bims-proteo Biomed News
on Proteostasis
Issue of 2025–01–19
forty-nine papers selected by
Eric Chevet, INSERM
  1. mTOR signalling controls protein aggregation during heat stress and cellular aging in a translation- and Hsf1-independent manner.
  2. IRE1 is a Promising Therapeutic Target in Pancreatic Cancer.
  3. Mass Spectrometry Analysis of Chemically and Collisionally Dissociated Molecular Glue- and PROTAC-Mediated Protein Complexes Informs on Disassembly Pathways.
  4. Unfolded protein response: An essential element of intestinal homeostasis and a potential therapeutic target for inflammatory bowel disease.
  5. TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells.
  6. Girolline is a sequence context-selective modulator of eIF5A activity.
  7. Development of the First-in-Class FEM1B-Recruiting Histone Deacetylase Degraders.
  8. E3 ligase substrate adaptor SPOP fine-tunes the UPR of pancreatic β cells.
  9. Spatial covariance reveals isothiocyanate natural products adjust redox stress to restore function in alpha-1-antitrypsin deficiency.
  10. Engineering Covalent Aptamer Chimeras for Enhanced Autophagic Degradation of Membrane Proteins.
  11. Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition.
  12. A STING-CASM-GABARAP pathway activates LRRK2 at lysosomes.
  13. mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase.
  14. 40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion.
  15. Discovering genetic modulators of the protein homeostasis system through multilevel analysis.
  16. Activation of the 26S Proteasome to Reduce Proteotoxic Stress and Improve the Efficacy of PROTACs.
  17. Artificial enforcement of the unfolded protein response (UPR) reduces disease features in multiple preclinical models of ALS/FTD.
  18. Proteasome inhibition induces microtubule-dependent changes in nuclear morphology.
  19. A truncated variant of the ribosome-associated trigger factor specifically contributes to plant chloroplast ribosome biogenesis.
  20. The regulatory landscape of 5' UTRs in translational control during zebrafish embryogenesis.
  21. Loss of correlated proteasomal subunit expression selectively promotes the 20SHigh state which underlies luminal breast tumorigenicity.
  22. The pentose phosphate pathway controls oxidative protein folding and prevents ferroptosis in chondrocytes.
  23. Reacting to reductive stress at the mitochondrial import gate.
  24. Unfolded protein response modulates Tyrosinase levels and melanin production during melanogenesis.
  25. Enantioselective OTUD7B fragment discovery through chemoproteomics screening and high-throughput optimisation.
  26. Structure of E6AP in complex with HPV16-E6 and p53 reveals a novel ordered domain important for E3 ligase activation.
  27. Macrocyclic peptides as a new class of targeted protein degraders.
  28. De novo designed proteins neutralize lethal snake venom toxins.
  29. Autophagy related 7 (ATG7) regulates food intake and liver health during asparaginase exposure.
  30. Active learning-assisted directed evolution.
  31. Simulating 500 million years of evolution with a language model.
  32. Male sex determination maintains proteostasis and extends lifespan of daf-18/PTEN deficient C. elegans.
  33. Regulation of stress granule maturation and dynamics by poly(ADP-ribose) interaction with PARP13.
  34. The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity.
  35. The ER protein CANX (calnexin)-mediated autophagy protects against alzheimer disease.
  36. Rapid discovery of cyclic peptide protein aggregation inhibitors by continuous selection.
  37. Mutations in histones dysregulate copper homeostasis leading to defect in Sec61 dependent protein translocation mechanism in Saccharomyces cerevisiae.
  38. Targeting protein-ligand neosurfaces with a generalizable deep learning tool.
  39. NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.
  40. Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity.
  41. Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation.
  42. Dense-core vesicles contain exosomes in secretory cells.
  43. Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis.
  44. Small protein ERSP encoded by LINC02870 promotes triple negative breast cancer progression via IRE1α/XBP1s activation.
  45. Protein-Based Degraders: From Chemical Biology Tools to Neo-Therapeutics.
  46. Structure of the human autophagy factor EPG5 and the molecular basis of its conserved mode of interaction with Atg8-family proteins.
  47. Multi-tissue characterization of the constitutive heterochromatin proteome in Drosophila identifies a link between satellite DNA organization and transposon repression.
  48. In situ quantification of ribosome number by electron tomography.
  49. Protocol for cell image-based spatiotemporal proteomics in budding yeast.
  1. J Biol Chem. 2025 Jan 09. pii: S0021-9258(25)00019-5. [Epub ahead of print] 108172
    mTOR signalling controls protein aggregation during heat stress and cellular aging in a translation- and Hsf1-independent manner.
    Arthur Fischbach, Per O Widlund, Xinxin Hao, Thomas Nyström.
      The mTOR (mechanistic target of rapamycin) signaling pathway appears central to the aging process as genetic or pharmacological inhibition of mTOR extends lifespan in most eukaryotes tested. While the regulation of protein synthesis by mTOR has been studied in great detail, its impact on protein misfolding and aggregation during stress and aging is less explored. In this study, we identified the mTOR signaling pathway and the linked SEA complex as central nodes of protein aggregation during heat stress and cellular aging, using Saccharomyces cerevisiae as a model organism. Based on a synthetic genetic array (SGA) screen, we found that reduced mTOR activity, achieved through deletion of TCO89, an mTORC1 subunit, almost completely prevents protein aggregation during heat stress and aging without reducing global translation rates and independently of an Hsf1-dependent stress response. Conversely, increased mTOR activity, achieved through deletion of NPR3, a SEA complex subunit, exacerbates protein aggregation, but not by over-activating translation. In summary, our work demonstrates that mTOR signaling is a central contributor to age-associated and heat shock-induced protein aggregation and that this is unlinked to quantitatively discernable effects on translation and Hsf1.
    Keywords:  Aging; mTOR; protein aggregation; proteinopathy; proteostasis; spatial protein quality control
    DOI:  https://doi.org/10.1016/j.jbc.2025.108172
  2. Am J Physiol Cell Physiol. 2025 Jan 16.
    IRE1 is a Promising Therapeutic Target in Pancreatic Cancer.
    Denise Lucas, Tamal Sarkar, Clara Y Niemeyer, Julian C Harnoss, Martin Schneider, Moritz J Strowitzki, Jonathan M Harnoss.
      Pancreatic cancer (PC) is one of the most aggressive malignancies, characterized by an increasing incidence and unfavorable prognosis. Despite recent advances, surgical resection combined with chemotherapy remains the only potentially curative therapeutic option. Therefore, it is of paramount importance to identify novel therapeutic targets and develop effective treatment strategies. Pancreatic ductal adenocarcinoma (PDAC), the most prevalent form of PC, originates from exocrine cells and is subjected to both intrinsic and extrinsic cellular stresses, including oncogene activation, loss of tumor suppressors, a hypoxic and immunosuppressive tumor microenvironment (TME), and chemotherapy, causing an accumulation of misfolded proteins within the endoplasmic reticulum (ER). The loss of ER proteostasis activates the unfolded protein response (UPR), an intracellular sensing-signaling network that enables cancer cells to alleviate ER stress and restore cellular proteostasis. The key UPR sensor Inositol-Requiring Enzyme 1 (IRE1) is an ER membrane protein that activates the transcription factor X-Box Protein 1 Spliced (XBP1s) through its cytoplasmic kinase-RNase module, promoting protein folding, secretion capacity, and proteasomal degradation of misfolded proteins. Additionally, it regulates IRE1-dependent decay (RIDD) of various mRNA and functions through scaffold interactions. In this review, we synthesize current evidence on the cell-autonomous and cell-non-autonomous roles of IRE1 in tumor initiation, progression, metastasis, and drug resistance in PDAC and outline key research directions to investigate IRE1 as a potential therapeutic target.
    Keywords:  IRE1; PDAC; endoplasmic reticulum stress; pancreatic cancer; unfolded protein response
    DOI:  https://doi.org/10.1152/ajpcell.00551.2024
  3. J Am Soc Mass Spectrom. 2025 Jan 15.
    Mass Spectrometry Analysis of Chemically and Collisionally Dissociated Molecular Glue- and PROTAC-Mediated Protein Complexes Informs on Disassembly Pathways.
    Edvaldo V S Maciel, Jonathan Eisert, Julian Müller, Tanja Habeck, Frederik Lermyte.
      Molecular glues (MGs) and proteolysis-targeting chimeras (PROTACs) are used to modulate protein-protein interactions (PPIs), via induced proximity between compounds that have little or no affinity for each other naturally. They promote either reversible inhibition or selective degradation of a target protein, including ones deemed undruggable by traditional therapeutics. Though native MS (nMS) is capable of analyzing multiprotein complexes, the behavior of these artificially induced compounds in the gas phase is still not fully understood, and the number of publications over the past few years is still rather limited. Here, we studied two MG-induced complexes between mTORFRB and FKBP12 as well as a PROTAC-induced complex between FKBP51FK1 and the von Hippel-Lindau E3 ligase (VHL). Native MS combined with collision-induced dissociation (CID) provided a way of measuring not only the formation of these complexes but also their dissociation pathways. Both protein complexes seem to eject preferably the centrally located small (compared to the mass of the proteins) ligand upon CID, rather than dissociating a peripheral subunit, as is often observed for naturally occurring protein complexes. In contrast, chemically induced dissociation in solution generated complementary data to CID, by disrupting the PPI surface, which resulted in more diverse MS spectra that preserved the stronger interactions in solution.
    DOI:  https://doi.org/10.1021/jasms.4c00429
  4. Acta Physiol (Oxf). 2025 Feb;241(2): e14284
    Unfolded protein response: An essential element of intestinal homeostasis and a potential therapeutic target for inflammatory bowel disease.
    Miriam Di Mattia, Michele Sallese, Loris Riccardo Lopetuso.
