking of plasma membrane and endolysosomal TLRs. CCDC134 controlled Gp96 stability as its loss led to Gp96 hyperglycosylation and ER-associated protein degradation (ERAD)-mediated clearance. Accordingly, CCDC134 deficiency impaired the folding, maturation, and trafficking of TLRs, resulting in blunted inflammatory responses upon stimulation. Altogether, this study reveals CCDC134 as a central regulator of the chaperone Gp96, thereby controlling TLR biogenesis and responses.
    DOI:  https://doi.org/10.1084/jem.20240825
  19. Protein Sci. 2025 Jan;34(1): e5239
    Amino acid sequence encodes protein abundance shaped by protein stability at reduced synthesis cost.
    Filip Buric, Sandra Viknander, Xiaozhi Fu, Oliver Lemke, Oriol Gracia Carmona, Jan Zrimec, Lukasz Szyrwiel, Michael Mülleder, Markus Ralser, Aleksej Zelezniak.
      Understanding what drives protein abundance is essential to biology, medicine, and biotechnology. Driven by evolutionary selection, an amino acid sequence is tailored to meet the required abundance of a proteome, underscoring the intricate relationship between sequence and functional demand. Yet, the specific role of amino acid sequences in determining proteome abundance remains elusive. Here we show that the amino acid sequence alone encodes over half of protein abundance variation across all domains of life, ranging from bacteria to mouse and human. With an attempt to go beyond predictions, we trained a manageable-size Transformer model to interpret latent factors predictive of protein abundances. Intuitively, the model's attention focused on the protein's structural features linked to stability and metabolic costs related to protein synthesis. To probe these relationships, we introduce MGEM (Mutation Guided by an Embedded Manifold), a methodology for guiding protein abundance through sequence modifications. We find that mutations which increase predicted abundance have significantly altered protein polarity and hydrophobicity, underscoring a connection between protein structural features and abundance. Through molecular dynamics simulations we revealed that abundance-enhancing mutations possibly contribute to protein thermostability by increasing rigidity, which occurs at a lower synthesis cost.
    Keywords:  deep learning; explainable machine learning; language models; molecular dynamics; protein engineering; protein expression; protein sequence; protein stability; proteome
    DOI:  https://doi.org/10.1002/pro.5239
  20. bioRxiv. 2024 Nov 26. pii: 2024.11.26.625518. [Epub ahead of print]
    Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs.
    Chotiwat Seephetdee, Daniel L Kiss.
      The COVID-19 mRNA vaccines demonstrated the power of mRNA medicines. Despite advancements in sequence design, evidence regarding the preferential use of synonymous codons on cellular stress and innate immune responses is lacking. To this end, we developed a proprietary codon optimality matrix to re-engineer the coding sequences of three luciferase reporters. We demonstrate that optimal mRNAs elicited dramatic increases in luciferase activities compared to non-optimal sequences. Notably, transfecting an optimal RNA affects the translation of other RNAs in the cell including control transcripts in dual luciferase assays. This held true in multiple cell lines and for an unrelated reporter. Further, non-optimal mRNAs preferentially activated innate immune pathways and the phosphorylation of the translation initiation factor eIF2α, a central event of the integrated stress response. Using nucleoside-modified or circular RNAs partially or fully abrogated these responses. Finally, we show that circularizing RNAs enhances both RNA lifespan and durability of protein expression. Our results show that RNA sequence, composition, and structure all govern RNA translatability. However, we also show that RNA sequences with poor codon optimality are immunogenic and induce cellular stress. Hence, RNA sequence engineering, chemical, and topological modifications must all be combined to elicit favorable therapeutic outcomes.
    DOI:  https://doi.org/10.1101/2024.11.26.625518
  21. Protein Sci. 2025 Jan;34(1): e5255
    Ordered ATP hydrolysis in the Hsp90 chaperone is regulated by Aha1 and a conserved post-translational modification.
    Desmond Prah Amoah, Solomon K Hussein, Jill L Johnson, Paul LaPointe.
      Hsp90 is a dimeric molecular chaperone that is important for the folding, stabilization, activation, and maturation of hundreds of protein substrates called "clients" in cells. Dozens of co-chaperones and hundreds of post-translational modifications (PTMs) regulate the ATP-dependent client activation cycle. The Aha1 co-chaperone is the most potent stimulator of the ATPase cycle of Hsp90 and phosphorylation of threonine 22 in Hsp90 can regulate the recruitment of Aha1 in cells. We report here that phosphorylation of threonine 22 regulates specific aspects of Aha1 function after recruitment occurs. The phosphomimetic substitution, T22E, neutralizes the action of the Aha1 NxNNWHW motif. Moreover, this substitution can exert this effect from only one protomer of the Hsp90 dimer. This work sheds light on how asymmetric modifications in the Hsp90 dimer can functionalize individual protomers and fine-tune the Hsp90 cycle.
    Keywords:  ATPase; Aha1; Hsp90; chaperones; protein folding; yeast genetics
    DOI:  https://doi.org/10.1002/pro.5255
  22. Cell Rep. 2024 Dec 05. pii: S2211-1247(24)01384-6. [Epub ahead of print]43(12): 115033
    The ATAC complex represses the transcriptional program of the autophagy-lysosome pathway via its E3 ubiquitin ligase activity.
    Xiaolu Wang, Lingling Wang, Zhili Zhou, Chenhao Jiang, Ziyu Bao, Yuexin Wang, Ying Zhang, Lili Song, Yueling Zhao, Xinying Li, Qianqian Li, Yujun Shen, Ying Yu, Wenyi Mi.
