bims-proteo Biomed News
on Proteostasis
Issue of 2024–06–09
thirty-six papers selected by
Eric Chevet, INSERM
  1. The HERCulean task of recognizing, ubiquitinating, and shielding misfolded integral membrane proteins.
  2. eIF4F complex dynamics are important for the activation of the integrated stress response.
  3. eIF4E integrates into stress response.
  4. Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.
  5. Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.
  6. IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.
  7. Different ER-plasma membrane tethers play opposing roles in autophagy of the cortical ER.
  8. Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast.
  9. ATG16L1 induces the formation of phagophore-like membrane cups.
  10. Protein translation rate determines neocortical neuron fate.
  11. Depletion of cap-binding protein eIF4E dysregulates amino acid metabolic gene expression.
  12. ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.
  13. VCP regulates early tau seed amplification via specific cofactors.
  14. Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.
  15. Profiling Cellular Morphological Changes Induced by Dual-targeting PROTACs of Aurora Kinase and RNA-binding Protein YTHDF2.
  16. The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.
  17. Xbp1 targets canonical UPRER and non-canonical pathways in separate tissues to promote longevity.
  18. Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response.
  19. Non-canonical mRNA translation initiation in cell stress and cancer.
  20. The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT.
  21. Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.
  22. Targeted protein degradation systems to enhance Wnt signaling.
  23. A disease-associated gene desert directs macrophage inflammation through ETS2.
  24. Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance.
  25. The DNA damage response and autophagy during cancer development: an antagonistic pleiotropy entanglement.
  26. Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.
  27. Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.
  28. Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms.
  29. PPIA dictates NRF2 stability to promote lung cancer progression.
  30. The shaping of mRNA translation plasticity by RNA G-quadruplexes in cancer progression and therapy resistance.
  31. Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.
  32. The ribotoxic stress response drives UV-mediated cell death.
  33. Selective Removal of Denatured Proteins Using MOF Nanopores.
  34. The tidyomics ecosystem: Enhancing omic data analyses.
  35. Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.
  36. A targetable PRR11-DHODH axis drives ferroptosis- and temozolomide-resistance in glioblastoma.
  1. J Cell Biol. 2024 Jul 01. pii: e202405160. [Epub ahead of print]223(7):
    The HERCulean task of recognizing, ubiquitinating, and shielding misfolded integral membrane proteins.
    Christopher J Guerriero, Jeffrey L Brodsky.
      During ER-associated decay, unfolded membrane-resident proteins are targeted for removal and degradation by ubiquitin ligases whose identities and precise operations remain unclear. In this issue, Guerriero and Brodsky discuss new results from Kamada et al. (https://doi.org/10.1083/jcb.202308003) showing the clearance of misfolded CFTR by the E3 ligase HERC3.
    DOI:  https://doi.org/10.1083/jcb.202405160
  2. Mol Cell. 2024 Jun 06. pii: S1097-2765(24)00331-9. [Epub ahead of print]84(11): 2135-2151.e7
    eIF4F complex dynamics are important for the activation of the integrated stress response.
    Kyusik Q Kim, Ankanahalli N Nanjaraj Urs, Victor Lasehinde, Alison C Greenlaw, Benjamin H Hudson, Hani S Zaher.
      In response to stress, eukaryotes activate the integrated stress response (ISR) via phosphorylation of eIF2α to promote the translation of pro-survival effector genes, such as GCN4 in yeast. Complementing the ISR is the target of rapamycin (TOR) pathway, which regulates eIF4E function. Here, we probe translational control in the absence of eIF4E in Saccharomyces cerevisiae. Intriguingly, we find that loss of eIF4E leads to de-repression of GCN4 translation. In addition, we find that de-repression of GCN4 translation is accompanied by neither eIF2α phosphorylation nor reduction in initiator ternary complex (TC). Our data suggest that when eIF4E levels are depleted, GCN4 translation is de-repressed via a unique mechanism that may involve faster scanning by the small ribosome subunit due to increased local concentration of eIF4A. Overall, our findings suggest that relative levels of eIF4F components are key to ribosome dynamics and may play important roles in translational control of gene expression.
    Keywords:  Gcn4; TOR; eIF4A; eIF4E; eIF4F complex; initiation; integrated stress response; ribosome; translation
    DOI:  https://doi.org/10.1016/j.molcel.2024.04.016
  3. Mol Cell. 2024 Jun 06. pii: S1097-2765(24)00399-X. [Epub ahead of print]84(11): 2009-2010
    eIF4E integrates into stress response.
    Xincheng Wu, Shu-Bing Qian.
      In this issue, Diamond et al.1 and Kim et al.2 report that depletion of eIF4E leads to translational upregulation of GCN4, a key player in the integrated stress response, in an eIF2α phosphorylation-independent manner, suggesting a new mode of translational adaptation.
    DOI:  https://doi.org/10.1016/j.molcel.2024.05.006
  4. Proc Natl Acad Sci U S A. 2024 Jun 11. 121(24): e2316892121
    Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry.
    Natalie H Jones, Qiwen Liu, Linas Urnavicius, Noa E Dahan, Lauren E Vostal, Tarun M Kapoor.
      The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.
    Keywords:  AAA protein; activator; cryo-EM; mechanoenzyme; small molecule
    DOI:  https://doi.org/10.1073/pnas.2316892121
  5. bioRxiv. 2024 May 23. pii: 2024.05.21.595139. [Epub ahead of print]
    Autophagy suppression in DNA damaged cells occurs through a newly identified p53-proteasome-LC3 axis.
