bims-proteo Biomed News
on Proteostasis
Issue of 2022‒11‒06
twenty-two papers selected by
Eric Chevet
INSERM
  1. Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.
  2. cIAP1-based degraders induce degradation via branched ubiquitin architectures.
  3. Elements of the ERAD ubiquitin ligase Doa10 regulating sequential poly-ubiquitylation of its targets.
  4. SCF-SKP2 E3 ubiquitin ligase links mTORC1-ER stress-ISR with YAP activation in murine renal cystogenesis.
  5. Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging.
  6. Cooperative assembly of p97 complexes involved in replication termination.
  7. Intramembrane client recognition potentiates the chaperone functions of calnexin.
  8. A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation.
  9. Regulated degradation of the inner nuclear membrane protein SUN2 maintains nuclear envelope architecture and function.
  10. Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders.
  11. Ablation of EWS-FLI1 by USP9X inhibition suppresses cancer cell growth in Ewing sarcoma.
  12. ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag.
  13. Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.
  14. Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins.
  15. Translational buffering by ribosome stalling in upstream open reading frames.
  16. A non-canonical RNA-binding domain of the Fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes.
  17. A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.
  18. Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.
  19. Intestinal Apc-inactivation induces HSP25 dependency.
  20. Subcellular location of source proteins improves prediction of neoantigens for immunotherapy.
  21. Translational alterations in pancreatic cancer: a central role for the integrated stress response.
  22. Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes.
  1. J Cell Sci. 2022 Nov 03. pii: jcs.260294. [Epub ahead of print]
    Stress-induced phase separation of ERES components into Sec bodies precedes ER exit inhibition in mammalian cells.
    Wessel van Leeuwen, Dan T M Nguyen, Rianne Grond, Tineke Veenendaal, Catherine Rabouille, Ginny G Farías.
      Phase separation of ER-exit-sites (ERES) components into membraneless compartments, the Sec bodies, occurs in Drosophila cells upon specific cellular stressors, i.e., salt stress and amino acid starvation, and their formation is linked to the early secretory pathway inhibition. Here, we show Sec bodies also form in secretory mammalian cells upon the same stress. These reversible and membraneless structures are positive for ERES components, including both Sec16A and Sec16B isoforms and COPII subunits. We find that Sec16A, but not Sec16B, is a driver for Sec body formation, and that the coalescence of ERES components into Sec bodies occurs by fusion. Lastly, we show that the stress-induced coalescence of ERES components into Sec bodies precedes ER-exit inhibition, leading to their progressive depletion from ERES that become non-functional. Stress-relief causes an immediate dissolution of Sec bodies and the concomitant restoration of ER-exit. We propose that the dynamic conversion between ERES and Sec body assembly, driven by Sec16A, regulates protein exit from the ER during stress and upon stress-relief in mammalian cells, thus providing a conserved pro-survival mechanism in response to stress.
    Keywords:  ER exit sites; ERES remodeling; Early secretory pathway; Mammalian cells; Phase separation; Protein transport; Sec body; Sec16; Stress
    DOI:  https://doi.org/10.1242/jcs.260294
  2. Nat Chem Biol. 2022 Oct 31.
    cIAP1-based degraders induce degradation via branched ubiquitin architectures.
    Yoshino Akizuki, Mai Morita, Yuki Mori, Ai Kaiho-Soma, Shivani Dixit, Akinori Endo, Marie Shimogawa, Gosuke Hayashi, Mikihiko Naito, Akimitsu Okamoto, Keiji Tanaka, Yasushi Saeki, Fumiaki Ohtake.
      Targeted protein degradation through chemical hijacking of E3 ubiquitin ligases is an emerging concept in precision medicine. The ubiquitin code is a critical determinant of the fate of substrates. Although two E3s, CRL2VHL and CRL4CRBN, frequently assemble with proteolysis-targeting chimeras (PROTACs) to attach lysine-48 (K48)-linked ubiquitin chains, the diversity of the ubiquitin code used for chemically induced degradation is largely unknown. Here we show that the efficacy of cIAP1-targeting degraders depends on the K63-specific E2 enzyme UBE2N. UBE2N promotes degradation of cIAP1 induced by cIAP1 ligands and subsequent cancer cell apoptosis. Mechanistically, UBE2N-catalyzed K63-linked ubiquitin chains facilitate assembly of highly complex K48/K63 and K11/K48 branched ubiquitin chains, thereby recruiting p97/VCP, UCH37 and the proteasome. Degradation of neo-substrates directed by cIAP1-recruiting PROTACs also depends on UBE2N. These results reveal an unexpected role for K63-linked ubiquitin chains and UBE2N in degrader-induced proteasomal degradation and demonstrate the diversity of the ubiquitin code used for chemical hijacking.
