bims-proteo 2023-10-08 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023‒10‒08
29 papers selected by
Eric Chevet, INSERM

UFMylation of HRD1 regulates endoplasmic reticulum homeostasis.
Proteostasis is differentially modulated by inhibition of translation initiation or elongation.
Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy.
Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.
Proteomic approaches advancing targeted protein degradation.
S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.
De novo designed Hsp70 activator dissolves intracellular condensates.
Discovery of a Potent Deubiquitinase (DUB) Small Molecule Activity-based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.
Ubiquitin specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.
The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites.
Gcn5- and Bre1-mediated Set2 degradation promotes chronological aging of Saccharomyces cerevisiae.
The USP46 complex deubiquitylates LRP6 to promote Wnt/β-catenin signaling.
A highly efficient human cell-free translation system.
Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells.
Assigning functionality to cysteines by base editing of cancer dependency genes.
Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.
UBE3B promotes breast cancer progression by antagonizing HIF-2α degradation.
Characterizing genetic variation in the regulation of the ER stress response through computational and cis-eQTL analyses.
Site-Specific Crosslinking Reveals Phosphofructokinase-L Inhibition Drives Self-Assembly and Attenuation of Protein Interactions.
GCN2 is required to maintain core body temperature in mice during acute cold.
The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.
Physiological ER stress caused by amylase production induces regulated Ire1-dependent mRNA decay in Aspergillus oryzae.
cIAPs control RIPK1 kinase activity-dependent and -independent cell death and tissue inflammation.
Anticodon sequence determines the impact of mistranslating tRNAAla variants.
Single-cell mapping identifies MSI+ cells as a common origin for diverse subtypes of pancreatic cancer.
An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.
Loss of DDRGK1 impairs IRE1α UFMylation in spondyloepiphyseal dysplasia.
Spatom: a graph neural network for structure-based protein-protein interaction site prediction.
Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites.

FASEB J. 2023 Nov;37(11): e23221

UFMylation of HRD1 regulates endoplasmic reticulum homeostasis.

Hui Luo, Qi-Bin Jiao, Chuan-Bin Shen, Wen-Yan Gong, Jing-Hua Yuan, Ying-Ying Liu, Zhen Chen, Jiang Liu, Xiao-Ling Xu, Yu-Sheng Cong, Xing-Wei Zhang.

  Ubiquitin fold modifier 1 is a small ubiquitin-like protein modifier that is essential for embryonic development of metazoans. Although UFMylation has been connected to endoplasmic reticulum homeostasis, the underlying mechanisms and the relevant cellular targets are largely unknown. Here, we show that HRD1, a ubiquitin ligase of ER-associated protein degradation (ERAD), is a novel substrate of UFM1 conjugation. HRD1 interacts with UFMylation components UFL1 and DDRGK1 and is UFMylated at Lys610 residue. In UFL1-depleted cells, the stability of HRD1 is increased and its ubiquitination modification is reduced. In the event of ER stress, the UFMylation and ubiquitination modification of HRD1 is gradually inhibited over time. Alteration of HRD1 Lys610 residue to arginine impairs its ability to degrade unfolded or misfolded proteins to disturb protein processing in ER. These results suggest that UFMylation of HRD1 facilitates ERAD function to maintain ER homeostasis.

Keywords:  ER homeostasis; ER-associated protein degradation; HRD1; UFL1; UFMylation

DOI:  https://doi.org/10.1096/fj.202300004RRRR
Elife. 2023 Oct 05. pii: e76465. [Epub ahead of print]12

Proteostasis is differentially modulated by inhibition of translation initiation or elongation.

Khalyd J Clay, Yongzhi Yang, Christina Clark, Michael Petrascheck.

  Recent work has revealed an increasingly important role for mRNA translation in maintaining proteostasis. Here, we use chemical inhibitors targeting discreet steps of translation to compare how lowering the concentration of all or only translation initiation-dependent proteins rescues Caenorhabditis elegans from proteotoxic stress. We systematically challenge proteostasis and show that pharmacologically inhibiting translation initiation or elongation elicits a distinct protective profile. Inhibiting elongation protects from heat and proteasome dysfunction independently from HSF-1 but does not protect from age-associated protein aggregation. Conversely, inhibition of initiation protects from heat and age-associated protein aggregation and increases lifespan, dependent on hsf-1, but does not protect from proteotoxicity caused by proteasome dysfunction. Surprisingly, we find that the ability of the translation initiation machinery to control the concentration of newly synthesized proteins depends on HSF-1. Inhibition of translation initiation in wild-type animals reduces the concentration of newly synthesized proteins but increases it in hsf-1 mutants. Our findings suggest that the HSF-1 pathway is not only a downstream target of translation but also directly cooperates with the translation initiation machinery to control the concentration of newly synthesized proteins to restore proteostasis.

