bims-proteo Biomed News
on Proteostasis
Issue of 2024‒10‒13
48 papers selected by
Eric Chevet, INSERM
  1. Chemical inhibition of the integrated stress response impairs the ubiquitin-proteasome system.
  2. Mechanism of degrader-targeted protein ubiquitinability.
  3. SIGMAR1/Sigma-1 receptor: a key regulator in stabilizing and translating LC3B mRNA for autophagosome formation.
  4. Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging.
  5. Ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant.
  6. HSP70 inhibits CHIP E3 ligase activity to maintain germline function in Caenorhabditis elegans.
  7. Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins.
  8. Rationalize the Functional Roles of Protein-Protein Interactions in Targeted Protein Degradation by Kinetic Monte-Carlo Simulations.
  9. Discovery of electrophilic degraders that exploit SNAr chemistry.
  10. The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.
  11. Trim21 modulates endoplasmic reticulum-associated degradation and sensitizes cancer cells to ER stress-induced apoptosis by inhibiting VCP/Npl4/UFD1 assembly.
  12. The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.
  13. The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy.
  14. Structural basis of mRNA decay by the human exosome-ribosome supercomplex.
  15. Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress.
  16. CYpHER: catalytic extracellular targeted protein degradation with high potency and durable effect.
  17. The hGIDGID4 E3 ubiquitin ligase complex targets ARHGAP11A to regulate cell migration.
  18. DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids.
  19. The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.
  20. Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability.
  21. Advances in molecular glues: exploring chemical space and design principles for targeted protein degradation.
  22. PROTAC-mediated activation, rather than degradation, of a nuclear receptor reveals complex ligand-receptor interaction network.
  23. The endoplasmic reticulum as an active liquid network.
  24. Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.
  25. An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.
  26. Advances in designing ternary complexes: Integrating in-silico and biochemical methods for PROTAC optimisation in target protein degradation.
  27. Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.
  28. Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion.
  29. Better Together: Interorganellar Communication in the Regulation of Proteostasis.
  30. DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.
  31. Phospholipase D3 regulates TFEB/TFE3 metabolism to maintain lysosomal homeostasis.
  32. Organellular imaging in vivo reveals a depletion of endoplasmic reticular calcium during post-ictal cortical spreading depolarization.
  33. How ribosomes shape protein folding.
  34. Mechanism of phospho-Ubls' specificity and conformational changes that regulate Parkin activity.
  35. Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.
  36. Lysosome-targeting chimeras containing an endocytic signaling motif trigger endocytosis and lysosomal degradation of cell-surface proteins.
  37. Biomolecular condensates mediate bending and scission of endosome membranes.
  38. Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs.
  39. Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis.
  40. Dual-ligand PROTACS mediate superior target protein degradation in vitro and therapeutic efficacy in vivo.
  41. Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection.
  42. Ribosomes hibernate on mitochondria during cellular stress.
  43. Protocols for identifying endogenous interactors of RNA-binding proteins in mammalian cells using the peroxidase APEX2 biotin-labeling method.
  44. Multi-omic human pancreatic islet endoplasmic reticulum and cytokine stress response mapping provides type 2 diabetes genetic insights.
  45. Mechanisms for assembly of the nucleoplasmic reticulum.
  46. Unmasking AlphaFold to integrate experiments and predictions in multimeric complexes.
  47. Stress granules are not present in Kras mutant cancers and do not control tumor growth.
  48. Coronavirus envelope protein activates TMED10-mediated unconventional secretion of inflammatory factors.
  1. Commun Biol. 2024 Oct 08. 7(1): 1282
    Chemical inhibition of the integrated stress response impairs the ubiquitin-proteasome system.
    Shanshan Xu, Maria E Gierisch, Enrica Barchi, Ina Poser, Simon Alberti, Florian A Salomons, Nico P Dantuma.
      Inhibitors of the integrated stress response (ISR) have been used to explore the potential beneficial effects of reducing the activation of this pathway in diseases. As the ISR is in essence a protective response, there is, however, a risk that inhibition may compromise the cell's ability to restore protein homeostasis. Here, we show that the experimental compound ISRIB impairs degradation of proteins by the ubiquitin-proteasome system (UPS) during proteotoxic stress in the cytosolic, but not nuclear, compartment. Accumulation of a UPS reporter substrate that is intercepted by ribosome quality control was comparable to the level observed after blocking the UPS with a proteasome inhibitor. Consistent with impairment of the cytosolic UPS, ISRIB treatment caused an accumulation of polyubiquitylated and detergent insoluble defective ribosome products (DRiPs) in the presence of puromycin. Our data suggest that the persistent protein translation during proteotoxic stress in the absence of a functional ISR increases the pool of DRiPs, thereby hindering the efficient clearance of cytosolic substrates by the UPS.
    DOI:  https://doi.org/10.1038/s42003-024-06974-0
  2. Sci Adv. 2024 Oct 11. 10(41): eado6492
    Mechanism of degrader-targeted protein ubiquitinability.
    Charlotte Crowe, Mark A Nakasone, Sarah Chandler, Conner Craigon, Gajanan Sathe, Michael H Tatham, Nikolai Makukhin, Ronald T Hay, Alessio Ciulli.
      Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4BD2 toward UBE2R1-ubiquitin, and Lys456 at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4BD2, with cellular degradation and ubiquitinomics confirming the importance of Lys456 and nearby Lys368/Lys445, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4BD2 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.
    DOI:  https://doi.org/10.1126/sciadv.ado6492
  3. Autophagy. 2024 Oct 10. 1-3
    SIGMAR1/Sigma-1 receptor: a key regulator in stabilizing and translating LC3B mRNA for autophagosome formation.
    Yu-Jie Chen, Jeffrey Knupp, Emily Wang, Peter Arvan, Billy Tsai.
      Macroautophagy/autophagy degrades and recycles cellular constituents via the lysosome to maintain cellular homeostasis. Our study identified the endoplasmic reticulum (ER)-resident SIGMAR1 (sigma non-opioid intracellular receptor 1) as a critical regulator of the biosynthesis of Atg8-family proteins that leads to the lipidation that is essential during autophagosome formation. We demonstrate that SIGMAR1 stabilizes MAP1LC3B/LC3B and GABARAP mRNAs, promoting their localized translation proximal to the ER for efficient lipidation. Using single-molecule fluorescence in situ hybridization/smFISH and co-immunoprecipitation, we found that SIGMAR1 directly binds to a conserved region in the 3' UTR of LC3B mRNA, facilitating its translation, efficient lipidation, and proper integration into the phagophore membrane. Cells lacking SIGMAR1 show reduced levels of many Atg8-family proteins and impaired autophagic flux. Our model suggests that SIGMAR1-mediated localized translation of Atg8-family proteins at the ER promotes efficient autophagosome formation, in contrast to recruiting preexisting cytosolic Atg8-family proteins to the lipidation machinery. Elucidating the role of SIGMAR1 in autophagy may provide better therapeutic strategies to prevent or treat autophagy-dependent neurodegenerative diseases, particularly given the highly druggable nature of SIGMAR1.
    Keywords:  ATG8; LC3; SIGMAR1; autophagy; lipidation; localized translation
    DOI:  https://doi.org/10.1080/15548627.2024.2413313
  4. Cell Rep Methods. 2024 Oct 03. pii: S2667-2375(24)00258-3. [Epub ahead of print] 100875
    Transgenic sensors reveal compartment-specific effects of aggregation-prone proteins on subcellular proteostasis during aging.
    Michelle Curley, Mamta Rai, Chia-Lung Chuang, Vishwajeeth Pagala, Anna Stephan, Zane Coleman, Maricela Robles-Murguia, Yong-Dong Wang, Junmin Peng, Fabio Demontis.
      Loss of proteostasis is a hallmark of aging that underlies many age-related diseases. Different cell compartments experience distinctive challenges in maintaining protein quality control, but how aging regulates subcellular proteostasis remains underexplored. Here, by targeting the misfolding-prone FlucDM luciferase to the cytoplasm, mitochondria, and nucleus, we established transgenic sensors to examine subcellular proteostasis in Drosophila. Analysis of detergent-insoluble and -soluble levels of compartment-targeted FlucDM variants indicates that thermal stress, cold shock, and pro-longevity inter-organ signaling differentially affect subcellular proteostasis during aging. Moreover, aggregation-prone proteins that cause different neurodegenerative diseases induce a diverse range of outcomes on FlucDM insolubility, suggesting that subcellular proteostasis is impaired in a disease-specific manner. Further analyses with FlucDM and mass spectrometry indicate that pathogenic tauV337M produces an unexpectedly complex regulation of solubility for different FlucDM variants and protein subsets. Altogether, compartment-targeted FlucDM sensors pinpoint a diverse modulation of subcellular proteostasis by aging regulators.
    Keywords:  CP: cell biology; CP: molecular biology; cell compartments; inter-tissue signaling; myokines; organelles; protein quality control; subcellular proteostasis; tools for aging research
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100875
  5. Proc Natl Acad Sci U S A. 2024 Oct 15. 121(42): e2414768121
    Ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant.
