bims-proteo Biomed News
on Proteostasis
Issue of 2024–08–25
forty-one papers selected by
Eric Chevet, INSERM
  1. The unfolded protein response regulates ER exit sites via SNRPB-dependent RNA splicing and contributes to bone development.
  2. Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.
  3. Myotubularin 2 interacts with SEC23A and negatively regulates autophagy at ER exit sites in arabidopsis.
  4. DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair.
  5. SKN-1 Activation During Infection of C. elegans Requires CDC-48 and ER Proteostasis.
  6. Proteostasis is a key driver of the pathogenesis in Apicomplexa.
  7. A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.
  8. Recruitment of Cdc48 to chloroplasts by a UBX-domain protein in chloroplast-associated protein degradation.
  9. NAC guides a ribosomal multienzyme complex for nascent protein processing.
  10. Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response.
  11. Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.
  12. The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS.
  13. BCL2L13 at endoplasmic reticulum-mitochondria contact sites regulates calcium homeostasis to maintain skeletal muscle function.
  14. Protein disulfide isomerase PDI8 is indispensable for parasite growth and associated with secretory protein processing in Toxoplasma gondii.
  15. Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability.
  16. Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro.
  17. Visualizing chaperonin function in situ by cryo-electron tomography.
  18. Mechanism and regulation of cargo entry into the Commander endosomal recycling pathway.
  19. Cellular oxidants and the proteostasis network: balance between activation and destruction.
  20. Protocol for quantifying LC3B FRET biosensor activity in living cells using a broad-to-sensitive data analysis pipeline.
  21. CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115.
  22. Small Molecule RNA Degraders.
  23. TRIM21-mediated ubiquitination of SQSTM1/p62 abolishes its Ser403 phosphorylation and enhances palmitic acid cytotoxicity.
  24. Coordination of force-generating actin-based modules stabilizes and remodels membranes in vivo.
  25. The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis and maintain translational repression of maternal mRNA and oocyte quality.
  26. The central role of transfer RNAs in mistranslation.
  27. WIPI2b recruitment to phagophores and ATG16L1 binding are regulated by ULK1 phosphorylation.
  28. Lysosomes drive the piecemeal removal of mitochondrial inner membrane.
  29. De novo design of miniprotein antagonists of cytokine storm inducers.
  30. ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes.
  31. Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex.
  32. Feel the breeze: Opening the therapeutic window with RIPTACs and induced proximity.
  33. Structural basis of sugar recognition by SCFFBS2 ubiquitin ligase involved in NGLY1 deficiency.
  34. The unfolded protein response sensor PERK mediates mechanical stress-induced maturation of focal adhesion complexes in glioblastoma cells.
  35. Local protein synthesis at neuromuscular synapses is required for motor functions.
  36. High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.
  37. Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.
  38. Conserved 5-methyluridine tRNA modification modulates ribosome translocation.
  39. The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.
  40. Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.
  41. Proteasome resides in and dismantles plant heat stress granules constitutively.
  1. EMBO J. 2024 Aug 19.
    The unfolded protein response regulates ER exit sites via SNRPB-dependent RNA splicing and contributes to bone development.
    Muhammad Zahoor, Yanchen Dong, Marco Preussner, Veronika Reiterer, Sabrina Shameen Alam, Margot Haun, Utku Horzum, Yannick Frey, Renata Hajdu, Stephan Geley, Valerie Cormier-Daire, Florian Heyd, Loydie A Jerome-Majewska, Hesso Farhan.
      Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology.
    Keywords:  COPII; Endoplasmic Reticulum; Proteostasis; Sec16A; Splicing
    DOI:  https://doi.org/10.1038/s44318-024-00208-z
  2. Mol Cell. 2024 Aug 19. pii: S1097-2765(24)00659-2. [Epub ahead of print]
    Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.
    Katharine R Page, Vy N Nguyen, Tino Pleiner, Giovani Pinton Tomaleri, Maxine L Wang, Alina Guna, Masami Hazu, Ting-Yu Wang, Tsui-Fen Chou, Rebecca M Voorhees.
      Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein-coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of Sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC⋅BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, the multipass translocon, and Sec61 for the biogenesis of diverse membrane proteins in human cells.
    Keywords:  ER membrane protein complex; GPCRs; biogenesis; endoplasmic reticulum; membrane proteins
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.005
  3. Autophagy. 2024 Aug 23.
    Myotubularin 2 interacts with SEC23A and negatively regulates autophagy at ER exit sites in arabidopsis.
    Xinjing Li, Jing Zheng, Jing Su, Lin Wang, Lin Luan, Taotao Wang, Fang Bai, Qing Zhong, Qingqiu Gong.
      Starvation- or stress-induced phosphatidylinositol 3-phosphate (PtdIns3P/PI3P) production at the endoplasmic reticulum (ER) subdomains organizes phagophore assembly and autophagosome formation. Coat protein complex II (COPII) vesicles budding from ER exit site (ERES) also contribute to autophagosome formation. Whether any PtdIns3P phosphatase functions at ERES to inhibit macroautophagy/autophagy is unknown. Here we report Myotubularin 2 (MTM2) of Arabidopsis as a PtdIns3P phosphatase that localizes to ERES and negatively regulates autophagy. MTM2 binds PtdIns3P with its PH-GRAM domain in vitro and acts toward PtdIns3P in vivo. Transiently expressed MTM2 colocalizes with ATG14b, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex, and overexpression of MTM2 blocks autophagic flux and causes over-accumulation of ATG18a, ATG5, and ATG8a. The mtm2 mutant has higher levels of autophagy and is more tolerant to starvation, whereas MTM2 overexpression leads to reduced autophagy and sensitivity to starvation. The phenotypes of mtm2 are suppressed by ATG2 mutation, suggesting that MTM2 acts upstream of ATG2. Importantly, MTM2 does not affect the endosomal functions of PtdIns3P. Instead, MTM2 specifically colocalizes with COPII coat proteins and is cradled by the ERES-defining protein SEC16. MTM2 interacts with SEC23A with its phosphatase domain and inhibits COPII-mediated protein secretion. Finally, a role for MTM2 in salt stress response is uncovered. mtm2 resembles the halophyte Thellungiella salsuginea in its efficient vacuolar compartmentation of Na+, maintenance of chloroplast integrity, and timely regulation of autophagy-related genes. Our findings reveal a balance between PtdIns3P synthesis and turnover in autophagosome formation, and provide a new link between autophagy and COPII function.
    Keywords:  Autophagy; COPII; ER exit site; PtdIns3P; myotubularin; salt stress
    DOI:  https://doi.org/10.1080/15548627.2024.2394302
  4. Proc Natl Acad Sci U S A. 2024 Aug 27. 121(35): e2321204121
    DNA damage-induced proteasome phosphorylation controls substrate recognition and facilitates DNA repair.
    Xiaomei Zhang, Tianyi Zhu, Xuemei Li, Hongxia Zhao, Shixian Lin, Jun Huang, Bing Yang, Xing Guo.
