bims-proteo Biomed News
on Proteostasis
Issue of 2024–01–28
forty-nine papers selected by
Eric Chevet, INSERM
  1. Excess dietary sodium partially restores salt and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron.
  2. Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.
  3. Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation.
  4. CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5.
  5. Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.
  6. Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response.
  7. Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.
  8. Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.
  9. Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis in C. elegans.
  10. HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.
  11. SEL1L-HRD1 ER-associated degradation regulates leptin receptor maturation and signaling in POMC neurons in diet-induced obesity.
  12. Computational inference of eIF4F complex function and structure in human cancers.
  13. Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.
  14. A new role for IFRD1 in regulation of ER stress in bladder epithelial homeostasis.
  15. LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α.
  16. LUBAC-mediated M1 Ub regulates necroptosis by segregating the cellular distribution of active MLKL.
  17. CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.
  18. Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.
  19. The unfolded protein response of the endoplasmic reticulum protects Caenorhabditis elegans against DNA damage caused by stalled replication forks.
  20. Methionine aminopeptidase 2 and its autoproteolysis product have different binding sites on the ribosome.
  21. Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.
  22. Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay.
  23. MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans.
  24. N6-methyladenosine in 5' UTR does not promote translation initiation.
  25. Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1.
  26. Protein SUMOylation promotes cAMP-independent EPAC1 activation.
  27. Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.
  28. Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.
  29. Single-cell expression predicts neuron-specific protein homeostasis networks.
  30. Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain.
  31. Multi-monoubiquitylation controls VASP-mediated actin dynamics.
  32. Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.
  33. TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation.
  34. E3 ubiquitin ligase Hul6 modulates iron-dependent metabolism by regulating Php4 stability.
  35. VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.
  36. Beyond antibiotic resistance: The whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria.
  37. ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.
  38. ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.
  39. Heat shock response during the resolution of inflammation and its progressive suppression in chronic-degenerative inflammatory diseases.
  40. ATG14 targets lipid droplets and acts as an autophagic receptor for syntaxin18-regulated lipid droplet turnover.
  41. The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics.
  42. Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.
  43. Loss of the E3 ubiquitin ligase TRIM67 alters the post-synaptic density proteome.
  44. Inhibition of ADAM9 promotes the selective degradation of KRAS and sensitizes pancreatic cancers to chemotherapy.
  45. Folding pathway of a discontinuous two-domain protein.
  46. Emerging roles of O-GlcNAcylation in protein trafficking and secretion.
  47. Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.
  48. Dihydroceramide desaturase governs endoplasmic reticulum and lipid droplet homeostasis to promote glial function in the nervous system.
  49. Selective autophagy in cancer: mechanisms, therapeutic implications, and future perspectives.
  1. bioRxiv. 2024 Jan 14. pii: 2024.01.13.575426. [Epub ahead of print]
    Excess dietary sodium partially restores salt and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron.
    Aidan Porter, Hannah E Vorndran, Allison L Marciszyn, Stephanie M Mutchler, Arohan R Subramanya, Thomas R Kleyman, Linda M Hendershot, Jeffrey L Brodsky, Teresa M Buck.
      The maintenance of fluid and electrolyte homeostasis by the kidney requires proper folding and trafficking of ion channels and transporters in kidney epithelia. Each of these processes requires a specific subset of a diverse class of proteins termed molecular chaperones. One such chaperone is GRP170, which is an Hsp70-like, endoplasmic reticulum (ER)-localized chaperone that plays roles in protein quality control and protein folding in the ER. We previously determined that loss of GRP170 in the mouse nephron leads to hypovolemia, electrolyte imbalance, and rapid weight loss. In addition, GRP170-deficient mice develop an AKI-like phenotype, typified by tubular injury, elevation of clinical kidney injury markers, and induction of the unfolded protein response (UPR). By using an inducible GRP170 knockout cellular model, we confirmed that GRP170 depletion induces the UPR, triggers an apoptotic response, and disrupts protein homeostasis. Based on these data, we hypothesized that UPR induction underlies hyponatremia and volume depletion in rodents, but that these and other phenotypes might be rectified by supplementation with high salt. To test this hypothesis, control and GRP170 tubule-specific knockout mice were provided with a diet containing 8% sodium chloride. We discovered that sodium supplementation improved electrolyte imbalance and reduced clinical kidney injury markers, but was unable to restore weight or tubule integrity. These results are consistent with UPR induction contributing to the kidney injury phenotype in the nephron-specific GR170 knockout model, and that the role of GRP170 in kidney epithelia is essential to both maintain electrolyte balance and cellular protein homeostasis.
    DOI:  https://doi.org/10.1101/2024.01.13.575426
  2. bioRxiv. 2024 Jan 09. pii: 2024.01.09.574907. [Epub ahead of print]
    Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10.
    Kevin Wu, Samuel Itskanov, Diane L Lynch, Yuanyuan Chen, Aasha Turner, James C Gumbart, Eunyong Park.
      Doa10 (MARCH6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we defined the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its well-defined degron Deg1. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrate's degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel.
    DOI:  https://doi.org/10.1101/2024.01.09.574907
  3. bioRxiv. 2024 Jan 02. pii: 2024.01.02.573950. [Epub ahead of print]
    Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation.
    Julia M Flynn, Margot E Joyce, Daniel N A Bolon.
      Most of the fundamental processes of cells are mediated by proteins. However, the biologically- relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating mechanism, but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co- chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. Our analysis of all point mutations in Hsp90 identified 205 that dramatically slowed the growth of yeast harboring a second WT copy of Hsp90. 75% of the dominant negative mutations that we identified were located in a loop that closes over bound ATP. We analyzed a small panel of individual dominant mutations in this loop in detail. In this panel, addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. We examined the stability to proteolysis of glucocorticoid receptor (GR) as a model Hsp90 substrate. Upon expression of dominant negative Hsp90 variants, GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.
    DOI:  https://doi.org/10.1101/2024.01.02.573950
  4. J Biol Chem. 2024 Jan 23. pii: S0021-9258(24)00049-8. [Epub ahead of print] 105673
    CHIP suppresses the proliferation and migration of A549 cells by mediating the ubiquitination of eIF2α and upregulation of tumor suppressor RBM5.
    Bo Jin, Mengran Wang, Yiheng Sun, Priscilla Ann Hweek Lee, Xiangqi Zhang, Yao Lu, Bo Zhao.
      The PERK-eIF2α pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase CHIP both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the transcription factor ATF4 under ER stress conditions. ATF4 induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression.
    Keywords:  CHIP; ER stress; PERK; RBM5; eIF2; phosphorylation; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2024.105673
  5. Nat Commun. 2024 Jan 22. 15(1): 659
    Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.
    Xiaoqiong Wei, You Lu, Liangguang Leo Lin, Chengxin Zhang, Xinxin Chen, Siwen Wang, Shuangcheng Alivia Wu, Zexin Jason Li, Yujun Quan, Shengyi Sun, Ling Qi.
      Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins.
    DOI:  https://doi.org/10.1038/s41467-024-44948-2
  6. Cold Spring Harb Perspect Biol. 2024 Jan 22. pii: a041400. [Epub ahead of print]
    Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response.
    Robert Ernst, Mike F Renne, Aamna Jain, Alexander von der Malsburg.
      The endoplasmic reticulum (ER) is the key organelle for membrane biogenesis. Most lipids are synthesized in the ER, and most membrane proteins are first inserted into the ER membrane before they are transported to their target organelle. The composition and properties of the ER membrane must be carefully controlled to provide a suitable environment for the insertion and folding of membrane proteins. The unfolded protein response (UPR) is a powerful signaling pathway that balances protein and lipid production in the ER. Here, we summarize our current knowledge of how aberrant compositions of the ER membrane, referred to as lipid bilayer stress, trigger the UPR.
    DOI:  https://doi.org/10.1101/cshperspect.a041400
  7. Elife. 2024 Jan 24. pii: RP92916. [Epub ahead of print]12
    Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.
    Swati Gaikwad, Fardin Ghobakhlou, Hongen Zhang, Alan G Hinnebusch.
      Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.
    Keywords:  IRES; S. cerevisiae; eIF2; eIF2A; genetics; genomics; translation; uORF; yeast
    DOI:  https://doi.org/10.7554/eLife.92916
  8. bioRxiv. 2024 Jan 01. pii: 2023.12.31.573735. [Epub ahead of print]
    Rapid turnover of CTLA4 is associated with a complex architecture of reversible ubiquitylation.
    Pei Yee Tey, Almut Dufner, Klaus-Peter Knobeloch, Jonathan N Pruneda, Michael J Clague, Sylvie Urbé.