    Patrick J Carmody, Francis J Roushar, Austin Tedman, Wei Wang, Madeline Herwig, Minsoo Kim, Eli F McDonald, Karen Noguera, Jennifer Wong-Roushar, Jon-Luc Poirier, Nathan B Zelt, Ben T Pockrass, Andrew G McKee, Charles P Kuntz, S Vamsee Raju, Lars Plate, Wesley D Penn, Jonathan P Schlebach.
      The cotranslational misfolding of the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) plays a central role in the molecular basis of CF. The misfolding of the most common CF variant (ΔF508) remodels both the translational regulation and quality control of CFTR. Nevertheless, it is unclear how the misassembly of the nascent polypeptide may directly influence the activity of the translation machinery. In this work, we identify a structural motif within the CFTR transcript that stimulates efficient -1 ribosomal frameshifting and triggers the premature termination of translation. Though this motif does not appear to impact the interactome of wild-type CFTR, silent mutations that disrupt this RNA structure alter the association of nascent ΔF508 CFTR with numerous translation and quality control proteins. Moreover, disrupting this RNA structure enhances the functional gating of the ΔF508 CFTR channel at the plasma membrane and its pharmacological rescue by the CFTR modulators contained in the CF drug Trikafta. The effects of the RNA structure on ΔF508 CFTR appear to be attenuated in the absence of the ER membrane protein complex, which was previously found to modulate ribosome collisions during "preemptive quality control" of a misfolded CFTR homolog. Together, our results reveal that ribosomal frameshifting selectively modulates the assembly, function, and pharmacological rescue of a misfolded CFTR variant. These findings suggest that interactions between the nascent chain, quality control machinery, and ribosome may dynamically modulate ribosomal frameshifting in order to tune the processivity of translation in response to cotranslational misfolding.
    Keywords:  CFTR; membrane protein folding; programmed ribosomal frameshifting; proteostasis; ribosome
    DOI:  https://doi.org/10.1073/pnas.2414768121
  6. J Biol Chem. 2024 Oct 07. pii: S0021-9258(24)02366-4. [Epub ahead of print] 107864
    HSP70 inhibits CHIP E3 ligase activity to maintain germline function in Caenorhabditis elegans.
    Pankaj Thapa, Rupesh V Chikale, Natalia A Szulc, Maria-Teodora Pandrea, Agnieszka Sztyler, Khushboo Jaggi, Marta Niklewicz, Remigiusz A Serwa, Thorsten Hoppe, Wojciech Pokrzywa.
      The ubiquitin-proteasome system is crucial for proteostasis, particularly during proteotoxic stress. The interaction between heat shock protein 70 (HSP70) and the ubiquitin ligase CHIP plays a key role in this process. Our study investigates the C. elegans orthologs HSP-1 and CHN-1, demonstrating that HSP-1 binding decreases CHN-1 E3 ligase activity, aligning with the inhibitory effects observed in human HSP70-CHIP interactions. To explore the physiological significance of this inhibition, we utilized the HSP-1EEYD mutant, which binds CHN-1 without reducing its activity, expressed in C. elegans. Our results reveal that the HSP-1-CHN-1 interaction is critical for maintaining germline integrity under heat stress by preventing excessive turnover of essential reproductive proteins. In HSP-1EEYD nematodes, this protective mechanism is impaired, leading to disrupted stress-induced apoptosis, which is restored by CHN-1 depletion. Additionally, proteomic analysis identified DAF-18/PTEN as a potential CHN-1 substrate, which becomes destabilized when CHN-1 activity is not downregulated by HSP-1 during stress. Depleting DAF-18 significantly compromises the reproductive benefits observed from CHN-1 knockout in HSP-1EEYD mutants, suggesting that the maintenance of DAF-18 plays a role in the observed phenotypes. These findings highlight the importance of HSP-1 in regulating CHN-1 E3 ligase activity to preserve germline function under stress conditions.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107864
  7. Mol Cell Proteomics. 2024 Oct 08. pii: S1535-9476(24)00144-0. [Epub ahead of print] 100854
    Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins.
    Simone Bonelli, Margot Lo Pinto, Yihong Ye, Stephan A Mueller, Stefan F Lichtenthaler, Simone D Scilabra.
      Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.
    Keywords:  legumain; lysosomal exocytosis; proteomics; secretory autophagy; ubiquitin carboxyl-terminal hydrolase 19; unconventional secretion
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100854
  8. bioRxiv. 2024 Sep 28. pii: 2024.09.26.615190. [Epub ahead of print]
    Rationalize the Functional Roles of Protein-Protein Interactions in Targeted Protein Degradation by Kinetic Monte-Carlo Simulations.
    Zhaoqian Su, Shanye Yin, Yinghao Wu.
      Targeted protein degradation is a promising therapeutic strategy to tackle disease-causing proteins that lack binding pockets for traditional small-molecule inhibitors. Its first step is to trigger the proximity between a ubiquitin ligase complex and a target protein through a heterobifunctional molecule, such as proteolysis targeting chimeras (PROTACs), leading to the formation of a ternary complex. The properties of protein-protein interactions play an important regulatory role during this process, which can be reflected by binding cooperativity. Unfortunately, although computer-aided drug design has become a cornerstone of modern drug development, the endeavor to model targeted protein degradation is still in its infancy. The development of computational tools to understand the impacts of protein-protein interactions on targeted protein degradation, therefore, is highly demanded. To reach this goal, we constructed a non-redundant structural benchmark of the most updated ternary complexes and applied a kinetic Monte-Carlo method to simulate the association between ligases and PROTAC-targeted proteins in the benchmark. Our results show that proteins in most complexes with positive cooperativity tend to associate into native-like configurations more often. In contrast, proteins very likely failed to associate into native-like configurations in complexes with negative cooperativity. Moreover, we compared the protein-protein association through different interfaces generated from molecular docking. The native-like binding interface shows a higher association probability than all the other alternative interfaces only in the complex with positive cooperativity. These observations support the idea that the formation of ternary complexes is closely regulated by the binary interactions between proteins. Finally, we applied our method to cyclin-dependent kinases 4 and 6 (CDK4/6). We found that their interactions with the ligase are not as similar as their structures. Altogether, our study paves the way for understanding the role of protein-protein interactions in PROTACE-induced ternary complex formation. It can potentially help in searching for degraders that selectively target specific proteins.
    DOI:  https://doi.org/10.1101/2024.09.26.615190
  9. bioRxiv. 2024 Sep 27. pii: 2024.09.25.615094. [Epub ahead of print]
    Discovery of electrophilic degraders that exploit SNAr chemistry.
    Zhe Zhuang, Woong Sub Byun, Zuzanna Kozicka, Brendan G Dwyer, Katherine A Donovan, Zixuan Jiang, Hannah M Jones, Dinah M Abeja, Meredith N Nix, Jianing Zhong, Mikolaj Slabicki, Eric S Fischer, Benjamin L Ebert, Nathanael S Gray.
      Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly 'undruggable' targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.
    DOI:  https://doi.org/10.1101/2024.09.25.615094
  10. J Cell Sci. 2024 Oct 07. pii: jcs.261810. [Epub ahead of print]
    The lysosomal lipid transporter LIMP-2/SCARB2 is part of lysosome-endoplasmic reticulum STARD3-VAPB-dependent contact sites.
    Sönke Rudnik, Saskia Heybrock, Etienne Coyaud, Zizhen Xu, Dante Neculai, Brian Raught, Viola Oorschot, Cecilia Heus, Judith Klumperman, Paul Saftig.
      SCARB2/LIMP-2 is an abundant lysosomal membrane protein. Previous studies have shown LIMP-2 functions as a virus receptor, a chaperone for lysosomal enzyme targeting, and a lipid transporter. The large luminal domain of LIMP-2 contains a hydrophobic tunnel that enables transport of phospholipids, sphingosine and cholesterol from the lysosomal lumen to the membrane. The question about the fate of the lipids after LIMP-2-mediated transport is largely unexplored. To elucidate whether LIMP-2 is part of contact sites between lysosomes and the endoplasmic reticulum (ER), we performed a proximity-based interaction screen. This revealed that LIMP-2 interacts with the endosomal protein STARD3 and the ER-resident protein VAPB. Using imaging and co-immunoprecipitation, we demonstrated colocalization and physical interaction between LIMP-2 and these proteins. Moreover, we found that interaction of LIMP-2 with VAPB required the presence of STARD3. Our findings suggest that LIMP-2 is part of ER-lysosome contact sites, possibly facilitating cholesterol transport from the lysosomal to the ER membrane. This suggests a novel mechanism for inter-organelle communication and lipid trafficking mediated by LIMP-2.
    Keywords:  Endoplasmic reticulum; LIMP-2; Lysosome; Membrane contact sites; SCARB2; STARD3; VAPB
    DOI:  https://doi.org/10.1242/jcs.261810
  11. Biochim Biophys Acta Mol Basis Dis. 2024 Oct 03. pii: S0925-4439(24)00527-1. [Epub ahead of print]1871(1): 167533
    Trim21 modulates endoplasmic reticulum-associated degradation and sensitizes cancer cells to ER stress-induced apoptosis by inhibiting VCP/Npl4/UFD1 assembly.
    Chao Yuan, Yanli Liao, WenXia Si, Mi Huang, Duanzhuo Li, Fuqing Wang, Yi Quan, Xin Yu, Shengjie Liao.
