bims-proteo Biomed News
on Proteostasis
Issue of 2024–06–23
48 papers selected by
Eric Chevet, INSERM
  1. Deciphering ER stress-unfolded protein response relationship by visualizing unfolded proteins in the ER.
  2. Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity.
  3. Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.
  4. Structural analysis of the dynamic ribosome-translocon complex.
  5. The ufmylation cascade controls COPII recruitment, anterograde transport, and sorting of nascent GPCRs at ER.
  6. Less is better: various means to reduce protein load in the endoplasmic reticulum.
  7. Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.
  8. UFMylation: An integral post-translational modification for the regulation of proteostasis and cellular functions.
  9. HLH-30/TFEB rewires the chaperone network to promote proteostasis under conditions of Coenzyme A and Iron-Sulfur Cluster Deficiency.
  10. Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.
  11. The integrated stress response in brain diseases: A double-edged sword for proteostasis and synapses.
  12. Structural transitions modulate the chaperone activities of Grp94.
  13. C. elegans LIN-66 mediates EIF-3/eIF3-dependent protein translation via a cold-shock domain.
  14. Age-dependent heat shock hormesis to HSF-1 deficiency suggests a compensatory mechanism mediated by the unfolded protein response and innate immunity in young Caenorhabditis elegans.
  15. Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.
  16. Inhibition of the eukaryotic initiation factor-2-α kinase PERK decreases risk of autoimmune diabetes in mice.
  17. Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.
  18. The hexosamine biosynthetic pathway rescues lysosomal dysfunction in Parkinson's disease patient iPSC derived midbrain neurons.
  19. Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.
  20. Ribosome-Associated Vesicles promote activity-dependent local translation.
  21. Endoplasmic reticulum-plasma membrane contact gradients direct cell migration.
  22. An initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal.
  23. Impacts of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on translation reinitiation and ribosome recycling.
  24. The maintenance of oocytes in the mammalian ovary involves extreme protein longevity.
  25. Secretory autophagy - a new paradigm regulating synaptic plasticity.
  26. Oxidative protein folding in the intermembrane space of human mitochondria.
  27. Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism.
  28. Muscle-specific lack of GFPT1 in knock-in mice triggers ER stress to alleviate misfolded proteins.
  29. Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.
  30. CCDC50 mediates the clearance of protein aggregates to prevent cellular proteotoxicity.
  31. LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes.
  32. Augmented microglial endoplasmic reticulum-mitochondria contacts mediate depression-like behavior in mice induced by chronic social defeat stress.
  33. Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.
  34. Sanglifehrin A mitigates multi-organ fibrosis by targeting the collagen chaperone cyclophilin B.
  35. Computational Modeling of the Synergistic Role of GCN2 and HPA Axis in Regulating the Integrated Stress Response in the Central Circadian Timing System.
  36. RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.
  37. Endoplasmic reticulum stress potentiates the immunosuppressive microenvironment in hepatocellular carcinoma by promoting the release of SNHG6-enriched small extracellular vesicles.
  38. A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.
  39. ProteoCure : an European network to fine-tune the proteome.
  40. The UFMylation pathway is impaired in Alzheimer's disease.
  41. The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.
  42. Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.
  43. Computational design of soluble and functional membrane protein analogues.
  44. Nlp-dependent ER-to-Golgi transport.
  45. Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.
  46. StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol.
  47. Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5.
  48. Chemogenomics for NR1 nuclear hormone receptors.
  1. Cell Rep. 2024 Jun 11. pii: S2211-1247(24)00686-7. [Epub ahead of print]43(6): 114358
    Deciphering ER stress-unfolded protein response relationship by visualizing unfolded proteins in the ER.
    Fenfen Xu, Likun Wang.
      Despite the consensus that accumulation of unfolded proteins in the endoplasmic reticulum (ER) lumen, i.e. ER stress, activates the unfolded protein response (UPR), studies under physiological and pathophysiological conditions suggest that ER stress may not always trigger the UPR, and the UPR can be activated in an ER stress-independent way. To better understand how the UPR is regulated and its relationship with ER stress requires direct detection of unfolded proteins in the ER, a method that is still lacking. Here, we report a strategy of visualizing unfolded protein accumulation in the ER lumen in living cells by employing an engineered ER stress sensor, PERK, which forms fluorescence puncta upon unfolded protein binding, in a fast and reversible way. Our reporter enables us to clarify the involvement of unfolded proteins in UPR activation under several physiological conditions and suggests that persistent unfolded protein accumulation in the ER despite UPR attenuation predicts cell death.
    Keywords:  CP: Cell biology; ER stress; PERK; live-cell imaging; unfolded protein; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2024.114358
  2. Life Sci Alliance. 2024 Sep;pii: e202302562. [Epub ahead of print]7(9):
    Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity.
    Andrea Orsi, Eelco van Anken, Milena Vitale, Moreno Zamai, Valeria R Caiolfa, Roberto Sitia, Anush Bakunts.
      The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1α sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1α endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1α dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1α dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1α dimers in the ER membrane is a key event. Formation of stable IRE1α clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1α to phosphatases. The stepwise activation of IRE1α molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.
    DOI:  https://doi.org/10.26508/lsa.202302562
  3. Nat Commun. 2024 Jun 19. 15(1): 5188
    Arabidopsis CaLB1 undergoes phase separation with the ESCRT protein ALIX and modulates autophagosome maturation.
    Niccolò Mosesso, Niharika Savant Lerner, Tobias Bläske, Felix Groh, Shane Maguire, Marie Laura Niedermeier, Eliane Landwehr, Karin Vogel, Konstanze Meergans, Marie-Kristin Nagel, Malte Drescher, Florian Stengel, Karin Hauser, Erika Isono.
      Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca2+-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
    DOI:  https://doi.org/10.1038/s41467-024-49485-6
  4. Elife. 2024 Jun 18. pii: RP95814. [Epub ahead of print]13
    Structural analysis of the dynamic ribosome-translocon complex.
    Aaron J O Lewis, Frank Zhong, Robert J Keenan, Ramanujan S Hegde.
      The protein translocon at the endoplasmic reticulum comprises the Sec61 translocation channel and numerous accessory factors that collectively facilitate the biogenesis of secretory and membrane proteins. Here, we leveraged recent advances in cryo-electron microscopy (cryo-EM) and structure prediction to derive insights into several novel configurations of the ribosome-translocon complex. We show how a transmembrane domain (TMD) in a looped configuration passes through the Sec61 lateral gate during membrane insertion; how a nascent chain can bind and constrain the conformation of ribosomal protein uL22; and how the translocon-associated protein (TRAP) complex can adjust its position during different stages of protein biogenesis. Most unexpectedly, we find that a large proportion of translocon complexes contains RAMP4 intercalated into Sec61's lateral gate, widening Sec61's central pore and contributing to its hydrophilic interior. These structures lead to mechanistic hypotheses for translocon function and highlight a remarkably plastic machinery whose conformations and composition adjust dynamically to its diverse range of substrates.
    Keywords:  cell biology; endoplasmic reticulum; mammal; membrane protein insertion; membrane transport; molecular biophysics; secretion; structural biology; translocon
    DOI:  https://doi.org/10.7554/eLife.95814
  5. Sci Adv. 2024 Jun 21. 10(25): eadm9216
    The ufmylation cascade controls COPII recruitment, anterograde transport, and sorting of nascent GPCRs at ER.
    Xin Xu, Wei Huang, Christian N Bryant, Zheng Dong, Honglin Li, Guangyu Wu.
      Ufmylation is implicated in multiple cellular processes, but little is known about its functions and regulation in protein trafficking. Here, we demonstrate that the genetic depletion of core components of the ufmylation cascade, including ubiquitin-fold modifier 1 (UFM1), UFM1 activation enzyme 5, UFM1-specific ligase 1 (UFL1), UFM1-specific protease 2, and UFM1-binding protein 1 (UFBP1) each markedly inhibits the endoplasmic reticulum (ER)-Golgi transport, surface delivery, and recruitment to COPII vesicles of a subset of G protein-coupled receptors (GPCRs) and UFBP1's function partially relies on UFM1 conjugation. We also show that UFBP1 and UFL1 interact with GPCRs and UFBP1 localizes at COPII vesicles coated with specific Sec24 isoforms. Furthermore, the UFBP1/UFL1-binding domain identified in the receptors effectively converts non-GPCR protein transport into the ufmylation-dependent pathway. Collectively, these data reveal important functions for the ufmylation system in GPCR recruitment to COPII vesicles, biosynthetic transport, and sorting at ER via UFBP1 ufmylation and interaction directly.
    DOI:  https://doi.org/10.1126/sciadv.adm9216
  6. FEBS J. 2024 Jun 12.
    Less is better: various means to reduce protein load in the endoplasmic reticulum.
