bims-proteo Biomed News
on Proteostasis
Issue of 2021–07–11
forty-one papers selected by
Eric Chevet, INSERM
  1. Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β- selection.
  2. Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology.
  3. Avadomide Induces Degradation of ZMYM2 Fusion Oncoproteins in Hematologic Malignancies.
  4. Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.
  5. K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.
  6. ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6.
  7. Ribosome-associated quality control and CAT tailing.
  8. AgDD System: A Chemical Controllable Protein Aggregates in Cells.
  9. The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.
  10. Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1.
  11. Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.
  12. Assessment of mammalian endosomal microautophagy.
  13. Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.
  14. Binding Features and Functions of ATG3.
  15. Autophagic flux assessment by immunoblot.
  16. Friend or Foe: UCHL3 Mediated Carcinogenesis and Current Approaches in Small Molecule Inhibitors' Development.
  17. RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging.
  18. OASIS/CREB3L1 is a factor that responds to nuclear envelope stress.
  19. A model of the aged lung epithelium in idiopathic pulmonary fibrosis.
  20. Degradation of lipid droplets by chimeric autophagy-tethering compounds.
  21. c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability.
  22. Identification of a class of non-conventional ER-stress-response-derived immunogenic peptides.
  23. ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.
  24. Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.
  25. Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.
  26. Targeting autophagy in disease: established and new strategies.
  27. zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.
  28. Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.
  29. The E3 ubiquitin ligase TRIM31 is involved in cerebral ischemic injury by promoting degradation of TIGAR.
  30. Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation.
  31. Loss of TRIM21 alleviates cardiotoxicity by suppressing ferroptosis induced by the chemotherapeutic agent doxorubicin.
  32. Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes.
  33. Human AGR2 Deficiency Causes Mucus Barrier Dysfunction and Infantile Inflammatory Bowel Disease.
  34. SUMO Paralog Specific Functions Revealed through Systematic Analysis of Human Knockout Cell Lines and Gene Expression Data.
  35. Bortezomib Induces Anti-Multiple Myeloma Immune Response Mediated by cGAS/STING Pathway Activation.
  36. Molecular basis for RASSF10/NPM/RNF2 feedback cascade-mediated regulation of gastric cancer cell proliferation.
  37. The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.
  38. A quantitative yeast aging proteomics analysis reveals novel aging regulators.
  39. Chaperone-like protein DAY plays critical roles in photomorphogenesis.
  40. Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer.
  41. Large organellar changes occur during mild heat shock in yeast.
  1. Elife. 2021 Jul 09. pii: e69975. [Epub ahead of print]10
    Notch-induced endoplasmic reticulum-associated degradation governs mouse thymocyte β- selection.
    Xia Liu, Jingjing Yu, Longyong Xu, Katharine Umphred-Wilson, Fanglue Peng, Yao Ding, Brendan M Barton, Xiangdong Lv, Michael Y Zhao, Shengyi Sun, Yuning Hong, Ling Qi, Stanley Adoro, Xi Chen.
      Signals from the pre-T cell receptor and Notch coordinately instruct b-selection of CD4-CD8- double negative (DN) thymocytes to generate ab T cells in the thymus. However, how these signals ensure a high-fidelity proteome and safeguard the clonal diversification of the pre-selection TCR repertoire given the considerable translational activity imposed by b-selection is largely unknown. Here, we identify the endoplasmic reticulum (ER)-associated degradation (ERAD) machinery as a critical proteostasis checkpoint during b-selection. Expression of the SEL1L-HRD1 complex, the most conserved branch of ERAD, is directly regulated by the transcriptional activity of the Notch intracellular domain. Deletion of Sel1l impaired DN3 to DN4 thymocyte transition and severely impaired mouse ab T cell development. Mechanistically, Sel1l deficiency induced unresolved ER stress that triggered thymocyte apoptosis through the PERK pathway. Accordingly, genetically inactivating PERK rescued T cell development from Sel1l-deficient thymocytes. In contrast, IRE1a/XBP1 pathway was induced as a compensatory adaptation to alleviate Sel1l-deficiency induced ER stress. Dual loss of Sel1l and Xbp1 markedly exacerbated the thymic defect. Our study reveals a critical developmental signal controlled proteostasis mechanism that enforces T cell development to ensure a healthy adaptive immunity.
    Keywords:  developmental biology; immunology; inflammation; mouse
    DOI:  https://doi.org/10.7554/eLife.69975
  2. J Biol Chem. 2021 Jul 02. pii: S0021-9258(21)00737-7. [Epub ahead of print] 100937
    Nodal modulator (NOMO) is required to sustain endoplasmic reticulum morphology.
    Catherine Amaya, Christopher Jf Cameron, Swapnil C Devarkar, Sebastian Jh Seager, Mark B Gerstein, Yong Xiong, Christian Schlieker.
      The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for protein folding, lipid synthesis, and calcium homeostasis. Maintenance of ER structural integrity is crucial for proper function, but much remains to be learned about the molecular players involved. To identify proteins that support the structure of the ER, we performed a proteomic screen and identified Nodal modulator (NOMO), a widely conserved type I transmembrane protein of unknown function, with three nearly identical orthologs specified in the human genome. We found that overexpression of NOMO1 imposes a sheet morphology on the ER, while depletion of NOMO1 and its orthologs causes a collapse of ER morphology concomitant with the formation of membrane-delineated holes in the ER network positive for the lysosomal marker LAMP1. In addition, the levels of key players of autophagy including LC3-II and p62 strongly increase upon NOMO depletion. In vitro reconstitution of NOMO1 revealed a "beads on a string" structure likely representing consecutive immunoglobulin (Ig)-like domains. Extending NOMO1 by insertion of additional Ig folds results in a correlative increase in the ER intermembrane distance. Based on these observations and a genetic epistasis analysis including the known ER-shaping proteins Atlastin2 and Climp63, we propose a role for NOMO1 in the functional network of ER-shaping proteins.