    DOI:  https://doi.org/10.1038/s41589-022-01178-1
  3. iScience. 2022 Nov 18. 25(11): 105351
    Elements of the ERAD ubiquitin ligase Doa10 regulating sequential poly-ubiquitylation of its targets.
    Adrian B Mehrtash, Mark Hochstrasser.
      In ER-associated degradation (ERAD), misfolded ER proteins are degraded by the proteasome after undergoing ubiquitylation. Yeast Doa10 (human MARCHF6/TEB4) is a membrane-embedded E3 ubiquitin ligase that functions with E2s Ubc6 and Ubc7. Ubc6 attaches a single ubiquitin to substrates, which is extended by Ubc7 to form a polyubiquitin chain. We show the conserved C-terminal element (CTE) of Doa10 promotes E3-mediated Ubc6 activity. Doa10 substrates undergoing an alternative ubiquitylation mechanism are still degraded in CTE-mutant cells. Structure prediction by AlphaFold2 suggests the CTE binds near the catalytic RING-CH domain, implying a direct role in substrate ubiquitylation, and we confirm this interaction using intragenic suppression. Truncation analysis defines a minimal E2-binding region of Doa10; structural predictions suggest that Doa10 forms a retrotranslocation channel and that E2s bind within the cofactor-binding region defined here. These results provide mechanistic insight into how Doa10, and potentially other ligases, interact with their cofactors and mediate ERAD.
    Keywords:  Biological sciences; Molecular biology; Molecular interaction; Natural sciences
    DOI:  https://doi.org/10.1016/j.isci.2022.105351
  4. J Clin Invest. 2022 Nov 03. pii: e153943. [Epub ahead of print]
    SCF-SKP2 E3 ubiquitin ligase links mTORC1-ER stress-ISR with YAP activation in murine renal cystogenesis.
    Dibyendu K Panda, Xiuying Bai, Yan Zhang, Nicholas A Stylianesis, Antonis E Koromilas, Mark L Lipman, Andrew C Karaplis.
      The Hippo pathway nuclear effector Yes-associated protein 1 (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1) driven by ERK1/2 and PI3K/AKT cascades induced endoplasmic reticulum (ER) proteotoxic stress. To reduce it by reprogramming translation, the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF-S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α-phosphorylation-deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress-ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan, impeded these processes. Restoring ER homeostasis, and/or interfering with the SKP2-YAP interaction represent novel potential therapeutic avenues for stemming the progression of renal cystogenesis.
    Keywords:  Chronic kidney disease; Nephrology
    DOI:  https://doi.org/10.1172/JCI153943
  5. EMBO J. 2022 Oct 31. e111952
    Unfolded protein response IRE1/XBP1 signaling is required for healthy mammalian brain aging.
    Felipe Cabral-Miranda, Giovanni Tamburini, Gabriela Martinez, Alvaro O Ardiles, Danilo B Medinas, Yannis Gerakis, Mei-Li Diaz Hung, René Vidal, Matias Fuentealba, Tim Miedema, Claudia Duran-Aniotz, Javier Diaz, Cristobal Ibaceta-Gonzalez, Carleen M Sabusap, Francisca Bermedo-Garcia, Paula Mujica, Stuart Adamson, Kaitlyn Vitangcol, Hernan Huerta, Xu Zhang, Tomohiro Nakamura, Sergio Pablo Sardi, Stuart A Lipton, Brian K Kennedy, Juan Pablo Henriquez, J Cesar Cárdenas, Lars Plate, Adrian G Palacios, Claudio Hetz.
