bims-proteo 2023-06-04 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–06–04
29 papers selected by
Eric Chevet, INSERM

Structural basis of ubiquitin-independent PP1 complex disassembly by p97.
The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes.
The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins.
Aggrephagy at a glance.
ER-to-Lysosome Associated Degradation (ERLAD) in a Nutshell: mammalian, yeast and plant ER-phagy as induced by misfolded proteins.
Bone and the Unfolded Protein Response: In Sickness and in Health.
A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response.
Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.
Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.
eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity.
Enzyme-Mediated Proximity Labeling Identifies Small RNAs in the Endoplasmic Reticulum Lumen.
Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.
The Scap-SREBP1-S1P/S2P lipogenesis signal orchestrates the homeostasis and spatiotemporal activation of NF-κB.
Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins.
LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.
Rational Chemical Design of Molecular Glue Degraders.
PhosY-secretome profiling combined with kinase-substrate interaction screening defines active c-Src-driven extracellular signaling.
Pancreatic ductal adenocarcinoma ubiquitination profiling reveals specific prognostic and theranostic markers.
Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling.
Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and vacuole.
eIF4E phosphorylation recruits β-catenin to mRNA cap and promotes Wnt pathway translation in dentate gyrus LTP maintenance.
Nucleophilic covalent ligand discovery for the cysteine redoxome.
Buffering of genetic dominance by allele-specific protein complex assembly.
LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models.
Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.
Learning consistent subcellular landmarks to quantify changes in multiplexed protein maps.
Discovery of Small-Molecule Degraders for Alpha-Synuclein Aggregates.
Allosteric regulation of the 20S proteasome by the Catalytic Core Regulators (CCRs) family.
What Makes a Good Protein-Protein Interaction Stabilizer: Analysis and Application of the Dual-Binding Mechanism.

EMBO J. 2023 Jun 02. e113110

Structural basis of ubiquitin-independent PP1 complex disassembly by p97.

Johannes van den Boom, Guendalina Marini, Hemmo Meyer, Helen R Saibil.

  The AAA+-ATPase p97 (also called VCP or Cdc48) unfolds proteins and disassembles protein complexes in numerous cellular processes, but how substrate complexes are loaded onto p97 and disassembled is unclear. Here, we present cryo-EM structures of p97 in the process of disassembling a protein phosphatase-1 (PP1) complex by extracting an inhibitory subunit from PP1. We show that PP1 and its partners SDS22 and inhibitor-3 (I3) are loaded tightly onto p97, surprisingly via a direct contact of SDS22 with the p97 N-domain. Loading is assisted by the p37 adapter that bridges two adjacent p97 N-domains underneath the substrate complex. A stretch of I3 is threaded into the central channel of the spiral-shaped p97 hexamer, while other elements of I3 are still attached to PP1. Thus, our data show how p97 arranges a protein complex between the p97 N-domain and central channel, suggesting a hold-and-extract mechanism for p97-mediated disassembly.

Keywords:  AAA ATPase; cryo-EM; protein phosphatase-1; protein unfolding

DOI:  https://doi.org/10.15252/embj.2022113110
Nat Commun. 2023 May 30. 14(1): 3132

The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes.

Shuangcheng Alivia Wu, Chenchen Shen, Xiaoqiong Wei, Xiawei Zhang, Siwen Wang, Xinxin Chen, Mauricio Torres, You Lu, Liangguang Leo Lin, Huilun Helen Wang, Allen H Hunter, Deyu Fang, Shengyi Sun, Magdalena I Ivanova, Yi Lin, Ling Qi.

Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.

DOI: https://doi.org/10.1038/s41467-023-38690-4
Cell Rep. 2023 May 31. pii: S2211-1247(23)00590-9. [Epub ahead of print]42(6): 112579

The E3 ubiquitin ligase FBXL6 controls the quality of newly synthesized mitochondrial ribosomal proteins.

Julie Lavie, Claude Lalou, Walid Mahfouf, Jean-William Dupuy, Aurélie Lacaule, Agata Ars Cywinska, Didier Lacombe, Anne-Marie Duchêne, Anne-Aurélie Raymond, Hamid Reza Rezvani, Richard Patryk Ngondo, Giovanni Bénard.

  In mammals, about 99% of mitochondrial proteins are synthesized in the cytosol as precursors that are subsequently imported into the organelle. The mitochondrial health and functions rely on an accurate quality control of these imported proteins. Here, we show that the E3 ubiquitin ligase F box/leucine-rich-repeat protein 6 (FBXL6) regulates the quality of cytosolically translated mitochondrial proteins. Indeed, we found that FBXL6 substrates are newly synthesized mitochondrial ribosomal proteins. This E3 binds to chaperones involved in the folding and trafficking of newly synthesized peptide and to ribosomal-associated quality control proteins. Deletion of these interacting partners is sufficient to hamper interactions between FBXL6 and its substrate. Furthermore, we show that cells lacking FBXL6 fail to degrade specifically mistranslated mitochondrial ribosomal proteins. Finally, showing the role of FBXL6-dependent mechanism, FBXL6-knockout (KO) cells display mitochondrial ribosomal protein aggregations, altered mitochondrial metabolism, and inhibited cell cycle in oxidative conditions.