      Endoplasmic reticulum-associated degradation (ERAD) serves as a crucial quality and quantity control system that removes misfolded or unassembled proteins from the Endoplasmic Reticulum (ER) through the cytoplasmic ubiquitin-proteasome system (UPS), which is critical for cell fate decision. ER stress arises when misfolded proteins accumulated within the ER lumen, potentially leading to cell death via proapoptotic unfolded protein response (UPR). UFD1 in associated with VCP-Npl4, is recognized as a key regulator of protein homeostasis in ERAD. However, the factors that control VCP complex assembly remain unclear. The study elucidates the function of Trim21, an E3 ubiquitin ligase, through its interaction with UFD1, facilitating K27-linkage ubiquitination of UFD1 and inhibiting its incorporation into the VCP complex. This results in the suppression of ERAD substrates degradation and the activation of a proapoptotic unfolded protein response in cancer cells. Additionally, Trim21 over-expression enhances ER stress response and promotes apoptosis upon expose to the ER inducer Tunicamycin. Notably, elevated Trim21 expression correlates with improved overall survival in various tumor types. Overall, the findings highlight the critical role of Trim21 in regulating ERAD progression and cell fate determination in cancer cells through modulation of VCP/Npl4/UFD1 complex assembly.
    Keywords:  Endoplasmic reticulum-associated degradation; Proapoptotic unfolded protein response; Trim21; UFD1; Ubiquitination
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167533
  12. Nat Commun. 2024 Oct 05. 15(1): 8655
    The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.
    Yoko Shibata, Emily E Mazur, Buyan Pan, Joao A Paulo, Steven P Gygi, Suyog Chavan, L Sebastian Alexis Valerio, Jiuchun Zhang, Tom A Rapoport.
      The endoplasmic reticulum (ER) is shaped by abundant membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of four proteins in mammals (REEP1-4), is less abundant and lack a N-terminal region. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but the function of these four REEP proteins remains enigmatic. Here we show that REEP1-4 reside in a unique vesicular compartment and identify features that determine their localization. Mutations in REEP1-4 that compromise curvature generation, including those causing disease, relocalize the proteins to the bulk ER. These mutants interact with wild-type proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1 vesicles contain the membrane fusogen atlastin-1, but not general ER proteins. We propose that REEP1-4 generate these vesicles themselves by budding from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics.
    DOI:  https://doi.org/10.1038/s41467-024-52901-6
  13. bioRxiv. 2024 Sep 26. pii: 2024.09.24.614749. [Epub ahead of print]
    The p97-UBXD8 complex maintains peroxisome abundance by suppressing pexophagy.
    Iris D Montes, Suganthan Amirthagunanathan, Amit S Joshi, Malavika Raman.
      Peroxisomes are vital organelles involved in key metabolic functions in eukaryotic cells. Their significance is highlighted by peroxisome biogenesis disorders; severe childhood diseases marked by disrupted lipid metabolism. One mechanism regulating peroxisome abundance is through selective ubiquitylation of peroxisomal membrane proteins that triggers peroxisome degradation via selective autophagy (pexophagy). However, the mechanisms regulating pexophagy remain poorly understood in mammalian cells. Here we show that the evolutionarily conserved AAA-ATPase p97 and its membrane embedded adaptor UBXD8 are essential for maintaining peroxisome abundance. From quantitative proteomic studies we reveal that loss of UBXD8 affects many peroxisomal proteins. We find depletion of UBXD8 results in a loss of peroxisomes in a manner that is independent of the known role of UBXD8 in ER associated degradation (ERAD). Loss of UBXD8 or inhibition of p97 increases peroxisomal turnover through autophagy and can be rescued by depleting key autophagy proteins or overexpressing the deubiquitylating enzyme USP30. Furthermore, we find increased ubiquitylation of the peroxisomal membrane protein PMP70 in cells lacking UBXD8 or p97. Collectively, our findings identify a new role for the p97-UBXD8 complex in regulating peroxisome abundance by suppressing pexophagy.
    DOI:  https://doi.org/10.1101/2024.09.24.614749
  14. Nature. 2024 Oct 09.
    Structural basis of mRNA decay by the human exosome-ribosome supercomplex.
    Alexander Kögel, Achim Keidel, Matina-Jasemi Loukeri, Christopher C Kuhn, Lukas M Langer, Ingmar B Schäfer, Elena Conti.
      The interplay between translation and mRNA decay is widespread in human cells1-3. In quality-control pathways, exonucleolytic degradation of mRNA associated with translating ribosomes is mediated largely by the cytoplasmic exosome4-9, which includes the exoribonuclease complex EXO10 and the helicase complex SKI238 (refs. 10-16). The helicase can extract mRNA from the ribosome and is expected to transfer it to the exoribonuclease core through a bridging factor, HBS1L3 (also known as SKI7), but the mechanisms of this molecular handover remain unclear7,17,18. Here we reveal how human EXO10 is recruited by HBS1L3 (SKI7) to an active ribosome-bound SKI238 complex. We show that rather than a sequential handover, a direct physical coupling mechanism takes place, which culminates in the formation of a cytoplasmic exosome-ribosome supercomplex. Capturing the structure during active decay reveals a continuous path in which an RNA substrate threads from the 80S ribosome through the SKI2 helicase into the exoribonuclease active site of the cytoplasmic exosome complex. The SKI3 subunit of the complex directly binds to HBS1L3 (SKI7) and also engages a surface of the 40S subunit, establishing a recognition platform in collided disomes. Exosome and ribosome thus work together as a single structural and functional unit in co-translational mRNA decay, coordinating their activities in a transient supercomplex.
    DOI:  https://doi.org/10.1038/s41586-024-08015-6
  15. J Biol Chem. 2024 Oct 07. pii: S0021-9258(24)02372-X. [Epub ahead of print] 107870
    Redox control of the deubiquitinating enzyme Ubp2 regulates translation during stress.
    Clara M Santos, Blanche K Cizubu, Dinachi A Okonkwo, Chia-Yu Chen, Natori Maske, Nathan A Snyder, Vanessa Simoes, Erica J Washington, Gustavo M Silva.
      Protein ubiquitination is essential to govern cells' ability to cope with harmful environments by regulating many aspects of protein dynamics from synthesis to degradation. As important as the ubiquitination process, the reversal of ubiquitin chains mediated by deubiquitinating enzymes (DUBs) is critical for proper recovery from stress and re-establishment of proteostasis. Although it is known that ribosomes are decorated with K63-linked polyubiquitin (K63-ub) chains that control protein synthesis under stress, the mechanisms by which these ubiquitin chains are reversed and regulate proteostasis during stress recovery remain elusive. Here, we showed in budding yeast that the DUB Ubp2 is redox-regulated during oxidative stress in a reversible manner, which determines the levels of K63-ub chains present on ribosomes. We also demonstrate that Ubp2 can cleave single ubiquitin moieties out of chain and its activity is modulated by a series of repeated domains and the formation of disulfide bonds. By combining cellular, biochemical, and proteomics analyses, we showed that Ubp2 is crucial for restoring translation after stress cessation, indicating an important role in determining the cellular response to oxidative stress. Our work demonstrates a novel role for Ubp2, revealing that a range of signaling pathways can be controlled by redox regulation of DUB activity in eukaryotes, which in turn will define cellular states of health and diseases.
    Keywords:  deubiquitylation (deubiquitination); oxidative stress; redox regulation; translation control; yeast
    DOI:  https://doi.org/10.1016/j.jbc.2024.107870
  16. Nat Commun. 2024 Oct 09. 15(1): 8731
    CYpHER: catalytic extracellular targeted protein degradation with high potency and durable effect.
    Zachary R Crook, Gregory P Sevilla, Pamela Young, Emily J Girard, Tinh-Doan Phi, Monique L Howard, Jason Price, James M Olson, Natalie W Nairn.
      Many disease-causing proteins have multiple pathogenic mechanisms, and conventional inhibitors struggle to reliably disrupt more than one. Targeted protein degradation (TPD) can eliminate the protein, and thus all its functions, by directing a cell's protein turnover machinery towards it. Two established strategies either engage catalytic E3 ligases or drive uptake towards the endolysosomal pathway. Here we describe CYpHER (CatalYtic pH-dependent Endolysosomal delivery with Recycling) technology with potency and durability from a catalytic mechanism that shares the specificity and straightforward modular design of endolysosomal uptake. By bestowing pH-dependent release on the target engager and using the rapid-cycling transferrin receptor as the uptake receptor, CYpHER induces endolysosomal delivery of surface and extracellular targets while re-using drug, potentially yielding increased potency and reduced off-target tissue exposure risks. The TfR-based approach allows targeting to tumors that overexpress this receptor and offers the potential for transport to the CNS. CYpHER function was demonstrated in vitro with EGFR and PD-L1, and in vivo with EGFR in a model of EGFR-driven non-small cell lung cancer.
    DOI:  https://doi.org/10.1038/s41467-024-52975-2
  17. Life Sci Alliance. 2024 Dec;pii: e202403046. [Epub ahead of print]7(12):
    The hGIDGID4 E3 ubiquitin ligase complex targets ARHGAP11A to regulate cell migration.