      Aging is a major risk factor for neurodegenerative diseases and is associated with decreased buffering capacity of the proteostasis network. We investigated the significance of the unfolded protein response (UPR), a major signaling pathway activated to cope with endoplasmic reticulum (ER) stress, in the functional deterioration of the mammalian brain during aging. We report that genetic disruption of the ER stress sensor IRE1 accelerated age-related cognitive decline. In mouse models, overexpressing an active form of the UPR transcription factor XBP1 restored synaptic and cognitive function, in addition to reducing cell senescence. Proteomic profiling of hippocampal tissue showed that XBP1 expression significantly restore changes associated with aging, including factors associated with synaptic function and pathways linked to neurodegenerative diseases. Similar changes were observed in human brain aging. Collectively, our results demonstrate that strategies to manipulate the UPR in mammals may help sustain healthy brain aging.
    Keywords:  ER stress; UPR; XBP1s; aging brain; proteostasis
    DOI:  https://doi.org/10.15252/embj.2022111952
  6. Nat Commun. 2022 Nov 03. 13(1): 6591
    Cooperative assembly of p97 complexes involved in replication termination.
    Olga V Kochenova, Sirisha Mukkavalli, Malavika Raman, Johannes C Walter.
      The p97 ATPase extracts polyubiquitylated proteins from diverse cellular structures in preparation for destruction by the proteasome. p97 functions with Ufd1-Npl4 and a variety of UBA-UBX co-factors, but how p97 complexes assemble on ubiquitylated substrates is unclear. To address this, we investigated how p97 disassembles the CMG helicase after it is ubiquitylated during replication termination. We show that p97Ufd1-Npl4 recruitment to CMG requires the UBA-UBX protein Ubxn7, and conversely, stable Ubxn7 binding to CMG requires p97Ufd1-Npl4. This cooperative assembly involves interactions between Ubxn7, p97, Ufd1-Npl4, and ubiquitin. Another p97 co-factor, Faf1, partially compensates for the loss of Ubxn7. Surprisingly, p97Ufd1-Npl4-Ubxn7 and p97Ufd1-Npl4-Faf1 also assemble cooperatively on unanchored ubiquitin chains. We propose that cooperative and substrate-independent recognition of ubiquitin chains allows p97 to recognize an unlimited number of polyubiquitylated proteins while avoiding the formation of partial, inactive complexes.
    DOI:  https://doi.org/10.1038/s41467-022-34210-y
  7. EMBO J. 2022 Oct 31. e110959
    Intramembrane client recognition potentiates the chaperone functions of calnexin.
    Nicolas Bloemeke, Kevin Meighen-Berger, Manuel Hitzenberger, Nina C Bach, Marina Parr, Joao Pl Coelho, Dmitrij Frishman, Martin Zacharias, Stephan A Sieber, Matthias J Feige.
      One-third of the human proteome is comprised of membrane proteins, which are particularly vulnerable to misfolding and often require folding assistance by molecular chaperones. Calnexin (CNX), which engages client proteins via its sugar-binding lectin domain, is one of the most abundant ER chaperones, and plays an important role in membrane protein biogenesis. Based on mass spectrometric analyses, we here show that calnexin interacts with a large number of nonglycosylated membrane proteins, indicative of additional nonlectin binding modes. We find that calnexin preferentially bind misfolded membrane proteins and that it uses its single transmembrane domain (TMD) for client recognition. Combining experimental and computational approaches, we systematically dissect signatures for intramembrane client recognition by calnexin, and identify sequence motifs within the calnexin TMD region that mediate client binding. Building on this, we show that intramembrane client binding potentiates the chaperone functions of calnexin. Together, these data reveal a widespread role of calnexin client recognition in the lipid bilayer, which synergizes with its established lectin-based substrate binding. Molecular chaperones thus can combine different interaction modes to support the biogenesis of the diverse eukaryotic membrane proteome.
    Keywords:  calnexin; chaperone; membrane protein
    DOI:  https://doi.org/10.15252/embj.2022110959
  8. Genes Dev. 2022 Nov 03.
    A synthetic KLHL20 ligand to validate CUL3KLHL20 as a potent E3 ligase for targeted protein degradation.
