bims-proteo Biomed News
on Proteostasis
Issue of 2024‒05‒26
forty-six papers selected by
Eric Chevet, INSERM
  1. ER-to-lysosome-associated degradation acts as failsafe mechanism upon ERAD dysfunction.
  2. ENDOPLASMIC RETICULUM: Monitoring and maintaining protein and membrane homeostasis in the endoplasmic reticulum by the unfolded protein response.
  3. mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.
  4. Upregulation of neuronal ER-phagy improves organismal fitness and alleviates APP toxicity.
  5. Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.
  6. ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.
  7. VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models.
  8. Circadian regulation of macromolecular complex turnover and proteome renewal.
  9. Ca2+-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.
  10. The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.
  11. Linear ubiquitin chains remodel the proteome and influence the levels of hundreds of regulators in Drosophila.
  12. The integrated stress response in pancreatic development, tissue homeostasis, and cancer.
  13. Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.
  14. Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.
  15. Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.
  16. Mitochondrial Chaperone Code: Just Warming Up.
  17. LC3A-mediated autophagy elicits PERK-eIF2α-ATF4 axis activation and mitochondrial dysfunction: Exposing vulnerability in aggresome positive cancer cells.
  18. A chemical probe to modulate human GID4 Pro/N-degron interactions.
  19. AMPK regulates phagophore-to-autophagosome maturation.
  20. eIF4A1 enhances LARP1-mediated translational repression during mTORC1 inhibition.
  21. Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.
  22. Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1.
  23. An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.
  24. Ribosome profiling reveals the role of yeast RNA-binding proteins Cth1 and Cth2 in translational regulation.
  25. Collagen fibril formation at the plasma membrane occurs independently from collagen secretion.
  26. Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.
  27. Legionella uses host Rab GTPases and BAP31 to create a unique ER niche.
  28. Mitochondria-ER-PM contacts regulate mitochondrial division and PI(4)P distribution.
  29. Apolipoprotein E2 Expression Alters Endosomal Pathways in a Mouse Model With Increased Brain Exosome Levels During Aging.
  30. Soft extracellular matrix drives endoplasmic reticulum stress-dependent S quiescence underlying molecular traits of pulmonary basal cells.
  31. Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.
  32. MLKL promotes hepatocarcinogenesis through inhibition of AMPK-mediated autophagy.
  33. Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.
  34. Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension.
  35. VCP/p97 UFMylation stabilizes BECN1 and facilitates the initiation of autophagy.
  36. Full-length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles.
  37. Upstream open reading frames: new players in the landscape of cancer gene regulation.
  38. The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.
  39. Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology.
  40. Natural proteome diversity links aneuploidy tolerance to protein turnover.
  41. Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.
  42. KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.
  43. An E3 ligase TRIM1 promotes colorectal cancer progression via K63-linked ubiquitination and activation of HIF1α.
  44. Pan-cancer analysis of the interplay between mutational signatures and cellular signaling.
  45. The impact of E3 ligase choice on PROTAC effectiveness in protein kinase degradation.
  46. TRIM9 controls growth cone responses to netrin through DCC and UNC5C.
  1. EMBO Rep. 2024 May 21.
    ER-to-lysosome-associated degradation acts as failsafe mechanism upon ERAD dysfunction.
    Elisa Fasana, Ilaria Fregno, Carmela Galli, Tatiana Soldà, Maurizio Molinari.
      The endoplasmic reticulum (ER) produces proteins destined to organelles of the endocytic and secretory pathways, the plasma membrane, and the extracellular space. While native proteins are transported to their intra- or extracellular site of activity, folding-defective polypeptides are retro-translocated across the ER membrane into the cytoplasm, poly-ubiquitylated and degraded by 26 S proteasomes in a process called ER-associated degradation (ERAD). Large misfolded polypeptides, such as polymers of alpha1 antitrypsin Z (ATZ) or mutant procollagens, fail to be dislocated across the ER membrane and instead enter ER-to-lysosome-associated degradation (ERLAD) pathways. Here, we show that pharmacological or genetic inhibition of ERAD components, such as the α1,2-mannosidase EDEM1 or the OS9 ERAD lectins triggers the delivery of the canonical ERAD clients Null Hong Kong (NHK) and BACE457Δ to degradative endolysosomes under control of the ER-phagy receptor FAM134B and the LC3 lipidation machinery. Our results reveal that ERAD dysfunction is compensated by the activation of FAM134B-driven ERLAD pathways that ensure efficient lysosomal clearance of orphan ERAD clients.
    Keywords:  ER-associated Degradation (ERAD); ER-phagy; ER-to-Lysosome-associated Degradation (ERLAD); Endoplasmic Reticulum (ER); Protein Quality Control
    DOI:  https://doi.org/10.1038/s44319-024-00165-y
  2. Int J Biochem Cell Biol. 2024 May 18. pii: S1357-2725(24)00090-6. [Epub ahead of print] 106598
    ENDOPLASMIC RETICULUM: Monitoring and maintaining protein and membrane homeostasis in the endoplasmic reticulum by the unfolded protein response.
    Paulina Kettel, G Elif Karagöz.
      The endoplasmic reticulum (ER) regulates essential cellular processes, including protein folding, lipid synthesis, and calcium homeostasis. The ER homeostasis is maintained by a conserved set of signaling cascades called the Unfolded Protein Response (UPR). How the UPR senses perturbations in ER homeostasis has been the subject of active research for decades. In metazoans, the UPR consists of three ER-membrane embedded sensors: IRE1, PERK and ATF6. These sensors detect the accumulation of misfolded proteins in the ER lumen and adjust protein folding capacity according to cellular needs. Early work revealed that the ER-resident chaperone BiP binds to all three UPR sensors in higher eukaryotes and BiP binding was suggested to regulate their activity. More recent data have shown that in higher eukaryotes the interaction of the UPR sensors with a complex network of chaperones and misfolded proteins modulates their activation and deactivation dynamics. Furthermore, emerging evidence suggests that the UPR monitors ER membrane integrity beyond protein folding defects. However, the mechanistic and structural basis of UPR activation by proteotoxic and lipid bilayer stress in higher eukaryotes remains only partially understood. Here, we review the current understanding of novel protein interaction networks and the contribution of the lipid membrane environment to UPR activation.
    Keywords:  Chaperone; Unfolded Protein Response; disordered regions; lipid bilayer stress
    DOI:  https://doi.org/10.1016/j.biocel.2024.106598
  3. Mol Cell. 2024 May 20. pii: S1097-2765(24)00393-9. [Epub ahead of print]
    mTORC1-CTLH E3 ligase regulates the degradation of HMG-CoA synthase 1 through the Pro/N-degron pathway.
    Sang Ah Yi, Sara Sepic, Brenda A Schulman, Alban Ordureau, Heeseon An.
      Mammalian target of rapamycin (mTOR) senses changes in nutrient status and stimulates the autophagic process to recycle amino acids. However, the impact of nutrient stress on protein degradation beyond autophagic turnover is incompletely understood. We report that several metabolic enzymes are proteasomal targets regulated by mTOR activity based on comparative proteome degradation analysis. In particular, 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) synthase 1 (HMGCS1), the initial enzyme in the mevalonate pathway, exhibits the most significant half-life adaptation. Degradation of HMGCS1 is regulated by the C-terminal to LisH (CTLH) E3 ligase through the Pro/N-degron motif. HMGCS1 is ubiquitylated on two C-terminal lysines during mTORC1 inhibition, and efficient degradation of HMGCS1 in cells requires a muskelin adaptor. Importantly, modulating HMGCS1 abundance has a dose-dependent impact on cell proliferation, which is restored by adding a mevalonate intermediate. Overall, our unbiased degradomics study provides new insights into mTORC1 function in cellular metabolism: mTORC1 regulates the stability of limiting metabolic enzymes through the ubiquitin system.
    Keywords:  CTLH; GID; HMGCS1; degradomics; mTOR; mTORC1; mevalonate pathway; sterol; ubiquitin; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.molcel.2024.04.026
  4. Cell Rep. 2024 May 17. pii: S2211-1247(24)00583-7. [Epub ahead of print]43(5): 114255
    Upregulation of neuronal ER-phagy improves organismal fitness and alleviates APP toxicity.
