bims-proteo Biomed News
on Proteostasis
Issue of 2023‒12‒24
29 papers selected by
Eric Chevet, INSERM
  1. Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies.
  2. Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
  3. Unlocking the plant ER stress code: IRE1-proteasome signaling cohort takes the lead.
  4. Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons.
  5. The lipid flippase Drs2 regulates anterograde transport of Atg9 during autophagy.
  6. Proteolysis-targeting chimeras with reduced off-targets.
  7. NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.
  8. The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways.
  9. Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.
  10. Direct-to-biology, automated, nano-scale synthesis, and phenotypic screening-enabled E3 ligase modulator discovery.
  11. Listerin promotes cGAS protein degradation through the ESCRT pathway to negatively regulate cGAS-mediated immune response.
  12. Subcytoplasmic location of translation controls protein output.
  13. Reprogramming of tumor-associated macrophages via NEDD4-mediated CSF1R degradation by targeting USP18.
  14. Mitochondrial quality control pathways sense mitochondrial protein import.
  15. baz-2 enhances systemic proteostasis in vivo by regulating acetylcholine metabolism.
  16. Lysosomal microautophagy: an emerging dimension in mammalian autophagy.
  17. Membrane contacts with the endoplasmic reticulum modulate plastid morphology and behaviour.
  18. The yeast dynamin-like GTPase Vps1 mediates Atg9 transport to the phagophore assembly site in Saccharomyces cerevisiae.
  19. Glucose stress causes mRNA retention in nuclear Nab2 condensates.
  20. Transcriptional reprogramming at the intersection of the heat shock response and proteostasis.
  21. Accelerating multiplexed profiling of protein-ligand interactions: High-throughput plate-based reactive cysteine profiling with minimal input.
  22. Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity.
  23. Atg15 is a vacuolar phospholipase that disintegrates organelle membranes.
  24. Dissecting ubiquitylation and DNA damage response pathways in the yeast Saccharomyces cerevisiae using a proteome-wide approach.
  25. BCL-2 Modulates IRE1α Activation to Attenuate ER Stress and Pulmonary Fibrosis.
  26. hGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.
  27. Distinct secretomes in p16- and p21- positive senescent cells across tissues.
  28. The energetic and allosteric landscape for KRAS inhibition.
  29. Machine-learning-based Structural Analysis of Interactions between Antibodies and Antigens.
  1. Mol Cell. 2023 Dec 08. pii: S1097-2765(23)00972-3. [Epub ahead of print]
    Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies.
    Jakub Chrustowicz, Dawafuti Sherpa, Jerry Li, Christine R Langlois, Eleftheria C Papadopoulou, D Tung Vu, Laura A Hehl, Özge Karayel, Viola Beier, Susanne von Gronau, Judith Müller, J Rajan Prabu, Matthias Mann, Gary Kleiger, Arno F Alpi, Brenda A Schulman.
      Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryoelectron microscopy (cryo-EM), biochemistry, and cell biology reveal this exquisitely specific E3-E2 pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 multisite phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data show that phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, rigidifies the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.
    Keywords:  CK2; CTLH complex; E2 ubiquitin conjugating enzyme; E3 ubiquitin ligase; GID complex; UBE2H; Ubc8; cryo-EM; phosphorylation; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.027
  2. Mol Cell. 2023 Dec 11. pii: S1097-2765(23)00961-9. [Epub ahead of print]
    Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.
    Christian Renz, Evrydiki Asimaki, Cindy Meister, Véronique Albanèse, Kirill Petriukov, Nils C Krapoth, Sabrina Wegmann, Hans-Peter Wollscheid, Ronald P Wong, Amitkumar Fulzele, Jia-Xuan Chen, Sébastien Léon, Helle D Ulrich.
      The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
    Keywords:  E3 ligase; EGFR; degron; endocytosis; monoubiquitylation; polyubiquitin chain linkage; proteostasis; synthetic biology; targeted protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.016
  3. Trends Plant Sci. 2023 Dec 14. pii: S1360-1385(23)00388-6. [Epub ahead of print]
    Unlocking the plant ER stress code: IRE1-proteasome signaling cohort takes the lead.
    Vishal Varshney, Jawahar Singh, Vishnu Mishra.
      In the intricate landscape of cellular function, proper protein folding is pivotal for cellular processes, particularly within the endoplasmic reticulum (ER). In a recent study, Ko et al. reveal a signaling role for inositol-requiring enzyme 1 (IRE1) in ER stress and identify PHOSPHATASE TYPE 2CA (PP2CA)-INTERACTING RING FINGER PROTEIN 1 (PIR1) as a crucial plant-specific regulator, balancing the unfolded protein response (UPR) and ubiquitin-proteasome system (UPS) by modulating ABI5 stability, unveiling intricate stress response connections.
    Keywords:  ABI5; E3 ligase; ER stress; PIR1; ubiquitin-proteasome system; unfolded protein response
    DOI:  https://doi.org/10.1016/j.tplants.2023.12.003
  4. Nat Commun. 2023 Dec 15. 14(1): 8363
    Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons.