      The immune checkpoint regulator CTLA4 is an unusually short-lived membrane protein. Here we show that its lysosomal degradation is dependent on ubiquitylation at Lysine residues 203 and 213. Inhibition of the v-ATPase partially restores CTLA4 levels following cycloheximide treatment, but also reveals a fraction that is secreted in exosomes. The endosomal deubiquitylase, USP8, interacts with CTLA4 and its loss enhances CTLA4 ubiquitylation in cancer cells, mouse CD4 + T cells and in cancer cell-derived exosomes. Depletion of the USP8 adapter protein, HD-PTP, but not ESCRT-0 recapitulates this cellular phenotype, but shows distinct properties vis-à-vis exosome incorporation. Re-expression of wild-type USP8, but neither a catalytically inactive, nor a localisation-compromised ΔMIT domain mutant can rescue delayed degradation of CTLA4, or counteract its accumulation in clustered endosomes. UbiCRest analysis of CTLA4-associated ubiquitin chain linkages identifies a complex mixture of conventional Lys63- and more unusual Lys27- and Lys29-linked polyubiquitin chains that may underly the rapidity of protein turnover.
    DOI:  https://doi.org/10.1101/2023.12.31.573735
  9. bioRxiv. 2024 Jan 04. pii: 2023.07.31.551399. [Epub ahead of print]
    Neurotrophic factor MANF regulates autophagy and lysosome function to promote proteostasis in C. elegans.
    Shane K B Taylor, Jessica H Hartman, Bhagwati P Gupta.
      The conserved mesencephalic astrocyte-derived neurotrophic factor (MANF) protects dopaminergic neurons but also functions in several other tissues. Previously, we showed that Caenorhabditis elegans manf-1 null mutants have increased ER stress, dopaminergic neurodegeneration, protein aggregation, slower growth, and a reduced lifespan. The multiple requirements of MANF in different systems suggest its essential role in regulating cellular processes. However, how intracellular and extracellular MANF regulates broader cellular function remains unknown. Here, we report a novel mechanism of action for manf-1 that involves the autophagy transcription factor HLH-30/TFEB-mediated signaling to regulate lysosomal function and aging. We generated multiple transgenic strains overexpressing MANF-1 and found that animals had extended lifespan, reduced protein aggregation, and improved neuronal health. Using a fluorescently tagged MANF-1, we observed different tissue localization of MANF-1 depending on the ER retention signal. Further subcellular analysis showed that MANF-1 localizes within cells to the lysosomes. These findings were consistent with our transcriptomic studies and, together with analysis of autophagy regulators, demonstrate that MANF-1 regulates protein homeostasis through increased autophagy and lysosomal activity. Collectively, our findings establish MANF as a critical regulator of the stress response, proteostasis, and aging.
    DOI:  https://doi.org/10.1101/2023.07.31.551399
  10. Mol Cell. 2024 Jan 19. pii: S1097-2765(24)00002-9. [Epub ahead of print]
    HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.
    Sergio Leone, Avinash Srivastava, Andrés Herrero-Ruiz, Barbara Hummel, Lena Tittel, Roberto Campalastri, Fernando Aprile-Garcia, Jun Hao Tan, Prashant Rawat, Patrik Andersson, Anne E Willis, Ritwick Sawarkar.
      Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
    Keywords:  HSP70; RNA polymerase III; RNA-binding proteome; RNA-protein interaction; heat-shock proteins; molecular chaperones; non-coding RNA; proteostasis; tRNA; transcription
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.001
  11. Res Sq. 2024 Jan 12. pii: rs.3.rs-3768472. [Epub ahead of print]
    SEL1L-HRD1 ER-associated degradation regulates leptin receptor maturation and signaling in POMC neurons in diet-induced obesity.
    Ling Qi, Hancheng Mao, Geun Hyang Kim.
      Endoplasmic reticulum (ER) homeostasis in the hypothalamus has been implicated in the pathogenesis of certain patho-physiological conditions such as diet-induced obesity (DIO) and type 2 diabetes; however, the significance of ER quality control mechanism(s) and its underlying mechanism remain largely unclear and highly controversial in some cases. Moreover, how the biogenesis of nascent leptin receptor in the ER is regulated remains largely unexplored. Here we report that the SEL1L-HRD1 protein complex of the highly conserved ER-associated protein degradation (ERAD) machinery in POMC neurons is indispensable for leptin signaling in diet-induced obesity. SEL1L-HRD1 ERAD is constitutively expressed in hypothalamic POMC neurons. Loss of SEL1L in POMC neurons attenuates leptin signaling and predisposes mice to HFD-associated pathologies including leptin resistance. Mechanistically, newly synthesized leptin receptors, both wildtype and disease-associated human mutant Cys604Ser (Cys602Ser in mice), are misfolding prone and bona fide substrates of SEL1L-HRD1 ERAD. Indeed, defects in SEL1L-HRD1 ERAD markedly impair the maturation of these receptors and causes their ER retention. This study not only uncovers a new role of SEL1L-HRD1 ERAD in the pathogenesis of diet-induced obesity and central leptin resistance, but a new regulatory mechanism for leptin signaling.
    DOI:  https://doi.org/10.21203/rs.3.rs-3768472/v1
  12. Proc Natl Acad Sci U S A. 2024 Jan 30. 121(5): e2313589121
    Computational inference of eIF4F complex function and structure in human cancers.
    Su Wu, Gerhard Wagner.
      The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.
    Keywords:  cap-independent translation initiation; computational analysis; eIF3e; eIF4F dysregulation; eukaryotic initiation factor 4F (eIF4F)
    DOI:  https://doi.org/10.1073/pnas.2313589121
  13. Traffic. 2024 Jan;25(1): e12927
    Rescue of secretion of rare-disease-associated misfolded mutant glycoproteins in UGGT1 knock-out mammalian cells.
    Gabor Tax, Kevin P Guay, Ludovica Pantalone, Martina Ceci, Tatiana Soldà, Charlie J Hitchman, Johan C Hill, Snežana Vasiljević, Andrea Lia, Carlos P Modenutti, Kees R Straatman, Angelo Santino, Maurizio Molinari, Nicole Zitzmann, Daniel N Hebert, Pietro Roversi, Marco Trerotola.
      Endoplasmic reticulum (ER) retention of misfolded glycoproteins is mediated by the ER-localized eukaryotic glycoprotein secretion checkpoint, UDP-glucose glycoprotein glucosyl-transferase (UGGT). The enzyme recognizes a misfolded glycoprotein and flags it for ER retention by re-glucosylating one of its N-linked glycans. In the background of a congenital mutation in a secreted glycoprotein gene, UGGT-mediated ER retention can cause rare disease, even if the mutant glycoprotein retains activity ("responsive mutant"). Using confocal laser scanning microscopy, we investigated here the subcellular localization of the human Trop-2-Q118E, E227K and L186P mutants, which cause gelatinous drop-like corneal dystrophy (GDLD). Compared with the wild-type Trop-2, which is correctly localized at the plasma membrane, these Trop-2 mutants are retained in the ER. We studied fluorescent chimeras of the Trop-2 Q118E, E227K and L186P mutants in mammalian cells harboring CRISPR/Cas9-mediated inhibition of the UGGT1 and/or UGGT2 genes. The membrane localization of the Trop-2 Q118E, E227K and L186P mutants was successfully rescued in UGGT1-/- cells. UGGT1 also efficiently reglucosylated Trop-2-Q118E-EYFP in cellula. The study supports the hypothesis that UGGT1 modulation would constitute a novel therapeutic strategy for the treatment of pathological conditions associated to misfolded membrane glycoproteins (whenever the mutation impairs but does not abrogate function), and it encourages the testing of modulators of ER glycoprotein folding quality control as broad-spectrum rescue-of-secretion drugs in rare diseases caused by responsive secreted glycoprotein mutants.
    Keywords:  GDLD; TACSTD2; Trop-2; UGGT; UGGT1; UGGT2; glycoprotein secretion; responsive mutant
    DOI:  https://doi.org/10.1111/tra.12927
  14. bioRxiv. 2024 Jan 10. pii: 2024.01.09.574887. [Epub ahead of print]
    A new role for IFRD1 in regulation of ER stress in bladder epithelial homeostasis.
    Bisiayo E Fashemi, Amala K Rougeau, Arnold M Salazar, Steven J Bark, Rayvanth Chappidi, Jeffrey W Brown, Charles J Cho, Jason C Mills, Indira U Mysorekar.
      A healthy bladder requires the homeostatic maintenance of and rapid regeneration of urothelium upon stress/injury/infection. Several factors have been identified to play important roles in urothelial development, injury and disease response, however, little is known about urothelial regulation at homeostasis. Here, we identify a new role for IFRD1, a stress-induced gene that has recently been demonstrated to play a critical role in adult tissue proliferation and regeneration, in maintenance of urothelial function/ homeostasis in a mouse model. We show that the mouse bladder expresses IFRD1 at homeostasis and its loss alters the global transcriptome of the bladder with significant accumulation of cellular organelles including multivesicular bodies with undigested cargo, lysosomes and mitochondria. We demonstrate that IFRD1 interacts with several mRNA-translation-regulating factors in human urothelial cells and that the urothelium of Ifrd1 -/- mice reveal decreased global translation and enhanced endoplasmic reticulum (ER) stress response. Ifrd1 -/- bladders have activation of the unfolded protein response (UPR) pathway, specifically the PERK arm, with a concomitant increase in oxidative stress and spontaneous exfoliation of urothelial cells. Further, we show that such increase in cell shedding is associated with a compensatory proliferation of the basal cells but impaired regeneration of superficial cells. Finally, we show that upon loss of IFRD1, mice display aberrant voiding behavior. Thus, we propose that IFRD1 is at the center of many crucial cellular pathways that work together to maintain urothelial homeostasis, highlighting its importance as a target for diagnosis and/or therapy in bladder conditions.