    Keywords:  Endoplasmic reticulum; cell biology; imaging; membrane protein; single particle analysis; structural model
    DOI:  https://doi.org/10.1016/j.jbc.2021.100937
  3. Cancer Discov. 2021 May;2(3): 250-265
    Avadomide Induces Degradation of ZMYM2 Fusion Oncoproteins in Hematologic Malignancies.
    Aline Renneville, Jessica A Gasser, Daniel E Grinshpun, Pierre M Jean Beltran, Namrata D Udeshi, Mary E Matyskiela, Thomas Clayton, Marie McConkey, Kaushik Viswanathan, Alexander Tepper, Andrew A Guirguis, Rob S Sellar, Sophie Cotteret, Christophe Marzac, Véronique Saada, Stéphane De Botton, Jean-Jacques Kiladjian, Jean-Michel Cayuela, Mark Rolfe, Philip P Chamberlain, Steven A Carr, Benjamin L Ebert.
      Thalidomide analogues exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment. SIGNIFICANCE: We extend the potential clinical scope of thalidomide analogues by the identification of a novel avadomide-dependent CRL4CRBN substrate, ZMYM2. Avadomide induces ubiquitination and degradation of ZMYM2-FGFR1 and ZMYM2-FLT3, two chimeric oncoproteins involved in hematologic malignancies, providing a proof of concept for drug-induced degradation of transcription factor fusion proteins by thalidomide analogues.
    DOI:  https://doi.org/10.1158/2643-3230.BCD-20-0105
  4. J Am Chem Soc. 2021 Jul 08.
    Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.
    Willem den Besten, Kshitij Verma, Sayumi Yamazoe, Nicole Blaquiere, Wilson Phung, Anita Izrael-Tomasevic, Melinda M Mulvihill, Elizabeth Helgason, Sumit Prakash, Tatiana Goncharov, Domagoj Vucic, Erin Dueber, Wayne J Fairbrother, Ingrid Wertz, Kebing Yu, Steven T Staben.
      We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.
    DOI:  https://doi.org/10.1021/jacs.1c05269
  5. Nat Chem Biol. 2021 Jul 08.
    K29-linked ubiquitin signaling regulates proteotoxic stress response and cell cycle.
    Yuanyuan Yu, Qingyun Zheng, Satchal K Erramilli, Man Pan, Seongjin Park, Yuan Xie, Jingxian Li, Jingyi Fei, Anthony A Kossiakoff, Lei Liu, Minglei Zhao.
      Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.
    DOI:  https://doi.org/10.1038/s41589-021-00823-5
  6. EMBO J. 2021 Jul 05. e107403
    ER-anchored CRTH2 antagonizes collagen biosynthesis and organ fibrosis via binding LARP6.
    Shengkai Zuo, Bei Wang, Jiao Liu, Deping Kong, Hui Cui, Yaonan Jia, Chenyao Wang, Xin Xu, Guilin Chen, Yuanyang Wang, Linlin Yang, Kai Zhang, Ding Ai, Jie Du, Yujun Shen, Ying Yu.
      Excessive deposition of extracellular matrix, mainly collagen protein, is the hallmark of organ fibrosis. The molecular mechanisms regulating fibrotic protein biosynthesis are unclear. Here, we find that chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2), a plasma membrane receptor for prostaglandin D2, is trafficked to the endoplasmic reticulum (ER) membrane in fibroblasts in a caveolin-1-dependent manner. ER-anchored CRTH2 binds the collagen mRNA recognition motif of La ribonucleoprotein domain family member 6 (LARP6) and promotes the degradation of collagen mRNA in these cells. In line, CRTH2 deficiency increases collagen biosynthesis in fibroblasts and exacerbates injury-induced organ fibrosis in mice, which can be rescued by LARP6 depletion. Administration of CRTH2 N-terminal peptide reduces collagen production by binding to LARP6. Similar to CRTH2, bumetanide binds the LARP6 mRNA recognition motif, suppresses collagen biosynthesis, and alleviates bleomycin-triggered pulmonary fibrosis in vivo. These findings reveal a novel anti-fibrotic function of CRTH2 in the ER membrane via the interaction with LARP6, which may represent a therapeutic target for fibrotic diseases.
    Keywords:  CRTH2; LARP6; collagen synthesis; organ fibrosis
    DOI:  https://doi.org/10.15252/embj.2020107403
  7. Crit Rev Biochem Mol Biol. 2021 Jul 07. 1-18
    Ribosome-associated quality control and CAT tailing.
    Conor J Howard, Adam Frost.