    Ka-Yiu Edwin Kong, Susmitha Shankar, Frank Rühle, Anton Khmelinskii.
      Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C‑terminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.
    DOI:  https://doi.org/10.1038/s41467-023-44096-z
  5. Autophagy Rep. 2022 Aug 09. 1(1): 345-367
    The lipid flippase Drs2 regulates anterograde transport of Atg9 during autophagy.
    Franziska Kriegenburg, Wouter Huiting, Fleur van Buuren-Broek, Emma Zwilling, Ralph Hardenberg, Muriel Mari, Claudine Kraft, Fulvio Reggiori.
      Macroautophagy/autophagy is a conserved catabolic pathway during which cellular material is sequestered within newly formed double-membrane vesicles called autophagosomes and delivered to the lytic compartment of eukaryotic cells for degradation. Autophagosome biogenesis depends on the core autophagy-related (Atg) machinery, and involves a massive supply and remodelling of membranes. To gain insight into the lipid remodelling mechanisms during autophagy, we have systematically investigated whether lipid flippases are required for this pathway in the yeast Saccharomyces cerevisiae. We found that the flippase Drs2, which transfers phosphatidylserine and phosphatidylethanolamine from the lumenal to the cytosolic leaflet of the limiting membrane at the trans-Golgi network, is required for normal progression of autophagy. We also show that Drs2 is important for the trafficking of the core Atg protein Atg9. Atg9 is a transmembrane protein important for autophagosome biogenesis and its anterograde transport from its post-Golgi reservoirs to the site of autophagosome formation is severely impaired in the absence of Drs2. Thus, our results identify a novel autophagy player and highlight that membrane asymmetry regulates early autophagy steps.
    Keywords:  Aminophospholipid; Atg protein; autophagosome; flippase; lipid asymmetry; phagophore; phagophore assembly site
    DOI:  https://doi.org/10.1080/27694127.2022.2104781
  6. Nat Chem. 2023 Dec 18.
    Proteolysis-targeting chimeras with reduced off-targets.
    Tuan M Nguyen, Vedagopuram Sreekanth, Arghya Deb, Praveen Kokkonda, Praveen K Tiwari, Katherine A Donovan, Veronika Shoba, Santosh K Chaudhary, Jaron A M Mercer, Sophia Lai, Ananthan Sadagopan, Max Jan, Eric S Fischer, David R Liu, Benjamin L Ebert, Amit Choudhary.
      Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a high-throughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.
    DOI:  https://doi.org/10.1038/s41557-023-01379-8
  7. Nat Commun. 2023 Dec 19. 14(1): 8368
    NEMO reshapes the α-Synuclein aggregate interface and acts as an autophagy adapter by co-condensation with p62.
    Nikolas Furthmann, Verian Bader, Lena Angersbach, Alina Blusch, Simran Goel, Ana Sánchez-Vicente, Laura J Krause, Sarah A Chaban, Prerna Grover, Victoria A Trinkaus, Eva M van Well, Maximilian Jaugstetter, Kristina Tschulik, Rune Busk Damgaard, Carsten Saft, Gisa Ellrichmann, Ralf Gold, Arend Koch, Benjamin Englert, Ana Westenberger, Christine Klein, Lisa Jungbluth, Carsten Sachse, Christian Behrends, Markus Glatzel, F Ulrich Hartl, Ken Nakamura, Chadwick W Christine, Eric J Huang, Jörg Tatzelt, Konstanze F Winklhofer.
      NEMO is a ubiquitin-binding protein which regulates canonical NF-κB pathway activation in innate immune signaling, cell death regulation and host-pathogen interactions. Here we identify an NF-κB-independent function of NEMO in proteostasis regulation by promoting autophagosomal clearance of protein aggregates. NEMO-deficient cells accumulate misfolded proteins upon proteotoxic stress and are vulnerable to proteostasis challenges. Moreover, a patient with a mutation in the NEMO-encoding IKBKG gene resulting in defective binding of NEMO to linear ubiquitin chains, developed a widespread mixed brain proteinopathy, including α-synuclein, tau and TDP-43 pathology. NEMO amplifies linear ubiquitylation at α-synuclein aggregates and promotes the local concentration of p62 into foci. In vitro, NEMO lowers the threshold concentrations required for ubiquitin-dependent phase transition of p62. In summary, NEMO reshapes the aggregate surface for efficient autophagosomal clearance by providing a mobile phase at the aggregate interphase favoring co-condensation with p62.
    DOI:  https://doi.org/10.1038/s41467-023-44033-0
  8. Nat Commun. 2023 Dec 15. 14(1): 8364
    The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways.
