bims-proteo Biomed News
on Proteostasis
Issue of 2024‒07‒07
twenty-six papers selected by
Eric Chevet, INSERM
  1. Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress.
  2. An APEX2-based proximity-dependent biotinylation assay with temporal specificity to study protein interactions during autophagy in the yeast Saccharomyces cerevisiae.
  3. UPS-dependent strategies of protein quality control degradation.
  4. Translocational attenuation mediated by the PERK-SRP14 axis is a protective mechanism of unfolded protein response.
  5. Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.
  6. Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.
  7. Tailored assemblies of COPII proteins in secretion.
  8. Confounding Factors in Targeted Degradation of Short-Lived Proteins.
  9. ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes.
  10. Intrinsic signaling pathways modulate targeted protein degradation.
  11. Recruitment of FBXO22 for targeted degradation of NSD2.
  12. A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.
  13. Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.
  14. Dysregulated ribosome quality control in human diseases.
  15. Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.
  16. Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.
  17. Next-generation Drosophila protein interactome map and its functional implications.
  18. Valosin-Containing Protein (VCP)/p97 Oligomerization.
  19. A little ER stress isn't bad: the IRE1α/XBP1 pathway shapes ILC3 functions during intestinal inflammation.
  20. E3 ligases RNF43 and ZNRF3 display differential specificity for endocytosis of Frizzled receptors.
  21. Single-nucleus proteomics identifies regulators of protein transport.
  22. A proximity proteomics pipeline with improved reproducibility and throughput.
  23. VAMP2 regulates phase separation of α-synuclein.
  24. Machine-learning-based Structural Analysis of Interactions between Antibodies and Antigens.
  25. TRMT10A dysfunction perturbs codon translation of initiator methionine and glutamine and impairs brain functions in mice.
  26. Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor.
  1. bioRxiv. 2024 Jun 21. pii: 2024.06.20.598218. [Epub ahead of print]
    Localized K63 ubiquitin signaling is regulated by VCP/p97 during oxidative stress.
    Austin O Maduka, Sandhya Manohar, Matthew W Foster, Gustavo M Silva.
      Under stress conditions, cells reprogram their molecular machineries to mitigate damage and promote survival. Ubiquitin signaling is globally increased during oxidative stress, controlling protein fate and supporting stress defenses at several subcellular compartments. However, the rules driving subcellular ubiquitin localization to promote these concerted response mechanisms remain understudied. Here, we show that K63-linked ubiquitin chains, known to promote proteasome-independent pathways, accumulate primarily in non-cytosolic compartments during oxidative stress induced by sodium arsenite in mammalian cells. Our subcellular ubiquitin proteomic analyses of non-cytosolic compartments expanded 10-fold the pool of proteins known to be ubiquitinated during arsenite stress (2,046) and revealed their involvement in pathways related to immune signaling and translation control. Moreover, subcellular proteome analyses revealed proteins that are recruited to non-cytosolic compartments under stress, including a significant enrichment of helper ubiquitin-binding adaptors of the ATPase VCP that processes ubiquitinated substrates for downstream signaling. We further show that VCP recruitment to non-cytosolic compartments under arsenite stress occurs in a ubiquitin-dependent manner mediated by its adaptor NPLOC4. Additionally, we show that VCP and NPLOC4 activities are critical to sustain low levels of non-cytosolic K63-linked ubiquitin chains, supporting a cyclical model of ubiquitin conjugation and removal that is disrupted by cellular exposure to reactive oxygen species. This work deepens our understanding of the role of localized ubiquitin and VCP signaling in the basic mechanisms of stress response and highlights new pathways and molecular players that are essential to reshape the composition and function of the human subcellular proteome under dynamic environments.
    Keywords:  K63 ubiquitin; VCP; human cells; oxidative stress; subcellular localization
    DOI:  https://doi.org/10.1101/2024.06.20.598218
  2. Autophagy. 2024 Jul 03. 1-16
    An APEX2-based proximity-dependent biotinylation assay with temporal specificity to study protein interactions during autophagy in the yeast Saccharomyces cerevisiae.
    Yasmina Filali-Mouncef, Alexandre Leytens, Prado Vargas Duarte, Mattia Zampieri, Jörn Dengjel, Fulvio Reggiori.
      Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast Saccharomyces cerevisiae, one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism.Abbreviations: APEX2, ascorbate peroxidase 2, Atg, autophagy-related; BP, biotin phenol; Cvt, cytoplasm-to-vacuole targeting; ER, endoplasmic reticulum; LN2, liquid nitrogen; MS, mass spectrometry; PAS, phagophore assembly site; PL, proximity labeling; PE, phosphatidylethanolamine; PPINs, protein-protein interaction networks; PPIs, protein-protein interactions; RT, room temperature; SARs, selective autophagy receptors; WT, wild-type.
    Keywords:  Atg proteins; Atg8; Atg9; mass spectrometry; proteomics; proximity labeling
    DOI:  https://doi.org/10.1080/15548627.2024.2366749
  3. Trends Biochem Sci. 2024 Jun 29. pii: S0968-0004(24)00149-X. [Epub ahead of print]
    UPS-dependent strategies of protein quality control degradation.
    Leonie Müller, Thorsten Hoppe.
      The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.
    Keywords:  E3 ubiquitin ligase; degron; molecular chaperone; protein damage; proteostasis; ubiquitin–proteasome system (UPS)
    DOI:  https://doi.org/10.1016/j.tibs.2024.06.006
  4. Cell Rep. 2024 Jun 27. pii: S2211-1247(24)00730-7. [Epub ahead of print]43(7): 114402
    Translocational attenuation mediated by the PERK-SRP14 axis is a protective mechanism of unfolded protein response.
    Yaofu Liu, Yuexi Gu, Ying Chen, Xuan Wang, Guangfeng Zhou, Jing Li, Mu Wang, Shengyun Fang, Yili Yang.
      The unfolded protein response (UPR) relieves endoplasmic reticulum (ER) stress through multiple strategies, including reducing protein synthesis, increasing protein folding capabilities, and enhancing misfolded protein degradation. After a multi-omics analysis, we find that signal recognition particle 14 (SRP14), an essential component of the SRP, is markedly reduced in cells undergoing ER stress. Further experiments indicate that SRP14 reduction requires PRKR-like ER kinase (PERK)-mediated eukaryotic translation initiation factor 2α (eIF2α) phosphorylation but is independent of ATF4 or ATF3 transcription factors. The decrease of SRP14 correlates with reduced translocation of fusion proteins and endogenous cathepsin D. Enforced expression of an SRP14 variant with elongation arrest capability prevents the reduced translocation of cathepsin D in stressed cells, whereas an SRP14 mutant without the activity does not. Finally, overexpression of SRP14 augments the UPR and aggravates ER-stress-induced cell death. These data suggest that translocational attenuation mediated by the PERK-SRP14 axis is a protective measure for the UPR to mitigate ER stress.
    Keywords:  CP: Molecular biology; ER stress; PERK-eIF2α; SRP14; UPR; cathepsin D; signal peptide; signal recognition particle; translocational attenuation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114402
  5. Mol Cancer Ther. 2024 Jul 05. OF1-OF13
    Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.
    Gregory S Parker, Julia I Toth, Sarah Fish, Gabrielle Blanco, Taylor Kampert, Xiaoming Li, Linette Yang, Craig R Stumpf, Kenneth Steadman, Aleksandar Jamborcic, Stephen Chien, Elizabeth Daniele, Alejandro Dearie, Geoffray Leriche, Simon Bailey, Peggy A Thompson.
      Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-24-0219
  6. Elife. 2024 Jul 04. pii: e84798. [Epub ahead of print]13
    Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.
    Ya-Juan Wang, Xiao-Jing Di, Pei-Pei Zhang, Xi Chen, Marnie P Williams, Dong-Yun Han, Raad Nashmi, Brandon J Henderson, Fraser J Moss, Ting-Wei Mu.
      Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.
    Keywords:  biochemistry; cell biology; chemical biology; human; mouse
    DOI:  https://doi.org/10.7554/eLife.84798
  7. J Cell Biol. 2024 Aug 05. pii: e202404013. [Epub ahead of print]223(8):
    Tailored assemblies of COPII proteins in secretion.
    Vivek Malhotra.
      Export of secretory cargoes from the endoplasmic reticulum (ER) requires COPII proteins, which were first identified for their ability to coat small vesicles that bud from the ER. Recent data indicate that COPII proteins can also organize into a collar at the necks of tubules, as well as phase-separate into liquid-like condensates. Thus, COPII assemblies seem to be tailored to accommodate variations in the size and quantities of cargo secreted.