      The Ada two A-containing (ATAC) complex, containing histone acetyltransferases general control non-derepressible 5 (GCN5) or p300/CBP-associated factor (PCAF), has gained recognition as a prominent transcriptional coactivator. Recent revelations unveiled E3 ligase activity present in both GCN5 and PCAF; however, how the dual enzymatic activities of the ATAC complex orchestrate distinct transcriptional programs and signaling networks remains largely elusive. Our study unveils the function of the ATAC complex as a negative regulator of the autophagy-lysosome pathway's transcriptional program by modulating the stability of transcription factors TFE3 and TFEB. The ATAC complex primarily impacts TFE3/TFEB destabilization through its E3 ligase activity rather than its acetyltransferase function. GCN5/PCAF-mediated ubiquitination prompts the proteasome-dependent degradation of TFE3 and TFEB. Furthermore, inactivation of the ATAC complex amplifies TFE3/TFEB-mediated autophagy-lysosome functions, thereby promoting cell survival during nutrient deprivation. In summary, our findings establish the "ATAC complex-TFE3/TFEB-autophagy-lysosome" axis as an intrinsic regulatory pathway for resisting starvation-induced cell death.
    Keywords:  ATAC complex; CP: Molecular biology; TFE3/TFEB stability; acetylation; autophagy-lysosome pathway; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2024.115033
  23. Cell Rep. 2024 Dec 12. pii: S2211-1247(24)01356-1. [Epub ahead of print]43(12): 115005
    Design principles to tailor Hsp104 therapeutics.
    JiaBei Lin, Peter J Carman, Craig W Gambogi, Nathan M Kendsersky, Edward Chuang, Stephanie N Gates, Adam L Yokom, Alexandrea N Rizo, Daniel R Southworth, James Shorter.
      The hexameric AAA+ disaggregase, Hsp104, collaborates with Hsp70 and Hsp40 via its autoregulatory middle domain (MD) to solubilize aggregated proteins. However, how ATP- or ADP-specific MD configurations regulate Hsp104 hexamers remains poorly understood. Here, we define an ATP-specific network of interprotomer contacts between nucleotide-binding domain 1 (NBD1) and MD helix L1, which tunes Hsp70 collaboration. Manipulating this network can (1) reduce Hsp70 collaboration without enhancing activity, (2) generate Hsp104 hypomorphs that collaborate selectively with class B Hsp40s, (3) produce Hsp70-independent potentiated variants, or (4) create species barriers between Hsp104 and Hsp70. Conversely, ADP-specific intraprotomer contacts between MD helix L2 and NBD1 restrict activity, and their perturbation frequently potentiates Hsp104. Importantly, adjusting an NBD1:MD helix L1 rheostat via rational design enables finely tuned collaboration with Hsp70 to safely potentiate Hsp104, minimize off-target toxicity, and counteract FUS and TDP-43 proteinopathies in human cells. Thus, we establish design principles to tailor Hsp104 therapeutics.
    Keywords:  ALS/FTD; CP: Molecular biology; FUS; Hsp104; Hsp70; TDP-43; disaggregase; neurodegeneration; protein engineering
    DOI:  https://doi.org/10.1016/j.celrep.2024.115005
  24. Nature. 2024 Dec 11.
    The mechanism of mRNA cap recognition.
    Riley C Gentry, Nicholas A Ide, Victoria M Comunale, Erik W Hartwick, Colin D Kinz-Thompson, Ruben L Gonzalez.
      During translation initiation, mRNA molecules must be identified and activated for loading into a ribosome1-3. In this rate-limiting step, the heterotrimeric protein eukaryotic initiation factor eIF4F must recognize and productively interact with the 7-methylguanosine cap at the 5' end of the mRNA and subsequently activate the message1-3. Despite its fundamental, regulatory role in gene expression, the molecular events underlying cap recognition and mRNA activation remain unclear3. Here we generate a single-molecule fluorescence imaging system to examine the dynamics with which eIF4F discriminates productive and non-productive locations on full-length, native mRNA molecules. At the single-molecule level, we observe stochastic sampling of eIF4F along the length of the mRNA and identify allosteric communication between the eIF4F subunits that ultimately drive cap-recognition and subsequent activation of the message. Our experiments uncover functions for each subunit of eIF4F and we conclude by presenting a model for mRNA activation that precisely defines the composition of the activated message. This model provides a general framework for understanding how mRNA molecules may be discriminated from one another and how other RNA-binding proteins may control the efficiency of translation initiation.
    DOI:  https://doi.org/10.1038/s41586-024-08304-0
  25. bioRxiv. 2024 Nov 29. pii: 2024.11.28.625971. [Epub ahead of print]
    GABRA1 frameshift variants impair GABAA receptor proteostasis.
    Marnie P Williams, Ya-Juan Wang, Jing-Qiong Kang, Ting-Wei Mu.
      The gamma-aminobutyric acid type A receptor (GABA A R) is the most common inhibitory neurotransmitter-gated ion channel in the central nervous system. Pathogenic variants in genes encoding GABA A R subunits can cause receptor dysfunction and lead to genetic epilepsy. Frameshift variants in these genes can result in a premature termination codon, producing truncated receptor subunit variants. However, the molecular mechanism as well as functional implications of these frameshift variants remains inadequately characterized. This study focused on four clinical frameshift variants of the α 1 subunit of GABA A R (encoded by the GABRA1 gene): K401fs (c.1200del), S326fs (c.975del), V290fs (c.869_888del), and F272fs (c.813del). These variants result in the loss of one to three transmembrane helices, whereas wild type α1 has four transmembrane helices. Therefore, these variants serve as valuable models to evaluate membrane protein biogenesis and proteostasis deficiencies. In HEK293T cells, all four frameshift variants exhibit significantly reduced trafficking to the cell surface, resulting in essentially non-functional ion channels. However, the severity of proteostasis deficiency varied among these four frameshift variants, presumably due to their specific transmembrane domain deletions. The variant α1 subunits exhibited endoplasmic reticulum (ER) retention and activated the unfolded protein response (UPR) to varying extents. Our findings revealed that these frameshift variants of GABRA1 utilize overlapping yet distinct molecular mechanisms to impair proteostasis, providing insights into the pathogenesis of GABA A R-associated epilepsy.