    Salam Dabsan, Gal Twito, Suma Biadsy, Aeid Igbaria.
      The endoplasmic reticulum (ER) is an important organelle that controls the intracellular and extracellular environments. The ER is responsible for folding almost one-third of the total protein population in the eukaryotic cell. Disruption of ER-protein folding is associated with numerous human diseases, including metabolic disorders, neurodegenerative diseases, and cancer. During ER perturbations, the cells deploy various mechanisms to increase the ER-folding capacity and reduce ER-protein load by minimizing the number of substrates entering the ER to regain homeostasis. These mechanisms include signaling pathways, degradation mechanisms, and other processes that mediate the reflux of ER content to the cytosol. In this review, we will discuss the recent discoveries of five different ER quality control mechanisms, including the unfolded protein response (UPR), ER-associated-degradation (ERAD), pre-emptive quality control, ER-phagy and ER to cytosol signaling (ERCYS). We will discuss the roles of these processes in decreasing ER-protein load and inter-mechanism crosstalk.
    Keywords:  COPII; ERAD; ERCYS; ER‐Phagy; ER‐pQC; UPR
    DOI:  https://doi.org/10.1111/febs.17201
  7. Proc Natl Acad Sci U S A. 2024 Jun 25. 121(26): e2317945121
    Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.
    Maryam Jafari, Adrián Macho-González, Antonio Diaz, Kristen Lindenau, Olaya Santiago-Fernández, Mei Zeng, Ashish C Massey, Rafael de Cabo, Susmita Kaushik, Ana Maria Cuervo.
      Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.
    Keywords:  aging; autophagy; dietary restriction; gerotherapeutics; lysosomes
    DOI:  https://doi.org/10.1073/pnas.2317945121
  8. Pharmacol Ther. 2024 Jun 13. pii: S0163-7258(24)00100-1. [Epub ahead of print]260 108680
    UFMylation: An integral post-translational modification for the regulation of proteostasis and cellular functions.
    Xiaohui Wang, Xiaowei Lv, Jingjing Ma, Guoqiang Xu.
      Ubiquitin-fold modifier 1 (UFM1) is covalently conjugated to protein substrates via a cascade of enzymatic reactions, a process known as UFMylation. UFMylation orchestrates an array of vital biological functions, including maintaining endoplasmic reticulum (ER) homeostasis, facilitating protein biogenesis, promoting cellular differentiation, regulating DNA damage response, and participating in cancer-associated signaling pathways. UFMylation has rapidly evolved into one of the forefront research areas within the last few years, yet much remains to be uncovered. In this review, first, UFMylation and its cellular functions associated with diseases are briefly introduced. Then, we summarize the proteomic approaches for identifying UFMylation substrates and explore the impact of UFMylation on gene transcription, protein translation, and maintenance of ER homeostasis. Next, we highlight the intricate regulation between UFMylation and two protein degradation pathways, the ubiquitin-proteasome system and the autophagy-lysosome pathway, and explore the potential of UFMylation system as a drug target. Finally, we discuss emerging perspectives in the UFMylation field. This review may provide valuable insights for drug discovery targeting the UFMylation system.
    Keywords:  ALP; ER homeostasis; Gene transcription; Protein translation; UFMylation; UPS
    DOI:  https://doi.org/10.1016/j.pharmthera.2024.108680
  9. bioRxiv. 2024 Jun 06. pii: 2024.06.05.597553. [Epub ahead of print]
    HLH-30/TFEB rewires the chaperone network to promote proteostasis under conditions of Coenzyme A and Iron-Sulfur Cluster Deficiency.
    Rewayd Shalash, Mor Levi-Ferber, Henrik von Chrzanowski, Mohammad Khaled Atrash, Yaron Shav-Tal, Sivan Henis-Korenblit.
      The maintenance of a properly folded proteome is critical for cellular function and organismal health, and its age-dependent collapse is associated with a wide range of diseases. Here, we find that despite the central role of Coenzyme A as a molecular cofactor in hundreds of cellular reactions, limiting Coenzyme A levels in C. elegans and in human cells, by inhibiting the conserved pantothenate kinase, promotes proteostasis. Impairment of the cytosolic iron-sulfur clusters formation pathway, which depends on Coenzyme A, similarly promotes proteostasis and acts in the same pathway. Proteostasis improvement by Coenzyme A/iron-sulfur cluster deficiencies are dependent on the conserved HLH-30/TFEB transcription factor. Strikingly, under these conditions, HLH-30 promotes proteostasis by potentiating the expression of select chaperone genes providing a chaperone-mediated proteostasis shield, rather than by its established role as an autophagy and lysosome biogenesis promoting factor. This reflects the versatile nature of this conserved transcription factor, that can transcriptionally activate a wide range of protein quality control mechanisms, including chaperones and stress response genes alongside autophagy and lysosome biogenesis genes. These results highlight TFEB as a key proteostasis-promoting transcription factor and underscore it and its upstream regulators as potential therapeutic targets in proteostasis-related diseases.
    DOI:  https://doi.org/10.1101/2024.06.05.597553
  10. bioRxiv. 2024 Jun 03. pii: 2023.10.06.561126. [Epub ahead of print]
    Inhibition of the Eukaryotic Initiation Factor-2-α Kinase PERK Decreases Risk of Autoimmune Diabetes in Mice.
    Charanya Muralidharan, Fei Huang, Jacob R Enriquez, Jiayi E Wang, Jennifer B Nelson, Titli Nargis, Sarah C May, Advaita Chakraborty, Kayla T Figatner, Svetlana Navitskaya, Cara M Anderson, Veronica Calvo, David Surguladze, Mark J Mulvihill, Xiaoyan Yi, Soumyadeep Sarkar, Scott A Oakes, Bobbie-Jo M Webb-Robertson, Emily K Sims, Kirk A Staschke, Decio L Eizirik, Ernesto S Nakayasu, Michael E Stokes, Sarah A Tersey, Raghavendra G Mirmira.
      Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing β cells renders these cells susceptible to autoimmunity. We show that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reverses the mRNA translation block in stressed human islets and delays the onset of diabetes, reduces islet inflammation, and preserves β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice shows reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice shows an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increase following PERK inhibition in human islets and EndoC-βH1 human β cells, interacts with and stabilizes PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.
    DOI:  https://doi.org/10.1101/2023.10.06.561126
  11. Curr Opin Neurobiol. 2024 Jun 19. pii: S0959-4388(24)00048-5. [Epub ahead of print]87 102886
    The integrated stress response in brain diseases: A double-edged sword for proteostasis and synapses.
    Elana R Lockshin, Nicole Calakos.
      The integrated stress response (ISR) is a highly conserved biochemical pathway that regulates protein synthesis. The ISR is activated in response to diverse stressors to restore cellular homeostasis. As such, the ISR is implicated in a wide range of diseases, including brain disorders. However, in the brain, the ISR also has potent influence on processes beyond proteostasis, namely synaptic plasticity, learning and memory. Thus, in the setting of brain diseases, ISR activity may have dual effects on proteostasis and synaptic function. In this review, we consider the ISR's contribution to brain disorders through the lens of its potential effects on synaptic plasticity. From these examples, we illustrate that at times ISR activity may be a "double-edged sword". We also highlight its potential as a therapeutic target to improve circuit function in brain diseases independent of its role in disease pathogenesis.
    DOI:  https://doi.org/10.1016/j.conb.2024.102886
  12. Trends Biochem Sci. 2024 Jun 20. pii: S0968-0004(24)00150-6. [Epub ahead of print]
    Structural transitions modulate the chaperone activities of Grp94.
    Duhita Mirikar, Yevheniia Bushman, Andrew W Truman.
      A recent study by Amankwah et al. reports how co-chaperone proteins and ATP hydrolysis fine-tune the function of endoplasmic reticulum (ER)-resident Hsp90 paralog Grp94.
    Keywords:  BiP; DEER; Grp94; Hsp90; endoplasmic reticulum (ER); protein folding
    DOI:  https://doi.org/10.1016/j.tibs.2024.06.007
  13. Life Sci Alliance. 2024 Sep;pii: e202402673. [Epub ahead of print]7(9):
    C. elegans LIN-66 mediates EIF-3/eIF3-dependent protein translation via a cold-shock domain.
    Stephen M Blazie, Daniel Fortunati, Yan Zhao, Yishi Jin.