    DOI:  https://doi.org/10.1083/jcb.202404013
  8. ACS Chem Biol. 2024 Jul 03.
    Confounding Factors in Targeted Degradation of Short-Lived Proteins.
    Vesna Vetma, Laura Casares Perez, Ján Eliaš, Andrea Stingu, Anju Kombara, Teresa Gmaschitz, Nina Braun, Tuncay Ciftci, Georg Dahmann, Emelyne Diers, Thomas Gerstberger, Peter Greb, Giorgia Kidd, Christiane Kofink, Ilaria Puoti, Valentina Spiteri, Nicole Trainor, Harald Weinstabl, Yvonne Westermaier, Claire Whitworth, Alessio Ciulli, William Farnaby, Kirsten McAulay, Aileen B Frost, Nicola Chessum, Manfred Koegl.
      Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.
    DOI:  https://doi.org/10.1021/acschembio.4c00152
  9. bioRxiv. 2024 Jun 20. pii: 2024.06.19.599784. [Epub ahead of print]
    ER-GUARD: an evolutionarily conserved antioxidant defense system at ER membranes.
    Zhijian Ji, Taruna Pandey, Henry de Belly, Bingying Wang, Orion D Weiner, Yao Tang, Shouhong Guang, Thomas D Goddard, Dengke K Ma.
      Oxidative protein folding in the endoplasmic reticulum (ER) is essential for all eukaryotic cells yet generates hydrogen peroxide (H2O2), a reactive oxygen species (ROS). The ER-transmembrane protein that provides reducing equivalents to ER and guards the cytosol for antioxidant defense remains unidentified. Here we combine AlphaFold2-based and functional reporter screens in C. elegans to identify a previously uncharacterized and evolutionarily conserved protein ERGU-1 that fulfills these roles. Deleting C. elegans ERGU-1 causes excessive H2O2 and transcriptional gene up-regulation through SKN-1, homolog of mammalian antioxidant master regulator NRF2. ERGU-1 deficiency also impairs organismal reproduction and behaviors. Both C. elegans and human ERGU-1 proteins localize to ER membranes and form network reticulum structures. We name this system ER-GUARD, Endoplasmic Reticulum Guardian Aegis of Redox Defense. Human and Drosophila homologs of ERGU-1 can rescue C. elegans mutant phenotypes, demonstrating evolutionarily ancient and conserved functions. Together, our results reveal an ER-membrane-specific protein machinery and defense-net system ER-GUARD for peroxide detoxification and suggest a previously unknown but conserved pathway for antioxidant defense in animal cells.
    DOI:  https://doi.org/10.1101/2024.06.19.599784
  10. Nat Commun. 2024 Jul 02. 15(1): 5379
    Intrinsic signaling pathways modulate targeted protein degradation.
    Yuki Mori, Yoshino Akizuki, Rikuto Honda, Miyu Takao, Ayaka Tsuchimoto, Sota Hashimoto, Hiroaki Iio, Masakazu Kato, Ai Kaiho-Soma, Yasushi Saeki, Jun Hamazaki, Shigeo Murata, Toshikazu Ushijima, Naoko Hattori, Fumiaki Ohtake.
      Targeted protein degradation is a groundbreaking modality in drug discovery; however, the regulatory mechanisms are still not fully understood. Here, we identify cellular signaling pathways that modulate the targeted degradation of the anticancer target BRD4 and related neosubstrates BRD2/3 and CDK9 induced by CRL2VHL- or CRL4CRBN -based PROTACs. The chemicals identified as degradation enhancers include inhibitors of cellular signaling pathways such as poly-ADP ribosylation (PARG inhibitor PDD00017273), unfolded protein response (PERK inhibitor GSK2606414), and protein stabilization (HSP90 inhibitor luminespib). Mechanistically, PARG inhibition promotes TRIP12-mediated K29/K48-linked branched ubiquitylation of BRD4 by facilitating chromatin dissociation of BRD4 and formation of the BRD4-PROTAC-CRL2VHL ternary complex; by contrast, HSP90 inhibition promotes BRD4 degradation after the ubiquitylation step. Consequently, these signal inhibitors sensitize cells to the PROTAC-induced apoptosis. These results suggest that various cell-intrinsic signaling pathways spontaneously counteract chemically induced target degradation at multiple steps, which could be liberated by specific inhibitors.
    DOI:  https://doi.org/10.1038/s41467-024-49519-z
  11. Nat Chem Biol. 2024 Jul 04.
    Recruitment of FBXO22 for targeted degradation of NSD2.