    DOI:  https://doi.org/10.1101/2024.01.09.574887
  15. Oncogenesis. 2024 Jan 25. 13(1): 6
    LUBAC promotes angiogenesis and lung tumorigenesis by ubiquitinating and antagonizing autophagic degradation of HIF1α.
    Ying Jin, Yazhi Peng, Jie Xu, Ye Yuan, Nan Yang, Zemei Zhang, Lei Xu, Lin Li, Yulian Xiong, Dejiao Sun, Yamu Pan, Ruiqing Wu, Jian Fu.
      Hypoxia-inducible factor 1 (HIF1) is critically important for driving angiogenesis and tumorigenesis. Linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin ligase capable of catalyzing protein linear ubiquitination to date, is implicated in cell signaling and associated with cancers. However, the role and mechanism of LUBAC in regulating the expression and function of HIF1α, the labile subunit of HIF1, remain to be elucidated. Herein we showed that LUBAC increases HIF1α protein expression in cultured cells and tissues of human lung cancer and enhances HIF1α DNA-binding and transcriptional activities, which are dependent upon LUBAC enzymatic activity. Mechanistically, LUBAC increases HIF1α stability through antagonizing HIF1α decay by the chaperone-mediated autophagy (CMA)-lysosome pathway, thereby potentiating HIF1α activity. We further demonstrated that HIF1α selectively interacts with HOIP (the catalytic subunit of LUBAC) primarily in the cytoplasm. LUBAC catalyzes linear ubiquitination of HIF1α at lysine 362. Linear ubiquitination shields HIF1α from interacting with heat-shock cognate protein of 70 kDa and lysosome-associated membrane protein type 2 A, two components of CMA. Consequently, linear ubiquitination confers protection against CMA-mediated destruction of HIF1α, increasing HIF1α stability and activity. We found that prolyl hydroxylation is not a perquisite for LUBAC's effects on HIF1α. Functionally, LUBAC facilitates proliferation, clonogenic formation, invasion and migration of lung cancer cells. LUBAC also boosts angiogenesis and exacerbates lung cancer growth in mice, which are greatly compromised by inhibition of HIF1α. This work provides novel mechanistic insights into the role of LUBAC in regulating HIF1α homeostasis, tumor angiogenesis and tumorigenesis of lung cancer, making LUBAC an attractive therapeutic target for cancers.
    DOI:  https://doi.org/10.1038/s41389-024-00508-3
  16. Cell Death Dis. 2024 01 20. 15(1): 77
    LUBAC-mediated M1 Ub regulates necroptosis by segregating the cellular distribution of active MLKL.
    Nadine Weinelt, Kaja Nicole Wächtershäuser, Gulustan Celik, Birte Jeiler, Isabelle Gollin, Laura Zein, Sonja Smith, Geoffroy Andrieux, Tonmoy Das, Jens Roedig, Leonard Feist, Björn Rotter, Melanie Boerries, Francesco Pampaloni, Sjoerd J L van Wijk.
      Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endo- and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in cells of human origin. Loss of LUBAC activity inhibits tumor necrosis factor α (TNFα)-mediated necroptosis, not by affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Finally, we confirm LUBAC-dependent activation of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as novel regulator of necroptosis which promotes MLKL membrane accumulation in human cells and pioneer primary human organoids to model necroptosis in near-physiological settings.
    DOI:  https://doi.org/10.1038/s41419-024-06447-6
  17. J Cell Biol. 2024 Mar 04. pii: e202305087. [Epub ahead of print]223(3):
    CLPB disaggregase dysfunction impacts the functional integrity of the proteolytic SPY complex.
    Megan J Baker, Kai Uwe Blau, Alexander J Anderson, Catherine S Palmer, Laura F Fielden, Jordan J Crameri, Dusanka Milenkovic, David R Thorburn, Ann E Frazier, Thomas Langer, Diana Stojanovski.
      CLPB is a mitochondrial intermembrane space AAA+ domain-containing disaggregase. CLPB mutations are associated with 3-methylglutaconic aciduria and neutropenia; however, the molecular mechanism underscoring disease and the contribution of CLPB substrates to disease pathology remains unknown. Interactions between CLPB and mitochondrial quality control (QC) factors, including PARL and OPA1, have been reported, hinting at dysregulation of organelle QC in disease. Utilizing proteomic and biochemical approaches, we show a stress-specific aggregation phenotype in a CLPB-null environment and define the CLPB substrate profile. We illustrate an interplay between intermembrane space proteins including CLPB, HAX1, HTRA2, and the inner membrane quality control proteins (STOML2, PARL, YME1L1; SPY complex), with CLPB deficiency impeding SPY complex function by virtue of protein aggregation in the intermembrane space. We conclude that there is an interdependency of mitochondrial QC components at the intermembrane space/inner membrane interface, and perturbations to this network may underscore CLPB disease pathology.
    DOI:  https://doi.org/10.1083/jcb.202305087
  18. Elife. 2024 Jan 22. pii: RP88579. [Epub ahead of print]12
    Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.
    Fabio Lolicato, Julia P Steringer, Roberto Saleppico, Daniel Beyer, Jaime Fernandez-Sobaberas, Sebastian Unger, Steffen Klein, Petra Riegerová, Sabine Wegehingel, Hans-Michael Müller, Xiao J Schmitt, Shreyas Kaptan, Christian Freund, Martin Hof, Radek Šachl, Petr Chlanda, Ilpo Vattulainen, Walter Nickel.
      Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.
    Keywords:  E. coli; FGF2; Protein trafficking; Protein-lipid interaction; Protein-protein interaction; Unconventional protein secretion; biochemistry; chemical biology; cho; cho k1; hela s3
    DOI:  https://doi.org/10.7554/eLife.88579
  19. G3 (Bethesda). 2024 Jan 24. pii: jkae017. [Epub ahead of print]
    The unfolded protein response of the endoplasmic reticulum protects Caenorhabditis elegans against DNA damage caused by stalled replication forks.
    Jiaming Xu, Brendil Sabatino, Junran Yan, Glafira Ermakova, Kelsie R S Doering, Stefan Taubert.
      All animals must maintain genome and proteome integrity, especially when experiencing endogenous or exogenous stress. To cope, organisms have evolved sophisticated and conserved response systems: unfolded protein responses (UPRs) ensure proteostasis while DNA damage responses (DDRs) maintains genome integrity. Emerging evidence suggests that UPRs and DDRs crosstalk, but this remains poorly understood. Here, we demonstrate that depletion of the DNA primases pri-1 or pri-2, which synthesize RNA primers at replication forks and whose inactivation causes DNA damage, activates the UPR of the endoplasmic reticulum (UPR-ER) in Caenorhabditis elegans, with especially strong activation in the germline. We observed activation of both the inositol-requiring-enzyme 1 (ire-1) and the protein kinase RNA-like ER kinase (pek-1) branches of the UPR-ER. Interestingly, activation of the UPR-ER output gene heat shock protein 4 (hsp-4) was partially independent of its canonical activators, ire-1 and X-box binding protein (xbp-1), and instead required the third branch of the UPR-ER, activating transcription factor 6 (atf-6), suggesting functional redundancy. We further found that primase depletion specifically induces the UPR-ER, but not the distinct cytosolic or mitochondrial UPRs, suggesting that primase inactivation causes compartment-specific rather than global stress. Functionally, loss of ire-1 or pek-1 sensitized animals to replication stress caused by hydroxyurea. Finally, transcriptome analysis of pri-1 embryos revealed several deregulated processes that could cause UPR-ER activation, including protein glycosylation, calcium signaling, and fatty acid desaturation. Together, our data show that the UPR-ER, but not other UPRs, responds to replication fork stress and that the UPR-ER is required to alleviate this stress.
    Keywords:  BiP; DNA damage; DNA replication; UPR; primase
    DOI:  https://doi.org/10.1093/g3journal/jkae017
  20. Nat Commun. 2024 Jan 24. 15(1): 716
    Methionine aminopeptidase 2 and its autoproteolysis product have different binding sites on the ribosome.
    Marius A Klein, Klemens Wild, Miglė Kišonaitė, Irmgard Sinning.