Keywords:  CP: Cell biology; F box leucin-rich repeat E3 ubiquitin ligase; FBXL6; mitochondria; protein quality control; ribosomal proteins

DOI:  https://doi.org/10.1016/j.celrep.2023.112579
J Cell Sci. 2023 May 15. pii: jcs260888. [Epub ahead of print]136(10):

Aggrephagy at a glance.

Bernd Bauer, Sascha Martens, Luca Ferrari.

  Cells keep their proteome functional by the action of the proteostasis network, composed of the chaperones, the ubiquitin-proteasome system and autophagy. The decline of this network results in the accumulation of protein aggregates and is associated with aging and disease. In this Cell Science at a Glance and accompanying poster, we provide an overview of the molecular mechanisms of the removal of protein aggregates by a selective autophagy pathway, termed aggrephagy. We outline how aggrephagy is regulated by post-translational modifications and via auxiliary proteins. We further describe alternative aggrephagy pathways in physiology and their disruption in pathology. In particular, we discuss aggrephagy pathways in neurons and accumulation of protein aggregates in a wide range of diseases. Finally, we highlight strategies to reprogram aggrephagy to treat protein aggregation diseases.

Keywords:  Autophagy; Neurodegeneration; Proteostasis network

DOI:  https://doi.org/10.1242/jcs.260888
FEBS Lett. 2023 Jun 01.

ER-to-Lysosome Associated Degradation (ERLAD) in a Nutshell: mammalian, yeast and plant ER-phagy as induced by misfolded proteins.

Mikhail Rudinskiy, Maurizio Molinari.

  Conserved catabolic pathways operate to remove aberrant polypeptides from the endoplasmic reticulum (ER), the major biosynthetic organelle of eukaryotic cells. The best known are the ER-associated degradation (ERAD) pathways that control retro-translocation of terminally misfolded proteins across the ER membrane for clearance by the cytoplasmic ubiquitin/proteasome system. In this review, we catalogue folding-defective mammalian, yeast, and plant proteins that fail to engage ERAD machineries. We describe that they rather segregate in ER subdomains that eventually vesiculate. These ER-derived vesicles are captured by double membrane autophagosomes, engulfed by endolysosomes/vacuoles, or fuse with degradative organelles to clear cells from their toxic cargo. These client-specific, mechanistically diverse ER-phagy pathways are grouped under the umbrella term of ER-to-Lysosome-Associated Degradation (ERLAD) for description in this essay.

Keywords:  ER-Associated Degradation (ERAD); ER-phagy; ER-to-Lysosome-Associated degradation (ERLAD); Lysosome/vacuole; Ubiquitin/proteasome system

DOI:  https://doi.org/10.1002/1873-3468.14674
Calcif Tissue Int. 2023 May 27.

Bone and the Unfolded Protein Response: In Sickness and in Health.

Srividhya Iyer, Douglas J Adams.

  Differentiation and optimal function of osteoblasts and osteoclasts are contingent on synthesis and maintenance of a healthy proteome. Impaired and/or altered secretory capacity of these skeletal cells is a primary driver of most skeletal diseases. The endoplasmic reticulum (ER) orchestrates the folding and maturation of membrane as well as secreted proteins at high rates within a calcium rich and oxidative organellar niche. Three ER membrane proteins monitor fidelity of protein processing in the ER and initiate an intricate signaling cascade known as the Unfolded Protein Response (UPR) to remediate accumulation of misfolded proteins in its lumen, a condition referred to as ER stress. The UPR aids in fine-tuning, expanding and/or modifying the cellular proteome, especially in specialized secretory cells, to match everchanging physiologic cues and metabolic demands. Sustained activation of the UPR due to chronic ER stress, however, is known to hasten cell death and drive pathophysiology of several diseases. A growing body of evidence suggests that ER stress and an aberrant UPR may contribute to poor skeletal health and the development of osteoporosis. Small molecule therapeutics that target distinct components of the UPR may therefore have implications for developing novel treatment modalities relevant to the skeleton. This review summarizes the complexity of UPR actions in bone cells in the context of skeletal physiology and osteoporotic bone loss, and highlights the need for future mechanistic studies to develop novel UPR therapeutics that mitigate adverse skeletal outcomes.

Keywords:  ATF6; ER stress; IRE1; PERK; Protein misfolding; Proteostasis

DOI:  https://doi.org/10.1007/s00223-023-01096-x
iScience. 2023 Jun 16. 26(6): 106808

A LY6E-PHB1-TRIM21 assembly degrades CD14 protein to mitigate LPS-induced inflammatory response.

Xinyu Zhu, Linxia Zhang, Daobin Feng, Lang Jiang, Peng Sun, Chen Zhao, Xiaoyan Zhang, Jianqing Xu.