Keywords:  C. elegans; biochemistry; cell biology; chemical biology

DOI:  https://doi.org/10.7554/eLife.76465
J Cell Biol. 2023 Dec 04. pii: e202303078. [Epub ahead of print]222(12):

Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy.

Jake Cross, Joanne Durgan, David G McEwan, Matthew Tayler, Kevin M Ryan, Oliver Florey.

Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.

DOI: https://doi.org/10.1083/jcb.202303078
Cell Rep. 2023 Sep 25. pii: S2211-1247(23)01172-5. [Epub ahead of print] 113160

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Konstantinos Tsioras, Kevin C Smith, Seby L Edassery, Mehraveh Garjani, Yichen Li, Chloe Williams, Elizabeth D McKenna, Wenxuan Guo, Anika P Wilen, Timothy J Hark, Stefan L Marklund, Lyle W Ostrow, Jonathan D Gilthorpe, Justin K Ichida, Robert G Kalb, Jeffrey N Savas, Evangelos Kiskinis.

  Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Keywords:  ALS; CP: Neuroscience; CP: Stem cell research; SILAC-based mass spectrometry; SOD1; VCP/p97; amyotrophic lateral sclerosis; iPSCs; motor neurons; protein degradation; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2023.113160
Trends Pharmacol Sci. 2023 Sep 29. pii: S0165-6147(23)00177-3. [Epub ahead of print]

Proteomic approaches advancing targeted protein degradation.

Gajanan Sathe, Gopal P Sapkota.

  Targeted protein degradation (TPD) is an emerging modality for research and therapeutics. Most TPD approaches harness cellular ubiquitin-dependent proteolytic pathways. Proteolysis-targeting chimeras (PROTACs) and molecular glue (MG) degraders (MGDs) represent the most advanced TPD approaches, with some already used in clinical settings. Despite these advances, TPD still faces many challenges, pertaining to both the development of effective, selective, and tissue-penetrant degraders and understanding their mode of action. In this review, we focus on progress made in addressing these challenges. In particular, we discuss the utility and application of recent proteomic approaches as indispensable tools to enable insights into degrader development, including target engagement, degradation selectivity, efficacy, safety, and mode of action.

Keywords:  PROTAC; chemoproteomics; molecular glue; proteomics; targeted protein degradation

DOI:  https://doi.org/10.1016/j.tips.2023.08.007
Mol Cell. 2023 Oct 05. pii: S1097-2765(23)00699-8. [Epub ahead of print]83(19): 3485-3501.e11

S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.

Xue Huang, Jia Yao, Lu Liu, Jing Chen, Ligang Mei, Jingjing Huangfu, Dong Luo, Xinyi Wang, Changhai Lin, Xiaorong Chen, Yi Yang, Sheng Ouyang, Fujing Wei, Zhuolin Wang, Shaolin Zhang, Tingxiu Xiang, Dante Neculai, Qiming Sun, Eryan Kong, Edward W Tate, Aimin Yang.

  p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Keywords:  APT1; S-acylation; ZDHHC19; autophagy; autophagy receptor; liquid-liquid phase separation; p62 droplet; p62 protein; protein posttranslational modification; selective autophagy

DOI:  https://doi.org/10.1016/j.molcel.2023.09.004
bioRxiv. 2023 Sep 19. pii: 2023.09.18.558356. [Epub ahead of print]

De novo designed Hsp70 activator dissolves intracellular condensates.

Jason Z Zhang, Nathan Greenwood, Jason Hernandez, Josh T Cuperus, Buwei Huang, Bryan D Ryder, Christine Queitsch, Jason E Gestwicki, David Baker.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70's ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro , similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

DOI: https://doi.org/10.1101/2023.09.18.558356
Angew Chem Int Ed Engl. 2023 Oct 01. e202311190

Discovery of a Potent Deubiquitinase (DUB) Small Molecule Activity-based Probe Enables Broad Spectrum DUB Activity Profiling in Living Cells.

Dan Conole, Fangyuan Cao, Christopher W Am Ende, Liang Xue, Sheila Kantesaria, Dahye Kang, Jun Jin, Dafydd Owen, Linda Lohr, Monica Schenone, Jaimeen D Majmudar, Edward William Tate.

