bims-proteo Biomed News
on Proteostasis
Issue of 2023–08–13
thirteen papers selected by
Eric Chevet, INSERM
  1. An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum.
  2. Protein disorder in the regulatory control of mitophagy.
  3. ER-associated degradation in cystinosis pathogenesis and the prospects of precision medicine.
  4. DELE1 oligomerization promotes integrated stress response activation.
  5. Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.
  6. Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.
  7. Feedback regulation of ubiquitination and phase separation of HECT E3 ligases.
  8. Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry.
  9. Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate.
  10. Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system.
  11. New Insights into RNA processing by the eukaryotic tRNA Splicing Endonuclease.
  12. A membrane-associated MHC-I inhibitory axis for cancer immune evasion.
  13. Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses.
  1. Nat Plants. 2023 Aug 10.
    An IRE1-proteasome system signalling cohort controls cell fate determination in unresolved proteotoxic stress of the plant endoplasmic reticulum.
    Dae Kwan Ko, Joo Yong Kim, Ethan A Thibault, Federica Brandizzi.
      Excessive accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, which is an underlying cause of major crop losses and devastating human conditions. ER proteostasis surveillance is mediated by the conserved master regulator of the unfolded protein response (UPR), Inositol Requiring Enzyme 1 (IRE1), which determines cell fate by controlling pro-life and pro-death outcomes through as yet largely unknown mechanisms. Here we report that Arabidopsis IRE1 determines cell fate in ER stress by balancing the ubiquitin-proteasome system (UPS) and UPR through the plant-unique E3 ligase, PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1). Indeed, PIR1 loss leads to suppression of pro-death UPS and the lethal phenotype of an IRE1 loss-of-function mutant in unresolved ER stress in addition to activating pro-survival UPR. Specifically, in ER stress, PIR1 loss stabilizes ABI5, a basic leucine zipper (bZIP) transcription factor, that directly activates expression of the critical UPR regulator gene, bZIP60, triggering transcriptional cascades enhancing pro-survival UPR. Collectively, our results identify new cell fate effectors in plant ER stress by showing that IRE1's coordination of cell death and survival hinges on PIR1, a key pro-death component of the UPS, which controls ABI5, a pro-survival transcriptional activator of bZIP60.
    DOI:  https://doi.org/10.1038/s41477-023-01480-3
  2. Autophagy Rep. 2023 ;pii: 2242054. [Epub ahead of print]2(1):
    Protein disorder in the regulatory control of mitophagy.
    Sheridan Mikhail, Scott A Soleimanpour.
      Mitophagy is a central component of the mitochondrial quality control machinery, which is necessary for cellular viability and bioenergetics. The E3 ubiquitin ligase CLEC16A (C-type lectin domain containing 16A) forms a tripartite mitophagy regulatory complex together with the E3 ligase RNF41 (ring finger protein 41) and the ubiquitin-specific peptidase USP8 (ubiquitin specific peptidase 8), yet CLEC16A structural/functional domains relevant for mitophagy are unknown. We identify that CLEC16A contains an internal intrinsically disordered region (IDR), which is important for CLEC16A function and stability. IDRs are flexible domains lacking fixed secondary structure and regulate an emerging number of diverse processes, yet they have been largely unstudied in mitophagy. We observe that the internal CLEC16A IDR is essential for CLEC16A degradation and is bound by RNF41 to promote CLEC16A turnover. This IDR also promotes assembly of the CLEC16A-RNF41-USP8 mitophagy regulatory complex. Thus, our study revealed the importance of IDRs in mitophagy via the regulation of CLEC16A abundance by RNF41, opening new structural insights into mitochondrial quality control.
    Keywords:  autophagy; clec16a; intrinsically disordered protein; mitochondria; ubiquitin
    DOI:  https://doi.org/10.1080/27694127.2023.2242054
  3. J Clin Invest. 2023 Aug 10. pii: e169551. [Epub ahead of print]
    ER-associated degradation in cystinosis pathogenesis and the prospects of precision medicine.
    Varsha Venkatarangan, Weichao Zhang, Xi Yang, Jess G Thoene, Si H Hahn, Ming Li.