      Translation is the set of mechanisms by which ribosomes decode genetic messages as they synthesize polypeptides of a defined amino acid sequence. While the ribosome has been honed by evolution for high-fidelity translation, errors are inevitable. Aberrant mRNAs, mRNA structure, defective ribosomes, interactions between nascent proteins and the ribosomal exit tunnel, and insufficient cellular resources, including low tRNA levels, can lead to functionally irreversible stalls. Life thus depends on quality control mechanisms that detect, disassemble and recycle stalled translation intermediates. Ribosome-associated Quality Control (RQC) recognizes aberrant ribosome states and targets their potentially toxic polypeptides for degradation. Here we review recent advances in our understanding of RQC in bacteria, fungi, and metazoans. We focus in particular on an unusual modification made to the nascent chain known as a "CAT tail", or Carboxy-terminal Alanine and Threonine tail, and the mechanisms by which ancient RQC proteins catalyze CAT-tail synthesis.
    Keywords:  CAT tails; LUCA; RQC; Ribosome-associated quality control; origins of life; peptide synthesis; protein translation; proteostasis
    DOI:  https://doi.org/10.1080/10409238.2021.1938507
  8. Methods Mol Biol. 2021 ;2312 277-285
    AgDD System: A Chemical Controllable Protein Aggregates in Cells.
    Yusuke Miyazaki.
      There are increasing evidence and growing interest in the relationship between protein aggregates/phase separation and various human diseases, especially neurodegenerative diseases. However, we do not entirely comprehend how aggregates generate or the clearance network of chaperones, proteasomes, ubiquitin ligases, and other factors interact with aggregates. Here, we describe chemically controllable systems compose with a genetically engineered cell and a small drug that enables us to rapidly induce protein aggregates' formation by withdrawing the small molecule. This trigger does not activate global stress responses induced by stimuli, such as proteasome inhibitors or heat shock. This method can produce aggregates in a specific compartment and diverse experimental systems, including live animals.
    Keywords:  Chaperones; Chemical biology; Destabilizing domain; Phase separation; Proteasome; Protein aggregates
    DOI:  https://doi.org/10.1007/978-1-0716-1441-9_16
  9. Front Mol Biosci. 2021 ;8 697913
    The Mitochondrial Hsp90 TRAP1 and Alzheimer's Disease.
    Françoise A Dekker, Stefan G D Rüdiger.
      Alzheimer's Disease (AD) is the most common form of dementia, characterised by intra- and extracellular protein aggregation. In AD, the cellular protein quality control (PQC) system is derailed and fails to prevent the formation of these aggregates. Especially the mitochondrial paralogue of the conserved Hsp90 chaperone class, tumour necrosis factor receptor-associated protein 1 (TRAP1), is strongly downregulated in AD, more than other major PQC factors. Here, we review molecular mechanism and cellular function of TRAP1 and subsequently discuss possible links to AD. TRAP1 is an interesting paradigm for the Hsp90 family, as it chaperones proteins with vital cellular function, despite not being regulated by any of the co-chaperones that drive its cytosolic paralogues. TRAP1 encloses late folding intermediates in a non-active state. Thereby, it is involved in the assembly of the electron transport chain, and it favours the switch from oxidative phosphorylation to glycolysis. Another key function is that it ensures mitochondrial integrity by regulating the mitochondrial pore opening through Cyclophilin D. While it is still unclear whether TRAP1 itself is a driver or a passenger in AD, it might be a guide to identify key factors initiating neurodegeneration.
    Keywords:  mitochondria; molecular chaperones; neurodegeneration; protein aggregation; protein folding; protein quality control; proteostasis
    DOI:  https://doi.org/10.3389/fmolb.2021.697913
  10. J Genet Genomics. 2021 May 26. pii: S1673-8527(21)00128-4. [Epub ahead of print]
    Molecular basis for the selective recognition and ubiquitination of centromeric histone H3 by yeast E3 ligase Psh1.
    Ning Zhou, Liuxin Shi, Shan Shan, Zheng Zhou.
      Centromeres are chromosomal loci marked by histone variant CenH3 (centromeric histone H3) and essential for genomic stability and cell division. The budding yeast E3 ubiquitin ligase Psh1 selectively recognizes the yeast CenH3 (Cse4) for ubiquitination and controls the cellular level of Cse4 for proteolysis, but the underlying mechanism remains largely unknown. Here, we show that Psh1 uses a Cse4-binding domain (CBD, residues 1-211) to interact with Cse4-H4 instead of H3-H4, yielding a dissociation constant (Kd) of 27 nM. Psh1 recognizes Cse4-specific residues in the L1 loop and α2 helix to ensure Cse4 binding and ubiquitination. We map the Psh1-binding region of Cse4-H4 and identify a wide range of Cse4-specific residues required for the Psh1-mediated Cse4 recognition and ubiquitination. Further analyses reveal that histone chaperone Scm3 can impair Cse4 ubiquitination by abrogating Psh1-Cse4 binding. Together, our study reveals a novel Cse4-binding mode distinct from those of known CenH3 chaperones and elucidates the mechanism by which Scm3 competes with Psh1 for Cse4 binding.
    Keywords:  CENP-A; Cse4; Psh1; Scm3; Selective recognition; Ubiquitination
    DOI:  https://doi.org/10.1016/j.jgg.2021.04.007
  11. Dev Cell. 2021 Jun 28. pii: S1534-5807(21)00516-5. [Epub ahead of print]
    Proteostasis failure and mitochondrial dysfunction leads to aneuploidy-induced senescence.
    Jery Joy, Lara Barrio, Celia Santos-Tapia, Daniela Romão, Nikolaos Nikiforos Giakoumakis, Marta Clemente-Ruiz, Marco Milán.