  Deubiquitinases (DUBs) are a family of >100 proteases that hydrolyze isopeptide bonds linking ubiquitin to protein substrates. This leads to reduced substrate degradation through the ubiquitin proteasome system. Deregulation of DUB activity has been implicated in many diseases, including cancer, neurodegeneration and auto-inflammation, and several have been recognized as attractive targets for therapeutic intervention. Ubiquitin-derived covalent activity-based probes (ABPs) provide a powerful tool for DUB activity profiling, but their large recognition element impedes cellular permeability and presents an unmet need for small molecule ABPs which can account for regulation of DUB activity in intact cells or organisms. Here, through comprehensive chemoproteomic warhead profiling, we identify cyanopyrrolidine (CNPy) probe IMP-2373 (12) as a small molecule pan-DUB ABP to monitor DUB activity in physiologically relevant live cells. Through proteomics and targeted assays, we demonstrate that IMP-2373 quantitatively engages more than 35 DUBs across a range of non-toxic concentrations in diverse cell lines. We further demonstrate its application to quantification of changes in intracellular DUB activity during pharmacological inhibition and during MYC deregulation in a model of B cell lymphoma. IMP-2373 thus offers a complementary tool to ubiquitin ABPs to monitor dynamic DUB activity in the context of disease-relevant phenotypes.

Keywords:  Deubiquitinase, ubiquitin activity-based probe, chemical probe, electrophilic warhead, MYC-deregulated cancer

DOI:  https://doi.org/10.1002/anie.202311190
J Biol Chem. 2023 Sep 28. pii: S0021-9258(23)02328-1. [Epub ahead of print] 105300

Ubiquitin specific protease 11 structure in complex with an engineered substrate mimetic reveals a molecular feature for deubiquitination selectivity.

Sigrun K Maurer, Matthias P Mayer, Stephanie J Ward, Sana Boudjema, Mohamed Halawa, Jiatong Zhang, Simon G Caulton, Jonas Emsley, Ingrid Dreveny.

  Ubiquitin specific proteases (USPs) are crucial for controlling cellular proteostasis and signaling pathways but how deubiquitination is selective remains poorly understood, in particular between paralogues. Here, we developed a fusion tag method by mining the Protein Data Bank and trapped USP11, a key regulator of DNA double-strand break repair, in complex with a novel engineered substrate mimetic. Together, this enabled structure determination of USP11 as a Michaelis-like complex that revealed key S1 and S1' binding site interactions with a substrate. Combined mutational, enzymatic, and binding experiments identified Met77 in linear di-ubiquitin as a significant residue that leads to substrate discrimination. We identified an aspartate 'gatekeeper' residue in the S1' site of USP11 as a contributing feature for discriminating against linear di-ubiquitin. When mutated to a glycine, the corresponding residue in paralogue USP15, USP11 acquired elevated activity towards linear di-ubiquitin in gel shift assays, but not controls. The reverse mutation in USP15 confirmed that this position confers paralogue-specific differences impacting di-ubiquitin cleavage rates. The results advance our understanding of the molecular basis for the higher selectivity of USP11 compared to USP15 and may aid targeted inhibitor development. Moreover, the reported carrier-based crystallization strategy may be applicable to other challenging targets.

Keywords:  crystal structure; cysteine protease; deubiquitylation (deubiquitination); protease; selectivity; ubiquitin; ubiquitin specific protease

DOI:  https://doi.org/10.1016/j.jbc.2023.105300
bioRxiv. 2023 Sep 18. pii: 2023.09.18.557036. [Epub ahead of print]

The sterol transporter STARD3 transports sphingosine at ER-lysosome contact sites.

Pia Hempelmann, Fabio Lolicato, Andrea Graziadei, Ryan D R Brown, Sarah Spiegel, Juri Rappsilber, Walter Nickel, Doris Höglinger, Denisa Jamecna.

Sphingolipids are important structural components of membranes. Additionally, simple sphingolipids such as sphingosine are highly bioactive and participate in complex subcellular signaling. Sphingolipid deregulation is associated with many severe diseases including diabetes, Parkinson's and cancer. Here, we focus on how sphingosine, generated from sphingolipid catabolism in late endosomes/lysosomes, is reintegrated into the biosynthetic machinery at the endoplasmic reticulum (ER). We characterized the sterol transporter STARD3 as a sphingosine transporter acting at lysosome-ER contact sites. Experiments featuring crosslinkable sphingosine probes, supported by unbiased molecular dynamics simulations, exposed how sphingosine binds to the lipid-binding domain of STARD3. Following the metabolic fate of pre-localized lysosomal sphingosine showed the importance of STARD3 and its actions at contact sites for the integration of sphingosine into ceramide in a cellular context. Our findings provide the first example of inter-organellar sphingosine transfer and pave the way for a better understanding of sphingolipid - sterol co-regulation.