    DOI:  https://doi.org/10.1101/2024.11.28.625971
  26. bioRxiv. 2024 Dec 01. pii: 2024.12.01.626216. [Epub ahead of print]
    The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity.
    Andrew C Fleming, Nalini R Rao, Matthew Wright, Jeffrey N Savas, Evangelos Kiskinis.
      The degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in the DNAJC7 gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X in DNAJC7 exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.
    DOI:  https://doi.org/10.1101/2024.12.01.626216
  27. bioRxiv. 2024 Nov 25. pii: 2024.11.25.625235. [Epub ahead of print]
    Dynamic regulation of the oxidative stress response by the E3 ligase TRIP12.
    Andrew J Ingersoll, Devlon M McCloud, Jenny Y Hu, Michael Rape.
      The oxidative stress response is centered on the transcription factor NRF2 and protects cells from reactive oxygen species (ROS). While ROS inhibit the E3 ligase CUL3 KEAP1 to stabilize NRF2 and elicit antioxidant gene expression, cells recovering from stress must rapidly reactivate CUL3 KEAP1 to prevent reductive stress and oxeiptosis-dependent cell death. How cells restore efficient NRF2-degradation upon ROS clearance remains poorly understood. Here, we identify TRIP12, an E3 ligase dysregulated in Clark-Baraitser Syndrome and Parkinson's Disease, as a component of the oxidative stress response. TRIP12 is a ubiquitin chain elongation factor that cooperates with CUL3 KEAP1 to ensure robust NRF2 degradation. In this manner, TRIP12 accelerates stress response silencing as ROS are being cleared, but limits NRF2 activation during stress. The need for dynamic control of NRF2-degradation therefore comes at the cost of diminished stress signaling, suggesting that TRIP12 inhibition could be used to treat degenerative pathologies characterized by ROS accumulation.
    DOI:  https://doi.org/10.1101/2024.11.25.625235
  28. EMBO J. 2024 Dec 12.
    qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.
    Reuben Philip, Amit Sharma, Laura Matellan, Anna C Erpf, Wen-Hsin Hsu, Johnny M Tkach, Haley D M Wyatt, Laurence Pelletier.
      Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.
    Keywords:  CRISPR; Endogenous Tagging; Gene-editing
    DOI:  https://doi.org/10.1038/s44318-024-00337-5
  29. Mol Syst Biol. 2024 Dec 09.
    Redesigning error control in cross-linking mass spectrometry enables more robust and sensitive protein-protein interaction studies.
    Boris Bogdanow, Max Ruwolt, Julia Ruta, Lars Mühlberg, Cong Wang, Wen-Feng Zeng, Arne Elofsson, Fan Liu.
      Cross-linking mass spectrometry (XL-MS) allows characterizing protein-protein interactions (PPIs) in native biological systems by capturing cross-links between different proteins (inter-links). However, inter-link identification remains challenging, requiring dedicated data filtering schemes and thorough error control. Here, we benchmark existing data filtering schemes combined with error rate estimation strategies utilizing concatenated target-decoy protein sequence databases. These workflows show shortcomings either in sensitivity (many false negatives) or specificity (many false positives). To ameliorate the limited sensitivity without compromising specificity, we develop an alternative target-decoy search strategy using fused target-decoy databases. Furthermore, we devise a different data filtering scheme that takes the inter-link context of the XL-MS dataset into account. Combining both approaches maintains low error rates and minimizes false negatives, as we show by mathematical simulations, analysis of experimental ground-truth data, and application to various biological datasets. In human cells, inter-link identifications increase by 75% and we confirm their structural accuracy through proteome-wide comparisons to AlphaFold2-derived models. Taken together, target-decoy fusion and context-sensitive data filtering deepen and fine-tune XL-MS-based interactomics.
    Keywords:  Cross-linking Mass Spectrometry; Error Control; False-Discovery Rate; Proteomics; Structure Modeling
    DOI:  https://doi.org/10.1038/s44320-024-00079-w
  30. Autophagy. 2024 Dec 12. 1-3
    Rubicon regulates exosome secretion via the non-autophagic pathway.
    Kyosuke Yanagawa, Tamotsu Yoshimori.
      Exosomes are small extracellular vesicles (EVs), which have the diameter of 50-150 nm and originate from intralumenal vesicles in multivesicular endosomes (MVBs). Exosomes secreted from donor cells are delivered to recipient cells for transferring of exosome cargos, such as proteins, lipids and nucleic acids. The cargo transfer by exosomes has a pivotal role in cell-to-cell communication for many cellular processes; however, the detailed mechanism remains largely elusive. In our recent study, we found that RUBCN/rubicon regulates exosome secretion through endosomal recruitment of WIPI2, which promotes ESCRT-dependent MVB formation. We further showed that this pathway is essential for age-dependent increasing of exosomes, which transfer the pro-senescent microRNAs, including Mir26a and Mir486a, and accelerate cellular senescence in the recipient cells. Our findings highlight RUBCN's key role in exosome secretion and its impact on cellular senescence, providing insights into its potential contributions to aging.
    Keywords:  Aging; autophagy; exosome; extracellular vesicle; microRNA; senescence
    DOI:  https://doi.org/10.1080/15548627.2024.2437653
  31. Nature. 2024 Dec 11.
    Crosslinking intermodular condensation in non-ribosomal peptide biosynthesis.
    Graham W Heberlig, James J La Clair, Michael D Burkart.