    Halil Bagci, Martin Winkler, Benjamin Grädel, Federico Uliana, Jonathan Boulais, Weaam I Mohamed, Sophia L Park, Jean-François Côté, Olivier Pertz, Matthias Peter.
      The human CTLH/GID (hGID) complex emerged as an important E3 ligase regulating multiple cellular processes, including cell cycle progression and metabolism. However, the range of biological functions controlled by hGID remains unexplored. Here, we used proximity-dependent biotinylation (BioID2) to identify proteins interacting with the hGID complex, among them, substrate candidates that bind GID4 in a pocket-dependent manner. Biochemical and cellular assays revealed that the hGIDGID4 E3 ligase binds and ubiquitinates ARHGAP11A, thereby targeting this RhoGAP for proteasomal degradation. Indeed, GID4 depletion or impeding the GID4 substrate binding pocket with the PFI-7 inhibitor stabilizes ARHGAP11A protein amounts, although it carries no functional N-terminal degron. Interestingly, GID4 inactivation impairs cell motility and directed cell movement by increasing ARHGAP11A levels at the cell periphery, where it inactivates RhoA. Together, we identified a wide range of hGIDGID4 E3 ligase substrates and uncovered a unique function of the hGIDGID4 E3 ligase regulating cell migration by targeting ARHGAP11A.
    DOI:  https://doi.org/10.26508/lsa.202403046
  18. Elife. 2024 Oct 08. pii: RP98070. [Epub ahead of print]13
    DTX3L ubiquitin ligase ubiquitinates single-stranded nucleic acids.
    Emily L Dearlove, Chatrin Chatrin, Lori Buetow, Syed F Ahmed, Tobias Schmidt, Martin Bushell, Brian O Smith, Danny T Huang.
      Ubiquitination typically involves covalent linking of ubiquitin (Ub) to a lysine residue on a protein substrate. Recently, new facets of this process have emerged, including Ub modification of non-proteinaceous substrates like ADP-ribose by the DELTEX E3 ligase family. Here, we show that the DELTEX family member DTX3L expands this non-proteinaceous substrate repertoire to include single-stranded DNA and RNA. Although the N-terminal region of DTX3L contains single-stranded nucleic acid binding domains and motifs, the minimal catalytically competent fragment comprises the C-terminal RING and DTC domains (RD). DTX3L-RD catalyses ubiquitination of the 3'-end of single-stranded DNA and RNA, as well as double-stranded DNA with a 3' overhang of two or more nucleotides. This modification is reversibly cleaved by deubiquitinases. NMR and biochemical analyses reveal that the DTC domain binds single-stranded DNA and facilitates the catalysis of Ub transfer from RING-bound E2-conjugated Ub. Our study unveils the direct ubiquitination of nucleic acids by DTX3L, laying the groundwork for understanding its functional implications.
    Keywords:  DTX3L; biochemistry; chemical biology; human; nucleic aicds; ubiquitin; ubiquitin ligase
    DOI:  https://doi.org/10.7554/eLife.98070
  19. bioRxiv. 2024 Sep 26. pii: 2024.09.24.614781. [Epub ahead of print]
    The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.
    Kentson Lam, Yoon Joon Kim, Carlo M Ong, Andrea Z Liu, Fanny J Zhou, Mary Jean Sunshine, Bernadette A Chua, Silvia Vicenzi, Pierce W Ford, Jie-Hua Zhou, Yuning Hong, Eric J Bennett, Leslie A Crews, Edward D Ball, Robert A J Signer.
      Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.
    DOI:  https://doi.org/10.1101/2024.09.24.614781
  20. bioRxiv. 2024 Sep 26. pii: 2024.09.24.614842. [Epub ahead of print]
    Bulk and selective autophagy cooperate to remodel a fungal proteome in response to changing nutrient availability.
    Bertina Telusma, Jean-Claude Farre, Danica S Cui, Suresh Subramani, Joseph H Davis.
      Cells remodel their proteomes in response to changing environments by coordinating changes in protein synthesis and degradation. In yeast, such degradation involves both proteasomal and vacuolar activity, with a mixture of bulk and selective autophagy delivering many of the vacuolar substrates. Although these pathways are known to be generally important for such remodeling, their relative contributions have not been reported on a proteome-wide basis. To assess this, we developed a method to pulse-label the methylotrophic yeast Komagataella phaffii ( i . e. Pichia pastoris ) with isotopically labeled nutrients, which, when coupled to quantitative proteomics, allowed us to globally monitor protein degradation on a protein-by-protein basis following an environmental perturbation. Using genetic ablations, we found that a targeted combination of bulk and selective autophagy drove the vast majority of the observed proteome remodeling activity, with minimal non-autophagic contributions. Cytosolic proteins and protein complexes, including ribosomes, were degraded via Atg11-independent bulk autophagy, whereas proteins targeted to the peroxisome and mitochondria were primarily degraded in an Atg11-dependent manner. Notably, these degradative pathways were independently regulated by environmental cues. Taken together, our new approach greatly increases the range of known autophagic substrates and highlights the outsized impact of autophagy on proteome remodeling. Moreover, the resulting datasets, which we have packaged in an accessible online database, constitute a rich resource for identifying proteins and pathways involved in fungal proteome remodeling.
    DOI:  https://doi.org/10.1101/2024.09.24.614842
  21. Drug Discov Today. 2024 Oct 09. pii: S1359-6446(24)00330-1. [Epub ahead of print] 104205
    Advances in molecular glues: exploring chemical space and design principles for targeted protein degradation.
    S Hemant Kumar, Venkatachalapathy Muthukumaran, Ramesh Sistla, Vasanthanathan Poongavanam.
      The discovery of the E3 ligase cereblon (CRBN) as the target of thalidomide and its analogs revolutionized the field of targeted protein degradation (TPD). This ubiquitin-mediated degradation pathway was first harnessed by bivalent degraders. Recently, the emergence of low-molecular-weight molecular glue degraders (MGDs) has expanded the TPD landscape, because MGDs operate via the same mechanism while offering attractive physicochemical properties that are consistent with small-molecule therapeutics. This review delves into the discovery and advancement of MGDs, with case studies on cyclin K and the zinc finger protein IKZF2, highlighting the design principles, biological assays and therapeutic applications. Additionally, it examines the chemical space of molecular glues and outlines the collaborative efforts that are fueling innovation in this field.
    Keywords:  artificial intelligence; cereblon; cyclosporin A; molecular docking; molecule glue; protein degradation; protein–protein interactions; ubiquitination
    DOI:  https://doi.org/10.1016/j.drudis.2024.104205
  22. Structure. 2024 Sep 28. pii: S0969-2126(24)00385-X. [Epub ahead of print]
    PROTAC-mediated activation, rather than degradation, of a nuclear receptor reveals complex ligand-receptor interaction network.
    Andrew D Huber, Wenwei Lin, Shyaron Poudel, Darcie J Miller, Taosheng Chen.
      Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules containing a ligand for a protein of interest linked to an E3 ubiquitin ligase ligand that induce protein degradation through E3 recruitment to the target protein. Small changes in PROTAC linkers can have drastic consequences, including loss of degradation activity, but the structural mechanisms governing such changes are unclear. To study this phenomenon, we screened PROTACs of diverse targeting modalities and identified dTAG-13 as an activator of the xenobiotic-sensing pregnane X receptor (PXR), which promiscuously binds various ligands. Characterization of dTAG-13 analogs and precursors revealed interplay between the PXR-binding moiety, linker, and E3 ligand that altered PXR activity without inducing degradation. A crystal structure of PXR ligand binding domain bound to a precursor ligand showed ligand-induced binding pocket distortions and a linker-punctured tunnel to the protein exterior at a region incompatible with E3 complex formation, highlighting the effects of linker environment on PROTAC activity.
    Keywords:  PROTAC; cytochrome P450; drug design; metabolism; nuclear receptor; pregnane X receptor; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.str.2024.09.016
  23. Proc Natl Acad Sci U S A. 2024 Oct 15. 121(42): e2409755121
    The endoplasmic reticulum as an active liquid network.
    Zubenelgenubi C Scott, Samuel B Steen, Greg Huber, Laura M Westrate, Elena F Koslover.
      The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While individual structural elements and the morphogens that stabilize them have been described, a quantitative understanding of the dynamic large-scale network topology remains elusive. We develop a physical model of the ER as an active liquid network, governed by a balance of tension-driven shrinking and new tubule growth. This minimalist model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Several parameter-independent geometric features of the liquid network model are shown to be representative of ER architecture in live mammalian cells. The liquid network model connects the timescales of distinct dynamic features such as ring closure and new tubule growth in the ER. Furthermore, it demonstrates how the steady-state network morphology on a cellular scale arises from the balance of microscopic dynamic rearrangements.
    Keywords:  endoplasmic reticulum; networks; organelle structure; physical modeling; subcellular dynamics
    DOI:  https://doi.org/10.1073/pnas.2409755121
  24. Proc Natl Acad Sci U S A. 2024 Oct 15. 121(42): e2408249121
    Biochemical and structural insights into a 5' to 3' RNA ligase reveal a potential role in tRNA ligation.
