bims-proteo Biomed News
on Proteostasis
Issue of 2023–07–30
38 papers selected by
Eric Chevet, INSERM
  1. Ubiquitin-mediated degradation at the Golgi apparatus.
  2. Mechanism of orphan subunit recognition during assembly quality control.
  3. ER membrane curvature and ubiquitin as drivers of ER-phagy.
  4. SARS-CoV-2 nonstructural protein 6 triggers endoplasmic reticulum stress-induced autophagy to degrade STING1.
  5. Proteostasis regulation through ribosome quality control and no-go-decay.
  6. Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.
  7. Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.
  8. mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
  9. Lipid homeostasis is essential for a maximal ER stress response.
  10. Olfactory chemosensation extends lifespan through TGF-β signaling and UPR activation.
  11. Evolutionarily divergent mTOR remodels translatome for tissue regeneration.
  12. Stem-loop induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control.
  13. ER stress activates TAp73α to promote colon cancer cell apoptosis via the PERK-ATF4 pathway.
  14. DNA framework-engineered chimeras platform enables selectively targeted protein degradation.
  15. Restoration of β-GC trafficking improves the lysosome function in Gaucher disease.
  16. Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7.
  17. mRNA location and translation rate determine protein targeting to dual destinations.
  18. DIRAS3 enhances RNF19B-mediated RAC1 ubiquitination and degradation in non-small-cell lung cancer cells.
  19. Control of protein synthesis through mRNA pseudouridylation by dyskerin.
  20. Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.
  21. DNAJC10 maintains survival and self-renewal of leukemia stem cells through PERK branch of the unfolded protein response.
  22. Mitochondrial proteostasis mediated by CRL5 Ozz and Alix maintains skeletal muscle function.
  23. Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses.
  24. COPI-regulated mitochondria-ER contact site formation maintains axonal integrity.
  25. Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics.
  26. A small molecule inhibitor of PTP1B and PTPN2 enhances T cell anti-tumor immunity.
  27. Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex.
  28. Monitoring integrated stress response in live Drosophila.
  29. ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator.
  30. Deploying synthetic coevolution and machine learning to engineer protein-protein interactions.
  31. Delivering on Cell-Selective Protein Degradation Using Chemically Tailored PROTACs.
  32. A proximity labeling strategy enables proteomic analysis of inter-organelle membrane contacts.
  33. microRNAs slow translating ribosomes to prevent protein misfolding in eukaryotes.
  34. Nascent mitochondrial proteins initiate the localized condensation of cytosolic protein aggregates on the mitochondrial surface.
  35. Calnexin controls TrkB cell surface transport and ER-phagy in mouse cerebral cortex development.
  36. UBQLN2 and HSP70 participate in Parkin-mediated mitophagy by facilitating outer mitochondrial membrane rupture.
  37. Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP βCTF.
  38. 14-3-3σ-NEDD4L axis promotes ubiquitination and degradation of HIF-1α in colorectal cancer.
  1. Front Mol Biosci. 2023 ;10 1197921
    Ubiquitin-mediated degradation at the Golgi apparatus.
    Lana Buzuk, Doris Hellerschmied.
      The Golgi apparatus is an essential organelle of the secretory pathway in eukaryotic cells. It processes secretory and transmembrane proteins and orchestrates their transport to other endomembrane compartments or the plasma membrane. The Golgi apparatus thereby shapes the cell surface, controlling cell polarity, cell-cell communication, and immune signaling. The cytosolic face of the Golgi hosts and regulates signaling cascades, impacting most notably the DNA damage response and mitosis. These essential functions strongly depend on Golgi protein homeostasis and Golgi integrity. Golgi fragmentation and consequent malfunction is associated with neurodegenerative diseases and certain cancer types. Recent studies provide first insight into the critical role of ubiquitin signaling in maintaining Golgi integrity and in Golgi protein quality control. Similar to well described pathways at the endoplasmic reticulum, ubiquitin-dependent degradation of non-native proteins prevents the accumulation of toxic protein aggregates at the Golgi. Moreover, ubiquitination regulates Golgi structural rearrangements in response to cellular stress. Advances in elucidating ubiquitination and degradation events at the Golgi are starting to paint a picture of the molecular machinery underlying Golgi (protein) homeostasis.
    Keywords:  Golgi fragmentation; Golgi homeostasis; Golgi protein quality control; PROTAC; transmembrane protein degradation; ubiquitin E3 ligase; ubiquitin-mediated protein degradation
    DOI:  https://doi.org/10.3389/fmolb.2023.1197921
  2. Cell. 2023 Jul 19. pii: S0092-8674(23)00694-3. [Epub ahead of print]
    Mechanism of orphan subunit recognition during assembly quality control.
    Yuichi Yagita, Eszter Zavodszky, Sew-Yeu Peak-Chew, Ramanujan S Hegde.
      Cells contain numerous abundant molecular machines assembled from multiple subunits. Imbalances in subunit production and failed assembly generate orphan subunits that are eliminated by poorly defined pathways. Here, we determined how orphan subunits of the cytosolic chaperonin CCT are recognized. Several unassembled CCT subunits recruited the E3 ubiquitin ligase HERC2 using ZNRD2 as an adaptor. Both factors were necessary for orphan CCT subunit degradation in cells, sufficient for CCT subunit ubiquitination with purified factors, and necessary for optimal cell fitness. Domain mapping and structure prediction defined the molecular features of a minimal HERC2-ZNRD2-CCT module. The structural model, whose key elements were validated in cells using point mutants, shows why ZNRD2 selectively recognizes multiple orphaned CCT subunits without engaging assembled CCT. Our findings reveal how failures during CCT assembly are monitored and provide a paradigm for the molecular recognition of orphan subunits, the largest source of quality control substrates in cells.
    Keywords:  E3 ligase; chaperonin; degradation; protein homeostasis; protein quality control; ubiquitination
    DOI:  https://doi.org/10.1016/j.cell.2023.06.016
  3. Dev Cell. 2023 Jul 24. pii: S1534-5807(23)00306-4. [Epub ahead of print]58(14): 1219-1220
    ER membrane curvature and ubiquitin as drivers of ER-phagy.
    Melissa J Hoyer, J Wade Harper.
      In a recent issue of Nature, González et al. and Foronda et al. examine the role of ubiquitin in autophagic capture of ER by ER-phagy. They propose that ubiquitylation of ER-phagy receptor FAM134B and ER-shaping protein ARL61PL1 promotes receptor clustering in nanodomains, which generates membrane curvature, facilitating autophagosomal capture.
    DOI:  https://doi.org/10.1016/j.devcel.2023.06.008
  4. Autophagy. 2023 Jul 23. 1-19
    SARS-CoV-2 nonstructural protein 6 triggers endoplasmic reticulum stress-induced autophagy to degrade STING1.
    Pengtao Jiao, Wenhui Fan, Xiaoya Ma, Runshan Lin, Yuna Zhao, Yabo Li, He Zhang, Xiaojuan Jia, Yuhai Bi, Xiaoli Feng, Minghua Li, Wenjun Liu, Ke Zhang, Lei Sun.