DOI: https://doi.org/10.1101/2023.09.18.557036
Cell Rep. 2023 Oct 03. pii: S2211-1247(23)01198-1. [Epub ahead of print]42(10): 113186

Gcn5- and Bre1-mediated Set2 degradation promotes chronological aging of Saccharomyces cerevisiae.

Yu-Min Li, Yu-Chao Mei, Ao-Hui Liu, Ru-Xin Wang, Runfa Chen, Hai-Ning Du.

  Loss of transcription-coupled histone H3 lysine 36 trimethylation (H3K36me3) contributes to shorter lifespans in eukaryotes. However, the molecular mechanism of the decline of H3K36me3 during aging remains poorly understood. Here, we report that the degradation of the methyltransferase Set2 is the cause of decreased H3K36me3 levels during chronological aging in budding yeast. We show that Set2 protein degradation during cellular senescence and chronological aging is mainly mediated by the ubiquitin-conjugating E2 enzyme Ubc3 and the E3 ligase Bre1. Lack of Bre1 or abolishment of the ubiquitination stabilizes Set2 protein, sustains H3K36me3 levels at the aging-related gene loci, and upregulates their gene expression, thus leading to extended chronological lifespan. We further illustrate that Gcn5-mediated Set2 acetylation is a prerequisite for Bre1-catalyzed Set2 polyubiquitination and proteolysis during aging. We propose that two sequential post-translational modifications regulate Set2 homeostasis, suggesting a potential strategy to target the Gcn5-Bre1-Set2 axis for intervention of longevity.

Keywords:  Bre1; CP: Molecular biology; Gcn5; H3K36 methylation; Set2; budding yeast; chronological aging; cryptic transcripts; modifications

DOI:  https://doi.org/10.1016/j.celrep.2023.113186
Nat Commun. 2023 Oct 05. 14(1): 6173

The USP46 complex deubiquitylates LRP6 to promote Wnt/β-catenin signaling.

Victoria H Ng, Zachary Spencer, Leif R Neitzel, Anmada Nayak, Matthew A Loberg, Chen Shen, Sara N Kassel, Heather K Kroh, Zhenyi An, Christin C Anthony, Jamal M Bryant, Amanda Lawson, Lily Goldsmith, Hassina Benchabane, Amanda G Hansen, Jingjing Li, Starina D'Souza, Andres M Lebensohn, Rajat Rohatgi, William A Weiss, Vivian L Weiss, Charles Williams, Charles C Hong, David J Robbins, Yashi Ahmed, Ethan Lee.

The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.

DOI: https://doi.org/10.1038/s41467-023-41836-z
RNA. 2023 Oct 04. pii: rna.079825.123. [Epub ahead of print]

A highly efficient human cell-free translation system.

Nikolay Aleksashin, Stacey Tsai-Lan Chang, Jamie Cate.

  Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation significantly simplifies lysate preparation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.

Keywords:  cell-free translation system; eEF2; eIF2; protein synthesis; ribosome

DOI:  https://doi.org/10.1261/rna.079825.123
Nat Methods. 2023 Oct 02.

Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells.

Lucas Ferguson, Heather E Upton, Sydney C Pimentel, Amanda Mok, Liana F Lareau, Kathleen Collins, Nicholas T Ingolia.

Ribosome profiling has unveiled diverse regulation and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected messenger RNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with ordered two-template relay, a single-tube protocol for sequencing library preparation that incorporates adaptors by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling and termination. Our optimized methods for mRNA footprint generation and capture provide a richer translatome profile with low input and fewer technical challenges.

DOI: https://doi.org/10.1038/s41592-023-02028-1
Nat Chem Biol. 2023 Oct 02.

Assigning functionality to cysteines by base editing of cancer dependency genes.

Haoxin Li, Tiantai Ma, Jarrett R Remsberg, Sang Joon Won, Kristen E DeMeester, Evert Njomen, Daisuke Ogasawara, Kevin T Zhao, Tony P Huang, Bingwen Lu, Gabriel M Simon, Bruno Melillo, Stuart L Schreiber, Jens Lykke-Andersen, David R Liu, Benjamin F Cravatt.

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential.

DOI: https://doi.org/10.1038/s41589-023-01428-w
Mol Cell. 2023 Sep 29. pii: S1097-2765(23)00734-7. [Epub ahead of print]

Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

Meiling Wang, Wenjing Li, Nozomi Tomimatsu, Corey H Yu, Jae-Hoon Ji, Salvador Alejo, Samuel R Witus, Dauren Alimbetov, O'Taveon Fitzgerald, Bo Wu, Qijing Wang, Yuxin Huang, Yaqi Gan, Felix Dong, Youngho Kwon, Gangadhara R Sareddy, Tyler J Curiel, Amyn A Habib, Robert Hromas, Carolina Dos Santos Passos, Tingting Yao, Dmitri N Ivanov, Peter S Brzovic, Sandeep Burma, Rachel E Klevit, Weixing Zhao.