      Different physiological and pathological situations can produce alterations in the cell's endoplasmic reticulum (ER), leading to a condition known as ER stress, which can trigger an intricate intracellular signal transduction system known as the unfolded protein response (UPR). UPR is primarily tailored to restore proteostasis and ER equilibrium; otherwise, if ER stress persists, it can cause programmed cell death as a cytoprotective mechanism and drive inflammatory processes. Therefore, since intestinal cells strongly rely on UPR for their biological functions and unbalanced UPR has been linked to inflammatory, metabolic, and immune disorders, here we discussed the role of the UPR within the intestinal tract, focusing on the UPR contribution to inflammatory bowel disease development. Importantly, we also highlighted the promising potential of UPR components as therapeutic targets for intestinal inflammatory diseases.
    Keywords:  ER stress; IBD; KDEL receptors; autophagy; inflammation; molecular chaperones; proteostasis
    DOI:  https://doi.org/10.1111/apha.14284
  5. J Cell Biol. 2025 Mar 03. pii: e202109010. [Epub ahead of print]224(3):
    TanGIBLE: A selective probe for evaluating hydrophobicity-exposed defective proteins in live cells.
    Yasuyuki Iwasa, Sohtaroh Miyata, Takuya Tomita, Naoto Yokota, Maho Miyauchi, Ruka Mori, Shin Matsushita, Rigel Suzuki, Yasushi Saeki, Hiroyuki Kawahara.
      The accumulation of defective polypeptides in cells is a major cause of various diseases. However, probing defective proteins is difficult because no currently available method can retrieve unstable defective translational products in a soluble state. To overcome this issue, there is a need for a molecular device specific to structurally defective polypeptides. In this study, we developed an artificial protein architecture comprising tandemly aligned BAG6 Domain I, a minimum substrate recognition platform responsible for protein quality control. This tandem-aligned entity shows enhanced affinity not only for model defective polypeptides but also for endogenous polyubiquitinated proteins, which are sensitive to translational inhibition. Mass-spectrometry analysis with this probe enabled the identification of endogenous defective proteins, including orphaned subunits derived from multiprotein complexes and misassembled transmembrane proteins. This probe is also useful for the real-time visualization of protein foci derived from defective polypeptides in stressed cells. Therefore, this "new molecular trap" is a versatile tool for evaluating currently "invisible" pools of defective polypeptides as tangible entities.
    DOI:  https://doi.org/10.1083/jcb.202109010
  6. Nat Commun. 2025 Jan 10. 16(1): 223
    Girolline is a sequence context-selective modulator of eIF5A activity.
    Tilman Schneider-Poetsch, Yongjun Dang, Wakana Iwasaki, Mayumi Arata, Yuichi Shichino, Ali Al Mourabit, Celine Moriou, Daniel Romo, Jun O Liu, Takuhiro Ito, Shintaro Iwasaki, Minoru Yoshida.
      Natural products have a long history of providing probes into protein biosynthesis, with many of these compounds serving as therapeutics. The marine natural product girolline has been described as an inhibitor of protein synthesis. Its precise mechanism of action, however, has remained unknown. The data we present here suggests that girolline is a sequence-selective modulator of translation factor eIF5A. Girolline interferes with ribosome-eIF5A interaction and induces ribosome stalling where translational progress is impeded, including on AAA-encoded lysine. Our data furthermore indicate that eIF5A plays a physiological role in ribosome-associated quality control and in maintaining the efficiency of translational progress. Girolline helped to deepen our understanding of the interplay between protein production and quality control in a physiological setting and offers a potent chemical tool to selectively modulate gene expression.
    DOI:  https://doi.org/10.1038/s41467-024-54838-2
  7. J Med Chem. 2025 Jan 13.
    Development of the First-in-Class FEM1B-Recruiting Histone Deacetylase Degraders.
    Felix Feller, Irina Honin, Martina Miranda, Heiko Weber, Svenja Henze, Maria Hanl, Finn K Hansen.
      Targeted protein degradation (TPD) represents a promising alternative to conventional occupancy-driven protein inhibition. Despite the existence of more than 600 E3 ligases in the human proteome, so far only a few have been utilized for TPD of histone deacetylases (HDACs), which represent important epigenetic anticancer drug targets. In this study, we disclose the first-in-class Fem-1 homologue B (FEM1B)-recruiting HDAC degraders. A set of 12 proteolysis targeting chimeras (PROTACs) was synthesized using a solid-phase supported parallel synthesis approach utilizing a covalent FEM1B ligand as an E3 ligase warhead. The evaluation of the HDAC degradation efficiency revealed substantial HDAC1 degradation by the top-performing degrader FF2049 (1g: Dmax = 85%; DC50 = 257 nM). Unlike our previously published cereblon-recruiting selective HDAC6 degrader, A6, which uses the same HDAC ligand, the FEM1B-based PROTACs achieved selective HDAC1-3 degradation. This unexpected change in the HDAC isoform degradation profile was accompanied by significant enhancement of the antiproliferative properties.
    DOI:  https://doi.org/10.1021/acs.jmedchem.4c02569
  8. Genes Dev. 2024 Dec 30.
    E3 ligase substrate adaptor SPOP fine-tunes the UPR of pancreatic β cells.
    Alexis U Oguh, Matthew W Haemmerle, Sabyasachi Sen, Andrea V Rozo, Shristi Shrestha, Jean-Philippe Cartailler, Hossein Fazelinia, Hua Ding, Sam Preza, Juxiang Yang, Xiaodun Yang, Lori Sussel, Juan R Alvarez-Dominguez, Nicolai Doliba, Lynn A Spruce, Rafael Arrojo E Drigo, Doris A Stoffers.
      The Cullin-3 E3 ligase adaptor protein SPOP targets proteins for ubiquitination and proteasomal degradation. We previously established the β-cell transcription factor (TF) and human diabetes gene PDX1 as an SPOP substrate, suggesting a functional role for SPOP in the β cell. Here, we generated a β-cell-specific Spop deletion mouse strain (Spop βKO) and found that Spop is necessary to prevent aberrant basal insulin secretion and for maintaining glucose-stimulated insulin secretion through impacts on glycolysis and glucose-stimulated calcium flux. Integration of proteomic, TF-regulatory gene network, and biochemical analyses identified XBP1 as a functionally important SPOP substrate in pancreatic β cells. Furthermore, loss of SPOP strengthened the IRE1α-XBP1 axis of unfolded protein response (UPR) signaling. ER stress promoted proteasomal degradation of SPOP, supporting a model whereby SPOP fine-tunes XBP1 activation during the UPR. These results position SPOP as a regulator of β-cell function and proper UPR activation.
    Keywords:  diabetes; proteasome; ubiquitin; unfolded protein response; β cells
    DOI:  https://doi.org/10.1101/gad.352010.124
  9. Cell Rep Med. 2025 Jan 07. pii: S2666-3791(24)00688-8. [Epub ahead of print] 101917
    Spatial covariance reveals isothiocyanate natural products adjust redox stress to restore function in alpha-1-antitrypsin deficiency.
    Shuhong Sun, Chao Wang, Junyan Hu, Pei Zhao, Xi Wang, William E Balch.
      Alpha-1 antitrypsin (AAT) deficiency (AATD) is a monogenic disease caused by misfolding of AAT variants resulting in gain-of-toxic aggregation in the liver and loss of monomer activity in the lung leading to chronic obstructive pulmonary disease (COPD). Using high-throughput screening, we discovered a bioactive natural product, phenethyl isothiocyanate (PEITC), highly enriched in cruciferous vegetables, including watercress and broccoli, which improves the level of monomer secretion and neutrophil elastase (NE) inhibitory activity of AAT-Z through the endoplasmic reticulum (ER) redox sensor protein disulfide isomerase (PDI) A4 (PDIA4). The intracellular polymer burden of AAT-Z can be managed by combination treatment of PEITC and an autophagy activator. Using Gaussian process (GP)-based spatial covariance (SCV) (GP-SCV) machine learning to map on a residue-by-residue basis at atomic resolution all variants in the worldwide AATD clinical population, we reveal a global rescue of monomer secretion and NE inhibitory activity for most variants triggering disease. We present a proof of concept that GP-SCV mapping of restoration of AAT variant function serves as a standard model to discover natural products such as the anti-oxidant PEITC that could potentially impact the redox/inflammatory environment of the ER to provide a nutraceutical approach to help minimize disease in AATD patients.
    Keywords:  AATD; COPD; Gaussian process machine learning; PDIA4; alpha-1 antitrypsin deficiency; chronic obstructive pulmonary disease; genetic variation; isothiocyanate; nutraceutical; oxidative stress; protein aggregation; protein disulfide isomerase A4; proteostasis
    DOI:  https://doi.org/10.1016/j.xcrm.2024.101917
  10. Angew Chem Int Ed Engl. 2025 Jan 17. e202425123
    Engineering Covalent Aptamer Chimeras for Enhanced Autophagic Degradation of Membrane Proteins.
    Yang Shi, Yangfang Yun, Rong Wang, Zheng Liu, Zhenkun Wu, Yu Xiang, Jingjing Zhang.
      Targeted degradation of membrane proteins represents an attractive strategy for eliminating pathogenesis-related proteins. Aptamer-based chimeras hold great promise as membrane protein degraders, however, their degradation efficacy is often hindered by the limited structural stability and the risk of off-target effects due to the non-covalent interaction with target proteins. We here report the first design of a covalent aptamer-based autophagosome-tethering chimera (CApTEC) for the enhanced autophagic degradation of cell-surface proteins, including transferrin receptor 1 (TfR1) and nucleolin (NCL). This strategy relies on the site-specific incorporation of sulfonyl fluoride groups onto aptamers to enable the cross-linking with target proteins, coupled with the conjugation of an LC3 ligand to hijack the autophagy-lysosomal pathway for targeted protein degradation. The chemically engineered CApTECs exhibit enhanced on-target retention and improved structural stability. Our results also demonstrate that CApTECs achieve remarkably enhanced and prolonged degradation of membrane proteins compared to the non-covalent designs. Furthermore, the CApTEC targeting TfR1 is combined with 5-fluorouracil (5-FU) for synergistic tumor therapy in a mouse model, leading to substantial suppression of tumor growth. Our strategy may provide deep insights into the LC3-mdiated autophagic degradation, affording a modular and effective strategy for membrane protein degradation and precise therapeutic applications.