    David Y Nie, John R Tabor, Jianping Li, Maria Kutera, Jonathan St-Germain, Ronan P Hanley, Esther Wolf, Ethan Paulakonis, Tristan M G Kenney, Shili Duan, Suman Shrestha, Dominic D G Owens, Matthew E R Maitland, Ailing Pon, Magdalena Szewczyk, Anthony Joseph Lamberto, Michael Menes, Fengling Li, Linda Z Penn, Dalia Barsyte-Lovejoy, Nicholas G Brown, Anthony M Barsotti, Andrew W Stamford, Jon L Collins, Derek J Wilson, Brian Raught, Jonathan D Licht, Lindsey I James, Cheryl H Arrowsmith.
      Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.
    DOI:  https://doi.org/10.1038/s41589-024-01660-y
  12. Nat Chem Biol. 2024 Jul 04.
    A CRISPR activation screen identifies FBXO22 supporting targeted protein degradation.
    Ananya A Basu, Chenlu Zhang, Isabella A Riha, Assa Magassa, Miguel A Campos, Alana G Caldwell, Felicia Ko, Xiaoyu Zhang.
      Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. Here we describe a clustered regularly interspaced short palindromic repeats (CRISPR)-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FK506-binding protein 12 when the transcription of FBXO22 gene is activated. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in F-box protein 22 (FBXO22) to achieve target degradation. Lastly, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading additional endogenous proteins, including bromodomain-containing protein 4 and the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase fusion protein.
    DOI:  https://doi.org/10.1038/s41589-024-01655-9
  13. Cell Rep. 2024 Jun 25. pii: S2211-1247(24)00685-5. [Epub ahead of print] 114357
    Endoplasmic reticulum morphology regulation by RTN4 modulates neuronal regeneration by curbing luminal transport.
    Tasuku Konno, Pierre Parutto, Cécile C Crapart, Valentina Davì, David M D Bailey, Mosab Ali Awadelkareem, Colin Hockings, Aidan I Brown, Katherine M Xiang, Anamika Agrawal, Joseph E Chambers, Molly J Vander Werp, Katherine M Koning, Louis Mounir Elfari, Sam Steen, Emmanouil Metzakopian, Laura M Westrate, Elena F Koslover, Edward Avezov.
      Cell functions rely on intracellular transport systems distributing bioactive molecules with high spatiotemporal accuracy. The endoplasmic reticulum (ER) tubular network constitutes a system for delivering luminal solutes, including Ca2+, across the cell periphery. How the ER structure enables this nanofluidic transport system is unclear. Here, we show that ER membrane-localized reticulon 4 (RTN4/Nogo) is sufficient to impose neurite outgrowth inhibition in human cortical neurons while acting as an ER morphoregulator. Improving ER transport visualization methodologies combined with optogenetic Ca2+ dynamics imaging and in silico modeling, we observed that ER luminal transport is modulated by ER tubule narrowing and dilation, proportional to the amount of RTN4. Excess RTN4 limited ER luminal transport and Ca2+ release, while RTN4 elimination reversed the effects. The described morphoregulatory effect of RTN4 defines the capacity of the ER for peripheral Ca2+ delivery for physiological releases and thus may constitute a mechanism for controlling the (re)generation of neurites.
    Keywords:  CP: Cell biology; CP: Neuroscience
    DOI:  https://doi.org/10.1016/j.celrep.2024.114357
  14. FEBS J. 2024 Jul 01.
    Dysregulated ribosome quality control in human diseases.
    Tom McGirr, Okan Onar, Seyed Mehdi Jafarnejad.
      Precise regulation of mRNA translation is of fundamental importance for maintaining homeostasis. Conversely, dysregulated general or transcript-specific translation, as well as abnormal translation events, have been linked to a multitude of diseases. However, driven by the misconception that the transient nature of mRNAs renders their abnormalities inconsequential, the importance of mechanisms that monitor the quality and fidelity of the translation process has been largely overlooked. In recent years, there has been a dramatic shift in this paradigm, evidenced by several seminal discoveries on the role of a key mechanism in monitoring the quality of mRNA translation - namely, Ribosome Quality Control (RQC) - in the maintenance of homeostasis and the prevention of diseases. Here, we will review recent advances in the field and emphasize the biological significance of the RQC mechanism, particularly its implications in human diseases.