      Non-ribosomal peptide synthetases are assembly line biosynthetic pathways that are used to produce critical therapeutic drugs and are typically arranged as large multi-domain proteins called megasynthetases1. They synthesize polypeptides using peptidyl carrier proteins that shuttle each amino acid through modular loading, modification and elongation2 steps, and remain challenging to structurally characterize, owing in part to the inherent dynamics of their multi-domain and multi-modular architectures3. Here we have developed site-selective crosslinking probes to conformationally constrain and resolve the interactions between carrier proteins and their partner enzymatic domains4,5. We apply tetrazine click chemistry to trap the condensation of two carrier protein substrates within the active site of the condensation domain that unites the first two modules of tyrocidine biosynthesis and report the high-resolution cryo-EM structure of this complex. Together with the X-ray crystal structure of the first carrier protein crosslinked to its epimerization domain, these structures highlight captured intermodular recognition events and define the processive movement of a carrier protein from one catalytic step to the next. Characterization of these structural relationships remains central to understanding the molecular details of these unique synthetases and critically informs future synthetic biology design of these pathways.
    DOI:  https://doi.org/10.1038/s41586-024-08306-y
  32. iScience. 2024 Dec 20. 27(12): 111372
    Single-nucleus transcriptomic analysis reveals the regulatory circuitry of myofiber XBP1 during regenerative myogenesis.
    Aniket S Joshi, Micah B Castillo, Meiricris Tomaz da Silva, Anh Tuan Vuong, Preethi H Gunaratne, Radbod Darabi, Yu Liu, Ashok Kumar.
      Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is activated in skeletal muscle under multiple conditions. However, the role of the UPR in the regulation of muscle regeneration remains less understood. We demonstrate that gene expression of various markers of the UPR is induced in both myogenic and non-myogenic cells in regenerating muscle. Genetic ablation of X-box binding protein 1 (XBP1), a downstream target of the Inositol requiring enzyme 1α (IRE1α) arm of the UPR, in myofibers attenuates muscle regeneration in adult mice. Single nucleus RNA sequencing (snRNA-seq) analysis showed that deletion of XBP1 in myofibers perturbs proteolytic systems and mitochondrial function in myogenic cells. Trajectory analysis of snRNA-seq dataset showed that XBP1 regulates the abundance of satellite cells and the formation of new myofibers in regenerating muscle. In addition, ablation of XBP1 disrupts the composition of non-myogenic cells in injured muscle microenvironment. Collectively, our study suggests that myofiber XBP1 regulates muscle regeneration through both cell-autonomous and -non-autonomous mechanisms.
    Keywords:  Biochemistry; Cell biology; Genetics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2024.111372
  33. Cell. 2024 Dec 06. pii: S0092-8674(24)01322-9. [Epub ahead of print]
    Dual BACH1 regulation by complementary SCF-type E3 ligases.
    Benedikt Goretzki, Maryam Khoshouei, Martin Schröder, Patrick Penner, Luca Egger, Christine Stephan, Dayana Argoti, Nele Dierlamm, Jimena Maria Rada, Sandra Kapps, Catrin Swantje Müller, Zacharias Thiel, Merve Mutlu, Claude Tschopp, David Furkert, Felix Freuler, Simon Haenni, Laurent Tenaillon, Britta Knapp, Alexandra Hinniger, Philipp Hoppe, Enrico Schmidt, Sascha Gutmann, Mario Iurlaro, Grigory Ryzhakov, César Fernández.
      Broad-complex, tramtrack, and bric-à-brac domain (BTB) and CNC homolog 1 (BACH1) is a key regulator of the cellular oxidative stress response and an oncogene that undergoes tight post-translational control by two distinct F-box ubiquitin ligases, SCFFBXO22 and SCFFBXL17. However, how both ligases recognize BACH1 under oxidative stress is unclear. In our study, we elucidate the mechanism by which FBXO22 recognizes a quaternary degron in a domain-swapped β-sheet of the BACH1 BTB dimer. Cancer-associated mutations and cysteine modifications destabilize the degron and impair FBXO22 binding but simultaneously expose an otherwise shielded degron in the dimer interface, allowing FBXL17 to recognize BACH1 as a monomer. These findings shed light on a ligase switch mechanism that enables post-translational regulation of BACH1 by complementary ligases depending on the stability of its BTB domain. Our results provide mechanistic insights into the oxidative stress response and may spur therapeutic approaches for targeting oxidative stress-related disorders and cancer.
    Keywords:  BACH1; Cullin-RING ligases; F-box; FBXL17; FBXO22; S-nitrosylation heme; cysteine modification; ligase switch; oxidative stress
    DOI:  https://doi.org/10.1016/j.cell.2024.11.006
  34. bioRxiv. 2024 Nov 28. pii: 2024.11.28.625793. [Epub ahead of print]
    Design of high specificity binders for peptide-MHC-I complexes.
    Bingxu Liu, Nathan F Greenwood, Julia E Bonzanini, Amir Motmaen, Jazmin Sharp, Chunyu Wang, Gian Marco Visani, Dionne K Vafeados, Nicole Roullier, Armita Nourmohammad, K Christopher Garcia, David Baker.
      Class I MHC molecules present peptides derived from intracellular antigens on the cell surface for immune surveillance, and specific targeting of these peptide-MHC (pMHC) complexes could have considerable utility for treating diseases. Such targeting is challenging as it requires readout of the few outward facing peptide antigen residues and the avoidance of extensive contacts with the MHC carrier which is present on almost all cells. Here we describe the use of deep learning-based protein design tools to de novo design small proteins that arc above the peptide binding groove of pMHC complexes and make extensive contacts with the peptide. We identify specific binders for ten target pMHCs which when displayed on yeast bind the on-target pMHC tetramer but not closely related peptides. For five targets, incorporation of designs into chimeric antigen receptors leads to T-cell activation by the cognate pMHC complexes well above the background from complexes with peptides derived from proteome. Our approach can generate high specificity binders starting from either experimental or predicted structures of the target pMHC complexes, and should be widely useful for both protein and cell based pMHC targeting.