      Excision of the initiator methionine is among the first co-translational processes that occur at the ribosome. While this crucial step in protein maturation is executed by two types of methionine aminopeptidases in eukaryotes (MAP1 and MAP2), additional roles in disease and translational regulation have drawn more attention to MAP2. Here, we report several cryo-EM structures of human and fungal MAP2 at the 80S ribosome. Irrespective of nascent chains, MAP2 can occupy the tunnel exit. On nascent chain displaying ribosomes, the MAP2-80S interaction is highly dynamic and the MAP2-specific N-terminal extension engages in stabilizing interactions with the long rRNA expansion segment ES27L. Loss of this extension by autoproteolytic cleavage impedes interactions at the tunnel, while promoting MAP2 to enter the ribosomal A-site, where it engages with crucial functional centers of translation. These findings reveal that proteolytic remodeling of MAP2 severely affects ribosome binding, and set the stage for targeted functional studies.
    DOI:  https://doi.org/10.1038/s41467-024-44862-7
  21. FASEB J. 2024 Feb;38(2): e23442
    Cleavage efficiency of the intramembrane protease γ-secretase is reduced by the palmitoylation of a substrate's transmembrane domain.
    Marlene Aßfalg, Gökhan Güner, Stephan A Müller, Stephan Breimann, Dieter Langosch, Claudia Muhle-Goll, Dmitrij Frishman, Harald Steiner, Stefan F Lichtenthaler.
      The intramembrane protease γ-secretase has broad physiological functions, but also contributes to Notch-dependent tumors and Alzheimer's disease. While γ-secretase cleaves numerous membrane proteins, only few nonsubstrates are known. Thus, a fundamental open question is how γ-secretase distinguishes substrates from nonsubstrates and whether sequence-based features or post-translational modifications of membrane proteins contribute to substrate recognition. Using mass spectrometry-based proteomics, we identified several type I membrane proteins with short ectodomains that were inefficiently or not cleaved by γ-secretase, including 'pituitary tumor-transforming gene 1-interacting protein' (PTTG1IP). To analyze the mechanism preventing cleavage of these putative nonsubstrates, we used the validated substrate FN14 as a backbone and replaced its transmembrane domain (TMD), where γ-cleavage occurs, with the one of nonsubstrates. Surprisingly, some nonsubstrate TMDs were efficiently cleaved in the FN14 backbone, demonstrating that a cleavable TMD is necessary, but not sufficient for cleavage by γ-secretase. Cleavage efficiencies varied by up to 200-fold. Other TMDs, including that of PTTG1IP, were still barely cleaved within the FN14 backbone. Pharmacological and mutational experiments revealed that the PTTG1IP TMD is palmitoylated, which prevented cleavage by γ-secretase. We conclude that the TMD sequence of a membrane protein and its palmitoylation can be key factors determining substrate recognition and cleavage efficiency by γ-secretase.
    Keywords:  FXYD3; FXYD6; LYRIC; PMEPA1; TNR12; Tweak receptor; intramembrane proteolysis; protease substrate specificity
    DOI:  https://doi.org/10.1096/fj.202302152R
  22. bioRxiv. 2024 Jan 11. pii: 2024.01.10.575080. [Epub ahead of print]
    Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay.
    Divya Kolakada, Rui Fu, Nikita Biziaev, Alexey Shuvalov, Mlana Lore, Amy E Campbell, Michael A Cortázar, Marcin P Sajek, Jay R Hesselberth, Neelanjan Mukherjee, Elena Alkalaeva, Sujatha Jagannathan.
      Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that prevents the accumulation of harmful truncated proteins by degrading transcripts with premature termination codons (PTCs). NMD efficiency varies across many contexts, but the factors that influence this variability remain poorly understood. Here, we find an enrichment of glycine (Gly) codons preceding a PTC in common nonsense variants in contrast with a depletion of Gly codons preceding a normal termination codon (NTC). Gly-PTC contexts have higher NMD activity compared to an alanine-PTC context, and this effect is stronger on NMD substrates with long 3'UTRs. We used a massively parallel reporter assay to test all possible combinations of -2 and -1 codons, the PTC, and the +4 nucleotide to assess comprehensively how PTC sequence context affects NMD efficiency. A random forest classifier revealed that peptidyl-tRNA hydrolysis rate during translation termination was the most important feature in discriminating high and low NMD activity. We show with in vitro biochemical assays that Gly-TC contexts have the slowest termination rate compared to other codons. Furthermore, Gly-PTC enrichment is most pronounced in genes that tolerate loss-of-function variants, suggesting that enhanced NMD of Gly-PTC context has shaped the evolution of PTCs. Based on these findings, we propose that NMD efficiency is modulated by the "window of opportunity" offered by peptidyl tRNA hydrolysis rate and thus, translation termination kinetics.
    DOI:  https://doi.org/10.1101/2024.01.10.575080
  23. Nat Commun. 2024 Jan 26. 15(1): 792
    MARC-3, a membrane-associated ubiquitin ligase, is required for fast polyspermy block in Caenorhabditis elegans.
    Ichiro Kawasaki, Kenta Sugiura, Taeko Sasaki, Noriyuki Matsuda, Miyuki Sato, Ken Sato.
      In many sexually reproducing organisms, oocytes are fundamentally fertilized with one sperm. In Caenorhabditis elegans, chitin layer formation after fertilization by the EGG complex is one of the mechanisms of polyspermy block, but other mechanisms remain unknown. Here, we demonstrate that MARC-3, a membrane-associated RING-CH-type ubiquitin ligase that localizes to the plasma membrane and cortical puncta in oocytes, is involved in fast polyspermy block. During polyspermy, the second sperm entry occurs within approximately 10 s after fertilization in MARC-3-deficient zygotes, whereas it occurs approximately 200 s after fertilization in egg-3 mutant zygotes defective in the chitin layer formation. MARC-3 also functions in the selective degradation of maternal plasma membrane proteins and the transient accumulation of endosomal lysine 63-linked polyubiquitin after fertilization. The RING-finger domain of MARC-3 is required for its in vitro ubiquitination activity and polyspermy block, suggesting that a ubiquitination-mediated mechanism sequentially regulates fast polyspermy block and maternal membrane protein degradation during the oocyte-to-embryo transition.
    DOI:  https://doi.org/10.1038/s41467-024-44928-6
  24. Mol Cell. 2024 Jan 15. pii: S1097-2765(23)01074-2. [Epub ahead of print]
    N6-methyladenosine in 5' UTR does not promote translation initiation.
    Ewelina Guca, Rodrigo Alarcon, Michael Z Palo, Leonardo Santos, Santiago Alonso-Gil, Marcos Davyt, Leonardo H F de Lima, Fanny Boissier, Sarada Das, Bojan Zagrovic, Joseph D Puglisi, Yaser Hashem, Zoya Ignatova.
      The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.
    Keywords:  cryo-EM; deep-sequencing; initiation; m(6)A modification; post-transcriptional mRNA modification; single-molecule FRET; translation
    DOI:  https://doi.org/10.1016/j.molcel.2023.12.028
  25. Nat Chem Biol. 2024 Jan 24.
    Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1.
    Mayuki Tanaka, Takeshi Yokoyama, Hironori Saito, Madoka Nishimoto, Kengo Tsuda, Naoyuki Sotta, Hideki Shigematsu, Mikako Shirouzu, Shintaro Iwasaki, Takuhiro Ito, Toru Fujiwara.
      In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.
    DOI:  https://doi.org/10.1038/s41589-023-01513-0
  26. bioRxiv. 2024 Jan 09. pii: 2024.01.08.574738. [Epub ahead of print]
    Protein SUMOylation promotes cAMP-independent EPAC1 activation.
    Wenli Yang, Fang C Mei, Wei Lin, Mark A White, Li Li, Yue Li, Sheng Pan, Xiaodong Cheng.
      Exchange protein directly activated by cAMP (EPAC1) mediates the intracellular functions of a critical stress-response second messenger, cAMP. Herein, we report that EPAC1 is a cellular substrate of protein SUMOylation, a prevalent stress-response posttranslational modification. Site-specific mapping of SUMOylation by mass spectrometer leads to identifying K561 as a primary SUMOylation site in EPAC1. Sequence and site-directed mutagenesis analyses reveal a functional SUMO-interacting motif required for cellular SUMOylation of EPAC1. SUMO modification of EPAC1 mediates its heat shock-induced Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.
    DOI:  https://doi.org/10.1101/2024.01.08.574738
  27. Nature. 2024 Jan 24.
    Motion of VAPB molecules reveals ER-mitochondria contact site subdomains.
    Christopher J Obara, Jonathon Nixon-Abell, Andrew S Moore, Federica Riccio, David P Hoffman, Gleb Shtengel, C Shan Xu, Kathy Schaefer, H Amalia Pasolli, Jean-Baptiste Masson, Harald F Hess, Christopher P Calderon, Craig Blackstone, Jennifer Lippincott-Schwartz.