  A major theme of host against invading pathogens lies in multiple regulatory nodes that ensure sufficient signals for protection while avoiding excessive signals toward over-inflammation. The TLR4/MD-2/CD14 complex receptor-mediated response to bacterial lipopolysaccharide (LPS) represents a paradigm for understanding the proper control of anti-pathogen innate immunity. In this study, we studied the mechanism by which the glycosylphosphatidylinositol (GPI)-linked LY6E protein constrains LPS response via downregulating CD14. We first showed that LY6E downregulated CD14 via ubiquitin-dependent proteasomal degradation. The subsequent profiling of LY6E protein interactome led to the revelation that the degradation of CD14 by LY6E requires PHB1, which interacts with CD14 in a LY6E-dependent manner. Finally, we identified the PHB1-interacting TRIM21 as the major ubiquitin E3 ligase for the LY6E-mediated ubiquitination of CD14. Together, our study elucidated the molecular basis of LY6E-mediated governance of LPS response, alongside providing new insights to regulatory mechanisms controlling the homeostasis of membrane proteins.

Keywords:  Immunology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2023.106808
Nat Commun. 2023 May 29. 14(1): 3086

Multi-omic approach characterises the neuroprotective role of retromer in regulating lysosomal health.

James L Daly, Chris M Danson, Philip A Lewis, Lu Zhao, Sara Riccardo, Lucio Di Filippo, Davide Cacchiarelli, Daehoon Lee, Stephen J Cross, Kate J Heesom, Wen-Cheng Xiong, Andrea Ballabio, James R Edgar, Peter J Cullen.

Retromer controls cellular homeostasis through regulating integral membrane protein sorting and transport and by controlling maturation of the endo-lysosomal network. Retromer dysfunction, which is linked to neurodegenerative disorders including Parkinson's and Alzheimer's diseases, manifests in complex cellular phenotypes, though the precise nature of this dysfunction, and its relation to neurodegeneration, remain unclear. Here, we perform an integrated multi-omics approach to provide precise insight into the impact of Retromer dysfunction on endo-lysosomal health and homeostasis within a human neuroglioma cell model. We quantify widespread changes to the lysosomal proteome, indicative of broad lysosomal dysfunction and inefficient autophagic lysosome reformation, coupled with a reconfigured cell surface proteome and secretome reflective of increased lysosomal exocytosis. Through this global proteomic approach and parallel transcriptomic analysis, we provide a holistic view of Retromer function in regulating lysosomal homeostasis and emphasise its role in neuroprotection.

DOI: https://doi.org/10.1038/s41467-023-38719-8
Mol Cell. 2023 Jun 01. pii: S1097-2765(23)00332-5. [Epub ahead of print]83(11): 1903-1920.e12

Irisin acts through its integrin receptor in a two-step process involving extracellular Hsp90α.

Mu A, Thomas E Wales, Haixia Zhou, Sorin-Valeriu Draga-Coletă, Christoph Gorgulla, Katherine A Blackmore, Melanie J Mittenbühler, Caroline R Kim, Dina Bogoslavski, Qiuyang Zhang, Zi-Fu Wang, Mark P Jedrychowski, Hyuk-Soo Seo, Kijun Song, Andrew Z Xu, Luke Sebastian, Steven P Gygi, Haribabu Arthanari, Sirano Dhe-Paganon, Patrick R Griffin, John R Engen, Bruce M Spiegelman.

  Exercise benefits the human body in many ways. Irisin is secreted by muscle, increased with exercise, and conveys physiological benefits, including improved cognition and resistance to neurodegeneration. Irisin acts via αV integrins; however, a mechanistic understanding of how small polypeptides like irisin can signal through integrins is poorly understood. Using mass spectrometry and cryo-EM, we demonstrate that the extracellular heat shock protein 90α (eHsp90α) is secreted by muscle with exercise and activates integrin αVβ5. This allows for high-affinity irisin binding and signaling through an Hsp90α/αV/β5 complex. By including hydrogen/deuterium exchange data, we generate and experimentally validate a 2.98 Å RMSD irisin/αVβ5 complex docking model. Irisin binds very tightly to an alternative interface on αVβ5 distinct from that used by known ligands. These data elucidate a non-canonical mechanism by which a small polypeptide hormone like irisin can function through an integrin receptor.

Keywords:  HDX-MS; RGD motif; exercise; extracellular Hsp90; fibronectin III domain; integrin; integrin activation; irisin; ligand binding; protein-protein docking

DOI:  https://doi.org/10.1016/j.molcel.2023.05.008
Nucleic Acids Res. 2023 May 29. pii: gkad461. [Epub ahead of print]

eEF2 diphthamide modification restrains spurious frameshifting to maintain translational fidelity.

Byung-Sik Shin, Ivaylo P Ivanov, Joo-Ran Kim, Chune Cao, Terri G Kinzy, Thomas E Dever.