    Keywords:  Functional nucleic acid, Covalent conjugation, Protein degradation, Aptamer, Tumor therapy
    DOI:  https://doi.org/10.1002/anie.202425123
  11. STAR Protoc. 2025 Jan 10. pii: S2666-1667(24)00712-3. [Epub ahead of print]6(1): 103547
    Protocol to identify endogenous proximal proteins using biotinylation by antibody recognition.
    Camilla Rega, Mercedes Pardo, Lesley-Ann Martin, Jyoti Choudhary.
      Biotinylation by antibody recognition (BAR) is an antibody-based approach for mapping proximal protein interactions in cells. Here, we present a protocol to biotinylate and identify proximal proteins using BAR. We describe steps for defining proximity labeling reaction conditions, assessing enrichment using western blot, and sample preparation for mass spectroscopy analysis. We then detail procedures for data analysis and identifying proximal proteins. This approach differs from standard proximity labeling techniques, which rely on genetically engineered enzymes fused to the target protein. For complete details on the use and execution of this protocol, please refer to Rega et al.1.
    Keywords:  cell biology; protein biochemistry; proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2024.103547
  12. J Cell Biol. 2025 Feb 03. pii: e202310150. [Epub ahead of print]224(2):
    A STING-CASM-GABARAP pathway activates LRRK2 at lysosomes.
    Amanda Bentley-DeSousa, Agnes Roczniak-Ferguson, Shawn M Ferguson.
      Mutations that increase LRRK2 kinase activity have been linked to Parkinson's disease and Crohn's disease. LRRK2 is also activated by lysosome damage. However, the endogenous cellular mechanisms that control LRRK2 kinase activity are not well understood. In this study, we identify signaling through stimulator of interferon genes (STING) as an activator of LRRK2 via the conjugation of ATG8 to single membranes (CASM) pathway. We furthermore establish that multiple chemical stimuli that perturb lysosomal homeostasis also converge on CASM to activate LRRK2. Although CASM results in the lipidation of multiple ATG8 protein family members, we establish that LRRK2 lysosome recruitment and kinase activation are highly dependent on interactions with the GABARAP member of this family. Collectively, these results define a pathway that integrates multiple stimuli at lysosomes to control the kinase activity of LRRK2. Aberrant activation of LRRK2 via this pathway may be of relevance in both Parkinson's and Crohn's diseases.
    DOI:  https://doi.org/10.1083/jcb.202310150
  13. Cell Rep. 2025 Jan 13. pii: S2211-1247(24)01530-4. [Epub ahead of print]44(1): 115179
    mTORC1 regulates the pyrimidine salvage pathway by controlling UCK2 turnover via the CTLH-WDR26 E3 ligase.
    Brittany Q Pham, Sang Ah Yi, Alban Ordureau, Heeseon An.
      One critical aspect of cell proliferation is increased nucleotide synthesis, including pyrimidines. Pyrimidines are synthesized through de novo and salvage pathways. Prior studies established that the mammalian target of rapamycin complex 1 (mTORC1) promotes pyrimidine synthesis by activating the de novo pathway for cell proliferation. However, the involvement of mTORC1 in regulating the salvage pathway remains unclear. Here, we report that mTORC1 controls the half-life of uridine cytidine kinase 2 (UCK2), the rate-limiting enzyme in the salvage pathway. Specifically, UCK2 is degraded via the CTLH-WDR26 E3 complex during mTORC1 inhibition, which is prevented when mTORC1 is active. We also find that UCK1, an isoform of UCK2, affects the turnover of UCK2 by influencing its cellular localization. Importantly, altered UCK2 levels through the mTORC1-CTLH E3 pathway affect pyrimidine salvage and the efficacy of pyrimidine analog prodrugs. Therefore, mTORC1-CTLH E3-mediated degradation of UCK2 adds another layer of complexity to mTORC1's role in regulating pyrimidine metabolism.
    Keywords:  CP: Metabolism; CP: Molecular biology; CTLH; UCK2; WDR26; YPEL5; degradomics; mTOR; mTORC1; pyrimidine; pyrimidine salvage; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2024.115179
  14. bioRxiv. 2025 Jan 04. pii: 2024.12.30.630811. [Epub ahead of print]
    40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion.
    Hironao Wakabayashi, Mingyi Zhu, Elizabeth J Grayhack, David H Mathews, Dmitri N Ermolenko.
      During eukaryotic translation initiation, the small (40S) ribosomal subunit is recruited to the 5' cap and subsequently scans the 5' untranslated region (5' UTR) of mRNA in search of the start codon. The molecular mechanism of mRNA scanning remains unclear. Here, using GFP reporters in Saccharomyces cerevisiae cells, we show that order-of-magnitude variations in the lengths of unstructured 5' UTRs have a modest effect on protein synthesis. These observations indicate that mRNA scanning is not rate limiting in yeast cells. Conversely, the presence of secondary structures in the 5' UTR strongly inhibits translation. Loss-of-function mutations in translational RNA helicases eIF4A and Ded1, as well as mutations in other initiation factors implicated in mRNA scanning, namely eIF4G, eIF4B, eIF3g and eIF3i, produced a similar decrease in translation of GFP reporters with short and long unstructured 5' UTRs. As expected, mutations in Ded1, eIF4B and eIF3i severely diminished translation of the reporters with structured 5' UTRs. Evidently, while RNA helicases eIF4A and Ded1 facilitate 40S recruitment and secondary structure unwinding, they are not rate-limiting for the 40S movement along the 5' UTR. Hence, our data indicate that, instead of helicase-driven translocation, one-dimensional diffusion predominately drives mRNA scanning by the 40S subunits in yeast cells.
    DOI:  https://doi.org/10.1101/2024.12.30.630811
  15. PNAS Nexus. 2025 Jan;4(1): pgae574
    Discovering genetic modulators of the protein homeostasis system through multilevel analysis.
    Vishal Sarsani, Berent Aldikacti, Tingting Zhao, Shai He, Peter Chien, Patrick Flaherty.
      Every protein progresses through a natural lifecycle from birth to maturation to death; this process is coordinated by the protein homeostasis system. Environmental or physiological conditions trigger pathways that maintain the homeostasis of the proteome. An open question is how these pathways are modulated to respond to the many stresses that an organism encounters during its lifetime. To address this question, we tested how the fitness landscape changes in response to environmental and genetic perturbations using directed and massively parallel transposon mutagenesis in Caulobacter crescentus. We developed a general computational pipeline for the analysis of gene-by-environment interactions in transposon mutagenesis experiments. This pipeline uses a combination of general linear models, statistical knockoffs, and a nonparametric Bayesian statistical model to identify essential genetic network components that are shared across environmental perturbations. This analysis allows us to quantify the similarity of proteotoxic environmental perturbations from the perspective of the fitness landscape. We find that essential genes vary more by genetic background than by environmental conditions, with limited overlap among mutant strains targeting different facets of the protein homeostasis system. We also identified 146 unique fitness determinants across different strains, with 19 genes common to at least two strains, showing varying resilience to proteotoxic stresses. Experiments exposing cells to a combination of genetic perturbations and dual environmental stressors show that perturbations that are quantitatively dissimilar from the perspective of the fitness landscape are likely to have a synergistic effect on the growth defect.
    Keywords:  conditionally essential networks; fitness; proteotoxic stress; transposon mutagenesis
    DOI:  https://doi.org/10.1093/pnasnexus/pgae574
  16. ACS Pharmacol Transl Sci. 2025 Jan 10. 8(1): 21-35
    Activation of the 26S Proteasome to Reduce Proteotoxic Stress and Improve the Efficacy of PROTACs.
    Jindrich Sedlacek.
      The 26S proteasome degrades the majority of cellular proteins and affects all aspects of cellular life. Therefore, the 26S proteasome abundance, proper assembly, and activity in different life contexts need to be precisely controlled. Impaired proteasome activity is considered a causative factor in several serious disorders. Recent advances in proteasome biology have revealed that the proteasome can be activated by different factors or small molecules. Thus, activated ubiquitin-dependent proteasome degradation has effects such as extending the lifespan in different models, preventing the accumulation of protein aggregates, and reducing their negative impact on cells. Increased 26S proteasome-mediated degradation reduces proteotoxic stress and can potentially improve the efficacy of engineered degraders, such as PROTACs, particularly in situations characterized by proteasome malfunction. Here, emerging ideas and recent insights into the pharmacological activation of the proteasome at the transcriptional and posttranslational levels are summarized.
    DOI:  https://doi.org/10.1021/acsptsci.4c00408
  17. Mol Ther. 2025 Jan 10. pii: S1525-0016(25)00008-5. [Epub ahead of print]
    Artificial enforcement of the unfolded protein response (UPR) reduces disease features in multiple preclinical models of ALS/FTD.
    Vicente Valenzuela, Daniela Becerra, José I Astorga, Matías Fuentealba, Guillermo Diaz, Leslie Bargsted, Carlos Chacón, Alexis Martinez, Romina Gozalvo, Kasey Jackson, Vania Morales, Macarena Las Heras, Giovanni Tamburini, Leonard Petrucelli, Pablo Sardi, Lars Plate, Claudio Hetz.
      Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) are part of a spectrum of diseases that share several causative genes, resulting in a combinatory of motor and cognitive symptoms and abnormal protein aggregation. Multiple unbiased studies have revealed that proteostasis impairment at the level of the endoplasmic reticulum (ER) is a transversal pathogenic feature of ALS/FTD. The transcription factor XBP1s is a master regulator of the unfolded protein response (UPR), the main adaptive pathway to cope with ER stress. Here we provide evidence of suboptimal activation of the UPR in ALS/FTD models under experimental ER stress. To artificially engage the UPR, we intracerebroventricularly administrated adeno-associated viruses (AAV) to express the active form of XBP1 (XBP1s) in the nervous system of ALS/FTD models. XBP1s expression improved motor performance and extended life span of mutant SOD1 mice, associated with reduced protein aggregation. AAV-XBP1s administration also attenuated disease progression in models of TDP-43 and C9orf72 pathogenesis. Proteomic profiling of spinal cord tissue revealed that XBP1s overexpression improved proteostasis and modulated the expression of a cluster of synaptic and cell morphology proteins. Our results suggest that strategies to improve ER proteostasis may serve as a pan-therapeutic strategy to treat ALS/FTD.