    Brian M Farrell, Fabian Gerth, Cheng-Hao R Yang, Johannes T-H Yeh.
      Targeted protein degradation (TPD) has risen as a promising therapeutic modality. Leveraging the catalytic nature of the ubiquitin-proteasome enzymatic machinery, TPD exhibits higher potency to eliminate disease-causing target proteins such as oncogenic transcription factors that may otherwise be difficult to abrogate by conventional inhibitors. However, there are challenges that remain. Currently, nearly all degraders engage CUL4CRBN or CUL2VHL as the E3 ligase for target ubiquitination. While their immediate efficacies are evident, the narrowed E3 ligase options make TPD vulnerable to potential drug resistance. In addition, E3 ligases show differential tissue expression and have intrinsic limitations in accessing varying types of disease-relevant targets. As the success of TPD is closely associated with the ability of E3 ligases to efficiently polyubiquitinate the target of interest, the long-term outlook of TPD drug development will depend on whether E3 ligases such as CUL4CRBN and CUL2VHL are accessible to the targets of interest. To overcome these potential caveats, a broad collection of actionable E3 ligases is required. Here, we designed a macrocyclic degrader engaging CUL3KLHL20 for targeting BET proteins and validated CUL3KLHL20 as an E3 ligase system suitable for TPD. This work thus contributes to the expansion of usable E3 ligases for potential drug development.
    Keywords:  CUL3; KLHL20; PROTAC; macrocycles; targeted protein degradation
    DOI:  https://doi.org/10.1101/gad.349717.122
  9. Elife. 2022 Nov 01. pii: e81573. [Epub ahead of print]11
    Regulated degradation of the inner nuclear membrane protein SUN2 maintains nuclear envelope architecture and function.
    Logesvaran Krshnan, Wingyan Skyla Siu, Michael Van de Weijer, Daniel Hayward, Elena Navarro Guerrero, Ulrike Gruneberg, Pedro Carvalho.
      Nuclear architecture and functions depend on dynamic interactions between nuclear components (such as chromatin) and inner nuclear membrane (INM) proteins. Mutations in INM proteins interfering with these interactions result in disease. However, mechanisms controlling the levels and turnover of INM proteins remain unknown. Here, we describe a mechanism of regulated degradation of the INM SUN domain-containing protein 2 (SUN2). We show that Casein Kinase II and the C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) have opposing effects on SUN2 levels by regulating SUN2 binding to the ubiquitin ligase Skp/Cullin1/F-BoxβTrCP (SCFβTrCP). Upon binding to phosphorylated SUN2, SCFβTrCP promotes its ubiquitination. Ubiquitinated SUN2 is membrane extracted by the AAA ATPase p97 and delivered to the proteasome for degradation. Importantly, accumulation of non-degradable SUN2 results in aberrant nuclear architecture, vulnerability to DNA damage and increased lagging chromosomes in mitosis. These findings uncover a central role of proteolysis in INM protein homeostasis.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.81573
  10. Nat Chem Biol. 2022 Nov 03.
    Functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders.
    Alexander Hanzl, Ryan Casement, Hana Imrichova, Scott J Hughes, Eleonora Barone, Andrea Testa, Sophie Bauer, Jane Wright, Matthias Brand, Alessio Ciulli, Georg E Winter.
      Targeted protein degradation is a novel pharmacology established by drugs that recruit target proteins to E3 ubiquitin ligases. Based on the structure of the degrader and the target, different E3 interfaces are critically involved, thus forming defined 'functional hotspots'. Understanding disruptive mutations in functional hotspots informs on the architecture of the assembly, and highlights residues susceptible to acquire resistance phenotypes. Here we employ haploid genetics to show that hotspot mutations cluster in substrate receptors of hijacked ligases, where mutation type and frequency correlate with gene essentiality. Intersection with deep mutational scanning revealed hotspots that are conserved or specific for chemically distinct degraders and targets. Biophysical and structural validation suggests that hotspot mutations frequently converge on altered ternary complex assembly. Moreover, we validated hotspots mutated in patients that relapse from degrader treatment. In sum, we present a fast and widely accessible methodology to characterize small-molecule degraders and associated resistance mechanisms.