    D'Feau J Lieu, Molly K Crowder, Jordan R Kryza, Batcha Tamilselvam, Paul J Kaminski, Ik-Jung Kim, Yingxing Li, Eunji Jeong, Michidmaa Enkhbaatar, Henry Chen, Sophia B Son, Hanlin Mok, Kenneth A Bradley, Heidi Phillips, Steven R Blanke.
      Macroautophagy is thought to have a critical role in shaping and refining cellular proteostasis in eukaryotic cells recovering from DNA damage. Here, we report a mechanism by which autophagy is suppressed in cells exposed to bacterial toxin-, chemical-, or radiation-mediated sources of genotoxicity. Autophagy suppression is directly linked to cellular responses to DNA damage, and specifically the stabilization of the tumor suppressor p53, which is both required and sufficient for regulating the ubiquitination and proteasome-dependent reduction in cellular pools of microtubule-associated protein 1 light chain 3 (LC3A/B), a key precursor of autophagosome biogenesis and maturation, in both epithelial cells and an ex vivo organoid model. Our data indicate that suppression of autophagy, through a newly identified p53-proteasome-LC3 axis, is a conserved cellular response to multiple sources of genotoxicity. Such a mechanism could potentially be important for realigning proteostasis in cells undergoing DNA damage repair.
    DOI:  https://doi.org/10.1101/2024.05.21.595139
  6. Circulation. 2024 Jun 05.
    IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.
    Chao Li, Shiqian Li, Guangyu Zhang, Qinfeng Li, Weidan Song, Xiaoding Wang, Jane A Cook, Miesje van der Stoel, Bradley W Wright, Francisco Altamirano, Erica L Niewold, Jungsoo Han, Garrett Kimble, Pengfei Zhang, Xiang Luo, Hery Urra, Herman I May, Anwarul Ferdous, Xue-Nan Sun, Yingfeng Deng, Elina Ikonen, Claudio Hetz, Randal J Kaufman, Kezhong Zhang, Thomas G Gillette, Philipp E Scherer, Joseph A Hill, Jin Chen, Zhao V Wang.
       BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms.
    METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control.
    RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo.
    CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.
    Keywords:  EGFR protein, mouse; heart failure; hypertrophy; protein synthesis inhibitors
    DOI:  https://doi.org/10.1161/CIRCULATIONAHA.123.067606
  7. Proc Natl Acad Sci U S A. 2024 Jun 11. 121(24): e2321991121
    Different ER-plasma membrane tethers play opposing roles in autophagy of the cortical ER.
    Dongmei Liu, Hua Yuan, Shuliang Chen, Susan Ferro-Novick, Peter Novick.
      The endoplasmic reticulum (ER) undergoes degradation by selective macroautophagy (ER-phagy) in response to starvation or the accumulation of misfolded proteins within its lumen. In yeast, actin assembly at sites of contact between the cortical ER (cER) and endocytic pits acts to displace elements of the ER from their association with the plasma membrane (PM) so they can interact with the autophagosome assembly machinery near the vacuole. A collection of proteins tether the cER to the PM. Of these, Scs2/22 and Ist2 are required for cER-phagy, most likely through their roles in lipid transport, while deletion of the tricalbins, TCB1/2/3, bypasses those requirements. An artificial ER-PM tether blocks cER-phagy in both the wild type (WT) and a strain lacking endogenous tethers, supporting the importance of cER displacement from the PM. Scs2 and Ist2 can be cross-linked to the selective cER-phagy receptor, Atg40. The COPII cargo adaptor subunit, Lst1, associates with Atg40 and is required for cER-phagy. This requirement is also bypassed by deletion of the ER-PM tethers, suggesting a role for Lst1 prior to the displacement of the cER from the PM during cER-phagy. Although pexophagy and mitophagy also require actin assembly, deletion of ER-PM tethers does not bypass those requirements. We propose that within the context of rapamycin-induced cER-phagy, Scs2/22, Ist2, and Lst1 promote the local displacement of an element of the cER from the cortex, while Tcb1/2/3 act in opposition, anchoring the cER to the plasma membrane.
    Keywords:  autophagy; endoplasmic reticulum; lipid transfer; membrane tether
    DOI:  https://doi.org/10.1073/pnas.2321991121
  8. iScience. 2024 Jun 21. 27(6): 109810
    Atg45 is an autophagy receptor for glycogen, a non-preferred cargo of bulk autophagy in yeast.
    Takahiro Isoda, Eigo Takeda, Sachiko Hosokawa, Shukun Hotta-Ren, Yoshinori Ohsumi.
      The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
    Keywords:  Biomolecules; Cell biology; Glycobiology; Model organism
    DOI:  https://doi.org/10.1016/j.isci.2024.109810
  9. Nat Struct Mol Biol. 2024 Jun 04.
    ATG16L1 induces the formation of phagophore-like membrane cups.
    Jagan Mohan, Satish B Moparthi, Christine Girard-Blanc, Daniele Campisi, Stéphane Blanchard, Charlotte Nugues, Sowmya Rama, Audrey Salles, Esthel Pénard, Stéphane Vassilopoulos, Thomas Wollert.
      The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12-ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12-ATG5-ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.
    DOI:  https://doi.org/10.1038/s41594-024-01300-y
  10. Nat Commun. 2024 Jun 07. 15(1): 4879
    Protein translation rate determines neocortical neuron fate.
    Ekaterina Borisova, Andrew G Newman, Marta Couce Iglesias, Rike Dannenberg, Theres Schaub, Bo Qin, Alexandra Rusanova, Marisa Brockmann, Janina Koch, Marieatou Daniels, Paul Turko, Olaf Jahn, David R Kaplan, Marta Rosário, Takao Iwawaki, Christian M T Spahn, Christian Rosenmund, David Meierhofer, Matthew L Kraushar, Victor Tarabykin, Mateusz C Ambrozkiewicz.