      To coordinate cellular physiology, eukaryotic cells rely on the rapid exchange of molecules at specialized organelle-organelle contact sites1,2. Endoplasmic reticulum-mitochondrial contact sites (ERMCSs) are particularly vital communication hubs, playing key roles in the exchange of signalling molecules, lipids and metabolites3,4. ERMCSs are maintained by interactions between complementary tethering molecules on the surface of each organelle5,6. However, due to the extreme sensitivity of these membrane interfaces to experimental perturbation7,8, a clear understanding of their nanoscale organization and regulation is still lacking. Here we combine three-dimensional electron microscopy with high-speed molecular tracking of a model organelle tether, Vesicle-associated membrane protein (VAMP)-associated protein B (VAPB), to map the structure and diffusion landscape of ERMCSs. We uncovered dynamic subdomains within VAPB contact sites that correlate with ER membrane curvature and undergo rapid remodelling. We show that VAPB molecules enter and leave ERMCSs within seconds, despite the contact site itself remaining stable over much longer time scales. This metastability allows ERMCSs to remodel with changes in the physiological environment to accommodate metabolic needs of the cell. An amyotrophic lateral sclerosis-associated mutation in VAPB perturbs these subdomains, likely impairing their remodelling capacity and resulting in impaired interorganelle communication. These results establish high-speed single-molecule imaging as a new tool for mapping the structure of contact site interfaces and reveal that the diffusion landscape of VAPB at contact sites is a crucial component of ERMCS homeostasis.
    DOI:  https://doi.org/10.1038/s41586-023-06956-y
  28. Traffic. 2024 Jan;25(1): e12930
    Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.
    Tamara Diamanti, Laura Trobiani, Lorenza Mautone, Federica Serafini, Roberta Gioia, Laura Ferrucci, Clotilde Lauro, Sara Bianchi, Camilla Perfetto, Stefano Guglielmo, Raimondo Sollazzo, Ezio Giorda, Andrea Setini, Davide Ragozzino, Elena Miranda, Davide Comoletti, Silvia Di Angelantonio, Emanuele Cacci, Antonella De Jaco.
      Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.
    Keywords:  ER stress; autism spectrum disorders; dexamethasone; neural stem progenitor cells; neurodevelopmental disorder; neurons; synaptic contacts; synaptic protein; unfolded protein response
    DOI:  https://doi.org/10.1111/tra.12930
  29. Open Biol. 2024 Jan;14(1): 230386
    Single-cell expression predicts neuron-specific protein homeostasis networks.
    Sebastian Pechmann.
      The protein homeostasis network keeps proteins in their correct shapes and avoids unwanted aggregation. In turn, the accumulation of aberrantly misfolded proteins has been directly associated with the onset of ageing-associated neurodegenerative diseases such as Alzheimer's and Parkinson's. However, a detailed and rational understanding of how protein homeostasis is achieved in health, and how it can be targeted for therapeutic intervention in diseases remains missing. Here, large-scale single-cell expression data from the Allen Brain Map are analysed to investigate the transcription regulation of the core protein homeostasis network across the human brain. Remarkably, distinct expression profiles suggest specialized protein homeostasis networks with systematic adaptations in excitatory neurons, inhibitory neurons and non-neuronal cells. Moreover, several chaperones and Ubiquitin ligases are found transcriptionally coregulated with genes important for synapse formation and maintenance, thus linking protein homeostasis to the regulation of neuronal function. Finally, evolutionary analyses highlight the conservation of an elevated interaction density in the chaperone network, suggesting that one of the most exciting aspects of chaperone action may yet be discovered in their collective action at the systems level. More generally, our work highlights the power of computational analyses for breaking down complexity and gaining complementary insights into fundamental biological problems.
    Keywords:  Ubiquitin ligases; chaperones; neurons; protein homeostasis
    DOI:  https://doi.org/10.1098/rsob.230386
  30. bioRxiv. 2024 Jan 09. pii: 2023.07.20.549210. [Epub ahead of print]
    Impaired biogenesis of basic proteins impacts multiple hallmarks of the aging brain.
    Domenico Di Fraia, Antonio Marino, Jae Ho Lee, Erika Kelmer Sacramento, Mario Baumgart, Sara Bagnoli, Pedro Tomaz da Silva, Amit Kumar Sahu, Giacomo Siano, Max Tiessen, Eva Terzibasi-Tozzini, Julien Gagneur, Judith Frydman, Alessandro Cellerino, Alessandro Ori.
      Aging and neurodegeneration entail diverse cellular and molecular hallmarks. Here, we studied the effects of aging on the transcriptome, translatome, and multiple layers of the proteome in the brain of a short-lived killifish. We reveal that aging causes widespread reduction of proteins enriched in basic amino acids that is independent of mRNA regulation, and it is not due to impaired proteasome activity. Instead, we identify a cascade of events where aberrant translation pausing leads to reduced ribosome availability resulting in proteome remodeling independently of transcriptional regulation. Our research uncovers a vulnerable point in the aging brain's biology - the biogenesis of basic DNA/RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity and the biosynthesis of macromolecules.
    One-Sentence Summary: Translation pausing reshapes the aging brain proteome, revealing vulnerabilities in the biogenesis of nucleic-acid protein.
    DOI:  https://doi.org/10.1101/2023.07.20.549210
  31. J Cell Sci. 2024 Jan 15. pii: jcs261527. [Epub ahead of print]137(2):
    Multi-monoubiquitylation controls VASP-mediated actin dynamics.
    Laura E McCormick, Cristian Suarez, Laura E Herring, Kevin S Cannon, David R Kovar, Nicholas G Brown, Stephanie L Gupton.
      The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.
    Keywords:  Actin; Filopodia; Nondegradative; TRIM9; Ubiquitylation; VASP
    DOI:  https://doi.org/10.1242/jcs.261527
  32. ACS Chem Biol. 2024 Jan 25.
    Ubiquitin-Derived Fragment as a Peptide Linker for the Efficient Cleavage of a Target Protein from a Degron.
    Yuki Utsugi, Ken Nishimura, Satoshi Yamanaka, Kohei Nishino, Hidetaka Kosako, Tatsuya Sawasaki, Hideyuki Shigemori, Thomas J Wandless, Yusaku Miyamae.
      The chemogenetic control of cellular protein stability using degron tags is a powerful experimental strategy in biomedical research. However, this technique requires permanent fusion of the degron to a target protein, which may interfere with the proper function of the protein. Here, we report a peptide fragment from the carboxyl terminus of ubiquitin as a cleavable linker that exhibits the slow but efficient cleavage of a degron tag via cellular deubiquitinating enzymes (DUBs). We designed a fusion protein consisting of a cleavable linker and a destabilizing domain (DD), which conditionally controls the expression and release of a target protein in a ligand-induced state, allowing the free unmodified protein to perform its function. Insertion of an AGIA epitope at the carboxyl terminus of the linker made space for the DUBs to access the site to assist the cleavage reaction when the amino terminus of the target protein caused steric hindrance. The developed system, termed a cleavable degron using ubiquitin-derived linkers (c-DUB), provides robust and tunable regulation of target proteins in their native forms. The c-DUB system is a useful tool for the regulation of proteins that have terminal sites that are essential for the proper localization and function. In addition, a mechanistic investigation using proximity labeling showed that DUBs associate with the refolded DD to reverse ubiquitination, suggesting a cellular surveillance system for distinguishing the refolded DD from misfolded proteins. The c-DUB method may benefit from this machinery so that DUBs subsequently cleave the neighboring linker.
    DOI:  https://doi.org/10.1021/acschembio.3c00670
  33. Nat Aging. 2024 Jan 24.
    TXNRD1 drives the innate immune response in senescent cells with implications for age-associated inflammation.
    Xue Hao, Bo Zhao, Martina Towers, Liping Liao, Edgar Luzete Monteiro, Xin Xu, Christina Freeman, Hongzhuang Peng, Hsin-Yao Tang, Aaron Havas, Andrew V Kossenkov, Shelley L Berger, Peter D Adams, David W Speicher, David Schultz, Ronen Marmorstein, Kenneth S Zaret, Rugang Zhang.
      Sterile inflammation, also known as 'inflammaging', is a hallmark of tissue aging. Cellular senescence contributes to tissue aging, in part, through the secretion of proinflammatory factors collectively known as the senescence-associated secretory phenotype (SASP). The genetic variability of thioredoxin reductase 1 (TXNRD1) is associated with aging and age-associated phenotypes such as late-life survival, activity of daily living and physical performance in old age. TXNRD1's role in regulating tissue aging has been attributed to its enzymatic role in cellular redox regulation. Here, we show that TXNRD1 drives the SASP and inflammaging through the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway independently of its enzymatic activity. TXNRD1 localizes to cytoplasmic chromatin fragments and interacts with cGAS in a senescence-status-dependent manner, which is necessary for the SASP. TXNRD1 enhances the enzymatic activity of cGAS. TXNRD1 is required for both the tumor-promoting and immune surveillance functions of senescent cells, which are mediated by the SASP in vivo in mouse models. Treatment of aged mice with a TXNRD1 inhibitor that disrupts its interaction with cGAS, but not with an inhibitor of its enzymatic activity alone, downregulated markers of inflammaging in several tissues. In summary, our results show that TXNRD1 promotes the SASP through the innate immune response, with implications for inflammaging. This suggests that the TXNRD1-cGAS interaction is a relevant target for selectively suppressing inflammaging.