    Wenqing Mou, Yinglu Tang, Yunpeng Huang, Zhihao Wu, Yixian Cui.
      ER-phagy, a selective autophagy targeting the endoplasmic reticulum (ER) for lysosomal degradation through cargo receptors, plays a critical role in ER quality control and is linked to various diseases. However, its physiological and pathological roles remain largely unclear due to a lack of animal model studies. This study establishes Drosophila as an in vivo ER-phagy model. Starvation triggers ER-phagy across multiple fly tissues. Disturbing ER-phagy by either globally upregulating or downregulating ER-phagy receptors, Atl or Rtnl1, harms the fly. Notably, moderate upregulation of ER-phagy in fly brains by overexpressing Atl or Rtnl1 significantly attenuates age-associated neurodegenerations. Furthermore, in a Drosophila model of Alzheimer's disease expressing human amyloid precursor protein (APP), impaired ER-phagy is observed. Enhancing ER-phagy in the APP-expressing fly brain facilitates APP degradation, significantly alleviating disease symptoms. Therefore, our findings suggest that modulating ER-phagy may offer a therapeutic strategy to treat aging and diseases associated with ER protein aggregation.
    Keywords:  APP; Atg8a; Atl; CP: Cell biology; CP: Molecular biology; ER-phagy; Rtnl1; aging; cargo receptor; macro-autophagy; neurodegeneration
    DOI:  https://doi.org/10.1016/j.celrep.2024.114255
  5. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2321167121
    Structure and mechanism of the human CTDNEP1-NEP1R1 membrane protein phosphatase complex necessary to maintain ER membrane morphology.
    Shujuan Gao, Jake W Carrasquillo Rodríguez, Shirin Bahmanyar, Michael V Airola.
      C-terminal Domain Nuclear Envelope Phosphatase 1 (CTDNEP1) is a noncanonical protein serine/threonine phosphatase that has a conserved role in regulating ER membrane biogenesis. Inactivating mutations in CTDNEP1 correlate with the development of medulloblastoma, an aggressive childhood cancer. The transmembrane protein Nuclear Envelope Phosphatase 1 Regulatory Subunit 1 (NEP1R1) binds CTDNEP1, but the molecular details by which NEP1R1 regulates CTDNEP1 function are unclear. Here, we find that knockdown of NEP1R1 generates identical phenotypes to reported loss of CTDNEP1 in mammalian cells, establishing CTDNEP1-NEP1R1 as an evolutionarily conserved membrane protein phosphatase complex that restricts ER expansion. Mechanistically, NEP1R1 acts as an activating regulatory subunit that directly binds and increases the phosphatase activity of CTDNEP1. By defining a minimal NEP1R1 domain sufficient to activate CTDNEP1, we determine high-resolution crystal structures of the CTDNEP1-NEP1R1 complex bound to a peptide sequence acting as a pseudosubstrate. Structurally, NEP1R1 engages CTDNEP1 at a site distant from the active site to stabilize and allosterically activate CTDNEP1. Substrate recognition is facilitated by a conserved Arg residue in CTDNEP1 that binds and orients the substrate peptide in the active site. Together, this reveals mechanisms for how NEP1R1 regulates CTDNEP1 and explains how cancer-associated mutations inactivate CTDNEP1.
    DOI:  https://doi.org/10.1073/pnas.2321167121
  6. Life Sci Alliance. 2024 Aug;pii: e202402620. [Epub ahead of print]7(8):
    ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.
    Matteo Veronese, Sebastian Kallabis, Alexander Tobias Kaczmarek, Anushka Das, Lennart Robers, Simon Schumacher, Alessia Lofrano, Susanne Brodesser, Stefan Müller, Kay Hofmann, Marcus Krüger, Elena I Rugarli.
      Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.
    DOI:  https://doi.org/10.26508/lsa.202402620
  7. J Clin Invest. 2024 May 23. pii: e169039. [Epub ahead of print]
    VCP activator reverses nuclear proteostasis defects and enhances TDP-43 aggregate clearance in multisystem proteinopathy models.
    Jessica M Phan, Benjamin C Creekmore, Aivi T Nguyen, Darya D Bershadskaya, Nabil F Darwich, Carolyn N Mann, Edward B Lee.
      Pathogenic variants in VCP cause multisystem proteinopathy (MSP), a disease characterized by multiple clinical phenotypes including inclusion body myopathy, Paget's disease of the bone, and frontotemporal dementia (FTD). How such diverse phenotypes are driven by pathogenic VCP variants is not known. We found that these diseases exhibit a common pathologic feature, ubiquitinated intranuclear inclusions affecting myocytes, osteoclasts and neurons. Moreover, knock-in cell lines harboring MSP variants show a reduction in nuclear VCP. Given that MSP is associated with neuronal intranuclear inclusions comprised of TDP-43 protein, we developed a cellular model whereby proteostatic stress results in the formation of insoluble intranuclear TDP-43 aggregates. Consistent with a loss of nuclear VCP function, cells harboring MSP variants or cells treated with VCP inhibitor exhibited decreased clearance of insoluble intranuclear TDP-43 aggregates. Moreover, we identified four compounds that activate VCP primarily by increasing D2 ATPase activity whereby pharmacologic VCP activation appears to enhance clearance of insoluble intranuclear TDP-43 aggregate. Our findings suggest that VCP function is important for nuclear protein homeostasis, that impaired nuclear proteostasis may contribute to MSP, and that VCP activation may be potential therapeutic by virtue of enhancing the clearance of intranuclear protein aggregates.
    Keywords:  Genetic diseases; Neurodegeneration; Neuroscience; Pharmacology; Therapeutics
    DOI:  https://doi.org/10.1172/JCI169039
  8. EMBO J. 2024 May 22.
    Circadian regulation of macromolecular complex turnover and proteome renewal.
    Estere Seinkmane, Anna Edmondson, Sew Y Peak-Chew, Aiwei Zeng, Nina M Rzechorzek, Nathan R James, James West, Jack Munns, David Cs Wong, Andrew D Beale, John S O'Neill.
      Although costly to maintain, protein homeostasis is indispensable for normal cellular function and long-term health. In mammalian cells and tissues, daily variation in global protein synthesis has been observed, but its utility and consequences for proteome integrity are not fully understood. Using several different pulse-labelling strategies, here we gain direct insight into the relationship between protein synthesis and abundance proteome-wide. We show that protein degradation varies in-phase with protein synthesis, facilitating rhythms in turnover rather than abundance. This results in daily consolidation of proteome renewal whilst minimising changes in composition. Coupled rhythms in synthesis and turnover are especially salient to the assembly of macromolecular protein complexes, particularly the ribosome, the most abundant species of complex in the cell. Daily turnover and proteasomal degradation rhythms render cells and mice more sensitive to proteotoxic stress at specific times of day, potentially contributing to daily rhythms in the efficacy of proteasomal inhibitors against cancer. Our findings suggest that circadian rhythms function to minimise the bioenergetic cost of protein homeostasis through temporal consolidation of protein turnover.
    Keywords:  Circadian; Proteostasis; Ribosome; SILAC
    DOI:  https://doi.org/10.1038/s44318-024-00121-5
  9. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2318412121
    Ca2+-sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.
    Wei Chen, Madeline M Motsinger, Jiaqian Li, Kevin P Bohannon, Phyllis I Hanson.
      Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that low concentrations of GPN rupture a small fraction of lysosomes, but surprisingly trigger Ca2+ release from nearly all. Chelating cytoplasmic Ca2+ makes lysosomes more sensitive to GPN-induced rupture, suggesting a role for Ca2+ in lysosomal membrane resilience. GPN-elicited Ca2+ release causes the Ca2+-sensor Apoptosis Linked Gene-2 (ALG-2), along with Endosomal Sorting Complex Required for Transport (ESCRT) proteins it interacts with, to redistribute onto lysosomes. Functionally, ALG-2, but not its ESCRT binding-disabled ΔGF122 splice variant, increases lysosomal resilience to osmotic stress. Importantly, elevating juxta-lysosomal Ca2+ without membrane damage by activating TRPML1 also recruits ALG-2 and ESCRTs, protecting lysosomes from subsequent osmotic rupture. These findings reveal that Ca2+, through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.