      Upon DNA damage, numerous proteins are targeted for ubiquitin-dependent proteasomal degradation, which is an integral part of the DNA repair program. Although details of the ubiquitination processes have been intensively studied, little is known about whether and how the 26S proteasome is regulated in the DNA damage response (DDR). Here, we show that human Rpn10/PSMD4, one of the three ubiquitin receptors of the 26S proteasome, is rapidly phosphorylated in response to different types of DNA damage. The phosphorylation occurs at Rpn10-Ser266 within a conserved SQ motif recognized by ATM/ATR/DNA-PK. Blockade of S266 phosphorylation attenuates homologous recombination-mediated DNA repair and sensitizes cells to genotoxic insults. In vitro and in cellulo experiments indicate that phosphorylation of S266, located in the flexible linker between the two ubiquitin-interacting motifs (UIMs) of Rpn10, alters the configuration of UIMs, and actually reduces ubiquitin chain (substrate) binding. As a result, essential DDR proteins such as BRCA1 are spared from premature degradation and allowed sufficient time to engage in DNA repair, a scenario supported by proximity labeling and quantitative proteomic studies. These findings reveal an inherent self-limiting mechanism of the proteasome that, by controlling substrate recognition through Rpn10 phosphorylation, fine-tunes protein degradation for optimal responses under stress.
    Keywords:  DNA damage; Rpn10; phosphorylation; proteasome; ubiquitination
    DOI:  https://doi.org/10.1073/pnas.2321204121
  5. Genetics. 2024 Aug 21. pii: iyae131. [Epub ahead of print]
    SKN-1 Activation During Infection of C. elegans Requires CDC-48 and ER Proteostasis.
    Carolaing Gabaldón, Ozgur Karakuzu, Danielle A Garsin.
      During challenge of Caenorhabditis elegans with human bacterial pathogens such as Pseudomonas aeruginosa and Enterococcus faecalis, the elicited host response can be damaging if not properly controlled. The activation of Nrf (nuclear factor erythroid-related factor)/CNC (Cap-n-collar) transcriptional regulators modulates the response by upregulating genes that neutralize damaging molecules and promote repair processes. Activation of the C. elegans Nrf ortholog, SKN-1, is tightly controlled by a myriad of regulatory mechanisms, but a central feature is an activating phosphorylation accomplished by the p38 mitogen-activated kinase (MAPK) cascade. In this work, loss of CDC-48, an AAA+ ATPase was observed to severely compromise SKN-1 activation on pathogen and we sought to understand the mechanism. CDC-48 is part of the endoplasmic reticulum (ER) - associated degradation (ERAD) complex where it functions as a remodeling chaperone enabling the translocation of proteins from the ER to the cytoplasm for degradation by the proteosome. Interestingly, one of the proteins retrotranslocated by ERAD, a process necessary for its activation, is SKN-1A, the ER isoform of SKN-1. However, we discovered that SKN-1A is not activated by pathogen exposure in marked contrast to the cytoplasmic associated isoform, SKN-1C. Rather, loss of CDC-48 blocks the antioxidant response normally orchestrated by SKN-1C by strongly inducing the unfolded protein response (UPRER). The data is consistent with the model of these two pathways being mutually inhibitory and support the emerging paradigm in the field of coordinated cooperation between different stress responses.
    Keywords:   Caenorhabditis elegans ; CDC-48; Nrf; SKN-1; infection; stress response
    DOI:  https://doi.org/10.1093/genetics/iyae131
  6. Biochim Biophys Acta Mol Cell Res. 2024 Aug 19. pii: S0167-4889(24)00167-8. [Epub ahead of print] 119824
    Proteostasis is a key driver of the pathogenesis in Apicomplexa.
    Pallabi Mitra, Abhijit S Deshmukh.
      Proteostasis, including protein folding mediated by molecular chaperones, protein degradation, and stress response pathways in organelles like ER (unfolded protein response: UPR), are responsible for cellular protein quality control. This is essential for cell survival as it regulates and reprograms cellular processes. Here, we underscore the role of the proteostasis pathway in Apicomplexan parasites with respect to their well-characterized roles as well as potential roles in many parasite functions, including survival, multiplication, persistence, and emerging drug resistance. In addition to the diverse physiological importance of proteostasis in Apicomplexa, we assess the potential of the pathway's components as chemotherapeutic targets.
    Keywords:  Apicomplexa; Chaperones; ER stress; Heat shock response (HSR); Plasmodium; Proteasome; Proteostasis; Toxoplasma; UPR; UPS
    DOI:  https://doi.org/10.1016/j.bbamcr.2024.119824
  7. Bioconjug Chem. 2024 Aug 21.
    A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.
    Joanna Koszela, Nhan T Pham, Steven Shave, Daniel St-Cyr, Derek F Ceccarelli, Steven Orlicky, Anne Marinier, Frank Sicheri, Mike Tyers, Manfred Auer.
      Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay. We demonstrate the capabilities of PPI-CONA by detecting low affinity ternary complex formation between the human CDC34A ubiquitin-conjugating (E2) enzyme, ubiquitin, and CC0651, a small molecule enhancer of the CDC34A-ubiquitin interaction. We further exemplify PPI-CONA with an E2 enzyme binding study on CC0651 and a CDC34A binding specificity study of a series of CC0651 analogues. Our results indicate that CC0651 is highly selective toward CDC34A. We further demonstrate how PPI-CONA can be applied to screening very low affinity interactions. PPI-CONA holds potential for high-throughput screening for modulators of PPI targets and characterization of their affinity, specificity, and selectivity.
    DOI:  https://doi.org/10.1021/acs.bioconjchem.4c00345
  8. Nat Plants. 2024 Aug 19.
    Recruitment of Cdc48 to chloroplasts by a UBX-domain protein in chloroplast-associated protein degradation.
    Na Li, R Paul Jarvis.
      The translocon at the outer chloroplast membrane (TOC) is the gateway for chloroplast protein import and so is vital for photosynthetic establishment and plant growth. Chloroplast-associated protein degradation (CHLORAD) is a ubiquitin-dependent proteolytic system that regulates TOC. In CHLORAD, cytosolic Cdc48 provides motive force for the retrotranslocation of ubiquitinated TOC proteins to the cytosol but how Cdc48 is recruited is unknown. Here, we identify plant UBX-domain protein PUX10 as a component of the CHLORAD machinery. We show that PUX10 is an integral chloroplast outer membrane protein that projects UBX and ubiquitin-associated domains into the cytosol. It interacts with Cdc48 via its UBX domain, bringing it to the chloroplast surface, and with ubiquitinated TOC proteins via its ubiquitin-associated domain. Genetic analyses in Arabidopsis revealed a requirement for PUX10 during CHLORAD-mediated regulation of TOC function and plant development. Thus, PUX10 coordinates ubiquitination and retrotranslocation activities of CHLORAD to enable efficient TOC turnover.
    DOI:  https://doi.org/10.1038/s41477-024-01769-x
  9. Nature. 2024 Aug 21.
    NAC guides a ribosomal multienzyme complex for nascent protein processing.
    Alfred M Lentzsch, Denis Yudin, Martin Gamerdinger, Sowmya Chandrasekar, Laurenz Rabl, Alain Scaiola, Elke Deuerling, Nenad Ban, Shu-Ou Shan.
      Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
    DOI:  https://doi.org/10.1038/s41586-024-07846-7
  10. Proc Natl Acad Sci U S A. 2024 Aug 27. 121(35): e2409628121
    Gcn2 structurally mimics and functionally repurposes the HisRS enzyme for the integrated stress response.
    Charles Bou-Nader, Swati Gaikwad, Soheila Bahmanjah, Fan Zhang, Alan G Hinnebusch, Jinwei Zhang.
      Protein kinase Gcn2 attenuates protein synthesis in response to amino acid starvation while stimulating translation of a transcriptional activator of amino acid biosynthesis. Gcn2 activation requires a domain related to histidyl-tRNA synthetase (HisRS), the enzyme that aminoacylates tRNAHis. While evidence suggests that deacylated tRNA binds the HisRS domain for kinase activation, ribosomal P-stalk proteins have been implicated as alternative activating ligands on stalled ribosomes. We report crystal structures of the HisRS domain of Chaetomium thermophilum Gcn2 that reveal structural mimicry of both catalytic (CD) and anticodon-binding (ABD) domains, which in authentic HisRS bind the acceptor stem and anticodon loop of tRNAHis. Elements for forming histidyl adenylate and aminoacylation are lacking, suggesting that Gcn2HisRS was repurposed for kinase activation, consistent with mutations in the CD that dysregulate yeast Gcn2 function. Substituting conserved ABD residues well positioned to contact the anticodon loop or that form a conserved ABD-CD interface impairs Gcn2 function in starved cells. Mimicry in Gcn2HisRS of two highly conserved structural domains for binding both ends of tRNA-each crucial for Gcn2 function-supports that deacylated tRNAs activate Gcn2 and exemplifies how a metabolic enzyme is repurposed to host new local structures and sequences that confer a novel regulatory function.
    Keywords:  GCN4 translation; Gcn2; eIF2α phosphorylation; histidyl-tRNA synthetase; yeast S. cerevisiae
    DOI:  https://doi.org/10.1073/pnas.2409628121
  11. Nat Commun. 2024 Aug 21. 15(1): 7194
    Palmitoylation of ULK1 by ZDHHC13 plays a crucial role in autophagy.
    Keisuke Tabata, Kenta Imai, Koki Fukuda, Kentaro Yamamoto, Hayato Kunugi, Toshiharu Fujita, Tatsuya Kaminishi, Christian Tischer, Beate Neumann, Sabine Reither, Fatima Verissimo, Rainer Pepperkok, Tamotsu Yoshimori, Maho Hamasaki.
      Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy.
    DOI:  https://doi.org/10.1038/s41467-024-51402-w
  12. Cell Rep. 2024 Aug 22. pii: S2211-1247(24)01018-0. [Epub ahead of print]43(9): 114667
    The TRIM4 E3 ubiquitin ligase degrades TPL2 and is modulated by oncogenic KRAS.
    Sapana Bansod, Paarth B Dodhiawala, Yutong Geng, Ashenafi Bulle, Peng Liu, Lin Li, Reid Townsend, Patrick M Grierson, Jason M Held, Hema Adhikari, Kian-Huat Lim.
      Loss-of-function mutations in the C terminus of TPL2 kinase promote oncogenesis by impeding its proteasomal degradation, leading to sustained protein expression. However, the degradation mechanism for TPL2 has remained elusive. Through proximity-dependent biotin identification (BioID), we uncovered tripartite motif-containing 4 (TRIM4) as the E3 ligase that binds and degrades TPL2 by polyubiquitination of lysines 415 and 439. The naturally occurring TPL2 mutants R442H and E188K exhibit impaired TRIM4 binding, enhancing their stability. We further discovered that TRIM4 itself is stabilized by another E3 ligase, TRIM21, which in turn is regulated by KRAS. Mutant KRAS recruits RNF185 to degrade TRIM21 and subsequently TRIM4, thereby stabilizing TPL2. In the presence of mutant KRAS, TPL2 phosphorylates and degrades GSK3β, resulting in β-catenin stabilization and activation of the Wnt pathway. These findings elucidate the physiological mechanisms regulating TPL2 and its exploitation by mutant KRAS, underscoring the need to develop TPL2 inhibitors for KRAS-mutant cancers.
    Keywords:  COT; CP: Cancer; CP: Molecular biology; KRAS; MAP3K8; RNF185; TPL2; TRIM21; TRIM4; Wnt/β-catenin; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2024.114667
  13. iScience. 2024 Aug 16. 27(8): 110510
    BCL2L13 at endoplasmic reticulum-mitochondria contact sites regulates calcium homeostasis to maintain skeletal muscle function.
    Dogan Grepper, Cassandra Tabasso, Nadège Zanou, Axel K F Aguettaz, Mauricio Castro-Sepulveda, Dorian V Ziegler, Sylviane Lagarrigue, Yoan Arribat, Adrien Martinotti, Ammar Ebrahimi, Jean Daraspe, Lluis Fajas, Francesca Amati.
      The physical connection between mitochondria and endoplasmic reticulum (ER) is an essential signaling hub to ensure organelle and cellular functions. In skeletal muscle, ER-mitochondria calcium (Ca2+) signaling is crucial to maintain cellular homeostasis during physical activity. High expression of BCL2L13, a member of the BCL-2 family, was suggested as an adaptive response in endurance-trained human subjects. In adult zebrafish, we found that the loss of Bcl2l13 impairs skeletal muscle structure and function. Ca2+ signaling is altered in Bcl2l13 knockout animals and mitochondrial complexes activity is decreased. Organelle fractioning in mammalian cells shows BCL2L13 at mitochondria, ER, and mitochondria-associated membranes. ER-mitochondria contact sites number is not modified by BCL2L13 modulation, but knockdown of BCL2L13 in C2C12 cells changes cytosolic Ca2+ release and mitochondrial Ca2+ uptake. This suggests that BCL2L13 interaction with mitochondria and ER, and its role in Ca2+ signaling, contributes to proper skeletal muscle function.
    Keywords:  cell biology; pharmacology
    DOI:  https://doi.org/10.1016/j.isci.2024.110510
  14. mBio. 2024 Aug 20. e0205124
    Protein disulfide isomerase PDI8 is indispensable for parasite growth and associated with secretory protein processing in Toxoplasma gondii.
    Chaoyue Wang, Pei Sun, Yonggen Jia, Xinming Tang, Xianyong Liu, Xun Suo, Hongjuan Peng.
      Protein disulfide isomerase, containing thioredoxin (Trx) domains, serves as a vital enzyme responsible for oxidative protein folding (the formation, reduction, and isomerization of disulfide bonds in newly synthesized proteins) in the endoplasmic reticulum (ER). However, the role of ER-localized PDI proteins in parasite growth and their interaction with secretory proteins remain poorly understood. In this study, we identified two ER-localized PDI proteins, TgPDI8 and TgPDI6, in Toxoplasma gondii. Conditional knockdown of TgPDI8 resulted in a significant reduction in intracellular proliferation and invasion abilities, leading to a complete block in plaque formation on human foreskin fibroblast monolayers, whereas parasites lacking TgPDI6 did not exhibit any apparent fitness defects. The complementation of TgPDI8 with mutant variants highlighted the critical role of the CXXC active site cysteines within its Trx domains for its enzymatic activity. By utilizing TurboID-based proximity labeling, we uncovered a close association between PDI proteins and canonical secretory proteins. Furthermore, parasites lacking TgPDI8 showed a significant reduction in the expression of secretory proteins, especially those from micronemes and dense granules. In summary, our study elucidates the roles of TgPDI8 and sets the stage for future drug discovery studies.