    DOI:  https://doi.org/10.1038/s43587-023-00564-1
  34. J Biol Chem. 2024 Jan 23. pii: S0021-9258(24)00046-2. [Epub ahead of print] 105670
    E3 ubiquitin ligase Hul6 modulates iron-dependent metabolism by regulating Php4 stability.
    Rui Yao, Rongrong Li, Xiaoyu Wu, Ting Jin, Ying Luo, Rong Li, Ying Huang.
      Schizosaccharomyces pombe Php4 is the regulatory subunit of the CCAAT-binding complexes and plays an important role in the regulation of iron homeostasis and iron-dependent metabolism. Here we show that Php4 undergoes ubiquitin-dependent degradation in the late logarithmic and stationary phases. The degradation and ubiquitination of Php4 could be attenuated by deletion of hul6, a gene encoding a putative HECT-type E3 ubiquitin ligase. The expression levels of Hul6 and Php4 are oppositely regulated during cell growth. Hul6 interacts with the C-terminal region of Php4. Two lysine residues (K217 and K274) located in the C-terminal region of Php4 are required for its polyubiquitination. Increasing the levels of Php4 by deletion of hul6 or overexpression of php4 decreased expression of Php4 target proteins involved in iron-dependent metabolic pathways such as the tricarboxylic cycle (TCA cycle) and mitochondrial oxidative phosphorylation (OXPHOS), thus causing increased sensitivity to high-iron and reductions in succinate dehydrogenase (SDH) and mitochondrial complex II activities. Hul6 is located primarily in the mitochondrial outer membrane and most likely targets cytosolic Php4 for ubiquitination and degradation. Taken together, our data suggest that Hul6 regulates iron-dependent metabolism through degradation of Php4 under normal growth conditions. Our results also suggest that Hul6 promotes iron-dependent metabolism to help the cell to adapt to a nutrient-starved growth phase.
    Keywords:  E3 ubiquitin ligase; OXPHOS; iron-regulatory factor; protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2024.105670
  35. Mol Biol Cell. 2024 Jan 24. mbcE23040149
    VAMP5 promotes Fcγ receptor-mediated phagocytosis and regulates phagosome maturation in macrophages.
    Chiye Sakurai, Natsumi Yamashita, Kento Azuma, Kiyotaka Hatsuzawa.
      Phagosome formation and maturation reportedly occur via sequential membrane fusion events mediated by synaptosomal-associated protein of 23 kDa (SNAP23), a plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. Vesicle-associated membrane protein 5 (VAMP5), also a plasmalemma SNARE, interacts with SNAP23; however, its precise function in phagocytosis in macrophages remains elusive. To elucidate this aspect, we investigated the characteristics of macrophages in the presence of VAMP5 overexpression or knockdown and found that VAMP5 participates in Fcγ receptor-mediated phagosome formation, although not directly in phagosome maturation. Overexpressed VAMP5 was localized to the early phagosomal membrane but no longer localized to the lysosomal-associated membrane protein 1-positive maturing phagosomal membrane. Analyses using compound-based selective inhibitors demonstrated that VAMP5 dissociation from early phagosomes occurs in a clathrin- and dynamin-dependent manner and is indispensable for SNAP23 function in subsequent membrane fusion during phagosome maturation. Accordingly, to the best of our knowledge, we demonstrate, for the first time, that VAMP5 exerts an immunologically critical function during phagosome formation and maturation via SNARE-based membrane trafficking in macrophages.
    DOI:  https://doi.org/10.1091/mbc.E23-04-0149
  36. Cell Chem Biol. 2024 Jan 09. pii: S2451-9456(23)00477-4. [Epub ahead of print]
    Beyond antibiotic resistance: The whiB7 transcription factor coordinates an adaptive response to alanine starvation in mycobacteria.
    Nicholas C Poulton, Michael A DeJesus, Vanisha Munsamy-Govender, Mariko Kanai, Cameron G Roberts, Zachary A Azadian, Barbara Bosch, Karl Matthew Lin, Shuqi Li, Jeremy M Rock.
      Pathogenic mycobacteria are a significant cause of morbidity and mortality worldwide. The conserved whiB7 stress response reduces the effectiveness of antibiotic therapy by activating several intrinsic antibiotic resistance mechanisms. Despite our comprehensive biochemical understanding of WhiB7, the complex set of signals that induce whiB7 expression remain less clear. We employed a reporter-based, genome-wide CRISPRi epistasis screen to identify a diverse set of 150 mycobacterial genes whose inhibition results in constitutive whiB7 expression. We show that whiB7 expression is determined by the amino acid composition of the 5' regulatory uORF, thereby allowing whiB7 to sense amino acid starvation. Although deprivation of many amino acids can induce whiB7, whiB7 specifically coordinates an adaptive response to alanine starvation by engaging in a feedback loop with the alanine biosynthetic enzyme, aspC. These findings describe a metabolic function for whiB7 and help explain its evolutionary conservation across mycobacterial species occupying diverse ecological niches.
    Keywords:  CRISPRi; WhiB7; amino acids; antibiotics; mycobacteria; stress response; translation; tuberculosis
    DOI:  https://doi.org/10.1016/j.chembiol.2023.12.020
  37. Antioxidants (Basel). 2024 Jan 08. pii: 81. [Epub ahead of print]13(1):
    ADP-Ribosylation Factor-Interacting Protein 2 Acts as a Novel Regulator of Mitophagy and Autophagy in Podocytes in Diabetic Nephropathy.
    Haihua Guo, Manuel Rogg, Julia Keller, Ann-Kathrin Scherzinger, Julia Jäckel, Charlotte Meyer, Alena Sammarco, Martin Helmstädter, Oliver Gorka, Olaf Groß, Christoph Schell, Wibke Bechtel-Walz.
      (1) Background: Differentiated podocytes are particularly vulnerable to oxidative stress and cellular waste products. The disease-related loss of postmitotic podocytes is a direct indicator of renal disease progression and aging. Podocytes use highly specific regulated networks of autophagy and endocytosis that counteract the increasing number of damaged protein aggregates and help maintain cellular homeostasis. Here, we demonstrate that ARFIP2 is a regulator of autophagy and mitophagy in podocytes both in vitro and in vivo. (2) Methods: In a recent molecular regulatory network analysis of mouse glomeruli, we identified ADP-ribosylation factor-interacting protein 2 (Arfip2), a cytoskeletal regulator and cofactor of ATG9-mediated autophagosome formation, to be differentially expressed with age. We generated an Arfip2-deficient immortalized podocyte cell line using the CRISPR/Cas technique to investigate the significance of Arfip2 for renal homeostasis in vitro. For the in vivo analyses of Arfip2 deficiency, we used a mouse model of Streptozotozin-induced type I diabetes and investigated physiological data and (patho)histological (ultra)structural modifications. (3) Results: ARFIP2 deficiency in immortalized human podocytes impedes autophagy. Beyond this, ARFIP2 deficiency in human podocytes interferes with ATG9A trafficking and the PINK1-Parkin pathway, leading to the compromised fission of mitochondria and short-term increase in mitochondrial respiration and induction of mitophagy. In diabetic mice, Arfip2 deficiency deteriorates autophagy and leads to foot process effacement, histopathological changes, and early albuminuria. (4) Conclusions: In summary, we show that ARFIP2 is a novel regulator of autophagy and mitochondrial homeostasis in podocytes by facilitating ATG9A trafficking during PINK1/Parkin-regulated mitophagy.
    Keywords:  ADP-ribosylation factor-interacting protein 2; Arfip2; CKD; autophagy; diabetes; mitophagy; podocyte
    DOI:  https://doi.org/10.3390/antiox13010081
  38. EMBO Rep. 2024 Jan 23.
    ChemRAP uncovers specific mRNA translation regulation via RNA 5' phospho-methylation.
    Hélène Ipas, Ellen B Gouws, Nathan S Abell, Po-Chin Chiou, Sravan K Devanathan, Solène Hervé, Sidae Lee, Marvin Mercado, Calder Reinsborough, Levon Halabelian, Cheryl H Arrowsmith, Blerta Xhemalçe.
      5'-end modifications play key roles in determining RNA fates. Phospho-methylation is a noncanonical cap occurring on either 5'-PPP or 5'-P ends. We used ChemRAP, in which affinity purification of cellular proteins with chemically synthesized modified RNAs is coupled to quantitative proteomics, to identify 5'-Pme "readers". We show that 5'-Pme is directly recognized by EPRS, the central subunit of the multisynthetase complex (MSC), through its linker domain, which has previously been involved in key noncanonical EPRS and MSC functions. We further determine that the 5'-Pme writer BCDIN3D regulates the binding of EPRS to specific mRNAs, either at coding regions rich in MSC codons, or around start codons. In the case of LRPPRC (leucine-rich pentatricopeptide repeat containing), a nuclear-encoded mitochondrial protein associated with the French Canadian Leigh syndrome, BCDIN3D deficiency abolishes binding of EPRS around its mRNA start codon, increases its translation but ultimately results in LRPPRC mislocalization. Overall, our results suggest that BCDIN3D may regulate the translation of specific mRNA via RNA-5'-Pme.