    DOI:  https://doi.org/10.1016/j.ymthe.2025.01.004
  18. iScience. 2025 Jan 17. 28(1): 111550
    Proteasome inhibition induces microtubule-dependent changes in nuclear morphology.
    Sourabh Sengupta, Abdullah Bashar Sami, Jesse C Gatlin, Daniel L Levy.
      Cancers and neurodegenerative disorders are associated with both disrupted proteostasis and altered nuclear morphology. Determining if changes in nuclear morphology contribute to pathology requires an understanding of the underlying mechanisms, which are difficult to elucidate in cells where pleiotropic effects of altering proteostasis might indirectly influence nuclear morphology. To investigate direct effects, we studied nuclei assembled in Xenopus egg extract where potentially confounding effects of transcription, translation, cell cycle progression, and actin dynamics are absent. We report that proteasome inhibition causes acute microtubule-dependent changes in nuclear morphology and stability and altered microtubule dynamics and organization. Proteomic analysis of proteasome-inhibited extracts identified an increased abundance of microtubule nucleator TubGCP6, and TubGCP6 depletion partially rescued nuclear morphology. Key results were confirmed in HeLa cells. We propose that accumulation of TubGCP6 leads to altered microtubule dynamics proximal to the nucleus, producing forces that deform the nucleus and impact nuclear morphology and integrity.
    Keywords:  Biological sciences; Cell biology; Functional aspects of cell biology
    DOI:  https://doi.org/10.1016/j.isci.2024.111550
  19. Nat Commun. 2025 Jan 13. 16(1): 629
    A truncated variant of the ribosome-associated trigger factor specifically contributes to plant chloroplast ribosome biogenesis.
    Fabian Ries, Jasmin Gorlt, Sabrina Kaiser, Vanessa Scherer, Charlotte Seydel, Sandra Nguyen, Andreas Klingl, Julia Legen, Christian Schmitz-Linneweber, Hinrik Plaggenborg, Jediael Z Y Ng, Dennis Wiens, Georg K A Hochberg, Markus Räschle, Torsten Möhlmann, David Scheuring, Felix Willmund.
      Molecular chaperones are essential throughout a protein's life and act already during protein synthesis. Bacteria and chloroplasts of plant cells share the ribosome-associated chaperone trigger factor (Tig1 in plastids), facilitating maturation of emerging nascent polypeptides. While typical trigger factor chaperones employ three domains for their task, the here described truncated form, Tig2, contains just the ribosome binding domain. Tig2 is widely present in green plants and appears to have acquired an entirely different task than co-translational nascent polypeptide folding. Tig2 deletion results in remarkable leaf developmental defects of cold-exposed Arabidopsis thaliana plants and specific defects in plastidic ribosomes. Our data indicate that Tig2 functions during ribosome biogenesis by promoting the maturation of the large subunit. We hypothesize that Tig2 binding to the ribosomal tunnel-exit surface aids protecting this sensitive surface during assembly. Tig2 illustrates a fascinating concept of how a chaperone domain evolved individually, serving a completely different molecular task.
    DOI:  https://doi.org/10.1038/s41467-025-55813-1
  20. Dev Cell. 2025 Jan 13. pii: S1534-5807(24)00777-9. [Epub ahead of print]
    The regulatory landscape of 5' UTRs in translational control during zebrafish embryogenesis.
    Madalena M Reimão-Pinto, Sebastian M Castillo-Hair, Georg Seelig, Alexander F Schier.
      The 5' UTRs of mRNAs are critical for translation regulation during development, but their in vivo regulatory features are poorly characterized. Here, we report the regulatory landscape of 5' UTRs during early zebrafish embryogenesis using a massively parallel reporter assay of 18,154 sequences coupled to polysome profiling. We found that the 5' UTR suffices to confer temporal dynamics to translation initiation and identified 86 motifs enriched in 5' UTRs with distinct ribosome recruitment capabilities. A quantitative deep learning model, Danio Optimus 5-Prime (DaniO5P), identified a combined role for 5' UTR length, translation initiation site context, upstream AUGs, and sequence motifs on ribosome recruitment. DaniO5P predicts the activities of maternal and zygotic 5' UTR isoforms and indicates that modulating 5' UTR length and motif grammar contributes to translation initiation dynamics. This study provides a first quantitative model of 5' UTR-based translation regulation in development and lays the foundation for identifying the underlying molecular effectors.
    Keywords:  5′ UTR; deep learning model; massively parallel reporter assay; translation initiation control; zebrafish embryogenesis
    DOI:  https://doi.org/10.1016/j.devcel.2024.12.038
  21. Commun Biol. 2025 Jan 15. 8(1): 55
    Loss of correlated proteasomal subunit expression selectively promotes the 20SHigh state which underlies luminal breast tumorigenicity.
    Rangapriya Sundararajan, Shubhada R Hegde, Ashish Kumar Panda, Joel Christie, Nikhil Gadewal, Prasanna Venkatraman.
      Why cancer cells disproportionately accumulate polyubiquitinated proteotoxic proteins despite high proteasomal activity is an outstanding question. While mis-regulated ubiquitination is a contributing factor, here we show that a structurally-perturbed and sub-optimally functioning proteasome is at the core of altered proteostasis in tumors. By integrating the gene coexpression signatures of proteasomal subunits in breast cancer (BrCa) patient tissues with the atomistic details of 26S holocomplex, we find that the transcriptional deregulation induced-stoichiometric imbalances perpetuate with disease severity. As seen in luminal BrCa cell lines, this imbalance limits the number of double-capped 19S-20S-19S holocomplexes (30S) formed and promotes free 20S catalytic core accumulation that is widely-believed to confer survival advantage to tumors. By retaining connectivity with key tumor 19S:20S interface nodes, the PSMD9 19S subunit chaperone emerges as a crucial regulator of 26S/30S:20S ratios sustaining tumor cell proteasome function. Disrupting this connectivity by depleting PSMD9 in MCF7 cells introduces structural anomalies in the proteasome, and shifts dependence from 20SHigh to a deregulated 26SHigh state invoking anti-tumor responses which opens up clinically-relevant therapeutic possibilities.
    DOI:  https://doi.org/10.1038/s42003-024-07432-7
  22. Nat Metab. 2025 Jan 10.
    The pentose phosphate pathway controls oxidative protein folding and prevents ferroptosis in chondrocytes.
    Shauni Loopmans, Katerina Rohlenova, Thomas van Brussel, Ingrid Stockmans, Karen Moermans, Nicolas Peredo, Peter Carmeliet, Diether Lambrechts, Steve Stegen, Geert Carmeliet.
      Bone lengthening and fracture repair depend on the anabolic properties of chondrocytes that function in an avascular milieu. The limited supply of oxygen and nutrients calls into question how biosynthesis and redox homeostasis are guaranteed. Here we show that glucose metabolism by the pentose phosphate pathway (PPP) is essential for endochondral ossification. Loss of glucose-6-phosphate dehydrogenase in chondrocytes does not affect cell proliferation because reversal of the non-oxidative PPP produces ribose-5-phosphate. However, the decreased NADPH production reduces glutathione recycling, resulting in decreased protection against the reactive oxygen species (ROS) produced during oxidative protein folding. The disturbed proteostasis activates the unfolded protein response and protein degradation. Moreover, the oxidative stress induces ferroptosis, which, together with altered matrix properties, results in a chondrodysplasia phenotype. Collectively, these data show that in hypoxia, the PPP is crucial to produce reducing power that confines ROS generated by oxidative protein folding and thereby controls proteostasis and prevents ferroptosis.
    DOI:  https://doi.org/10.1038/s42255-024-01187-5
  23. Trends Cell Biol. 2025 Jan 13. pii: S0962-8924(24)00281-2. [Epub ahead of print]
    Reacting to reductive stress at the mitochondrial import gate.
    Sylvie Callegari.
      A byproduct of mitochondrial energy production is the generation of reactive oxygen species (ROS). Too much ROS is toxic, but ROS deficiency is equally deleterious (reductive stress). In a recent study, McMinimy et al. uncovered a ubiquitin proteasome-mediated mechanism at the translocase of the outer membrane (TOM) complex, which senses ROS depletion and adjusts mitochondrial protein import accordingly.
    Keywords:  TOM complex; mitochondrial import; proteasome; reactive oxygen species; reductive stress; ubiquitin
    DOI:  https://doi.org/10.1016/j.tcb.2024.12.013
  24. J Dermatol Sci. 2025 Jan 08. pii: S0923-1811(25)00001-5. [Epub ahead of print]
    Unfolded protein response modulates Tyrosinase levels and melanin production during melanogenesis.
    Akari Yamazaki, Issei Omura, Yasunao Kamikawa, Michihiro Hide, Akio Tanaka, Masayuki Kaneko, Kazunori Imaizumi, Atsushi Saito.
       BACKGROUND: Melanocytes protect the body from ultraviolet radiation by synthesizing melanin. Tyrosinase, a key enzyme in melanin production, accumulates in the endoplasmic reticulum (ER) during melanin synthesis, potentially causing ER stress. However, regulating ER function for melanin synthesis has been less studied than controlling Tyrosinase activity.
    OBJECTIVE: This study investigates the regulatory mechanisms of melanin production, focusing on ER stress and the ER stress-induced response.
    METHODS: B16 mouse melanoma cells induced to undergo melanogenesis were treated with unfolded protein response (UPR) inhibitors or chemical chaperones, and their effects on melanogenesis were analyzed.
    RESULTS: During melanogenesis in B16 cells stimulated by alpha-melanocyte-stimulating hormone (α-MSH), ER stress and UPR activation occurred, accompanied by increased Tyrosinase protein. Reducing IRE1 and ATF6 branch activity lowered melanin levels, while chemical chaperone treatment restored melanin production and increased Tyrosinase levels.
    CONCLUSION: UPR activation, linked to elevated Tyrosinase levels, influences melanin production during melanogenesis. Modulating UPR can regulate melanin synthesis and provides a potential new approach for treating pigmentation disorders.