      Protein translation initiation is a conserved process involving many proteins acting in concert. The 13 subunit eukaryotic initiation factor 3 (eIF3) complex is essential for assembly of the pre-initiation complex that scans mRNA and positions ribosome at the initiation codon. We previously reported that a gain-of-function (gf) mutation affecting the G subunit of the Caenorhabditis elegans eIF3 complex, eif-3.g(gf), selectively modulates protein translation in the ventral cord cholinergic motor neurons. Here, through unbiased genetic suppressor screening, we identified that the gene lin-66 mediates eif-3.g(gf)-dependent protein translation in motor neurons. LIN-66 is composed largely of low-complexity amino acid sequences with unknown functional domains. We combined bioinformatics analysis with in vivo functional dissection and identified a cold-shock domain in LIN-66 critical for its function. In cholinergic motor neurons, LIN-66 shows a close association with EIF-3.G in the cytoplasm. The low-complexity amino acid sequences of LIN-66 modulate its subcellular pattern. As cold-shock domains function broadly in RNA regulation, we propose that LIN-66 mediates stimulus-dependent protein translation by facilitating the interaction of mRNAs with EIF-3.G.
    DOI:  https://doi.org/10.26508/lsa.202402673
  14. Aging Cell. 2024 Jun 19. e14246
    Age-dependent heat shock hormesis to HSF-1 deficiency suggests a compensatory mechanism mediated by the unfolded protein response and innate immunity in young Caenorhabditis elegans.
    Dániel Kovács, János Barnabás Biró, Saqib Ahmed, Márton Kovács, Tímea Sigmond, Bernadette Hotzi, Máté Varga, Viktor Vázsony Vincze, Umar Mohammad, Tibor Vellai, János Barna.
      The transcription factor HSF-1 (heat shock factor 1) acts as a master regulator of heat shock response in eukaryotic cells to maintain cellular proteostasis. The protein has a protective role in preventing cells from undergoing ageing, and neurodegeneration, and also mediates tumorigenesis. Thus, modulating HSF-1 activity in humans has a promising therapeutic potential for treating these pathologies. Loss of HSF-1 function is usually associated with impaired stress tolerance. Contrary to this conventional knowledge, we show here that inactivation of HSF-1 in the nematode Caenorhabditis elegans results in increased thermotolerance at young adult stages, whereas HSF-1 deficiency in animals passing early adult stages indeed leads to decreased thermotolerance, as compared to wild-type. Furthermore, a gene expression analysis supports that in young adults, distinct cellular stress response and immunity-related signaling pathways become induced upon HSF-1 deficiency. We also demonstrate that increased tolerance to proteotoxic stress in HSF-1-depleted young worms requires the activity of the unfolded protein response of the endoplasmic reticulum and the SKN-1/Nrf2-mediated oxidative stress response pathway, as well as an innate immunity-related pathway, suggesting a mutual compensatory interaction between HSF-1 and these conserved stress response systems. A similar compensatory molecular network is likely to also operate in higher animal taxa, raising the possibility of an unexpected outcome when HSF-1 activity is manipulated in humans.
    Keywords:   C. Elegans ; skn‐1 ; autophagy; cellular stress response; heat shock factor 1; heat shock proteins; heat shock response; hormesis; innate immunity; insulin‐like signaling pathway; intracellular pathogen response; proteostasis; thermotolerance; unfolded protein response
    DOI:  https://doi.org/10.1111/acel.14246
  15. Elife. 2024 Jun 20. pii: e98649. [Epub ahead of print]13
    Novel autophagy inducers by accelerating lysosomal clustering against Parkinson's disease.
    Yuki Date, Yukiko Sasazawa, Mitsuhiro Kitagawa, Kentaro Gejima, Ayami Suzuki, Hideyuki Saya, Yasuyuki Kida, Masaya Imoto, Eisuke Itakura, Nobutaka Hattori, Shinji Saiki.
      The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.
    Keywords:  cell biology
    DOI:  https://doi.org/10.7554/eLife.98649
  16. J Clin Invest. 2024 Jun 18. pii: e176136. [Epub ahead of print]
    Inhibition of the eukaryotic initiation factor-2-α kinase PERK decreases risk of autoimmune diabetes in mice.
    Charanya Muralidharan, Fei Huang, Jacob R Enriquez, Jiayi E Wang, Jennifer B Nelson, Titli Nargis, Sarah C May, Advaita Chakraborty, Kayla T Figatner, Svetlana Navitskaya, Cara M Anderson, Veronica Calvo, David Surguladze, Mark J Mulvihill, Xiaoyan Yi, Soumyadeep Sarkar, Scott A Oakes, Bobbie-Jo M Webb-Robertson, Emily K Sims, Kirk A Staschke, Decio L Eizirik, Ernesto S Nakayasu, Michael E Stokes, Sarah A Tersey, Raghavendra G Mirmira.
      Preventing the onset of autoimmune type 1 diabetes (T1D) is feasible through pharmacological interventions that target molecular stress-responsive mechanisms. Cellular stresses, such as nutrient deficiency, viral infection, or unfolded proteins, trigger the integrated stress response (ISR), which curtails protein synthesis by phosphorylating eIF2α. In T1D, maladaptive unfolded protein response (UPR) in insulin-producing beta cells renders these cells susceptible to autoimmunity. We found that inhibition of the eIF2α kinase PERK, a common component of the UPR and ISR, reversed the mRNA translation block in stressed human islets and delayed the onset of diabetes, reduced islet inflammation, and preserved β cell mass in T1D-susceptible mice. Single-cell RNA sequencing of islets from PERK-inhibited mice showed reductions in the UPR and PERK signaling pathways and alterations in antigen processing and presentation pathways in β cells. Spatial proteomics of islets from these mice showed an increase in the immune checkpoint protein PD-L1 in β cells. Golgi membrane protein 1, whose levels increased following PERK inhibition in human islets and EndoC-βH1 human β cells, interacted with and stabilized PD-L1. Collectively, our studies show that PERK activity enhances β cell immunogenicity, and inhibition of PERK may offer a strategy to prevent or delay the development of T1D.
    Keywords:  Beta cells; Diabetes; Endocrinology; Pharmacology
    DOI:  https://doi.org/10.1172/JCI176136
  17. Dev Cell. 2024 May 20. pii: S1534-5807(24)00295-8. [Epub ahead of print]
    Suppressed basal mitophagy drives cellular aging phenotypes that can be reversed by a p62-targeting small molecule.
    George Kelly, Tetsushi Kataura, Johan Panek, Gailing Ma, Hanna Salmonowicz, Ashley Davis, Hannah Kendall, Charlotte Brookes, Daniel Moscoh Ayine-Tora, Peter Banks, Glyn Nelson, Laura Dobby, Patricia R Pitrez, Laura Booth, Lydia Costello, Gavin D Richardson, Penny Lovat, Stefan Przyborski, Lino Ferreira, Laura Greaves, Karolina Szczepanowska, Thomas von Zglinicki, Satomi Miwa, Max Brown, Michael Flagler, John E Oblong, Charles C Bascom, Bernadette Carroll, Jóhannes Reynisson, Viktor I Korolchuk.
      Selective degradation of damaged mitochondria by autophagy (mitophagy) is proposed to play an important role in cellular homeostasis. However, the molecular mechanisms and the requirement of mitochondrial quality control by mitophagy for cellular physiology are poorly understood. Here, we demonstrated that primary human cells maintain highly active basal mitophagy initiated by mitochondrial superoxide signaling. Mitophagy was found to be mediated by PINK1/Parkin-dependent pathway involving p62 as a selective autophagy receptor (SAR). Importantly, this pathway was suppressed upon the induction of cellular senescence and in naturally aged cells, leading to a robust shutdown of mitophagy. Inhibition of mitophagy in proliferating cells was sufficient to trigger the senescence program, while reactivation of mitophagy was necessary for the anti-senescence effects of NAD precursors or rapamycin. Furthermore, reactivation of mitophagy by a p62-targeting small molecule rescued markers of cellular aging, which establishes mitochondrial quality control as a promising target for anti-aging interventions.
    Keywords:  PINK1; Parkin; aging; autophagy; mitophagy; nicotinamide; nicotinamide riboside; p62; rapamycin; redox; senescence
    DOI:  https://doi.org/10.1016/j.devcel.2024.04.020
  18. Nat Commun. 2024 Jun 19. 15(1): 5206
    The hexosamine biosynthetic pathway rescues lysosomal dysfunction in Parkinson's disease patient iPSC derived midbrain neurons.
    Willayat Y Wani, Friederike Zunke, Nandkishore R Belur, Joseph R Mazzulli.
      Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson's disease, however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5'-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson's patient midbrain cultures accumulate glucose and uridine-5'-triphosphate, while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme, GFPT2, through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate, while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological α-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity, and its failure in Parkinson's disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement.
    DOI:  https://doi.org/10.1038/s41467-024-49256-3
  19. ACS Synth Biol. 2024 Jun 18.
    Protease-Responsive Toolkit for Conditional Targeted Protein Degradation.
    Hopen Yang, Wilfred Chen.