       ABBREVIATIONS: aa: amino acid; ATF6: activating transcription factor 6; ATG5: autophagy related 5; CCPG1: cell cycle progression 1; CFTR: CF transmembrane conductance regulator; cGAMP: cyclic GMP-AMP; CGAS: cyclic GMP-AMP synthase; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CQ: chloroquine; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HSV-1: herpes simplex virus type 1; IFIT1: interferon induced protein with tetratricopeptide repeats 1; IFNB1/IFN-β: interferon beta 1; IRF3: interferon regulatory factor 3; ISG15: ISG15 ubiquitin like modifier; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP3K7/TAK1: mitogen-activated protein kinase kinase kinase 7; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NFKB/NF-κB: nuclear factor kappa B; NSP6: non-structural protein 6; Δ106-108: deletion of amino acids 106-108 in NSP6 of SARS-CoV-2; Δ105-107: deletion of amino acids 105-107 in NSP6 of SARS-CoV-2; RETREG1/FAM134B: reticulophagy regulator 1; RIGI/DDX58: RNA sensor RIG-I; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1.
    Keywords:  Autophagy; HSPA5; NSP6; SARS-CoV-2; STING1; endoplasmic reticulum stress
    DOI:  https://doi.org/10.1080/15548627.2023.2238579
  5. Wiley Interdiscip Rev RNA. 2023 Jul 24. e1809
    Proteostasis regulation through ribosome quality control and no-go-decay.
    Lokha Ranjani Alagar Boopathy, Emma Beadle, Aitana Garcia-Bueno Rico, Maria Vera.
      Cell functionality relies on the existing pool of proteins and their folding into functional conformations. This is achieved through the regulation of protein synthesis, which requires error-free mRNAs and ribosomes. Ribosomes are quality control hubs for mRNAs and proteins. Problems during translation elongation slow down the decoding rate, leading to ribosome halting and the eventual collision with the next ribosome. Collided ribosomes form a specific disome structure recognized and solved by ribosome quality control (RQC) mechanisms. RQC pathways orchestrate the degradation of the problematic mRNA by no-go decay and the truncated nascent peptide, the repression of translation initiation, and the recycling of the stalled ribosomes. All these events maintain protein homeostasis and return valuable ribosomes to translation. As such, cell homeostasis and function are maintained at the mRNA level by preventing the production of aberrant or unnecessary proteins. It is becoming evident that the crosstalk between RQC and the protein homeostasis network is vital for cell function, as the absence of RQC components leads to the activation of stress response and neurodegenerative diseases. Here, we review the molecular events of RQC discovered through well-designed stalling reporters. Given the impact of RQC in proteostasis, we discuss the relevance of identifying endogenous mRNA regulated by RQC and their preservation in stress conditions. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms Translation > Regulation.
    Keywords:  no-go decay; ribosome quality control; stress response
    DOI:  https://doi.org/10.1002/wrna.1809
  6. bioRxiv. 2023 Jul 20. pii: 2023.07.19.549095. [Epub ahead of print]
    Quantitative Measurement of Secretory Protein Mistargeting by Proximity Labeling and Parallel Reaction Monitoring.
    Ziqi Lyu, Joseph C Genereux.
      Proximity labeling is a powerful approach for characterizing subcellular proteomes. We recently demonstrated that proximity labeling can be used to identify mistrafficking of secretory proteins, such as occurs during pre-emptive quality control (pre-QC) following endoplasmic reticulum (ER) stress. This assay depends on protein quantification by immunoblotting and densitometry, which is only semi-quantitative and suffers from poor sensitivity. Here, we integrate parallel reaction monitoring mass spectrometry to enable a more quantitative platform for ER import. PRM as opposed to densitometry improves quantification of transthyretin mistargeting while also achieving at least a ten-fold gain in sensitivity. The multiplexing of PRM also enabled us to evaluate a series of normalization approaches, revealing that normalization to auto-labeled APEX2 peroxidase is necessary to account for drug treatment-dependent changes in labeling efficiency. We apply this approach to systematically characterize the relationship between chemical ER stressors and ER pre-QC induction in HEK293T cells. Using dual-FLAG-tagged transthyretin ( FLAG TTR) as a model secretory protein, we find that Brefeldin A treatment as well as ER calcium depletion cause pre-QC, while tunicamycin and dithiothreitol do not, indicating ER stress alone is not sufficient. This finding contrasts with the canonical model of pre-QC induction, and establishes the utility of our platform.
    TOC graph:
    DOI:  https://doi.org/10.1101/2023.07.19.549095
  7. Dev Cell. 2023 Jul 18. pii: S1534-5807(23)00331-3. [Epub ahead of print]
    Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.
    Melissa A Roberts, Kirandeep K Deol, Alyssa J Mathiowetz, Mike Lange, Dara E Leto, Julian Stevenson, Sayed Hadi Hashemi, David W Morgens, Emilee Easter, Kartoosh Heydari, Mike A Nalls, Michael C Bassik, Martin Kampmann, Ron R Kopito, Faraz Faghri, James A Olzmann.
      Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.
    Keywords:  CRISPR; ERAD; MARCH6; PLIN2; endoplasmic reticulum; genetic screen; lipid droplet; metabolism; perilipin; resource
    DOI:  https://doi.org/10.1016/j.devcel.2023.07.001
  8. Cell Rep. 2023 Jul 25. pii: S2211-1247(23)00879-3. [Epub ahead of print]42(8): 112868
    mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching.
    Sejeong Shin, Min-Joon Han, Mark P Jedrychowski, Ziyang Zhang, Kevan M Shokat, David R Plas, Noah Dephoure, Sang-Oh Yoon.
      Cells maintain and dynamically change their proteomes according to the environment and their needs. Mechanistic target of rapamycin (mTOR) is a key regulator of proteostasis, homeostasis of the proteome. Thus, dysregulation of mTOR leads to changes in proteostasis and the consequent progression of diseases, including cancer. Based on the physiological and clinical importance of mTOR signaling, we investigated mTOR feedback signaling, proteostasis, and cell fate. Here, we reveal that mTOR targeting inhibits eIF4E-mediated cap-dependent translation, but feedback signaling activates a translation initiation factor, eukaryotic translation initiation factor 3D (eIF3D), to sustain alternative non-canonical translation mechanisms. Importantly, eIF3D-mediated protein synthesis enables cell phenotype switching from proliferative to more migratory. eIF3D cooperates with mRNA-binding proteins such as heterogeneous nuclear ribonucleoprotein F (hnRNPF), heterogeneous nuclear ribonucleoprotein K (hnRNPK), and Sjogren syndrome antigen B (SSB) to support selective mRNA translation following mTOR inhibition, which upregulates and activates proteins involved in insulin receptor (INSR)/insulin-like growth factor 1 receptor (IGF1R)/insulin receptor substrate (IRS) and interleukin 6 signal transducer (IL-6ST)/Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling. Our study highlights the mechanisms by which cells establish the dynamic change of proteostasis and the resulting phenotype switch.
    Keywords:  CP: Cell biology; CP: Molecular biology; eIF3D; eIF4E; mRNA translation; mTOR; proteome; proteostasis
    DOI:  https://doi.org/10.1016/j.celrep.2023.112868
  9. Elife. 2023 Jul 25. pii: e83884. [Epub ahead of print]12
    Lipid homeostasis is essential for a maximal ER stress response.
    Gilberto Garcia, Hanlin Zhang, Sophia Moreno, C Kimberly Tsui, Brant Michael Webster, Ryo Higuchi-Sanabria, Andrew Dillin.