    DOI:  https://doi.org/10.1101/2024.11.28.625793
  35. bioRxiv. 2024 Nov 26. pii: 2024.11.24.624947. [Epub ahead of print]
    Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function.
    David R Rawnsley, Moydul Islam, Chen Zhao, Yasaman Kargar Gaz Kooh, Adelita Mendoza, Honora Navid, Minu Kumari, Xumin Guan, John T Murphy, Jess Nigro, Attila Kovacs, Kartik Mani, Nathaniel Huebsch, Xiucui Ma, Abhinav Diwan.
       Background: Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiac myocytes, and mutations in PQC pathway proteins, such as CRYAB (arginine to glycine at position 120, R120G) and BAG3 (proline to lysine at position 209, P209L) induce protein aggregate pathology with cardiomyopathy in humans. Novel observations in yeast and mammalian cells demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway essential for myocardial homeostasis, facilitates cytosolic protein quality control in cardiac myocytes.
    Methods: Mice with inducible cardiac myocyte specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging in comparison to floxed controls. Induced pluripotent stem cell (iPSC)-derived cardiac myocytes with R120G knock-in to the CRYAB locus were assessed for localization of the CRYAB protein. Transgenic mice expressing R120G CRYAB protein (R120G-TG) were subjected to both TRAF2 gain-of-function (with AAV9-cardiac Troponin T promoter-driven TRAF2 transduction) and TRAF2 loss-of-function (with tamoxifen-inducible ablation of one Traf2 allele) in cardiac myocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology.
    Results: Cardiomyocyte-specific ablation of TRAF2 results accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic chaperone proteins, namely arginine to glycine (R120G) CRYAB mutant and proline to lysine (P209L) BAG3 mutant. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of TRAF2 in the mitochondrial fraction with increased mitophagy as compared with wild type. Adult-onset inducible haplo-insufficiency of TRAF2 resulted in accelerated mortality, impaired left ventricular systolic function and increased protein aggregates in R120G-TG mice as compared with controls. Conversely, AAV9-mediated TRAF2 transduction in R120G-TG mice reduced mortality and attenuated left ventricular systolic dysfunction, with reduced protein aggregates and restoration of normal localization of DESMIN, a cytosolic scaffolding protein chaperoned by CRYAB, as compared with control AAV9-GFP group.
    Conclusions: TRAF2-mediated mitophagy in cardiac myocytes facilitates removal of cytosolic protein aggregates and can be stimulated to ameliorate proteotoxic cardiomyopathy.
    DOI:  https://doi.org/10.1101/2024.11.24.624947
  36. PLoS Genet. 2024 Dec 09. 20(12): e1011338
    Acetylation of the yeast Hsp40 chaperone protein Ydj1 fine-tunes proteostasis and translational fidelity.
    Siddhi Omkar, Megan M Mitchem, Joel R Hoskins, Courtney Shrader, Jake T Kline, Nitika, Luca Fornelli, Sue Wickner, Andrew W Truman.
      Proteostasis, the maintenance of cellular protein balance, is essential for cell viability and is highly conserved across all organisms. Newly synthesized proteins, or "clients," undergo sequential processing by Hsp40, Hsp70, and Hsp90 chaperones to achieve proper folding and functionality. Despite extensive characterization of post-translational modifications (PTMs) on Hsp70 and Hsp90, the modifications on Hsp40 remain less understood. This study aims to elucidate the role of lysine acetylation on the yeast Hsp40, Ydj1. By mutating acetylation sites on Ydj1's J-domain to either abolish or mimic constitutive acetylation, we observed that preventing acetylation had no noticeable phenotypic impact, whereas acetyl-mimic mutants exhibited various defects indicative of impaired Ydj1 function. Proteomic analysis revealed several Ydj1 interactions affected by J-domain acetylation, notably with proteins involved in translation. Further investigation uncovered a novel role for Ydj1 acetylation in stabilizing ribosomal subunits and ensuring translational fidelity. Our data suggest that acetylation may facilitate the transfer of Ydj1 between Ssa1 and Hsp82. Collectively, this work highlights the critical role of Ydj1 acetylation in proteostasis and translational fidelity.
    DOI:  https://doi.org/10.1371/journal.pgen.1011338
  37. Cell Signal. 2024 Dec 08. pii: S0898-6568(24)00535-7. [Epub ahead of print]127 111560
    Inhibition of ERO1L induces autophagy and apoptosis via endoplasmic reticulum stress in colorectal cancer.
    Peng Chen, Yinhao Chen, Amit Sharma, A Gonzalez-Carmona Maria, Ingo G H Schmidt-Wolf.
      Colorectal cancer (CRC) is one of the most common types of cancer with high incidence and mortality. Endoplasmic reticulum oxidoreductase 1 alpha (ERO1L) is overexpressed in CRC. This study aims to explore the role of ERO1L in CRC progression and evaluate the anti-tumor efficacy of the combination treatment of ERO1L inhibition with endoplasmic reticulum (ER) stress-inducing therapies. Herein, we found that ERO1L was elevated in CRC cell lines and patients. ER stress upregulated the expression of ERO1L, and ERO1L deficiency induced ER stress in CRC. ERO1L knockdown increased the susceptibility of CRC cells to ER stress. ERO1L contributed to the malignant phenotypes of CRC cells. Inhibition of ERO1L induced autophagy and caspase-dependent apoptosis by the induction of ER stress in CRC cells. Mechanically, the ERK1/2 pathway was involved in ERO1L knockdown-mediated apoptosis and autophagy. Combination treatment of ERO1L inhibition with ER stress-inducing agents, such as unfolded protein response (UPR)-targeting inhibitors and proteasome inhibitors, demonstrated enhanced anti-tumor capacity. In conclusion, ERO1L is overexpressed in CRC, and ERO1L deficiency induces apoptosis and autophagy via ER stress. ERO1L inhibition combined with ER stress-inducing therapies exhibits more effective anti-tumor activity against CRC. ERO1L may serve as a biomarker and therapeutic target for CRC treatment.