      BioPROTACs are heterobifunctional proteins designed for targeted protein degradation. While they offer a potential therapeutic avenue for modulating disease-related proteins, the current strategies are static in nature and lack the ability to modulate protein degradation dynamically. Here, we introduce a synthetic framework for dynamic fine-tuning of target protein levels using protease control switches. The idea is to utilize proteases as an interfacing layer between exogenous inputs and protein degradation by modulating the recruitment of target proteins to E3 ligase by separating the two binding domains on bioPROTACs. By decoupling the external inputs from the primary protease layer, new conditional degradation phenotypes can be readily adapted with minimal modifications to the design. We demonstrate the adaptability of this approach using two highly efficient "bioPROTAC" systems: AdPROM and IpaH9.8-based Ubiquibodies. Using the TEV protease as the transducer, we can interface small-molecule and optogenetic inputs for conditional targeted protein degradation. Our findings highlight the potential of bioPROTACs with protease-responsive linkers as a versatile tool for conditional targeted protein degradation.
    Keywords:  E3 ligase; modular; synthetic biology; targeted protein degradation
    DOI:  https://doi.org/10.1021/acssynbio.4c00014
  20. bioRxiv. 2024 Jun 10. pii: 2024.06.07.598007. [Epub ahead of print]
    Ribosome-Associated Vesicles promote activity-dependent local translation.
    Eva Martin-Solana, Stephen D Carter, Eric K F Donahue, Jiying Ning, Jill R Glausier, Matias A Preisegger, Leanna Eisenman, Paul N Joseph, Cedric Bouchet-Marquis, Ken Wu, Catherina L Mobini, Amber N Frantz, Stephanie Puig, Cheri M Hampton, Nadine Kabbani, Grant J Jensen, Simon C Watkins, Karl Deisseroth, Lief E Fenno, Michael S Gold, Zachary P Wills, Kristopher Burkewitz, Sulagna Das, Zachary Freyberg.
      Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo , identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.
    DOI:  https://doi.org/10.1101/2024.06.07.598007
  21. Nature. 2024 Jun 12.
    Endoplasmic reticulum-plasma membrane contact gradients direct cell migration.
    Bo Gong, Jake D Johnston, Alexander Thiemicke, Alex de Marco, Tobias Meyer.
      Directed cell migration is driven by the front-back polarization of intracellular signalling1-3. Receptor tyrosine kinases and other inputs activate local signals that trigger membrane protrusions at the front2,4-6. Equally important is a long-range inhibitory mechanism that suppresses signalling at the back to prevent the formation of multiple fronts7-9. However, the identity of this mechanism is unknown. Here we report that endoplasmic reticulum-plasma membrane (ER-PM) contact sites are polarized in single and collectively migrating cells. The increased density of these ER-PM contacts at the back provides the ER-resident PTP1B phosphatase more access to PM substrates, which confines receptor signalling to the front and directs cell migration. Polarization of the ER-PM contacts is due to microtubule-regulated polarization of the ER, with more RTN4-rich curved ER at the front and more CLIMP63-rich flattened ER at the back. The resulting ER curvature gradient leads to small and unstable ER-PM contacts only at the front. These contacts flow backwards and grow to large and stable contacts at the back to form the front-back ER-PM contact gradient. Together, our study suggests that the structural polarity mediated by ER-PM contact gradients polarizes cell signalling, directs cell migration and prolongs cell migration.
    DOI:  https://doi.org/10.1038/s41586-024-07527-5
  22. Autophagy. 2024 Jun 20. 1-22
    An initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal.
    Serena R Wisner, Madison Chlebowski, Amrita Mandal, Don Mai, Chris Stein, Ronald S Petralia, Ya-Xian Wang, Catherine M Drerup.
      In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport. During retrograde transport these autophagosomes mature through repetitive fusion events. Complete lysosomal cargo degradation occurs largely in the cell body. The precipitating events to stimulate retrograde autophagosome transport have been debated but their importance is clear: disrupting neuronal autophagy or autophagosome transport is detrimental to neuronal health and function. We have identified the HOPS complex as essential for early autophagosome maturation and consequent initiation of retrograde transport from the axon terminal. In yeast and mammalian cells, HOPS controls fusion between autophagosomes and late endosomes with lysosomes. Using zebrafish strains with loss-of-function mutations in vps18 and vps41, core components of the HOPS complex, we found that disruption of HOPS eliminates autophagosome maturation and disrupts retrograde autophagosome transport initiation from the axon terminal. We confirmed this phenotype was due to loss of HOPS complex formation using an endogenous deletion of the HOPS binding domain in Vps18. Finally, using pharmacological inhibition of lysosomal proteases, we show that initiation of autophagosome retrograde transport requires autophagosome maturation. Together, our data demonstrate that HOPS-mediated fusion events are critical for retrograde autophagosome transport initiation through promoting autophagosome maturation. This reveals critical roles for the HOPS complex in neuronal autophagy which deepens our understanding of the cellular pathology of HOPS-complex linked neurodegenerative diseases.Abbreviations: CORVET: Class C core vacuole/endosome tethering; gRNA: guide RNA; HOPS: homotypic fusion and protein sorting; pLL: posterior lateral line; Vps18: VPS18 core subunit of CORVET and HOPS complexes; Vps41: VPS41 subunit of HOPS complex.
    Keywords:  Axon terminal; Vps18; autophagy; axonal transport; lysosome; neuron
    DOI:  https://doi.org/10.1080/15548627.2024.2366122
  23. bioRxiv. 2024 Mar 07. pii: 2024.03.06.583729. [Epub ahead of print]
    Impacts of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on translation reinitiation and ribosome recycling.
    Kristína Jendruchová, Swati Gaikwad, Kristýna Poncová, Stanislava Gunišová, Leoš Shivaya Valášek, Alan G Hinnebusch.
      Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.
    DOI:  https://doi.org/10.1101/2024.03.06.583729
  24. Nat Cell Biol. 2024 Jun 20.
    The maintenance of oocytes in the mammalian ovary involves extreme protein longevity.
    Katarina Harasimov, Rebecca L Gorry, Luisa M Welp, Sarah Mae Penir, Yehor Horokhovskyi, Shiya Cheng, Katsuyoshi Takaoka, Alexandra Stützer, Ann-Sophie Frombach, Ana Lisa Taylor Tavares, Monika Raabe, Sara Haag, Debojit Saha, Katharina Grewe, Vera Schipper, Silvio O Rizzoli, Henning Urlaub, Juliane Liepe, Melina Schuh.
      Women are born with all of their oocytes. The oocyte proteome must be maintained with minimal damage throughout the woman's reproductive life, and hence for decades. Here we report that oocyte and ovarian proteostasis involves extreme protein longevity. Mouse ovaries had more extremely long-lived proteins than other tissues, including brain. These long-lived proteins had diverse functions, including in mitochondria, the cytoskeleton, chromatin and proteostasis. The stable proteins resided not only in oocytes but also in long-lived ovarian somatic cells. Our data suggest that mammals increase protein longevity and enhance proteostasis by chaperones and cellular antioxidants to maintain the female germline for long periods. Indeed, protein aggregation in oocytes did not increase with age and proteasome activity did not decay. However, increasing protein longevity cannot fully block female germline senescence. Large-scale proteome profiling of ~8,890 proteins revealed a decline in many long-lived proteins of the proteostasis network in the aging ovary, accompanied by massive proteome remodeling, which eventually leads to female fertility decline.
    DOI:  https://doi.org/10.1038/s41556-024-01442-7
  25. Autophagy. 2024 Jun 20.
    Secretory autophagy - a new paradigm regulating synaptic plasticity.
    Yen-Ching Chang, Karen T Chang.
      When exposed to new experiences or changes in the environment, neurons rapidly remodel their synaptic structure and function in a process called activity-induced synaptic remodeling. This process is necessary for transforming transient experiences into stable, lasting memories. The molecular mechanisms underlying acute, activity-dependent synaptic changes are not well understood, partly because processes regulating synaptic plasticity and neurodevelopment are intricately linked. By using an RNAi screen in Drosophila targeting genes associated with human nervous system function, we found that while macroautophagy (referred to as autophagy) is fundamental for both synapse development and synaptic plasticity, activity-induced synaptic remodeling does not rely on genes associated with lysosomal degradation. These findings suggest a requirement for the unconventional secretory autophagy pathway in regulating synaptic plasticity, wherein autophagosomes, instead of fusing with lysosomes for degradation, fuse with the plasma membrane to release their contents extracellularly. To test this hypothesis, we knocked down Sec22, Snap29, and Rab8, molecular components required for secretory autophagy, all of which disrupted structural and functional plasticity. Additionally, by monitoring autophagy, we demonstrated that neuronal activity suppresses degradative autophagy to shift the pathway toward secretory autophagy release. Our work unveils secretory autophagy as a novel trans-synaptic signaling mechanism crucial for activity-induced synaptic remodeling.