      The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1α as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1α regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5'-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1α-safeguarded proteostasis serving as an essential regulator of brain development.
    DOI:  https://doi.org/10.1038/s41467-024-49198-w
  11. Mol Cell. 2024 Jun 06. pii: S1097-2765(24)00430-1. [Epub ahead of print]84(11): 2119-2134.e5
    Depletion of cap-binding protein eIF4E dysregulates amino acid metabolic gene expression.
    Paige D Diamond, Nicholas J McGlincy, Nicholas T Ingolia.
      Protein synthesis is metabolically costly and must be tightly coordinated with changing cellular needs and nutrient availability. The cap-binding protein eIF4E makes the earliest contact between mRNAs and the translation machinery, offering a key regulatory nexus. We acutely depleted this essential protein and found surprisingly modest effects on cell growth and recovery of protein synthesis. Paradoxically, impaired protein biosynthesis upregulated genes involved in the catabolism of aromatic amino acids simultaneously with the induction of the amino acid biosynthetic regulon driven by the integrated stress response factor GCN4. We further identified the translational control of Pho85 cyclin 5 (PCL5), a negative regulator of Gcn4, that provides a consistent protein-to-mRNA ratio under varied translation environments. This regulation depended in part on a uniquely long poly(A) tract in the PCL5 5' UTR and poly(A) binding protein. Collectively, these results highlight how eIF4E connects protein synthesis to metabolic gene regulation, uncovering mechanisms controlling translation during environmental challenges.
    Keywords:  amino acid metabolism; cap-binding protein; translation
    DOI:  https://doi.org/10.1016/j.molcel.2024.05.008
  12. J Cell Biol. 2024 Sep 02. pii: e202405025. [Epub ahead of print]223(9):
    ESCRT disruption provides evidence against trans-synaptic signaling via extracellular vesicles.
    Erica C Dresselhaus, Kathryn P Harris, Cassandra R Blanchette, Kate Koles, Steven J Del Signore, Matthew F Pescosolido, Biljana Ermanoska, Mark Rozencwaig, Rebecca C Soslowsky, Michael J Parisi, Bryan A Stewart, Timothy J Mosca, Avital A Rodal.
      Extracellular vesicles (EVs) are released by many cell types, including neurons, carrying cargoes involved in signaling and disease. It is unclear whether EVs promote intercellular signaling or serve primarily to dispose of unwanted materials. We show that loss of multivesicular endosome-generating endosomal sorting complex required for transport (ESCRT) machinery disrupts release of EV cargoes from Drosophila motor neurons. Surprisingly, ESCRT depletion does not affect the signaling activities of the EV cargo Synaptotagmin-4 (Syt4) and disrupts only some signaling activities of the EV cargo evenness interrupted (Evi). Thus, these cargoes may not require intercellular transfer via EVs, and instead may be conventionally secreted or function cell-autonomously in the neuron. We find that EVs are phagocytosed by glia and muscles, and that ESCRT disruption causes compensatory autophagy in presynaptic neurons, suggesting that EVs are one of several redundant mechanisms to remove cargoes from synapses. Our results suggest that synaptic EV release serves primarily as a proteostatic mechanism for certain cargoes.
    DOI:  https://doi.org/10.1083/jcb.202405025
  13. Res Sq. 2024 May 22. pii: rs.3.rs-4307848. [Epub ahead of print]
    VCP regulates early tau seed amplification via specific cofactors.
    Sushobhna Batra, Jaime Iii Vaquer-Alicea, Clarissa Valdez, Skyler P Taylor, Victor A Manon, Anthony R Vega, Omar M Kashmer, Sourav Kolay, Andrew Lemoff, Nigel J Cairns, Charles L White, Marc I Diamond.
      Background Neurodegenerative tauopathies may progress based on seeding by pathological tau assemblies, whereby an aggregate is released from one cell, gains entry to an adjacent or connected cell, and serves as a specific template for its own replication in the cytoplasm. In vitro seeding reactions typically take days, yet seeding into the complex cytoplasmic milieu happens within hours, implicating a machinery with unknown players that controls this process in the acute phase. Methods We used proximity labeling to identify factors that control seed amplification within 5h of seed exposure. We fused split-APEX2 to the C-terminus of tau repeat domain (RD) to reconstitute peroxidase activity 5h after seeded intracellular tau aggregation. Valosin containing protein (VCP/p97) was the top hit. VCP harbors dominant mutations that underlie two neurodegenerative diseases, multisystem proteinopathy and vacuolar tauopathy, but its mechanistic role is unclear. We used immortalized cells and human neurons to study the effects of VCP on tau seeding. We exposed cells to fibrils or brain homogenates in cell culture media and measured effects on uptake and induction of intracellular tau aggregation following various genetic and chemical manipulations of VCP. Results VCP knockdown reduced tau seeding. Chemical inhibitors had opposing effects on aggregation in HEK293T tau biosensor cells and human neurons alike: ML-240 increased seeding efficiency, whereas NMS-873 decreased it. The inhibitors were effective only when administered within 8h of seed exposure, indicating a role for VCP early in seed processing. We screened 30 VCP co-factors in HEK293T biosensor cells by genetic knockout or knockdown. Reduction of ATXN3, NSFL1C, UBE4B, NGLY1, and OTUB1 decreased tau seeding, as did NPLOC4, which also uniquely increased soluble tau levels. By contrast, reduction of FAF2 increased tau seeding. Conclusions Divergent effects on tau seeding of chemical inhibitors and cofactor reduction indicate that VCP regulates this process. This is consistent with a dedicated cytoplasmic processing complex based on VCP that directs seeds acutely towards degradation vs. amplification.