    Keywords:  ALG-2; ESCRTs; lysosome; membrane resilience; osmotic stress
    DOI:  https://doi.org/10.1073/pnas.2318412121
  10. bioRxiv. 2024 May 09. pii: 2024.05.09.593318. [Epub ahead of print]
    The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.
    Brian J North, Amelia E Ohnstad, Michael J Ragusa, Christopher J Shoemaker.
      During autophagy, potentially toxic cargo is enveloped by a newly formed autophagosome and trafficked to the lysosome for degradation. Ubiquitinated protein aggregates, a key target for autophagy, are identified by multiple autophagy receptors. NBR1 is an archetypal autophagy receptor and an excellent model for deciphering the role of the multivalent, heterotypic interactions made by cargo-bound receptors. Using NBR1 as a model, we find that three critical binding partners - ATG8-family proteins, FIP200, and TAX1BP1 - each bind to a short linear interaction motif (SLiM) within NBR1. Mutational peptide arrays indicate that these binding events are mediated by distinct overlapping determinants, rather than a single, convergent, SLiM. AlphaFold modeling underlines the need for conformational flexibility within the NBR1 SLiM, as distinct conformations mediate each binding event. To test the extent to which overlapping SLiMs exist beyond NBR1, we performed peptide binding arrays on >100 established LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Comparative analysis of phosphomimetic peptides highlights that while FIP200 and Atg8-family binding are generally augmented by phosphorylation, TAX1BP1 binding is nonresponsive, suggesting differential regulation of these binding events. In vivo studies confirm that LIR-mediated interactions with TAX1BP1 enhance NBR1 activity, increasing autophagosomal delivery by leveraging an additional LIR from TAX1BP1. In sum, these results reveal a one-to-many binding modality in NBR1, providing key insights into the cooperative mechanisms among autophagy receptors. Furthermore, these findings underscore the pervasive role of multifunctional SLiMs in autophagy, offering substantial avenues for further exploration into their regulatory functions.
    DOI:  https://doi.org/10.1101/2024.05.09.593318
  11. bioRxiv. 2024 May 10. pii: 2024.05.09.593206. [Epub ahead of print]
    Linear ubiquitin chains remodel the proteome and influence the levels of hundreds of regulators in Drosophila.
    Oluwademilade Nuga, Kristin Richardson, Nikhil Patel, Xusheng Wang, Vishwajeeth Pagala, Anna Stephan, Junmin Peng, Fabio Demontis, Sokol V Todi.
      Ubiquitin controls many cellular processes via its post-translational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their own ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express non-cleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin with distinct lengths and suggest that the ubiquitin system is exceedingly pliable.
    DOI:  https://doi.org/10.1101/2024.05.09.593206
  12. Gastroenterology. 2024 May 18. pii: S0016-5085(24)04931-X. [Epub ahead of print]
    The integrated stress response in pancreatic development, tissue homeostasis, and cancer.
    Greg Malnassy, Leah Ziolkowski, Kay F Macleod, Scott A Oakes.
      Present in all eukaryotic cells, the integrated stress response (ISR) is a highly coordinated signaling network that controls cellular behavior, metabolism and survival in response to diverse stresses. The ISR is initiated when any one of four stress sensing kinases (PERK, GCN2, PKR, HRI) becomes activated to phosphorylate the protein translation initiation factor eIF2α, shifting gene expression toward a comprehensive rewiring of cellular machinery to promote adaptation. While the ISR has been shown to play an important role in the homeostasis of multiple tissues, evidence suggests that it is particularly crucial for the development and ongoing health of the pancreas. Among the most synthetically dynamic tissues in the body, the exocrine and endocrine pancreas relies heavily on the ISR to rapidly adjust cell function to meet the metabolic demands of the organism. The hardwiring of the ISR into normal pancreatic functions and adaptation to stress may explain why it is a commonly utilized pro-oncogenic and therapy-resistance mechanism in both pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumors (PanNETs). Here we review what is known about the key roles that the ISR plays in the development, homeostasis, and neoplasia of the pancreas.
    Keywords:  diabetes; pancreatic ductal adenocarcinoma; pancreatic neuroendocrine tumors; pancreatitis
    DOI:  https://doi.org/10.1053/j.gastro.2024.05.009
  13. J Cell Sci. 2024 May 20. pii: jcs.261977. [Epub ahead of print]
    Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.
    Shotaro Namba, Hisao Moriya.
      While protein aggregation can cause cytotoxicity, it also forms to mitigate cytotoxicity from misfolded proteins, though the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein linked protein (3×GFP) induces giant aggregates, and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbs the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70, causing the heat shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat shock response while preventing an overload to the degradation system.
    Keywords:  Aggregation; Fluorocent protein; Hsp70; Overproduction; Toxicity; Yeast
    DOI:  https://doi.org/10.1242/jcs.261977
  14. J Gen Virol. 2024 May;105(5):
    Japanese encephalitis virus hijacks ER-associated degradation regulators for its replication.
    Riya Sarkar, Simran Chhabra, Mukesh Tanwar, Nisheeth Agarwal, Manjula Kalia.
      Flaviviruses target their replication on membranous structures derived from the ER, where both viral and host proteins play crucial structural and functional roles. Here, we have characterized the involvement of the ER-associated degradation (ERAD) pathway core E3 ligase complex (SEL1L-HRD1) regulator proteins in the replication of Japanese encephalitis virus (JEV). Through high-resolution immunofluorescence imaging of JEV-infected HeLa cells, we observe that the virus replication complexes marked by NS1 strongly colocalize with the ERAD adapter SEL1L, lectin OS9, ER-membrane shuttle factor HERPUD1, E3 ubiquitin ligase HRD1 and rhomboid superfamily member DERLIN1. NS5 positive structures also show strong overlap with SEL1L. While these effectors show significant transcriptional upregulation, their protein levels remain largely stable in infected cells. siRNA mediated depletion of OS9, SEL1L, HERPUD1 and HRD1 significantly inhibit viral RNA replication and titres, with SEL1L depletion showing the maximum attenuation of replication. By performing protein translation arrest experiments, we show that SEL1L, and OS9 are stabilised upon JEV infection. Overall results from this study suggest that these ERAD effector proteins are crucial host-factors for JEV replication.
    Keywords:  DERLIN1; ERAD; HERPUD1; HRD1; OS9; SEL1L; flavivirus; retrotranslocon; ubiquitin proteasome
    DOI:  https://doi.org/10.1099/jgv.0.001995
  15. Sci Rep. 2024 05 22. 14(1): 11718
    Deletion of IRE1α in podocytes exacerbates diabetic nephropathy in mice.
    Andrey V Cybulsky, Joan Papillon, Julie Guillemette, José R Navarro-Betancourt, Chen-Fang Chung, Takao Iwawaki, I George Fantus.
      Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.
    Keywords:  Albuminuria; Autophagy; Endoplasmic reticulum; Gene expression; Glomerulopathy; Unfolded protein response
    DOI:  https://doi.org/10.1038/s41598-024-62599-7
  16. Cell Stress Chaperones. 2024 May 17. pii: S1355-8145(24)00074-9. [Epub ahead of print]
    Mitochondrial Chaperone Code: Just Warming Up.
    R Felipe Perez, Gianna Mochi, Ariba Khan, Mark Woodford.
      More than 99% of the mitochondrial proteome is encoded by the nucleus and requires refolding following import. Therefore, mitochondrial proteins require the coordinated action of molecular chaperones for their folding and activation. Several heat shock protein (Hsp) molecular chaperones, including members of the Hsp27, Hsp40/70, and Hsp90 families, as well as the chaperonin complex Hsp60/10 have an established role in mitochondrial protein import and folding. The 'Chaperone Code' describes the regulation of chaperone activity by dynamic post-translational modifications; however, little is known about post-translational regulation of mitochondrial chaperones. Dissecting the regulation of chaperone function is essential for understanding their differential regulation in pathogenic conditions and the potential development of efficacious therapeutic strategies. Here, we summarize the recent literature on post-translational regulation of mitochondrial chaperones, the consequences for mitochondrial function, and potential implications for disease.