    Rayene Berkane, Hung Ho-Xuan, Marius Glogger, Pablo Sanz-Martinez, Lorène Brunello, Tristan Glaesner, Santosh Kumar Kuncha, Katharina Holzhüter, Sara Cano-Franco, Viviana Buonomo, Paloma Cabrerizo-Poveda, Ashwin Balakrishnan, Georg Tascher, Koraljka Husnjak, Thomas Juretschke, Mohit Misra, Alexis González, Volker Dötsch, Paolo Grumati, Mike Heilemann, Alexandra Stolz.
      Selective autophagy of the endoplasmic reticulum (ER), known as ER-phagy, is an important regulator of ER remodeling and essential to maintain cellular homeostasis during environmental changes. We recently showed that members of the FAM134 family play a critical role during stress-induced ER-phagy. However, the mechanisms on how they are activated remain largely unknown. In this study, we analyze phosphorylation of FAM134 as a trigger of FAM134-driven ER-phagy upon mTOR (mechanistic target of rapamycin) inhibition. An unbiased screen of kinase inhibitors reveals CK2 to be essential for FAM134B- and FAM134C-driven ER-phagy after mTOR inhibition. Furthermore, we provide evidence that ER-phagy receptors are regulated by ubiquitination events and that treatment with E1 inhibitor suppresses Torin1-induced ER-phagy flux. Using super-resolution microscopy, we show that CK2 activity is essential for the formation of high-density FAM134B and FAM134C clusters. In addition, dense clustering of FAM134B and FAM134C requires phosphorylation-dependent ubiquitination of FAM134B and FAM134C. Treatment with the CK2 inhibitor SGC-CK2-1 or mutation of FAM134B and FAM134C phosphosites prevents ubiquitination of FAM134 proteins, formation of high-density clusters, as well as Torin1-induced ER-phagy flux. Therefore, we propose that CK2-dependent phosphorylation of ER-phagy receptors precedes ubiquitin-dependent activation of ER-phagy flux.
    DOI:  https://doi.org/10.1038/s41467-023-44101-5
  9. Life Sci Alliance. 2024 Mar;pii: e202302338. [Epub ahead of print]7(3):
    Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.
    Sara Meril, Marcela Bahlsen, Miriam Eisenstein, Alon Savidor, Yishai Levin, Shani Bialik, Shmuel Pietrokovski, Adi Kimchi.
      Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.
    DOI:  https://doi.org/10.26508/lsa.202302338
  10. Nat Commun. 2023 Dec 19. 14(1): 8437
    Direct-to-biology, automated, nano-scale synthesis, and phenotypic screening-enabled E3 ligase modulator discovery.
    Zefeng Wang, Shabnam Shaabani, Xiang Gao, Yuen Lam Dora Ng, Valeriia Sapozhnikova, Philipp Mertins, Jan Krönke, Alexander Dömling.
      Thalidomide and its analogs are molecular glues (MGs) that lead to targeted ubiquitination and degradation of key cancer proteins via the cereblon (CRBN) E3 ligase. Here, we develop a direct-to-biology (D2B) approach for accelerated discovery of MGs. In this platform, automated, high throughput, and nano scale synthesis of hundreds of pomalidomide-based MGs was combined with rapid phenotypic screening, enabling an unprecedented fast identification of potent CRBN-acting MGs. The small molecules were further validated by degradation profiling and anti-cancer activity. This revealed E14 as a potent MG degrader targeting IKZF1/3, GSPT1 and 2 with profound effects on a panel of cancer cells. In a more generalized view, integration of automated, nanoscale synthesis with phenotypic assays has the potential to accelerate MGs discovery.
    DOI:  https://doi.org/10.1038/s41467-023-43614-3
  11. Proc Natl Acad Sci U S A. 2023 Dec 26. 120(52): e2308853120
    Listerin promotes cGAS protein degradation through the ESCRT pathway to negatively regulate cGAS-mediated immune response.
    Fei Qin, Baoshan Cai, Runyu Cao, Xuemei Bai, Jiahua Yuan, Yuling Zhang, Yaxing Liu, Tian Chen, Feng Liu, Wanwei Sun, Yi Zheng, Xiaopeng Qi, Wei Zhao, Bingyu Liu, Chengjiang Gao.
      The enzyme cyclic GMP-AMP synthase (cGAS) is a key sensor for detecting misplaced double-stranded DNA (dsDNA) of genomic, mitochondrial, and microbial origin. It synthesizes 2'3'-cGAMP, which in turn activates the stimulator of interferon genes pathway, leading to the initiation of innate immune responses. Here, we identified Listerin as a negative regulator of cGAS-mediated innate immune response. We found that Listerin interacts with cGAS on endosomes and promotes its K63-linked ubiquitination through recruitment of the E3 ligase TRIM27. The polyubiquitinated cGAS is then recognized by the endosomal sorting complexes required for transport machinery and sorted into endosomes for degradation. Listerin deficiency enhances the innate antiviral response to herpes simplex virus 1 infection. Genetic deletion of Listerin also deteriorates the neuroinflammation and the ALS disease progress in an ALS mice model; overexpression of Listerin can robustly ameliorate disease progression in ALS mice. Thus, our work uncovers a mechanism for cGAS regulation and suggests that Listerin may be a promising therapeutic target for ALS disease.