  The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

Keywords:  BARD1; BRCA1; DNA damage response; DNA end resection; HDR; histone; homology-directed repair; later stages for HDR completion; substrates; tumor suppression; ubiquitin E3 ligase

DOI:  https://doi.org/10.1016/j.molcel.2023.09.015
Oncogene. 2023 Oct 02.

UBE3B promotes breast cancer progression by antagonizing HIF-2α degradation.

Yijie Wang, Xiong Liu, Min Wang, Yu Wang, Shuo Wang, Lai Jin, Min Liu, Jun Zhou, Yan Chen.

Mutations in E3 ubiquitin ligase UBE3B have been linked to Kaufman Oculocerebrofacial Syndrome (KOS). Accumulating evidence indicates that UBE3B may play an important role in cancer. However, the precise role of UBE3B in cancer and the underlying mechanism remain largely uncharted. Here, we reported that UBE3B is an E3 ligase for hypoxia-inducible factor 2α (HIF-2α). Mechanically, UBE3B physically interacts with HIF-2α and promotes its lysine 63 (K63)-linked polyubiquitination, thereby inhibiting the Von Hippel-Lindau (VHL) E3 ligase complex-mediated HIF-2α degradation. UBE3B depletion inhibits breast cancer cell proliferation, colony formation, migration, and invasion in vitro and suppresses breast tumor growth and lung metastasis in vivo. We further identified K394, K497, and K503 of HIF-2α as key ubiquitination sites for UBE3B. K394/497/503R mutation of HIF-2α dramatically abolishes UBE3B-mediated breast cancer growth and lung metastasis. Intriguingly, the protein levels of UBE3B are upregulated and positively correlated with HIF-2α protein levels in breast cancer tissues. These findings uncover a critical mechanism underlying the role of UBE3B in HIF-2α regulation and breast cancer progression.

DOI: https://doi.org/10.1038/s41388-023-02842-z
G3 (Bethesda). 2023 Oct 04. pii: jkad229. [Epub ahead of print]

Characterizing genetic variation in the regulation of the ER stress response through computational and cis-eQTL analyses.

Nikki D Russell, Lynn B Jorde, Clement Y Chow.

  Misfolded proteins in the endoplasmic reticulum (ER) elicit the ER stress response, a large transcriptional response driven by three well-characterized transcription factors. This transcriptional response is variable across different genetic backgrounds. One mechanism in which genetic variation can lead to transcriptional variability in the ER stress response is through altered binding and activity of the three main transcription factors: XBP1, ATF6, and ATF4. This work attempts to better understand this mechanism by first creating a computational pipeline to identify potential binding sites throughout the human genome. We utilized GTEx datasets to identify cis- eQTLs that fall within predicted transcription factor binding sites (TFBSs). We also utilized the ClinVar database to compare the number of pathogenic versus benign variants at different positions of the binding motifs. Finally, we performed a cis- eQTL analysis on human cell lines experiencing ER stress to identify cis- eQTLs that regulate the variable ER stress response. The majority of these cis- eQTLs are unique to a given condition: control or ER stress. Some of these stress-specific cis- eQTLs fall within putative binding sites of the three main ER stress response transcription factors, providing a potential mechanism by which these cis- eQTLs might be impacting gene expression under ER stress conditions through altered TF binding. This study represents the first cis- eQTL analysis on human samples experiencing ER stress and is a vital step towards identifying the genetic components responsible for the variable ER stress response.

Keywords:   cis- eQTL; ER stress; genetic resource; genetic variation; transcriptional variability

DOI:  https://doi.org/10.1093/g3journal/jkad229
bioRxiv. 2023 Sep 20. pii: 2023.09.19.558525. [Epub ahead of print]

Site-Specific Crosslinking Reveals Phosphofructokinase-L Inhibition Drives Self-Assembly and Attenuation of Protein Interactions.

Athira Sivadas, Eli Fritz McDonald, Sydney O Shuster, Caitlin M Davis, Lars Plate.