    Keywords:  chaperones; heat shock proteins; metabolism; mitochondria; post-translational modification
    DOI:  https://doi.org/10.1016/j.cstres.2024.05.002
  17. J Biol Chem. 2024 May 20. pii: S0021-9258(24)01899-4. [Epub ahead of print] 107398
    LC3A-mediated autophagy elicits PERK-eIF2α-ATF4 axis activation and mitochondrial dysfunction: Exposing vulnerability in aggresome positive cancer cells.
    Nada Amer, Dina Hesham, Nouran Al-Shehaby, Hisham A Elshoky, May Amer, Sameh Magdeldin, Manar Mansour, Khaled Abou-Aisha, Shahenda El-Naggar.
      The unfolded protein response pathways (UPR), autophagy, and compartmentalization of misfolded proteins into inclusion bodies are critical components of the protein quality control network. Among inclusion bodies, aggresomes are particularly intriguing due to their association with cellular survival, drug resistance, and cancer-aggressive behavior. Aggresomes are molecular condensates formed when collapsed vimentin cages encircle misfolded proteins before final removal by autophagy. Yet significant gaps persist in the mechanisms governing aggresome formation and elimination in cancer cells. Understanding these mechanisms is crucial, especially considering the involvement of LC3A, a member of the MAP1LC3 family, which plays a unique role in autophagy regulation and has been reported to be epigenetically silenced in many cancers. Herein, we utilized tetracycline-inducible expression of LC3A to investigate its role in choroid plexus carcinoma cells, which inherently exhibit the presence of aggresomes. Live cell imaging was employed to demonstrate the effect of LC3A expression on aggresome-positive cells, while SILAC-based proteomics identified LC3A-induced protein and pathway alterations. Our findings demonstrate that extended expression of LC3A is associated with cellular senescence. However, the obstruction of lysosomal degradation in this context has a deleterious effect on cellular viability. In response to LC3A-induced autophagy, we observed significant alterations in mitochondrial morphology, reflected by mitochondrial dysfunction and increased ROS production. Furthermore, LC3A expression elicited the activation of the PERK-eIF2α-ATF4 axis of the UPR, underscoring a significant change in protein quality control network. In conclusion, our results elucidate that LC3A-mediated autophagy alters the protein quality control network, exposing a vulnerability in aggresome-positive cancer cells.
    Keywords:  Aggresomes; Autophagy; Endoplasmic reticulum; Inclusion bodies; MAP1LC3A; Protein quality control; Proteostasis; Senescence
    DOI:  https://doi.org/10.1016/j.jbc.2024.107398
  18. Nat Chem Biol. 2024 May 21.
    A chemical probe to modulate human GID4 Pro/N-degron interactions.
    Dominic D G Owens, Matthew E R Maitland, Aliakbar Khalili Yazdi, Xiaosheng Song, Viviane Reber, Martin P Schwalm, Raquel A C Machado, Nicolas Bauer, Xu Wang, Magdalena M Szewczyk, Cheng Dong, Aiping Dong, Peter Loppnau, Matthew F Calabrese, Matthew S Dowling, Jisun Lee, Justin I Montgomery, Thomas N O'Connell, Chakrapani Subramanyam, Feng Wang, Ella C Adamson, Matthieu Schapira, Matthias Gstaiger, Stefan Knapp, Masoud Vedadi, Jinrong Min, Gilles A Lajoie, Dalia Barsyte-Lovejoy, Dafydd R Owen, Caroline Schild-Poulter, Cheryl H Arrowsmith.
      The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.
    DOI:  https://doi.org/10.1038/s41589-024-01618-0
  19. J Cell Biol. 2024 Aug 05. pii: e202309145. [Epub ahead of print]223(8):
    AMPK regulates phagophore-to-autophagosome maturation.
    Carlo Barnaba, David G Broadbent, Emily G Kaminsky, Gloria I Perez, Jens C Schmidt.
      Autophagy is an important metabolic pathway that can non-selectively recycle cellular material or lead to targeted degradation of protein aggregates or damaged organelles. Autophagosome formation starts with autophagy factors accumulating on lipid vesicles containing ATG9. These phagophores attach to donor membranes, expand via ATG2-mediated lipid transfer, capture cargo, and mature into autophagosomes, ultimately fusing with lysosomes for their degradation. Autophagy can be activated by nutrient stress, for example, by a reduction in the cellular levels of amino acids. In contrast, how autophagy is regulated by low cellular ATP levels via the AMP-activated protein kinase (AMPK), an important therapeutic target, is less clear. Using live-cell imaging and an automated image analysis pipeline, we systematically dissect how nutrient starvation regulates autophagosome biogenesis. We demonstrate that glucose starvation downregulates autophagosome maturation by AMPK-mediated inhibition of phagophore tethering to donor membrane. Our results clarify AMPKs regulatory role in autophagy and highlight its potential as a therapeutic target to reduce autophagy.
    DOI:  https://doi.org/10.1083/jcb.202309145
  20. Nat Struct Mol Biol. 2024 May 21.
    eIF4A1 enhances LARP1-mediated translational repression during mTORC1 inhibition.
    Yuichi Shichino, Tomokazu Yamaguchi, Kazuhiro Kashiwagi, Mari Mito, Mari Takahashi, Takuhiro Ito, Nicholas T Ingolia, Keiji Kuba, Shintaro Iwasaki.
      Eukaryotic translation initiation factor (eIF)4A-a DEAD-box RNA-binding protein-plays an essential role in translation initiation. Recent reports have suggested helicase-dependent and helicase-independent functions for eIF4A, but the multifaceted roles of eIF4A have not been fully explored. Here we show that eIF4A1 enhances translational repression during the inhibition of mechanistic target of rapamycin complex 1 (mTORC1), an essential kinase complex controlling cell proliferation. RNA pulldown followed by sequencing revealed that eIF4A1 preferentially binds to mRNAs containing terminal oligopyrimidine (TOP) motifs, whose translation is rapidly repressed upon mTORC1 inhibition. This selective interaction depends on a La-related RNA-binding protein, LARP1. Ribosome profiling revealed that deletion of EIF4A1 attenuated the translational repression of TOP mRNAs upon mTORC1 inactivation. Moreover, eIF4A1 increases the interaction between TOP mRNAs and LARP1 and, thus, ensures stronger translational repression upon mTORC1 inhibition. Our data show the multimodality of eIF4A1 in modulating protein synthesis through an inhibitory binding partner and provide a unique example of the repressive role of a universal translational activator.
    DOI:  https://doi.org/10.1038/s41594-024-01321-7
  21. PLoS One. 2024 ;19(5): e0287877
    Proinsulin degradation and presentation of a proinsulin B-chain autoantigen involves ER-associated protein degradation (ERAD)-enzyme UBE2G2.
    Tom Cremer, Hanneke Hoelen, Michael L van de Weijer, George M Janssen, Ana I Costa, Peter A van Veelen, Robert Jan Lebbink, Emmanuel J H J Wiertz.
      Type 1 diabetes (T1D) is characterized by HLA class I-mediated presentation of autoantigens on the surface of pancreatic β-cells. Recognition of these autoantigens by CD8+ T cells results in the destruction of pancreatic β-cells and, consequently, insulin deficiency. Most epitopes presented at the surface of β-cells derive from the insulin precursor molecule proinsulin. The intracellular processing pathway(s) involved in the generation of these peptides are poorly defined. In this study, we show that a proinsulin B-chain antigen (PPIB5-14) originates from proinsulin molecules that are processed by ER-associated protein degradation (ERAD) and thus originate from ER-resident proteins. Furthermore, screening genes encoding for E2 ubiquitin conjugating enzymes, we identified UBE2G2 to be involved in proinsulin degradation and subsequent presentation of the PPIB10-18 autoantigen. These insights into the pathway involved in the generation of insulin-derived peptides emphasize the importance of proinsulin processing in the ER to T1D pathogenesis and identify novel targets for future T1D therapies.
    DOI:  https://doi.org/10.1371/journal.pone.0287877
  22. EMBO J. 2024 May 20.
    Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1.