    Keywords:  Endoplasmic reticulum stress; Melanin production; Pigmentation disorders; Tyrosinase; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.jdermsci.2025.01.001
  25. Commun Chem. 2025 Jan 15. 8(1): 12
    Enantioselective OTUD7B fragment discovery through chemoproteomics screening and high-throughput optimisation.
    Aini Vuorinen, Cassandra R Kennedy, Katherine A McPhie, William McCarthy, Jonathan Pettinger, J Mark Skehel, David House, Jacob T Bush, Katrin Rittinger.
      Deubiquitinating enzymes (DUBs) are key regulators of cellular homoeostasis, and their dysregulation is associated with several human diseases. The ovarian tumour protease (OTU) family of DUBs are biochemically well-characterised and of therapeutic interest, yet only a few tool compounds exist to study their cellular function and therapeutic potential. Here we present a chemoproteomics fragment screening platform for identifying novel DUB-specific hit matter, that combines activity-based protein profiling with high-throughput chemistry direct-to-biology optimisation to enable rapid elaboration of initial fragment hits against OTU DUBs. Applying these approaches, we identify an enantioselective covalent fragment for OTUD7B, and validate it using chemoproteomics and biochemical DUB activity assays.
    DOI:  https://doi.org/10.1038/s42004-025-01410-8
  26. Structure. 2025 Jan 04. pii: S0969-2126(24)00548-3. [Epub ahead of print]
    Structure of E6AP in complex with HPV16-E6 and p53 reveals a novel ordered domain important for E3 ligase activation.
    Sebastian Kenny, Shalini Iyer, Clinton A Gabel, Natalia Tegenfeldt, Andrew G DeMarco, Mark C Hall, Leifu Chang, V Jo Davisson, Scott Vande Pol, Chittaranjan Das.
      High-risk human papillomavirus E6 oncoprotein is a model system for the recognition and degradation of cellular p53 tumor suppressor protein. There remains a gap in the understanding of the ubiquitin transfer reaction, including placement of the E6AP catalytic HECT domain of the ligase concerning the p53 substrate and how E6 itself is protected from ubiquitination. We determined the cryoelectron microscopy (cryo-EM) structure of the E6AP/E6/p53 complex, related the structure to in vivo modeling of the tri-molecular complex, and identified structural interactions associated with activation of the ubiquitin ligase function. The structure reveals that the N-terminal ordered domain (NOD) in E6AP has a terminal alpha helix that mediates the interaction of the NOD with the HECT domain of E6AP and protects the HPV-E6 protein from ubiquitination. In addition, this NOD helix is required for E6AP ligase function by contributing to the affinity of the E6-E6AP association, modulating E6 substrate recognition, while displacing UbcH7.
    Keywords:  E3 ligase; E6AP; HPV; cryo-EM; ligase activation; targeted degradation; ubiquitin; viral factor; yeast hybrid
    DOI:  https://doi.org/10.1016/j.str.2024.12.013
  27. RSC Chem Biol. 2025 Jan 15.
    Macrocyclic peptides as a new class of targeted protein degraders.
    Xuefei Jing, Joel P Mackay, Toby Passioura.
      Targeted protein degraders, in the form of proteolysis targeting chimaeras (PROTACs) and molecular glues, leverage the ubiquitin-proteasome system to catalytically degrade specific target proteins of interest. Because such molecules can be extremely potent, they have attracted considerable attention as a therapeutic modality in recent years. However, while targeted degraders have great potential, they are likely to face many of the same challenges as more traditional small molecules when it comes to their development as therapeutics. In particular, existing targeted degrader design is largely only applicable to the same set of protein targets as traditional small molecules (i.e., ∼15% of the human proteome). Here, we consider the potential of macrocyclic peptides to overcome this limitation. Such molecules possess several features that make them well-suited for the role, including the ability to induce the formation of ternary protein complexes that can involve relatively flat surfaces and their structural commonality with E3 ligase-recruiting peptide degrons. For these reasons, macrocyclic peptides provide the opportunity both to broaden the number of targets accessible to degrader activity and to broaden the number of E3 ligases that can be harnessed to mediate that activity.
    DOI:  https://doi.org/10.1039/d4cb00199k
  28. Nature. 2025 Jan 15.
    De novo designed proteins neutralize lethal snake venom toxins.
    Susana Vázquez Torres, Melisa Benard Valle, Stephen P Mackessy, Stefanie K Menzies, Nicholas R Casewell, Shirin Ahmadi, Nick J Burlet, Edin Muratspahić, Isaac Sappington, Max D Overath, Esperanza Rivera-de-Torre, Jann Ledergerber, Andreas H Laustsen, Kim Boddum, Asim K Bera, Alex Kang, Evans Brackenbrough, Iara A Cardoso, Edouard P Crittenden, Rebecca J Edge, Justin Decarreau, Robert J Ragotte, Arvind S Pillai, Mohamad Abedi, Hannah L Han, Stacey R Gerben, Analisa Murray, Rebecca Skotheim, Lynda Stuart, Lance Stewart, Thomas J A Fryer, Timothy P Jenkins, David Baker.
      Snakebite envenoming remains a devastating and neglected tropical disease, claiming over 100,000 lives annually and causing severe complications and long-lasting disabilities for many more1,2. Three-finger toxins (3FTx) are highly toxic components of elapid snake venoms that can cause diverse pathologies, including severe tissue damage3 and inhibition of nicotinic acetylcholine receptors, resulting in life-threatening neurotoxicity4. At present, the only available treatments for snakebites consist of polyclonal antibodies derived from the plasma of immunized animals, which have high cost and limited efficacy against 3FTxs5-7. Here we used deep learning methods to de novo design proteins to bind short-chain and long-chain α-neurotoxins and cytotoxins from the 3FTx family. With limited experimental screening, we obtained protein designs with remarkable thermal stability, high binding affinity and near-atomic-level agreement with the computational models. The designed proteins effectively neutralized all three 3FTx subfamilies in vitro and protected mice from a lethal neurotoxin challenge. Such potent, stable and readily manufacturable toxin-neutralizing proteins could provide the basis for safer, cost-effective and widely accessible next-generation antivenom therapeutics. Beyond snakebite, our results highlight how computational design could help democratize therapeutic discovery, particularly in resource-limited settings, by substantially reducing costs and resource requirements for the development of therapies for neglected tropical diseases.
    DOI:  https://doi.org/10.1038/s41586-024-08393-x
  29. J Biol Chem. 2025 Jan 09. pii: S0021-9258(25)00018-3. [Epub ahead of print] 108171
    Autophagy related 7 (ATG7) regulates food intake and liver health during asparaginase exposure.
    Brian A Zalma, Maria Ibrahim, Flavio C Rodriguez-Polanco, Chintan T Bhavsar, Esther M Rodriguez, Eduardo Cararo-Lopes, Saad A Farooq, Jordan L Levy, Ronald C Wek, Eileen White, Tracy G Anthony.
      Amino acid starvation by the chemotherapy agent asparaginase is a potent activator of the integrated stress response (ISR) in liver and can upregulate autophagy in some cell types. We hypothesized that autophagy related 7 (ATG7), a protein that is essential for autophagy and an ISR target gene, was necessary during exposure to asparaginase to maintain liver health. We knocked down Atg7 systemically (Atg7Δ/Δ) or in hepatocytes only (ls-Atg7KO) in mice before exposure to pegylated asparaginase for 5 d. Intact mice injected with asparaginase lost body weight due to reduced food intake and increased energy expenditure. Systemic Atg7 ablation reduced liver protein synthesis and increased liver injury in vehicle-injected mice, but did not further reduce liver protein synthesis, exacerbate steatosis or liver injury, or alter energy expenditure following 5 d asparaginase exposure. Atg7Δ/Δ mice were unexpectantly protected from asparaginase-induced anorexia and weight loss. This protection corresponded with reduced phosphorylation of hepatic GCN2 and blunted increases in ISR gene targets including growth differentiation factor 15 (GDF15), a negative regulator of food intake. Interestingly, asparaginase elevated serum GDF15 and reduced food intake in ls-Atg7KO mice, similar to intact mice. Liver triglycerides and production of the hepatokine fibroblast growth factor 21, another ISR gene target, were suppressed in asparaginase-exposed Atg7Δ/Δ and ls-Atg7KO mice. This work identifies a bidirectional relationship between autophagy and the ISR in the liver during asparaginase, affecting food intake and liver health.
    Keywords:  FGF21; GDF15; amino acid; body composition; eukaryotic initiation factor 2 (eIF2); gene expression; polysome profiling; protein synthesis; translation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108171
  30. Nat Commun. 2025 Jan 16. 16(1): 714
    Active learning-assisted directed evolution.
    Jason Yang, Ravi G Lal, James C Bowden, Raul Astudillo, Mikhail A Hameedi, Sukhvinder Kaur, Matthew Hill, Yisong Yue, Frances H Arnold.
      Directed evolution (DE) is a powerful tool to optimize protein fitness for a specific application. However, DE can be inefficient when mutations exhibit non-additive, or epistatic, behavior. Here, we present Active Learning-assisted Directed Evolution (ALDE), an iterative machine learning-assisted DE workflow that leverages uncertainty quantification to explore the search space of proteins more efficiently than current DE methods. We apply ALDE to an engineering landscape that is challenging for DE: optimization of five epistatic residues in the active site of an enzyme. In three rounds of wet-lab experimentation, we improve the yield of a desired product of a non-native cyclopropanation reaction from 12% to 93%. We also perform computational simulations on existing protein sequence-fitness datasets to support our argument that ALDE can be more effective than DE. Overall, ALDE is a practical and broadly applicable strategy to unlock improved protein engineering outcomes.
    DOI:  https://doi.org/10.1038/s41467-025-55987-8
  31. Science. 2025 Jan 16. eads0018
    Simulating 500 million years of evolution with a language model.