      Changes in lipid metabolism are associated with aging and age-related diseases, including proteopathies. The endoplasmic reticulum (ER) is uniquely a major hub for protein and lipid synthesis, making its function essential for both protein and lipid homeostasis. However, it is less clear how lipid metabolism and protein quality may impact each other. Here, we identified let-767, a putative hydroxysteroid dehydrogenase in Caenorhabditis elegans, as an essential gene for both lipid and ER protein homeostasis. Knockdown of let-767 reduces lipid stores, alters ER morphology in a lipid-dependent manner, and blocks induction of the Unfolded Protein Response of the ER (UPRER). Interestingly, a global reduction in lipogenic pathways restores UPRER induction in animals with reduced let-767. Specifically, we find that supplementation of 3-oxoacyl, the predicted metabolite directly upstream of let-767, is sufficient to block induction of the UPRER. This study highlights a novel interaction through which changes in lipid metabolism can alter a cell's response to protein-induced stress.
    Keywords:  C. elegans; ER UPR; cell biology; genetics; genomics; lipid homeostasis; proteostasis; stress response
    DOI:  https://doi.org/10.7554/eLife.83884
  10. Nat Aging. 2023 Jul 27.
    Olfactory chemosensation extends lifespan through TGF-β signaling and UPR activation.
    Evandro A De-Souza, Maximillian A Thompson, Rebecca C Taylor.
      Animals rely on chemosensory cues to survive in pathogen-rich environments. In Caenorhabditis elegans, pathogenic bacteria trigger aversive behaviors through neuronal perception and activate molecular defenses throughout the animal. This suggests that neurons can coordinate the activation of organism-wide defensive responses upon pathogen perception. In this study, we found that exposure to volatile pathogen-associated compounds induces activation of the endoplasmic reticulum unfolded protein response (UPRER) in peripheral tissues after xbp-1 splicing in neurons. This odorant-induced UPRER activation is dependent upon DAF-7/transforming growth factor beta (TGF-β) signaling and leads to extended lifespan and enhanced clearance of toxic proteins. Notably, rescue of the DAF-1 TGF-β receptor in RIM/RIC interneurons is sufficient to significantly recover UPRER activation upon 1-undecene exposure. Our data suggest that the cell non-autonomous UPRER rewires organismal proteostasis in response to pathogen detection, pre-empting proteotoxic stress. Thus, chemosensation of particular odors may be a route to manipulation of stress responses and longevity.
    DOI:  https://doi.org/10.1038/s43587-023-00467-1
  11. Nature. 2023 Jul 26.
    Evolutionarily divergent mTOR remodels translatome for tissue regeneration.
    Olena Zhulyn, Hannah D Rosenblatt, Leila Shokat, Shizhong Dai, Duygu Kuzuoglu-Öztürk, Zijian Zhang, Davide Ruggero, Kevan M Shokat, Maria Barna.
      An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.
    DOI:  https://doi.org/10.1038/s41586-023-06365-1
  12. bioRxiv. 2023 Jul 12. pii: 2023.07.12.548609. [Epub ahead of print]
    Stem-loop induced ribosome queuing in the uORF2/ ATF4 overlap fine-tunes stress-induced human ATF4 translational control.
    Anna M Smirnova, Vladislava Hronová, Stanislava Gunišová, Pasha Mahabub Mohammad, Denisa Petráčková, Petr Halada, Justin Rendleman, Štěpán Coufal, Anna Herrmannová, Petra Beznosková, Kristína Jendruchová, Maria Hatzoglou, Christine Vogel, Leoš Shivaya Valášek.
      ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. Since its discovery, the delayed translation reinitiation mechanism, with reinitiation-permissive uORF1 playing a key role, was thought to underlie the ATF4 stress-induced upregulation. However, mounting evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional means of its regulation. Contrasting the original model, we show that inhibitory uORF2 is inducible and translated even under stress; that it encompasses two post-transcriptionally modified adenines performing antagonistic roles; and that the canonical ATF4 translation start site is robustly leaky-scanned. Most importantly, we demonstrate that the presence of a stable stem-loop in the uORF2/ ATF4 overlap, immediately preceded by near-cognate CUG, introduces another layer of regulation in the form of ribosome queuing. Thus, ATF4 translational control is much more complex than originally described underpinning its key role in diverse biological processes.
    DOI:  https://doi.org/10.1101/2023.07.12.548609
  13. J Cancer. 2023 ;14(11): 1946-1955
    ER stress activates TAp73α to promote colon cancer cell apoptosis via the PERK-ATF4 pathway.
    Shengnan Sun, Yong Yi, Zhi-Xiong Jim Xiao, Hu Chen.
      Colorectal cancer (CRC) is the fourth most diagnosed cancer worldwide. 43% of CRCs harbor p53 mutations. The tumor suppressor p53 induces cell growth arrest and/or apoptosis in response to stress, including endoplasmic reticulum (ER) stress. It has been documented that the p53 gene is mutated in more than 50% of human tumors and loses its tumor suppressor function, suggesting that ER stress-induced apoptosis might not rely on p53. In this study, we found that activation of ER stress promotes p53 null colon cancer cell apoptosis concomitant with an increased level of the TAp73α protein, a homologue of p53 in vitro and in vivo. Knockdown of TAp73α partially restores ER stress-induced apoptosis, indicating that ER stress stimulates apoptosis in a manner dependent on TAp73α, but not p53. Furthermore, we found that ER stress activates TAp73α mRNA and protein expression through PERK signalling, a branch of the unfolded protein response (UPR). Moreover, PERK promotes TAp73α expression by upregulating the expression of the transcription factor ATF4. ATF4 directly activates the transcription of TAp73α. Consistent with this finding, ATF4 knockdown inhibited PERK- or ER stress-induced TAp73α expression. Our findings reveal that ER stress activates TAp73α to promote colon cancer cell apoptosis via the PERK-ATF4 signalling. Therefore, prolonged ER stress or upregulation of TAp73α might be a therapeutic strategy for colon cancer.
    Keywords:  ATF4; Cell apoptosis; ER stress; PERK; TAp73α
    DOI:  https://doi.org/10.7150/jca.84170
  14. Nat Commun. 2023 07 27. 14(1): 4510
    DNA framework-engineered chimeras platform enables selectively targeted protein degradation.
    Li Zhou, Bin Yu, Mengqiu Gao, Rui Chen, Zhiyu Li, Yueqing Gu, Jinlei Bian, Yi Ma.
      A challenge in developing proteolysis targeting chimeras (PROTACs) is the establishment of a universal platform applicable in multiple scenarios for precise degradation of proteins of interest (POIs). Inspired by the addressability, programmability, and rigidity of DNA frameworks, we develop covalent DNA framework-based PROTACs (DbTACs), which can be synthesized in high-throughput via facile bioorthogonal chemistry and self-assembly. DNA tetrahedra are employed as templates and the spatial position of each atom is defined. Thus, by precisely locating ligands of POI and E3 ligase on the templates, ligand spacings can be controllably manipulated from 8 Å to 57 Å. We show that DbTACs with the optimal linker length between ligands achieve higher degradation rates and enhanced binding affinity. Bispecific DbTACs (bis-DbTACs) with trivalent ligand assembly enable multi-target depletion while maintaining highly selective degradation of protein subtypes. When employing various types of warheads (small molecules, antibodies, and DNA motifs), DbTACs exhibit robust efficacy in degrading diverse targets, including protein kinases and transcription factors located in different cellular compartments. Overall, utilizing modular DNA frameworks to conjugate substrates offers a universal platform that not only provides insight into general degrader design principles but also presents a promising strategy for guiding drug discovery.