      Aneuploidy, an unbalanced number of chromosomes, is highly deleterious at the cellular level and leads to senescence, a stress-induced response characterized by permanent cell-cycle arrest and a well-defined associated secretory phenotype. Here, we use a Drosophila epithelial model to delineate the pathway that leads to the induction of senescence as a consequence of the acquisition of an aneuploid karyotype. Whereas aneuploidy induces, as a result of gene dosage imbalance, proteotoxic stress and activation of the major protein quality control mechanisms, near-saturation functioning of autophagy leads to compromised mitophagy, accumulation of dysfunctional mitochondria, and the production of radical oxygen species (ROS). We uncovered a role of c-Jun N-terminal kinase (JNK) in driving senescence as a consequence of dysfunctional mitochondria and ROS. We show that activation of the major protein quality control mechanisms and mitophagy dampens the deleterious effects of aneuploidy, and we identify a role of senescence in proteostasis and compensatory proliferation for tissue repair.
    Keywords:  Drosophila; aneuploidy; autophagy; chromosomal instability; mitochondrial dysfunction; mitophagy; proteotoxic stress; senescence; tissue repair
    DOI:  https://doi.org/10.1016/j.devcel.2021.06.009
  12. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30192-8. [Epub ahead of print]164 167-185
    Assessment of mammalian endosomal microautophagy.
    Gregory J Krause, Ana Maria Cuervo.
      Endosomal microautophagy (eMI) is a type of autophagy that allows for the selective uptake and degradation of cytosolic proteins in late endosome/multi-vesicular bodies (LE/MVB). This process starts with the recognition of a pentapeptide amino acid KFERQ-like targeting motif in the substrate protein by the hsc70 chaperone, which then enables binding and subsequent uptake of the protein into the LE/MVB compartment. The recognition of a KFERQ-like motif by hsc70 is the same initial step in chaperone-mediated autophagy (CMA), a form of selective autophagy that degrades the hsc70-targeted proteins in lysosomes in a LAMP-2A dependent manner. The shared step of substrate recognition by hsc70, originally identified for CMA, makes it now necessary to differentiate between the two pathways. Here, we detail biochemical and imaging-based methods to track eMI activity in vitro with isolated LE/MVBs and in cells in culture using fluorescent reporters and highlight approaches to distinguish whether a protein is a substrate of eMI or CMA.
    Keywords:  Autophagy; Chaperones; Late endosomes; Multi-vesicular bodies; Organelle isolation; Protein degradation; Protein targeting; Proteostasis
    DOI:  https://doi.org/10.1016/bs.mcb.2020.10.009
  13. Front Cell Dev Biol. 2021 ;9 669086
    Junctional ER Organization Affects Mechanotransduction at Cadherin-Mediated Adhesions.
    Michelle Joy-Immediato, Manuel J Ramirez, Mauricio Cerda, Yusuke Toyama, Andrea Ravasio, Pakorn Kanchanawong, Cristina Bertocchi.
      Cadherin-mediated adhesions (also known as adherens junctions) are adhesive complexes that connect neighboring cells in a tissue. While the role of the actin cytoskeleton in withstanding tension at these sites of contact is well documented, little is known about the involvement of microtubules and the associated endoplasmic reticulum (ER) network in cadherin mechanotransduction. Therefore, we investigated how the organization of ER extensions in close proximity of cadherin-mediated adhesions can affect such complexes, and vice versa. Here, we show that the extension of the ER to cadherin-mediated adhesions is tension dependent and appears to be cadherin-type specific. Furthermore, the different structural organization of the ER/microtubule network seems to affect the localization of ER-bound PTP1B at cadherin-mediated adhesions. This phosphatase is involved in the modulation of vinculin, a molecular clutch which enables differential engagement of the cadherin-catenin layer with the actomyosin cytoskeleton in response to tension. This suggests a link between structural organization of the ER/microtubule network around cadherin-specific adhesions, to control the mechanotransduction of adherens junctions by modulation of vinculin conformational state.
    Keywords:  cadherin-mediated adhesion; endoplasmic reticulum; mechanotransduction; microtubules; vinculin
    DOI:  https://doi.org/10.3389/fcell.2021.669086
  14. Front Cell Dev Biol. 2021 ;9 685625
    Binding Features and Functions of ATG3.
    Dongmei Fang, Huazhong Xie, Tao Hu, Hao Shan, Min Li.
      Autophagy is an evolutionarily conserved catabolic process that is essential for maintaining cellular, tissue, and organismal homeostasis. Autophagy-related (ATG) genes are indispensable for autophagosome formation. ATG3 is one of the key genes involved in autophagy, and its homologs are common in eukaryotes. During autophagy, ATG3 acts as an E2 ubiquitin-like conjugating enzyme in the ATG8 conjugation system, contributing to phagophore elongation. ATG3 has also been found to participate in many physiological and pathological processes in an autophagy-dependent manner, such as tumor occurrence and progression, ischemia-reperfusion injury, clearance of pathogens, and maintenance of organelle homeostasis. Intriguingly, a few studies have recently discovered the autophagy-independent functions of ATG3, including cell differentiation and mitosis. Here, we summarize the current knowledge of ATG3 in autophagosome formation, highlight its binding partners and binding sites, review its autophagy-dependent functions, and provide a brief introduction into its autophagy-independent functions.
    Keywords:  ATG3; autophagy; binding feature; cancer; function; homeostasis; phosphatidylethanolamine; post-translational modification
    DOI:  https://doi.org/10.3389/fcell.2021.685625
  15. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30188-6. [Epub ahead of print]164 63-72
    Autophagic flux assessment by immunoblot.