Phosphofructokinase is the central enzyme in glycolysis and constitutes a highly regulated step. The liver isoform (PFKL) compartmentalizes during activation and inhibition in vitro and in vivo respectively. Compartmentalized PFKL is hypothesized to modulate metabolic flux consistent with its central role as the rate limiting step in glycolysis. PFKL tetramers self-assemble at two interfaces in the monomer (interface 1 and 2), yet how these interfaces contribute to PFKL compartmentalization and drive protein interactions remains unclear. Here, we used site-specific incorporation of noncanonical photocrosslinking amino acids to identify PFKL interactors at interface 1, 2, and the active site. Tandem mass tag-based quantitative interactomics reveals interface 2 as a hotspot for PFKL interactions, particularly with cytoskeletal, glycolytic, and carbohydrate derivative metabolic proteins. Furthermore, PFKL compartmentalization into puncta was observed in human cells using citrate inhibition. Puncta formation attenuated crosslinked protein-protein interactions with the cytoskeleton at interface 2. This result suggests that PFKL compartmentalization sequesters interface 2, but not interface 1, and may modulate associated protein assemblies with the cytoskeleton.

DOI: https://doi.org/10.1101/2023.09.19.558525
Am J Physiol Endocrinol Metab. 2023 Oct 04.

GCN2 is required to maintain core body temperature in mice during acute cold.

Jordan L Levy, Emily T Mirek, Esther M Rodriguez, Brian Zalma, Jeffrey Burns, William O Jonsson, Harini Sampath, Kirk A Staschke, Ronald C Wek, Tracy G Anthony.

  Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. GCN2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, GCN2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.

Keywords:  Activating Transcription Factor 4 (ATF4); Energy expenditure; Eukaryotic initiation factor 2 (eIF2); Hypothermia; Mechanistic target of rapamycin complex 1 (mTORC1)

DOI:  https://doi.org/10.1152/ajpendo.00181.2023
J Cell Biol. 2023 Dec 04. pii: e202204048. [Epub ahead of print]222(12):

The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.

Takayuki Shima, Monami Ogura, Ruriko Matsuda, Shuhei Nakamura, Natsuko Jin, Tamotsu Yoshimori, Akiko Kuma.

Membrane rupture of lysosomes results in leakage of their contents, which is harmful to cells. Recent studies have reported that several systems contribute to the repair or elimination of damaged lysosomes. Lysophagy is a type of selective autophagy that plays a crucial role in the lysosomal damage response. Because multiple pathways are involved in this response, an assay that specifically evaluates lysophagy is needed. Here, we developed the TMEM192-mKeima probe to evaluate lysophagy. By comparing the use of this probe with the conventional galectin-3 assay, we showed that this probe is more specific to lysophagy. Using TMEM192-mKeima, we showed that TFEB and p62 are important for the lysosomal damage response but not for lysophagy, although they have previously been considered to be involved in lysophagy. We further investigated the initial steps in lysophagy and identified UBE2L3, UBE2N, TRIM10, 16, and 27 as factors involved in it. Our results demonstrate that the TMEM192-mKeima probe is a useful tool for investigating lysophagy.

DOI: https://doi.org/10.1083/jcb.202204048
Commun Biol. 2023 Oct 04. 6(1): 1009

Physiological ER stress caused by amylase production induces regulated Ire1-dependent mRNA decay in Aspergillus oryzae.

Mizuki Tanaka, Silai Zhang, Shun Sato, Jun-Ichi Yokota, Yuko Sugiyama, Yasuaki Kawarasaki, Youhei Yamagata, Katsuya Gomi, Takahiro Shintani.

Regulated Ire1-dependent decay (RIDD) is a feedback mechanism in which the endoribonuclease Ire1 cleaves endoplasmic reticulum (ER)-localized mRNAs encoding secretory and membrane proteins in eukaryotic cells under ER stress. RIDD is artificially induced by chemicals that generate ER stress; however, its importance under physiological conditions remains unclear. Here, we demonstrate the occurrence of RIDD in filamentous fungus using Aspergillus oryzae as a model, which secretes copious amounts of amylases. α-Amylase mRNA was rapidly degraded by IreA, an Ire1 ortholog, depending on its ER-associated translation when mycelia were treated with dithiothreitol, an ER-stress inducer. The mRNA encoding maltose permease MalP, a prerequisite for the induction of amylolytic genes, was also identified as an RIDD target. Importantly, RIDD of malP mRNA is triggered by inducing amylase production without any artificial ER stress inducer. Our data provide the evidence that RIDD occurs in eukaryotic microorganisms under physiological ER stress.

DOI: https://doi.org/10.1038/s42003-023-05386-w
EMBO J. 2023 Oct 04. e113614

cIAPs control RIPK1 kinase activity-dependent and -independent cell death and tissue inflammation.

Fabian Schorn, J Paul Werthenbach, Mattes Hoffmann, Mila Daoud, Johanna Stachelscheid, Lars M Schiffmann, Ximena Hildebrandt, Su Ir Lyu, Nieves Peltzer, Alexander Quaas, Domagoj Vucic, John Silke, Manolis Pasparakis, Hamid Kashkar.