    DOI:  https://doi.org/10.1038/s41467-023-40244-7
  15. Traffic. 2023 Jul 25.
    Restoration of β-GC trafficking improves the lysosome function in Gaucher disease.
    Saloni Patel, Dhwani Radhakrishnan, Darpan Kumari, Priyanka Bhansali, Subba Rao Gangi Setty.
      Lysosomes function as a primary site for catabolism and cellular signaling. These organelles digest a variety of substrates received through endocytosis, secretion and autophagy with the help of resident acid hydrolases. Lysosomal enzymes are folded in the endoplasmic reticulum (ER) and trafficked to lysosomes via Golgi and endocytic routes. The inability of hydrolase trafficking due to mutations or mutations in its receptor or cofactor leads to cargo accumulation (storage) in lysosomes, resulting in lysosome storage disorder (LSD). In Gaucher disease (GD), the lysosomes accumulate glucosylceramide because of low β-glucocerebrosidase (β-GC) activity that causes lysosome enlargement/dysfunction. We hypothesize that improving the trafficking of mutant β-GC to lysosomes may improve the lysosome function in GD. RNAi screen using high throughput based β-GC activity assay followed by reporter trafficking assay utilizing β-GC-mCherry led to the identification of nine potential phosphatases. Depletion of these phosphatases in HeLa cells enhanced the β-GC activity by increasing the folding and trafficking of Gaucher mutants to the lysosomes. Consistently, the lysosomes in primary fibroblasts from GD patients restored their β-GC activity upon the knockdown of these phosphatases. Thus, these studies provide evidence that altering phosphatome activity is an alternative therapeutic strategy to restore the lysosome function in GD.
    Keywords:  Gaucher disease; lysosome; lysosome storage diseases; phosphatome; β-glucocerebrosidase
    DOI:  https://doi.org/10.1111/tra.12911
  16. Traffic. 2023 Jul 26.
    Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7.
    Julia E J Holm, Sandro G Soares, Martyn F Symmons, Afiqah Saleh Huddin, Martin C Moncrieffe, Nicholas J Gay.
      Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.
    Keywords:  COP-coated vesicles; TLRs; endoplasmic reticulum; p24; protein transport
    DOI:  https://doi.org/10.1111/tra.12912
  17. Mol Cell. 2023 Jul 20. pii: S1097-2765(23)00514-2. [Epub ahead of print]
    mRNA location and translation rate determine protein targeting to dual destinations.
    Alexander N Gasparski, Konstadinos Moissoglu, Sandeep Pallikkuth, Sezen Meydan, Nicholas R Guydosh, Stavroula Mili.
      Numerous proteins are targeted to two or multiple subcellular destinations where they exert distinct functional consequences. The balance between such differential targeting is thought to be determined post-translationally, relying on protein sorting mechanisms. Here, we show that mRNA location and translation rate can also determine protein targeting by modulating protein binding to specific interacting partners. Peripheral localization of the NET1 mRNA and fast translation lead to higher cytosolic retention of the NET1 protein by promoting its binding to the membrane-associated scaffold protein CASK. By contrast, perinuclear mRNA location and/or slower translation rate favor nuclear targeting by promoting binding to importins. This mRNA location-dependent mechanism is modulated by physiological stimuli and profoundly impacts NET1 function in cell motility. These results reveal that the location of protein synthesis and the rate of translation elongation act in coordination as a "partner-selection" mechanism that robustly influences protein distribution and function.
    Keywords:  CASK; NET1; RNA localization; RhoA activity; cell migration; elongation rate; local translation; nuclear import; ribosome stalling
    DOI:  https://doi.org/10.1016/j.molcel.2023.06.036
  18. iScience. 2023 Jul 21. 26(7): 107157
    DIRAS3 enhances RNF19B-mediated RAC1 ubiquitination and degradation in non-small-cell lung cancer cells.
    Yingying Wang, Minli Wei, Min Su, Zhiyuan Du, Jiaxi Dong, Yu Zhang, Yingdi Wu, Xiaopeng Li, Ling Su, Xiangguo Liu.
      Distant metastasis remains the leading cause of high mortality in patients with non-small-cell lung cancer (NSCLC). DIRAS3 is a candidate tumor suppressor protein that is decreased in various tumors. However, the regulatory mechanism of DIRAS3 on metastasis of NSCLC remains unclear. Here, we found that DIRAS3 suppressed the migration of NSCLC cells. Besides, DIRAS3 stimulated the polyubiquitination of RAC1 and suppressed its protein expression. Furthermore, RNF19B, a member of the RBR E3 ubiquitin ligase family, was observed to be the E3 ligase involved in the DIRAS3-induced polyubiquitination of RAC1. DIRAS3 could promote the binding of RAC1 and RNF19B, thus enhancing the degradation of RAC1 by the ubiquitin-proteasome pathway. Finally, the DIRAS3-RNF19B-RAC1 axis was confirmed to be associated with the malignant progression of NSCLC. These findings may be beneficial for developing potential prognostic markers of NSCLC and may provide an effective treatment strategy.
    Keywords:  Biotechnology; Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2023.107157
  19. Sci Adv. 2023 Jul 28. 9(30): eadg1805
    Control of protein synthesis through mRNA pseudouridylation by dyskerin.
    Chiara Pederiva, Davide M Trevisan, Dimitra Peirasmaki, Shan Chen, Sharon A Savage, Ola Larsson, Jernej Ule, Laura Baranello, Federico Agostini, Marianne Farnebo.
      Posttranscriptional modifications of mRNA have emerged as regulators of gene expression. Although pseudouridylation is the most abundant, its biological role remains poorly understood. Here, we demonstrate that the pseudouridine synthase dyskerin associates with RNA polymerase II, binds to thousands of mRNAs, and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a guide RNA with full complementarity. In cells lacking dyskerin, mRNA pseudouridylation is reduced, while at the same time, de novo protein synthesis is enhanced, indicating that this modification interferes with translation. Accordingly, mRNAs with fewer pseudouridines due to knockdown of dyskerin are translated more efficiently. Moreover, mRNA pseudouridylation is severely reduced in patients with dyskeratosis congenita caused by inherited mutations in the gene encoding dyskerin (i.e., DKC1). Our findings demonstrate that pseudouridylation by dyskerin modulates mRNA translatability, with important implications for both normal development and disease.
    DOI:  https://doi.org/10.1126/sciadv.adg1805
  20. bioRxiv. 2023 Jul 20. pii: 2023.07.19.549693. [Epub ahead of print]
    Pancreatic islet cell stress induced by insulin-degrading enzyme deficiency promotes islet regeneration and protection from autoimmune diabetes.
    Shuaishuai Zhu, Emmanuelle Waeckel-Énée, Anna Moser, Marie-Andrée Bessard, Kevin Roger, Joanna Lipecka, Ayse Yilmaz, Barbara Bertocci, Julien Diana, Benjamin Saintpierre, Ida Chiara Guerrera, Stefania Francesconi, François-Xavier Mauvais, Peter van Endert.