    Keywords:  ALS; ESCRT; antiviral innate immunity; cGAS; listerin
    DOI:  https://doi.org/10.1073/pnas.2308853120
  12. Mol Cell. 2023 Dec 21. pii: S1097-2765(23)00970-X. [Epub ahead of print]83(24): 4509-4523.e11
    Subcytoplasmic location of translation controls protein output.
    Ellen L Horste, Mervin M Fansler, Ting Cai, Xiuzhen Chen, Sibylle Mitschka, Gang Zhen, Flora C Y Lee, Jernej Ule, Christine Mayr.
      The cytoplasm is highly compartmentalized, but the extent and consequences of subcytoplasmic mRNA localization in non-polarized cells are largely unknown. We determined mRNA enrichment in TIS granules (TGs) and the rough endoplasmic reticulum (ER) through particle sorting and isolated cytosolic mRNAs by digitonin extraction. When focusing on genes that encode non-membrane proteins, we observed that 52% have transcripts enriched in specific compartments. Compartment enrichment correlates with a combinatorial code based on mRNA length, exon length, and 3' UTR-bound RNA-binding proteins. Compartment-biased mRNAs differ in the functional classes of their encoded proteins: TG-enriched mRNAs encode low-abundance proteins with strong enrichment of transcription factors, whereas ER-enriched mRNAs encode large and highly expressed proteins. Compartment localization is an important determinant of mRNA and protein abundance, which is supported by reporter experiments showing that redirecting cytosolic mRNAs to the ER increases their protein expression. In summary, the cytoplasm is functionally compartmentalized by local translation environments.
    Keywords:  3′ UTR; CDS exon length; RNA-binding proteins; TIAL1; TIS granules; TIS11B; condensates; cytoplasmic organization; endoplasmic reticulum; gene architecture; mRNA length; mRNA localization; spatial regulation of protein synthesis; translation environment
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.025
  13. Cell Rep. 2023 Dec 13. pii: S2211-1247(23)01572-3. [Epub ahead of print]42(12): 113560
    Reprogramming of tumor-associated macrophages via NEDD4-mediated CSF1R degradation by targeting USP18.
    Sayuri Miyauchi, Kei-Ichiro Arimoto, Mengdan Liu, Yue Zhang, Dong-Er Zhang.
      Tumor-associated myeloid cells modulate the tumor microenvironment and affect tumor progression. Type I interferon (IFN-I) has multiple effects on tumors and immune response, and ubiquitin-specific peptidase 18 (USP18) functions as a negative regulator of IFN-I signal transduction. This study aims to examine the function of IFN-I in myeloid cells during tumor progression. Here, we show that deletion of USP18 in myeloid cells suppresses tumor progression. Enhanced IFN-I signaling and blocked USP18 expression prompt downregulation of colony stimulating factor 1 receptor (CSF1R) and polarization of tumor-associated macrophages toward pro-inflammatory phenotypes. Further in vitro experiments reveal that downregulation of CSF1R is mediated by ubiquitin-proteasome degradation via E3 ligase neural precursor cell-expressed, developmentaly downregulated 4 (NEDD4) and the IFN-induced increase in ubiquitin E2 ubiquitin-conjugating enzyme H5. USP18 impairs ubiquitination and subsequent degradation of CSF1R by interrupting NEDD4 binding to CSF1R. These results reveal a previously unappreciated role of IFN-I in macrophage polarization by regulating CSF1R via USP18 and suggest targeting USP18 in myeloid-lineage cells as an effective strategy for IFN-based therapies.
    Keywords:  CP: Cancer; CSF1R; NEDD4; USP18; macrophage polarization; tumor microenvironment; tumor-associated macrophages; type I interferon; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2023.113560
  14. Trends Endocrinol Metab. 2023 Dec 15. pii: S1043-2760(23)00243-6. [Epub ahead of print]
    Mitochondrial quality control pathways sense mitochondrial protein import.
    Laurie P Lee-Glover, Timothy E Shutt.
      Mitochondrial quality control (MQC) mechanisms are required to maintain a functional proteome, which enables mitochondria to perform a myriad of important cellular functions from oxidative phosphorylation to numerous other metabolic pathways. Mitochondrial protein homeostasis begins with the import of over 1000 nuclear-encoded mitochondrial proteins and the synthesis of 13 mitochondrial DNA-encoded proteins. A network of chaperones and proteases helps to fold new proteins and degrade unnecessary, damaged, or misfolded proteins, whereas more extensive damage can be removed by mitochondrial-derived vesicles (MDVs) or mitochondrial autophagy (mitophagy). Here, focusing on mechanisms in mammalian cells, we review the importance of mitochondrial protein import as a sentinel of mitochondrial function that activates multiple MQC mechanisms when impaired.