    Keywords:  Activity-induced synaptic remodeling; Drosophila; secretory autophagy; synaptic plasticity
    DOI:  https://doi.org/10.1080/15548627.2024.2370179
  26. FEBS Open Bio. 2024 Jun 12.
    Oxidative protein folding in the intermembrane space of human mitochondria.
    Christine Zarges, Jan Riemer.
      The mitochondrial intermembrane space hosts a machinery for oxidative protein folding, the mitochondrial disulfide relay. This machinery imports a large number of soluble proteins into the compartment, where they are retained through oxidative folding. Additionally, the disulfide relay enhances the stability of many proteins by forming disulfide bonds. In this review, we describe the mitochondrial disulfide relay in human cells, its components, and their coordinated collaboration in mechanistic detail. We also discuss the human pathologies associated with defects in this machinery and its protein substrates, providing a comprehensive overview of its biological importance and implications for health.
    Keywords:  ALR; IMS; MIA40; mitochondria; oxidative protein folding; protein import
    DOI:  https://doi.org/10.1002/2211-5463.13839
  27. Nat Commun. 2024 Jun 19. 15(1): 5179
    Broad-spectrum activity against mosquito-borne flaviviruses achieved by a targeted protein degradation mechanism.
    Han-Yuan Liu, Zhengnian Li, Theresia Reindl, Zhixiang He, Xueer Qiu, Ryan P Golden, Katherine A Donovan, Adam Bailey, Eric S Fischer, Tinghu Zhang, Nathanael S Gray, Priscilla L Yang.
      Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4CRBN E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.
    DOI:  https://doi.org/10.1038/s41467-024-49161-9
  28. Dis Model Mech. 2024 Jun 21. pii: dmm.050768. [Epub ahead of print]
    Muscle-specific lack of GFPT1 in knock-in mice triggers ER stress to alleviate misfolded proteins.
    Ruchen Zhang, Paniz Farshadyeganeh, Bisei Ohkawara, Kazuki Nakajima, Jun-Ichi Takeda, Mikako Ito, Shaochuan Zhang, Yuki Miyasaka, Tamio Ohno, Madoka Mori-Yoshimura, Akio Masuda, Kinji Ohno.
      Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9 simulating a CMS patient. As Gfpt1 exon 9 is exclusively included in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (i) UDP-HexNAc, CMP-NeuAc, and protein O-GlcNAcylations were reduced in skeletal muscles; (ii) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (iii) markers for unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of ER stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation, and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.
    Keywords:  And unfolded protein response (UPR); Congenital myasthenic syndrome (CMS); Endoplasmic reticulum (ER) stress; Glutamine fructose-6-phosphate transaminase 1 (GFPT1); Hexosamine biosynthesis pathway (HBP); Neuromuscular junction (NMJ)
    DOI:  https://doi.org/10.1242/dmm.050768
  29. PLoS Comput Biol. 2024 Jun 17. 20(6): e1012212
    Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.
    Jack M O'Shea, Peter Doerner, Annis Richardson, Christopher W Wood.
      Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.
    DOI:  https://doi.org/10.1371/journal.pcbi.1012212
  30. Autophagy. 2024 Jun 13.
    CCDC50 mediates the clearance of protein aggregates to prevent cellular proteotoxicity.
    Yu Ye, Penghui Jia, Jiafan Miao, Yicheng Wang, Zibo Li, Yuxin Lin, Miao He, Shurui Liu, Bi-Rong Zheng, Junyu Wu, Ji'an Pan, Chun-Mei Li, Panpan Hou, Deyin Guo.
      Protein aggregation caused by the disruption of proteostasis will lead to cellular cytotoxicity and even cell death, which is implicated in multiple neurodegenerative diseases. The elimination of aggregated proteins is mediated by selective macroautophagy receptors, which is termed aggrephagy. However, the identity and redundancy of aggrephagy receptors in recognizing substrates remain largely unexplored. Here, we find that CCDC50, a highly expressed autophagy receptor in brain, is recruited to proteotoxic stresses-induced polyubiquitinated protein aggregates and ectopically expressed aggregation-prone proteins. CCDC50 recognizes and further clears these cytotoxic aggregates through autophagy. The ectopic expression of CCDC50 increases the tolerance to stress-induced proteotoxicity and hence improved cell survival in neuron cells, whereas CCDC50 deficiency caused accumulation of lipid deposits and polyubiquitinated protein conjugates in the brain of one-year-old mice. Our study illustrates how aggrephagy receptor CCDC50 combats proteotoxic stress for the benefit of neuronal cell survival, thus suggesting a protective role in neurotoxic proteinopathy.
    Keywords:  Aggrephagy receptor; CCDC50; neurodegenerative diseases; protein aggregation; proteostasis
    DOI:  https://doi.org/10.1080/15548627.2024.2367183
  31. bioRxiv. 2024 Jun 03. pii: 2024.06.03.597215. [Epub ahead of print]
    LONP1 regulation of mitochondrial protein folding provides insight into beta cell failure in type 2 diabetes.
    Jin Li, Jie Zhu, Yamei Deng, Emma C Reck, Emily M Walker, Vaibhav Sidarala, Dre L Hubers, Mabelle B Pasmooij, Chun-Shik Shin, Khushdeep Bandesh, Eftyhmios Motakis, Siddhi Nargund, Romy Kursawe, Venkatesha Basrur, Alexey I Nesvizhskii, Michael L Stitzel, David C Chan, Scott A Soleimanpour.
      Proteotoxicity is a contributor to the development of type 2 diabetes (T2D), but it is unknown whether protein misfolding in T2D is generalized or has special features. Here, we report a robust accumulation of misfolded proteins within the mitochondria of human pancreatic islets in T2D and elucidate its impact on β cell viability. Surprisingly, quantitative proteomics studies of protein aggregates reveal that human islets from donors with T2D have a signature more closely resembling mitochondrial rather than ER protein misfolding. The matrix protease LonP1 and its chaperone partner mtHSP70 were among the proteins enriched in protein aggregates. Deletion of LONP1 in mice yields mitochondrial protein misfolding and reduced respiratory function, ultimately leading to β cell apoptosis and hyperglycemia. Intriguingly, LONP1 gain of function ameliorates mitochondrial protein misfolding and restores human β cell survival following glucolipotoxicity via a protease-independent effect requiring LONP1-mtHSP70 chaperone activity. Thus, LONP1 promotes β cell survival and prevents hyperglycemia by facilitating mitochondrial protein folding. These observations may open novel insights into the nature of impaired proteostasis on β cell loss in the pathogenesis of T2D that could be considered as future therapeutic targets.
    DOI:  https://doi.org/10.1101/2024.06.03.597215
  32. Nat Commun. 2024 Jun 18. 15(1): 5199
    Augmented microglial endoplasmic reticulum-mitochondria contacts mediate depression-like behavior in mice induced by chronic social defeat stress.
    Jia-Rui Zhang, Shi-Yu Shen, Meng-Ying Zhai, Zu-Qi Shen, Wei Li, Ling-Feng Liang, Shu-Yuan Yin, Qiu-Qin Han, Bing Li, Yu-Qiu Zhang, Jin Yu.
      Extracellular ATP (eATP) signaling through the P2X7 receptor pathway is widely believed to trigger NLRP3 inflammasome assembly in microglia, potentially contributing to depression. However, the cellular stress responses of microglia to both eATP and stress itself remain largely unexplored. Mitochondria-associated membranes (MAMs) is a platform facilitating calcium transport between the endoplasmic reticulum (ER) and mitochondria, regulating ER stress responses and mitochondrial homeostasis. This study aims to investigate how MAMs influence microglial reaction and their involvement in the development of depression-like symptoms in response to chronic social defeat stress (CSDS). CSDS induced ER stress, MAMs' modifications, mitochondrial damage, and the formation of the IP3R3-GRP75-VDAC1 complex at the ER-mitochondria interface in hippocampal microglia, all concomitant with depression-like behaviors. Additionally, exposing microglia to eATP to mimic CSDS conditions resulted in analogous outcomes. Furthermore, knocking down GRP75 in BV2 cells impeded ER-mitochondria contact, calcium transfer, ER stress, mitochondrial damage, mitochondrial superoxide production, and NLRP3 inflammasome aggregation induced by eATP. In addition, reduced GRP75 expression in microglia of Cx3cr1CreER/+Hspa9f/+ mice lead to reduce depressive behaviors, decreased NLRP3 inflammasome aggregation, and fewer ER-mitochondria contacts in hippocampal microglia during CSDS. Here, we show the role of MAMs, particularly the formation of a tripartite complex involving IP3R3, GRP75, and VDAC1 within MAMs, in facilitating communication between the ER and mitochondria in microglia, thereby contributing to the development of depression-like phenotypes in male mice.