    Wanqi Zhan, Zhiyang Li, Jie Zhang, Yongfeng Liu, Guanglong Liu, Bingsong Li, Rong Shen, Yi Jiang, Wanjing Shang, Shenjia Gao, Han Wu, Ya'nan Wang, Wankun Chen, Zhizhang Wang.
      Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.
    Keywords:  Energy Stress; HAX1; P-bodies; TRIM23; Translation Inhibition
    DOI:  https://doi.org/10.1038/s44318-024-00120-6
  23. Dev Cell. 2024 May 21. pii: S1534-5807(24)00271-5. [Epub ahead of print]
    An evolutionarily conserved mechanism controls reversible amyloids of pyruvate kinase via pH-sensing regions.
    Gea Cereghetti, Vera M Kissling, Lisa M Koch, Alexandra Arm, Claudia C Schmidt, Yannik Thüringer, Nicola Zamboni, Pavel Afanasyev, Miriam Linsenmeier, Cédric Eichmann, Sonja Kroschwald, Jiangtao Zhou, Yiping Cao, Dorota M Pfizenmaier, Thomas Wiegand, Riccardo Cadalbert, Govind Gupta, Daniel Boehringer, Tuomas P J Knowles, Raffaele Mezzenga, Paolo Arosio, Roland Riek, Matthias Peter.
      Amyloids are known as irreversible aggregates associated with neurodegenerative diseases. However, recent evidence shows that a subset of amyloids can form reversibly and fulfill essential cellular functions. Yet, the molecular mechanisms regulating functional amyloids and distinguishing them from pathological aggregates remain unclear. Here, we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that yeast PK (Cdc19) and human PK (PKM2) form reversible amyloids through a pH-sensitive amyloid core. Stress-induced cytosolic acidification promotes aggregation via protonation of specific glutamate (yeast) or histidine (human) residues within the amyloid core. Mutations mimicking protonation cause constitutive PK aggregation, while non-protonatable PK mutants remain soluble even upon stress. Physiological PK aggregation is coupled to metabolic rewiring and glycolysis arrest, causing severe growth defects when misregulated. Our work thus identifies an evolutionarily conserved, potentially widespread mechanism regulating functional amyloids during stress.
    Keywords:  aggregate disassembly; aggregate regulation; amyloid regulation; functional amyloids; isoforms; protein aggregation; pyruvate kinase; reversible amyloids; stress response; stress-induced pH changes
    DOI:  https://doi.org/10.1016/j.devcel.2024.04.018
  24. iScience. 2024 Jun 21. 27(6): 109868
    Ribosome profiling reveals the role of yeast RNA-binding proteins Cth1 and Cth2 in translational regulation.
    Hanna Barlit, Antonia M Romero, Ali Gülhan, Praveen K Patnaik, Alexander Tyshkovskiy, María T Martínez-Pastor, Vadim N Gladyshev, Sergi Puig, Vyacheslav M Labunskyy.
      Iron serves as a cofactor for enzymes involved in several steps of protein translation, but the control of translation during iron limitation is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis and leads to translational repression of multiple genes involved in iron-related processes. Furthermore, we demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in this translational regulation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor and inhibiting mitochondrial translation and heme biosynthesis. Additionally, we found that iron deficiency represses MRS3 mRNA translation through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron, demonstrating how this important metal affects protein translation at multiple levels.
    Keywords:  Molecular biology; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2024.109868
  25. bioRxiv. 2024 May 10. pii: 2024.05.09.593302. [Epub ahead of print]
    Collagen fibril formation at the plasma membrane occurs independently from collagen secretion.
    Adam Pickard, Richa Garva, Antony Adamson, Ben C Calverley, Anna Hoyle, Christina E Hayward, David Spiller, Yinhui Lu, Nigel Hodson, Oriana Mandolfo, Kevin K Kim, George Bou-Gharios, Joe Swift, Brian Bigger, Karl E Kadler.
      Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.
    DOI:  https://doi.org/10.1101/2024.05.09.593302
  26. PLoS Biol. 2024 May;22(5): e3002550
    Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.
    Ying Wang, Tianzi Wei, Man Zhao, Aima Huang, Fan Sun, Lu Chen, Risheng Lin, Yubao Xie, Ming Zhang, Shiyu Xu, Zhihui Sun, Liang Hong, Rui Wang, Ruilin Tian, Guofeng Li.
      Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.
    DOI:  https://doi.org/10.1371/journal.pbio.3002550
  27. bioRxiv. 2024 May 12. pii: 2024.05.10.593622. [Epub ahead of print]
    Legionella uses host Rab GTPases and BAP31 to create a unique ER niche.
    Attinder Chadha, Yu Yanai, Hiromu Oide, Yuichi Wakana, Hiroki Inoue, Saradindu Saha, Mitsuo Tagaya, Kohei Arasaki, Shaeri Mukherjee.
      Upon entry into host cells, the facultative intracellular bacterium Legionella pneumophila ( L.p .) uses its type IV secretion system, Dot/Icm, to secrete ~330 bacterial effector proteins into the host cell. Some of these effectors hijack endoplasmic reticulum (ER)-derived vesicles to form the Legionella -containing vacuole (LCV). Despite extensive investigation over decades, the fundamental question persists: Is the LCV membrane distinct from or contiguous with the host ER network? Here, we employ advanced photobleaching techniques, revealing a temporal acquisition of both smooth and rough ER (sER and rER) markers on the LCV. In the early stages of infection, the sER intimately associates with the LCV. Remarkably, as the infection progresses, the LCV evolves into a distinct niche comprising an rER membrane that is independent of the host ER network. We discover that the L.p. effector LidA binds to and recruits two host proteins of the Rab superfamily, Rab10, and Rab4, that play significant roles in acquiring sER and rER membranes, respectively. Additionally, we identify the pivotal role of a host ER-resident protein, BAP31, in orchestrating the transition from sER to rER. While previously recognized for shuttling between sER and rER, we demonstrate BAP31's role as a Rab effector, mediating communication between these ER sub-compartments. Furthermore, using genomic deletion strains, we uncover a novel L.p. effector, Lpg1152, essential for recruiting BAP31 to the LCV and facilitating its transition from sER to rER. Depletion of BAP31 or infection with an isogenic L.p. strain lacking Lpg1152 results in a growth defect. Collectively, our findings illuminate the intricate interplay between molecular players from both host and pathogen, elucidating how L.p. orchestrates the transformation of its residing vacuole membrane from a host-associated sER to a distinct rER membrane that is not contiguous with the host ER network.
    DOI:  https://doi.org/10.1101/2024.05.10.593622
  28. J Cell Biol. 2024 Sep 02. pii: e202308144. [Epub ahead of print]223(9):
    Mitochondria-ER-PM contacts regulate mitochondrial division and PI(4)P distribution.
    Jason C Casler, Clare S Harper, Antoineen J White, Heidi L Anderson, Laura L Lackner.
      The mitochondria-ER-cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1's functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1's role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.
    DOI:  https://doi.org/10.1083/jcb.202308144
  29. Traffic. 2024 May;25(5): e12937
    Apolipoprotein E2 Expression Alters Endosomal Pathways in a Mouse Model With Increased Brain Exosome Levels During Aging.
    Katherine Y Peng, Braison Liemisa, Jonathan Pasato, Pasquale D'Acunzo, Monika Pawlik, Adriana Heguy, Sai C Penikalapati, Amanda Labuza, Harshitha Pidikiti, Melissa J Alldred, Stephen D Ginsberg, Efrat Levy, Paul M Mathews.
      The polymorphic APOE gene is the greatest genetic determinant of sporadic Alzheimer's disease risk: the APOE4 allele increases risk, while the APOE2 allele is neuroprotective compared with the risk-neutral APOE3 allele. The neuronal endosomal system is inherently vulnerable during aging, and APOE4 exacerbates this vulnerability by driving an enlargement of early endosomes and reducing exosome release in the brain of humans and mice. We hypothesized that the protective effects of APOE2 are, in part, mediated through the endosomal pathway. Messenger RNA analyses showed that APOE2 leads to an enrichment of endosomal pathways in the brain when compared with both APOE3 and APOE4. Moreover, we show age-dependent alterations in the recruitment of key endosomal regulatory proteins to vesicle compartments when comparing APOE2 to APOE3. In contrast to the early endosome enlargement previously shown in Alzheimer's disease and APOE4 models, we detected similar morphology and abundance of early endosomes and retromer-associated vesicles within cortical neurons of aged APOE2 targeted-replacement mice compared with APOE3. Additionally, we observed increased brain extracellular levels of endosome-derived exosomes in APOE2 compared with APOE3 mice during aging, consistent with enhanced endosomal cargo clearance by exosomes to the extracellular space. Our findings thus demonstrate that APOE2 enhances an endosomal clearance pathway, which has been shown to be impaired by APOE4 and which may be protective due to APOE2 expression during brain aging.