    IMPORTANCE: Apicomplexans, a phylum of intracellular parasites, encompass various zoonotic pathogens, including Plasmodium, Cryptosporidium, Toxoplasma, and Babesia, causing a significant economic burden on human populations. These parasites exhibit hypersensitivity to disruptions in endoplasmic reticulum (ER) redox homeostasis, necessitating the presence of ER-localized thioredoxin (Trx) superfamily proteins, particularly protein disulfide isomerase (PDI), for proper oxidative folding. However, the functional characteristics of ER-localized PDI proteins in Toxoplasma gondii remain largely unexplored. In this study, we identified two ER-localized proteins, namely, TgPDI8 and TgPDI6, and demonstrated the indispensable role of TgPDI8 in parasite survival. Through a comprehensive multi-omics analysis, we elucidated the crucial role of TgPDI8 in the processing of secretory proteins in T. gondii. Additionally, we introduced a novel ER-anchored TurboID method to label and identify canonical secretory proteins in T. gondii. This research opens up new avenues for understanding oxidative folding and the secretory pathway in apicomplexan parasites, laying the groundwork for future advancements in antiparasitic drug development.
    Keywords:  Toxoplasma gondii; invasion; protein disulfide isomerase; protein processing; secretory protein
    DOI:  https://doi.org/10.1128/mbio.02051-24
  15. PNAS Nexus. 2024 Aug;3(8): pgae321
    Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability.
    Tejinder Pal Khaket, Suman Rimal, Xingjun Wang, Sunil Bhurtel, Yen-Chi Wu, Bingwei Lu.
      Myc is a major driver of tumor initiation, progression, and maintenance. Up-regulation of Myc protein level rather than acquisition of neomorphic properties appears to underlie most Myc-driven cancers. Cellular mechanisms governing Myc expression remain incompletely defined. In this study, we show that ribosome-associated quality control (RQC) plays a critical role in maintaining Myc protein level. Ribosomes stall during the synthesis of the N-terminal portion of cMyc, generating aberrant cMyc species and necessitating deployment of the early RQC factor ZNF598 to handle translational stress and restore cMyc translation. ZNF598 expression is up-regulated in human glioblastoma (GBM), and its expression positively correlates with that of cMyc. ZNF598 knockdown inhibits human GBM neurosphere formation in cell culture and Myc-dependent tumor growth in vivo in Drosophila. Intriguingly, the SARS-COV-2-encoded translational regulator Nsp1 impinges on ZNF598 to restrain cMyc translation and consequently cMyc-dependent cancer growth. Remarkably, Nsp1 exhibits synthetic toxicity with the translation and RQC-related factor ATP-binding cassette subfamily E member 1, which, despite its normally positive correlation with cMyc in cancer cells, is co-opted by Nsp1 to down-regulate cMyc and inhibit tumor growth. Ribosome stalling during c-myc translation thus offers actionable cancer cell vulnerability.
    Keywords:  Nsp1; cMyc; cancer; ribosome-associated quality control (RQC); translation stalling
    DOI:  https://doi.org/10.1093/pnasnexus/pgae321
  16. STAR Protoc. 2024 Aug 21. pii: S2666-1667(24)00440-4. [Epub ahead of print]5(3): 103275
    Protocol to reconstitute translationally arrested heat shock mRNPs and condensates in vitro.
    Christine Desroches Altamirano, Simon Alberti.
      Heat shock (HS) coincides with the assembly of translationally arrested heat shock messenger ribonucleoprotein particles (HS-mRNPs) and condensates. Here, we present a protocol to reconstitute HS-mRNPs and HS condensates with eIF4G, eIF4E, Pab1p, and mRNA in vitro. In addition, we describe the necessary steps to measure the effect of HS-mRNPs and HS condensates on translation in yeast extracts. The protocol can be modified to study mRNPs and condensates assembled with other proteins and to study translation in extracts prepared from different cells. For complete details on the use and execution of this protocol, please refer to Desroches Altamirano et al.1.
    Keywords:  Cell Biology; Protein Biochemistry; Protein expression and purification
    DOI:  https://doi.org/10.1016/j.xpro.2024.103275
  17. Nature. 2024 Aug 21.
    Visualizing chaperonin function in situ by cryo-electron tomography.
    Jonathan Wagner, Alonso I Carvajal, Andreas Bracher, Florian Beck, William Wan, Stefan Bohn, Roman Körner, Wolfgang Baumeister, Ruben Fernandez-Busnadiego, F Ulrich Hartl.
      Chaperonins are large barrel-shaped complexes that mediate ATP-dependent protein folding1-3. The bacterial chaperonin GroEL forms juxtaposed rings that bind unfolded protein and the lid-shaped cofactor GroES at their apertures. In vitro analyses of the chaperonin reaction have shown that substrate protein folds, unimpaired by aggregation, while transiently encapsulated in the GroEL central cavity by GroES4-6. To determine the functional stoichiometry of GroEL, GroES and client protein in situ, here we visualized chaperonin complexes in their natural cellular environment using cryo-electron tomography. We find that, under various growth conditions, around 55-70% of GroEL binds GroES asymmetrically on one ring, with the remainder populating symmetrical complexes. Bound substrate protein is detected on the free ring of the asymmetrical complex, defining the substrate acceptor state. In situ analysis of GroEL-GroES chambers, validated by high-resolution structures obtained in vitro, showed the presence of encapsulated substrate protein in a folded state before release into the cytosol. Based on a comprehensive quantification and conformational analysis of chaperonin complexes, we propose a GroEL-GroES reaction cycle that consists of linked asymmetrical and symmetrical subreactions mediating protein folding. Our findings illuminate the native conformational and functional chaperonin cycle directly within cells.
    DOI:  https://doi.org/10.1038/s41586-024-07843-w
  18. Nat Commun. 2024 Aug 21. 15(1): 7180
    Mechanism and regulation of cargo entry into the Commander endosomal recycling pathway.
    Rebeka Butkovič, Alexander P Walker, Michael D Healy, Kerrie E McNally, Meihan Liu, Tineke Veenendaal, Kohji Kato, Nalan Liv, Judith Klumperman, Brett M Collins, Peter J Cullen.
      Commander is a multiprotein complex that orchestrates endosomal recycling of integral cargo proteins and is essential for normal development. While the structure of this complex has recently been described, how cargo proteins are selected for Commander-mediated recycling remains unclear. Here we identify the mechanism through which the unstructured carboxy-terminal tail of the cargo adaptor sorting nexin-17 (SNX17) directly binds to the Retriever sub-complex of Commander. SNX17 adopts an autoinhibited conformation where its carboxy-terminal tail occupies the cargo binding groove. Competitive cargo binding overcomes this autoinhibition, promoting SNX17 endosomal residency and the release of the tail for Retriever association. Furthermore, our study establishes the central importance of SNX17-Retriever association in the handover of integrin and lipoprotein receptor cargoes into pre-existing endosomal retrieval sub-domains. In describing the principal mechanism of cargo entry into the Commander recycling pathway we provide key insight into the function and regulation of this evolutionary conserved sorting pathway.