    DOI:  https://doi.org/10.1038/s41467-024-49597-z
  33. bioRxiv. 2024 May 16. pii: 2024.05.15.594402. [Epub ahead of print]
    Chaperoning the chaperones: Proteomic analysis of the SMN complex reveals conserved and etiologic connections to the proteostasis network.
    A Gregory Matera, Rebecca E Steiner, C Alison Mills, Laura E Herring, Eric L Garcia.
      Molecular chaperones and co-chaperones are highly conserved cellular components that perform variety of duties related to the proper three-dimensional folding of the proteome. The web of factors that carries out this essential task is called the proteostasis network (PN). Ribonucleoproteins (RNPs) represent an underexplored area in terms of the connections they make with the PN. The Survival Motor Neuron (SMN) complex is an RNP assembly chaperone and serves as a paradigm for studying how specific small nuclear (sn)RNAs are identified and paired with their client substrate proteins. SMN protein is the eponymous component of a large complex required for the biogenesis of uridine-rich small nuclear ribonucleoproteins (U-snRNPs) and localizes to distinct membraneless organelles in both the nucleus and cytoplasm of animal cells. SMN forms the oligomeric core of this complex, and missense mutations in its YG box self-interaction domain are known to cause Spinal Muscular Atrophy (SMA). The basic framework for understanding how snRNAs are assembled into U-snRNPs is known, the pathways and mechanisms used by cells to regulate their biogenesis are poorly understood. Given the importance of these processes to normal development as well as neurodegenerative disease, we set out to identify and characterize novel SMN binding partners. Here, we carried out affinity purification mass spectrometry (AP-MS) of SMN using stable fly lines exclusively expressing either wildtype or SMA-causing missense alleles. Bioinformatic analyses of the pulldown data, along with comparisons to proximity labeling studies carried out in human cells, revealed conserved connections to at least two other major chaperone systems including heat shock folding chaperones (HSPs) and histone/nucleosome assembly chaperones. Notably, we found that heat shock cognate protein Hsc70-4 and other HspA family members preferentially interacted with SMA-causing alleles of SMN. Hsc70-4 is particularly interesting because its mRNA is aberrantly sequestered by a mutant form of TDP-43 in mouse and Drosophila ALS (Amyotrophic Lateral Sclerosis) disease models. Most important, a missense allele of Hsc70-4 (HspA8 in mammals) was recently identified as a bypass suppressor of the SMA phenotype in mice. Collectively, these findings suggest that chaperone-related dysfunction lies at the etiological root of both ALS and SMA.
    DOI:  https://doi.org/10.1101/2024.05.15.594402
  34. JCI Insight. 2024 Jun 20. pii: e171162. [Epub ahead of print]
    Sanglifehrin A mitigates multi-organ fibrosis by targeting the collagen chaperone cyclophilin B.
    Hope A Flaxman, Maria-Anna Chrysovergi, Hongwei Han, Farah Kabir, Rachael T Lister, Chia-Fu Chang, Robert Yvon, Katharine E Black, Andreas Weigert, Rajkumar Savai, Alejandro Egea-Zorrilla, Ana Pardo-Saganta, David Lagares, Christina M Woo.
      Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as anti-fibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photo-affinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-β1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress, affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-β1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct anti-fibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.
    Keywords:  Collagens; Drug therapy; Fibrosis; Inflammation; Therapeutics
    DOI:  https://doi.org/10.1172/jci.insight.171162
  35. Physiol Genomics. 2024 Jun 17.
    Computational Modeling of the Synergistic Role of GCN2 and HPA Axis in Regulating the Integrated Stress Response in the Central Circadian Timing System.
    Yannuo Li, Lingjun Lu, Jordan L Levy, Tracy G Anthony, Ioannis P Androulakis.
      The circadian timing system and integrated stress response (ISR) systems are fundamental regulatory mechanisms that maintain body homeostasis. The central circadian pacemaker in the suprachiasmatic nucleus (SCN) governs daily rhythms through interactions with peripheral oscillators via the hypothalamus-pituitary-adrenal (HPA) axis. On the other hand, ISR signaling is pivotal for preserving cellular homeostasis in response to physiological changes. Notably, disrupted circadian rhythms are observed in cases of impaired ISR signaling. In this work, we examine the potential interplay between the central circadian system and the ISR, mainly through the SCN and HPA axis. We introduce a semi-mechanistic mathematical model to delineate the suprachiasmatic nucleus (SCN)'s capacity for indirectly perceiving physiological stress through glucocorticoid-mediated feedback from the HPA axis and orchestrating a cellular response via the ISR mechanism. Key components of our investigation include evaluating general control nonderepressible 2 (GCN2) expression in the SCN, the effect of physiological stress stimuli on the HPA axis, and the interconnected feedback between the HPA and SCN. Simulation reveals a critical role for GCN2 in linking ISR with circadian rhythms. Experimental findings have demonstrated that a Gcn2 deletion in mice leads to rapid re-entrainment of the circadian clock following jetlag, as well as to an elongation of the circadian period. These.
    Keywords:  Circadian rhythms; GCN2; HPA; ISR; chronobiology
    DOI:  https://doi.org/10.1152/physiolgenomics.00030.2024
  36. Proc Natl Acad Sci U S A. 2024 Jun 18. 121(25): e2320995121
    RNF212B E3 ligase is essential for crossover designation and maturation during male and female meiosis in the mouse.
    Yazmine B Condezo, Raquel Sainz-Urruela, Laura Gomez-H, Daniel Salas-Lloret, Natalia Felipe-Medina, Rachel Bradley, Ian D Wolff, Stephanie Tanis, Jose Luis Barbero, Manuel Sánchez-Martín, Dirk de Rooij, Ivo A Hendriks, Michael L Nielsen, Román Gonzalez-Prieto, Paula E Cohen, Alberto M Pendas, Elena Llano.
      Meiosis, a reductional cell division, relies on precise initiation, maturation, and resolution of crossovers (COs) during prophase I to ensure the accurate segregation of homologous chromosomes during metaphase I. This process is regulated by the interplay of RING-E3 ligases such as RNF212 and HEI10 in mammals. In this study, we functionally characterized a recently identified RING-E3 ligase, RNF212B. RNF212B colocalizes and interacts with RNF212, forming foci along chromosomes from zygonema onward in a synapsis-dependent and DSB-independent manner. These consolidate into larger foci at maturing COs, colocalizing with HEI10, CNTD1, and MLH1 by late pachynema. Genetically, RNF212B foci formation depends on Rnf212 but not on Msh4, Hei10, and Cntd1, while the unloading of RNF212B at the end of pachynema is dependent on Hei10 and Cntd1. Mice lacking RNF212B, or expressing an inactive RNF212B protein, exhibit modest synapsis defects, a reduction in the localization of pro-CO factors (MSH4, TEX11, RPA, MZIP2) and absence of late CO-intermediates (MLH1). This loss of most COs by diakinesis results in mostly univalent chromosomes. Double mutants for Rnf212b and Rnf212 exhibit an identical phenotype to that of Rnf212b single mutants, while double heterozygous demonstrate a dosage-dependent reduction in CO number, indicating a functional interplay between paralogs. SUMOylome analysis of testes from Rnf212b mutants and pull-down analysis of Sumo- and Ubiquitin-tagged HeLa cells, suggest that RNF212B is an E3-ligase with Ubiquitin activity, serving as a crucial factor for CO maturation. Thus, RNF212 and RNF212B play vital, yet overlapping roles, in ensuring CO homeostasis through their distinct E3 ligase activities.
    Keywords:  crossing over; fertility; meiosis; mouse; recombination
    DOI:  https://doi.org/10.1073/pnas.2320995121
  37. Cancer Immunol Res. 2024 Jun 20.
    Endoplasmic reticulum stress potentiates the immunosuppressive microenvironment in hepatocellular carcinoma by promoting the release of SNHG6-enriched small extracellular vesicles.
    Chengdong Liu, Xiaohan Zhou, Hanyi Zeng, Jiaping Yu, Wenwen Li, Wanli Zhang, Yanxia Liao, Haijian Wang, Li Liu.