Diphthamide (DPH), a conserved amino acid modification on eukaryotic translation elongation factor eEF2, is synthesized via a complex, multi-enzyme pathway. While DPH is non-essential for cell viability and its function has not been resolved, diphtheria and other bacterial toxins ADP-ribosylate DPH to inhibit translation. Characterizing Saccharomyces cerevisiae mutants that lack DPH or show synthetic growth defects in the absence of DPH, we show that loss of DPH increases resistance to the fungal translation inhibitor sordarin and increases -1 ribosomal frameshifting at non-programmed sites during normal translation elongation and at viral programmed frameshifting sites. Ribosome profiling of yeast and mammalian cells lacking DPH reveals increased ribosomal drop-off during elongation, and removal of out-of-frame stop codons restores ribosomal processivity on the ultralong yeast MDN1 mRNA. Finally, we show that ADP-ribosylation of DPH impairs the productive binding of eEF2 to elongating ribosomes. Our results reveal that loss of DPH impairs the fidelity of translocation during translation elongation resulting in increased rates of ribosomal frameshifting throughout elongation and leading to premature termination at out-of-frame stop codons. We propose that the costly, yet non-essential, DPH modification has been conserved through evolution to maintain translational fidelity despite being a target for inactivation by bacterial toxins.

DOI: https://doi.org/10.1093/nar/gkad461
Biochemistry. 2023 May 30.

Enzyme-Mediated Proximity Labeling Identifies Small RNAs in the Endoplasmic Reticulum Lumen.

Ziqi Ren, Ran Li, Xinyue Zhou, Yu Chen, Yuxin Fang, Peng Zou.

In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. Despite broad distributions throughout the cytoplasm, RNA molecules are conventionally believed to be excluded from the secretory pathway compartments including the endoplasmic reticulum (ER). Recent discovery of RNA N-glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons. Our data set reveals the presence of small non-coding RNAs in the ER lumen, including U RNAs and Y RNAs, which raises interesting questions regarding their transport mechanism and biological functions in the ER.

DOI: https://doi.org/10.1021/acs.biochem.3c00142
EMBO J. 2023 May 30. e112534

Selective autophagy regulates chloroplast protein import and promotes plant stress tolerance.

Chen Wan, Hui Zhang, Hongying Cheng, Robert G Sowden, Wenjuan Cai, R Paul Jarvis, Qihua Ling.

  Chloroplasts are plant organelles responsible for photosynthesis and environmental sensing. Most chloroplast proteins are imported from the cytosol through the translocon at the outer envelope membrane of chloroplasts (TOC). Previous work has shown that TOC components are regulated by the ubiquitin-proteasome system (UPS) to control the chloroplast proteome, which is crucial for the organelle's function and plant development. Here, we demonstrate that the TOC apparatus is also subject to K63-linked polyubiquitination and regulation by selective autophagy, potentially promoting plant stress tolerance. We identify NBR1 as a selective autophagy adaptor targeting TOC components, and mediating their relocation into vacuoles for autophagic degradation. Such selective autophagy is shown to control TOC protein levels and chloroplast protein import and to influence photosynthetic activity as well as tolerance to UV-B irradiation and heat stress in Arabidopsis plants. These findings uncover the vital role of selective autophagy in the proteolytic regulation of specific chloroplast proteins, and how dynamic control of chloroplast protein import is critically important for plants to cope with challenging environments.

Keywords:  NBR1; autophagy; chloroplast; protein import; stress response

DOI:  https://doi.org/10.15252/embj.2022112534
Cell Rep. 2023 Jun 01. pii: S2211-1247(23)00597-1. [Epub ahead of print]42(6): 112586

The Scap-SREBP1-S1P/S2P lipogenesis signal orchestrates the homeostasis and spatiotemporal activation of NF-κB.

Xia Fei, Jiaqi Huang, Fei Li, Yuejue Wang, Zhehua Shao, Lingling Dong, Yinfang Wu, Boran Li, Xue Zhang, Baihui Lv, Yun Zhao, Qingyu Weng, Kaijun Chen, Min Zhang, Shiyi Yang, Chao Zhang, Min Zhang, Wen Li, Songmin Ying, Qiming Sun, Zhihua Chen, Huahao Shen.

  The nuclear factor κB (NF-κB) pathway plays essential roles in innate and adaptive immunity, but little is known how NF-κB signaling is compartmentalized and spatiotemporally activated in the cytoplasm. Here, we show that the lipogenesis signal cascade Scap-SREBP1-S1P/S2P orchestrates the homeostasis and spatiotemporal activation of NF-κB. SREBP cleavage-activating protein (Scap) and sterol regulatory element-binding protein 1 (SREBP1) form a super complex with inhibitors of NF-κB α (IκBα) to associate NF-κB close to the endoplasmic reticulum (ER). Upon lipopolysaccharide (LPS) stimulation, Scap transports the complex to the Golgi apparatus, where SREBP1 is cleaved by site-1 protease (S1P)/S2P, liberating IκBα for IκB kinase (Ikk)-mediated phosphorylation and subsequent activation of NF-κB. Loss of Scap or inhibition of S1P or S2P diminishes, while SREBP1 deficiency augments, LPS-induced NF-κB activation and subsequent inflammatory responses. Our results reveal the Scap-SREBP1 complex as an additional cytoplasmic checkpoint for NF-κB homeostasis and unveil the Golgi apparatus as the optimal cellular platform for NF-κB activation, providing insights into the crosstalk between lipogenesis signaling and immunity.