    DOI:  https://doi.org/10.1038/s41467-024-50971-0
  19. Trends Biochem Sci. 2024 Aug 20. pii: S0968-0004(24)00164-6. [Epub ahead of print]
    Cellular oxidants and the proteostasis network: balance between activation and destruction.
    Agnes Ulfig, Ursula Jakob.
      Loss of protein homeostasis (proteostasis) is a common hallmark of aging and age-associated diseases. Considered as the guardian of proteostasis, the proteostasis network (PN) acts to preserve the functionality of proteins during their lifetime. However, its activity declines with age, leading to disease manifestation. While reactive oxygen species (ROS) were traditionally considered culprits in this process, recent research challenges this view. While harmful at high concentrations, moderate ROS levels protect the cell against age-mediated onset of proteotoxicity by activating molecular chaperones, stress response pathways, and autophagy. This review explores the nuanced roles of ROS in proteostasis and discusses the most recent findings regarding the redox regulation of the PN and its potential in extending healthspan and delaying age-related pathologies.
    Keywords:  aging; neurodegeneration; oxidative stress; protein aggregation; protein homeostasis; protein misfolding
    DOI:  https://doi.org/10.1016/j.tibs.2024.07.001
  20. STAR Protoc. 2024 Aug 22. pii: S2666-1667(24)00346-0. [Epub ahead of print]5(3): 103181
    Protocol for quantifying LC3B FRET biosensor activity in living cells using a broad-to-sensitive data analysis pipeline.
    Elif Begüm Gökerküçük, Marc Tramier, Giulia Bertolin.
      Here, we present a protocol to comprehensively quantify autophagy initiation using the readout of the microtubule associated protein 1 light chain 3 beta (LC3B) Förster's resonance energy transfer (FRET) biosensor. We describe steps for cell seeding, transfection, FRET/FLIM (fluorescence lifetime imaging microscopy) imaging, and image analysis. This protocol can be useful in any physiology- or disease-related paradigm where the LC3B biosensor can be expressed to determine whether autophagy has been initiated or is stalled. The analysis pipeline presented here can be applied to any other genetically encoded FRET sensor imaged using FRET/FLIM. For complete details on the use and execution of this protocol, please refer to Gökerküçük et al.1.
    Keywords:  Cell-based Assays; Microscopy; Molecular/Chemical Probes; Single Cell
    DOI:  https://doi.org/10.1016/j.xpro.2024.103181
  21. EMBO J. 2024 Aug 19.
    CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115.
    Joshua Greig, George T Bates, Daowen I Yin, Kit Briant, Boris Simonetti, Peter J Cullen, Frances M Brodsky.
      The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, shuttles the glucose transporter GLUT4 from the ERGIC (endoplasmic-reticulum-to-Golgi intermediate compartment) directly to an intracellular GLUT4 storage compartment (GSC), from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. We show that the C-terminal trimerization domain of CHC22 interacts with SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both N- and C-termini of the heavy chain, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi-bypass route for GLUT4 trafficking. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.
    Keywords:  CHC22; Clathrin; Golgi bypass; Sorting nexin 5; p115
    DOI:  https://doi.org/10.1038/s44318-024-00198-y
  22. Angew Chem Int Ed Engl. 2024 Aug 20. e202412925
    Small Molecule RNA Degraders.
    Javier Bonet-Aleta, Tomoaki Maehara, Benjamin A Craig, Gonçalo J L Bernardes.
      RNA is a central molecule in life, involved in a plethora of biological processes and playing a key role in many diseases. Targeting RNA emerges as a significant endeavor in drug discovery, diverging from conventional protein-centric approaches to tackle various pathologies. Whilst identifying small molecules that bind to specific RNA regions is the first step, the abundance of non-functional RNA segments renders many interactions biologically inert. Consequently, small molecule binding does not necessarily meet stringent criteria for clinical translation, calling for solutions to push the field forward. Converting RNA-binders into RNA-degraders presents a promising avenue to enhance RNA-targeting. This mini-review outlines strategies and exemplars wherein simple small molecule RNA binders are reprogrammed into active degraders through the linkage of functional groups. These approaches encompass mechanisms that induce degradation via endogenous enzymes, termed RIBOTACs, as well as those with functional moieties acting autonomously to degrade RNA. Through this exploration, we aim to offer insights into advancing RNA-targeted therapeutic strategies.
    Keywords:  Degradation; RNA; proximity-induced; small-molecule; therapy
    DOI:  https://doi.org/10.1002/anie.202412925
  23. Autophagy. 2024 Aug 22.
    TRIM21-mediated ubiquitination of SQSTM1/p62 abolishes its Ser403 phosphorylation and enhances palmitic acid cytotoxicity.
    Peng Yang, Shenglan Gao, Jianliang Shen, Tong Liu, Kevin Lu, Xinlu Han, Jun Wang, Hong-Min Ni, Wen-Xing Ding, Hong Li, Ji-An Pan, Kesong Peng, Wei-Xing Zong.
      Long-chain free fatty acids (FFAs) accumulation and oxidative toxicity is a major cause for several pathological conditions. The mechanisms underlying FFA cytotoxicity remain elusive. Here we show that palmitic acid (PA), the most abundant FFA in the circulation, induces S403 phosphorylation of SQSTM1/p62 (sequestosome 1) and its aggregation, which sequesters KEAP1 and activates the non-canonical SQSTM1-KEAP1-NFE2L2 antioxidant pathway. The PA-induced SQSTM1 S403 phosphorylation and aggregation are dependent on SQSTM1 K7-D69 hydrogen bond formation and dimerization in the Phox and Bem1 (PB1) domain, which facilitates the recruitment of TBK1 that phosphorylates SQSTM1 S403. The ubiquitin E3 ligase TRIM21 ubiquitinates SQSTM1 at the K7 residue and abolishes the PB1 dimerization, S403 phosphorylation, and SQSTM1 aggregation. TRIM21 is oxidized at C92, C111, and C114 to form disulfide bonds that lead to its oligomerization and decreased E3 activity. Mutagenizing the three C residues to S (3CS) abolishes TRIM21 oligomerization and increases its E3 activity. TRIM21 ablation leads to decreased SQSTM1 K7 ubiquitination, hence elevated SQSTM1 S403 phosphorylation and aggregation, which confers protection against PA-induced oxidative stress and cytotoxicity. Therefore, TRIM21 is a negative regulator of SQSTM1 phosphorylation, aggregation, and the antioxidant sequestration function. TRIM21 is oxidized to reduce its E3 activity that helps enhance the SQSTM1-KEAP1-NFE2L2 antioxidant pathway. Inhibition of TRIM21 May be a viable strategy to protect tissues from lipotoxicity resulting from long-chain FFAs.