    Keywords:  RQC; proteostasis; ribosome collisions; ribosome quality control; ribosome stalling
    DOI:  https://doi.org/10.1111/febs.17217
  15. bioRxiv. 2024 Jun 23. pii: 2024.06.22.600216. [Epub ahead of print]
    Premature aging in aneuploid yeast is caused in part by aneuploidy-induced defects in Ribosome Quality Control.
    Leah E Escalante, James Hose, Hollis Howe, Norah Paulsen, Michael Place, Audrey P Gasch.
      Premature aging is a hallmark of Down syndrome, caused by trisomy of human chromosome 21, but the reason is unclear and difficult to study in humans. We used an aneuploid model in wild yeast to show that chromosome amplification disrupts nutrient-induced cell-cycle arrest, quiescence entry, and healthy aging, across genetic backgrounds and amplified chromosomes. We discovered that these defects are due in part to aneuploidy-induced dysfunction in Ribosome Quality Control (RQC). Compared to euploids, aneuploids entering quiescence display aberrant ribosome profiles, accumulate RQC intermediates, and harbor an increased load of protein aggregates. Although they have normal proteasome capacity, aneuploids show signs of ubiquitin dysregulation, which impacts cyclin abundance to disrupt arrest. Remarkably, inducing ribosome stalling in euploids produces similar aberrations, while up-regulating limiting RQC subunits or proteins in ubiquitin metabolism alleviates many of the aneuploid defects. Our results provide implications for other aneuploidy disorders including Down syndrome.
    DOI:  https://doi.org/10.1101/2024.06.22.600216
  16. bioRxiv. 2024 Jun 20. pii: 2024.06.19.599770. [Epub ahead of print]
    Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.
    Shruti Kolli, Cassidy J Kline, Kimya M Rad, Ann M Wehman.
      Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we show that the LC3 ortholog LGG-2 is enriched in the polar body phagolysosome independent of membrane association or autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in C. elegans embryos. Determining how membrane association of Atg8/LC3 promotes the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.
    DOI:  https://doi.org/10.1101/2024.06.19.599770
  17. Dev Cell. 2024 Jun 27. pii: S1534-5807(24)00380-0. [Epub ahead of print]
    Next-generation Drosophila protein interactome map and its functional implications.
    Guruharsha Bhat, Kejie Li, George Locke, Marina Theodorou, Krishna Kilambi, Kazuya Hori, Diana Ho, Robert Obar, Leah Williams, Hannah Parzen, Noah Dephoure, Craig Braun, Marc Muskavitch, Susan E Celniker, Steven Gygi, Spyros Artavanis-Tsakonas.
      We describe a next-generation Drosophila protein interaction map-"DPIM2"-established from affinity purification-mass spectrometry of 5,805 baits, covering the largest fraction of the Drosophila proteome. The network contains 32,668 interactions among 3,644 proteins, organized into 632 clusters representing putative functional modules. Our analysis expands the pool of known protein interactions in Drosophila, provides annotation for poorly studied genes, and postulates previously undescribed protein interaction relationships. The predictive power and functional relevance of this network are probed through the lens of the Notch signaling pathway, and we find that newly identified members of complexes that include known Notch modifiers can also modulate Notch signaling. DPIM2 allows direct comparisons with a recently published human protein interaction network, defining the existence of functional interactions conserved across species. Thus, DPIM2 defines a valuable resource for predicting protein co-complex memberships and functional associations as well as generates functional hypotheses regarding specific protein interactions.
    Keywords:  affinity purification; interactome; interologs; modifiers; notch pathway; protein interaction; proteome
    DOI:  https://doi.org/10.1016/j.devcel.2024.06.002
  18. Subcell Biochem. 2024 ;104 485-501
    Valosin-Containing Protein (VCP)/p97 Oligomerization.
    Guimei Yu, Yunpeng Bai, Zhong-Yin Zhang.
      Valosin-containing protein (VCP), also known as p97, is an evolutionarily conserved AAA+ ATPase essential for cellular homeostasis. Cooperating with different sets of cofactors, VCP is involved in multiple cellular processes through either the ubiquitin-proteasome system (UPS) or the autophagy/lysosomal route. Pathogenic mutations frequently found at the interface between the NTD domain and D1 ATPase domain have been shown to cause malfunction of VCP, leading to degenerative disorders including the inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD), amyotrophic lateral sclerosis (ALS), and cancers. Therefore, VCP has been considered as a potential therapeutic target for neurodegeneration and cancer. Most of previous studies found VCP predominantly exists and functions as a hexamer, which unfolds and extracts ubiquitinated substrates from protein complexes for degradation. However, recent studies have characterized a new VCP dodecameric state and revealed a controlling mechanism of VCP oligomeric states mediated by the D2 domain nucleotide occupancy. Here, we summarize our recent knowledge on VCP oligomerization, regulation, and potential implications of VCP in cellular function and pathogenic progression.