    Keywords:  Apoptosis; Autophagy; Colorectal cancer; Combination treatment; ER stress; ERO1L
    DOI:  https://doi.org/10.1016/j.cellsig.2024.111560
  38. Proc Natl Acad Sci U S A. 2024 Dec 17. 121(51): e2402040121
    Kinetic principles of chemical cross-link formation for protein-protein interactions.
    Kai-Michael Kammer, Terese Eisgruber, Peter Heid, Riccardo Pellarin, Florian Stengel.
      Proteins play a central role in most biological processes within the cell, and deciphering how they interact is key to understand their function. Cross-linking coupled with mass spectrometry is an essential tool for elucidating protein-protein interactions (PPIs). Despite its importance, we still know surprisingly little about the principles that underlie the process of chemical cross-link formation itself and how it is influenced by different physicochemical factors. To understand the molecular details of cross-link formation, we have set up a comprehensive kinetic model and carried out simulations of protein cross-linking on large protein complexes. We dissect the contribution on the cross-link yield of parameters such as amino acid reactivity, cross-linker concentration, and hydrolysis rate. Our model can compute cross-link formation based solely on the structure of a protein complex, thereby enabling realistic predictions for a diverse set of systems. We quantitatively show how cross-links and mono-links are in direct competition and how the hydrolysis rate and abundance of cross-linker and proteins directly influence their relative formation. We show how cross-links and mono-links exist in an "all-against-all" competition due to their simultaneous formation, resulting in a nonintuitive network of interdependence. We show that this interdependence is locally confined and mainly limited to direct neighbors or residues in direct vicinity. These results enable us to identify the optimal cross-linker concentration at which the maximal number of cross-links is formed. Taken together, our study establishes a comprehensive kinetic model to quantitatively describe cross-link formation for PPIs.
    Keywords:  chemical kinetics; cross-link reaction; cross-linking; mass spectrometry; protein–protein interactions
    DOI:  https://doi.org/10.1073/pnas.2402040121
  39. Mol Cell. 2024 Dec 05. pii: S1097-2765(24)00949-3. [Epub ahead of print]
    CRL3ARMC5 ubiquitin ligase and Integrator phosphatase form parallel mechanisms to control early stages of RNA Pol II transcription.
    Roberta Cacioppo, Alexander Gillis, Iván Shlamovitz, Andrew Zeller, Daniela Castiblanco, Alastair Crisp, Benjamin Haworth, Angela Arabiotorre, Pegah Abyaneh, Yu Bao, Julian E Sale, Scott Berry, Ana Tufegdžić Vidaković.
      Control of RNA polymerase II (RNA Pol II) through ubiquitylation is essential for the DNA-damage response. Here, we reveal a distinct ubiquitylation pathway in human cells, mediated by CRL3ARMC5, that targets excessive and defective RNA Pol II molecules at the initial stages of the transcription cycle. Upon ARMC5 loss, RNA Pol II accumulates in the free pool and in the promoter-proximal zone but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess RNA Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has detrimental effects on cell growth and results in the uncontrolled release of excessive RNA Pol II complexes into early elongation, many of which are transcriptionally incompetent and fail to reach the ends of genes. These findings uncover CRL3ARMC5 and Integrator as two distinct pathways acting in parallel to monitor the quantity and quality of transcription complexes before they are licensed into elongation.
    Keywords:  ARMC5; CUL3; INTS8; Integrator; RNA polymerase II; elongation; pausing; promoter-proximal; transcription; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.024
  40. EMBO Rep. 2024 Dec 10.
    RudLOV is an optically synchronized cargo transport method revealing unexpected effects of dynasore.
    Tatsuya Tago, Takumi Ogawa, Yumi Goto, Kiminori Toyooka, Takuro Tojima, Akihiko Nakano, Takunori Satoh, Akiko K Satoh.
      Live imaging of secretory cargoes is a powerful method for understanding the mechanisms of membrane trafficking. Inducing the synchronous release of cargoes from an organelle is key for enhancing microscopic observation. We developed an optical cargo-releasing method, 'retention using dark state of LOV2' (RudLOV), which enables precise spatial, temporal, and quantity control during cargo release. A limited amount of cargo-release using RudLOV is able to visualize cargo cisternal-movement and cargo-specific exit sites on the Golgi/trans-Golgi network. Moreover, by controlling the timing of cargo-release using RudLOV, we reveal the canonical and non-canonical effects of the well-known dynamin inhibitor dynasore, which inhibits early- but not late-Golgi transport and exits from the trans-Golgi network where dynamin-2 is active. Accumulation of COPI vesicles at the cis-side of the Golgi stacks in dynasore-treated cells suggests that dynasore targets COPI-uncoating/tethering/fusion machinery in the early-Golgi cisternae or endoplasmic reticulum but not in the late-Golgi cisternae. These results provide insight into the cisternal maturation of Golgi stacks.
    Keywords:   trans-Golgi Network; Cisternal Maturation; Dynasore; Live Imaging; RudLOV
    DOI:  https://doi.org/10.1038/s44319-024-00342-z
  41. Autophagy. 2024 Dec 09.
    Janus-like behavior of intrinsically disordered regions in reticulophagy.
    Sergio Alejandro Poveda Cuevas, Kateryna Lohachova, Borna Markusic, Ivan Dikic, Gerhard Hummer, Ramachandra Bhaskara.