      Cystinosis is a lysosomal storage disease that is characterized by the accumulation of di-peptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7∆). We demonstrated that this mutant is retained in the endoplasmic reticulum (ER) and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the role of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7∆) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones, previously used for treating CFTR F508∆, and demonstrated that they could facilitate the folding and trafficking of cystinosin(7∆). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by ~70%. Conclusively, our study establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7∆).
    Keywords:  Cell Biology; Chronic kidney disease; Genetic diseases; Nephrology; Ubiquitin-proteosome system
    DOI:  https://doi.org/10.1172/JCI169551
  4. Nat Struct Mol Biol. 2023 Aug 07.
    DELE1 oligomerization promotes integrated stress response activation.
    Jie Yang, Kelsey R Baron, Daniel E Pride, Anette Schneemann, Xiaoyan Guo, Wenqian Chen, Albert S Song, Giovanni Aviles, Martin Kampmann, R Luke Wiseman, Gabriel C Lander.
      Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.
    DOI:  https://doi.org/10.1038/s41594-023-01061-0
  5. Nat Commun. 2023 08 09. 14(1): 4798
    Auranofin targets UBA1 and enhances UBA1 activity by facilitating ubiquitin trans-thioesterification to E2 ubiquitin-conjugating enzymes.
    Wenjing Yan, Yongwang Zhong, Xin Hu, Tuan Xu, Yinghua Zhang, Stephen Kales, Yanyan Qu, Daniel C Talley, Bolormaa Baljinnyam, Christopher A LeClair, Anton Simeonov, Brian M Polster, Ruili Huang, Yihong Ye, Ganesha Rai, Mark J Henderson, Dingyin Tao, Shengyun Fang.
      UBA1 is the primary E1 ubiquitin-activating enzyme responsible for generation of activated ubiquitin required for ubiquitination, a process that regulates stability and function of numerous proteins. Decreased or insufficient ubiquitination can cause or drive aging and many diseases. Therefore, a small-molecule enhancing UBA1 activity could have broad therapeutic potential. Here we report that auranofin, a drug approved for the treatment of rheumatoid arthritis, is a potent UBA1 activity enhancer. Auranofin binds to the UBA1's ubiquitin fold domain and conjugates to Cys1039 residue. The binding enhances UBA1 interactions with at least 20 different E2 ubiquitin-conjugating enzymes, facilitating ubiquitin charging to E2 and increasing the activities of seven representative E3s in vitro. Auranofin promotes ubiquitination and degradation of misfolded ER proteins during ER-associated degradation in cells at low nanomolar concentrations. It also facilitates outer mitochondrial membrane-associated degradation. These findings suggest that auranofin can serve as a much-needed tool for UBA1 research and therapeutic exploration.
    DOI:  https://doi.org/10.1038/s41467-023-40537-x
  6. STAR Protoc. 2023 Aug 09. pii: S2666-1667(23)00456-2. [Epub ahead of print]4(3): 102489
    Profiling and verifying the substrates of E3 ubiquitin ligase Rsp5 in yeast cells.
    Shuai Fang, Geng Chen, Yiyang Wang, Rakhee Ganti, Tatiana A Chernova, Li Zhou, Savannah E Jacobs, Duc Duong, Hiroaki Kiyokawa, Yury O Chernoff, Ming Li, Natalia Shcherbik, Bo Zhao, Jun Yin.
      Yeast is an essential model organism for studying protein ubiquitination pathways; however, identifying the direct substrates of E3 in the cell presents a challenge. Here, we present a protocol for using the orthogonal ubiquitin transfer (OUT) cascade to profile the substrate specificity of yeast E3 Rsp5. We describe steps for OUT profiling, proteomics analysis, in vitro and in cell ubiquitination, and stability assay. The protocol can be adapted for identifying and verifying the ubiquitination targets of other E3s in yeast. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
    Keywords:  Cell Biology; Cell-based Assays; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry
    DOI:  https://doi.org/10.1016/j.xpro.2023.102489
  7. Proc Natl Acad Sci U S A. 2023 08 15. 120(33): e2302478120
    Feedback regulation of ubiquitination and phase separation of HECT E3 ligases.