    Keywords:  Alzheimer's disease; apolipoprotein E; early endosome; endosome; exosome; mouse model
    DOI:  https://doi.org/10.1111/tra.12937
  30. Acta Biomater. 2024 May 22. pii: S1742-7061(24)00272-1. [Epub ahead of print]
    Soft extracellular matrix drives endoplasmic reticulum stress-dependent S quiescence underlying molecular traits of pulmonary basal cells.
    Pierre-Alexandre Laval, Marie Piecyk, Paul Le Guen, Mirela-Diana Ilie, Aubepart Marion, Joelle Fauvre, Isabelle Coste, Toufic Renno, Nicolas Aznar, Celine Hadji, Camille Migdal, Cedric Duret, Philippe Bertolino, Carole Ferraro-Peyret, Alice Nicolas, Cedric Chaveroux.
      Cell culture on soft matrix, either in 2D and 3D, preserves the characteristics of progenitors. However, the mechanism by which the mechanical microenvironment determines progenitor phenotype, and its relevance to human biology, remains poorly described. Here we designed multi-well hydrogel plates with a high degree of physico-chemical uniformity to reliably address the molecular mechanism underlying cell state modification driven by physiological stiffness. Cell cycle, differentiation and metabolic activity could be studied in parallel assays, showing that the soft environment promotes an atypical S-phase quiescence and prevents cell drift, while preserving the differentiation capacities of human bronchoepithelial cells. These softness-sensitive responses are associated with calcium leakage from the endoplasmic reticulum (ER) and defects in proteostasis and enhanced basal ER stress. The analysis of available single cell data of the human lung also showed that this non-conventional state coming from the soft extracellular environment is indeed consistent with molecular feature of pulmonary basal cells. Overall, this study demonstrates that mechanical mimicry in 2D culture supports allows to maintain progenitor cells in a state of high physiological relevance for characterizing the molecular events that govern progenitor biology in human tissues. STATEMENT OF SIGNIFICANCE: This study focuses on the molecular mechanism behind the progenitor state induced by a soft environment. Using innovative hydrogel supports mimicking normal human lung stiffness, the data presented demonstrate that lung mechanics prevent drift while preserving the differentiation capabilities of lung epithelial cells. Furthermore, we show that the cells are positioned in a quiescent state in the atypical S phase. Mechanistically, we demonstrate that this quiescence: i) is driven by calcium leakage from the endoplasmic reticulum (ER) and basal activation of the PERK branch of ER stress signalling, and ii) protects cells from lethal ER stress caused by metabolic stress. Finally, we validate using human single-cell data that these molecular features identified on the soft matrix are found in basal lung cells. Our results reveal original and relevant molecular mechanisms orchestrating cell fate in a soft environment and resistance to exogenous stresses, thus providing new fundamental and clinical insights into basal cell biology.
    Keywords:  S-phase quiescence; Stiffness; cell drift and differentiation capability; physiological ER stress; resistance to metabolic stress
    DOI:  https://doi.org/10.1016/j.actbio.2024.05.033
  31. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2315690121
    Tau fibrils induce nanoscale membrane damage and nucleate cytosolic tau at lysosomes.
    Kevin Rose, Tyler Jepson, Sankalp Shukla, Alex Maya-Romero, Martin Kampmann, Ke Xu, James H Hurley.
      The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.
    Keywords:  Alzheimer’s disease; ESCRT; STORM; astrocyte; lysosome
    DOI:  https://doi.org/10.1073/pnas.2315690121
  32. Cell Death Differ. 2024 May 23.
    MLKL promotes hepatocarcinogenesis through inhibition of AMPK-mediated autophagy.
    Xianjun Yu, Mengyuan Feng, Jian Guo, Haoyu Wang, Jun Yu, Anjie Zhang, Jingyi Wu, Yamei Han, Zequn Sun, Yingying Liao, Qun Zhao.
      The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential component of the activation of the necroptotic pathway. Emerging evidence suggests that MLKL plays a key role in liver disease. However, how MLKL contributes to hepatocarcinogenesis has not been fully elucidated. Herein, we report that MLKL is upregulated in a diethylnitrosamine (DEN)-induced murine HCC model and is associated with human hepatocellular carcinomas. Hepatocyte-specific MLKL knockout suppresses the progression of hepatocarcinogenesis. Conversely, MLKL overexpression aggravates the initiation and progression of DEN-induced HCC. Mechanistic study reveals that deletion of MLKL significantly increases the activation of autophagy, thereby protecting against hepatocarcinogenesis. MLKL directly interacts with AMPKα1 and inhibits its activity independent of its necroptotic function. Mechanistically, MLKL serves as a bridging molecule between AMPKα1 and protein phosphatase 1B (PPM1B), thus enhancing the dephosphorylation of AMPKα1. Consistently, MLKL expression correlates negatively with AMPKα1 phosphorylation in HCC patients. Taken together, our findings highlight MLKL as a novel AMPK gatekeeper that plays key roles in inhibiting autophagy and driving hepatocarcinogenesis, suggesting that the MLKL-AMPKα1 axis is a potential therapeutic target for HCC.
    DOI:  https://doi.org/10.1038/s41418-024-01314-5
  33. Proc Natl Acad Sci U S A. 2024 May 28. 121(22): e2321600121
    Dual role of the peptide-loading complex as proofreader and limiter of MHC-I presentation.
    Jamina Brunnberg, Martina Barends, Stefan Frühschulz, Christian Winter, Claire Battin, Ben de Wet, David K Cole, Peter Steinberger, Robert Tampé.
      Antigen presentation via major histocompatibility complex class I (MHC-I) molecules is essential for surveillance by the adaptive immune system. Central to this process is the peptide-loading complex (PLC), which translocates peptides from the cytosol to the endoplasmic reticulum and catalyzes peptide loading and proofreading of peptide-MHC-I (pMHC-I) complexes. Despite its importance, the impact of individual PLC components on the presented pMHC-I complexes is still insufficiently understood. Here, we used stoichiometrically defined antibody-nanobody complexes and engineered soluble T cell receptors (sTCRs) to quantify different MHC-I allomorphs and defined pMHC-I complexes, respectively. Thereby, we uncovered distinct effects of individual PLC components on the pMHC-I surface pool. Knockouts of components of the PLC editing modules, namely tapasin, ERp57, or calreticulin, changed the MHC-I surface composition to a reduced proportion of HLA-A*02:01 presentation compensated by a higher ratio of HLA-B*40:01 molecules. Intriguingly, these knockouts not only increased the presentation of suboptimally loaded HLA-A*02:01 complexes but also elevated the presentation of high-affinity peptides overexpressed in the cytosol. Our findings suggest that the components of the PLC editing module serve a dual role, acting not only as peptide proofreaders but also as limiters for abundant peptides. This dual function ensures the presentation of a broad spectrum of antigenic peptides.
    Keywords:  T cell receptor; antigen processing; peptide-loading complex; transporter associated with antigen processing TAP; tumor-associated antigen
    DOI:  https://doi.org/10.1073/pnas.2321600121
  34. Hum Mol Genet. 2024 May 22. pii: ddae082. [Epub ahead of print]
    Functional validation of EIF2AK4 (GCN2) missense variants associated with pulmonary arterial hypertension.
    Giulia Emanuelli, JiaYi Zhu, Wei Li, Nicholas W Morrell, Stefan J Marciniak.