    Keywords:  Free fatty acids; KEAP1; NFE2L2; ROS; lipotoxicity; liver damage
    DOI:  https://doi.org/10.1080/15548627.2024.2394308
  24. J Cell Biol. 2024 Nov 04. pii: e202401091. [Epub ahead of print]223(11):
    Coordination of force-generating actin-based modules stabilizes and remodels membranes in vivo.
    Marco Heydecker, Akiko Shitara, Desu Chen, Duy T Tran, Andrius Masedunskas, Muhibullah S Tora, Seham Ebrahim, Mark A Appaduray, Jorge Luis Galeano Niño, Abhishek Bhardwaj, Kedar Narayan, Edna C Hardeman, Peter W Gunning, Roberto Weigert.
      Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes.
    DOI:  https://doi.org/10.1083/jcb.202401091
  25. Mol Biol Cell. 2024 Aug 21. mbcE24050216
    The CCT chaperonin and actin modulate the ER and RNA-binding protein condensation during oogenesis and maintain translational repression of maternal mRNA and oocyte quality.
    Mohamed T Elaswad, Mingze Gao, Victoria E Tice, Cora G Bright, Grace M Thomas, Chloe Munderloh, Nicholas J Trombley, Christya N Haddad, Ulysses G Johnson, Ashley N Cichon, Jennifer A Schisa.
      The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer.
    DOI:  https://doi.org/10.1091/mbc.E24-05-0216
  26. J Biol Chem. 2024 Aug 16. pii: S0021-9258(24)02180-X. [Epub ahead of print] 107679
    The central role of transfer RNAs in mistranslation.
    Dominik B Schuntermann, Mateusz Jaskolowski, Noah M Reynolds, Oscar Vargas-Rodriguez.
      Transfer RNAs (tRNA) are essential small non-coding RNAs that enable the translation of genomic information into proteins in all life forms. The principal function of tRNAs is to bring amino acid building blocks to the ribosomes for protein synthesis. In the ribosome, tRNAs interact with messenger RNA (mRNA) to mediate the incorporation of amino acids into a growing polypeptide chain following the rules of the genetic code. Accurate interpretation of the genetic code requires tRNAs to carry amino acids matching their anticodon identity and decode the correct codon on mRNAs. Errors in these steps cause the translation of codons with the wrong amino acids (mistranslation), compromising the accurate flow of information from DNA to proteins. Accumulation of mutant proteins due to mistranslation jeopardizes proteostasis and cellular viability. However, the concept of mistranslation is evolving, with increasing evidence indicating that mistranslation can be used as a mechanism for survival and acclimatization to environmental conditions. In this review, we discuss the central role of tRNAs in modulating translational fidelity through their dynamic and complex interplay with translation factors. We summarize recent discoveries of mistranslating tRNAs and describe the underlying molecular mechanisms and the specific conditions and environments that enable and promote mistranslation.
    Keywords:  aminoacyl-tRNA synthetases; editing; genetic code; mistranslation; post-transcriptional modifications; protein synthesis; ribosome; tRNA
    DOI:  https://doi.org/10.1016/j.jbc.2024.107679
  27. EMBO Rep. 2024 Aug 16.
    WIPI2b recruitment to phagophores and ATG16L1 binding are regulated by ULK1 phosphorylation.
    Andrea Gubas, Eleanor Attridge, Harold Bj Jefferies, Taki Nishimura, Minoo Razi, Simone Kunzelmann, Yuval Gilad, Thomas J Mercer, Michael M Wilson, Adi Kimchi, Sharon A Tooze.
      One of the key events in autophagy is the formation of a double-membrane phagophore, and many regulatory mechanisms underpinning this remain under investigation. WIPI2b is among the first proteins to be recruited to the phagophore and is essential for stimulating autophagy flux by recruiting the ATG12-ATG5-ATG16L1 complex, driving LC3 and GABARAP lipidation. Here, we set out to investigate how WIPI2b function is regulated by phosphorylation. We studied two phosphorylation sites on WIPI2b, S68 and S284. Phosphorylation at these sites plays distinct roles, regulating WIPI2b's association with ATG16L1 and the phagophore, respectively. We confirm WIPI2b is a novel ULK1 substrate, validated by the detection of endogenous phosphorylation at S284. Notably, S284 is situated within an 18-amino acid stretch, which, when in contact with liposomes, forms an amphipathic helix. Phosphorylation at S284 disrupts the formation of the amphipathic helix, hindering the association of WIPI2b with membranes and autophagosome formation. Understanding these intricacies in the regulatory mechanisms governing WIPI2b's association with its interacting partners and membranes, holds the potential to shed light on these complex processes, integral to phagophore biogenesis.
    Keywords:  Amphipathic Helix; Autophagosome; Autophagy; Kinase; WIPIs
    DOI:  https://doi.org/10.1038/s44319-024-00215-5
  28. Nature. 2024 Aug 21.
    Lysosomes drive the piecemeal removal of mitochondrial inner membrane.
    Akriti Prashar, Claudio Bussi, Antony Fearns, Mariana I Capurro, Xiaodong Gao, Hiromi Sesaki, Maximiliano G Gutierrez, Nicola L Jones.
      Mitochondrial membranes define distinct structural and functional compartments. Cristae of the inner mitochondrial membrane (IMM) function as independent bioenergetic units that undergo rapid and transient remodelling, but the significance of this compartmentalized organization is unknown1. Using super-resolution microscopy, here we show that cytosolic IMM vesicles, devoid of outer mitochondrial membrane or mitochondrial matrix, are formed during resting state. These vesicles derived from the IMM (VDIMs) are formed by IMM herniation through pores formed by voltage-dependent anion channel 1 in the outer mitochondrial membrane. Live-cell imaging showed that lysosomes in proximity to mitochondria engulfed the herniating IMM and, aided by the endosomal sorting complex required for transport machinery, led to the formation of VDIMs in a microautophagy-like process, sparing the remainder of the organelle. VDIM formation was enhanced in mitochondria undergoing oxidative stress, suggesting their potential role in maintenance of mitochondrial function. Furthermore, the formation of VDIMs required calcium release by the reactive oxygen species-activated, lysosomal calcium channel, transient receptor potential mucolipin 1, showing an interorganelle communication pathway for maintenance of mitochondrial homeostasis. Thus, IMM compartmentalization could allow for the selective removal of damaged IMM sections via VDIMs, which should protect mitochondria from localized injury. Our findings show a new pathway of intramitochondrial quality control.
    DOI:  https://doi.org/10.1038/s41586-024-07835-w
  29. Nat Commun. 2024 Aug 16. 15(1): 7064
    De novo design of miniprotein antagonists of cytokine storm inducers.
    Buwei Huang, Brian Coventry, Marta T Borowska, Dimitrios C Arhontoulis, Marc Exposit, Mohamad Abedi, Kevin M Jude, Samer F Halabiya, Aza Allen, Cami Cordray, Inna Goreshnik, Maggie Ahlrichs, Sidney Chan, Hillary Tunggal, Michelle DeWitt, Nathaniel Hyams, Lauren Carter, Lance Stewart, Deborah H Fuller, Ying Mei, K Christopher Garcia, David Baker.