Keywords:  CP: Immunology; inflammation; metabolism

DOI:  https://doi.org/10.1016/j.celrep.2023.112586
Mol Cell. 2023 Jun 01. pii: S1097-2765(23)00330-1. [Epub ahead of print]83(11): 1936-1952.e7

Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins.

Eugene Serebryany, Victor Y Zhao, Kibum Park, Amir Bitran, Sunia A Trauger, Bogdan Budnik, Eugene I Shakhnovich.

  Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

Keywords:  Chaperone; Conformational landscape; Disulfide bond; Disulfide scanning; Kinetic trap; Misfolding avoidance; Periplasm; Protein folding; Protein sequencing; Proteotoxic stress

DOI:  https://doi.org/10.1016/j.molcel.2023.05.006
iScience. 2023 Jun 16. 26(6): 106880

LEAFY homeostasis is regulated via ubiquitin-dependent degradation and sequestration in cytoplasmic condensates.

Ulla Dolde, Fernando Muzzopappa, Charlotte Delesalle, Julie Neveu, Fabian Erdel, Grégory Vert.

  The transcription factor LEAFY (LFY) plays crucial roles in flower development by activating floral homeotic genes. Activation of LFY targets requires the combined action of LFY and the E3 ubiquitin ligase UFO, although the precise underlying mechanism remains unclear. Here, we show that LFY accumulates in biomolecular condensates within the cytoplasm, while recombinant LFY forms condensates with similar properties in vitro. UFO interacts with LFY within these condensates and marks it for degradation. LFY levels in the nucleus are buffered against changes in total LFY levels induced by proteasome inhibition, UFO overexpression, or mutation of lysine residues in a disordered region of LFY. Perturbation of cytoplasmic LFY condensates by 1,6-hexanediol treatment induces the relocalization of LFY to the nucleus and the subsequent activation of the LFY target AP3 in flowers. Our data suggest that nucleocytoplasmic partitioning, condensation, and ubiquitin-dependent degradation regulate LFY levels in the nucleus to control its activity.

Keywords:  Cell biology; Plant biochemistry; Plant development

DOI:  https://doi.org/10.1016/j.isci.2023.106880
ACS Cent Sci. 2023 May 24. 9(5): 915-926

Rational Chemical Design of Molecular Glue Degraders.

Ethan S Toriki, James W Papatzimas, Kaila Nishikawa, Dustin Dovala, Andreas O Frank, Matthew J Hesse, Daniela Dankova, Jae-Geun Song, Megan Bruce-Smythe, Heidi Struble, Francisco J Garcia, Scott M Brittain, Andrew C Kile, Lynn M McGregor, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura.

Targeted protein degradation with molecular glue degraders has arisen as a powerful therapeutic modality for eliminating classically undruggable disease-causing proteins through proteasome-mediated degradation. However, we currently lack rational chemical design principles for converting protein-targeting ligands into molecular glue degraders. To overcome this challenge, we sought to identify a transposable chemical handle that would convert protein-targeting ligands into molecular degraders of their corresponding targets. Using the CDK4/6 inhibitor ribociclib as a prototype, we identified a covalent handle that, when appended to the exit vector of ribociclib, induced the proteasome-mediated degradation of CDK4 in cancer cells. Further modification of our initial covalent scaffold led to an improved CDK4 degrader with the development of a but-2-ene-1,4-dione ("fumarate") handle that showed improved interactions with RNF126. Subsequent chemoproteomic profiling revealed interactions of the CDK4 degrader and the optimized fumarate handle with RNF126 as well as additional RING-family E3 ligases. We then transplanted this covalent handle onto a diverse set of protein-targeting ligands to induce the degradation of BRD4, BCR-ABL and c-ABL, PDE5, AR and AR-V7, BTK, LRRK2, HDAC1/3, and SMARCA2/4. Our study undercovers a design strategy for converting protein-targeting ligands into covalent molecular glue degraders.

DOI: https://doi.org/10.1021/acscentsci.2c01317
Cell Rep. 2023 May 25. pii: S2211-1247(23)00550-8. [Epub ahead of print]42(6): 112539

PhosY-secretome profiling combined with kinase-substrate interaction screening defines active c-Src-driven extracellular signaling.

Sarah J Backe, SarahBeth D Votra, Matthew P Stokes, Endre Sebestyén, Matteo Castelli, Luca Torielli, Giorgio Colombo, Mark R Woodford, Mehdi Mollapour, Dimitra Bourboulia.

  c-Src tyrosine kinase is a renowned key intracellular signaling molecule and a potential target for cancer therapy. Secreted c-Src is a recent observation, but how it contributes to extracellular phosphorylation remains elusive. Using a series of domain deletion mutants, we show that the N-proximal region of c-Src is essential for its secretion. The tissue inhibitor of metalloproteinases 2 (TIMP2) is an extracellular substrate of c-Src. Limited proteolysis-coupled mass spectrometry and mutagenesis studies verify that the Src homology 3 (SH3) domain of c-Src and the P31VHP34 motif of TIMP2 are critical for their interaction. Comparative phosphoproteomic analyses identify an enrichment of PxxP motifs in phosY-containing secretomes from c-Src-expressing cells with cancer-promoting roles. Inhibition of extracellular c-Src using custom SH3-targeting antibodies disrupt kinase-substrate complexes and inhibit cancer cell proliferation. These findings point toward an intricate role for c-Src in generating phosphosecretomes, which will likely influence cell-cell communication, particularly in c-Src-overexpressing cancers.