    Keywords:  mitochondria; mitochondrial protein import; mitochondrial quality control; mitochondrial unfolded protein response; mitochondrial-derived vesicles; mitophagy
    DOI:  https://doi.org/10.1016/j.tem.2023.11.004
  15. Cell Rep. 2023 Dec 14. pii: S2211-1247(23)01589-9. [Epub ahead of print]42(12): 113577
    baz-2 enhances systemic proteostasis in vivo by regulating acetylcholine metabolism.
    Christian Gallrein, Ashley B Williams, David H Meyer, Jan-Erik Messling, Antonio Garcia, Björn Schumacher.
      Neurodegenerative disorders, such as Alzheimer's disease (AD) or Huntington's disease (HD), are linked to protein aggregate neurotoxicity. According to the "cholinergic hypothesis," loss of acetylcholine (ACh) signaling contributes to the AD pathology, and therapeutic restoration of ACh signaling is a common treatment strategy. How disease causation and the effect of ACh are linked to protein aggregation and neurotoxicity remains incompletely understood, thus limiting the development of more effective therapies. Here, we show that BAZ-2, the Caenorhabditis elegans ortholog of human BAZ2B, limits ACh signaling. baz-2 mutations reverse aggregation and toxicity of amyloid-beta as well as polyglutamine peptides, thereby restoring health and lifespan in nematode models of AD and HD, respectively. The neuroprotective effect of Δbaz-2 is mediated by choline acetyltransferase, phenocopied by ACh-esterase depletion, and dependent on ACh receptors. baz-2 reduction or ectopic ACh treatment augments proteostasis via induction of the endoplasmic reticulum unfolded protein response and the ubiquitin proteasome system.
    Keywords:  Alzheimer's disease; CP: Metabolism; CP: Neuroscience; acetylcholine; amyloid-beta; baz-2; neurodegenerative disorders; non-cell-autonomous signaling; proteostasis regulation; ubiquitin proteasome system; unfolded protein response in the endoplasmic reticulum
    DOI:  https://doi.org/10.1016/j.celrep.2023.113577
  16. Trends Cell Biol. 2023 Dec 15. pii: S0962-8924(23)00238-6. [Epub ahead of print]
    Lysosomal microautophagy: an emerging dimension in mammalian autophagy.
    Yoshihiko Kuchitsu, Tomohiko Taguchi.
      Autophagy is a self-catabolic process through which cellular components are delivered to lysosomes for degradation. There are three types of autophagy, i.e., macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. In macroautophagy, a portion of the cytoplasm is wrapped by the autophagosome, which then fuses with lysosomes and delivers the engulfed cytoplasm for degradation. In CMA, the translocation of cytosolic substrates to the lysosomal lumen is directly across the limiting membrane of lysosomes. In microautophagy, lytic organelles, including endosomes or lysosomes, take up a portion of the cytoplasm directly. Although macroautophagy has been investigated extensively, microautophagy has received much less attention. Nonetheless, it has become evident that microautophagy plays a variety of cellular roles from yeast to mammals. Here we review the very recent updates of microautophagy. In particular, we focus on the feature of the degradative substrates and the molecular machinery that mediates microautophagy.
    Keywords:  ESCRT complex; K63-linked ubiquitination; lysosome; microautophagy; vacuole
    DOI:  https://doi.org/10.1016/j.tcb.2023.11.005
  17. Front Plant Sci. 2023 ;14 1293906
    Membrane contacts with the endoplasmic reticulum modulate plastid morphology and behaviour.
    Jaideep Mathur, Thomas Kadanthottu Kunjumon, Alena Mammone, Neeta Mathur.
      Plastid behaviour often occurs in tandem with endoplasmic reticulum (ER) dynamics. In order to understand the underlying basis for such linked behaviour we have used time-lapse imaging-based analysis of plastid movement and pleomorphy, including the extension and retraction of stromules. Stable transgenic plants that simultaneously express fluorescent fusion proteins targeted to the plastid stroma, and the ER along with BnCLIP1-eGFP, an independent plastid envelope localized membrane contact site (MCS) marker were utilized. Our experiments strongly suggest that transient MCS formed between the plastid envelope and the ER are responsible for their concomitant behaviour.
    Keywords:  BnCLIP1; chloroplasts; endoplasmic reticulum; lipases; membrane-contact-sites; stromules
    DOI:  https://doi.org/10.3389/fpls.2023.1293906
  18. Autophagy Rep. 2023 Aug 17. 2(1): 2247309
    The yeast dynamin-like GTPase Vps1 mediates Atg9 transport to the phagophore assembly site in Saccharomyces cerevisiae.
    Yan Hu, Fulvio Reggiori.
      Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.
    Keywords:  Autophagosome; DNM2; Saccharomyces cerevisiae; autophagy; phagophore; traffic
    DOI:  https://doi.org/10.1080/27694127.2023.2247309
  19. Cell Rep. 2023 Dec 18. pii: S2211-1247(23)01605-4. [Epub ahead of print]43(1): 113593
    Glucose stress causes mRNA retention in nuclear Nab2 condensates.