    Thomas Hayes, Roshan Rao, Halil Akin, Nicholas J Sofroniew, Deniz Oktay, Zeming Lin, Robert Verkuil, Vincent Q Tran, Jonathan Deaton, Marius Wiggert, Rohil Badkundri, Irhum Shafkat, Jun Gong, Alexander Derry, Raul S Molina, Neil Thomas, Yousuf A Khan, Chetan Mishra, Carolyn Kim, Liam J Bartie, Matthew Nemeth, Patrick D Hsu, Tom Sercu, Salvatore Candido, Alexander Rives.
      More than three billion years of evolution have produced an image of biology encoded into the space of natural proteins. Here we show that language models trained at scale on evolutionary data can generate functional proteins that are far away from known proteins. We present ESM3, a frontier multimodal generative language model that reasons over the sequence, structure, and function of proteins. ESM3 can follow complex prompts combining its modalities and is highly responsive to alignment to improve its fidelity. We have prompted ESM3 to generate fluorescent proteins. Among the generations that we synthesized, we found a bright fluorescent protein at a far distance (58% sequence identity) from known fluorescent proteins, which we estimate is equivalent to simulating five hundred million years of evolution.
    DOI:  https://doi.org/10.1126/science.ads0018
  32. EMBO Rep. 2025 Jan 16.
    Male sex determination maintains proteostasis and extends lifespan of daf-18/PTEN deficient C. elegans.
    Zhi Qu, Lu Zhang, Xue Yin, Fangzhou Dai, Wei Huang, Yutong Zhang, Dongyang Ran, Shanqing Zheng.
      Although females typically have a survival advantage, those with PTEN functional abnormalities face a higher risk of developing tumors than males. However, the differences in how each sex responds to PTEN dysfunction have rarely been studied. We use Caenorhabditis elegans to investigate how male and hermaphrodite worms respond to dysfunction of the PTEN homolog daf-18. Our study reveals that male worms can counterbalance the negative effects of daf-18 deficiency, resulting in longer adult lifespan. The survival advantage depends on the loss of DAF-18 protein phosphatase activity, while its lipid phosphatase activity is dispensable. The deficiency in DAF-18 protein phosphatase activity leads to the failure of dephosphorylation of the endoplasmic reticulum membrane protein C18E9.2/SEC62, causing increased levels of unfolded and aggregated proteins in hermaphrodites. In contrast, males maintain proteostasis through a UNC-23/NEF-mediated protein ubiquitination and degradation process, providing them with a survival advantage. We find that sex determination is a key factor in regulating the differential expression of unc-23 between sexes in response to daf-18 loss. These findings highlight the unique role of the male sex determination pathway in regulating protein degradation.
    Keywords:   C. elegans ; daf-18 ; unc-23 ; Longevity; Male Sex Determination
    DOI:  https://doi.org/10.1038/s44319-025-00368-x
  33. Nat Commun. 2025 Jan 13. 16(1): 621
    Regulation of stress granule maturation and dynamics by poly(ADP-ribose) interaction with PARP13.
    Shang-Jung Cheng, Temitope Gafaar, Jijin R A Kuttiyatveetil, Aleksandr Sverzhinsky, Carla Chen, Minghui Xu, Allison Lilley, John M Pascal, Anthony K L Leung.
      Non-covalent interactions of poly(ADP-ribose) (PAR) facilitate condensate formation, yet the impact of these interactions on condensate properties remains unclear. Here, we demonstrate that PAR-mediated interactions through PARP13, specifically the PARP13.2 isoform, are essential for modulating the dynamics of stress granules-a class of cytoplasmic condensates that form upon stress, including types frequently observed in cancers. Single amino acid mutations in PARP13, which reduce its PAR-binding activity, lead to the formation of smaller yet more numerous stress granules than observed in the wild-type. This fragmented stress granule phenotype is also apparent in PARP13 variants with cancer-associated single-nucleotide polymorphisms (SNPs) that disrupt PAR binding. Notably, this fragmented phenotype is conserved across a variety of stresses that trigger stress granule formation via diverse pathways. Furthermore, this PAR-binding mutant diminishes condensate dynamics and impedes fusion. Overall, our study uncovers the important role of PAR-protein interactions in stress granule dynamics and maturation, mediated through PARP13.
    DOI:  https://doi.org/10.1038/s41467-024-55666-0
  34. Cell Rep. 2025 Jan 13. pii: S2211-1247(24)01507-9. [Epub ahead of print]44(1): 115156
    The Golgi complex governs natural killer cell lytic granule positioning to promote directionality in cytotoxicity.
    Luis A Pedroza, Frederique van den Haak, Alexander Frumovitz, Evelyn Hernandez, Everardo Hegewisch-Solloa, Tabitha K Orange, Keri B Sheehan, Susan Prockop, Aaron Bodansky, Ivan K Chinn, James R Lupski, Jennifer E Posey, Emily M Mace, Yu Li, Jordan S Orange.
      Cytotoxic immune cells mediate precise attacks against diseased cells to maintain organismal health. Their operational unit of killing and host defense is lytic granules (LGs), which are specialized lysosomal-related organelles. Precision in cytotoxicity is achieved by converging the many LGs to the microtubule-organizing center (MTOC) and polarizing these to the diseased cell for secretion. We identify unappreciated intimate relationships between the Golgi, MTOC, and LGs after cytotoxic cell activation, as well as the trans-Golgin protein GCC2 on the LG surface. GCC2 serves to tether LGs to the Golgi following convergence, and both GCC2 and the Golgi are required for the persistence of convergence. GCC2 allows LGs to utilize the Golgi as a docking station preventing LG dispersion and innocent bystander killing in complex three-dimensional environments. We also identify GCC2 variants causing human natural killer cell deficiency, further emphasizing the importance of LG convergence and Golgi linkage in precision targeting for human immunity.
    Keywords:  CP: Immunology; GCC2; Golgi; Golgins; cancer; confocal microscopy; cytotoxicity; lytic granule convergence; natural killer cells
    DOI:  https://doi.org/10.1016/j.celrep.2024.115156
  35. Autophagy. 2025 Jan 15. 1-20
    The ER protein CANX (calnexin)-mediated autophagy protects against alzheimer disease.
    Hongtao Shen, Yuying Xie, Yan Wang, Yusheng Xie, Yongxiang Wang, Zhenyan Su, Laixi Zhao, Shi Yao, Xiaoling Cao, Jinglan Liang, Junrui Long, Rimei Zhong, Jinfeng Tang, Sijie Wang, Liangqing Zhang, Xiaojing Wang, Björn Stork, Lili Cui, Wenxian Wu.
      Although the relationship between macroautophagy/autophagy and Alzheimer disease (AD) is widely studied, the underlying mechanisms are poorly understood, especially the regulatory role of the initiation signaling of autophagy on AD. Here, we find that the ER transmembrane protein CANX (calnexin) is a novel interaction partner of the autophagy-inducing kinase ULK1 and is required for ULK1 recruitment to the ER under basal or starved conditions. Loss of CANX results in the inactivity of ULK1 kinase and inhibits autophagy flux. In the brains of people with AD and APP-PSEN1 mice, the interaction of CANX and ULK1 declines. In mice, the lack of CANX in hippocampal neurons causes the accumulation of autophagy receptors and neuron damage, which affects the cognitive function of C57BL/6 mice. Conversely, overexpression of CANX in hippocampal neurons enhances autophagy flux and partially contributes to improving cognitive function of APP-PSEN1 mice, but not the CANX variant lacking the interaction domain with ULK1. These findings reveal a novel role of CANX in autophagy activity and cognitive function by cooperating with ULK1.Abbreviation: AD: Alzheimer disease; APEX: ascorbate peroxidase; APP: amyloid beta precursor protein; APP-PSEN1 mice: amyloid beta precursor protein-presenilin 1 transgenic mice; ATG: autophagy related; Aβ: amyloid-β; BiFC: bimolecular fluorescence complementation; CANX: calnexin; EBSS: Earle's balanced salt solution; EM: electron microscopy; IP: immunopurification; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MWM: Morris water maze; PLA: proximity ligation assay; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62, sequestosome 1.
    Keywords:  Alzheimer disease; ER; ULK1; autophagy initiation; calnexin
    DOI:  https://doi.org/10.1080/15548627.2024.2447206
  36. Nat Chem Biol. 2025 Jan 13.
    Rapid discovery of cyclic peptide protein aggregation inhibitors by continuous selection.
    Linwei Yang, Jingwei Zhang, James S Andon, Lingjun Li, Tina Wang.
      Protein aggregates are associated with numerous diseases. Here we report a platform for the rapid phenotypic selection of protein aggregation inhibitors from genetically encoded cyclic peptide libraries in Escherichia coli based on phage-assisted continuous evolution (PACE). We developed a new PACE-compatible selection for protein aggregation inhibition and used it to identify cyclic peptides that suppress amyloid-β42 and human islet amyloid polypeptide aggregation. Additionally, we integrated a negative selection that removes false positives and off-target hits, greatly improving cyclic peptide selectivity. We show that selected inhibitors are active when chemically resynthesized in in vitro assays. Our platform provides a powerful approach for the rapid discovery of cyclic peptide inhibitors of protein aggregation and may serve as the basis for the future evolution of cyclic peptides with a broad spectrum of inhibitory activities.
    DOI:  https://doi.org/10.1038/s41589-024-01823-x
  37. J Biol Chem. 2025 Jan 08. pii: S0021-9258(25)00010-9. [Epub ahead of print] 108163
    Mutations in histones dysregulate copper homeostasis leading to defect in Sec61 dependent protein translocation mechanism in Saccharomyces cerevisiae.
    Santoshi Acharjee, Rajshree Pal, Smriti Anand, Prateeksha Thakur, Vandana Anjana, Ranu Singh, Mrittika Paul, Ashis Biswas, Raghuvir Singh Tomar.