    DOI:  https://doi.org/10.21203/rs.3.rs-4307848/v1
  14. Elife. 2024 Jun 04. pii: RP92189. [Epub ahead of print]12
    Syntaxin 17 recruitment to mature autophagosomes is temporally regulated by PI4P accumulation.
    Saori Shinoda, Yuji Sakai, Takahide Matsui, Masaaki Uematsu, Ikuko Koyama-Honda, Jun-Ichi Sakamaki, Hayashi Yamamoto, Noboru Mizushima.
      During macroautophagy, cytoplasmic constituents are engulfed by autophagosomes. Lysosomes fuse with closed autophagosomes but not with unclosed intermediate structures. This is achieved in part by the late recruitment of the autophagosomal SNARE syntaxin 17 (STX17) to mature autophagosomes. However, how STX17 recognizes autophagosome maturation is not known. Here, we show that this temporally regulated recruitment of STX17 depends on the positively charged C-terminal region of STX17. Consistent with this finding, mature autophagosomes are more negatively charged compared with unclosed intermediate structures. This electrostatic maturation of autophagosomes is likely driven by the accumulation of phosphatidylinositol 4-phosphate (PI4P) in the autophagosomal membrane. Accordingly, dephosphorylation of autophagosomal PI4P prevents the association of STX17 to autophagosomes. Furthermore, molecular dynamics simulations support PI4P-dependent membrane insertion of the transmembrane helices of STX17. Based on these findings, we propose a model in which STX17 recruitment to mature autophagosomes is temporally regulated by a PI4P-driven change in the surface charge of autophagosomes.
    Keywords:  SNARE; autophagosome; autophagy; cell biology; human; membrane charge; mouse; phosphatidylinositol 4-phosphate; syntaxin 17
    DOI:  https://doi.org/10.7554/eLife.92189
  15. Chembiochem. 2024 Jun 04. e202400183
    Profiling Cellular Morphological Changes Induced by Dual-targeting PROTACs of Aurora Kinase and RNA-binding Protein YTHDF2.
    Georg L Goebel, Nicole Giannino, Philipp Lampe, Xiaqiu Qiu, Jeffrey Schloßhauer, Jochen Imig, Sonja Sievers, Peng Wu.
      Proteolysis targeting chimeras (PROTACs) are new chemical modalities that degrade proteins of interest, including established kinase targets and emerging RNA-binding proteins (RBPs). Whereas diverse sets of biochemical, biophysical and cellular assays are available for the evaluation and optimizations of PROTACs in understanding the involved ubiquitin-proteasome-mediated degradation mechanism and the structure-degradation relationship, a phenotypic method profiling the cellular morphological changes is rarely used. In this study, first, we reported the only examples of PROTACs degrading the mRNA-binding protein YTHDF2 via screening of multikinase PROTACs. Second, we reported the profiling of cellular morphological changes of the dual kinase- and RBP-targeting PROTACs using the unbiased cell painting assay (CPA). The CPA analysis revealed the high biosimilarity with the established aurora kinase cluster and annotated aurora kinase inhibitors, which reflected the association between YTHDF2 and the aurora kinase signaling network. Broadly, the results demonstrated that the cell painting assay can be a straightforward and powerful approach to evaluate PROTACs. Complementary to the existing biochemical, biophysical and cellular assays, CPA provided a new perspective in characterizing PROTACs at the cellular morphology.
    Keywords:  Proteolysis targeting chimera (PROTAC); RNA-binding protein; YTHDF2; cell painting assay (CPA); cellular morphological change
    DOI:  https://doi.org/10.1002/cbic.202400183
  16. Biol Open. 2024 May 31. pii: bio.060412. [Epub ahead of print]
    The Endosomal Sorting Complex, ESCRT, has diverse roles in blood progenitor maintenance, lineage choice and immune response.
    Arindam Ray, Yashashwinee Rai, Maneesha S Inamdar.
      Most hematological malignancies are associated with reduced expression of one or more components of the Endosomal Sorting Complex Required for Transport (ESCRT). However, the roles of ESCRT in stem cell and progenitor maintenance are not resolved. Parsing signaling pathways in relation to the canonical role of ESCRT poses a challenge. The Drosophila hematopoietic organ, the larval lymph gland, provides a path to dissect the roles of cellular trafficking pathways such as ESCRT in blood development and maintenance. Drosophila has 13 core ESCRT components. Knockdown of individual ESCRTs showed that only Vps28 and Vp36 were required in all lymph gland progenitors. Using the well-conserved ESCRT-II complex as an example of the range of phenotypes seen upon ESCRT depletion, we show that ESCRTs have cell autonomous as well as non-autonomous roles in progenitor maintenance and differentiation. ESCRT depletion also sensitized posterior lobe progenitors to respond to immunogenic wasp infestation. We also identify key heterotypic roles for ESCRT in position-dependent control of Notch activation to suppress crystal cell differentiation. Our study shows that the cargo sorting machinery determines the identity of progenitors and their adaptability to the dynamic microenvironment. These mechanisms for control of cell fate may tailor developmental diversity in multiple contexts.
    Keywords:  Cargo sorting; ESCRT; Hematopoiesis; Lamellocytes; Lineage choice; Non-canonical function of ESCRT; Notch signaling; Ubiquitin accumulation
    DOI:  https://doi.org/10.1242/bio.060412
  17. iScience. 2024 Jun 21. 27(6): 109962
    Xbp1 targets canonical UPRER and non-canonical pathways in separate tissues to promote longevity.