    Qi Wu, Ai-Ling Tian, Hui Pan, Oliver Kepp, Guido Kroemer.
      Autophagy is one of the main adaptive mechanisms to maintain cellular homeostasis in response to multiple stresses. During autophagy diverse cellular components such as damaged organelles or superfluous proteins are targeted for lysosomal degradation. Importantly, during the initiation of autophagy MAP1LC3B (better known as LC3) lipidates into the membrane of the forming phagophore, which facilitates the formation and lengthening of autophagosomes. In addition, the autophagy receptor SQSTM1 (better known as p62) selectively recruits various cargos to autophagosomes for lysosomal degradation. Both, the conversion of LC3 as well as the degradation of p62 can be assessed as means of monitoring autophagy. Here we detail a protocol for assessing these key events of the autophagic flux via immunoblot.
    Keywords:  Autophagic cargo; Drug discovery; LC3; Lipidation; Lysosomal degradation
    DOI:  https://doi.org/10.1016/bs.mcb.2020.10.005
  16. Curr Med Chem. 2021 Jul 07.
    Friend or Foe: UCHL3 Mediated Carcinogenesis and Current Approaches in Small Molecule Inhibitors' Development.
    Mona Ahmed Samy, Nada Mohamady Abd El Fatah, Safa Elsayed Yahia, Reem K Arafa.
      As cancer continues to be one of the leading causes of death, various cancer treatments are being developed, from traditional surgery to the more recent emergence of target therapy. However, therapy resistance is a restricting problem that needs to be overcome. Henceforth, the field of research shifts to new plausible drug targets, among which is the ubiquitin-proteasome system. This review is focused on the ubiquitin carboxyl-terminal hydrolase (UCH) protease family, which are members of Deubiquitinating enzymes (DUBs), specifically Ubiquitin carboxyl-terminal hydrolase L3 (UCHL3). DUBs regulate a broad array of regulatory processes, including cell-cycle progression, tissue development, and differentiation. DUBs are classified into seven subfamilies, including ubiquitin-specific proteases (USPs), JAB1/MPN/Mov34 metalloenzyme, ovarian tumor proteases (OTUs), Josephin and JAB1/MPN+(MJP), MIU-containing novel DUB (MINDY), zinc finger-containing ubiquitin peptidase 1 (ZUP1), and ubiquitin C-terminal hydrolases (UCHs). Having a significant role in tumorigenesis, UCHL3 is thus emerging as a therapeutic target. Knowing its involvement in cancer, it's important to understand the structure of UCHL3, its substrate specificity, and its interaction to pave the way for the development of potential inhibitors. This review covers several directions of proteasome inhibitors drug discovery and small molecule inhibitors development.
    Keywords:  Cancer; DUBs; Proteosome; UCHL3 inhibitors; Ubiquitin carboxyl-terminal hydrolase L3; biochemical structure and conformation; drug discovery; small molecule inhibitors
    DOI:  https://doi.org/10.2174/0929867328666210708085544
  17. Autophagy. 2021 Jul 07. 1-18
    RAB7 activity is required for the regulation of mitophagy in oocyte meiosis and oocyte quality control during ovarian aging.
    Xin Jin, Kehan Wang, Lu Wang, Wenwen Liu, Chi Zhang, Yuexin Qiu, Wei Liu, Huiyu Zhang, Dong Zhang, Zhixia Yang, Tinghe Wu, Jing Li.
      There is increasing evidence that mitophagy, a specialized form of autophagy to degrade and clear long-lived or damaged mitochondria, is impaired in aging and age-related disease. Previous study has demonstrated the obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy. However, it remains unknown whether mitophagy functions in oocyte and what's the regulatory mechanism in oocyte aging. In the study, when fully grown oocytes were treated with CCCP, an uncoupling agent to induce mitophagy, we found the activation of the PRKN-mediated mitophagy pathway accompanied the blockage of meiosis at metaphase I stage. Our result then demonstrated its association with the decreased activity of RAB7 and all the observed defects in CCCP treated oocytes could be effectively rescued by microinjection of mRNA encoding active RAB7Q67L or treatment with the RAB7 activator ML098. Further study indicated PRKN protein level as a rate-limiting factor to facilitate degradation of RAB7 and its GEF (guanine nucleotide exchange factor) complex CCZ1-MON1 through the ubiquitin-proteasome system. In GV oocytes collected during ovarian aging, we found the age-related increase of PINK1 and PRKN proteins and a significant decrease of RAB7 which resulted in defects of mitophagosome formation and the accumulation of damaged mitochondria. The age-related retardation of female fertility was improved after in vivo treatment of ML098. Thus, RAB7 activity is required to maintain the balance between mitophagy and chromosome stability and RAB7 activator is a good candidate to ameliorate age-related deterioration of oocyte quality.Abbreviations: ATG9: autophagy related 9A; ATP: adenosine triphosphate; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CCZ1: CCZ1 vacuolar protein trafficking and biogenesis associated; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAPs: GTPase-activating proteins; GEF: guanine nucleotide exchange factor; GV: germinal vesicle; GVBD: germinal vesicle breakdown; LAMP1: lysosomal-associated membrane protein 1; MI: metaphase I stage of meiosis; MII: metaphase II stage of meiosis; Mito: MitoTracker; mtDNA: mitochondrial DNA; MON1: MON1 homolog, secretory trafficking associated; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB7: RAB7, member RAS oncogene family; ROS: reactive oxygen species; TEM: transmission electron microscopy; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin, beta; UB: ubiquitin.