      Cytokine release syndrome (CRS), commonly known as cytokine storm, is an acute systemic inflammatory response that is a significant global health threat. Interleukin-6 (IL-6) and interleukin-1 (IL-1) are key pro-inflammatory cytokines involved in CRS and are hence critical therapeutic targets. Current antagonists, such as tocilizumab and anakinra, target IL-6R/IL-1R but have limitations due to their long half-life and systemic anti-inflammatory effects, making them less suitable for acute or localized treatments. Here we present the de novo design of small protein antagonists that prevent IL-1 and IL-6 from interacting with their receptors to activate signaling. The designed proteins bind to the IL-6R, GP130 (an IL-6 co-receptor), and IL-1R1 receptor subunits with binding affinities in the picomolar to low-nanomolar range. X-ray crystallography studies reveal that the structures of these antagonists closely match their computational design models. In a human cardiac organoid disease model, the IL-1R antagonists demonstrated protective effects against inflammation and cardiac damage induced by IL-1β. These minibinders show promise for administration via subcutaneous injection or intranasal/inhaled routes to mitigate acute cytokine storm effects.
    DOI:  https://doi.org/10.1038/s41467-024-50919-4
  30. Cell Rep. 2024 Aug 19. pii: S2211-1247(24)00976-8. [Epub ahead of print]43(8): 114626
    ALS-FUS mutations cause abnormal PARylation and histone H1.2 interaction, leading to pathological changes.
    Hafiza Alirzayeva, Rute Loureiro, Seda Koyuncu, Franziska Hommen, Yara Nabawi, William Hongyu Zhang, Thien T P Dao, Markus Wehrmann, Hyun Ju Lee, David Vilchez.
      The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.
    Keywords:  CP: Cell biology; FUS; PARP1; PARylation; amyotrophic lateral sclerosis; histone H1.2; neurodegeneration; protein aggregation; proteostasis; stress granules
    DOI:  https://doi.org/10.1016/j.celrep.2024.114626
  31. Commun Chem. 2024 Aug 16. 7(1): 183
    Mechanistic insights into a heterobifunctional degrader-induced PTPN2/N1 complex.
    Qi Hao, Manoj K Rathinaswamy, Kelly L Klinge, Matthew Bratkowski, Amirhossein Mafi, Christina K Baumgartner, Keith M Hamel, Gesine K Veits, Rinku Jain, Claudio Catalano, Mark Fitzgerald, Alexander W Hird, Eunice Park, Harit U Vora, James A Henderson, Kenton Longenecker, Charles W Hutchins, Wei Qiu, Giovanna Scapin, Qi Sun, Vincent S Stoll, Chaohong Sun, Ping Li, Dan Eaton, David Stokoe, Stewart L Fisher, Christopher G Nasveschuk, Marcia Paddock, Michael E Kort.
      PTPN2 (protein tyrosine phosphatase non-receptor type 2, or TC-PTP) and PTPN1 are attractive immuno-oncology targets, with the deletion of Ptpn1 and Ptpn2 improving response to immunotherapy in disease models. Targeted protein degradation has emerged as a promising approach to drug challenging targets including phosphatases. We developed potent PTPN2/N1 dual heterobifunctional degraders (Cmpd-1 and Cmpd-2) which facilitate efficient complex assembly with E3 ubiquitin ligase CRL4CRBN, and mediate potent PTPN2/N1 degradation in cells and mice. To provide mechanistic insights into the cooperative complex formation introduced by degraders, we employed a combination of structural approaches. Our crystal structure reveals how PTPN2 is recognized by the tri-substituted thiophene moiety of the degrader. We further determined a high-resolution structure of DDB1-CRBN/Cmpd-1/PTPN2 using single-particle cryo-electron microscopy (cryo-EM). This structure reveals that the degrader induces proximity between CRBN and PTPN2, albeit the large conformational heterogeneity of this ternary complex. The molecular dynamic (MD)-simulations constructed based on the cryo-EM structure exhibited a large rigid body movement of PTPN2 and illustrated the dynamic interactions between PTPN2 and CRBN. Together, our study demonstrates the development of PTPN2/N1 heterobifunctional degraders with potential applications in cancer immunotherapy. Furthermore, the developed structural workflow could help to understand the dynamic nature of degrader-induced cooperative ternary complexes.
    DOI:  https://doi.org/10.1038/s42004-024-01263-7
  32. Cell Chem Biol. 2024 Aug 15. pii: S2451-9456(24)00315-5. [Epub ahead of print]31(8): 1391-1393
    Feel the breeze: Opening the therapeutic window with RIPTACs and induced proximity.
    Kyle Mangano, Patrick Ryan Potts.
      In this issue of Cell Chemical Biology, Raina et al.1 demonstrate proof of concept of a new chemical induced proximity strategy for targeted cancer therapeutics. Building on a recent surge in induced proximity modalities, RIPTACs represent a novel approach that offers promise in treating cancers with improved safety profiles.
    DOI:  https://doi.org/10.1016/j.chembiol.2024.07.013
  33. FEBS Lett. 2024 Aug 22.
    Structural basis of sugar recognition by SCFFBS2 ubiquitin ligase involved in NGLY1 deficiency.
    Tadashi Satoh, Maho Yagi-Utsumi, Nozomi Ishii, Tsunehiro Mizushima, Hirokazu Yagi, Ryuichi Kato, Yuriko Tachida, Hiroaki Tateno, Ichiro Matsuo, Koichi Kato, Tadashi Suzuki, Yukiko Yoshida.
      The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.
    Keywords:  FBS2; NGLY1; NMR; N‐glycan; SBDD; crystal structure
    DOI:  https://doi.org/10.1002/1873-3468.15003
  34. FEBS Lett. 2024 Aug 16.
    The unfolded protein response sensor PERK mediates mechanical stress-induced maturation of focal adhesion complexes in glioblastoma cells.
    Mohammad Khoonkari, Dong Liang, Marleen Kamperman, Patrick van Rijn, Frank A E Kruyt.
      Stiffening of the brain extracellular matrix (ECM) in glioblastoma promotes tumor progression. Previously, we discovered that protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) plays a role in glioblastoma stem cell (GSC) adaptation to matrix stiffness through PERK/FLNA-dependent F-actin remodeling. Here, we examined the involvement of PERK in detecting stiffness changes via focal adhesion complex (FAC) formation. Compared to control GSCs, PERK-deficient GSCs show decreased vinculin and tensin expression, while talin and integrin-β1 remain constant. Furthermore, vimentin was also reduced while tubulin increased, and a stiffness-dependent increase of the differentiation marker GFAP expression was absent in PERK-deficient GSCs. In conclusion, our study reveals a novel role for PERK in FAC formation during matrix stiffening, which is likely linked to its regulation of F-actin remodeling.
    Keywords:  extracellular matrix; focal adhesion complex; glioblastoma; mechanical stress; unfolded protein response
    DOI:  https://doi.org/10.1002/1873-3468.14996
  35. Cell Rep. 2024 Aug 22. pii: S2211-1247(24)01012-X. [Epub ahead of print]43(9): 114661
    Local protein synthesis at neuromuscular synapses is required for motor functions.