    Mengjia Li, Haocheng Shou, Guillermo Martínez Corrales, Tatiana Svermova, Alessandra Vieira Franco, Nazif Alic.
      Transcription factors can reprogram gene expression to promote longevity. Here, we investigate the role of Drosophila Xbp1. Xbp1 is activated by splicing of its primary transcript, Xbp1u, to generate Xbp1s, a key activator of the endoplasmic reticulum unfolded protein response (UPRER). We show that Xbp1s induces the conical UPRER in the gut, promoting longevity from the resident stem cells. In contrast, in the fat body, Xbp1s does not appear to trigger UPRER but alters metabolic gene expression and is still able to extend lifespan. In the fat body, Xbp1s and dFOXO impinge on the same target genes, including the PGC-1α orthologue Srl, and dfoxo requires Xbp1 to extend lifespan. Interestingly, unspliceable version of the Xbp1 mRNA, Xbp1u can also extend lifespan, hinting at roles in longevity for the poorly characterized Xbp1u transcription factor. These findings reveal the diverse functions of Xbp1 in longevity in the fruit fly.
    Keywords:  Biological sciences; Molecular biology; Molecular mechanism of gene regulation
    DOI:  https://doi.org/10.1016/j.isci.2024.109962
  18. Cell Rep. 2024 May 30. pii: S2211-1247(24)00615-6. [Epub ahead of print]43(6): 114287
    Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response.
    Agnes Karasik, Hernan A Lorenzi, Andrew V DePass, Nicholas R Guydosh.
      Viral infection triggers several double-stranded RNA (dsRNA) sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, ribonuclease L (RNase L), that cleaves single-stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here, we show that this fragmentation induces the ribotoxic stress response via ZAKα, potentially through stalled ribosomes and/or ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes. Intriguingly, we found that the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.
    Keywords:  CP: Immunology; CP: Molecular biology; RNase A; RNase L; ZAKα; endonuclease; integrated stress response; ribosome collisions
    DOI:  https://doi.org/10.1016/j.celrep.2024.114287
  19. NAR Cancer. 2024 Jun;6(2): zcae026
    Non-canonical mRNA translation initiation in cell stress and cancer.
    Mélanie Mahé, Tiffany Rios-Fuller, Olga Katsara, Robert J Schneider.
      The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.
    DOI:  https://doi.org/10.1093/narcan/zcae026
  20. J Biol Chem. 2024 Jun 04. pii: S0021-9258(24)01949-5. [Epub ahead of print] 107448
    The ubiquitin E3 ligase APC/CCdc20 mediates mitotic degradation of OGT.
    Li Meng, Rui Dong, Weixiao Mi, Ke Qin, Kunfu Ouyang, Jianwei Sun, Jing Li.
      O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.
    Keywords:  APC/C(Cdc20); OGT; cell cycle; ubiquitination; uterine carcinoma
    DOI:  https://doi.org/10.1016/j.jbc.2024.107448
  21. Elife. 2024 Jun 05. pii: RP89306. [Epub ahead of print]12
    Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.
    Ivan Castello-Serrano, Frederick A Heberle, Barbara Diaz-Rohrer, Rossana Ippolito, Carolyn R Shurer, Pablo Lujan, Felix Campelo, Kandice R Levental, Ilya Levental.
      The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
    Keywords:  cell biology; membrane structure; membrane transport; none; physics of living systems; secretory trafficking
    DOI:  https://doi.org/10.7554/eLife.89306
  22. Elife. 2024 Jun 07. pii: RP93908. [Epub ahead of print]13
    Targeted protein degradation systems to enhance Wnt signaling.
    Parthasarathy Sampathkumar, Heekyung Jung, Hui Chen, Zhengjian Zhang, Nicholas Suen, Yiran Yang, Zhong Huang, Tom Lopez, Robert Benisch, Sung-Jin Lee, Jay Ye, Wen-Chen Yeh, Yang Li.
      Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody-RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.
    Keywords:  ASGR1; LYTAC; PROTAC; R-spondin; SWEETS; Wnt; biochemistry; chemical biology; none; regenerative medicine; stem cells
    DOI:  https://doi.org/10.7554/eLife.93908
  23. Nature. 2024 Jun 05.
    A disease-associated gene desert directs macrophage inflammation through ETS2.
    C T Stankey, C Bourges, L M Haag, T Turner-Stokes, A P Piedade, C Palmer-Jones, I Papa, M Silva Dos Santos, Q Zhang, A J Cameron, A Legrini, T Zhang, C S Wood, F N New, L O Randzavola, L Speidel, A C Brown, A Hall, F Saffioti, E C Parkes, W Edwards, H Direskeneli, P C Grayson, L Jiang, P A Merkel, G Saruhan-Direskeneli, A H Sawalha, E Tombetti, A Quaglia, D Thorburn, J C Knight, A P Rochford, C D Murray, P Divakar, M Green, E Nye, J I MacRae, N B Jamieson, P Skoglund, M Z Cader, C Wallace, D C Thomas, J C Lee.