Keywords:  CP: Cancer; CP: Molecular biology; PxxP motif; Src tyrosine kinase; TIMP2; blocking antibodies; phosphoproteomic; post-translational modification; secretome; tissue inhibitors of metalloproteinases; tumor growth inhibition

DOI:  https://doi.org/10.1016/j.celrep.2023.112539
EBioMedicine. 2023 May 29. pii: S2352-3964(23)00199-8. [Epub ahead of print]92 104634

Pancreatic ductal adenocarcinoma ubiquitination profiling reveals specific prognostic and theranostic markers.

Abdessamad El Kaoutari, Nicolas A Fraunhoffer, Stéphane Audebert, Luc Camoin, Yolande Berthois, Odile Gayet, Julie Roques, Martin Bigonnet, Claire Bongrain, Joseph Ciccolini, Juan L Iovanna, Nelson J Dusetti, Philippe Soubeyran.

   BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been widely studied at multiomics level. However, little is known about its specific ubiquitination, a major post-translational modification (PTM). As PTMs regulate the final function of any gene, we decided to establish the ubiquitination profiles of 60 PDAC.
METHODS: We used specific proteomic tools to establish the ubiquitin dependent proteome (ubiquitinome) of frozen PDXs (Patients' derived xenographs). Then, we performed bioinformatics analysis to identify the possible associations of these ubiquitination profiles with tumour phenotype, patient survival and resistance to chemotherapies. Finally, we used proximity ligation assays (PLA) to detect and quantify the ubiquitination level of one identified marker.
FINDINGS: We identified 38 ubiquitination site profiles correlating with the transcriptomic phenotype of tumours and four had notable prognostic capabilities. Seventeen ubiquitination profiles displayed potential theranostic marker for gemcitabine, seven for 5-FU, six for oxaliplatin and thirteen for irinotecan. Using PLA, we confirmed the use of one ubiquitination profile as a drug-response marker, directly on paraffin embedded tissues, supporting the possible application of these biomarkers in the clinical setting.
INTERPRETATION: These findings bring new and important insights on the relationship between ubiquitination levels of proteins and different molecular and clinical features of PDAC patients. Markers identified in this study could have a potential application in clinical settings to help to predict response to chemotherapies thereby allowing the personalization of treatments.
FUNDING: Fondation ARC (PJA 20181208270 and PGA 12021010002840_3562); INCa; Canceropôle PACA; DGOS; Amidex Foundation; Fondation de France; and INSERM.

Keywords:  Pancreatic cancer; Ubiquitin profiling; Ubiquitin prognostic markers; Ubiquitin theranostic markers

DOI:  https://doi.org/10.1016/j.ebiom.2023.104634
Nat Commun. 2023 Jun 02. 14(1): 3200

Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling.

Pietro Fontana, Sara C Buch-Larsen, Osamu Suyari, Rebecca Smith, Marcin J Suskiewicz, Kira Schützenhofer, Antonio Ariza, Johannes Gregor Matthias Rack, Michael L Nielsen, Ivan Ahel.

In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.

DOI: https://doi.org/10.1038/s41467-023-38793-y
J Cell Sci. 2023 Jun 01. pii: jcs.260889. [Epub ahead of print]

Yck3 casein kinase-mediated phosphorylation determines Ivy1 localization and function at endosomes and vacuole.

Sophie Grziwa, Jan-Hannes Schäfer, Raffaele Nicastro, Annabel Arens, Claudio De Virgilio, Florian Fröhlich, Arne Moeller, Jieqiong Gao, Lars Langemeyer, Christian Ungermann.

  The casein kinase Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates. We localized endogenously tagged Yck3 not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localizes to both endosomes and vacuoles, and Yck3 controls this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, its non-phosphorylatable SA variant strongly localized to endosomes, similar to what is observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying Rab7-like Ypt7. Modelling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which are both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.

Keywords:  Endosome; HOPS; Ivy1; MVB; Phosphorylation; Signaling endosome; Yck3

DOI:  https://doi.org/10.1242/jcs.260889
iScience. 2023 May 19. 26(5): 106649

eIF4E phosphorylation recruits β-catenin to mRNA cap and promotes Wnt pathway translation in dentate gyrus LTP maintenance.

Sudarshan Patil, Kleanthi Chalkiadaki, Tadiwos F Mergiya, Konstanze Krimbacher, Inês S Amorim, Shreeram Akerkar, Christos G Gkogkas, Clive R Bramham.