    Wen-Yo Tu, Wentao Xu, Lei Bai, Jun Liu, Yi Han, Benyan Luo, Bingwei Wang, Kejing Zhang, Chengyong Shen.
      Motor neurons are highly polarized, and their axons extend over great distances to form connections with myofibers via neuromuscular junctions (NMJs). Local translation at the NMJs in vivo has not been identified. Here, we utilized motor neuron-labeled RiboTag mice and the TRAP (translating ribosome affinity purification) technique to spatiotemporally profile the translatome at NMJs. We found that mRNAs associated with glucose catabolism, synaptic connection, and protein homeostasis are enriched at presynapses. Local translation at the synapse shifts from the assembly of cytoskeletal components during early developmental stages to energy production in adulthood. The mRNA of neuronal Agrin (Agrn), the key molecule for NMJ assembly, is present at motor axon terminals and locally translated. Disrupting the axonal location of Agrn mRNA causes impairment of synaptic transmission and motor functions in adult mice. Our findings indicate that spatiotemporal regulation of mRNA local translation at NMJs plays critical roles in synaptic transmission and motor functions in vivo.
    Keywords:  CP: Neuroscience
    DOI:  https://doi.org/10.1016/j.celrep.2024.114661
  36. Nat Commun. 2024 Aug 16. 15(1): 7067
    High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.
    Qishan Liang, Tao Yu, Eric Kofman, Pratibha Jagannatha, Kevin Rhine, Brian A Yee, Kevin D Corbett, Gene W Yeo.
      RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.
    DOI:  https://doi.org/10.1038/s41467-024-50363-4
  37. PLoS One. 2024 ;19(8): e0307312
    Proteome-scale prediction of molecular mechanisms underlying dominant genetic diseases.
    Mihaly Badonyi, Joseph A Marsh.
      Many dominant genetic disorders result from protein-altering mutations, acting primarily through dominant-negative (DN), gain-of-function (GOF), and loss-of-function (LOF) mechanisms. Deciphering the mechanisms by which dominant diseases exert their effects is often experimentally challenging and resource intensive, but is essential for developing appropriate therapeutic approaches. Diseases that arise via a LOF mechanism are more amenable to be treated by conventional gene therapy, whereas DN and GOF mechanisms may require gene editing or targeting by small molecules. Moreover, pathogenic missense mutations that act via DN and GOF mechanisms are more difficult to identify than those that act via LOF using nearly all currently available variant effect predictors. Here, we introduce a tripartite statistical model made up of support vector machine binary classifiers trained to predict whether human protein coding genes are likely to be associated with DN, GOF, or LOF molecular disease mechanisms. We test the utility of the predictions by examining biologically and clinically meaningful properties known to be associated with the mechanisms. Our results strongly support that the models are able to generalise on unseen data and offer insight into the functional attributes of proteins associated with different mechanisms. We hope that our predictions will serve as a springboard for researchers studying novel variants and those of uncertain clinical significance, guiding variant interpretation strategies and experimental characterisation. Predictions for the human UniProt reference proteome are available at https://osf.io/z4dcp/.
    DOI:  https://doi.org/10.1371/journal.pone.0307312
  38. Proc Natl Acad Sci U S A. 2024 Aug 27. 121(35): e2401743121
    Conserved 5-methyluridine tRNA modification modulates ribosome translocation.
    Joshua D Jones, Monika K Franco, Rachel N Giles, Daniel E Eyler, Mehmet Tardu, Tyler J Smith, Laura R Snyder, Yury S Polikanov, Robert T Kennedy, Rachel O Niederer, Kristin S Koutmou.
      While the centrality of posttranscriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one of the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA in Escherichia coli, Trm2 in Saccharomyces cerevisiae) exhibit altered tRNA modification patterns. Furthermore, m5U54-deficient tRNAs are desensitized to small molecules that prevent translocation in vitro. This finding is consistent with our observations that relative to wild-type cells, trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.
    Keywords:  modification; protein synthesis; tRNA; translation
    DOI:  https://doi.org/10.1073/pnas.2401743121
  39. Cell. 2024 Aug 15. pii: S0092-8674(24)00838-9. [Epub ahead of print]
    The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.
    Yixuan Xie, Peiyuan Chai, Nicholas A Till, Helena Hemberger, Charlotta G Lebedenko, Jennifer Porat, Christopher P Watkins, Reese M Caldwell, Benson M George, Jonathan Perr, Carolyn R Bertozzi, Benjamin A Garcia, Ryan A Flynn.
      GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.
    Keywords:  RNA modifications; acp3U; cell surface; glycoRNA
    DOI:  https://doi.org/10.1016/j.cell.2024.07.044
  40. Immunity. 2024 Aug 07. pii: S1074-7613(24)00368-6. [Epub ahead of print]
    Rapid phagosome isolation enables unbiased multiomic analysis of human microglial phagosomes.
    Emile Wogram, Felix Sümpelmann, Wentao Dong, Eshaan Rawat, Inés Fernández Maestre, Dongdong Fu, Brandyn Braswell, Andrew Khalil, Joerg M Buescher, Gerhard Mittler, Georg H H Borner, Andreas Vlachos, Stefan Tholen, Oliver Schilling, George W Bell, Angelika S Rambold, Asifa Akhtar, Oliver Schnell, Jürgen Beck, Monther Abu-Remaileh, Marco Prinz, Rudolf Jaenisch.
      Microglia are the resident macrophages of the central nervous system (CNS). Their phagocytic activity is central during brain development and homeostasis-and in a plethora of brain pathologies. However, little is known about the composition, dynamics, and function of human microglial phagosomes under homeostatic and pathological conditions. Here, we developed a method for rapid isolation of pure and intact phagosomes from human pluripotent stem cell-derived microglia under various in vitro conditions, and from human brain biopsies, for unbiased multiomic analysis. Phagosome profiling revealed that microglial phagosomes were equipped to sense minute changes in their environment and were highly dynamic. We detected proteins involved in synapse homeostasis, or implicated in brain pathologies, and identified the phagosome as the site where quinolinic acid was stored and metabolized for de novo nicotinamide adenine dinucleotide (NAD+) generation in the cytoplasm. Our findings highlight the central role of phagosomes in microglial functioning in the healthy and diseased brain.
    Keywords:  glioblastoma; human pluripotent stem cells; metabolomics; microglia; organelle; phagocytosis; phagosome; proteomics; quinolinic acid; synaptic pruning
    DOI:  https://doi.org/10.1016/j.immuni.2024.07.019
  41. Mol Cell. 2024 Aug 15. pii: S1097-2765(24)00631-2. [Epub ahead of print]
    Proteasome resides in and dismantles plant heat stress granules constitutively.
    Zhouli Xie, Shuai Zhao, Yuchen Tu, Enhui Liu, Ying Li, Xingwei Wang, Changtian Chen, Shuwei Zhai, Jie Qi, Chengyun Wu, Honghong Wu, Mian Zhou, Wei Wang.
      Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly.
    Keywords:  heat stress; proteasome; stress granule
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.033
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