    Keywords:  BCDIN3D; LRPPRC; Local Translation; RNA Modification Reader; RNA Phospho-methylation
    DOI:  https://doi.org/10.1038/s44319-024-00059-z
  39. Cell Stress Chaperones. 2024 Jan 18. pii: S1355-8145(24)00042-7. [Epub ahead of print]
    Heat shock response during the resolution of inflammation and its progressive suppression in chronic-degenerative inflammatory diseases.
    Helena Trevisan Schroeder, Carlos Henrique de Lemos Muller, Thiago Gomes Heck, Mauricio Krause, Paulo Ivo Homem de Bittencourt.
      The heat shock response (HSR) is a crucial biochemical pathway that orchestrates the resolution of inflammation, primarily under proteotoxic stress conditions. This process hinges on the upregulation of heat shock proteins (HSPs) and other chaperones, notably the 70kDa family of HSPs (HSP70s), under the command of the heat shock transcription factor-1 (HSF1). However, in the context of chronic degenerative disorders characterized by persistent low-grade inflammation - such as insulin resistance, obesity, type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases - a gradual suppression of the HSR does occur. This work delves into the mechanisms behind this phenomenon. It explores how the Western diet and sedentary lifestyle, culminating in the endoplasmic reticulum (ER) stress within adipose tissue cells, trigger a cascade of events. This cascade includes the unfolded protein response (UPR) and activation of the NLRP3 inflammasome, leading to the emergence of the senescence-associated secretory phenotype (SASP) and the propagation of inflammation throughout the body. Notably, the activation of the NLRP3 inflammasome not only fuels inflammation but also sabotages the HSR by degrading HuR, a crucial mRNA-binding protein responsible for maintaining HSF1 mRNA expression and stability on heat shock gene promoters. This paper underscores the imperative need to comprehend how chronic inflammation stifles the HSR and the clinical significance of evaluating the HSR using cost-effective and accessible tools. Such understanding is pivotal in the development of innovative strategies aimed at the prevention and treatment of these chronic inflammatory ailments, which continue to exact a heavy toll on global health and well-being.
    Keywords:  HSP70; chronic inflammatory diseases; exercise; heat shock response; insulin resistance; obesity
    DOI:  https://doi.org/10.1016/j.cstres.2024.01.002
  40. Nat Commun. 2024 Jan 20. 15(1): 631
    ATG14 targets lipid droplets and acts as an autophagic receptor for syntaxin18-regulated lipid droplet turnover.
    Zhen Yuan, Kun Cai, Jiajia Li, Ruifeng Chen, Fuhai Zhang, Xuan Tan, Yaming Jiu, Haishuang Chang, Bing Hu, Weiyi Zhang, Binbin Ding.
      Lipid droplets (LDs) are dynamic lipid storage organelles that can be degraded by autophagy machinery to release neutral lipids, a process called lipophagy. However, specific receptors and regulation mechanisms for lipophagy remain largely unknown. Here, we identify that ATG14, the core unit of the PI3KC3-C1 complex, also targets LD and acts as an autophagic receptor that facilitates LD degradation. A negative regulator, Syntaxin18 (STX18) binds ATG14, disrupting the ATG14-ATG8 family members interactions and subverting the PI3KC3-C1 complex formation. Knockdown of STX18 activates lipophagy dependent on ATG14 not only as the core unit of PI3KC3-C1 complex but also as the autophagic receptor, resulting in the degradation of LD-associated anti-viral protein Viperin. Furthermore, coronavirus M protein binds STX18 and subverts the STX18-ATG14 interaction to induce lipophagy and degrade Viperin, facilitating virus production. Altogether, our data provide a previously undescribed mechanism for additional roles of ATG14 in lipid metabolism and virus production.
    DOI:  https://doi.org/10.1038/s41467-024-44978-w
  41. bioRxiv. 2024 Jan 01. pii: 2023.12.31.573790. [Epub ahead of print]
    The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics.
    Laura E McCormick, Elliot B Evans, Natalie K Barker, Laura E Herring, Graham H Diering, Stephanie L Gupton.
      During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1 dependent axon guidance and branching. Here we demonstrate TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the post-synaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9 . In addition, we document changes in the synaptic receptors associated with loss of Trim9 . These defects converge on a loss of netrindependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.
    DOI:  https://doi.org/10.1101/2023.12.31.573790
  42. Traffic. 2024 Jan;25(1): e12928
    Endoglin mutants retained in the endoplasmic reticulum exacerbate loss of function in hereditary hemorrhagic telangiectasia type 1 (HHT1) by exerting dominant negative effects on the wild type allele.
    Nesrin Gariballa, Sally Badawi, Bassam R Ali.
      Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder affecting 1 in 5000-8000 individuals. Hereditary hemorrhagic telangiectasia type 1 (HHT1) is the most common HHT and manifests as diverse vascular malformations ranging from mild symptoms such as epistaxis and mucosal and cutaneous telangiectases to severe arteriovenous malformations (AVMs) in the lungs, brain or liver. HHT1 is caused by heterozygous mutations in the ENG gene, which encodes endoglin, the TGFβ homodimeric co-receptor. It was previously shown that some endoglin HHT1-causing variants failed to traffic to the plasma membrane due to their retention in the endoplasmic reticulum (ER) and consequent degradation by ER-associated degradation (ERAD). Endoglin is a homodimer formed in the ER, and we therefore hypothesized that mixed heterodimers might form between ER-retained variants and WT protein, thus hampering its maturation and trafficking to the plasma membrane causing dominant negative effects. Indeed, HA-tagged ER-retained mutants formed heterodimers with Myc-tagged WT endoglin. Moreover, variants L32R, V105D, P165L, I271N and C363Y adversely affected the trafficking of WT endoglin by reducing its maturation and plasma membrane localization. These results strongly suggest dominant negative effects exerted by these ER-retained variants aggravating endoglin loss of function in patients expressing them in the heterozygous state with the WT allele. Moreover, this study may help explain some of the variability observed among HHT1 patients due to the additional loss of function exerted by the dominant negative effects in addition to that due to haploinsufficiency. These findings might also have implications for some of the many conditions impacted by ERAD.
    Keywords:  AVMs; ER-retained variants; ERAD; HHT1; endoglin; haploinsufficiency; hereditary hemorrhagic telangiectasia; telangiectases
    DOI:  https://doi.org/10.1111/tra.12928
  43. bioRxiv. 2024 Jan 06. pii: 2024.01.05.574385. [Epub ahead of print]
    Loss of the E3 ubiquitin ligase TRIM67 alters the post-synaptic density proteome.
    Laura E McCormick, Natalie K Barker, Laura E Herring, Stephanie L Gupton.
      The E3 ubiquitin ligase TRIM67 is enriched in the central nervous system and is required for proper neuronal development. Previously we demonstrated TRIM67 coordinates with the closely related E3 ubiquitin ligase TRIM9 to regulate cytoskeletal dynamics downstream of the netrin-1 during axon guidance and axon branching in early neuronal morphogenesis. Interestingly, loss of Trim67 impacts cognitive flexibility in a spatial learning and memory task. Despite this behavioral phenotype, it was previously uninvestigated if TRIM67 was involved in synapse formation or function. Here we demonstrate TRIM67 localizes to the post-synaptic density (PSD) within dendritic spines. Furthermore, we show that loss of Trim67 significantly changes the PSD proteome, including changes in the regulation of the actin and microtubule cytoskeletons. Collectively, our data propose a synaptic role for TRIM67.
    DOI:  https://doi.org/10.1101/2024.01.05.574385
  44. Nat Cancer. 2024 Jan 24.
    Inhibition of ADAM9 promotes the selective degradation of KRAS and sensitizes pancreatic cancers to chemotherapy.
    Yu-Kai Huang, Wei-Chung Cheng, Ting-Ting Kuo, Juan-Cheng Yang, Yang-Chang Wu, Heng-Hsiung Wu, Chia-Chien Lo, Chih-Ying Hsieh, Sze-Ching Wong, Chih-Hao Lu, Wan-Ling Wu, Shih-Jen Liu, Yi-Chuan Li, Ching-Chan Lin, Chia-Ning Shen, Mien-Chie Hung, Jaw-Town Lin, Chun-Chieh Yeh, Yuh-Pyng Sher.
      Kirsten rat sarcoma virus (KRAS) signaling drives pancreatic ductal adenocarcinoma (PDAC) malignancy, which is an unmet clinical need. Here, we identify a disintegrin and metalloproteinase domain (ADAM)9 as a modulator of PDAC progression via stabilization of wild-type and mutant KRAS proteins. Mechanistically, ADAM9 loss increases the interaction of KRAS with plasminogen activator inhibitor 1 (PAI-1), which functions as a selective autophagy receptor in conjunction with light chain 3 (LC3), triggering lysosomal degradation of KRAS. Suppression of ADAM9 by a small-molecule inhibitor restricts disease progression in spontaneous models, and combination with gemcitabine elicits dramatic regression of patient-derived tumors. Our findings provide a promising strategy to target the KRAS signaling cascade and demonstrate a potential modality to enhance sensitivity to chemotherapy in PDAC.