  The mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), is crucial for translation and regulated by Ser209 phosphorylation. However, the biochemical and physiological role of eIF4E phosphorylation in translational control of long-term synaptic plasticity is unknown. We demonstrate that phospho-ablated Eif4eS209A Knockin mice are profoundly impaired in dentate gyrus LTP maintenance in vivo, whereas basal perforant path-evoked transmission and LTP induction are intact. mRNA cap-pulldown assays show that phosphorylation is required for synaptic activity-induced removal of translational repressors from eIF4E, allowing initiation complex formation. Using ribosome profiling, we identified selective, phospho-eIF4E-dependent translation of the Wnt signaling pathway in LTP. Surprisingly, the canonical Wnt effector, β-catenin, was massively recruited to the eIF4E cap complex following LTP induction in wild-type, but not Eif4eS209A, mice. These results demonstrate a critical role for activity-evoked eIF4E phosphorylation in dentate gyrus LTP maintenance, remodeling of the mRNA cap-binding complex, and specific translation of the Wnt pathway.

Keywords:  Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2023.106649
Nat Chem Biol. 2023 May 29.

Nucleophilic covalent ligand discovery for the cysteine redoxome.

Ling Fu, Youngeun Jung, Caiping Tian, Renan B Ferreira, Ruifeng Cheng, Fuchu He, Jing Yang, Kate S Carroll.

With an eye toward expanding chemistries used for covalent ligand discovery, we elaborated an umpolung strategy that exploits the 'polarity reversal' of sulfur when cysteine is oxidized to sulfenic acid, a widespread post-translational modification, for selective bioconjugation with C-nucleophiles. Here we present a global map of a human sulfenome that is susceptible to covalent modification by members of a nucleophilic fragment library. More than 500 liganded sulfenic acids were identified on proteins across diverse functional classes, and, of these, more than 80% were not targeted by electrophilic fragment analogs. We further show that members of our nucleophilic fragment library can impair functional protein-protein interactions involved in nuclear oncoprotein transport and DNA damage repair. Our findings reveal a vast expanse of ligandable sulfenic acids in the human proteome and highlight the utility of nucleophilic small molecules in the fragment-based covalent ligand discovery pipeline, presaging further opportunities using non-traditional chemistries for targeting proteins.

DOI: https://doi.org/10.1038/s41589-023-01330-5
Sci Adv. 2023 Jun 02. 9(22): eadf9845

Buffering of genetic dominance by allele-specific protein complex assembly.

Mihaly Badonyi, Joseph A Marsh.

Protein complex assembly often occurs while subunits are being translated, resulting in complexes whose subunits were translated from the same mRNA in an allele-specific manner. It has thus been hypothesized that such cotranslational assembly may counter the assembly-mediated dominant-negative effect, whereby co-assembly of mutant and wild-type subunits "poisons" complex activity. Here, we show that cotranslationally assembling subunits are much less likely to be associated with autosomal dominant relative to recessive disorders, and that subunits with dominant-negative disease mutations are significantly depleted in cotranslational assembly compared to those associated with loss-of-function mutations. We also find that complexes with known dominant-negative effects tend to expose their interfaces late during translation, lessening the likelihood of cotranslational assembly. Finally, by combining complex properties with other features, we trained a computational model for predicting proteins likely to be associated with non-loss-of-function disease mechanisms, which we believe will be of considerable utility for protein variant interpretation.

DOI: https://doi.org/10.1126/sciadv.adf9845
Nat Commun. 2023 May 29. 14(1): 3077

LC3-associated phagocytosis promotes glial degradation of axon debris after injury in Drosophila models.

Áron Szabó, Virág Vincze, Aishwarya Sanjay Chhatre, András Jipa, Sarolta Bognár, Katalin Eszter Varga, Poulami Banik, Adél Harmatos-Ürmösi, Lukas J Neukomm, Gábor Juhász.

Glial engulfment of neuron-derived debris after trauma, during development, and in neurodegenerative diseases supports nervous system functions. However, mechanisms governing the efficiency of debris degradation in glia have remained largely unexplored. Here we show that LC3-associated phagocytosis (LAP), an engulfment pathway assisted by certain autophagy factors, promotes glial phagosome maturation in the Drosophila wing nerve. A LAP-specific subset of autophagy-related genes is required in glia for axon debris clearance, encoding members of the Atg8a (LC3) conjugation system and the Vps34 lipid kinase complex including UVRAG and Rubicon. Phagosomal Rubicon and Atg16 WD40 domain-dependent conjugation of Atg8a mediate proper breakdown of internalized axon fragments, and Rubicon overexpression in glia accelerates debris elimination. Finally, LAP promotes survival following traumatic brain injury. Our results reveal a role of glial LAP in the clearance of neuronal debris in vivo, with potential implications for the recovery of the injured nervous system.

DOI: https://doi.org/10.1038/s41467-023-38755-4
Nat Cancer. 2023 May 29.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Pravat Kumar Parida, Mauricio Marquez-Palencia, Suvranil Ghosh, Nitin Khandelwal, Kangsan Kim, Vidhya Nair, Xiao-Zheng Liu, Hieu S Vu, Lauren G Zacharias, Paula I Gonzalez-Ericsson, Melinda E Sanders, Bret C Mobley, Jeffrey G McDonald, Andrew Lemoff, Yan Peng, Cheryl Lewis, Gonçalo Vale, Nils Halberg, Carlos L Arteaga, Ariella B Hanker, Ralph J DeBerardinis, Srinivas Malladi.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

DOI: https://doi.org/10.1038/s43018-023-00563-6
Nat Methods. 2023 May 29.