    DOI:  https://doi.org/10.1038/s41589-022-01177-2
  11. Cancer Lett. 2022 Oct 29. pii: S0304-3835(22)00471-2. [Epub ahead of print]552 215984
    Ablation of EWS-FLI1 by USP9X inhibition suppresses cancer cell growth in Ewing sarcoma.
    Shan Wang, Xiaofang Huo, Yiping Yang, Yingxi Mo, Rahul K Kollipara, Ralf Kittler.
      The neomorphic transcription factor EWS-FLI1 is a key driver of Ewing sarcoma. Ablation of EWS-FLI1 may present a promising therapeutic strategy for this malignancy. Here we found that the deubiquitinase, ubiquitin specific peptidase 9 X-linked (USP9X) stabilizes EWS-FLI1 protein expression in Ewing sarcoma. We show that USP9X binds the ETS domain of EWS-FLI1 in Ewing sarcoma cells and deubiquitinates EWS-FLI1 and that USP9X and EWS-FLI1 protein expression is correlated in clinical Ewing sarcoma specimens. We found that treatment of Ewing sarcoma cells with the USP9X inhibitor WP1130 mediates rapid EWS-FLI1 degradation in vitro and in vivo which coincides with reduced growth of Ewing sarcoma cells and tumors. Our results suggest that USP9X might be a potential therapeutic target to mediate EWS-FLI1 depletion in Ewing sarcoma.
    Keywords:  EWS-FLI1; Ewing sarcoma; USP9X; Ubiquitination; WP1130
    DOI:  https://doi.org/10.1016/j.canlet.2022.215984
  12. Cell Rep. 2022 Nov 01. pii: S2211-1247(22)01448-6. [Epub ahead of print]41(5): 111583
    ER-mitochondrial contact protein Miga regulates autophagy through Atg14 and Uvrag.
    Lingna Xu, Yunyi Qiu, Xufeng Wang, Weina Shang, Jian Bai, Kexin Shi, Hao Liu, Jun-Ping Liu, Liquan Wang, Chao Tong.
      Mitochondrial malfunction and autophagy defects are often concurrent phenomena associated with neurodegeneration. We show that Miga, a mitochondrial outer-membrane protein that regulates endoplasmic reticulum-mitochondrial contact sites (ERMCSs), is required for autophagy. Loss of Miga results in an accumulation of autophagy markers and substrates, whereas PI3P and Syx17 levels are reduced. Further experiments indicated that the fusion between autophagosomes and lysosomes is defective in Miga mutants. Miga binds to Atg14 and Uvrag; concordantly, Miga overexpression results in Atg14 and Uvrag recruitment to mitochondria. The heightened PI3K activity induced by Miga requires Uvrag, whereas Miga-mediated stabilization of Syx17 is dependent on Atg14. Miga-regulated ERMCSs are critical for PI3P formation but are not essential for the stabilization of Syx17. In summary, we identify a mitochondrial protein that regulates autophagy by recruiting two alternative components of the PI3K complex present at the ERMCSs.
    Keywords:  CP: Cell biology; Drosophila; ER–mitochondrial contact; autophagy; lysosome; mitochondria
    DOI:  https://doi.org/10.1016/j.celrep.2022.111583
  13. Nat Commun. 2022 Nov 03. 13(1): 6614
    Tumor suppressor mediated ubiquitylation of hnRNPK is a barrier to oncogenic translation.
    Bartosz Mucha, Shuo Qie, Sagar Bajpai, Vincenzo Tarallo, J Nathaniel Diehl, Frank Tedeschi, Gao Zhou, Zhaofeng Gao, Samuel Flashner, Andres J Klein-Szanto, Hanina Hibshoosh, Shimonosono Masataka, Olga S Chajewski, Ireneusz Majsterek, Dariusz Pytel, Maria Hatzoglou, Channing J Der, Hiroshi Nakagawa, Adam J Bass, Kwok-Kin Wong, Serge Y Fuchs, Anil K Rustgi, Eckhard Jankowsky, J Alan Diehl.
      Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCFFbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCFFbxo4-hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCFFbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCFFbxo4-hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCFFbxo4-hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis.