    Yingjie Hu, Victor A Lopez, Hengyi Xu, James P Pfister, Bing Song, Kelly A Servage, Masahiro Sakurai, Benjamin T Jones, Joshua T Mendell, Tao Wang, Jun Wu, Alan M Lambowitz, Diana R Tomchick, Krzysztof Pawłowski, Vincent S Tagliabracci.
      ATP-grasp superfamily enzymes contain a hand-like ATP-binding fold and catalyze a variety of reactions using a similar catalytic mechanism. More than 30 protein families are categorized in this superfamily, and they are involved in a plethora of cellular processes and human diseases. Here, we identify C12orf29 (RLIG1) as an atypical ATP-grasp enzyme that ligates RNA. Human RLIG1 and its homologs autoadenylate on an active site Lys residue as part of a reaction intermediate that specifically ligates RNA halves containing a 5'-phosphate and a 3'-hydroxyl. RLIG1 binds tRNA in cells and can ligate tRNA within the anticodon loop in vitro. Transcriptomic analyses of Rlig1 knockout mice revealed significant alterations in global tRNA levels in the brains of female mice, but not in those of male mice. Furthermore, crystal structures of a RLIG1 homolog from Yasminevirus bound to nucleotides revealed a minimal and atypical RNA ligase fold with a conserved active site architecture that participates in catalysis. Collectively, our results identify RLIG1 as an RNA ligase and suggest its involvement in tRNA biology.
    Keywords:  C12orf29; RLIG1; RNA ligase; tRNA
    DOI:  https://doi.org/10.1073/pnas.2408249121
  25. Cell Rep Med. 2024 Oct 03. pii: S2666-3791(24)00523-8. [Epub ahead of print] 101778
    An RNA damage response network mediates the lethality of 5-FU in colorectal cancer.
    Jung-Kuei Chen, Karl A Merrick, Yi Wen Kong, Anita Izrael-Tomasevic, George Eng, Erika D Handly, Jesse C Patterson, Ian G Cannell, Lucia Suarez-Lopez, Aaron M Hosios, Anh Dinh, Donald S Kirkpatrick, Kebing Yu, Christopher M Rose, Jonathan M Hernandez, Haeun Hwangbo, Adam C Palmer, Matthew G Vander Heiden, Ömer H Yilmaz, Michael B Yaffe.
      5-fluorouracil (5-FU), a major anti-cancer therapeutic, is believed to function primarily by inhibiting thymidylate synthase, depleting deoxythymidine triphosphate (dTTP), and causing DNA damage. Here, we show that clinical combinations of 5-FU with oxaliplatin or irinotecan show no synergy in human colorectal cancer (CRC) trials and sub-additive killing in CRC cell lines. Using selective 5-FU metabolites, phospho- and ubiquitin proteomics, and primary human CRC organoids, we demonstrate that 5-FU-mediated CRC cell killing primarily involves an RNA damage response during ribosome biogenesis, causing lysosomal degradation of damaged rRNAs and proteasomal degradation of ubiquitinated ribosomal proteins. Tumor types clinically responsive to 5-FU treatment show upregulated rRNA biogenesis while 5-FU clinically non-responsive tumor types do not, instead showing greater sensitivity to 5-FU's DNA damage effects. Finally, we show that treatments upregulating ribosome biogenesis, including KDM2A inhibition, promote RNA-dependent cell killing by 5-FU, demonstrating the potential for combinatorial targeting of this ribosomal RNA damage response for improved cancer therapy.
    Keywords:  5-FU; 5-FU-based chemotherapy; RNA damage; ribosomal RNA; ribosomal protein
    DOI:  https://doi.org/10.1016/j.xcrm.2024.101778
  26. Bioorg Chem. 2024 Oct 04. pii: S0045-2068(24)00773-9. [Epub ahead of print]153 107868
    Advances in designing ternary complexes: Integrating in-silico and biochemical methods for PROTAC optimisation in target protein degradation.
    Shareef Shaik, Prasanna Kumar Reddy Gayam, Manish Chaudhary, Gurvinder Singh, Aravinda Pai.
      Target protein degradation (TPD) is an emerging approach to mitigate disease-causing proteins. TPD contains several strategies, and one of the strategies that gained immersive importance in recent times is Proteolysis Targeting Chimeras (PROTACs); the PROTACs recruit small molecules to induce the poly-ubiquitination of disease-causing protein by hijacking the ubiquitin-proteasome system (UPS) by bringing the E3 ligase and protein of interest (POI) into appropriate proximity. The steps involved in designing and evaluating the PROTACs remain critical in optimising the PROTACs to degrade the POI. It is observed that using in-silico and biochemical methods to study the ternary complexes (TCs) of the POI-PROTAC-E3 ligase is essential to understanding the structural activity, cooperativity, and stability of formed TCs. A better understanding of the above-mentioned leads to an appropriate rationale for designing the PROTACs targeting the disease-causing proteins. In this review, we tried to summarise the approaches used to design the ternary complexes, i.e., in-silico and in-vitro methods, to understand the behaviour of the PROTAC-induced ternary complexes.
    Keywords:  Degradation; Poly-Ubiquitination; Protein Of Interest; Proteolysis Targeting Chimeras (PROTACs); Ternary complexes
    DOI:  https://doi.org/10.1016/j.bioorg.2024.107868
  27. EMBO Rep. 2024 Oct 07.
    Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.
    Ana Crespillo-Casado, Prathyush Pothukuchi, Katerina Naydenova, Matthew C J Yip, Janet M Young, Jerome Boulanger, Vimisha Dharamdasani, Ceara Harper, Pierre-Mehdi Hammoudi, Elsje G Otten, Keith Boyle, Mayuri Gogoi, Harmit S Malik, Felix Randow.
      Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown. Here we report that the evolutionary history of RNF213 is indicative of repeated adaptation to diverse pathogen target structures, especially in and around its newly identified CBM20 carbohydrate-binding domain, which we have resolved by cryo-EM. We find that RNF213 forms coats on phylogenetically distant pathogens. ATP hydrolysis by RNF213's dynein-like domain is essential for coat formation on all three pathogens studied as is RZ finger-mediated E3 ligase activity for bacteria. Coat formation is not diffusion-limited but instead relies on rate-limiting initiation events and subsequent cooperative incorporation of further RNF213 molecules. We conclude that RNF213 responds to evolutionarily distant pathogens through enzymatically amplified cooperative recruitment.
    Keywords:  Host-pathogen Interaction; Innate Immunity; Pattern Recognition Receptor; Positive Selection; Ubiquitylation
    DOI:  https://doi.org/10.1038/s44319-024-00280-w
  28. Nat Commun. 2024 Oct 10. 15(1): 8777
    Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion.
    Kosuke Kamemura, Rio Kozono, Mizuki Tando, Misako Okumura, Daisuke Koga, Satoshi Kusumi, Kanako Tamai, Aoi Okumura, Sayaka Sekine, Daichi Kamiyama, Takahiro Chihara.
      VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB (Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis.
    DOI:  https://doi.org/10.1038/s41467-024-53097-5
  29. Contact (Thousand Oaks). 2024 Jan-Dec;7:7 25152564241272245
    Better Together: Interorganellar Communication in the Regulation of Proteostasis.
    Andreas Kohler, Verena Kohler.
      An extensive network of chaperones and folding factors is responsible for maintaining a functional proteome, which is the basis for cellular life. The underlying proteostatic mechanisms are not isolated within organelles, rather they are connected over organellar borders via signalling processes or direct association via contact sites. This review aims to provide a conceptual understanding of proteostatic mechanisms across organelle borders, not focussing on individual organelles. This discussion highlights the precision of these finely tuned systems, emphasising the complicated balance between cellular protection and adaptation to stress. In this review, we discuss widely accepted aspects while shedding light on newly discovered perspectives.
    Keywords:  calcium; chaperones; protein homeostasis; protein misfolding; reactive oxygen species; stress response
    DOI:  https://doi.org/10.1177/25152564241272245
  30. EMBO J. 2024 Oct 08.
    DELE1 maintains muscle proteostasis to promote growth and survival in mitochondrial myopathy.
    Hsin-Pin Lin, Jennifer D Petersen, Alexandra J Gilsrud, Angelo Madruga, Theresa M D'Silva, Xiaoping Huang, Mario K Shammas, Nicholas P Randolph, Kory R Johnson, Yan Li, Drew R Jones, Michael E Pacold, Derek P Narendra.
      Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.
    Keywords:  Mitochondria Unfolded Protein Response (mt-UPR); Mitochondrial Disorders; Mitohormesis; Mitonuclear Communication; Mitophagy
    DOI:  https://doi.org/10.1038/s44318-024-00242-x
  31. bioRxiv. 2024 Sep 26. pii: 2024.09.26.615214. [Epub ahead of print]
    Phospholipase D3 regulates TFEB/TFE3 metabolism to maintain lysosomal homeostasis.
    Yongchao Wang, Paige Rieschick, Wilber Romero-Fernandez, Nathan Appelbaum, Cristian Carvajal-Tapia, Alena Shostak, Matthew Schrag.