Learning consistent subcellular landmarks to quantify changes in multiplexed protein maps.

Hannah Spitzer, Scott Berry, Mark Donoghoe, Lucas Pelkmans, Fabian J Theis.

Highly multiplexed imaging holds enormous promise for understanding how spatial context shapes the activity of the genome and its products at multiple length scales. Here, we introduce a deep learning framework called CAMPA (Conditional Autoencoder for Multiplexed Pixel Analysis), which uses a conditional variational autoencoder to learn representations of molecular pixel profiles that are consistent across heterogeneous cell populations and experimental perturbations. Clustering these pixel-level representations identifies consistent subcellular landmarks, which can be quantitatively compared in terms of their size, shape, molecular composition and relative spatial organization. Using high-resolution multiplexed immunofluorescence, this reveals how subcellular organization changes upon perturbation of RNA synthesis, RNA processing or cell size, and uncovers links between the molecular composition of membraneless organelles and cell-to-cell variability in bulk RNA synthesis rates. By capturing interpretable cellular phenotypes, we anticipate that CAMPA will greatly accelerate the systematic mapping of multiscale atlases of biological organization to identify the rules by which context shapes physiology and disease.

DOI: https://doi.org/10.1038/s41592-023-01894-z
J Med Chem. 2023 Jun 02.

Discovery of Small-Molecule Degraders for Alpha-Synuclein Aggregates.

Yichen Tong, Wentao Zhu, Jian Chen, Tianzhi Wen, Fang Xu, Jiyan Pang.

Alpha-synuclein (αSyn) species, especially the oligomers and fibers, are associated with multiple neurodegenerative diseases and cannot be directly targeted under the conventional pharmacological paradigm. Proteolysis-targeting chimera technology confers degradation of various "undruggable" targets; however, hardly any small-molecule degrader for αSyn aggregates has been reported yet. Herein, by using the probe molecule sery308 as a warhead, a series of small-molecule degraders for αSyn aggregates were designed and synthesized. Their degradation effects on αSyn aggregates were evaluated on a modified pre-formed fibril-seeding cell model. Compound 2b exhibited the highest degradation efficiency (DC50 = 7.51 ± 0.53 μM) with high selectivity. Mechanistic exploration revealed that both proteasomal and lysosomal pathways were involved in this kind of degradation. Moreover, the therapeutic effects of 2b were tested on SH-SY5Y (human neuroblastoma cell line) cells and Caenorhabditis elegans. Our results provided a new class of small-molecule candidates against synucleinopathies and broadened the substrate spectrum of PROTAC-based degraders.

DOI: https://doi.org/10.1021/acs.jmedchem.3c00274
Nat Commun. 2023 May 30. 14(1): 3126

Allosteric regulation of the 20S proteasome by the Catalytic Core Regulators (CCRs) family.

Fanindra Kumar Deshmukh, Gili Ben-Nissan, Maya A Olshina, Maria G Füzesi-Levi, Caley Polkinghorn, Galina Arkind, Yegor Leushkin, Irit Fainer, Sarel J Fleishman, Dan Tawfik, Michal Sharon.

Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 β-subunit with attenuation of the complex's three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome's enzymatic subunits but rather propagates through the non-catalytic subunit PSMB4. This way of 20S proteasome-specific attenuation opens avenues for decoupling the 20S and 26S proteasome degradation pathways as well as for developing selective 20S proteasome inhibitors.

DOI: https://doi.org/10.1038/s41467-023-38404-w
ACS Cent Sci. 2023 May 24. 9(5): 969-979

What Makes a Good Protein-Protein Interaction Stabilizer: Analysis and Application of the Dual-Binding Mechanism.

Shu-Yu Chen, Martin Zacharias.

Protein-protein interactions (PPIs) are essential for biological processes including immune reactions and diseases. Inhibition of PPIs by drug-like compounds is a common basis for therapeutic approaches. In many cases the flat interface of PP complexes prevents discovery of specific compound binding to cavities on one partner and PPI inhibition. However, frequently new pockets are formed at the PP interface that allow accommodation of stabilizers which is often as desirable as inhibition but a much less explored alternative strategy. Herein, we employ molecular dynamics simulations and pocket detection to investigate 18 known stabilizers and associated PP complexes. For most cases, we find that a dual-binding mechanism, a similar stabilizer interaction strength with each protein partner, is an important prerequisite for effective stabilization. A few stabilizers follow an allosteric mechanism by stabilizing the protein bound structure and/or increase the PPI indirectly. On 226 protein-protein complexes, we find in >75% of the cases interface cavities suitable for binding of drug-like compounds. We propose a computational compound identification workflow that exploits new PP interface cavities and optimizes the dual-binding mechanism and apply it to 5 PP complexes. Our study demonstrates a great potential for in silico PPI stabilizers discovery with a wide range of therapeutic applications.

DOI: https://doi.org/10.1021/acscentsci.3c00003