      Appropriate tuning of protein homeostasis through mobilization of the unfolded protein response (UPR) is key to the capacity of pancreatic beta cells to cope with highly variable demand for insulin synthesis. An efficient UPR ensures a sufficient beta cell mass and secretory output but can also affect beta cell resilience to autoimmune aggression. The factors regulating protein homeostasis in the face of metabolic and immune challenges are insufficiently understood. We examined beta cell adaptation to stress in mice deficient for insulin-degrading enzyme (IDE), a ubiquitous protease with high affinity for insulin and genetic association with type 2 diabetes. IDE deficiency induced a low-level UPR in both C57BL/6 and autoimmune non-obese diabetic (NOD) mice, associated with rapamycin-sensitive beta cell proliferation strongly enhanced by proteotoxic stress. Moreover, in NOD mice, IDE deficiency protected from spontaneous diabetes and triggered an additional independent pathway, conditional on the presence of islet inflammation but inhibited by proteotoxic stress, highlighted by strong upregulation of regenerating islet-derived protein 2, a protein attenuating autoimmune inflammation. Our findings establish a key role of IDE in islet cell protein homeostasis, identify a link between low-level UPR and proliferation, and reveal an UPR-independent anti-inflammatory islet cell response uncovered in the absence of IDE of potential interest in autoimmune diabetes.
    DOI:  https://doi.org/10.1101/2023.07.19.549693
  21. Haematologica. 2023 Jul 27.
    DNAJC10 maintains survival and self-renewal of leukemia stem cells through PERK branch of the unfolded protein response.
    Minjing Li, Xingli Wu, Meiyang Chen, Shiyu Hao, Yue Yu, Xiang Li, Erdi Zhao, Ming Xu, Zhenhai Yu, Zhiqiang Wang, Ning Xu, Changzhu Jin, Yancun Yin.
      Leukemia stem cells (LSCs) requires frequent adaptation to maintenance self-renewal ability despite they are longer exposure to cell-intrinsic and cell-extrinsic stresses. However, the mechanism by which LSCs maintain their leukemogenic activities and how individual LSCs respond to stress remain poorly understood. Herein, we found that DNAJC10, a member of HSP40 family, was frequently upregulated in various types of acute myeloid leukemia (AML) and in LSC-enriched cells. Deficiency of DNAJC10 leads to a dramatic increase in the apoptosis of both human leukemia cell lines and LSCs enriched populations. Although DNAJC10 is not required for normal hematopoiesis, deficiency of Dnajc10 significantly abrogated AML development and suppressed self-renewal of LSCs in the MLL-AF9-induced murine leukemia model. Mechanistically, inhibition of DNAJC10 specifically induces endoplasmic reticulum (ER) stress and promotes activating of PERK-EIF2α-ATF4 branch of unfolded protein response (UPR). Blocking PERK by GSK2606414 (PERKi) or shRNA rescued the loss of function of DNAJC10 both in vitro and in vivo. Importantly, deficiency of DNAJC10 increased sensitivity of AML cells to daunorubicin (DNR) and cytarabine (Ara-C). These data revealed that DNAJC10 functions as an oncogene in MLL-AF9-induced AML via regulating PERK branch of the UPR. DNAJC10 may be an ideal therapeutic target for eliminating LSCs and improving the effectiveness of DNR and Ara-C.
    DOI:  https://doi.org/10.3324/haematol.2023.282691
  22. bioRxiv. 2023 Jul 11. pii: 2023.07.11.548601. [Epub ahead of print]
    Mitochondrial proteostasis mediated by CRL5 Ozz and Alix maintains skeletal muscle function.
    Yvan Campos, Ricardo Rodriguez-Enriquez, Gustavo Palacios, Diantha Van de Vlekkert, Xiaohui Qiu, Jayce Weesner, Elida Gomero, Jeroen Demmers, Tulio Bertorini, Joseph T Opferman, Gerard C Grosveld, Alessandra d'Azzo.
      High energy-demanding tissues, such as skeletal muscle, require mitochondrial proteostasis to function properly. Two quality-control mechanisms, the ubiquitin proteasome system (UPS) and the release of mitochondria-derived vesicles, safeguard mitochondrial proteostasis. However, whether these processes interact is unknown. Here we show that the E3 ligase CRL5 Ozz , a member of the UPS, and its substrate Alix control the mitochondrial concentration of Slc25A4, a solute carrier that is essential for ATP production. The mitochondria in Ozz -/- or Alix -/- skeletal muscle share overt morphologic alterations (they are supernumerary, swollen, and dysmorphic) and have abnormal metabolomic profiles. We found that CRL5 Ozz ubiquitinates Slc25A4 and promotes its proteasomal degradation, while Alix facilitates SLC25A4 loading into exosomes destined for lysosomal destruction. The loss of Ozz or Alix offsets steady-state levels of Slc25A4, which disturbs mitochondrial metabolism and alters muscle fiber composition. These findings reveal hitherto unknown regulatory functions of Ozz and Alix in mitochondrial proteostasis.
    DOI:  https://doi.org/10.1101/2023.07.11.548601
  23. bioRxiv. 2023 Jul 14. pii: 2023.07.13.548611. [Epub ahead of print]
    Pooled tagging and hydrophobic targeting of endogenous proteins for unbiased mapping of unfolded protein responses.
    Stephanie E Sansbury, Yevgeniy V Serebrenik, Tomer Lapidot, George M Burslem, Ophir Shalem.
      System-level understanding of proteome organization and function requires methods for direct visualization and manipulation of proteins at scale. We developed an approach enabled by high-throughput gene tagging for the generation and analysis of complex cell pools with endogenously tagged proteins. Proteins are tagged with HaloTag to enable visualization or direct perturbation. Fluorescent labeling followed by in situ sequencing and deep learning-based image analysis identifies the localization pattern of each tag, providing a bird's-eye-view of cellular organization. Next, we use a hydrophobic HaloTag ligand to misfold tagged proteins, inducing spatially restricted proteotoxic stress that is read out by single cell RNA sequencing. By integrating optical and perturbation data, we map compartment-specific responses to protein misfolding, revealing inter-compartment organization and direct crosstalk, and assigning proteostasis functions to uncharacterized genes. Altogether, we present a powerful and efficient method for large-scale studies of proteome dynamics, function, and homeostasis.
    DOI:  https://doi.org/10.1101/2023.07.13.548611
  24. Cell Rep. 2023 Jul 26. pii: S2211-1247(23)00894-X. [Epub ahead of print]42(8): 112883
    COPI-regulated mitochondria-ER contact site formation maintains axonal integrity.
    Daniel C Maddison, Bilal Malik, Leonardo Amadio, Dana M Bis-Brewer, Stephan Züchner, Owen M Peters, Gaynor A Smith.