      The translocation of proteins from the cytoplasm to the endoplasmic reticulum occurs via a conserved Sec61 protein channel. Previously, we reported that mutations in histones cause downregulation of a CUP1 copper metallothionein and copper exposure inhibits the activity of Sec61. However, the role of epigenetic dysregulation on the activity of channel is not clear. Identification of cellular factors regulating copper metabolism and Sec61 activity is needed as the dysregulation can cause human diseases. In this study, we elucidate the intricate relationship between copper homeostasis and Sec61-mediated protein translocation. Utilizing copper-sensitive yeast histone mutants exhibiting deficiencies in the expression of CUP1, we uncover a copper-specific impairment of the protein translocation process, causing a reduction in the maturation of secretory proteins. Our findings highlight the inhibitory effect of copper on both co-translational and post-translational protein translocations. We demonstrate that supplementation with a copper-specific chelator, or amino acids such as cysteine, histidine, and reduced glutathione, zinc and overexpression of CUP1 restores the translocation process and growth. This study, for the first time provides a functional insight on epigenetic and metabolic regulation of copper homeostasis in governing Sec61-dependent protein translocation process and may be useful to understand human disorders of copper metabolism.
    Keywords:  Sec61; copper homeostasis; endoplasmic reticulum; protein translocation
    DOI:  https://doi.org/10.1016/j.jbc.2025.108163
  38. Nature. 2025 Jan 15.
    Targeting protein-ligand neosurfaces with a generalizable deep learning tool.
    Anthony Marchand, Stephen Buckley, Arne Schneuing, Martin Pacesa, Maddalena Elia, Pablo Gainza, Evgenia Elizarova, Rebecca M Neeser, Pao-Wan Lee, Luc Reymond, Yangyang Miao, Leo Scheller, Sandrine Georgeon, Joseph Schmidt, Philippe Schwaller, Sebastian J Maerkl, Michael Bronstein, Bruno E Correia.
      Molecular recognition events between proteins drive biological processes in living systems1. However, higher levels of mechanistic regulation have emerged, in which protein-protein interactions are conditioned to small molecules2-5. Despite recent advances, computational tools for the design of new chemically induced protein interactions have remained a challenging task for the field6,7. Here we present a computational strategy for the design of proteins that target neosurfaces, that is, surfaces arising from protein-ligand complexes. To develop this strategy, we leveraged a geometric deep learning approach based on learned molecular surface representations8,9 and experimentally validated binders against three drug-bound protein complexes: Bcl2-venetoclax, DB3-progesterone and PDF1-actinonin. All binders demonstrated high affinities and accurate specificities, as assessed by mutational and structural characterization. Remarkably, surface fingerprints previously trained only on proteins could be applied to neosurfaces induced by interactions with small molecules, providing a powerful demonstration of generalizability that is uncommon in other deep learning approaches. We anticipate that such designed chemically induced protein interactions will have the potential to expand the sensing repertoire and the assembly of new synthetic pathways in engineered cells for innovative drug-controlled cell-based therapies10.
    DOI:  https://doi.org/10.1038/s41586-024-08435-4
  39. EMBO J. 2025 Jan 13.
    NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.
    Chris A Brosey, Runze Shen, John A Tainer.
      Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4's redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF's cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.
    Keywords:  Apoptosis-inducing Factor (AIF); CHCHD4/MIA40; OXPHOS; Small-angle X-ray Scattering (SAXS); X-ray Crystallography
    DOI:  https://doi.org/10.1038/s44318-024-00360-6
  40. Nat Metab. 2025 Jan 15.
    Autophagy regulator ATG5 preserves cerebellar function by safeguarding its glycolytic activity.
    Janine Tutas, Marianna Tolve, Ebru Özer-Yildiz, Lotte Ickert, Ines Klein, Quinn Silverman, Filip Liebsch, Frederik Dethloff, Patrick Giavalisco, Heike Endepols, Theodoros Georgomanolis, Bernd Neumaier, Alexander Drzezga, Guenter Schwarz, Bernard Thorens, Graziana Gatto, Christian Frezza, Natalia L Kononenko.
      Dysfunctions in autophagy, a cellular mechanism for breaking down components within lysosomes, often lead to neurodegeneration. The specific mechanisms underlying neuronal vulnerability due to autophagy dysfunction remain elusive. Here we show that autophagy contributes to cerebellar Purkinje cell (PC) survival by safeguarding their glycolytic activity. Outside the conventional housekeeping role, autophagy is also involved in the ATG5-mediated regulation of glucose transporter 2 (GLUT2) levels during cerebellar maturation. Autophagy-deficient PCs exhibit GLUT2 accumulation on the plasma membrane, along with increased glucose uptake and alterations in glycolysis. We identify lysophosphatidic acid and serine as glycolytic intermediates that trigger PC death and demonstrate that the deletion of GLUT2 in ATG5-deficient mice mitigates PC neurodegeneration and rescues their ataxic gait. Taken together, this work reveals a mechanism for regulating GLUT2 levels in neurons and provides insights into the neuroprotective role of autophagy by controlling glucose homeostasis in the brain.
    DOI:  https://doi.org/10.1038/s42255-024-01196-4
  41. J Am Chem Soc. 2025 Jan 13.
    Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation.
    Yishu Bao, Zhiyi Xu, Kai Cheng, Xiaojing Li, Fangke Chen, Dingdong Yuan, Fang Zhang, Audrey Run-Yu Che, Xiangze Zeng, Yuan-Di Zhao, Jiang Xia.
      A low-molecular-weight compound whose structure strikes a fine balance between hydrophobicity and hydrophilicity may form coacervates via liquid-liquid phase separation in an aqueous solution. These coacervates may encapsulate and convoy proteins across the plasma membrane into the cell. However, releasing the cargo from the vehicle to the cytosol is challenging. Here, we address this issue by designing phase-separating coacervates, which are disassembled by the bioorthogonal Staudinger reaction. We constructed and selected triphenylphosphine-based compounds that formed phase-separated coacervates in an aqueous solution. Reacting the coacervates with azides resulted in microdroplet dissolution, so they received the name Staudinger Reaction-Responsive Coacervates, SR-Coa. SR-Coa could encapsulate proteins, including antibodies, and translocate them across the plasma membrane into the cell. Further treatment of the cell with ethyl azidoacetate induced the cargo dispersion from the puncta to the cytosolic distribution. We showcased an application of the SR-Coa/ethyl azidoacetate system in facilitating the translocation of the EGFR/antibody complex into the cell, which induced EGFR degradation via the TRIM21-dependent pathway both in vitro and in vivo. Besides the membrane protein EGFR, this system could also degrade endogenous protein EZH2. Taken together, here we report a strategy of controlling molecular coacervates by a bioorthogonal reaction in the cell for cytosolic protein delivery and demonstrate its use in promoting targeted protein degradation via the proteasome-dependent pathway.
    DOI:  https://doi.org/10.1021/jacs.4c17054
  42. Biophys J. 2025 Jan 13. pii: S0006-3495(25)00003-7. [Epub ahead of print]
    Dense-core vesicles contain exosomes in secretory cells.
    Xin Wang, Gianvito Arpino, Ammar Mohseni, Christopher K E Bleck, Ling-Gang Wu.
      Dense-core vesicles (DCVs) are found in various types of cells, such as neurons, pancreatic β-cells, and chromaffin cells. These vesicles release transmitters, peptides, and hormones to regulate diverse functions, such as the stress response, immune response, behavior, and blood glucose levels. In traditional electron microscopy after chemical fixation, it is often reported that the dense cores occupy a portion of the vesicle towards the center and are surrounded by a clear halo. With electron microscopy following cryo-fixation in adrenal chromaffin cells, we report here that we did not observe halos, but dense cores filling up the entire vesicles suggesting that halos are likely the product of chemical fixation. More importantly, we observed that a fraction of DCVs contained 36-168 nm clear-core vesicles. A similar fraction of DCVs labeled with fluorescent false neurotransmitter FFN 511 or the dense-core matrix protein chromogranin A (CGA) were colocalized with fluorescently labeled or endogenous CD63 or ALIX, the membrane or lumen marker of ∼40-160 nm exosomes. These results suggest that DCVs contain exosomes. Since exosomes are generally thought to reside within multivesicular bodies in the cytosol and are released to the extracellular space to mediate diverse cell-to-cell communications, our findings suggest that dense-core vesicle fusion from many cell types is a new source for releasing exosomes to mediate intercellular communications. Given that dense-core vesicle fusion mediates many physiological functions, such as stress responses, immune responses, behavior regulation, and blood glucose regulation, exosome release from dense-core vesicle fusion might contribute to mediating these important functions.
    DOI:  https://doi.org/10.1016/j.bpj.2025.01.003
  43. Elife. 2025 Jan 15. pii: RP95842. [Epub ahead of print]13
    Endocytic recycling is central to circadian collagen fibrillogenesis and disrupted in fibrosis.
    Joan Chang, Adam Pickard, Jeremy A Herrera, Sarah O'Keefe, Richa Garva, Matthew Hartshorn, Anna Hoyle, Lewis Dingle, John Knox, Thomas A Jowitt, Madeleine Coy, Jason Wong, Adam Reid, Yinhui Lu, Cédric Zeltz, Rajamiyer V Venkateswaran, Patrick T Caswell, Stephen High, Donald Gullberg, Karl E Kadler.
      Collagen-I fibrillogenesis is crucial to health and development, where dysregulation is a hallmark of fibroproliferative diseases. Here, we show that collagen-I fibril assembly required a functional endocytic system that recycles collagen-I to assemble new fibrils. Endogenous collagen production was not required for fibrillogenesis if exogenous collagen was available, but the circadian-regulated vacuolar protein sorting (VPS) 33b and collagen-binding integrin α11 subunit were crucial to fibrillogenesis. Cells lacking VPS33B secrete soluble collagen-I protomers but were deficient in fibril formation, thus secretion and assembly are separately controlled. Overexpression of VPS33B led to loss of fibril rhythmicity and overabundance of fibrils, which was mediated through integrin α11β1. Endocytic recycling of collagen-I was enhanced in human fibroblasts isolated from idiopathic pulmonary fibrosis, where VPS33B and integrin α11 subunit were overexpressed at the fibrogenic front; this correlation between VPS33B, integrin α11 subunit, and abnormal collagen deposition was also observed in samples from patients with chronic skin wounds. In conclusion, our study showed that circadian-regulated endocytic recycling is central to homeostatic assembly of collagen fibrils and is disrupted in diseases.