      Endoplasmic reticulum (ER) stress leads to hepatocellular carcinoma (HCC) progression. Small extracellular vesicles (sEVs) play a crucial role in modulating the tumor microenvironment (TME) by influencing cellular communication and immune responses. However, it is unclear whether ER stress modulates the TME through sEVs. In the current study, we investigated the effects and underlying mechanisms of ER stress on the HCC TME. In vivo and in vitro experiments showed that overactivated ER stress was a salient attribute of the immunosuppressive HCC TME. This was caused by the ATF4-promoted release of SNHG6-carrying sEVs, which attenuated T cell-mediated immune responses. Overall, SNHG6 modulated the immunosuppressive TME and aggravated ER stress. Meanwhile, targeting SNHG6 facilitated M1-like macrophage and CD8+ T-cell infiltration and decreased the proportion of M2-like macrophages. In addition, SNHG6 knockdown enhanced anti-PD-1 immunotherapeutic efficacy. Moreover, in HCC patients, overexpression of SNHG6 was associated with a lack of response to anti-PD-1 therapy and poor prognosis, whereas low SNHG6 expression was associated with improved therapeutic efficacy and prognoses. These data indicate that a correlation exists among ER stress, sEVs, an immunosuppressive HCC TME, and immunotherapeutic efficacy. Hence, SNHG6-targeted therapy may represent an effective strategy for patients with HCC.
    DOI:  https://doi.org/10.1158/2326-6066.CIR-23-0469
  38. Nat Commun. 2024 Jun 15. 15(1): 5119
    A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.
    Deborah Mesa, Elisa Barbieri, Andrea Raimondi, Stefano Freddi, Giorgia Miloro, Gorana Jendrisek, Giusi Caldieri, Micaela Quarto, Irene Schiano Lomoriello, Maria Grazia Malabarba, Arianna Bresci, Francesco Manetti, Federico Vernuccio, Hind Abdo, Giorgio Scita, Letizia Lanzetti, Dario Polli, Carlo Tacchetti, Paolo Pinton, Massimo Bonora, Pier Paolo Di Fiore, Sara Sigismund.
      One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.
    DOI:  https://doi.org/10.1038/s41467-024-49543-z
  39. Biochimie. 2024 Jun 18. pii: S0300-9084(24)00140-8. [Epub ahead of print]
    ProteoCure : an European network to fine-tune the proteome.
    Core Group of ProteoCure
      Proteins are essential molecular actors in every cellular process. From their synthesis to their degradation, they are subject to continuous quality control mechanisms to ensure that they fulfil cellular needs in proper and timely fashion. Proteostasis is a key process allowing cells or organisms to maintain an appropriate but dynamic equilibrium of their proteome (the ensemble of all their proteins). It relies on multiple mechanisms that together control the level, fate and function of individual proteins, and ensure elimination of abnormal ones. The proteostasis network is essential for development and adaptation to environmental changes or challenges. Its dysfunctions can lead to accumulation of deleterious proteins or, conversely, to excessive degradation of beneficial ones, and are implicated in many diseases such as cancers, neurodegeneration, or developmental and aging disorders. Manipulating this network to control abundance of selected target proteins is therefore a strategy with enormous therapeutic or biotechnological potential. The ProteoCure COST Action gathers more than 350 researchers and their teams (31 countries represented) from the academic, clinical, and industrial sectors, who share the conviction that our understanding of proteostasis is mature enough to develop novel and highly specific therapies based on selective tunning of protein levels. Towards this objective, the Action organizes community-building activities to foster synergies among its participants and reinforce training of the next generation of European researchers. Its ambition is to function as a knowledge-based network and a creative exchange hub on normal and pathologic proteostasis, focusing on developing innovative tools modulating the level of specific protein(s).
    Keywords:  COST Action; Europe; Proteolysis; Proteostasis; Targeted Protein Degradation
    DOI:  https://doi.org/10.1016/j.biochi.2024.06.004
  40. bioRxiv. 2024 May 26. pii: 2024.05.24.595755. [Epub ahead of print]
    The UFMylation pathway is impaired in Alzheimer's disease.
    Tingxiang Yan, Michael G Heckman, Emily C Craver, Chia-Chen Liu, Bailey D Rawlinson, Xue Wang, Melissa E Murray, Dennis W Dickson, Nilufer Ertekin-Taner, Zhenkun Lou, Guojun Bu, Wolfdieter Springer, Fabienne C Fiesel.
       Background: Alzheimer's disease (AD) is characterized by the presence of neurofibrillary tangles made of hyperphosphorylated tau and senile plaques composed of beta-amyloid. These pathognomonic deposits have been implicated in the pathogenesis, although the molecular mechanisms and consequences remain undetermined. UFM1 is an important, but understudied ubiquitin-like protein that is covalently attached to substrates. This UFMylation has recently been identified as major modifier of tau aggregation upon seeding in experimental models. However, potential alterations of the UFM1 pathway in human AD brain have not been investigated yet.
    Methods: Here we used frontal and temporal cortex samples from individuals with or without AD to measure the protein levels of the UFMylation pathway in human brain. We used multivariable regression analyses followed by Bonferroni correction for multiple testing to analyze associations of the UFMylation pathway with neuropathological characteristics, primary biochemical measurements of tau and additional biochemical markers from the same cases. We further studied associations of the UFMylation cascade with cellular stress pathways using Spearman correlations with bulk RNAseq expression data and functionally validated these interactions using gene-edited neurons that were generated by CRISPR-Cas9.
    Results: Compared to controls, human AD brain had increased protein levels of UFM1. Our data further indicates that this increase mainly reflects conjugated UFM1 indicating hyperUFMylation in AD. UFMylation was strongly correlated with pathological tau in both AD-affected brain regions. In addition, we found that the levels of conjugated UFM1 were negatively correlated with soluble levels of the deUFMylation enzyme UFSP2. Functional analysis of UFM1 and/or UFSP2 knockout neurons revealed that the DNA damage response as well as the unfolded protein response are perturbed by changes in neuronal UFM1 signaling.
    Conclusions: There are marked changes in the UFMylation pathway in human AD brain. These changes are significantly associated with pathological tau, supporting the idea that the UFMylation cascade might indeed act as a modifier of tau pathology in human brain. Our study further nominates UFSP2 as an attractive target to reduce the hyperUFMylation observed in AD brain but also underscores the critical need to identify risks and benefits of manipulating the UFMylation pathway as potential therapeutic avenue for AD.
    DOI:  https://doi.org/10.1101/2024.05.24.595755
  41. bioRxiv. 2024 Jun 03. pii: 2024.06.02.597051. [Epub ahead of print]
    The glycoprotein quality control factor Malectin promotes coronavirus replication and viral protein biogenesis.
    Jonathan P Davies, Lars Plate.
      Coronaviruses (CoV) rewire host protein homeostasis (proteostasis) networks through interactions between viral nonstructural proteins (nsps) and host factors to promote infection. With the emergence of SARS-CoV-2, it is imperative to characterize host interactors shared across nsp homologs. Using quantitative proteomics and functional genetic screening, we identify conserved proteostasis interactors of nsp2 and nsp4 that serve pro-viral roles during infection of murine hepatitis virus (MHV) - a model betacoronavirus. We uncover a glycoprotein quality control factor, Malectin (MLEC), which significantly reduces infectious titers when knocked down. During infection, nsp2 interacts with MLEC-associated proteins and the MLEC-interactome is drastically altered, stabilizing association with the Oligosaccheryltransferase (OST) complex, a crucial component of viral glycoprotein production. MLEC promotes viral protein levels and genome replication through its quality control activity. Lastly, we show MLEC promotes SARS-CoV-2 replication. Our results reveal a role for MLEC in mediating CoV infection and identify a potential target for pan-CoV antivirals.
    DOI:  https://doi.org/10.1101/2024.06.02.597051
  42. bioRxiv. 2024 May 02. pii: 2024.05.01.592080. [Epub ahead of print]
    Human ESCRT-I and ALIX function as scaffolding helical filaments in vivo.
    Stephanie J Spada, Kevin M Rose, Paola Sette, Sarah K O'Connor, Vincent Dussupt, V Siddartha Yerramilli, Kunio Nagashima, Virginie Helle Sjoelund, Phillip Cruz, Juraj Kabat, Sundar Ganesan, Margery Smelkinson, Aleksandra Nita-Lazar, Forrest Hoyt, Suzanne Scarlata, Vanessa Hirsch, Sonja M Best, Michael E Grigg, Fadila Bouamr.
      The Endosomal Sorting Complex Required for Transport (ESCRT) is an evolutionarily conserved machinery that performs reverse-topology membrane scission in cells universally required from cytokinesis to budding of enveloped viruses. Upstream acting ESCRT-I and ALIX control these events and link recruitment of viral and cellular partners to late-acting ESCRT-III CHMP4 through incompletely understood mechanisms. Using structure-function analyses combined with super-resolution imaging, we show that ESCRT-I and ALIX function as distinct helical filaments in vivo . Together, they are essential for optimal structural scaffolding of HIV-1 nascent virions, the retention of viral and human genomes through defined functional interfaces, and recruitment of CHMP4 that itself assembles into corkscrew-like filaments intertwined with ESCRT-I or ALIX helices. Disruption of filament assembly or their conformationally clustered RNA binding interfaces in human cells impaired membrane abscission, resulted in major structural instability and leaked nucleic acid from nascent virions and nuclear envelopes. Thus, ESCRT-I and ALIX function as helical filaments in vivo and serve as both nucleic acid-dependent structural scaffolds as well as ESCRT-III assembly templates.