      Coat protein complex I (COPI) is best known for its role in Golgi-endoplasmic reticulum (ER) trafficking, responsible for the retrograde transport of ER-resident proteins. The ER is crucial to neuronal function, regulating Ca2+ homeostasis and the distribution and function of other organelles such as endosomes, peroxisomes, and mitochondria via functional contact sites. Here we demonstrate that disruption of COPI results in mitochondrial dysfunction in Drosophila axons and human cells. The ER network is also disrupted, and the neurons undergo rapid degeneration. We demonstrate that mitochondria-ER contact sites (MERCS) are decreased in COPI-deficient axons, leading to Ca2+ dysregulation, heightened mitophagy, and a decrease in respiratory capacity. Reintroducing MERCS is sufficient to rescue not only mitochondrial distribution and Ca2+ uptake but also ER morphology, dramatically delaying neurodegeneration. This work demonstrates an important role for COPI-mediated trafficking in MERC formation, which is an essential process for maintaining axonal integrity.
    Keywords:  Axon transport; COPI; CP: Cell biology; Calcium homeostasis; Endoplasmic reticulum; Golgi; Mitochondria; Neuronal cell biology; Vesicle trafficking
    DOI:  https://doi.org/10.1016/j.celrep.2023.112883
  25. bioRxiv. 2023 Jul 14. pii: 2023.07.13.548759. [Epub ahead of print]
    Pooled endogenous protein tagging and recruitment for scalable discovery of effectors for induced proximity therapeutics.
    Yevgeniy V Serebrenik, Deepak Mani, Timothé Maujean, George M Burslem, Ophir Shalem.
      The field of induced proximity therapeutics is in its ascendancy but is limited by a lack of scalable tools to systematically explore effector-target protein pairs in an unbiased manner. Here, we combined Scalable POoled Targeting with a LIgandable Tag at Endogenous Sites (SPOTLITES) for the high-throughput tagging of endogenous proteins, with generic small molecule-based protein recruitment to screen for novel proximity-based effectors. We apply this methodology in two orthogonal screens for targeted protein degradation: the first using fluorescence to monitor target protein levels directly, and the second using a cellular growth phenotype that depends on the degradation of an essential protein. Our screens revealed a multitude of potential new effector proteins for degradation and converged on members of the CTLH complex which we demonstrate potently induce degradation. Altogether, we introduce a platform for pooled induction of endogenous protein-protein interactions that can be used to expand our toolset of effector proteins for targeted protein degradation and other forms of induced proximity.
    DOI:  https://doi.org/10.1101/2023.07.13.548759
  26. Nat Commun. 2023 Jul 27. 14(1): 4524
    A small molecule inhibitor of PTP1B and PTPN2 enhances T cell anti-tumor immunity.
    Shuwei Liang, Eric Tran, Xin Du, Jiajun Dong, Harrison Sudholz, Hao Chen, Zihan Qu, Nicholas D Huntington, Jeffrey J Babon, Nadia J Kershaw, Zhong-Yin Zhang, Jonathan B Baell, Florian Wiede, Tony Tiganis.
      The inhibition of protein tyrosine phosphatases 1B (PTP1B) and N2 (PTPN2) has emerged as an exciting approach for bolstering T cell anti-tumor immunity. ABBV-CLS-484 is a PTP1B/PTPN2 inhibitor in clinical trials for solid tumors. Here we have explored the therapeutic potential of a related small-molecule-inhibitor, Compound-182. We demonstrate that Compound-182 is a highly potent and selective active site competitive inhibitor of PTP1B and PTPN2 that enhances T cell recruitment and activation and represses the growth of tumors in mice, without promoting overt immune-related toxicities. The enhanced anti-tumor immunity in immunogenic tumors can be ascribed to the inhibition of PTP1B/PTPN2 in T cells, whereas in cold tumors, Compound-182 elicited direct effects on both tumor cells and T cells. Importantly, treatment with Compound-182 rendered otherwise resistant tumors sensitive to α-PD-1 therapy. Our findings establish the potential for small molecule inhibitors of PTP1B and PTPN2 to enhance anti-tumor immunity and combat cancer.
    DOI:  https://doi.org/10.1038/s41467-023-40170-8
  27. Cell Rep. 2023 Jul 25. pii: S2211-1247(23)00849-5. [Epub ahead of print]42(8): 112838
    Increased degradation of FMRP contributes to neuronal hyperexcitability in tuberous sclerosis complex.
    Kellen D Winden, Truc T Pham, Nicole A Teaney, Juan Ruiz, Ryan Chen, Cidi Chen, Mustafa Sahin.
      Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder, but new therapies have been impeded by a lack of understanding of the pathological mechanisms. Tuberous sclerosis complex (TSC) and fragile X syndrome are associated with alterations in the mechanistic target of rapamycin (mTOR) and fragile X messenger ribonucleoprotein 1 (FMRP), which have been implicated in the development of ASD. Previously, we observed that transcripts associated with FMRP were down-regulated in TSC2-deficient neurons. In this study, we find that FMRP turnover is dysregulated in TSC2-deficient rodent primary neurons and human induced pluripotent stem cell (iPSC)-derived neurons and is dependent on the E3 ubiquitin ligase anaphase-promoting complex. We also demonstrate that overexpression of FMRP can partially rescue hyperexcitability in TSC2-deficient iPSC-derived neurons. These data indicate that FMRP dysregulation represents an important pathological mechanism in the development of abnormal neuronal activity in TSC and illustrate a molecular convergence between these two neurogenetic disorders.
    Keywords:  CP: Neuroscience; autism spectrum disorder; fragile X messenger ribonucleoprotein 1; iPSC-derived neuron; tuberous sclerosis complex
    DOI:  https://doi.org/10.1016/j.celrep.2023.112838
  28. bioRxiv. 2023 Jul 14. pii: 2023.07.13.548942. [Epub ahead of print]
    Monitoring integrated stress response in live Drosophila.
    Peter V Lidsky, Jing Yuan, Kseniya A Lashkevich, Sergey E Dmitriev, Raul Andino.
      Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, our understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder our ability to track and manipulate the ISR in vivo. To overcome these limitations, we have successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables us to label Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, we have unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of our reporter, we have engineered ISR-null eIF2αS50A mutant flies for stress response analysis. Our results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.
    DOI:  https://doi.org/10.1101/2023.07.13.548942
  29. Proc Natl Acad Sci U S A. 2023 08;120(31): e2303448120
    ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator.
    Ze Liu, Guanlin Liu, Dat P Ha, Justin Wang, Min Xiong, Amy S Lee.
      Cancer cells are commonly subjected to endoplasmic reticulum (ER) stress. To gain survival advantage, cancer cells exploit the adaptive aspects of the unfolded protein response such as upregulation of the ER luminal chaperone GRP78. The finding that when overexpressed, GRP78 can escape to other cellular compartments to gain new functions regulating homeostasis and tumorigenesis represents a paradigm shift. Here, toward deciphering the mechanisms whereby GRP78 knockdown suppresses EGFR transcription, we find that nuclear GRP78 is prominent in cancer and stressed cells and uncover a nuclear localization signal critical for its translocation and nuclear activity. Furthermore, nuclear GRP78 can regulate expression of genes and pathways, notably those important for cell migration and invasion, by interacting with and inhibiting the activity of the transcriptional repressor ID2. Our study reveals a mechanism for cancer cells to respond to ER stress via transcriptional regulation mediated by nuclear GRP78 to adopt an invasive phenotype.
    Keywords:  ER stress; GRP78; lung cancer; nuclear translocation; transcriptional regulation
    DOI:  https://doi.org/10.1073/pnas.2303448120
  30. Science. 2023 Jul 28. 381(6656): eadh1720
    Deploying synthetic coevolution and machine learning to engineer protein-protein interactions.