    Keywords:  ALS; Cancer; Dodecamer; Hexamer; IBMPFD; Nucleotide binding; Oligomerization; Ubiquitination; VCP/p97
    DOI:  https://doi.org/10.1007/978-3-031-58843-3_18
  19. J Clin Invest. 2024 Jul 01. pii: e182204. [Epub ahead of print]134(13):
    A little ER stress isn't bad: the IRE1α/XBP1 pathway shapes ILC3 functions during intestinal inflammation.
    Cinzia Fionda, Giuseppe Sciumè.
      Type 3 innate lymphoid cells (ILC3s) are key regulators of intestinal homeostasis and epithelial barrier integrity. In this issue of the JCI, Cao and colleagues found that a sensor of endoplasmic reticulum (ER) stress, the inositol-requiring kinase 1α/X-box-binding protein 1 (IRE1α/XBP1) pathway, fine-tuned the functions of ILC3s. Activation of IRE1α and XBP1 in ILC3s limited intestinal inflammation in mice and correlated with the efficacy of ustekinumab, an IL-12/IL-23 blocker, in patients with Crohn's disease. These results advance our understanding in the use of ILCs as biomarkers not only to predict disease outcomes but also to indicate the response to biologicals in patients with inflammatory bowel disease.
    DOI:  https://doi.org/10.1172/JCI182204
  20. Life Sci Alliance. 2024 Sep;pii: e202402575. [Epub ahead of print]7(9):
    E3 ligases RNF43 and ZNRF3 display differential specificity for endocytosis of Frizzled receptors.
    Jeroen M Bugter, Peter van Kerkhof, Ingrid Jordens, Eline Janssen, Thi Tran Ngoc Minh, Daniel Iglesias van Montfort, Cara Jamieson, Madelon M Maurice.
      The transmembrane E3 ligases RNF43 and ZNRF3 perform key tumour suppressor roles by inducing endocytosis of members of the Frizzled (FZD) family, the primary receptors for WNT. Loss-of-function mutations in RNF43 and ZNRF3 mediate FZD stabilisation and a WNT-hypersensitive growth state in various cancer types. Strikingly, RNF43 and ZNRF3 mutations are differentially distributed across cancer types, raising questions about their functional redundancy. Here, we compare the efficacy of RNF43 and ZNRF3 of targeting different FZDs for endocytosis. We find that RNF43 preferentially down-regulates FZD1/FZD5/FZD7, whereas ZNRF3 displays a preference towards FZD6. We show that the RNF43 transmembrane domain (TMD) is a key molecular determinant for inducing FZD5 endocytosis. Furthermore, a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD5 down-regulation. We conclude that RNF43 and ZNRF3 preferentially down-regulate specific FZDs, in part by a TMD-dependent mechanism. In accordance, tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 cancer mutations in those tissues. Consequently, our data point to druggable vulnerabilities of specific FZD receptors in RNF43- or ZNRF3-mutant human cancers.
    DOI:  https://doi.org/10.26508/lsa.202402575
  21. bioRxiv. 2024 Jun 18. pii: 2024.06.17.599449. [Epub ahead of print]
    Single-nucleus proteomics identifies regulators of protein transport.
    Jason Derks, Tobias Jonson, Andrew Leduc, Saad Khan, Luke Khoury, Mahmoud-Reza Rafiee, Nikolai Slavov.