    Significance statement: When cellular membranes are dissolved or breached, ESCRT is rapidly deployed to repair membranes to restore the integrity of intracellular compartments. Membrane sealing is ensured by ESCRT-III filaments assembled on the inner face of membrane; a mechanism termed inverse topology membrane scission. This mechanism, initiated by ESCRT-I and ALIX, is universally necessary for cytokinesis, wound repair, budding of enveloped viruses, and more. We show ESCRT-I and ALIX individually oligomerize into helical filaments that cluster newly discovered nucleic acid-binding interfaces and scaffold-in genomes within nascent virions and nuclear envelopes. These oligomers additionally appear to serve as ideal templates for ESCRT-III polymerization, as helical filaments of CHMP4B were found intertwined ESCRT-I or ALIX filaments in vivo . Similarly, corkscrew-like filaments of ALIX are also interwoven with ESCRT-I, supporting a model of inverse topology membrane scission that is synergistically reinforced by inward double filament scaffolding.
    DOI:  https://doi.org/10.1101/2024.05.01.592080
  43. Nature. 2024 Jun 19.
    Computational design of soluble and functional membrane protein analogues.
    Casper A Goverde, Martin Pacesa, Nicolas Goldbach, Lars J Dornfeld, Petra E M Balbi, Sandrine Georgeon, Stéphane Rosset, Srajan Kapoor, Jagrity Choudhury, Justas Dauparas, Christian Schellhaas, Simon Kozlov, David Baker, Sergey Ovchinnikov, Alex J Vecchio, Bruno E Correia.
      De novo design of complex protein folds using solely computational means remains a substantial challenge1. Here we use a robust deep learning pipeline to design complex folds and soluble analogues of integral membrane proteins. Unique membrane topologies, such as those from G-protein-coupled receptors2, are not found in the soluble proteome, and we demonstrate that their structural features can be recapitulated in solution. Biophysical analyses demonstrate the high thermal stability of the designs, and experimental structures show remarkable design accuracy. The soluble analogues were functionalized with native structural motifs, as a proof of concept for bringing membrane protein functions to the soluble proteome, potentially enabling new approaches in drug discovery. In summary, we have designed complex protein topologies and enriched them with functionalities from membrane proteins, with high experimental success rates, leading to a de facto expansion of the functional soluble fold space.
    DOI:  https://doi.org/10.1038/s41586-024-07601-y
  44. Int J Biol Sci. 2024 ;20(8): 2881-2903
    Nlp-dependent ER-to-Golgi transport.
    Danna Yeerken, Wenchang Xiao, Jia Li, Yan Wang, Qingnan Wu, Jie Chen, Wei Gong, Mengzhu Lv, Ting Wang, Ying Gong, Rui Liu, Jiawen Fan, Jinting Li, Weimin Zhang, Qimin Zhan.
      The mechanism that maintains ER-to-Golgi vesicles formation and transport is complicated. As one of the adapters, Ninein-like protein (Nlp) participated in assembly and transporting of partial ER-to-Golgi vesicles that contained specific proteins, such as β-Catenin and STING. Nlp acted as a platform to sustain the specificity and continuity of cargoes during COPII and COPI-coated vesicle transition and transportation through binding directly with SEC31A as well as Rab1B. Thus, we proposed an integrated transport model that particular adapter participated in specific cargo selection or transportation through cooperating with different membrane associated proteins to ensure the continuity of cargo trafficking. Deficiency of Nlp led to vesicle budding failure and accumulation of unprocessed proteins in ER, which further caused ER stress as well as Golgi fragmentation, and PERK-eIF2α pathway of UPR was activated to reduce the synthesis of universal proteins. In contrast, upregulation of Nlp resulted in Golgi fragmentation, which enhanced the cargo transport efficiency between ER and Golgi. Moreover, Nlp deficient mice were prone to spontaneous B cell lymphoma, since the developments and functions of lymphocytes significantly depended on secretory proteins through ER-to-Golgi vesicle trafficking, including IL-13, IL-17 and IL-21. Thus, perturbations of Nlp altered ER-to-Golgi communication and cellular homeostasis, and might contribute to the pathogenesis of B cell lymphoma.
    Keywords:  Adapter; B cell lymphoma; ER stress; ER-to-Golgi cargo trafficking; Nlp
    DOI:  https://doi.org/10.7150/ijbs.91792
  45. bioRxiv. 2024 Apr 23. pii: 2024.04.19.590253. [Epub ahead of print]
    Yeast poly(A)-binding protein (Pab1) controls translation initiation in vivo primarily by blocking mRNA decapping and decay.
    Poonam Poonia, Vishalini Valabhoju, Tianwei Li, James Iben, Xiao Niu, Zhenguo Lin, Alan G Hinnebusch.
      Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in a manner suppressed by deleting the catalytic subunit of decapping enzyme ( dcp2 Δ), demonstrating that enhanced decapping/degradation is the major driver of reduced mRNA abundance and protein synthesis at limiting Pab1 levels. An increased median poly(A) tail length conferred by Pab1 depletion was also nullified by dcp2 Δ, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1. In contrast to findings on mammalian cells, the translational efficiencies (TEs) of many mRNAs were altered by Pab1 depletion; however, these changes were broadly diminished by dcp2 Δ, suggesting that reduced mRNA abundance is a major driver of translational reprogramming at limiting Pab1. Thus, assembly of the closed-loop mRNP via PABP-eIF4G interaction appears to be dispensable for normal translation of most yeast mRNAs in vivo. Interestingly, histone mRNAs and proteins are preferentially diminished on Pab1 depletion dependent on Dcp2, accompanied by activation of internal cryptic promoters in the manner expected for reduced nucleosome occupancies, revealing a new layer of post-transcriptional control of histone gene expression.
    DOI:  https://doi.org/10.1101/2024.04.19.590253
  46. Proc Natl Acad Sci U S A. 2024 Jun 25. 121(26): e2322927121
    StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol.
    Jinmo Koo, Subba Reddy Palli.
      RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.
    Keywords:  C-terminal KDEL motif; Colorado potato beetle; RNA interference; intracellular trafficking; protein localization
    DOI:  https://doi.org/10.1073/pnas.2322927121
  47. Cell Rep. 2024 Jun 14. pii: S2211-1247(24)00733-2. [Epub ahead of print]43(6): 114404
    Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5.
    Eiji Obayashi, Rafael E Luna, Takashi Nagata, Pilar Martin-Marcos, Hiroyuki Hiraishi, Chingakham Ranjit Singh, Jan Peter Erzberger, Fan Zhang, Haribabu Arthanari, Jacob Morris, Riccardo Pellarin, Chelsea Moore, Ian Harmon, Evangelos Papadopoulos, Hisashi Yoshida, Mahmoud L Nasr, Satoru Unzai, Bryttney Thompson, Eric Aube, Samantha Hustak, Florian Stengel, Eddie Dagraca, Asokan Ananbandam, Philip Gao, Takeshi Urano, Alan G Hinnebusch, Gerhard Wagner, Katsura Asano.
      
    DOI:  https://doi.org/10.1016/j.celrep.2024.114404
  48. Nat Commun. 2024 Jun 18. 15(1): 5201
    Chemogenomics for NR1 nuclear hormone receptors.
    Laura Isigkeit, Espen Schallmayer, Romy Busch, Lorene Brunello, Amelie Menge, Lewis Elson, Susanne Müller, Stefan Knapp, Alexandra Stolz, Julian A Marschner, Daniel Merk.
      Nuclear receptors (NRs) regulate transcription in response to ligand binding and NR modulation allows pharmacological control of gene expression. Although some NRs are relevant as drug targets, the NR1 family, which comprises 19 NRs binding to hormones, vitamins, and lipid metabolites, has only been partially explored from a translational perspective. To enable systematic target identification and validation for this protein family in phenotypic settings, we present an NR1 chemogenomic (CG) compound set optimized for complementary activity/selectivity profiles and chemical diversity. Based on broad profiling of candidates for specificity, toxicity, and off-target liabilities, sixty-nine comprehensively annotated NR1 agonists, antagonists and inverse agonists covering all members of the NR1 family and meeting potency and selectivity standards are included in the final NR1 CG set. Proof-of-concept application of this set reveals effects of NR1 members in autophagy, neuroinflammation and cancer cell death, and confirms the suitability of the set for target identification and validation.
    DOI:  https://doi.org/10.1038/s41467-024-49493-6
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