    Keywords:  Aging; PRKN; RAB7; meiosis; mitophagy; oocyte
    DOI:  https://doi.org/10.1080/15548627.2021.1946739
  18. Cell Death Discov. 2021 Jun 29. 7(1): 152
    OASIS/CREB3L1 is a factor that responds to nuclear envelope stress.
    Yasunao Kamikawa, Atsushi Saito, Koji Matsuhisa, Masayuki Kaneko, Rie Asada, Yasunori Horikoshi, Satoshi Tashiro, Kazunori Imaizumi.
      The nuclear envelope (NE) safeguards the genome and is pivotal for regulating genome activity as the structural scaffold of higher-order chromatin organization. NE had been thought as the stable during the interphase of cell cycle. However, recent studies have revealed that the NE can be damaged by various stresses such as mechanical stress and cellular senescence. These types of stresses are called NE stress. It has been proposed that NE stress is closely related to cellular dysfunctions such as genome instability and cell death. Here, we found that an endoplasmic reticulum (ER)-resident transmembrane transcription factor, OASIS, accumulates at damaged NE. Notably, the major components of nuclear lamina, Lamin proteins were depleted at the NE where OASIS accumulates. We previously demonstrated that OASIS is cleaved at the membrane domain in response to ER stress. In contrast, OASIS accumulates as the full-length form to damaged NE in response to NE stress. The accumulation to damaged NE is specific for OASIS among OASIS family members. Intriguingly, OASIS colocalizes with the components of linker of nucleoskeleton and cytoskeleton complexes, SUN2 and Nesprin-2 at the damaged NE. OASIS partially colocalizes with BAF, LEM domain proteins, and a component of ESCRT III, which are involved in the repair of ruptured NE. Furthermore, OASIS suppresses DNA damage induced by NE stress and restores nuclear deformation under NE stress conditions. Our findings reveal a novel NE stress response pathway mediated by OASIS.
    DOI:  https://doi.org/10.1038/s41420-021-00540-x
  19. Aging (Albany NY). 2021 Jul 08. 13
    A model of the aged lung epithelium in idiopathic pulmonary fibrosis.
    Hoora Shaghaghi, Karina Cuevas-Mora, Rachel Para, Cara Tran, Willy Roque, Matthew J Robertson, Ivan O Rosas, Ross Summer, Freddy Romero.
      Idiopathic pulmonary fibrosis (IPF) is an age-related disorder that carries a universally poor prognosis and is thought to arise from repetitive micro injuries to the alveolar epithelium. To date, a major factor limiting our understanding of IPF is a deficiency of disease models, particularly in vitro models that can recapitulate the full complement of molecular attributes in the human condition. In this study, we aimed to develop a model that more closely resembles the aberrant IPF lung epithelium. By exposing mouse alveolar epithelial cells to repeated, low doses of bleomycin, instead of usual one-time exposures, we uncovered changes strikingly similar to those in the IPF lung epithelium. This included the acquisition of multiple phenotypic and functional characteristics of senescent cells and the adoption of previously described changes in mitochondrial homeostasis, including alterations in redox balance, energy production and activity of the mitochondrial unfolded protein response. We also uncovered dramatic changes in cellular metabolism and detected a profound loss of proteostasis, as characterized by the accumulation of cytoplasmic protein aggregates, dysregulated expression of chaperone proteins and decreased activity of the ubiquitin proteasome system. In summary, we describe an in vitro model that closely resembles the aberrant lung epithelium in IPF. We propose that this simple yet powerful tool could help uncover new biological mechanisms and assist in developing new pharmacological tools to treat the disease.
    Keywords:  IPF; aging; epithelial cells; mitochondria; proteostasis
    DOI:  https://doi.org/10.18632/aging.203291
  20. Cell Res. 2021 Jul 08.
    Degradation of lipid droplets by chimeric autophagy-tethering compounds.
    Yuhua Fu, Ningxie Chen, Ziying Wang, Shouqing Luo, Yu Ding, Boxun Lu.
      Degrading pathogenic proteins by degrader technologies such as PROTACs (proteolysis-targeting chimeras) provides promising therapeutic strategies, but selective degradation of non-protein pathogenic biomolecules has been challenging. Here, we demonstrate a novel strategy to degrade non-protein biomolecules by autophagy-tethering compounds (ATTECs), using lipid droplets (LDs) as an exemplar target. LDs are ubiquitous cellular structures storing lipids and could be degraded by autophagy. We hypothesized that compounds interacting with both the LDs and the key autophagosome protein LC3 may enhance autophagic degradation of LDs. We designed and synthesized such compounds by connecting LC3-binding molecules to LD-binding probes via a linker. These compounds were capable of clearing LDs almost completely and rescued LD-related phenotypes in cells and in two independent mouse models with hepatic lipidosis. We further confirmed that the mechanism of action of these compounds was mediated through LC3 and autophagic degradation. Our proof-of-concept study demonstrates the capability of degrading LDs by ATTECs. Conceptually, this strategy could be applied to other protein and non-protein targets.
    DOI:  https://doi.org/10.1038/s41422-021-00532-7
  21. Cell Death Dis. 2021 Jul 08. 12(7): 686
    c-FLIP regulates autophagy by interacting with Beclin-1 and influencing its stability.