  Cellular inhibitor of apoptosis proteins (cIAPs) are RING-containing E3 ubiquitin ligases that ubiquitylate receptor-interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR ). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1-mediated apoptosis. While expression of kinase-inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N /cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF-induced apoptosis and necroptosis, implying additional kinase-independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock-out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1-mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF-signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1.

Keywords:  RIPK1; TNF; cIAP1; cIAP2; ubiquitin

DOI:  https://doi.org/10.15252/embj.2023113614
RNA Biol. 2023 Jan;20(1): 791-804

Anticodon sequence determines the impact of mistranslating tRNAAla variants.

Ecaterina Cozma, Megha Rao, Madison Dusick, Julie Genereaux, Ricard A Rodriguez-Mias, Judit Villén, Christopher J Brandl, Matthew D Berg.

  Transfer RNAs (tRNAs) maintain translation fidelity through accurate charging by their cognate aminoacyl-tRNA synthetase and codon:anticodon base pairing with the mRNA at the ribosome. Mistranslation occurs when an amino acid not specified by the genetic message is incorporated into proteins and has applications in biotechnology, therapeutics and is relevant to disease. Since the alanyl-tRNA synthetase uniquely recognizes a G3:U70 base pair in tRNAAla and the anticodon plays no role in charging, tRNAAla variants with anticodon mutations have the potential to mis-incorporate alanine. Here, we characterize the impact of the 60 non-alanine tRNAAla anticodon variants on the growth of Saccharomyces cerevisiae. Overall, 36 tRNAAla anticodon variants decreased growth in single- or multi-copy. Mass spectrometry analysis of the cellular proteome revealed that 52 of 57 anticodon variants, not decoding alanine or stop codons, induced mistranslation when on single-copy plasmids. Variants with G/C-rich anticodons resulted in larger growth deficits than A/U-rich variants. In most instances, synonymous anticodon variants impact growth differently, with anticodons containing U at base 34 being the least impactful. For anticodons generating the same amino acid substitution, reduced growth generally correlated with the abundance of detected mistranslation events. Differences in decoding specificity, even between synonymous anticodons, resulted in each tRNAAla variant mistranslating unique sets of peptides and proteins. We suggest that these differences in decoding specificity are also important in determining the impact of tRNAAla anticodon variants.

Keywords:  Mistranslation; expanded decoding; genetic code; tRNA biology; tRNAAla

DOI:  https://doi.org/10.1080/15476286.2023.2257471
Cancer Cell. 2023 Sep 25. pii: S1535-6108(23)00324-0. [Epub ahead of print]

Single-cell mapping identifies MSI+ cells as a common origin for diverse subtypes of pancreatic cancer.

Nirakar Rajbhandari, Michael Hamilton, Cynthia M Quintero, L Paige Ferguson, Raymond Fox, Christian M Schürch, Jun Wang, Mari Nakamura, Nikki K Lytle, Matthew McDermott, Emily Diaz, Hannah Pettit, Marcie Kritzik, Haiyong Han, Derek Cridebring, Kwun Wah Wen, Susan Tsai, Michael G Goggins, Andrew M Lowy, Robert J Wechsler-Reya, Daniel D Von Hoff, Aaron M Newman, Tannishtha Reya.

  Identifying the cells from which cancers arise is critical for understanding the molecular underpinnings of tumor evolution. To determine whether stem/progenitor cells can serve as cells of origin, we created a Msi2-CreERT2 knock-in mouse. When crossed to CAG-LSL-MycT58A mice, Msi2-CreERT2 mice developed multiple pancreatic cancer subtypes: ductal, acinar, adenosquamous, and rare anaplastic tumors. Combining single-cell genomics with computational analysis of developmental states and lineage trajectories, we demonstrate that MYC preferentially triggers transformation of the most immature MSI2+ pancreas cells into multi-lineage pre-cancer cells. These pre-cancer cells subsequently diverge to establish pancreatic cancer subtypes by activating distinct transcriptional programs and large-scale genomic changes, and enforced expression of specific signals like Ras can redirect subtype specification. This study shows that multiple pancreatic cancer subtypes can arise from a common pool of MSI2+ cells and provides a powerful model to understand and control the programs that shape divergent fates in pancreatic cancer.

Keywords:  Musashi; Myc; acinar cell carcinoma; adenosquamous carcinoma; cancer; cell of origin stem cells; pancreatic cancer; single cell; tumor evolution

DOI:  https://doi.org/10.1016/j.ccell.2023.09.008
Nat Cell Biol. 2023 Oct 02.