    Stephanie Heinrich, Maria Hondele, Désirée Marchand, Carina Patrizia Derrer, Mostafa Zedan, Alexandra Oswald, Liliana Malinovska, Federico Uliana, Sarah Khawaja, Roberta Mancini, David Grunwald, Karsten Weis.
      Nuclear mRNA export via nuclear pore complexes is an essential step in eukaryotic gene expression. Although factors involved in mRNA transport have been characterized, a comprehensive mechanistic understanding of this process and its regulation is lacking. Here, we use single-RNA imaging in yeast to show that cells use mRNA retention to control mRNA export during stress. We demonstrate that, upon glucose withdrawal, the essential RNA-binding factor Nab2 forms RNA-dependent condensate-like structures in the nucleus. This coincides with a reduced abundance of the DEAD-box ATPase Dbp5 at the nuclear pore. Depleting Dbp5, and consequently blocking mRNA export, is necessary and sufficient to trigger Nab2 condensation. The state of Nab2 condensation influences the extent of nuclear mRNA accumulation and can be recapitulated in vitro, where Nab2 forms RNA-dependent liquid droplets. We hypothesize that cells use condensation to regulate mRNA export and control gene expression during stress.
    Keywords:  CP: Cell biology; DEAD-box ATPase Dbp5; Nab2; RNA; condensation; glucose stress; mRNA export
    DOI:  https://doi.org/10.1016/j.celrep.2023.113593
  20. Mol Cell. 2023 Dec 11. pii: S1097-2765(23)00969-3. [Epub ahead of print]
    Transcriptional reprogramming at the intersection of the heat shock response and proteostasis.
    Jenny C Pessa, Jenny Joutsen, Lea Sistonen.
      Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
    Keywords:  HSF; HSR; PTM; Pol II; RNA polymerase II; chromatin; enhancer; heat shock factor; heat shock response; post-translational modification; proteostasis; transcription; transcription factor
    DOI:  https://doi.org/10.1016/j.molcel.2023.11.024
  21. Cell Chem Biol. 2023 Dec 12. pii: S2451-9456(23)00429-4. [Epub ahead of print]
    Accelerating multiplexed profiling of protein-ligand interactions: High-throughput plate-based reactive cysteine profiling with minimal input.
    Ka Yang, Rebecca L Whitehouse, Shane L Dawson, Lu Zhang, Jeffrey G Martin, Douglas S Johnson, Joao A Paulo, Steven P Gygi, Qing Yu.
      Chemoproteomics has made significant progress in investigating small-molecule-protein interactions. However, the proteome-wide profiling of cysteine ligandability remains challenging to adapt for high-throughput applications, primarily due to a lack of platforms capable of achieving the desired depth using low input in 96- or 384-well plates. Here, we introduce a revamped, plate-based platform which enables routine interrogation of either ∼18,000 or ∼24,000 reactive cysteines based on starting amounts of 10 or 20 μg, respectively. This represents a 5-10X reduction in input and 2-3X improved coverage. We applied the platform to screen 192 electrophiles in the native HEK293T proteome, mapping the ligandability of 38,450 reactive cysteines from 8,274 human proteins. We further applied the platform to characterize new cellular targets of established drugs, uncovering that ARS-1620, a KRASG12C inhibitor, binds to and inhibits an off-target adenosine kinase ADK. The platform represents a major step forward to high-throughput proteome-wide evaluation of reactive cysteines.
    Keywords:  ABPP; TMT; chemoproteomics; covalent electrophile; cysteine; high throughput
    DOI:  https://doi.org/10.1016/j.chembiol.2023.11.015
  22. bioRxiv. 2023 Dec 09. pii: 2023.12.09.569604. [Epub ahead of print]
    Genome-Wide Profiling of tRNA Using an Unexplored Reverse Transcriptase with High Processivity.
    Yuko Nakano, Howard Gamper, Henri McGuigan, Sunita Maharjan, Zhiyi Sun, Keerthana Krishnan, Erbay Yigit, Nan-Sheng Li, Joseph A Piccirilli, Ralph Kleiner, Nicole Nichols, Ya-Ming Hou.
      Monitoring the dynamic changes of cellular tRNA pools is challenging, due to the extensive post-transcriptional modifications of individual species. The most critical component in tRNAseq is a processive reverse transcriptase (RT) that can read through each modification with high efficiency. Here we show that the recently developed group-II intron RT Induro has the processivity and efficiency necessary to profile tRNA dynamics. Using our Induro-tRNAseq, simpler and more comprehensive than the best methods to date, we show that Induro progressively increases readthrough of tRNA over time and that the mechanism of increase is selective removal of RT stops, without altering the misincorporation frequency. We provide a parallel dataset of the misincorporation profile of Induro relative to the related TGIRT RT to facilitate the prediction of non-annotated modifications. We report an unexpected modification profile among human proline isoacceptors, absent from mouse and lower eukaryotes, that indicates new biology of decoding proline codons.