    DOI:  https://doi.org/10.1038/s41467-022-34402-6
  14. J Am Chem Soc. 2022 Nov 01.
    Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins.
    Jim Voorneveld, Max S Kloet, Sven Wijngaarden, Robbert Q Kim, Angeliki Moutsiopoulou, Marnix Verdegaal, Mohit Misra, Ivan Đikić, Gijsbert A van der Marel, Herman S Overkleeft, Dmitri V Filippov, Gerbrand J van der Heden van Noort.
      We describe the development and optimization of a methodology to prepare peptides and proteins modified on the arginine residue with an adenosine-di-phosphate-ribosyl (ADPr) group. Our method comprises reacting an ornithine containing polypeptide on-resin with an α-linked anomeric isothiourea N-riboside, ensuing installment of a phosphomonoester at the 5'-hydroxyl of the ribosyl moiety followed by the conversion into the adenosine diphosphate. We use this method to obtain four regioisomers of ADP-ribosylated ubiquitin (UbADPr), each modified with an ADP-ribosyl residue on a different arginine position within the ubiquitin (Ub) protein (Arg42, Arg54, Arg72, and Arg74) as the first reported examples of fully synthetic arginine-linked ADPr-modified proteins. We show the chemically prepared Arg-linked UbADPr to be accepted and processed by Legionella enzymes and compare the entire suite of four Arg-linked UbADPr regioisomers in a variety of biochemical experiments, allowing us to profile the activity and selectivity of Legionella pneumophila ligase and hydrolase enzymes.
    DOI:  https://doi.org/10.1021/jacs.2c06249
  15. PLoS Genet. 2022 Oct 31. 18(10): e1010460
    Translational buffering by ribosome stalling in upstream open reading frames.
    Ty A Bottorff, Heungwon Park, Adam P Geballe, Arvind Rasi Subramaniam.
      Upstream open reading frames (uORFs) are present in over half of all human mRNAs. uORFs can potently regulate the translation of downstream open reading frames through several mechanisms: siphoning away scanning ribosomes, regulating re-initiation, and allowing interactions between scanning and elongating ribosomes. However, the consequences of these different mechanisms for the regulation of protein expression remain incompletely understood. Here, we performed systematic measurements on the uORF-containing 5' UTR of the cytomegaloviral UL4 mRNA to test alternative models of uORF-mediated regulation in human cells. We find that a terminal diproline-dependent elongating ribosome stall in the UL4 uORF prevents decreases in main ORF protein expression when ribosome loading onto the mRNA is reduced. This uORF-mediated buffering is insensitive to the location of the ribosome stall along the uORF. Computational kinetic modeling based on our measurements suggests that scanning ribosomes dissociate rather than queue when they collide with stalled elongating ribosomes within the UL4 uORF. We identify several human uORFs that repress main ORF protein expression via a similar terminal diproline motif. We propose that ribosome stalls in uORFs provide a general mechanism for buffering against reductions in main ORF translation during stress and developmental transitions.
    DOI:  https://doi.org/10.1371/journal.pgen.1010460
  16. J Biol Chem. 2022 Oct 31. pii: S0021-9258(22)01103-6. [Epub ahead of print] 102660
    A non-canonical RNA-binding domain of the Fragile X protein, FMRP, elicits translational repression independent of mRNA G-quadruplexes.
    MaKenzie R Scarpitti, Julia E Warrick, Evelyn L Yoder, Michael G Kearse.
      Loss of functional fragile X mental retardation protein (FMRP) causes fragile X syndrome, the leading form of inherited intellectual disability and the most common monogenic cause of autism spectrum disorders. FMRP is an RNA-binding protein that controls neuronal mRNA localization and translation. FMRP is thought to inhibit translation elongation after being recruited to target transcripts via binding RNA G-quadruplexes (G4s) within the coding sequence. Here, we directly test this model and report that FMRP inhibits translation independent of mRNA G4s. Furthermore, we found that the RGG box motif together with its natural C-terminal domain forms a non-canonical RNA-binding domain (ncRBD) that is essential for translational repression. The ncRBD elicits broad RNA binding ability and binds to multiple reporter mRNAs and all four homopolymeric RNAs. Serial deletion analysis of the ncRBD identified that the regions required for mRNA-binding and translational repression overlap but are not identical. Consistent with FMRP stalling elongating ribosomes and causing the accumulation of slowed 80S ribosomes, transcripts bound by FMRP via the ncRBD co-sediment with heavier polysomes and were present in puromycin-resistant ribosome complexes. Together, this work identifies a ncRBD and translational repression domain that shifts our understanding of how FMRP inhibits translation independent of mRNA G4s.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102660
  17. Nat Protoc. 2022 Nov 02.
    A bifunctional molecule-assisted synthesis of mimics for use in probing the ubiquitination system.