      Pulmonary arterial hypertension (PAH) is a disorder with a large genetic component. Biallelic mutations of EIF2AK4, which encodes the kinase GCN2, are causal in two ultra-rare subtypes of PAH, pulmonary veno-occlusive disease and pulmonary capillary haemangiomatosis. EIF2AK4 variants of unknown significance have also been identified in patients with classical PAH, though their relationship to disease remains unclear. To provide patients with diagnostic information and enable family testing, the functional consequences of such rare variants must be determined, but existing computational methods are imperfect. We applied a suite of bioinformatic and experimental approaches to sixteen EIF2AK4 variants that had been identified in patients. By experimentally testing the functional integrity of the integrated stress response (ISR) downstream of GCN2, we determined that existing computational tools have insufficient sensitivity to reliably predict impaired kinase function. We determined experimentally that several EIF2AK4 variants identified in patients with classical PAH had preserved function and are therefore likely to be non-pathogenic. The dysfunctional variants of GCN2 that we identified could be subclassified into three groups: misfolded, kinase-dead, and hypomorphic. Intriguingly, members of the hypomorphic group were amenable to paradoxical activation by a type-1½ GCN2 kinase inhibitor. This experiment approach may aid in the clinical stratification of EIF2AK4 variants and potentially identify hypomorophic alleles receptive to pharmacological activation.
    Keywords:  EIF2AK4; GCN2; PVOD; missense variants; pulmonary hypertension
    DOI:  https://doi.org/10.1093/hmg/ddae082
  35. Autophagy. 2024 May 18.
    VCP/p97 UFMylation stabilizes BECN1 and facilitates the initiation of autophagy.
    Zhifeng Wang, Shuhui Xiong, Zhaoyi Wu, Xingde Wang, Yamin Gong, Wei-Guo Zhu, Xingzhi Xu.
      Macroautophagy/autophagy is essential for the degradation and recycling of cytoplasmic materials. The initiation of this process is determined by phosphatidylinositol-3-kinase (PtdIns3K) complex, which is regulated by factor BECN1 (beclin 1). UFMylation is a novel ubiquitin-like modification that has been demonstrated to modulate several cellular activities. However, the role of UFMylation in regulating autophagy has not been fully elucidated. Here, we found that VCP/p97 is UFMylated on K109 by the E3 UFL1 (UFM1 specific ligase 1) and this modification promotes BECN1 stabilization and assembly of the PtdIns3K complex, suggesting a role for VCP/p97 UFMylation in autophagy initiation. Mechanistically, VCP/p97 UFMylation stabilizes BECN1 through ATXN3 (ataxin 3)-mediated deubiquitination. As a key component of the PtdIns3K complex, stabilized BECN1 facilitates assembly of this complex. Re-expression of VCP/p97, but not the UFMylation-defective mutant, rescued the VCP/p97 depletion-induced increase in MAP1LC3B/LC3B protein expression. We also showed that several pathogenic VCP/p97 mutations identified in a variety of neurological disorders and cancers were associated with reduced UFMylation, thus implicating VCP/p97 UFMylation as a potential therapeutic target for these diseases.
    Keywords:  BECN1/beclin 1; PtdIns3K complex; UFL1; Ufmylation; Vcp/p97
    DOI:  https://doi.org/10.1080/15548627.2024.2356488
  36. bioRxiv. 2024 May 12. pii: 2024.05.12.593148. [Epub ahead of print]
    Full-length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles.
    Chantal Scheepbouwer, Ernesto Aparicio-Puerta, Cristina Gómez-Martin, Monique A J van Eijndhoven, Esther E E Drees, Leontien Bosch, Daphne de Jong, Thomas Wurdinger, Josée M Zijlstra, Michael Hackenberg, Alan Gerber, D Michiel Pegtel.
      Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.
    DOI:  https://doi.org/10.1101/2024.05.12.593148
  37. NAR Cancer. 2024 Jun;6(2): zcae023
    Upstream open reading frames: new players in the landscape of cancer gene regulation.
    Anwesha Dasgupta, John R Prensner.
      The translation of RNA by ribosomes represents a central biological process and one of the most dysregulated processes in cancer. While translation is traditionally thought to occur exclusively in the protein-coding regions of messenger RNAs (mRNAs), recent transcriptome-wide approaches have shown abundant ribosome activity across diverse stretches of RNA transcripts. The most common type of this kind of ribosome activity occurs in gene leader sequences, also known as 5' untranslated regions (UTRs) of the mRNA, that precede the main coding sequence. Translation of these upstream open reading frames (uORFs) is now known to occur in upwards of 25% of all protein-coding genes. With diverse functions from RNA regulation to microprotein generation, uORFs are rapidly igniting a new arena of cancer biology, where they are linked to cancer genetics, cancer signaling, and tumor-immune interactions. This review focuses on the contributions of uORFs and their associated 5'UTR sequences to cancer biology.
    DOI:  https://doi.org/10.1093/narcan/zcae023
  38. bioRxiv. 2024 May 12. pii: 2024.05.09.593285. [Epub ahead of print]
    The molecular mechanism of on-demand sterol biosynthesis at organelle contact sites.
    Naama Zung, Nitya Aravindan, Angela Boshnakovska, Rosario Valenti, Noga Preminger, Felix Jonas, Gilad Yaakov, Mathilda M Willoughby, Bettina Homberg, Jenny Keller, Meital Kupervaser, Nili Dezorella, Tali Dadosh, Sharon G Wolf, Maxim Itkin, Sergey Malitsky, Alexander Brandis, Naama Barkai, Rubén Fernández-Busnadiego, Amit R Reddi, Peter Rehling, Doron Rapaport, Maya Schuldiner.
      Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.
    DOI:  https://doi.org/10.1101/2024.05.09.593285
  39. Cell Chem Biol. 2024 May 17. pii: S2451-9456(24)00177-6. [Epub ahead of print]
    Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology.
    Lauren Kageler, Jonathan Perr, Ryan A Flynn.
      Proximity is a fundamental concept in chemistry and biology, referring to the convergence of molecules to facilitate new molecular interactions or reactions. Hybrid biopolymers like glycosylphosphatidylinositol (GPI)-anchored proteins, ubiquitinated proteins, glycosylated RNAs (glycoRNAs), and RNAylated proteins exemplify this by covalent bonding of moieties that are often orthogonally active. Hybrid molecules like glycoRNAs are localized to new physical spaces, generating new interfaces for biological functions. To fully investigate the compositional and spatial features of molecules like glycoRNAs, flexible genetic and chemical tools that encompass different encoding and targeting biopolymers are required. Here we discuss concepts of molecular proximity and explore newer proximity labeling technologies that facilitate applications in RNA biology, cell surface biology, and the interface therein with a particular focus on glycoRNA biology. We review the advantages and disadvantages of methods pertaining to cell surface RNA identification and provide insights into the vast opportunities for method development in this area.
    DOI:  https://doi.org/10.1016/j.chembiol.2024.04.015
  40. Nature. 2024 May 22.
    Natural proteome diversity links aneuploidy tolerance to protein turnover.
    Julia Muenzner, Pauline Trébulle, Federica Agostini, Henrik Zauber, Christoph B Messner, Martin Steger, Christiane Kilian, Kate Lau, Natalie Barthel, Andrea Lehmann, Kathrin Textoris-Taube, Elodie Caudal, Anna-Sophia Egger, Fatma Amari, Matteo De Chiara, Vadim Demichev, Toni I Gossmann, Michael Mülleder, Gianni Liti, Joseph Schacherer, Matthias Selbach, Judith Berman, Markus Ralser.
      Accessing the natural genetic diversity of species unveils hidden genetic traits, clarifies gene functions and allows the generalizability of laboratory findings to be assessed. One notable discovery made in natural isolates of Saccharomyces cerevisiae is that aneuploidy-an imbalance in chromosome copy numbers-is frequent1,2 (around 20%), which seems to contradict the substantial fitness costs and transient nature of aneuploidy when it is engineered in the laboratory3-5. Here we generate a proteomic resource and merge it with genomic1 and transcriptomic6 data for 796 euploid and aneuploid natural isolates. We find that natural and lab-generated aneuploids differ specifically at the proteome. In lab-generated aneuploids, some proteins-especially subunits of protein complexes-show reduced expression, but the overall protein levels correspond to the aneuploid gene dosage. By contrast, in natural isolates, more than 70% of proteins encoded on aneuploid chromosomes are dosage compensated, and average protein levels are shifted towards the euploid state chromosome-wide. At the molecular level, we detect an induction of structural components of the proteasome, increased levels of ubiquitination, and reveal an interdependency of protein turnover rates and attenuation. Our study thus highlights the role of protein turnover in mediating aneuploidy tolerance, and shows the utility of exploiting the natural diversity of species to attain generalizable molecular insights into complex biological processes.