      The physiological response of a cell to stimulation depends on its proteome configuration. Therefore, the abundance variation of regulatory proteins across unstimulated single cells can be associatively linked with their response to stimulation. Here we developed an approach that leverages this association across individual cells and nuclei to systematically identify potential regulators of biological processes, followed by targeted validation. Specifically, we applied this approach to identify regulators of nucleocytoplasmic protein transport in macrophages stimulated with lipopolysaccharide (LPS). To this end, we quantified the proteomes of 3,412 individual nuclei, sampling the dynamic response to LPS treatment, and linking functional variability to proteomic variability. Minutes after the stimulation, the protein transport in individual nuclei correlated strongly with the abundance of known protein transport regulators, thus revealing the impact of natural protein variability on functional cellular response. We found that simple biophysical constraints, such as the quantity of nuclear pores, partially explain the variability in LPS-induced nucleocytoplasmic transport. Among the many proteins newly identified to be associated with the response, we selected 16 for targeted validation by knockdown. The knockdown phenotypes confirmed the inferences derived from natural protein and functional variation of single nuclei, thus demonstrating the potential of (sub-)single-cell proteomics to infer functional regulation. We expect this approach to generalize to broad applications and enhance the functional interpretability of single-cell omics data.
    DOI:  https://doi.org/10.1101/2024.06.17.599449
  22. Mol Syst Biol. 2024 Jul 01.
    A proximity proteomics pipeline with improved reproducibility and throughput.
    Xiaofang Zhong, Qiongyu Li, Benjamin J Polacco, Trupti Patil, Aaron Marley, Helene Foussard, Prachi Khare, Rasika Vartak, Jiewei Xu, Jeffrey F DiBerto, Bryan L Roth, Manon Eckhardt, Mark von Zastrow, Nevan J Krogan, Ruth Hüttenhain.
      Proximity labeling (PL) via biotinylation coupled with mass spectrometry (MS) captures spatial proteomes in cells. Large-scale processing requires a workflow minimizing hands-on time and enhancing quantitative reproducibility. We introduced a scalable PL pipeline integrating automated enrichment of biotinylated proteins in a 96-well plate format. Combining this with optimized quantitative MS based on data-independent acquisition (DIA), we increased sample throughput and improved protein identification and quantification reproducibility. We applied this pipeline to delineate subcellular proteomes across various compartments. Using the 5HT2A serotonin receptor as a model, we studied temporal changes of proximal interaction networks induced by receptor activation. In addition, we modified the pipeline for reduced sample input to accommodate CRISPR-based gene knockout, assessing dynamics of the 5HT2A network in response to perturbation of selected interactors. This PL approach is universally applicable to PL proteomics using biotinylation-based PL enzymes, enhancing throughput and reproducibility of standard protocols.
    Keywords:  APEX2-based Proximity Labeling; G Protein-Coupled Receptor; Protein–Protein Interaction; Proximity Proteomics; Subcellular Proteomics
    DOI:  https://doi.org/10.1038/s44320-024-00049-2
  23. Nat Cell Biol. 2024 Jul 01.
    VAMP2 regulates phase separation of α-synuclein.
    Aishwarya Agarwal, Aswathy Chandran, Farheen Raza, Irina-Maria Ungureanu, Christine Hilcenko, Katherine Stott, Nicholas A Bright, Nobuhiro Morone, Alan J Warren, Janin Lautenschläger.
      α-Synuclein (αSYN), a pivotal synaptic protein implicated in synucleinopathies such as Parkinson's disease and Lewy body dementia, undergoes protein phase separation. We reveal that vesicle-associated membrane protein 2 (VAMP2) orchestrates αSYN phase separation both in vitro and in cells. Electrostatic interactions, specifically mediated by VAMP2 via its juxtamembrane domain and the αSYN C-terminal region, drive phase separation. Condensate formation is specific for R-SNARE VAMP2 and dependent on αSYN lipid membrane binding. Our results delineate a regulatory mechanism for αSYN phase separation in cells. Furthermore, we show that αSYN condensates sequester vesicles and attract complexin-1 and -2, thus supporting a role in synaptic physiology and pathophysiology.
    DOI:  https://doi.org/10.1038/s41556-024-01451-6
  24. Biosystems. 2024 Jul 02. pii: S0303-2647(24)00149-7. [Epub ahead of print] 105264
    Machine-learning-based Structural Analysis of Interactions between Antibodies and Antigens.
    Grace Zhang, Xiaohan Kuang, Yuhao Zhang, Yunchao Liu, Zhaoqian Su, Tom Zhang, Yinghao Wu.