      A coding variant in Phospholipase D3 ( PLD3 ) increases the risk of Alzheimer's disease (AD). PLD3 is a lysosomal protein, and endosomal and lysosomal abnormalities are linked to AD; however, the role of PLD3 in lysosomal homeostasis and its implications in AD remain poorly understood. To address this knowledge gap, we conducted comprehensive studies integrating transcriptomics, proteomics, and cell biology approaches. We observed significant enlargement of lysosomes in neurons lacking PLD3, accompanied by increased endocytosis and autophagy, but a decline in lysosomal proteolytic activity. Lysosomes of PLD3-deficient cells underwent proteome remodeling, manifested by an enrichment of proteins involved in lysosomal biogenesis, endocytosis and calcium signaling. Mechanistically, we discovered that PLD3 mediates TFEB/TFE3 degradation through the proteasome, and as a result, PLD3 deficiency leads to increased TFEB/TFE3 levels, nuclear translocation, and transcriptional activities. Notably, variants in PLD3, e.g., V232M or K486R, do not alter its impact on TFEB/TFE3 metabolism. Transcriptomic profiling further confirmed the enrichment of transcripts involved in lysosomal biogenesis, endocytosis, autophagy, mTOR signaling and AD in response to PLD3 loss. Additionally, PLD3 ablation has synergistic effects with β-amyloid in causing lysosomal abnormalities and modifying TFEB/TFE3 signaling. In conclusion, our findings demonstrate that PLD3 is involved in regulating lysosomal biogenesis via TFEB/TFE3 signaling, and lysosomal abnormalities resulting from PLD3 deficiency are potentially a risk factor for AD.
    DOI:  https://doi.org/10.1101/2024.09.26.615214
  32. bioRxiv. 2024 Sep 24. pii: 2024.09.21.614252. [Epub ahead of print]
    Organellular imaging in vivo reveals a depletion of endoplasmic reticular calcium during post-ictal cortical spreading depolarization.
    Matthew A Stern, Eric R Cole, Claire-Anne Gutekunst, Jenny J Yang, Ken Berglund, Robert E Gross.
      During cortical spreading depolarization (CSD), neurons exhibit a dramatic increase in cytosolic calcium, which may be integral to CSD-mediated seizure termination. This calcium increase greatly exceeds that during seizures, suggesting the calcium source may not be solely extracellular. Thus, we sought to determine if the endoplasmic reticulum (ER), the largest intracellular calcium store, is involved. We developed a two-photon calcium imaging paradigm to simultaneously record the cytosol and ER during seizures in awake mice. Paired with direct current recording, we reveal that CSD can manifest as a slow post-ictal cytosolic calcium wave with a concomitant depletion of ER calcium that is spatiotemporally consistent with a calcium-induced calcium release. Importantly, we observed both naturally occurring and electrically induced CSD suppressed post-ictal epileptiform activity. Collectively, this work links ER dynamics to CSD, which serves as an innate process for seizure suppression and a potential mechanism underlying therapeutic electrical stimulation for epilepsy.
    DOI:  https://doi.org/10.1101/2024.09.21.614252
  33. Nature. 2024 Oct 09.
    How ribosomes shape protein folding.
      
    Keywords:  Biophysics; Structural biology
    DOI:  https://doi.org/10.1038/d41586-024-03251-2
  34. Structure. 2024 Sep 26. pii: S0969-2126(24)00381-2. [Epub ahead of print]
    Mechanism of phospho-Ubls' specificity and conformational changes that regulate Parkin activity.
    Dipti Ranjan Lenka, Shradha Chaurasiya, Loknath Ratnakar, Atul Kumar.
      PINK1 and Parkin mutations lead to the early onset of Parkinson's disease. PINK1-mediated phosphorylation of ubiquitin (Ub), ubiquitin-like protein (NEDD8), and ubiquitin-like (Ubl) domain of Parkin activate autoinhibited Parkin E3 ligase. The mechanism of various phospho-Ubls' specificity and conformational changes leading to Parkin activation remain elusive. Herein, we show that compared to Ub, NEDD8 is a more robust binder and activator of Parkin. Structures and biophysical/biochemical data reveal specific recognition and underlying mechanisms of pUb/pNEDD8 and pUbl domain binding to the RING1 and RING0 domains, respectively. Also, pUb/pNEDD8 binding in the RING1 pocket promotes allosteric conformational changes in Parkin's catalytic domain (RING2), leading to Parkin activation. Furthermore, Parkinson's disease mutation K211N in the RING0 domain was believed to perturb Parkin activation due to loss of pUb binding. However, our data reveal allosteric conformational changes due to N211 that lock RING2 with RING0 to inhibit Parkin activity without disrupting pNEDD8/pUb binding.
    Keywords:  NEDD8; PINK1; Parkin; Parkinson's disease; RBR E3 ligase; X-ray crystallography; ubiquitin
    DOI:  https://doi.org/10.1016/j.str.2024.09.012
  35. bioRxiv. 2024 Sep 26. pii: 2024.09.24.614704. [Epub ahead of print]
    Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.
    Frances V Hundley, Miguel A Gonzalez-Lozano, Lena M Gottschalk, Aslan N K Cook, Jiuchun Zhang, Joao A Paulo, J Wade Harper.
      Plasma membrane protein degradation and recycling is regulated by the endolysosomal system, wherein endosomes bud from the plasma membrane into the cytosol and mature into degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface, influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we developed a toolkit for capturing EEA1-postive endosomes (Endo-IP) and TMEM192-positive lysosomes (Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrated its utility by revealing the endolysosomal protein landscapes for cortical-like iNeurons and stem cells. This allowed us to globally profile endocytic cargo, identifying hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.
    DOI:  https://doi.org/10.1101/2024.09.24.614704
  36. Chem Sci. 2024 Oct 01.
    Lysosome-targeting chimeras containing an endocytic signaling motif trigger endocytosis and lysosomal degradation of cell-surface proteins.
    Tong Fang, Zhenting Zheng, Na Li, Yishu Zhang, Jing Ma, Chengyu Yun, Xiaoqing Cai.
      Lysosome-targeting degradation technologies have emerged as a promising therapeutic strategy for the selective depletion of target extracellular and cell-surface proteins by harnessing a cell-surface effector protein such as lysosome-targeting receptors (LTRs) or transmembrane E3 ligases that direct lysosomal degradation. We recently developed a lysosome-targeting degradation platform termed signal-mediated lysosome-targeting chimeras (SignalTACs) that functions independently of an LTR or E3 ligase; these are engineered fusion proteins comprising a target binder, a cell-penetrating peptide (CPP), and a lysosomal sorting signal motif (P1). Herein, we present the next-generation SignalTACs containing a single endocytic signal that bypasses the need for a CPP. We demonstrate that the fusion with a 10-amino acid endocytic signaling peptide (P3) derived from the cation-independent mannose-6-phosphate receptor (CI-M6PR) induces robust internalization and lysosomal degradation of the target protein. The P3-based SignalTAC exhibited enhanced antitumor efficacy compared to the parent antibody. We envision that the fusion of the endocytic signaling peptide P3 to a target binder may allow the construction of an effective degrader for membrane-associated targets. Furthermore, mechanistic studies identified different drivers for the activities of the P3- and P1-based SignalTACs, which is expected to provide crucial insights toward the harnessing of the intrinsic signaling pathways to direct protein trafficking and degradation.
    DOI:  https://doi.org/10.1039/d4sc05093b
  37. Nature. 2024 Oct 09.
    Biomolecular condensates mediate bending and scission of endosome membranes.
    Yanning Wang, Shulin Li, Marcel Mokbel, Alexander I May, Zizhen Liang, Yonglun Zeng, Weiqi Wang, Honghong Zhang, Feifei Yu, Katharina Sporbeck, Liwen Jiang, Sebastian Aland, Jaime Agudo-Canalejo, Roland L Knorr, Xiaofeng Fang.
      Multivesicular bodies are key endosomal compartments implicated in cellular quality control through their degradation of membrane-bound cargo proteins1-3. The ATP-consuming ESCRT protein machinery mediates the capture and engulfment of membrane-bound cargo proteins through invagination and scission of multivesicular-body membranes to form intraluminal vesicles4,5. Here we report that the plant ESCRT component FREE16 forms liquid-like condensates that associate with membranes to drive intraluminal vesicle formation. We use a minimal physical model, reconstitution experiments and in silico simulations to identify the dynamics of this process and describe intermediate morphologies of nascent intraluminal vesicles. Furthermore, we find that condensate-wetting-induced line tension forces and membrane asymmetries are sufficient to mediate scission of the membrane neck without the ESCRT protein machinery or ATP consumption. Genetic manipulation of the ESCRT pathway in several eukaryotes provides additional evidence for condensate-mediated membrane scission in vivo. We find that the interplay between condensate and machinery-mediated scission mechanisms is indispensable for osmotic stress tolerance in plants. We propose that condensate-mediated scission represents a previously undescribed scission mechanism that depends on the physicomolecular properties of the condensate and is involved in a range of trafficking processes. More generally, FREE1 condensate-mediated membrane scission in multivesicular-body biogenesis highlights the fundamental role of wetting in intracellular dynamics and organization.
    DOI:  https://doi.org/10.1038/s41586-024-07990-0
  38. J Biol Chem. 2024 Oct 07. pii: S0021-9258(24)02368-8. [Epub ahead of print] 107866
    Mechanistic differences in eukaryotic initiation factor requirements for eIF4GI-driven cap-independent translation of structured mRNAs.