    DOI:  https://doi.org/10.1038/s43018-023-00720-x
  45. Nat Commun. 2024 Jan 23. 15(1): 690
    Folding pathway of a discontinuous two-domain protein.
    Ganesh Agam, Anders Barth, Don C Lamb.
      It is estimated that two-thirds of all proteins in higher organisms are composed of multiple domains, many of them containing discontinuous folds. However, to date, most in vitro protein folding studies have focused on small, single-domain proteins. As a model system for a two-domain discontinuous protein, we study the unfolding/refolding of a slow-folding double mutant of the maltose binding protein (DM-MBP) using single-molecule two- and three-color Förster Resonance Energy Transfer experiments. We observe a dynamic folding intermediate population in the N-terminal domain (NTD), C-terminal domain (CTD), and at the domain interface. The dynamic intermediate fluctuates rapidly between unfolded states and compact states, which have a similar FRET efficiency to the folded conformation. Our data reveals that the delayed folding of the NTD in DM-MBP is imposed by an entropic barrier with subsequent folding of the highly dynamic CTD. Notably, accelerated DM-MBP folding is routed through the same dynamic intermediate within the cavity of the GroEL/ES chaperone system, suggesting that the chaperonin limits the conformational space to overcome the entropic folding barrier. Our study highlights the subtle tuning and co-dependency in the folding of a discontinuous multi-domain protein.
    DOI:  https://doi.org/10.1038/s41467-024-44901-3
  46. J Biol Chem. 2024 Jan 23. pii: S0021-9258(24)00053-X. [Epub ahead of print] 105677
    Emerging roles of O-GlcNAcylation in protein trafficking and secretion.
    Jianchao Zhang, Yanzhuang Wang.
      The emerging roles of O-GlcNAcylation, a distinctive post-translational modification, are increasingly recognized for their involvement in the intricate processes of protein trafficking and secretion. This modification exerts its influence on both conventional and unconventional secretory pathways. Under healthy and stress conditions, such as during diseases, it orchestrates the transport of proteins within cells, ensuring timely delivery to their intended destinations. O-GlcNAcylation occurs on key factors like COPI, COPII, clathrin, SNAREs, and GRASP55 that control vesicle budding and fusion in anterograde and retrograde trafficking and unconventional secretion. The understanding of O-GlcNAcylation offers valuable insights into its critical functions in cellular physiology and the progression of diseases, including neurodegeneration, cancer, and metabolic disorders. In this review, we summarize and discuss the latest findings elucidating the involvement of O-GlcNAc in protein trafficking and its significance in various human disorders.
    Keywords:  COPI; COPII; GRASP55; O-GlcNAcylation; autophagy; cancer; clathrin; conventional secretion; exosome; metabolic disease; neurodegeneration; protein trafficking; unconventional secretion
    DOI:  https://doi.org/10.1016/j.jbc.2024.105677
  47. Traffic. 2024 Jan;25(1): e12925
    Mechanisms regulating the intracellular trafficking and release of CLN5 and CTSD.
    Robert J Huber, William D Kim, Morgan L D M Wilson-Smillie.
      Ceroid lipofuscinosis neuronal 5 (CLN5) and cathepsin D (CTSD) are soluble lysosomal enzymes that also localize extracellularly. In humans, homozygous mutations in CLN5 and CTSD cause CLN5 disease and CLN10 disease, respectively, which are two subtypes of neuronal ceroid lipofuscinosis (commonly known as Batten disease). The mechanisms regulating the intracellular trafficking of CLN5 and CTSD and their release from cells are not well understood. Here, we used the social amoeba Dictyostelium discoideum as a model system to examine the pathways and cellular components that regulate the intracellular trafficking and release of the D. discoideum homologs of human CLN5 (Cln5) and CTSD (CtsD). We show that both Cln5 and CtsD contain signal peptides for secretion that facilitate their release from cells. Like Cln5, extracellular CtsD is glycosylated. In addition, Cln5 release is regulated by the amount of extracellular CtsD. Autophagy induction promotes the release of Cln5, and to a lesser extent CtsD. Release of Cln5 requires the autophagy proteins Atg1, Atg5, and Atg9, as well as autophagosomal-lysosomal fusion. Atg1 and Atg5 are required for the release of CtsD. Together, these data support a model where Cln5 and CtsD are actively released from cells via their signal peptides for secretion and pathways linked to autophagy. The release of Cln5 and CtsD from cells also requires microfilaments and the D. discoideum homologs of human AP-3 complex mu subunit, the lysosomal-trafficking regulator LYST, mucopilin-1, and the Wiskott-Aldrich syndrome-associated protein WASH, which all regulate lysosomal exocytosis in this model organism. These findings suggest that lysosomal exocytosis also facilitates the release of Cln5 and CtsD from cells. In addition, we report the roles of ABC transporters, microtubules, osmotic stress, and the putative D. discoideum homologs of human sortilin and cation-independent mannose-6-phosphate receptor in regulating the intracellular/extracellular distribution of Cln5 and CtsD. In total, this study identifies the cellular mechanisms regulating the release of Cln5 and CtsD from D. discoideum cells and provides insight into how altered trafficking of CLN5 and CTSD causes disease in humans.
    Keywords:  ABC transporters; Batten disease; autophagy; cytoskeleton; exocytosis; glycosylation; lysosome; neuronal ceroid lipofuscinosis; osmoregulation
    DOI:  https://doi.org/10.1111/tra.12925
  48. bioRxiv. 2024 Jan 02. pii: 2024.01.01.573836. [Epub ahead of print]
    Dihydroceramide desaturase governs endoplasmic reticulum and lipid droplet homeostasis to promote glial function in the nervous system.
    Yuqing Zhu, Kevin Cho, Haluk Lacin, Yi Zhu, Jose DiPaola, Beth A Wilson, Gary J Patti, James B Skeath.
      Dihydroceramide desaturases convert dihydroceramides to ceramides in the last step of the de novo ceramide pathway. Reduction of DEGS1 dihydroceramide desaturase function in humans leads to the rare neurodegenerative disorder hypomyelinating leukodystrophy-18, but the exact mechanism that underlies the disease remains unclear. Through a forward genetic screen, we discovered that infertile crescent (ifc) , the sole Drosophila dihydroceramide desaturase, governs central nervous system development and morphology. Expressed and genetically active most prominently in glia rather than neurons, ifc primarily controls nervous system development through a cell autonomous function in glia. Within the nervous system, loss of ifc results in ceramide depletion, dihydroceramide accumulation, and increased saturation of major membrane phospholipids. At the cellular level, loss of ifc leads to severe glial defects, particularly in cortex glia, including expansion of the endoplasmic reticulum, cell swelling, failure to enwrap neurons, and lipid droplet depletion. Our research supports a model in which inappropriate retention of dihydroceramide in the endoplasmic reticulum (ER) of glial cells drives ER expansion and cell swelling, disrupting glial function and leading to subsequent nervous system dysfunction. Given the conserved nature of the de novo ceramide biosynthesis pathway, our findings in the fly system suggest that dihydroceramide-triggered expansion of the ER in the membrane-rich glial cells may be the proximal cause of hypomyelinating leukodystrophy-18 in humans.
    DOI:  https://doi.org/10.1101/2024.01.01.573836
  49. Mol Cancer. 2024 Jan 24. 23(1): 22
    Selective autophagy in cancer: mechanisms, therapeutic implications, and future perspectives.
    Jiaxi Liu, Yongya Wu, Sha Meng, Ping Xu, Shutong Li, Yong Li, Xiuying Hu, Liang Ouyang, Guan Wang.
      Eukaryotic cells engage in autophagy, an internal process of self-degradation through lysosomes. Autophagy can be classified as selective or non-selective depending on the way it chooses to degrade substrates. During the process of selective autophagy, damaged and/or redundant organelles like mitochondria, peroxisomes, ribosomes, endoplasmic reticulum (ER), lysosomes, nuclei, proteasomes, and lipid droplets are selectively recycled. Specific cargo is delivered to autophagosomes by specific receptors, isolated and engulfed. Selective autophagy dysfunction is closely linked with cancers, neurodegenerative diseases, metabolic disorders, heart failure, etc. Through reviewing latest research, this review summarized molecular markers and important signaling pathways for selective autophagy, and its significant role in cancers. Moreover, we conducted a comprehensive analysis of small-molecule compounds targeting selective autophagy for their potential application in anti-tumor therapy, elucidating the underlying mechanisms involved. This review aims to supply important scientific references and development directions for the biological mechanisms and drug discovery of anti-tumor targeting selective autophagy in the future.
    Keywords:  Cancer; Molecular mechanism; Selective autophagy; Small-molecule compounds; Targeted therapy
    DOI:  https://doi.org/10.1186/s12943-024-01934-y
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