    Jingyu Li, Kang Zhu, Aihong Gu, Yiqing Zhang, Shijing Huang, Ronggui Hu, Weiguo Hu, Qun-Ying Lei, Wenyu Wen.
      Lipid homeostasis is essential for normal cellular functions and dysregulation of lipid metabolism is highly correlated with human diseases including neurodegenerative diseases. In the ubiquitin-dependent autophagic degradation pathway, Troyer syndrome-related protein Spartin activates and recruits HECT-type E3 Itch to lipid droplets (LDs) to regulate their turnover. In this study, we find that Spartin promotes the formation of Itch condensates independent of LDs. Spartin activates Itch through its multiple PPAY-motif platform generated by self-oligomerization, which targets the WW12 domains of Itch and releases the autoinhibition of the ligase. Spartin-induced activation and subsequent autoubiquitination of Itch lead to liquid-liquid phase separation (LLPS) of the poly-, but not oligo-, ubiquitinated Itch together with Spartin and E2 both in vitro and in living cells. LLPS-mediated condensation of the reaction components further accelerates the generation of polyubiquitin chains, thus forming a positive feedback loop. Such Itch-Spartin condensates actively promote the autophagy-dependent turnover of LDs. Moreover, we show that the catalytic HECT domain of Itch is sufficient to interact and phase separate with poly-, but not oligo-ubiquitin chains. HECT domains from other HECT E3 ligases also exhibit LLPS-mediated the promotion of ligase activity. Therefore, LLPS and ubiquitination are mutually interdependent and LLPS promotes the ligase activity of the HECT family E3 ligases.
    Keywords:  HECT-type ligases; feedback regulation; liquid–liquid phase separation; turnover of lipid droplets; ubiquitination
    DOI:  https://doi.org/10.1073/pnas.2302478120
  8. Proc Natl Acad Sci U S A. 2023 08 15. 120(33): e2303167120
    Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry.
    Ruiyue Tan, Margaret Hoare, Kevin A Welle, Kyle Swovick, Jennifer R Hryhorenko, Sina Ghaemmaghami.
      The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.
    Keywords:  cotranslational protein folding; methionine oxidation; protein stability; proteomics; ribosome
    DOI:  https://doi.org/10.1073/pnas.2303167120
  9. Autophagy. 2023 Aug 06. 1-20
    Inhibition of autophagy and induction of glioblastoma cell death by NEO214, a perillyl alcohol-rolipram conjugate.
    Mengting Ou, Hee-Yeon Cho, Jie Fu, Thu Zan Thein, Weijun Wang, Stephen D Swenson, Radu O Minea, Apostolos Stathopoulos, Axel H Schönthal, Florence M Hofman, Liling Tang, Thomas C Chen.
      Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide.
    Keywords:  MTOR complex; NEO214; autophagic flux; chloroquine; glioblastoma; transcription factor EB
    DOI:  https://doi.org/10.1080/15548627.2023.2242696
  10. EMBO J. 2023 Aug 09. e114318
    Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system.
    Cameron G Roberts, Tyler G Franklin, Jonathan N Pruneda.
      Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.
    Keywords:  Ubiquitin; bacterial effector; bacterial pathogenesis; post-translational modification
    DOI:  https://doi.org/10.15252/embj.2023114318
  11. J Biol Chem. 2023 Aug 04. pii: S0021-9258(23)02166-X. [Epub ahead of print] 105138
    New Insights into RNA processing by the eukaryotic tRNA Splicing Endonuclease.
    Cassandra K Hayne, Samoil Sekulovski, Jennifer E Hurtig, Robin E Stanley, Simon Trowitzsch, Ambro van Hoof.
      Through its role in intron cleavage, tRNA splicing endonuclease (TSEN) plays a critical function in the maturation of intron-containing pre-tRNAs. The catalytic mechanism and core requirement for this process is conserved between archaea and eukaryotes, but for decades it has been known that eukaryotic TSENs have evolved additional modes of RNA recognition, which have remained poorly understood. Recent research identified new roles for eukaryotic TSEN, including processing or degradation of additional RNA substrates, and determined the first structures of pre-tRNA bound human TSEN complexes. These recent discoveries have changed our understanding of how the eukaryotic TSEN targets and recognizes substrates. Here we review these recent discoveries, their implications, and the new questions raised by these findings.