    Baishakhi Saha, Solomon A Haizel, Dixie J Goss.
      Protein translation is globally downregulated under stress conditions. Many proteins that are synthesized under stress conditions use a cap-independent translation initiation pathway. A subset of cellular mRNAs that encode for these proteins contain stable secondary structures within their 5' untranslated region (5'UTR), and initiate cap-independent translation using elements called Cap-Independent Translation Enhancers (CITEs) or Internal Ribosome Entry Sites (IRESs) within their 5'UTRs. The interaction among initiation factors such as eIF4E, eIF4A and eIF4GI, especially in regulating the eIF4F complex during non-canonical translation initiation of different 5'UTR mRNAs, is poorly understood. Here, equilibrium-binding assays, circular dichroism studies and in vitro translation assays were employed to elucidate the recruitment of these initiation factors to the highly structured 5'UTRs of fibroblast-growth factor 9 (FGF-9) and hypoxia inducible factor 1 subunit alpha (HIF-1α) encoding mRNAs. We showed that eIF4A and eIF4E enhanced eIF4GI's binding affinity to the uncapped 5'UTR of HIF-1α mRNA, inducing conformational changes in the protein/RNA complex. In contrast, these factors have no effect on the binding of eIF4GI to the 5'UTR of FGF-9 mRNA. Recently, Izidoro, M. S. et al. reported that the interaction of 42nt unstructured RNA to human eIF4F complex is dominated by eIF4E and ATP-bound state of eIF4A. Here we show that structured 5'UTR mRNA binding mitigates this requirement. Based on these observations, we describe two possible cap-independent translation mechanisms for FGF-9 and HIF-1α encoding mRNAs employed by cells to mitigate cellular stress conditions.
    Keywords:  5’ cap-independent translation enhancer (CITE); Cap-independent translation initiation; eIF4A; eIF4E; eIF4GI; internal ribosome entry site (IRES); structured 5’UTR mRNAs
    DOI:  https://doi.org/10.1016/j.jbc.2024.107866
  39. Cell Death Differ. 2024 Oct 09.
    Activating the NFE2L1-ubiquitin-proteasome system by DDI2 protects from ferroptosis.
    Anahita Ofoghi, Stefan Kotschi, Imke L Lemmer, Daniel T Haas, Nienke Willemsen, Batoul Bayer, Anna S Jung, Sophie Möller, Stefanie Haberecht-Müller, Elke Krüger, Natalie Krahmer, Alexander Bartelt.
      Ferroptosis is an iron-dependent, non-apoptotic form of cell death initiated by oxidative stress and lipid peroxidation. Recent evidence has linked ferroptosis to the action of the transcription factor Nuclear factor erythroid-2 derived,-like-1 (NFE2L1). NFE2L1 regulates proteasome abundance in an adaptive fashion, maintaining protein quality control to secure cellular homeostasis, but the regulation of NFE2L1 during ferroptosis and the role of the ubiquitin-proteasome system (UPS) herein are still unclear. In the present study, using an unbiased proteomic approach charting the specific ubiquitylation sites, we show that induction of ferroptosis leads to recalibration of the UPS. RSL3-induced ferroptosis inhibits proteasome activity and leads to global hyperubiquitylation, which is linked to NFE2L1 activation. As NFE2L1 resides in the endoplasmic reticulum tethered to the membrane, it undergoes complex posttranslational modification steps to become active and induce the expression of proteasome subunit genes. We show that proteolytic cleavage of NFE2L1 by the aspartyl protease DNA-damage inducible 1 homolog 2 (DDI2) is a critical step for the ferroptosis-induced feed-back loop of proteasome function. Cells lacking DDI2 cannot activate NFE2L1 in response to RSL3 and show global hyperubiquitylation. Genetic or chemical induction of ferroptosis in cells with a disrupted DDI2-NFE2L1 pathway diminishes proteasomal activity and promotes cell death. Also, treating cells with the clinical drug nelfinavir, which inhibits DDI2, sensitized cells to ferroptosis. In conclusion, our results provide new insight into the importance of the UPS in ferroptosis and highlight the role of the DDI2-NFE2L1 as a potential therapeutic target. Manipulating DDI2-NFE2L1 activity through chemical inhibition might help sensitizing cells to ferroptosis, thus enhancing existing cancer therapies.
    DOI:  https://doi.org/10.1038/s41418-024-01398-z
  40. Chem Sci. 2024 Oct 02.
    Dual-ligand PROTACS mediate superior target protein degradation in vitro and therapeutic efficacy in vivo.
    Yong Chen, Zihan Xia, Ujjwal Suwal, Pekka Rappu, Jyrki Heino, Olivier De Wever, Bruno G De Geest.
      Proteolysis targeting chimeras (PROTACs) are revolutionizing the drug development landscape due to their unique ability to selectively degrade disease-associated proteins. Conventional PROTACs are bivalent entities that induce ubiquitination and subsequent proteolysis of a chosen protein of interest (POI) by forming a ternary complex with an E3 ligase. We hypothesized that dual-ligand PROTACs, featuring two copies each of a POI ligand and an E3 ligase ligand, would facilitate the formation of high-avidity, long-lived ternary complexes inside cells, thereby increasing POI degradation potency. To this end, we developed a convergent synthesis route, using l-aspartic acid as a building block for homodimer synthesis, followed by copper-catalyzed azide-alkyne cycloaddition (CuAAC) to conjugate both dimers through a flexible linker. Dual-ligand PROTACs achieved up to a tenfold increase in degradation efficiency and a hundredfold increase in cytotoxicity in vitro across various cancer cell lines compared to their single-ligand counterparts. Furthermore, dual-ligand PROTACs sustain prolonged protein degradation, up to 60 hours after pulsing and washout. In vivo, in a mouse tumor model, the superior therapeutic activity of dual ligand PROTACs was observed.
    DOI:  https://doi.org/10.1039/d4sc03555k
  41. Mol Cell Proteomics. 2024 Oct 07. pii: S1535-9476(24)00146-4. [Epub ahead of print] 100856
    Profiling Cullin4-E3 ligases interactomes and their rewiring in influenza A virus infection.
    Guillaume Dugied, Thibaut Douche, Melanie Dos Santos, Quentin Giai Gianetto Q, Camille Cassonnet, Françoise Vuillier, Patricia Cassonnet, Yves Jacob, Sylvie van der Werf, Anastassia Komarova, Mariette Matondo, Marwah Karim, Caroline Demeret.
      Understanding the integrated regulation of cellular processes during viral infection is crucial for developing host-targeted approaches. We have previously reported that an optimal in vitro infection by influenza A (IAV) requires three components of Cullin 4-RING E3 ubiquitin ligases (CRL4) complexes, namely the DDB1 adaptor and two Substrate Recognition Factors (SRF), DCAF11 and DCAF12L1, which mediate non-degradative poly-ubiquitination of the PB2 subunit of the viral polymerase. However, the impact of IAV infection on the CRL4 interactome remains elusive. Here, using Affinity Purification coupled with Mass Spectrometry (AP-MS) approaches, we identified cellular proteins interacting with these CRL4 components in IAV-infected and non-infected contexts. IAV infection induces significant modulations in protein interactions, resulting in a global loss of DDB1 and DCAF11 interactions, and an increase in DCAF12L1-associated proteins. The distinct rewiring of CRL4's associations upon infection impacted cellular proteins involved in protein folding, ubiquitination, translation, splicing, and stress responses. Using a split-nanoluciferase-based assay, we identified direct partners of CRL4 components and via siRNA-mediated silencing validated their role in IAV infection, representing potential substrates or regulators of CRL4 complexes. Our findings unravel the dynamic remodeling of the proteomic landscape of CRL4's E3 ubiquitin ligases during IAV infection, likely involved in shaping a cellular environment conducive to viral replication and offer potential for the exploration of future host-targeted antiviral therapeutic strategies.
    Keywords:  Affinity Purification coupled with Mass Spectrometry; Cullin 4-RING E3 ubiquitin ligase; Influenza A virus; protein-protein interactions; split-nanoluciferase assay
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100856
  42. Nat Commun. 2024 Oct 08. 15(1): 8666
    Ribosomes hibernate on mitochondria during cellular stress.
    Olivier Gemin, Maciej Gluc, Higor Rosa, Michael Purdy, Moritz Niemann, Yelena Peskova, Simone Mattei, Ahmad Jomaa.
      Cell survival under nutrient-deprived conditions relies on cells' ability to adapt their organelles and rewire their metabolic pathways. In yeast, glucose depletion induces a stress response mediated by mitochondrial fragmentation and sequestration of cytosolic ribosomes on mitochondria. This cellular adaptation promotes survival under harsh environmental conditions; however, the underlying mechanism of this response remains unknown. Here, we demonstrate that upon glucose depletion protein synthesis is halted. Cryo-electron microscopy structure of the ribosomes show that they are devoid of both tRNA and mRNA, and a subset of the particles depicted a conformational change in rRNA H69 that could prevent tRNA binding. Our in situ structural analyses reveal that the hibernating ribosomes tether to fragmented mitochondria and establish eukaryotic-specific, higher-order storage structures by assembling into oligomeric arrays on the mitochondrial surface. Notably, we show that hibernating ribosomes exclusively bind to the outer mitochondrial membrane via the small ribosomal subunit during cellular stress. We identify the ribosomal protein Cpc2/RACK1 as the molecule mediating ribosomal tethering to mitochondria. This study unveils the molecular mechanism connecting mitochondrial stress with the shutdown of protein synthesis and broadens our understanding of cellular responses to nutrient scarcity and cell quiescence.