    Keywords:  cell biology; collagen; endocytosis; fibrillogenesis; fibrosis; human; integrin; mouse
    DOI:  https://doi.org/10.7554/eLife.95842
  44. Cell Death Differ. 2025 Jan 11.
    Small protein ERSP encoded by LINC02870 promotes triple negative breast cancer progression via IRE1α/XBP1s activation.
    Xiaolu Wang, Qianqian Wang, Hong Wang, Guodi Cai, Yana An, Peiqing Liu, Huihao Zhou, Hong-Wu Chen, Shufeng Ji, Jiantao Ye, Junjian Wang.
      Clinical treatment options for triple-negative breast cancer (TNBC) are currently limited to chemotherapy because of a lack of effective therapeutic targets. Recent evidence suggests that long noncoding RNAs (lncRNAs) encode bioactive peptides or proteins, thereby playing noncanonical yet significant roles in regulating cellular processes. However, the potential of lncRNA-translated products in cancer progression remains largely unknown. In this study, we identified a previously undocumented small protein encoded by the lncRNA LINC02870. This protein is localized at the endoplasmic reticulum (ER) and participates in ER stress, thus, we named it the endoplasmic reticulum stress protein (ERSP). ERSP was highly expressed in TNBC tissues, and elevated LINC02870 content was correlated with poor prognosis in TNBC patients. Loss of ERSP inhibited TNBC growth and metastasis both in vitro and in vivo. The pro-oncogenic effects of ERSP could be attributed to its selective activation of the IRE1α/XBP1s branch. ERSP enhances the unfolded protein response (UPR) by interacting with XBP1s, facilitating the nuclear accumulation of XBP1s, thereby promoting the expression of ER stress-related genes. These findings highlight the regulatory role of the lncRNA-encoded protein ERSP in ER stress and suggest that it is a potential therapeutic target for TNBC.
    DOI:  https://doi.org/10.1038/s41418-025-01443-5
  45. Chem Rev. 2025 Jan 17.
    Protein-Based Degraders: From Chemical Biology Tools to Neo-Therapeutics.
    Lisha Ou, Mekedlawit T Setegne, Jeandele Elliot, Fangfang Shen, Laura M K Dassama.
      The nascent field of targeted protein degradation (TPD) could revolutionize biomedicine due to the ability of degrader molecules to selectively modulate disease-relevant proteins. A key limitation to the broad application of TPD is its dependence on small-molecule ligands to target proteins of interest. This leaves unstructured proteins or those lacking defined cavities for small-molecule binding out of the scope of many TPD technologies. The use of proteins, peptides, and nucleic acids (otherwise known as "biologics") as the protein-targeting moieties in degraders addresses this limitation. In the following sections, we provide a comprehensive and critical review of studies that have used proteins and peptides to mediate the degradation and hence the functional control of otherwise challenging disease-relevant protein targets. We describe existing platforms for protein/peptide-based ligand identification and the drug delivery systems that might be exploited for the delivery of biologic-based degraders. Throughout the Review, we underscore the successes, challenges, and opportunities of using protein-based degraders as chemical biology tools to spur discoveries, elucidate mechanisms, and act as a new therapeutic modality.
    DOI:  https://doi.org/10.1021/acs.chemrev.4c00595
  46. Autophagy. 2025 Jan 14. 1-19
    Structure of the human autophagy factor EPG5 and the molecular basis of its conserved mode of interaction with Atg8-family proteins.
    Yiu Wing Sunny Cheung, Sung-Eun Nam, Gage M J Fairlie, Karlton Scheu, Jennifer M Bui, Hannah R Shariati, Jörg Gsponer, Calvin K Yip.
      The multi-step macroautophagy/autophagy process ends with the cargo-laden autophagosome fusing with the lysosome to deliver the materials to be degraded. The metazoan-specific autophagy factor EPG5 plays a crucial role in this step by enforcing fusion specificity and preventing mistargeting. How EPG5 exerts its critical function and how its deficiency leads to diverse phenotypes of the rare multi-system disorder Vici syndrome are not fully understood. Here, we report the first structure of human EPG5 (HsEPG5) determined by cryo-EM and AlphaFold2 modeling. Our structure revealed that HsEPG5 is constructed from helical bundles analogous to tethering factors in membrane trafficking pathways but contains a unique protruding thumb domain positioned adjacent to the atypical tandem LIR motifs involved in interaction with the GABARAP subfamily of Atg8-family proteins. Our NMR spectroscopic, molecular dynamics simulations and AlphaFold modeling studies showed that the HsEPG5 tandem LIR motifs only bind the canonical LIR docking site (LDS) on GABARAP without engaging in multivalent interaction. Our co-immunoprecipitation analysis further indicated that full-length HsEPG5-GABARAP interaction is mediated primarily by LIR1. Finally, our biochemical affinity isolation, X-ray crystallographic analysis, affinity measurement, and AlphaFold modeling demonstrated that this mode of binding is observed between Caenorhabditis elegans EPG-5 and its Atg8-family proteins LGG-1 and LGG-2. Collectively our work generated novel insights into the structural properties of EPG5 and how it potentially engages with the autophagosome to confer fusion specificity.ABBREVIATIONS: ATG: autophagy related; CSP: chemical shift perturbation; eGFP: enhanced green fluoresent protein; EM: electron microscopy; EPG5: ectopic P-granules 5 autophagy tethering factor; GST: glutathione S-transferase; HP: hydrophobic pocket; HSQC: heteronuclear single-quantum correlation; ITC: isothermal titration calorimetry; LDS: LC3 docking site; LIR: LC3-interacting region; MD: molecular dynamics; NMR: nuclear magnetic resonance; TEV: tobacco etch virus.
    Keywords:  Autophagy; EPG5; NMR spectroscopy; Vici syndrome; cryo-EM
    DOI:  https://doi.org/10.1080/15548627.2024.2447213
  47. PLoS Biol. 2025 Jan;23(1): e3002984
    Multi-tissue characterization of the constitutive heterochromatin proteome in Drosophila identifies a link between satellite DNA organization and transposon repression.
    Ankita Chavan, Lena Skrutl, Federico Uliana, Melanie Pfister, Franziska Brändle, Laszlo Tirian, Delora Baptista, Dominik Handler, David Burke, Anna Sintsova, Pedro Beltrao, Julius Brennecke, Madhav Jagannathan.
      Noncoding satellite DNA repeats are abundant at the pericentromeric heterochromatin of eukaryotic chromosomes. During interphase, sequence-specific DNA-binding proteins cluster these repeats from multiple chromosomes into nuclear foci known as chromocenters. Despite the pivotal role of chromocenters in cellular processes like genome encapsulation and gene repression, the associated proteins remain incompletely characterized. Here, we use 2 satellite DNA-binding proteins, D1 and Prod, as baits to characterize the chromocenter-associated proteome in Drosophila embryos, ovaries, and testes through quantitative mass spectrometry. We identify D1- and Prod-associated proteins, including known heterochromatin proteins as well as proteins previously unlinked to satellite DNA or chromocenters, thereby laying the foundation for a comprehensive understanding of cellular functions enabled by satellite DNA repeats and their associated proteins. Interestingly, we find that multiple components of the transposon-silencing piRNA pathway are associated with D1 and Prod in embryos. Using genetics, transcriptomics, and small RNA profiling, we show that flies lacking D1 during embryogenesis exhibit transposon expression and gonadal atrophy as adults. We further demonstrate that this gonadal atrophy can be rescued by mutating the checkpoint kinase, Chk2, which mediates germ cell arrest in response to transposon mobilization. Thus, we reveal that a satellite DNA-binding protein functions during embryogenesis to silence transposons, in a manner that is heritable across later stages of development.
    DOI:  https://doi.org/10.1371/journal.pbio.3002984
  48. J Microsc. 2025 Jan 15.
    In situ quantification of ribosome number by electron tomography.
    Mounir El Hankouri, Marco Nousch, Aayush Poddar, Thomas Müller-Reichert, Gunar Fabig.
      Ribosomes, discovered in 1955 by George Palade, were initially described as small cytoplasmic particles preferentially associated with the endoplasmic reticulum (ER). Over the years, extensive research has focused on both the structure and function of ribosomes. However, a fundamental question - how many ribosomes are present within whole cells - has remained largely unaddressed. In this study, we developed a microscopic method to quantify the total number of ribosomes in hTERT-RPE-1 cells and in nematode cells from various tissues of Caenorhabditis elegans hermaphrodites. Using electron tomography of high-pressure frozen, freeze-substituted and resin-embedded samples, we determined that the ribosome number in hTERT-RPE-1 cells is in the same order of magnitude as biochemical measurements obtained via RNA capillary electrophoresis. As expected, control worms exhibited a higher number of ribosomes compared to RNA polymerase I A subunit (RPOA-1)-depleted worms in two out of three analysed tissue types. Our imaging-based approach complements established biochemical methods by enabling direct quantification of ribosome numbers in specific samples. This method offers a powerful tool for advancing our understanding of ribosome localisation and distribution in cells and tissues across diverse model systems.
    Keywords:  C. elegans; electron tomography; hTERT‐RPE‐1 cells; high‐pressure freezing; ribosome quantification; ribosomes
    DOI:  https://doi.org/10.1111/jmi.13380
  49. STAR Protoc. 2025 Jan 16. pii: S2666-1667(24)00742-1. [Epub ahead of print]6(1): 103577
    Protocol for cell image-based spatiotemporal proteomics in budding yeast.
    Athanasios Litsios, Myra Paz David Masinas, Helena Friesen, Charles Boone, Brenda J Andrews.
      The eukaryotic cell division cycle is a highly conserved process, featuring fluctuations in protein localization and abundance required for key cell cycle transitions. Here, we present a protocol for the spatiotemporal analysis of the proteome during the budding yeast cell division cycle using live-cell imaging. We describe steps for strain construction, cell cultivation, microscopy, and image analysis. Variations of this protocol can be applied for the spatiotemporal analysis of the proteome in different contexts, such as genetic and environmental perturbations. For complete details on the use and execution of this protocol, please refer to Litsios et al.1.
    Keywords:  Cell Biology; Microscopy; Single Cell; Systems biology
    DOI:  https://doi.org/10.1016/j.xpro.2024.103577
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