      Increasing rates of autoimmune and inflammatory disease present a burgeoning threat to human health1. This is compounded by the limited efficacy of available treatments1 and high failure rates during drug development2, highlighting an urgent need to better understand disease mechanisms. Here we show how functional genomics could address this challenge. By investigating an intergenic haplotype on chr21q22-which has been independently linked to inflammatory bowel disease, ankylosing spondylitis, primary sclerosing cholangitis and Takayasu's arteritis3-6-we identify that the causal gene, ETS2, is a central regulator of human inflammatory macrophages and delineate the shared disease mechanism that amplifies ETS2 expression. Genes regulated by ETS2 were prominently expressed in diseased tissues and more enriched for inflammatory bowel disease GWAS hits than most previously described pathways. Overexpressing ETS2 in resting macrophages reproduced the inflammatory state observed in chr21q22-associated diseases, with upregulation of multiple drug targets, including TNF and IL-23. Using a database of cellular signatures7, we identified drugs that might modulate this pathway and validated the potent anti-inflammatory activity of one class of small molecules in vitro and ex vivo. Together, this illustrates the power of functional genomics, applied directly in primary human cells, to identify immune-mediated disease mechanisms and potential therapeutic opportunities.
    DOI:  https://doi.org/10.1038/s41586-024-07501-1
  24. Nucleic Acids Res. 2024 Jun 06. pii: gkae455. [Epub ahead of print]
    Cooperation of regulatory RNA and the RNA degradosome in transcript surveillance.
    Katarzyna J Bandyra, Kathrin S Fröhlich, Jörg Vogel, Marina Rodnina, Akanksha Goyal, Ben F Luisi.
      The ompD transcript, encoding an outer membrane porin in Salmonella, harbors a controlling element in its coding region that base-pairs imperfectly with a 'seed' region of the small regulatory RNA (sRNA) MicC. When tagged with the sRNA, the ompD mRNA is cleaved downstream of the pairing site by the conserved endoribonuclease RNase E, leading to transcript destruction. We observe that the sRNA-induced cleavage site is accessible to RNase E in vitro upon recruitment of ompD into the 30S translation pre-initiation complex (PIC) in the presence of the degradosome components. Evaluation of substrate accessibility suggests that the paused 30S PIC presents the mRNA for targeted recognition and degradation. Ribonuclease activity on PIC-bound ompD is critically dependent on the recruitment of RNase E into the multi-enzyme RNA degradosome, and our data suggest a process of substrate capture and handover to catalytic sites within the degradosome, in which sequential steps of seed matching and duplex remodelling contribute to cleavage efficiency. Our findings support a putative mechanism of surveillance at translation that potentially terminates gene expression efficiently and rapidly in response to signals provided by regulatory RNA.
    DOI:  https://doi.org/10.1093/nar/gkae455
  25. Autophagy. 2024 Jun 02.
    The DNA damage response and autophagy during cancer development: an antagonistic pleiotropy entanglement.
    Vassilis G Gorgoulis, Konstantinos Evangelou, Daniel J Klionsky.
      The DNA damage response (DDR) pathway is a cardinal cellular stress response mechanism that during cancer development follows an antagonistic pleiotropy mode of action. Given that DDR activation is an energy demanding process, interplay with macroautophagy/autophagy, a stress response and energy providing mechanism, is likely to take place. While molecular connections between both mechanisms have been reported, an open question regards whether autophagy activation follows solely or is entangled with DDR in a similar antagonistic pleiotropy pattern during cancer development. Combing evidence on the spatiotemporal relationship of DDR and autophagy in the entire spectrum of carcinogenesis from our previous studies, we discuss these issues in the current addendum.
    Keywords:  Disease; genomic instability; macroautophagy; stress; tumor
    DOI:  https://doi.org/10.1080/15548627.2024.2362121
  26. Dev Cell. 2024 Jun 04. pii: S1534-5807(24)00325-3. [Epub ahead of print]
    Neuronal activity-driven O-GlcNAcylation promotes mitochondrial plasticity.
    Seungyoon B Yu, Haoming Wang, Richard G Sanchez, Natasha M Carlson, Khanh Nguyen, Andrew Zhang, Zachary D Papich, Ahmed A Abushawish, Zachary Whiddon, Weronika Matysik, Jie Zhang, Thomas C Whisenant, Majid Ghassemian, John N Koberstein, Melissa L Stewart, Samuel A Myers, Gulcin Pekkurnaz.
      Neuronal activity is an energy-intensive process that is largely sustained by instantaneous fuel utilization and ATP synthesis. However, how neurons couple ATP synthesis rate to fuel availability is largely unknown. Here, we demonstrate that the metabolic sensor enzyme O-linked N-acetyl glucosamine (O-GlcNAc) transferase regulates neuronal activity-driven mitochondrial bioenergetics in hippocampal and cortical neurons. We show that neuronal activity upregulates O-GlcNAcylation in mitochondria. Mitochondrial O-GlcNAcylation is promoted by activity-driven glucose consumption, which allows neurons to compensate for high energy expenditure based on fuel availability. To determine the proteins that are responsible for these adjustments, we mapped the mitochondrial O-GlcNAcome of neurons. Finally, we determine that neurons fail to meet activity-driven metabolic demand when O-GlcNAcylation dynamics are prevented. Our findings suggest that O-GlcNAcylation provides a fuel-dependent feedforward control mechanism in neurons to optimize mitochondrial performance based on neuronal activity. This mechanism thereby couples neuronal metabolism to mitochondrial bioenergetics and plays a key role in sustaining energy homeostasis.
    Keywords:  ATP synthesis; O-GlcNAc transferase; O-GlcNAcylation; glycosylation; mitochondria; neuronal metabolism; synaptic activity
    DOI:  https://doi.org/10.1016/j.devcel.2024.05.008
  27. Sci Immunol. 2024 Jun 07. 9(96): eadl2388
    Plasma membrane abundance dictates phagocytic capacity and functional cross-talk in myeloid cells.