    Aerin Yang, Kevin M Jude, Ben Lai, Mason Minot, Anna M Kocyla, Caleb R Glassman, Daisuke Nishimiya, Yoon Seok Kim, Sai T Reddy, Aly A Khan, K Christopher Garcia.
      Fine-tuning of protein-protein interactions occurs naturally through coevolution, but this process is difficult to recapitulate in the laboratory. We describe a platform for synthetic protein-protein coevolution that can isolate matched pairs of interacting muteins from complex libraries. This large dataset of coevolved complexes drove a systems-level analysis of molecular recognition between Z domain-affibody pairs spanning a wide range of structures, affinities, cross-reactivities, and orthogonalities, and captured a broad spectrum of coevolutionary networks. Furthermore, we harnessed pretrained protein language models to expand, in silico, the amino acid diversity of our coevolution screen, predicting remodeled interfaces beyond the reach of the experimental library. The integration of these approaches provides a means of simulating protein coevolution and generating protein complexes with diverse molecular recognition properties for biotechnology and synthetic biology.
    DOI:  https://doi.org/10.1126/science.adh1720
  31. Chembiochem. 2023 Jul 26. e202300413
    Delivering on Cell-Selective Protein Degradation Using Chemically Tailored PROTACs.
    Rui Yao, Tianli Luo, Ming Wang.
      PROTACs (Proteolysis-Targeting Chimeras) have emerged as a groundbreaking class of chemical tools that facilitate the degradation of target proteins by leveraging the ubiquitin-proteasome system (UPS). However, the effective utilization of PROTACs in chemical biology studies and therapeutics encounters significant challenges when it comes to achieving cell-selective protein degradation and in vivo applications. This review article aims to shed light on recent advancements in the development of Pro-PROTACs, which exhibit controlled protein degradation capabilities in response to external stimuli or disease-related endogenous biochemical signals. The article delves into the specific chemical strategies employed to regulate the interaction between PROTACs and E3 ubiquitin ligases or target proteins. These strategies enable spatial and temporal control over the protein degradation potential of Pro-PROTACs. Furthermore, the review summarizes recent investigations regarding the delivery of PROTACs using biodegradable nanoparticles for in vivo applications and targeted protein degradation. Such delivery systems hold great promise for enabling efficient and selective protein degradation in vivo. Lastly, the article provides a perspective on the future design of multifunctional PROTACs and their intracellular delivery mechanisms, with a particular focus on achieving cell-selective protein degradation.
    Keywords:  PROTAC; biodegradable; drug delivery; nanoparticles; targeted protein degradation
    DOI:  https://doi.org/10.1002/cbic.202300413
  32. iScience. 2023 Jul 21. 26(7): 107159
    A proximity labeling strategy enables proteomic analysis of inter-organelle membrane contacts.
    Maoge Zhou, Bingjie Kong, Xiang Zhang, Ke Xiao, Jing Lu, Weixing Li, Min Li, Zonghong Li, Wei Ji, Junjie Hou, Tao Xu.
      Inter-organelle membrane contacts are highly dynamic and act as central hubs for many biological processes, but the protein compositions remain largely unknown due to the lack of efficient tools. Here, we developed BiFCPL to analyze the contact proteome in living cells by a bimolecular fluorescence complementation (BiFC)-based proximity labeling (PL) strategy. BiFCPL was applied to study mitochondria-endoplasmic reticulum contacts (MERCs) and mitochondria-lipid droplet (LD) contacts. We identified 403 highly confident MERC proteins, including many transiently resident proteins and potential tethers. Moreover, we demonstrated that mitochondria-LD contacts are sensitive to nutrient status. A comparative proteomic analysis revealed that 60 proteins are up- or downregulated at contact sites under metabolic challenge. We verified that SQLE, an enzyme for cholesterol synthesis, accumulates at mitochondria-LD contact sites probably to utilize local ATP for cholesterol synthesis. This work provides an efficient method to identify key proteins at inter-organelle membrane contacts in living cells.
    Keywords:  Biophysics; Membranes; Proteomics
    DOI:  https://doi.org/10.1016/j.isci.2023.107159
  33. EMBO J. 2023 Jul 26. e112469
    microRNAs slow translating ribosomes to prevent protein misfolding in eukaryotes.
    Hiroaki Sako, Mohieldin Youssef, Olga Elisseeva, Takayuki Akimoto, Katsuhiko Suzuki, Takashi Ushida, Tadashi Yamamoto.
      Slower translation rates reduce protein misfolding. Such reductions in speed can be mediated by the presence of non-optimal codons, which allow time for proper folding to occur. Although this phenomenon is conserved from bacteria to humans, it is not known whether there are additional eukaryote-specific mechanisms which act in the same way. MicroRNAs (miRNAs), not present in prokaryotes, target both coding sequences (CDS) and 3' untranslated regions (UTR). Given their low suppressive efficiency, it has been unclear why miRNAs are equally likely to bind to a CDS. Here, we show that miRNAs transiently stall translating ribosomes, preventing protein misfolding with little negative effect on protein abundance. We first analyzed ribosome profiles and miRNA binding sites to examine whether miRNAs stall ribosomes. Furthermore, either global or specific miRNA deficiency accelerated ribosomes and induced aggregation of a misfolding-prone polypeptide reporter. These defects were rescued by slowing ribosomes using non-cleaving shRNAs as miRNA mimics. We finally show that proinsulin misfolding, associated with type II diabetes, was resolved by non-cleaving shRNAs. Our findings provide a eukaryote-specific mechanism of co-translational protein folding and a previously unknown mechanism of action to target protein misfolding diseases.
    Keywords:  miRNA; misfolding; translational pause
    DOI:  https://doi.org/10.15252/embj.2022112469
  34. Proc Natl Acad Sci U S A. 2023 08;120(31): e2300475120
    Nascent mitochondrial proteins initiate the localized condensation of cytosolic protein aggregates on the mitochondrial surface.
    Qingqing Liu, Benjamin Fong, Seungmin Yoo, Jay R Unruh, Fengli Guo, Zulin Yu, Jingjing Chen, Kausik Si, Rong Li, Chuankai Zhou.
      Eukaryotes organize cellular contents into membrane-bound organelles and membrane-less condensates, for example, protein aggregates. An unsolved question is why the ubiquitously distributed proteins throughout the cytosol give rise to spatially localized protein aggregates on the organellar surface, like mitochondria. We report that the mitochondrial import receptor Tom70 is involved in the localized condensation of protein aggregates in budding yeast and human cells. This is because misfolded cytosolic proteins do not autonomously aggregate in vivo; instead, they are recruited to the condensation sites initiated by Tom70's substrates (nascent mitochondrial proteins) on the organellar membrane using multivalent hydrophobic interactions. Knocking out Tom70 partially impairs, while overexpressing Tom70 increases the formation and association between cytosolic protein aggregates and mitochondria. In addition, ectopic targeting Tom70 and its substrates to the vacuole surface is able to redirect the localized aggregation from mitochondria to the vacuolar surface. Although other redundant mechanisms may exist, this nascent mitochondrial proteins-based initiation of protein aggregation likely explains the localized condensation of otherwise ubiquitously distributed molecules on the mitochondria. Disrupting the mitochondrial association of aggregates impairs their asymmetric retention during mitosis and reduces the mitochondrial import of misfolded proteins, suggesting a proteostasis role of the organelle-condensate interactions.