    DOI:  https://doi.org/10.1016/j.jbc.2023.105138
  12. Cell. 2023 Aug 02. pii: S0092-8674(23)00783-3. [Epub ahead of print]
    A membrane-associated MHC-I inhibitory axis for cancer immune evasion.
    Xufeng Chen, Qiao Lu, Hua Zhou, Jia Liu, Bettina Nadorp, Audrey Lasry, Zhengxi Sun, Baoling Lai, Gergely Rona, Jiangyan Zhang, Michael Cammer, Kun Wang, Wafa Al-Santli, Zoe Ciantra, Qianjin Guo, Jia You, Debrup Sengupta, Ahmad Boukhris, Hongbing Zhang, Cheng Liu, Peter Cresswell, Patricia L M Dahia, Michele Pagano, Iannis Aifantis, Jun Wang.
      Immune-checkpoint blockade has revolutionized cancer treatment, but some cancers, such as acute myeloid leukemia (AML), do not respond or develop resistance. A potential mode of resistance is immune evasion of T cell immunity involving aberrant major histocompatibility complex class I (MHC-I) antigen presentation (AP). To map such mechanisms of resistance, we identified key MHC-I regulators using specific peptide-MHC-I-guided CRISPR-Cas9 screens in AML. The top-ranked negative regulators were surface protein sushi domain containing 6 (SUSD6), transmembrane protein 127 (TMEM127), and the E3 ubiquitin ligase WWP2. SUSD6 is abundantly expressed in AML and multiple solid cancers, and its ablation enhanced MHC-I AP and reduced tumor growth in a CD8+ T cell-dependent manner. Mechanistically, SUSD6 forms a trimolecular complex with TMEM127 and MHC-I, which recruits WWP2 for MHC-I ubiquitination and lysosomal degradation. Together with the SUSD6/TMEM127/WWP2 gene signature, which negatively correlates with cancer survival, our findings define a membrane-associated MHC-I inhibitory axis as a potential therapeutic target for both leukemia and solid cancers.
    Keywords:  MHC-I; SUSD6; T cell; TMEM127; WWP2; antigen presentation; cancer; immune evasion; lysosomal degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.cell.2023.07.016
  13. Cell Rep. 2023 Aug 07. pii: S2211-1247(23)00936-1. [Epub ahead of print]42(8): 112925
    Deneddylation of ribosomal proteins promotes synergy between MLN4924 and chemotherapy to elicit complete therapeutic responses.
    Arthur Aubry, Joel D Pearson, Jason Charish, Tao Yu, Jeremy M Sivak, Dimitris P Xirodimas, Hervé Avet-Loiseau, Jill Corre, Philippe P Monnier, Rod Bremner.
      The neddylation inhibitor MLN4924/Pevonedistat is in clinical trials for multiple cancers. Efficacy is generally attributed to cullin RING ligase (CRL) inhibition, but the contribution of non-CRL targets is unknown. Here, CRISPR screens map MLN4924-monotherapy sensitivity in retinoblastoma to a classic DNA damage-induced p53/E2F3/BAX-dependent death effector network, which synergizes with Nutlin3a or Navitoclax. In monotherapy-resistant cells, MLN4924 plus standard-of-care topotecan overcomes resistance, but reduces DNA damage, instead harnessing ribosomal protein nucleolar-expulsion to engage an RPL11/p21/MYCN/E2F3/p53/BAX synergy network that exhibits extensive cross-regulation. Strikingly, unneddylatable RPL11 substitutes for MLN4924 to perturb nucleolar function and enhance topotecan efficacy. Orthotopic tumors exhibit complete responses while preserving visual function. Moreover, MLN4924 plus melphalan deploy this DNA damage-independent strategy to synergistically kill multiple myeloma cells. Thus, MLN4924 synergizes with standard-of-care drugs to unlock a nucleolar death effector network across cancer types implying broad therapeutic relevance.
    Keywords:  CP: Cancer; MYCN; RPL11; melphalan; multiple myeloma; nucleoli; p53; pevonedistat; retinoblastoma; synergy; topotecan
    DOI:  https://doi.org/10.1016/j.celrep.2023.112925
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