    Qingyun Zheng, Tian Wang, Junxiong Mao, Guochao Chu, Lujun Liang, Yangwode Jing, Chong Zuo, Yuanyuan Yu, Honggang Hu, Man Pan.
      Ubiquitination regulates almost every life process of eukaryotes. The study of the ubiquitin (Ub) coupling or decoupling process and the interaction study of Ub-Ub binding protein have always been the central focus. However, such studies are challenging, owing to the transient nature of Ub-coupling enzymes and deubiquitinases in the reactions, as well as the difficulty in preparing large quantities of polyubiquitinated samples. Here we describe a recently developed strategy for the efficient preparation of analogs of Ub chains and analogs for Ub coupling and uncoupling intermediates, which facilitate the study of the ubiquitination process. The strategy includes mainly the following steps: (i) the bifunctional molecule conjugation on the only cysteine (Cys) residue of a target protein (usually a Ub or Ub-conjugating enzyme), exposing an orthogonal reactive site for native chemical ligation; (ii) covalent ligation with a Ub-derived thioester, exposing a free sulfhydryl; and (iii) (optional) a disulfide bond formation with a substrate protein (mainly with only one Cys protein) through nonactivity-based cross-linking or with a deubiquitinase (mainly with several Cys residues) through activity-based cross-linking. When the bifunctional molecule and target proteins are obtained, the final products can be prepared in milligram quantities within 2 weeks.
    DOI:  https://doi.org/10.1038/s41596-022-00761-z
  18. Nat Chem. 2022 Oct 31.
    Insights into the ribosome function from the structures of non-arrested ribosome-nascent chain complexes.
    Egor A Syroegin, Elena V Aleksandrova, Yury S Polikanov.
      During protein synthesis, the growing polypeptide threads through the ribosomal exit tunnel and modulates ribosomal activity by itself or by sensing various small molecules, such as metabolites or antibiotics, appearing in the tunnel. While arrested ribosome-nascent chain complexes (RNCCs) have been extensively studied structurally, the lack of a simple procedure for the large-scale preparation of peptidyl-tRNAs, intermediates in polypeptide synthesis that carry the growing chain, means that little attention has been given to RNCCs representing functionally active states of the ribosome. Here we report the facile synthesis of stably linked peptidyl-tRNAs through a chemoenzymatic approach based on native chemical ligation and use them to determine several structures of RNCCs in the functional pre-attack state of the peptidyl transferase centre. These structures reveal that C-terminal parts of the growing peptides adopt the same uniform β-strand conformation stabilized by an intricate network of hydrogen bonds with the universally conserved 23S rRNA nucleotides, and explain how the ribosome synthesizes growing peptides containing various sequences with comparable efficiencies.
    DOI:  https://doi.org/10.1038/s41557-022-01073-1
  19. EMBO Mol Med. 2022 Nov 02. e16194
    Intestinal Apc-inactivation induces HSP25 dependency.
    Sanne M van Neerven, Wouter L Smit, Milou S van Driel, Vaishali Kakkar, Nina E de Groot, Lisanne E Nijman, Clara C Elbers, Nicolas Léveillé, Jarom Heijmans, Louis Vermeulen.
      The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.
    Keywords:  Apc mutations; Wnt signaling; colorectal cancer; heat shock proteins; intestinal stem cells
    DOI:  https://doi.org/10.15252/emmm.202216194
  20. EMBO J. 2022 Oct 31. e111071
    Subcellular location of source proteins improves prediction of neoantigens for immunotherapy.