      Intrinsically disordered regions (IDRs) are crucial to homeostatic and organellar remodeling pathways. In reticulophagy/ER-phagy, long cytosolic IDR-containing receptors (e.g. RETREG1/FAM134B) house the LC3-interacting region (LIR) motif to recruit the phagophore. The precise functions of the IDR beyond engaging the autophagic machinery are unclear. Here, we comment on the role of the RETREG1-IDR based on our recent computer modeling and molecular dynamics (MD) simulations. Extensive analysis of the RETREG1-IDR indicates a continuum of conformations between expanded and compact structures, displaying a Janus-like feature. Using an adapted MARTINI model, we find that the IDR ensemble properties vary widely depending on the membrane anchor. IDRs alone are sufficient to promote and sense membrane curvature and can act as entropic tethers. When anchored to the RHD, they adopt compact collapsed conformations, acting as effector scaffolds that amplify RHD membrane remodeling properties, enhancing receptor-clustering and accelerating spontaneous budding. These findings expand the operational scope of IDRs within reticulophagy, offering fresh insights into a mechanistic understanding of membrane remodeling.
    Keywords:  Conformational heterogeneity; ER remodeling; IDRs; curvature induction; effector; entropic chain
    DOI:  https://doi.org/10.1080/15548627.2024.2437652
  42. Liver Int. 2025 Jan;45(1): e16207
    Alpha-1 Antitrypsin Inclusions Sequester GRP78 in a Bile Acid-Inducible Manner.
    Igor Spivak, Nurdan Guldiken, Valentyn Usachov, Frank Schaap, Steven W M Olde Damink, Marion Bouchecareilh, Alexandra Lehmann, Lei Fu, Fa-Rong Mo, Gökce Kobazi Ensari, Franziska Hufnagel, Malin Fromme, Christian Preisinger, Pavel Strnad.
       BACKGROUND AND AIMS: The homozygous PiZ mutation (PIZZ genotype) constitutes the predominant cause of severe alpha-1 antitrypsin (AAT) deficiency and leads to liver disease via hepatocellular AAT aggregation. We systematically analysed the composition of AAT aggregates and studied the impact of bile acids.
    METHODS: AAT inclusions were isolated from livers of PiZ overexpressing mice and PIZZ humans via fluorescence-activated and immunomagnetic sorting (FACS/MACS), while insoluble proteins were obtained via Triton-X extraction. Inclusion composition was evaluated through mass-spectrometry (MS), immunoblotting and immunostaining. Hepatocytes with versus without AAT aggregates were obtained via microdissection. Serum bile acids were assessed in 57 PIZZ subjects and 19 controls. Mice were administered 2% cholic acid (CA)-supplemented chow for 7 days.
    RESULTS: MS identified the key endoplasmic reticulum chaperone 78 kDa glucose-regulated protein (GRP78) in FACS/MACS pulldowns. GRP78 was also enriched in insoluble fractions from PiZ mice versus wild types and detected in insoluble fractions/MACS isolates from PIZZ liver explants. In cultured cells/primary hepatocytes, PiZ overexpression was associated with increased GRP78 mRNA/protein levels. In human livers, hepatocytes with AAT aggregates had higher GRP78 levels than hepatocytes without. PIZZ subjects displayed higher serum bile acid levels than controls and the highest levels were seen in individuals with liver injury/fibrosis. In PiZ mice, CA-mediated bile acid challenge resulted in increased liver injury and translocation of GRP78 into the aggregates.
    CONCLUSIONS: Our results demonstrate that GRP78 is sequestered within AAT inclusions. Bile acid accumulation, as seen in PIZZ subjects with liver disease, may promote GRP78 segregation and thereby augment liver damage.
    TRIAL REGISTRATION: NCT02929940.
    Keywords:  ER chaperone; cholestatic liver injury; genetic liver disease; rare liver disease; α‐1 antitrypsin deficiency
    DOI:  https://doi.org/10.1111/liv.16207
  43. EMBO Rep. 2024 Dec 11.
    Acquired resistance to PD-L1 inhibition enhances a type I IFN-regulated secretory program in tumors.
    Yuhao Shi, Amber McKenery, Melissa Dolan, Michalis Mastri, James W Hill, Adam Dommer, Sebastien Benzekry, Mark Long, Scott I Abrams, Igor Puzanov, John M L Ebos.
      Therapeutic inhibition of programmed cell death ligand (PD-L1) is linked to alterations in interferon (IFN) signaling. Since IFN-regulated intracellular signaling can control extracellular secretory programs in tumors to modulate immunity, we examined IFN-related secretory changes in tumor cells following resistance to PD-L1 inhibition. Here we report an anti-PD-L1 treatment-induced secretome (PTIS) in tumor models of acquired resistance that is regulated by type I IFNs. These secretory changes can suppress activation of T cells ex vivo while diminishing tumor cell cytotoxicity, revealing that tumor-intrinsic treatment adaptations can exert broad tumor-extrinsic effects. When reimplanted in vivo, resistant tumor growth can slow or stop when PTIS components are disrupted individually, or when type I IFN signaling machinery is blocked. Interestingly, genetic and therapeutic disruption of PD-L1 in vitro can only partially recapitulate the PTIS phenotype highlighting the importance of developing in vivo-based resistance models to more faithfully mimic clinically-relevant treatment failure. Together, this study shows acquired resistance to immune-checkpoint inhibitors 'rewires' tumor secretory programs controlled by type I IFNs that, in turn, can protect from immune cell attack.
    Keywords:  IFN; Immune-checkpoint; PD-L1; Resistance; Secretome
    DOI:  https://doi.org/10.1038/s44319-024-00333-0
  44. PNAS Nexus. 2024 Dec;3(12): pgae499
    Disagreement on foundational principles of biological aging.