    Benjamin Y Winer, Alexander H Settle, Alexandrina M Yakimov, Carlos Jeronimo, Tomi Lazarov, Murray Tipping, Michelle Saoi, Anjelique Sawh, Anna-Liisa L Sepp, Michael Galiano, Justin S A Perry, Yung Yu Wong, Frederic Geissmann, Justin Cross, Ting Zhou, Lance C Kam, H Amalia Pasolli, Tobias Hohl, Jason G Cyster, Orion D Weiner, Morgan Huse.
      Professional phagocytes like neutrophils and macrophages tightly control what they consume, how much they consume, and when they move after cargo uptake. We show that plasma membrane abundance is a key arbiter of these cellular behaviors. Neutrophils and macrophages lacking the G protein subunit Gβ4 exhibited profound plasma membrane expansion, accompanied by marked reduction in plasma membrane tension. These biophysical changes promoted the phagocytosis of bacteria, fungus, apoptotic corpses, and cancer cells. We also found that Gβ4-deficient neutrophils are defective in the normal inhibition of migration following cargo uptake. Sphingolipid synthesis played a central role in these phenotypes by driving plasma membrane accumulation in cells lacking Gβ4. In Gβ4 knockout mice, neutrophils not only exhibited enhanced phagocytosis of inhaled fungal conidia in the lung but also increased trafficking of engulfed pathogens to other organs. Together, these results reveal an unexpected, biophysical control mechanism central to myeloid functional decision-making.
    DOI:  https://doi.org/10.1126/sciimmunol.adl2388
  28. Res Sq. 2024 May 20. pii: rs.3.rs-4431542. [Epub ahead of print]
    Legionella maintains host ubiquitin homeostasis by effectors with unique catalytic mechanisms.
    Zhao-Qing Luo, Jiaqi Fu, Siying Li, Wei Xian, Zhengrui Zhang, Tao-Tao Chen, Yao Liu, Qiuhua Lu, Songying Ouyang, Chittaranjan Das, Xiaoyun Liu, Lei Song, Hongxin Guan, Chuang Li, Qingtian Guan, Jingting Wang, Jinyu Li, Lina Kang, Si-Ru Zheng, Shoujing Cao.
      The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.
    DOI:  https://doi.org/10.21203/rs.3.rs-4431542/v1
  29. Nat Commun. 2024 Jun 03. 15(1): 4703
    PPIA dictates NRF2 stability to promote lung cancer progression.
    Weiqiang Lu, Jiayan Cui, Wanyan Wang, Qian Hu, Yun Xue, Xi Liu, Ting Gong, Yiping Lu, Hui Ma, Xinyu Yang, Bo Feng, Qi Wang, Naixia Zhang, Yechun Xu, Mingyao Liu, Ruth Nussinov, Feixiong Cheng, Hongbin Ji, Jin Huang.
      Nuclear factor erythroid 2-related factor 2 (NRF2) hyperactivation has been established as an oncogenic driver in a variety of human cancers, including non-small cell lung cancer (NSCLC). However, despite massive efforts, no specific therapy is currently available to target NRF2 hyperactivation. Here, we identify peptidylprolyl isomerase A (PPIA) is required for NRF2 protein stability. Ablation of PPIA promotes NRF2 protein degradation and blocks NRF2-driven growth in NSCLC cells. Mechanistically, PPIA physically binds to NRF2 and blocks the access of ubiquitin/Kelch Like ECH Associated Protein 1 (KEAP1) to NRF2, thus preventing ubiquitin-mediated degradation. Our X-ray co-crystal structure reveals that PPIA directly interacts with a NRF2 interdomain linker via a trans-proline 174-harboring hydrophobic sequence. We further demonstrate that an FDA-approved drug, cyclosporin A (CsA), impairs the interaction of NRF2 with PPIA, inducing NRF2 ubiquitination and degradation. Interestingly, CsA interrupts glutamine metabolism mediated by the NRF2/KLF5/SLC1A5 pathway, consequently suppressing the growth of NRF2-hyperactivated NSCLC cells. CsA and a glutaminase inhibitor combination therapy significantly retard tumor progression in NSCLC patient-derived xenograft (PDX) models with NRF2 hyperactivation. Our study demonstrates that targeting NRF2 protein stability is an actionable therapeutic approach to treat NRF2-hyperactivated NSCLC.
    DOI:  https://doi.org/10.1038/s41467-024-48364-4
  30. NAR Cancer. 2024 Jun;6(2): zcae025
    The shaping of mRNA translation plasticity by RNA G-quadruplexes in cancer progression and therapy resistance.
    Anne Cammas, Alice Desprairies, Erik Dassi, Stefania Millevoi.
      Translational reprogramming in response to oncogenic signaling or microenvironmental stress factors shapes the proteome of cancer cells, enabling adaptation and phenotypic changes underlying cell plasticity, tumor progression and response to cancer therapy. Among the mechanisms regulating translation are RNA G-quadruplexes (RG4s), non-canonical four-stranded structures whose conformational modulation by small molecule ligands and RNA-binding proteins affects the expression of cancer proteins. Here, we discuss the role of RG4s in the regulation of mRNA translation by focusing on paradigmatic examples showing their contribution to adaptive mechanisms of mRNA translation in cancer.
    DOI:  https://doi.org/10.1093/narcan/zcae025
  31. Science. 2024 Jun 07. 384(6700): eadk0850
    Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.
    Jennifer E Klomp, J Nathaniel Diehl, Jeffrey A Klomp, A Cole Edwards, Runying Yang, Alexis J Morales, Khalilah E Taylor, Kristina Drizyte-Miller, Kirsten L Bryant, Antje Schaefer, Jared L Johnson, Emily M Huntsman, Tomer M Yaron, Mariaelena Pierobon, Elisa Baldelli, Alex W Prevatte, Natalie K Barker, Laura E Herring, Emanuel F Petricoin, Lee M Graves, Lewis C Cantley, Adrienne D Cox, Channing J Der, Clint A Stalnecker.