    DOI:  https://doi.org/10.1038/s41586-024-07442-9
  41. Nat Commun. 2024 May 18. 15(1): 4237
    Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.
    Xiaoyan Xu, Tingxue Xie, Mengxin Zhou, Yaqin Sun, Fengqi Wang, Yanan Tian, Ziyan Chen, Yanqi Xie, Ronghai Wu, Xufeng Cen, Jichun Zhou, Tingjun Hou, Lei Zhang, Chaoyang Huang, Qingwei Zhao, Dongrui Wang, Hongguang Xia.
      Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/β inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.
    DOI:  https://doi.org/10.1038/s41467-024-48597-3
  42. J Cell Sci. 2024 May 24. pii: jcs.261728. [Epub ahead of print]
    KRAP regulates mitochondrial Ca2+ uptake by licensing IP3 receptor activity and stabilizing ER-mitochondrial junctions.
    Peace Atakpa-Adaji, Adelina Ivanova, Karolina Kujawa, Colin W Taylor.
      Inositol 1,4,5-trisphosphate receptors (IP3Rs) are high-conductance channels that allow the regulated redistribution of Ca2+ from the ER to the cytosol and, at specialised membrane contact sites (MCS), to other organelles. Only a subset of IP3Rs release Ca2+ to the cytosol in response to IP3. These 'licensed' IP3Rs are associated with Kras-induced actin-interacting protein (KRAP) beneath the plasma membrane. It is unclear whether KRAP regulates IP3Rs at MCS. We show, using simultaneous measurements of Ca2+ concentration in the cytosol and mitochondrial matrix, that KRAP also licenses IP3Rs to release Ca2+ to mitochondria. Loss of KRAP abolishes cytosolic and mitochondrial Ca2+ signals evoked by stimulation of IP3Rs via endogenous receptors. KRAP is located at ER-mitochondria membrane contact sites (ERMCS) populated by IP3R clusters. Using a proximity ligation assay between IP3R and voltage-dependent anion channel 1 (VDAC1), we show that loss of KRAP reduces the number of ERMCS. We conclude that KRAP regulates Ca2+ transfer from IP3Rs to mitochondria by both licensing IP3R activity and stabilizing ERMCS.
    Keywords:  Ca2+; Endoplasmic reticulum; HeLa cell; Histamine; IP3 receptor; KRAP; MCU; Membrane contact site; Mitochondria; Proximity ligation assay; VDAC1
    DOI:  https://doi.org/10.1242/jcs.261728
  43. Oncogenesis. 2024 May 20. 13(1): 16
    An E3 ligase TRIM1 promotes colorectal cancer progression via K63-linked ubiquitination and activation of HIF1α.
    Liuliu Shi, Xianglan Fang, Lijie Du, Jin Yang, Juan Xue, Xiaokai Yue, Duoshuang Xie, Yuanjian Hui, Kun Meng.
      Accumulating studies have shown that E3 ligases play crucial roles in regulating cellular biological processes and signaling pathways during carcinogenesis via ubiquitination. Tripartite-motif (TRIM) ubiquitin E3 ligases consist of over 70 members. However, the clinical significance and their contributions to tumorigenesis remain largely unknown. In this study, we analyzed the RNA-sequencing expression of TRIM E3 ligases in colorectal cancer (CRC) and identified 10 differentially expressed genes, among which TRIM1 expression predicted poor prognosis of CRC patients. We demonstrated that TRIM1 expression is positively associated with CRC pathological stages, and higher expression is positively correlated with infiltrating levels of immune cells and immunotherapy biomarkers. TRIM1 expression promotes the proliferation and migration of colorectal cancer cells in vitro and in vivo. Transcriptional analysis showed that TRIM1 is responsible for metabolism promotion and immune suppression. Mechanistically, we found that TRIM1 binds HIF1α and mediates its K63-linked ubiquitination, which is required for HIF1α nuclear translocation and subsequent activation. Ubiquitination occurs at Lys214 in the loop between the two PAS domains of HIF1α, and mutation of Lys214 severely disturbs the function of HIF1α. Besides, HIF1α ubiquitination enhances its binding with proteins involved in cellular trafficking and nucleocytoplasmic transport pathway. Collectively, our results indicate TRIM1's role in predicting prognosis and reveal how TRIM1 functions to upregulate HIF1α expression and promote tumor cell proliferation.
    DOI:  https://doi.org/10.1038/s41389-024-00517-2
  44. iScience. 2024 Jun 21. 27(6): 109873
    Pan-cancer analysis of the interplay between mutational signatures and cellular signaling.
    Anna Hakobyan, Mathilde Meyenberg, Nelli Vardazaryan, Joel Hancock, Loan Vulliard, Joanna I Loizou, Jörg Menche.
      Cancer is a multi-faceted disease with intricate relationships between mutagenic processes, alterations in cellular signaling, and the tissue microenvironment. To date, these processes have been largely studied in isolation. A systematic understanding of how they interact and influence each other is lacking. Here, we present a framework for systematically characterizing the interaction between pairs of mutational signatures and between signatures and signaling pathway alterations. We applied this framework to large-scale data from TCGA and PCAWG and identified multiple positive and negative interactions, both cross֊tissue and tissue֊specific, that provide new insights into the molecular routes observed in tumorigenesis and their respective drivers. This framework allows for a more fine-grained dissection of common and distinct etiology of mutational signatures. We further identified several interactions with both positive and negative impacts on patient survival, demonstrating their clinical relevance and potential for improving personalized cancer care.
    Keywords:  Bioinformatics; Biological sciences; Cancer systems biology
    DOI:  https://doi.org/10.1016/j.isci.2024.109873
  45. Drug Discov Today. 2024 May 22. pii: S1359-6446(24)00157-0. [Epub ahead of print] 104032
    The impact of E3 ligase choice on PROTAC effectiveness in protein kinase degradation.
    Tomasz Sobierajski, Joanna Małolepsza, Marta Pichlakb, Edyta Gendaszewska-Darmach, Katarzyna M Błażewska.
      Proteolysis targeting chimera (PROTACs) provide a novel therapeutic approach that is revolutionizing drug discovery. The success of PROTACs largely depends on the combination of their three fragments: E3 ligase ligand, linker and protein of interest (POI)-targeting ligand. We summarize the pivotal significance of the precise combination of the E3 ligase ligand with the POI-recruiting warhead, which is crucial for the successful execution of cellular processes and achieving the desired outcomes. Therefore, the key to selection was the use of at least two ligands recruiting two different ligases. This approach enables a direct comparison of the impacts of the specific ligases on target degradation.
    Keywords:  E3 ubiquitin ligase; PROTAC; Proteolysis targeting chimera; drug discovery
    DOI:  https://doi.org/10.1016/j.drudis.2024.104032
  46. bioRxiv. 2024 May 08. pii: 2024.05.08.593135. [Epub ahead of print]
    TRIM9 controls growth cone responses to netrin through DCC and UNC5C.
    Sampada P Mutalik, Ellen C O'Shaughnessy, Chris T Ho, Stephanie L Gupton.
      The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits these diverse axonal responses, beyond engaging the attractive receptor DCC and repulsive receptors of the UNC5 family, remains elusive. Here we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C, and that deletion of murine Trim9 alters both attractive and repulsive responses to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in surface levels of DCC and total levels of UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of netrin-1. We investigate membrane dynamics of the UNC5C receptor using pH-mScarlet fused to the extracellular domain of UNC5C. Minutes after netrin addition, levels of UNC5C at the plasma membrane drop in a TRIM9-independent fashion, however TRIM9 regulated the mobility of UNC5C in the plasma membrane in the absence of netrin-1. Together this work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.
    DOI:  https://doi.org/10.1101/2024.05.08.593135
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