    DOI:  https://doi.org/10.1101/2023.12.09.569604
  23. Cell Rep. 2023 Dec 15. pii: S2211-1247(23)01579-6. [Epub ahead of print] 113567
    Atg15 is a vacuolar phospholipase that disintegrates organelle membranes.
    Yasunori Watanabe, Yurina Iwasaki, Kyoka Sasaki, Chie Motono, Kenichiro Imai, Kuninori Suzuki.
      Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar/intralysosomal autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here, we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity. The activity of CtAtg15(73-475) was markedly elevated by limited digestion. We inserted the human rhinovirus 3C protease recognition sequence and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). Our molecular dynamics simulation suggested that the cleavage facilitated conformational change around the active center of CtAtg15, resulting in an exposed state. We confirmed that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC of S. cerevisiae were disintegrated by CtAtg15. This study suggests Atg15 plays a role in disrupting any organelle membranes delivered to vacuoles by autophagy.
    Keywords:  CP: Cell biology; autophagosome; autophagy; autophagy-related 15; cytoplasm-to-vacuole targeting; molecular dynamics simulation; organelle membrane; phospholipase; phospholipid; vacuole; yeast
    DOI:  https://doi.org/10.1016/j.celrep.2023.113567
  24. Mol Cell Proteomics. 2023 Dec 13. pii: S1535-9476(23)00206-2. [Epub ahead of print] 100695
    Dissecting ubiquitylation and DNA damage response pathways in the yeast Saccharomyces cerevisiae using a proteome-wide approach.
    Ewa Blaszczak, Emeline Pasquier, Gaëlle Le Dez, Adrian Odrzywolski, Natalia Lazarewicz, Audrey Brossard, Emilia Fornal, Piotr Moskalek, Robert Wysocki, Gwenaël Rabut.
      In response to genotoxic stress, cells evolved with a complex signaling network referred to as the DNA damage response (DDR). It is now well established that the DDR depends upon various post-translational modifications; among them, ubiquitylation plays a key regulatory role. Here, we profiled ubiquitylation in response to the DNA alkylating agent methyl methanesulfonate (MMS) in the budding yeast Saccharomyces cerevisiae using quantitative proteomics. To discover new proteins ubiquitylated upon DNA replication stress, we used stable isotope labeling by amino acids in cell culture (SILAC) followed by an enrichment of ubiquitylated peptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, we identified 1853 ubiquitylated proteins, including 473 proteins that appeared upregulated more than 2-fold in response to MMS treatment. This enabled us to localize 519 ubiquitylation sites potentially regulated upon MMS in 435 proteins. We demonstrated that the overexpression of some of these proteins renders the cells sensitive to MMS. We also assayed the abundance change upon MMS treatment of a selection of yeast nuclear proteins. Several of them were differentially regulated upon MMS treatment. These findings corroborate the important role of ubiquitin-proteasome-mediated degradation in regulating the DDR.
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100695
  25. Am J Respir Cell Mol Biol. 2023 Dec 20.
    BCL-2 Modulates IRE1α Activation to Attenuate ER Stress and Pulmonary Fibrosis.
    Claude Jourdan Le Saux, Tsung Che Ho, Alexis M Brumwell, Jaymin J Kathiriya, Ying Wei, Jun-Wei B Hughes, Kiana Garakani, Kamran Atabai, Vincent C Auyeung, Feroz R Papa, Harold A Chapman.
      BCL-2 family members are known to be pro-survival agents in numerous biological settings. Here we provide evidence that in injury and repair processes in lungs, BCL-2 mainly acts to attenuate endoplasmic reticulum (ER) stress and limit extracellular matrix (ECM) accumulation. Days after intratracheal bleomycin mice lose a fraction of their alveolar type II epithelium from terminal ER stress driven by activation of the critical ER sensor and stress effector IRE1-α. This fraction is dramatically increased by BCL-2 inhibition because IRE1-α activation is dependent on its physical association with the BCL-2-pro-apoptotic family member BAX and we found BCL-2 to disrupt this association in vitro. In vivo, Navitoclax (a BCL-2/BCL-xL inhibitor) given 15-21 days after bleomycin challenge evoked strong activation of IRE-1α in mesenchymal cells and markers of ER stress but not apoptosis. Remarkably, following BCL-2 inhibition, bleomycin-exposed mice demonstrated persistent collagen accumulation at day 42 compared to resolution in controls. Enhanced fibrosis proved to be due to the RNAase activity of IRE1a downregulating MRC2 mRNA and protein, a mediator of collagen turnover. The critical role of MRC2 was confirmed in PCLS cultures of day 42 lungs from bleomycin-exposed WT and MRC2 null mice. Soluble and tissue collagens accumulated in PCLS cultures from Navitoclax-treated, bleomycin challenged mice compared to controls, nearly identical to that of challenged but untreated MRC2 nulls. Thus, apart from mitochondrial-based anti-apoptosis, BCL-2 functions to attenuate ER stress responses, fostering tissue homeostasis and injury repair.