    Andrea Castro, Saghar Kaabinejadian, Hooman Yari, William Hildebrand, Maurizio Zanetti, Hannah Carter.
      Antigen presentation via the major histocompatibility complex (MHC) is essential for anti-tumor immunity. However, the rules that determine which tumor-derived peptides will be immunogenic are still incompletely understood. Here, we investigated whether constraints on peptide accessibility to the MHC due to protein subcellular location are associated with peptide immunogenicity potential. Analyzing over 380,000 peptides from studies of MHC presentation and peptide immunogenicity, we find clear spatial biases in both eluted and immunogenic peptides. We find that including parent protein location improves the prediction of peptide immunogenicity in multiple datasets. In human immunotherapy cohorts, the location was associated with a neoantigen vaccination response, and immune checkpoint blockade responders generally had a higher burden of neopeptides from accessible locations. We conclude that protein subcellular location adds important information for optimizing cancer immunotherapies.
    Keywords:  Immunotherapy; immunogenicity; major histocompatibility complex; neoantigen; subcellular location
    DOI:  https://doi.org/10.15252/embj.2022111071
  21. NAR Cancer. 2022 Dec;4(4): zcac031
    Translational alterations in pancreatic cancer: a central role for the integrated stress response.
    Sauyeun Shin, Jacobo Solorzano, Mehdi Liauzun, Stéphane Pyronnet, Corinne Bousquet, Yvan Martineau.
      mRNA translation is a key mechanism for cancer cell proliferation and stress adaptation. Regulation of this machinery implicates upstream pathways such as PI3K/AKT/mTOR, RAS/MEK/ERK and the integrated stress response (ISR), principally coordinating the translation initiation step. During the last decade, dysregulation of the mRNA translation process in pancreatic cancer has been widely reported, and shown to critically impact on cancer initiation, development and survival. This includes translation dysregulation of mRNAs encoding oncogenes and tumor suppressors. Hence, cancer cells survive a stressful microenvironment through a flexible regulation of translation initiation for rapid adaptation. The ISR pathway has an important role in chemoresistance and shows high potential therapeutic interest. Despite the numerous translational alterations reported in pancreatic cancer, their consequences are greatly underestimated. In this review, we summarize the different translation dysregulations described in pancreatic cancer, which make it invulnerable, as well as the latest drug discoveries bringing a glimmer of hope.
    DOI:  https://doi.org/10.1093/narcan/zcac031
  22. Nature. 2022 Nov 02.
    Blocking PD-L1-PD-1 improves senescence surveillance and ageing phenotypes.
    Teh-Wei Wang, Yoshikazu Johmura, Narumi Suzuki, Satotaka Omori, Toshiro Migita, Kiyoshi Yamaguchi, Seira Hatakeyama, Satoshi Yamazaki, Eigo Shimizu, Seiya Imoto, Yoichi Furukawa, Akihiko Yoshimura, Makoto Nakanishi.
      The accumulation of senescent cells is a major cause of age-related inflammation and predisposes to a variety of age-related diseases1. However, little is known about the molecular basis underlying this accumulation and its potential as a target to ameliorate the ageing process. Here we show that senescent cells heterogeneously express the immune checkpoint protein programmed death-ligand 1 (PD-L1) and that PD-L1+ senescent cells accumulate with age in vivo. PD-L1- cells are sensitive to T cell surveillance, whereas PD-L1+ cells are resistant, even in the presence of senescence-associated secretory phenotypes (SASP). Single-cell analysis of p16+ cells in vivo revealed that PD-L1 expression correlated with higher levels of SASP. Consistent with this, administration of programmed cell death protein 1 (PD-1) antibody to naturally ageing mice or a mouse model with normal livers or induced nonalcoholic steatohepatitis reduces the total number of p16+ cells in vivo as well as the PD-L1+ population in an activated CD8+ T cell-dependent manner, ameliorating various ageing-related phenotypes. These results suggest that the heterogeneous expression of PD-L1 has an important role in the accumulation of senescent cells and inflammation associated with ageing, and the elimination of PD-L1+ senescent cells by immune checkpoint blockade may be a promising strategy for anti-ageing therapy.
    DOI:  https://doi.org/10.1038/s41586-022-05388-4
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