    DOI:  https://doi.org/10.1038/s41467-024-52911-4
  43. STAR Protoc. 2024 Oct 09. pii: S2666-1667(24)00533-1. [Epub ahead of print]5(4): 103368
    Protocols for identifying endogenous interactors of RNA-binding proteins in mammalian cells using the peroxidase APEX2 biotin-labeling method.
    Ryota Uozumi, Kohji Mori, Shoshin Akamine, Manabu Ikeda.
      Engineered ascorbate peroxidase, APEX2, is widely applied for the identification of intracellular molecule-molecule interaction analyses. Here, we present a protocol for identifying interactors of RNA-binding proteins (RBPs) in living HeLa cells using the APEX2 fusion construct. We describe steps for generation of RBP-APEX2, proximity biotin labeling, and preparation of labeled molecules for mass spectrometry analysis. This protocol may be applicable to other cell cultures and RBPs of interest. For complete details on the use and execution of this protocol, please refer to Uozumi et al.1.
    Keywords:  Bioinformatics; Cell Biology; Protein expression and purification; Proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2024.103368
  44. Cell Metab. 2024 Oct 04. pii: S1550-4131(24)00370-X. [Epub ahead of print]
    Multi-omic human pancreatic islet endoplasmic reticulum and cytokine stress response mapping provides type 2 diabetes genetic insights.
    Eishani K Sokolowski, Romy Kursawe, Vijay Selvam, Redwan M Bhuiyan, Asa Thibodeau, Chi Zhao, Cassandra N Spracklen, Duygu Ucar, Michael L Stitzel.
      Endoplasmic reticulum (ER) and inflammatory stress responses contribute to islet dysfunction in type 2 diabetes (T2D). Comprehensive genomic understanding of these human islet stress responses and whether T2D-associated genetic variants modulate them is lacking. Here, comparative transcriptome and epigenome analyses of human islets exposed ex vivo to these stressors revealed 30% of expressed genes and 14% of islet cis-regulatory elements (CREs) as stress responsive, modulated largely in an ER- or cytokine-specific fashion. T2D variants overlapped 86 stress-responsive CREs, including 21 induced by ER stress. We linked the rs6917676-T T2D risk allele to increased islet ER-stress-responsive CRE accessibility and allele-specific β cell nuclear factor binding. MAP3K5, the ER-stress-responsive putative rs6917676 T2D effector gene, promoted stress-induced β cell apoptosis. Supporting its pro-diabetogenic role, MAP3K5 expression correlated inversely with human islet β cell abundance and was elevated in T2D β cells. This study provides genome-wide insights into human islet stress responses and context-specific T2D variant effects.
    Keywords:  ER stress; GWAS; MAP3K5/ASK1; beta cell; cis-regulatory element; human islets; multiomics; proinflammatory cytokines; type 2 diabetes
    DOI:  https://doi.org/10.1016/j.cmet.2024.09.006
  45. Cell Mol Life Sci. 2024 Oct 05. 81(1): 415
    Mechanisms for assembly of the nucleoplasmic reticulum.
    Michael McPhee, Graham Dellaire, Neale D Ridgway.
      The nuclear envelope consists of an outer membrane connected to the endoplasmic reticulum, an inner membrane facing the nucleoplasm and a perinuclear space separating the two bilayers. The inner and outer nuclear membranes are physically connected at nuclear pore complexes that mediate selective communication and transfer of materials between the cytoplasm and nucleus. The spherical shape of the nuclear envelope is maintained by counterbalancing internal and external forces applied by cyto- and nucleo-skeletal networks, and the nuclear lamina and chromatin that underly the inner nuclear membrane. Despite its apparent rigidity, the nuclear envelope can invaginate to form an intranuclear membrane network termed the nucleoplasmic reticulum (NR) consisting of Type-I NR contiguous with the inner nuclear membrane and Type-II NR containing both the inner and outer nuclear membranes. The NR extends deep into the nuclear interior potentially facilitating communication and exchanges between the nuclear interior and the cytoplasm. This review details the evidence that NR intrusions that regulate cytoplasmic communication and genome maintenance are the result of a dynamic interplay between membrane biogenesis and remodelling, and physical forces exerted on the nuclear lamina derived from the cyto- and nucleo-skeletal networks.
    Keywords:  Calcium; DNA damage repair; Extracellular vesicles; Nuclear envelope; Nucleoplasmic reticulum; Phosphatidylcholine
    DOI:  https://doi.org/10.1007/s00018-024-05437-3
  46. Nat Commun. 2024 Oct 09. 15(1): 8724
    Unmasking AlphaFold to integrate experiments and predictions in multimeric complexes.
    Claudio Mirabello, Björn Wallner, Björn Nystedt, Stavros Azinas, Marta Carroni.
      Since the release of AlphaFold, researchers have actively refined its predictions and attempted to integrate it into existing pipelines for determining protein structures. These efforts have introduced a number of functionalities and optimisations at the latest Critical Assessment of protein Structure Prediction edition (CASP15), resulting in a marked improvement in the prediction of multimeric protein structures. However, AlphaFold's capability of predicting large protein complexes is still limited and integrating experimental data in the prediction pipeline is not straightforward. In this study, we introduce AF_unmasked to overcome these limitations. Our results demonstrate that AF_unmasked can integrate experimental information to build larger or hard to predict protein assemblies with high confidence. The resulting predictions can help interpret and augment experimental data. This approach generates high quality (DockQ score > 0.8) structures even when little to no evolutionary information is available and imperfect experimental structures are used as a starting point. AF_unmasked is developed and optimised to fill incomplete experimental structures (structural inpainting), which may provide insights into protein dynamics. In summary, AF_unmasked provides an easy-to-use method that efficiently integrates experiments to predict large protein complexes more confidently.
    DOI:  https://doi.org/10.1038/s41467-024-52951-w
  47. EMBO Rep. 2024 Oct 10.
    Stress granules are not present in Kras mutant cancers and do not control tumor growth.
    Maxime Libert, Sophie Quiquempoix, Jean S Fain, Sébastien Pyr Dit Ruys, Malak Haidar, Margaux Wulleman, Gaëtan Herinckx, Didier Vertommen, Christelle Bouchart, Tatjana Arsenijevic, Jean-Luc Van Laethem, Patrick Jacquemin.
      Stress granules (SG) are membraneless ribonucleoprotein-based cytoplasmic organelles that assemble in response to stress. Their formation is often associated with an almost global suppression of translation, and the aberrant assembly or disassembly of these granules has pathological implications in neurodegeneration and cancer. In cancer, and particularly in the presence of oncogenic KRAS mutations, in vivo studies concluded that SG increase the resistance of cancer cells to stress. Hence, SG have recently been considered a promising target for therapy. Here, starting from our observations that genes coding for SG proteins are stimulated during development of pancreatic ductal adenocarcinoma, we analyze the formation of SG during tumorigenesis. We resort to in vitro, in vivo and in silico approaches, using mouse models, human samples and human data. Our analyses do not support that SG are formed during tumorigenesis of KRAS-driven cancers, at least that their presence is not universal, leading us to propose that caution is required before considering SG as therapeutic targets.
    Keywords:  Cancer; KRAS; PDAC; Stress Granules
    DOI:  https://doi.org/10.1038/s44319-024-00284-6
  48. Nat Commun. 2024 Oct 08. 15(1): 8708
    Coronavirus envelope protein activates TMED10-mediated unconventional secretion of inflammatory factors.
    Lei Liu, Lijingyao Zhang, Xinyan Hao, Yang Wang, Xiaochun Zhang, Liang Ge, Peihui Wang, Boxue Tian, Min Zhang.
      The precise cellular mechanisms underlying heightened proinflammatory cytokine production during coronavirus infection remain incompletely understood. Here we identify the envelope (E) protein in severe coronaviruses (SARS-CoV-2, SARS, or MERS) as a potent inducer of interleukin-1 release, intensifying lung inflammation through the activation of TMED10-mediated unconventional protein secretion (UcPS). In contrast, the E protein of mild coronaviruses (229E, HKU1, or OC43) demonstrates a less pronounced effect. The E protein of severe coronaviruses contains an SS/DS motif, which is not present in milder strains and facilitates interaction with TMED10. This interaction enhances TMED10-oligomerization, facilitating UcPS cargo translocation into the ER-Golgi intermediate compartment (ERGIC)-a pivotal step in interleukin-1 UcPS. Progesterone analogues were identified as compounds inhibiting E-enhanced release of proinflammatory factors and lung inflammation in a Mouse Hepatitis Virus (MHV) infection model. These findings elucidate a molecular mechanism driving coronavirus-induced hyperinflammation, proposing the E-TMED10 interaction as a potential therapeutic target to counteract the adverse effects of coronavirus-induced inflammation.
    DOI:  https://doi.org/10.1038/s41467-024-52818-0
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