    Keywords:  Bcl-2; ER stress, fibrosis, apoptosis, fibroblast, alveolar epithelial cells
    DOI:  https://doi.org/10.1165/rcmb.2023-0109OC
  26. J Cell Biol. 2024 Feb 05. pii: e202304030. [Epub ahead of print]223(2):
    hGRAD: A versatile "one-fits-all" system to acutely deplete RNA binding proteins from condensates.
    Benjamin Arnold, Ricarda J Riegger, Ellen Kazumi Okuda, Irena Slišković, Mario Keller, Cem Bakisoglu, François McNicoll, Kathi Zarnack, Michaela Müller-McNicoll.
      Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and crossregulation. They are highly abundant and many localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a "one-fits-all" plasmid, and integration into any cell line with endogenously GFP-tagged proteins allows for an inducible, rapid, and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2, and NONO are completely cleared from nuclear speckles and paraspeckles within 2 h. hGRAD works in various cell types, is more efficient than previous methods, and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered transient transcript changes, compensatory mechanisms, and an effect of SRSF5 on transcript stability.
    DOI:  https://doi.org/10.1083/jcb.202304030
  27. bioRxiv. 2023 Dec 05. pii: 2023.12.05.569858. [Epub ahead of print]
    Distinct secretomes in p16- and p21- positive senescent cells across tissues.
    Dominik Saul, Diana Jurk, Madison L Doolittle, Robyn Laura Kosinsky, David G Monroe, Nathan K LeBrasseur, Paul D Robbins, Laura J Niedernhofer, Sundeep Khosla, João F Passos.
      Senescent cells drive age-related tissue dysfunction via the induction of a chronic senescence-associated secretory phenotype (SASP). The cyclin-dependent kinase inhibitors p21 Cip1 and p16 Ink4a have long served as markers of cellular senescence. However, their individual roles remain incompletely elucidated. Thus, we conducted a comprehensive examination of multiple single-cell RNA sequencing (scRNA-seq) datasets spanning both murine and human tissues during aging. Our analysis revealed that p21 Cip1 and p16 Ink4a transcripts demonstrate significant heterogeneity across distinct cell types and tissues, frequently exhibiting a lack of co-expression. Moreover, we identified tissue-specific variations in SASP profiles linked to p21 Cip1 or p16 Ink4a expression. Our study underscores the extraordinary diversity of cellular senescence and the SASP, emphasizing that these phenomena are inherently cell- and tissue-dependent. However, a few SASP factors consistently contribute to a shared "core" SASP. These findings highlight the need for a more nuanced investigation of senescence across a wide array of biological contexts.
    DOI:  https://doi.org/10.1101/2023.12.05.569858
  28. Nature. 2023 Dec 18.
    The energetic and allosteric landscape for KRAS inhibition.
    Chenchun Weng, Andre J Faure, Albert Escobedo, Ben Lehner.
      Thousands of proteins have now been genetically-validated as therapeutic targets in hundreds of human diseases1. However, very few have actually been successfully targeted and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions: direct inhibition of binding interfaces is difficult, requiring the identification of allosteric sites. However, most proteins have no known allosteric sites and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the impact of >26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants allowed us to perform biophysical measurements at scale, inferring >22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central beta sheet of KRAS and multiple surface pockets are genetically-validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.
    DOI:  https://doi.org/10.1038/s41586-023-06954-0
  29. bioRxiv. 2023 Dec 08. pii: 2023.12.06.570397. [Epub ahead of print]
    Machine-learning-based Structural Analysis of Interactions between Antibodies and Antigens.
    Grace Zhang, Zhaoqian Su, Tom Zhang, Yinghao Wu.
      Computational analysis of paratope-epitope interactions between antibodies and their corresponding antigens can facilitate our understanding of the molecular mechanism underlying humoral immunity and boost the design of new therapeutics for many diseases. The recent breakthrough in artificial intelligence has made it possible to predict protein-protein interactions and model their structures. Unfortunately, detecting antigen-binding sites associated with a specific antibody is still a challenging problem. To tackle this challenge, we implemented a deep learning model to characterize interaction patterns between antibodies and their corresponding antigens. With high accuracy, our model can distinguish between antibody-antigen complexes and other types of protein-protein complexes. More intriguingly, we can identify antigens from other common protein binding regions with an accuracy of higher than 70% even if we only have the epitope information. This indicates that antigens have distinct features on their surface that antibodies can recognize. Additionally, our model was unable to predict the partnerships between antibodies and their particular antigens. This result suggests that one antigen may be targeted by more than one antibody and that antibodies may bind to previously unidentified proteins. Taken together, our results support the precision of antibody-antigen interactions while also suggesting positive future progress in the prediction of specific pairing.
    DOI:  https://doi.org/10.1101/2023.12.06.570397
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