An ESCRT grommet cooperates with a diffusion barrier to maintain nuclear integrity.

Nicholas R Ader, Linda Chen, Ivan V Surovtsev, William L Chadwick, Elisa C Rodriguez, Megan C King, C Patrick Lusk.

The molecular mechanisms by which the endosomal sorting complexes required for transport (ESCRT) proteins contribute to the integrity of the nuclear envelope (NE) barrier are not fully defined. We leveraged the single NE hole generated by mitotic extrusion of the Schizosaccharomyces pombe spindle pole body to reveal two modes of ESCRT function executed by distinct complements of ESCRT-III proteins, both dependent on CHMP7/Cmp7. A grommet-like function is required to restrict the NE hole in anaphase B, whereas replacement of Cmp7 by a sealing module ultimately closes the NE in interphase. Without Cmp7, nucleocytoplasmic compartmentalization remains intact despite NE discontinuities of up to 540 nm, suggesting mechanisms to limit diffusion through these holes. We implicate spindle pole body proteins as key components of a diffusion barrier acting with Cmp7 in anaphase B. Thus, NE remodelling mechanisms cooperate with proteinaceous diffusion barriers beyond nuclear pore complexes to maintain the nuclear compartment.

DOI: https://doi.org/10.1038/s41556-023-01235-4
Int J Biol Sci. 2023 ;19(15): 4709-4725

Loss of DDRGK1 impairs IRE1α UFMylation in spondyloepiphyseal dysplasia.

Xiao Yang, Tangjun Zhou, Xin Wang, Ying Xia, Xiankun Cao, Xiaofei Cheng, Yu Cao, Peixiang Ma, Hui Ma, An Qin, Jie Zhao.

  Spondyloepiphyseal dysplasia (SEMD) is a rare disease in which cartilage growth is disrupted, and the DDRGK1 mutation is one of the causative genes. In our study, we established Ddrgk1fl/fl, Col2a1-ERT Cre mice, which showed a thickened hypertrophic zone (HZ) in the growth plate, simulating the previous reported SEMD pathology in vivo. Instead of the classical modulation mechanism towards SOX9, our further mechanism study found that DDRGK1 stabilizes the stress sensor endoplasmic reticulum-to-nucleus signaling 1 (IRE1α) to maintain endoplasmic reticulum (ER) homoeostasis. The loss of DDRGK1 decreased the UFMylation and subsequently led to increased ubiquitylation-mediated IRE1α degradation, causing ER dysfunction and activating the PERK/CHOP/Caspase3 apoptosis pathway. Further DDRGK1 K268R-mutant mice revealed the importance of K268 UFMylation site in IRE1α degradation and subsequent ER dysfunction. In conclusion, DDRGK1 stabilizes IRE1α to ameliorate ER stress and following apoptosis in chondrocytes, which finally promote the normal chondrogenesis.

Keywords:  DDRGK1; IRE1α; apoptosis; endoplasmic reticulum stress; spondyloepiphyseal dysplasia

DOI:  https://doi.org/10.7150/ijbs.82765
Brief Bioinform. 2023 Sep 22. pii: bbad345. [Epub ahead of print]24(6):

Spatom: a graph neural network for structure-based protein-protein interaction site prediction.

Haonan Wu, Jiyun Han, Shizhuo Zhang, Gaojia Xin, Chaozhou Mou, Juntao Liu.

  Accurate identification of protein-protein interaction (PPI) sites remains a computational challenge. We propose Spatom, a novel framework for PPI site prediction. This framework first defines a weighted digraph for a protein structure to precisely characterize the spatial contacts of residues, then performs a weighted digraph convolution to aggregate both spatial local and global information and finally adds an improved graph attention layer to drive the predicted sites to form more continuous region(s). Spatom was tested on a diverse set of challenging protein-protein complexes and demonstrated the best performance among all the compared methods. Furthermore, when tested on multiple popular proteins in a case study, Spatom clearly identifies the interaction interfaces and captures the majority of hotspots. Spatom is expected to contribute to the understanding of protein interactions and drug designs targeting protein binding.

Keywords:  continuous region; improved graph attention; protein–protein interaction; weighted digraph convolution

DOI:  https://doi.org/10.1093/bib/bbad345
Commun Biol. 2023 Oct 04. 6(1): 1008

Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites.

Nina Criado Santos, Samuel Bouvet, Maria Cruz Cobo, Marion Mandavit, Flavien Bermont, Cyril Castelbou, Farah Mansour, Maral Azam, Francesca Giordano, Paula Nunes-Hasler.

Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

DOI: https://doi.org/10.1038/s42003-023-05382-0