      To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.
    DOI:  https://doi.org/10.1126/science.adk0850
  32. Cell. 2024 May 31. pii: S0092-8674(24)00527-0. [Epub ahead of print]
    The ribotoxic stress response drives UV-mediated cell death.
    Niladri K Sinha, Connor McKenney, Zhong Y Yeow, Jeffrey J Li, Ki Hong Nam, Tomer M Yaron-Barir, Jared L Johnson, Emily M Huntsman, Lewis C Cantley, Alban Ordureau, Sergi Regot, Rachel Green.
      While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage.
    Keywords:  DNA damage response; GCN2; UV radiation; ZAK; apoptosis; collisions; phosphoproteomics; ribosomes; ribotoxic stress; signaling
    DOI:  https://doi.org/10.1016/j.cell.2024.05.018
  33. J Am Chem Soc. 2024 Jun 06.
    Selective Removal of Denatured Proteins Using MOF Nanopores.
    Hirotaka Taketomi, Nobuhiko Hosono, Takashi Uemura.
      Here we present, for the first time, the selective adsorption of denatured proteins using a metal-organic framework (MOF), demonstrating promising potential for protein purification. Typical proteins, such as lysozyme and carbonic anhydrase B, enter the pores of MIL-101 through their narrow apertures when they are denatured to an unfolded state. Selective adsorption is achieved by finely tuning two key features: the sizes of the aperture and cage of the MOF nanopores, which are responsible for sorting unfolded polypeptide chains and inhibiting the translocation of the native form into the pores, respectively. By leveraging this selective adsorption, we successfully purified a mixture of native and denatured proteins by adding MOF to the mixture, achieving a native purity of over 99%.
    DOI:  https://doi.org/10.1021/jacs.4c03886
  34. bioRxiv. 2024 May 22. pii: 2023.09.10.557072. [Epub ahead of print]
    The tidyomics ecosystem: Enhancing omic data analyses.
    tidyomics Consortium
      The growth of omic data presents evolving challenges in data manipulation, analysis, and integration. Addressing these challenges, Bioconductor1 provides an extensive community-driven biological data analysis platform. Meanwhile, tidy R programming2 offers a revolutionary standard for data organisation and manipulation. Here, we present the tidyomics software ecosystem, bridging Bioconductor to the tidy R paradigm. This ecosystem aims to streamline omic analysis, ease learning, and encourage cross-disciplinary collaborations. We demonstrate the effectiveness of tidyomics by analysing 7.5 million peripheral blood mononuclear cells from the Human Cell Atlas3, spanning six data frameworks and ten analysis tools.
    DOI:  https://doi.org/10.1101/2023.09.10.557072
  35. Nat Immunol. 2024 Jun;25(6): 969-980
    Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus.
    Mahmoud Al-Azab, Elina Idiiatullina, Ziyang Liu, Meng Lin, Katja Hrovat-Schaale, Huifang Xian, Jianheng Zhu, Mandy Yang, Bingtai Lu, Zhiyao Zhao, Yiyi Liu, Jingjie Chang, Xiaotian Li, Caiqin Guo, Yunfeng Liu, Qi Wu, Jiazhang Chen, Chaoting Lan, Ping Zeng, Jun Cui, Xia Gao, Wenhao Zhou, Yan Zhang, Yuxia Zhang, Seth L Masters.
      Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.
    DOI:  https://doi.org/10.1038/s41590-024-01846-5
  36. Redox Biol. 2024 May 30. pii: S2213-2317(24)00198-8. [Epub ahead of print]73 103220
    A targetable PRR11-DHODH axis drives ferroptosis- and temozolomide-resistance in glioblastoma.
    Zong Miao, Lei Xu, Wei Gu, Yimin Ren, Rong Li, Shuai Zhang, Chao Chen, Hongxiang Wang, Jing Ji, Juxiang Chen.
      Temozolomide (TMZ) is a widely utilized chemotherapy treatment for patients with glioblastoma (GBM), although drug resistance constitutes a major therapeutic hurdle. Emerging evidence suggests that ferroptosis-mediated therapy could offer an appropriate alternative treatment option against cancer cells that are resistant to certain drugs. However, recurrent gliomas display robust ferroptosis resistance, although the precise mechanism of resistance remains elusive. In the present work, we report that proline rich protein 11 (PRR11) depletion significantly sensitizes GBM cells to TMZ by inducing ferroptosis. Mechanistically, PRR11 directly binds to and stabilizes dihydroorotate dehydrogenase (DHODH), which leads to glioma ferroptosis-resistant in a DHODH-dependent manner in vivo and in vitro. Furthermore, PRR11 inhibits HERC4 and DHODH binding, by suppressing the recruitment of E3 ubiquitin ligase HERC4 and polyubiquitination degradation of DHODH at the K306 site, which maintains DHODH protein stability. Importantly, downregulated PRR11 increases lipid peroxidation and alters DHODH-mediated mitochondrial morphology, thereby promoting ferroptosis and increasing TMZ chemotherapy sensitivity. In conclusion, our results reveal a mechanism via which PRR11 drives ferroptosis resistance and identifies ferroptosis induction and TMZ as an attractive combined therapeutic strategy for GBM.
    Keywords:  DHODH; Ferroptosis; Glioblastoma; HERC4; PRR11; Ubiquitination
    DOI:  https://doi.org/10.1016/j.redox.2024.103220
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