    Keywords:  condensate; mitochondria; protein aggregation
    DOI:  https://doi.org/10.1073/pnas.2300475120
  35. Dev Cell. 2023 Jul 22. pii: S1534-5807(23)00334-9. [Epub ahead of print]
    Calnexin controls TrkB cell surface transport and ER-phagy in mouse cerebral cortex development.
    Patrick Lüningschrör, Thomas Andreska, Alexander Veh, Daniel Wolf, Neha Jadhav Giridhar, Mehri Moradi, Angela Denzel, Michael Sendtner.
      Transactivation of Tropomyosin receptor kinase B (TrkB) by EGF leads to cell surface transport of TrkB, promoting its signaling responsiveness to brain-derived neurotrophic factor (BDNF), a critical process for proper cortical plate development. However, the mechanisms that regulate the transport of TrkB to the cell surface are not fully understood. Here, we identified Calnexin as a regulator for targeting TrkB either to the cell surface or toward autophagosomal processing. Calnexin-deficient mouse embryos show impaired cortical plate formation and elevated levels of transactivated TrkB. In Calnexin-depleted mouse neuronal precursor cells, we detected an impaired cell surface transport of TrkB in response to EGF and an impaired delivery to autophagosomes. Mechanistically, we show that Calnexin facilitates the interaction of TrkB with the ER-phagy receptor Fam134b, thereby targeting TrkB to ER-phagy. This mechanism appears as a critical process for fine-tuning the sensitivity of neurons to BDNF.
    Keywords:  BDNF; Calnexin; EGF; ER-phagy; TrkB; autophagy; transactivation
    DOI:  https://doi.org/10.1016/j.devcel.2023.07.004
  36. EMBO Rep. 2023 Jul 28. e55859
    UBQLN2 and HSP70 participate in Parkin-mediated mitophagy by facilitating outer mitochondrial membrane rupture.
    Qilian Ma, Jiaqi Xin, Qiang Peng, Ningning Li, Shan Sun, Hongyu Hou, Guoqiang Ma, Nana Wang, Li Zhang, Kin Yip Tam, Heiko Dussmann, Jochen Hm Prehn, Hongfeng Wang, Zheng Ying.
      Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two aging-related neurodegenerative diseases that share common key features, including aggregation of pathogenic proteins, dysfunction of mitochondria, and impairment of autophagy. Mutations in ubiquilin 2 (UBQLN2), a shuttle protein in the ubiquitin-proteasome system (UPS), can cause ALS/FTD, but the mechanism underlying UBQLN2-mediated pathogenesis is still uncertain. Recent studies indicate that mitophagy, a selective form of autophagy which is crucial for mitochondrial quality control, is tightly associated with neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, and ALS. In this study, we show that after Parkin-dependent ubiquitination of damaged mitochondria, UBQLN2 is recruited to poly-ubiquitinated mitochondria through the UBA domain. UBQLN2 cooperates with the chaperone HSP70 to promote UPS-driven degradation of outer mitochondrial membrane (OMM) proteins. The resulting rupture of the OMM triggers the autophagosomal recognition of the inner mitochondrial membrane receptor PHB2. UBQLN2 is required for Parkin-mediated mitophagy and neuronal survival upon mitochondrial damage, and the ALS/FTD pathogenic mutations in UBQLN2 impair mitophagy in primary cultured neurons. Taken together, our findings link dysfunctional mitophagy to UBQLN2-mediated neurodegeneration.
    Keywords:  ALS; Parkin; UBQLN2; mitophagy; ubiquitin
    DOI:  https://doi.org/10.15252/embr.202255859
  37. Sci Adv. 2023 Jul 28. 9(30): eadg1925
    Lysosomal dysfunction in Down syndrome and Alzheimer mouse models is caused by v-ATPase inhibition by Tyr682-phosphorylated APP βCTF.
    Eunju Im, Ying Jiang, Philip H Stavrides, Sandipkumar Darji, Hediye Erdjument-Bromage, Thomas A Neubert, Jun Yong Choi, Jerzy Wegiel, Ju-Hyun Lee, Ralph A Nixon.
      Lysosome dysfunction arises early and propels Alzheimer's disease (AD). Herein, we show that amyloid precursor protein (APP), linked to early-onset AD in Down syndrome (DS), acts directly via its β-C-terminal fragment (βCTF) to disrupt lysosomal vacuolar (H+)-adenosine triphosphatase (v-ATPase) and acidification. In human DS fibroblasts, the phosphorylated 682YENPTY internalization motif of APP-βCTF binds selectively within a pocket of the v-ATPase V0a1 subunit cytoplasmic domain and competitively inhibits association of the V1 subcomplex of v-ATPase, thereby reducing its activity. Lowering APP-βCTF Tyr682 phosphorylation restores v-ATPase and lysosome function in DS fibroblasts and in vivo in brains of DS model mice. Notably, lowering APP-βCTF Tyr682 phosphorylation below normal constitutive levels boosts v-ATPase assembly and activity, suggesting that v-ATPase may also be modulated tonically by phospho-APP-βCTF. Elevated APP-βCTF Tyr682 phosphorylation in two mouse AD models similarly disrupts v-ATPase function. These findings offer previously unknown insight into the pathogenic mechanism underlying faulty lysosomes in all forms of AD.
    DOI:  https://doi.org/10.1126/sciadv.adg1925
  38. Cell Rep. 2023 Jul 25. pii: S2211-1247(23)00881-1. [Epub ahead of print]42(8): 112870
    14-3-3σ-NEDD4L axis promotes ubiquitination and degradation of HIF-1α in colorectal cancer.
    Sicheng Liu, Rui Guo, Hui Xu, Jinneng Yang, Haidan Luo, Sai-Ching Jim Yeung, Kai Li, Mong-Hong Lee, Runxiang Yang.
      A hypoxic microenvironment contributes to tumor progression, with hypoxia-inducible factor-1α (HIF-1α) being a critical regulator. We have reported that 14-3-3σ is negatively associated with HIF-1α expression; however, its role in hypoxia-induced tumor progression remains poorly characterized. Here we show that 14-3-3σ suppresses cancer hypoxia-induced metastasis and angiogenesis in colorectal cancer (CRC). 14-3-3σ opposes HIF-1α expression by regulating the protein stability of HIF-1α, thereby decreasing HIF-1α transcriptional activity and suppressing tumor progression. Mechanistic studies show that the 14-3-3σ-interacting protein neural precursor cell-expressed developmentally down-regulated 4-like (NEDD4L) is an E3 ligase that targets HIF-1α. 14-3-3σ promotes the binding of S448-phosphorylated NEDD4L to HIF-1α, thereby enhancing HIF-1α poly-ubiquitination and subsequent proteasome-mediated degradation. Consistent with this anti-tumorigenic function for 14-3-3σ, low 14-3-3σ expression levels correlate with poor CRC patient survival, and 14-3-3σ enhances the response of CRC to bevacizumab. These results reveal an important mechanism for 14-3-3σ in tumor suppression through HIF-1α regulation.
    Keywords:  14-3-3σ; CP: Cancer; HIF-1α; NEDD4L; bevacizumab; colorectal cancer; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2023.112870
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