      Computational analysis of paratope-epitope interactions between antibodies and their corresponding antigens can facilitate our understanding of the molecular mechanism underlying humoral immunity and boost the design of new therapeutics for many diseases. The recent breakthrough in artificial intelligence has made it possible to predict protein-protein interactions and model their structures. Unfortunately, detecting antigen-binding sites associated with a specific antibody is still a challenging problem. To tackle this challenge, we implemented a deep learning model to characterize interaction patterns between antibodies and their corresponding antigens. With high accuracy, our model can distinguish between antibody-antigen complexes and other types of protein-protein complexes. More intriguingly, we can identify antigens from other common protein binding regions with an accuracy of higher than 70% even if we only have the epitope information. This indicates that antigens have distinct features on their surface that antibodies can recognize. Additionally, our model was unable to predict the partnerships between antibodies and their particular antigens. This result suggests that one antigen may be targeted by more than one antibody and that antibodies may bind to previously unidentified proteins. Taken together, our results support the precision of antibody-antigen interactions while also suggesting positive future progress in the prediction of specific pairing.
    Keywords:  Antibody-antigen complex; Deep learning; Paratope-epitope interactions; Structure-based modeling
    DOI:  https://doi.org/10.1016/j.biosystems.2024.105264
  25. Nucleic Acids Res. 2024 Jul 02. pii: gkae520. [Epub ahead of print]
    TRMT10A dysfunction perturbs codon translation of initiator methionine and glutamine and impairs brain functions in mice.
    Roland Tresky, Yuta Miyamoto, Yu Nagayoshi, Yasushi Yabuki, Kimi Araki, Yukie Takahashi, Yoshihiro Komohara, Huicong Ge, Kayo Nishiguchi, Takaichi Fukuda, Hitomi Kaneko, Nobuko Maeda, Jin Matsuura, Shintaro Iwasaki, Kourin Sakakida, Norifumi Shioda, Fan-Yan Wei, Kazuhito Tomizawa, Takeshi Chujo.
      In higher eukaryotes, tRNA methyltransferase 10A (TRMT10A) is responsible for N1-methylguanosine modification at position nine of various cytoplasmic tRNAs. Pathogenic mutations in TRMT10A cause intellectual disability, microcephaly, diabetes, and short stature in humans, and generate cytotoxic tRNA fragments in cultured cells; however, it is not clear how TRMT10A supports codon translation or brain functions. Here, we generated Trmt10a null mice and showed that tRNAGln(CUG) and initiator methionine tRNA levels were universally decreased in various tissues; the same was true in a human cell line lacking TRMT10A. Ribosome profiling of mouse brain revealed that dysfunction of TRMT10A causes ribosome slowdown at the Gln(CAG) codon and increases translation of Atf4 due to higher frequency of leaky scanning of its upstream open reading frames. Broadly speaking, translation of a subset of mRNAs, especially those for neuronal structures, is perturbed in the mutant brain. Despite not showing discernable defects in the pancreas, liver, or kidney, Trmt10a null mice showed lower body weight and smaller hippocampal postsynaptic densities, which is associated with defective synaptic plasticity and memory. Taken together, our study provides mechanistic insight into the roles of TRMT10A in the brain, and exemplifies the importance of universal tRNA modification during translation of specific codons.
    DOI:  https://doi.org/10.1093/nar/gkae520
  26. bioRxiv. 2024 Jun 20. pii: 2024.06.11.598518. [Epub ahead of print]
    Actinomycin D and bortezomib disrupt protein homeostasis in Wilms tumor.
    Patricia D B Tiburcio, Kenian Chen, Lin Xu, Kenneth S Chen.
      Wilms tumor is the most common kidney cancer in children, and diffusely anaplastic Wilms tumor is the most chemoresistant histological subtype. Here we explore how Wilms tumor cells evade the common chemotherapeutic drug actinomycin D, which inhibits ribosomal RNA biogenesis. Using ribosome profiling, protein arrays, and a genome-wide knockout screen, we describe how actinomycin D disrupts protein homeostasis and blocks cell cycle progression. We found that, when ribosomal capacity is limited by actinomycin D treatment, anaplastic Wilms tumor cells preferentially translate proteasome components and upregulate proteasome activity. Furthermore, the proteasome inhibitor bortezomib sensitizes cells to actinomycin D treatment by inducing apoptosis both in vitro and in vivo . Lastly, we show that increased levels of proteasome components are associated with anaplastic histology and with worse prognosis in non-anaplastic Wilms tumor. In sum, maintaining protein homeostasis is critical for Wilms tumor proliferation, and it can be therapeutically disrupted by blocking protein synthesis or turnover.
    DOI:  https://doi.org/10.1101/2024.06.11.598518
This page is being maintained by Thomas Krichel. It was last updated on 2024‒07‒07 at 5:57.