    Vadim N Gladyshev, Benjamin Anderson, Hanna Barlit, Benjamin Barré, Samuel Beck, Bahareh Behrouz, Daniel W Belsky, Amandine Chaix, Manish Chamoli, Brian H Chen, Kaiyang Cheng, Jane Chuprin, Gary A Churchill, Andrea Cipriano, Alex Colville, Joris Deelen, Yuri Deigin, KeHuan K Edmonds, Bradley W English, Ruogu Fang, Michael Florea, Iosif M Gershteyn, Diljeet Gill, Laura H Goetz, Vera Gorbunova, Patrick T Griffin, Steve Horvath, Martin Borch Jensen, Xin Jin, Sara Jovanovska, Kathrin M Kajderowicz, Tomoko Kasahara, Csaba Kerepesi, Subhash Kulkarni, Vyacheslav M Labunskyy, Morgan E Levine, Sergiy Libert, J Yuyang Lu, Yuancheng Ryan Lu, Riccardo E Marioni, Brianah M McCoy, Wayne Mitchell, Mahdi Moqri, Farzaneh Nasirian, Peter Niimi, Hamilton Se-Hwee Oh, Brian Okundaye, Andrey A Parkhitko, Leonid Peshkin, Mia Petljak, Jesse R Poganik, Glen Pridham, Daniel E L Promislow, Weronika Prusisz, Margaux Quiniou, Ken Raj, Daniel Richard, Jose Luis Ricon, Jarod Rutledge, Morten Scheibye-Knudsen, Nicholas J Schork, Andrei Seluanov, Michael Shadpour, Anastasia V Shindyapina, Steven R Shuken, Sruthi Sivakumar, Thomas Stoeger, Ayumu Sugiura, Nadia R Sutton, Alexander Suvorov, Andrei E Tarkhov, Emma C Teeling, Alexandre Trapp, Alexander Tyshkovskiy, Maximilian Unfried, Cavin K Ward-Caviness, Sun Hee Yim, Kejun Ying, Jeffrey Yunes, Bohan Zhang, Alex Zhavoronkov.
      To gain insight into how researchers of aging perceive the process they study, we conducted a survey among experts in the field. While highlighting some common features of aging, the survey exposed broad disagreement on the foundational issues. What is aging? What causes it? When does it begin? What constitutes rejuvenation? Not only was there no consensus on these and other core questions, but none of the questions received a majority opinion-even regarding the need for consensus itself. Despite many researchers believing they understand aging, their understanding diverges considerably. Importantly, as different processes are labeled as "aging" by researchers, different experimental approaches are prioritized. The survey shed light on the need to better define which aging processes this field should target and what its goals are. It also allowed us to categorize contemporary views on aging and rejuvenation, revealing critical, yet largely unanswered, questions that appear disconnected from the current research focus. Finally, we discuss ways to address the disagreement, which we hope will ultimately aid progress in the field.
    DOI:  https://doi.org/10.1093/pnasnexus/pgae499
  45. Mol Syst Biol. 2024 Dec 12.
    A split intein and split luciferase-coupled system for detecting protein-protein interactions.
    Zhong Yao, Jiyoon Kim, Betty Geng, Jinkun Chen, Victoria Wong, Anna Lyakisheva, Jamie Snider, Marina Rudan Dimlić, Sanda Raić, Igor Stagljar.
      Elucidation of protein-protein interactions (PPIs) represents one of the most important methods in biomedical research. Recently, PPIs have started to be exploited for drug discovery purposes and have thus attracted much attention from both the academic and pharmaceutical sectors. We previously developed a sensitive method, Split Intein-Mediated Protein Ligation (SIMPL), for detecting binary PPIs via irreversible splicing of the interacting proteins being investigated. Here, we incorporated tripart nanoluciferase (tNLuc) into the system, providing a luminescence signal which, in conjunction with homogenous liquid phase operation, improves the quantifiability and operability of the assay. Using a reference PPI set, we demonstrated an improvement in both sensitivity and specificity over the original SIMPL assay. Moreover, we designed the new SIMPL-tNLuc ('SIMPL2') platform with an inherent modularity allowing for flexible measurement of molecular modulators of target PPIs, including inhibitors, molecular glues and PROTACs. Our results demonstrate that SIMPL2 is a sensitive, cost- and labor-effective tool suitable for high-throughput screening (HTS) in both PPI mapping and drug discovery applications.
    Keywords:  Drug Discovery; Inhibitor; Protein-protein Interaction; Split Intein; Tri-part Nanoluciferase
    DOI:  https://doi.org/10.1038/s44320-024-00081-2
  46. Mol Cell. 2024 Dec 03. pii: S1097-2765(24)00919-5. [Epub ahead of print]
    Epitranscriptomic rRNA fingerprinting reveals tissue-of-origin and tumor-specific signatures.
    Ivan Milenkovic, Sonia Cruciani, Laia Llovera, Morghan C Lucas, Rebeca Medina, Cornelius Pauli, Daniel Heid, Thomas Muley, Marc A Schneider, Laura V Klotz, Michael Allgäuer, Ruben Lattuca, Denis L J Lafontaine, Carsten Müller-Tidow, Eva Maria Novoa.
      Mammalian ribosomal RNA (rRNA) molecules are highly abundant RNAs, decorated with over 220 rRNA modifications. Previous works have shown that some rRNA modification types can be dynamically regulated; however, how and when the mammalian rRNA modification landscape is remodeled remains largely unexplored. Here, we employ direct RNA sequencing to chart the human and mouse rRNA epitranscriptome across tissues, developmental stages, cell types, and disease. Our analyses reveal multiple rRNA sites that are differentially modified in a tissue- and/or developmental stage-specific manner, including previously unannotated modified sites. We demonstrate that rRNA modification patterns can be used for tissue and cell-type identification, which we hereby term "epitranscriptomic fingerprinting." We then explore rRNA modification patterns in normal-tumor matched samples from lung cancer patients, finding that epitranscriptomic fingerprinting accurately classifies clinical samples into normal and tumor groups from only 250 reads per sample, demonstrating the potential of rRNA modifications as diagnostic biomarkers.
    Keywords:  RNA modifications; cancer; classification; direct RNA sequencing; epitranscriptome; fingerprinting; nanopore; pseudouridine; rRNA
    DOI:  https://doi.org/10.1016/j.molcel.2024.11.014
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