    Luana Tomaipitinca, Simonetta Petrungaro, Pasquale D'Acunzo, Angelo Facchiano, Amit Dubey, Salvatore Rizza, Federico Giulitti, Eugenio Gaudio, Antonio Filippini, Elio Ziparo, Francesco Cecconi, Claudia Giampietri.
      c-FLIP (cellular FLICE-like inhibitory protein) protein is mostly known as an apoptosis modulator. However, increasing data underline that c-FLIP plays multiple roles in cellular homoeostasis, influencing differently the same pathways depending on its expression level and isoform predominance. Few and controversial data are available regarding c-FLIP function in autophagy. Here we show that autophagic flux is less effective in c-FLIP-/- than in WT MEFs (mouse embryonic fibroblasts). Indeed, we show that the absence of c-FLIP compromises the expression levels of pivotal factors in the generation of autophagosomes. In line with the role of c-FLIP as a scaffold protein, we found that c-FLIPL interacts with Beclin-1 (BECN1: coiled-coil, moesin-like BCL2-interacting protein), which is required for autophagosome nucleation. By a combination of bioinformatics tools and biochemistry assays, we demonstrate that c-FLIPL interaction with Beclin-1 is important to prevent Beclin-1 ubiquitination and degradation through the proteasomal pathway. Taken together, our data describe a novel molecular mechanism through which c-FLIPL positively regulates autophagy, by enhancing Beclin-1 protein stability.
    DOI:  https://doi.org/10.1038/s41419-021-03957-5
  22. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00688-4. [Epub ahead of print]36(1): 109312
    Identification of a class of non-conventional ER-stress-response-derived immunogenic peptides.
    Alessia Melacarne, Valentina Ferrari, Luca Tiraboschi, Michele Mishto, Juliane Liepe, Marina Aralla, Laura Marconato, Michela Lizier, Chiara Pozzi, Offer Zeira, Giuseppe Penna, Maria Rescigno.
      Efforts to overcome resistance to immune checkpoint blockade therapy have focused on vaccination strategies using neoepitopes, although they cannot be applied on a large scale due to the "private" nature of cancer mutations. Here, we show that infection of tumor cells with Salmonella induces the opening of membrane hemichannels and the extracellular release of proteasome-generated peptides by the exacerbation of endoplasmic reticulum (ER) stress. Peptides released by cancer cells foster an antitumor response in vivo, both in mice bearing B16F10 melanomas and in dogs suffering from osteosarcoma. Mass spectrometry analysis on the supernatant of human melanoma cells revealed 12 peptides capable of priming healthy-donor CD8+ T cells that recognize and kill human melanoma cells in vitro and when xenotransplanted in vivo. Hence, we identified a class of shared tumor antigens that are generated in ER-stressed cells, such as tumor cells, that do not induce tolerance and are not presented by healthy cells.
    Keywords:  ER-stress response; Salmonella; cancer; immunotherapy; tumor antigens; vaccine
    DOI:  https://doi.org/10.1016/j.celrep.2021.109312
  23. Elife. 2021 Jul 05. pii: e57376. [Epub ahead of print]10
    ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.
    Brian D Alford, Eduardo Tassoni-Tsuchida, Danish Khan, Jeremy J Work, Gregory Valiant, Onn Brandman.
      Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.
    Keywords:  CRISPR; HSF1; S. cerevisiae; cell biology; chaperones; genetic screens; genetics; genomics; heat shock response; protein quality control
    DOI:  https://doi.org/10.7554/eLife.57376
  24. Cell Rep. 2021 Jul 06. pii: S2211-1247(21)00693-8. [Epub ahead of print]36(1): 109317
    Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.
    Mohinder Pal, Hugo Muñoz-Hernandez, Dennis Bjorklund, Lihong Zhou, Gianluca Degliesposti, J Mark Skehel, Emma L Hesketh, Rebecca F Thompson, Laurence H Pearl, Oscar Llorca, Chrisostomos Prodromou.
      The R2TP (RUVBL1-RUVBL2-RPAP3-PIH1D1) complex, in collaboration with heat shock protein 90 (HSP90), functions as a chaperone for the assembly and stability of protein complexes, including RNA polymerases, small nuclear ribonucleoprotein particles (snRNPs), and phosphatidylinositol 3-kinase (PI3K)-like kinases (PIKKs) such as TOR and SMG1. PIKK stabilization depends on an additional complex of TELO2, TTI1, and TTI2 (TTT), whose structure and function are poorly understood. The cryoelectron microscopy (cryo-EM) structure of the human R2TP-TTT complex, together with biochemical experiments, reveals the mechanism of TOR recruitment to the R2TP-TTT chaperone. The HEAT-repeat TTT complex binds the kinase domain of TOR, without blocking its activity, and delivers TOR to the R2TP chaperone. In addition, TTT regulates the R2TP chaperone by inhibiting RUVBL1-RUVBL2 ATPase activity and by modulating the conformation and interactions of the PIH1D1 and RPAP3 components of R2TP. Taken together, our results show how TTT couples the recruitment of TOR to R2TP with the regulation of this chaperone system.
    Keywords:  HSP90 chaperone; PIKK; R2TP; RUVBL1; RUVBL2; TELO2; TTI1; TTI2; TTT; mTOR
    DOI:  https://doi.org/10.1016/j.celrep.2021.109317
  25. Biochem Soc Trans. 2021 Jul 09. pii: BST20201136. [Epub ahead of print]
    Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.
    David Shore, Sevil Zencir, Benjamin Albert.
      Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1-Fhl1-Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1-Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1-Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.
    Keywords:   Saccharomyces cerevisiae yeast; gene regulation; growth; proteostasis; ribosome biogenesis; stress response
    DOI:  https://doi.org/10.1042/BST20201136
  26. Autophagy. 2021 Jul 09. 1-23
    Targeting autophagy in disease: established and new strategies.
    Muhammed Kocak, Saba Ezazi Erdi, Guillem Jorba, Inés Maestro, Judith Farrés, Vladimir Kirkin, Ana Martinez, Ole Pless.
      Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible.Abbreviations: ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.
    Keywords:  Autophagy activators; autophagy inhibitors; autophagy modulators; clinical trials; drug discovery
    DOI:  https://doi.org/10.1080/15548627.2021.1936359
  27. J Cell Biol. 2021 Sep 06. pii: e202103186. [Epub ahead of print]220(9):
    zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.
    Ashley M Bourke, Samantha L Schwartz, Aaron B Bowen, Mason S Kleinjan, Christina S Winborn, Dean J Kareemo, Amos Gutnick, Thomas L Schwarz, Matthew J Kennedy.
      Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.
    DOI:  https://doi.org/10.1083/jcb.202103186
  28. Nat Struct Mol Biol. 2021 Jul 08.
    Membrane perturbation by lipidated Atg8 underlies autophagosome biogenesis.
    Tatsuro Maruyama, Jahangir Md Alam, Tomoyuki Fukuda, Shun Kageyama, Hiromi Kirisako, Yuki Ishii, Ichio Shimada, Yoshinori Ohsumi, Masaaki Komatsu, Tomotake Kanki, Hitoshi Nakatogawa, Nobuo N Noda.
      Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.
    DOI:  https://doi.org/10.1038/s41594-021-00614-5
  29. Redox Biol. 2021 Jun 29. pii: S2213-2317(21)00217-2. [Epub ahead of print]45 102058
    The E3 ubiquitin ligase TRIM31 is involved in cerebral ischemic injury by promoting degradation of TIGAR.
    Shenglan Zeng, Ze Zhao, Shengnan Zheng, Mengting Wu, Xiaomeng Song, Yiquan Li, Yi Zheng, Bingyu Liu, Lin Chen, Chengjiang Gao, Huiqing Liu.
      Tripartite motif (TRIM) 31 has been implicated in diverse biological and pathological conditions. However, whether TRIM31 plays a role in ischemic stroke progression is not clarified. Here we demonstrated that TRIM31 was significantly downregulated in the ischemic brain and the deficiency of TRIM31 alleviated brain injury induced by middle cerebral artery occlusion by reducing reactive oxygen species production and maintaining mitochondrial homeostasis. Mechanistically, we found that TRIM31 is an E3 ubiquitin ligase for TP53-induced glycolysis and apoptosis regulator (TIGAR), which confers protection against brain ischemia by increasing the pentose phosphate pathway flux and preserving mitochondria function. TRIM31 interacted with TIGAR and promoted the polyubiquitination of TIGAR, consequently facilitated its degradation in a proteasome-dependent pathway. Furthermore, TIGAR knockdown effectively abolished the protective effect of TRIM31 deficiency after cerebral ischemia. In conclusion, we identified that TRIM31 was a novel E3 ubiquitin ligase for TIGAR, played a critical role in regulating its protein level, and subsequently involved in the ischemic brain injury, suggesting TRIM31 as a potential therapeutic target for ischemic stroke.
    Keywords:  Ischemic stroke; Mitochondria; Reactive oxygen species; TIGAR; TRIM31
    DOI:  https://doi.org/10.1016/j.redox.2021.102058
  30. ACS Chem Neurosci. 2021 Jul 07. 12(13): 2273-2279
    Domain-Independent Inhibition of CBP/p300 Attenuates α-Synuclein Aggregation.
    Irena Hlushchuk, Heikki Ruskoaho, Andrii Domanskyi, Mikko Airavaara, Mika J Välimäki.
      Neurodegenerative diseases are associated with failed proteostasis and accumulation of insoluble protein aggregates that compromise neuronal function and survival. In Parkinson's disease, a major pathological finding is Lewy bodies and neurites that are mainly composed of phosphorylated and aggregated α-synuclein and fragments of organelle membranes. Here, we analyzed a series of selective inhibitors acting on multidomain proteins CBP and p300 that contain both lysine acetyltransferase and bromodomains and are responsible for the recognition and enzymatic modification of lysine residues. By using high-affinity inhibitors, A-485, GNE-049, and SGC-CBP30, we explored the role of two closely related proteins, CBP and p300, as promising targets for selective attenuation of α-synuclein aggregation. Our data show that selective CBP/p300 inhibitors may alter the course of pathological α-synuclein accumulation in primary mouse embryonic dopaminergic neurons. Hence, drug-like CBP/p300 inhibitors provide an effective approach for the development of high-affinity drug candidates preventing α-synuclein aggregation via systemic administration.
    Keywords:  CBP; Parkinson’s disease; bromodomain; lysine acetyltransferase; p300; α-Synuclein
    DOI:  https://doi.org/10.1021/acschemneuro.1c00215
  31. EBioMedicine. 2021 Jul 04. pii: S2352-3964(21)00249-8. [Epub ahead of print]69 103456
    Loss of TRIM21 alleviates cardiotoxicity by suppressing ferroptosis induced by the chemotherapeutic agent doxorubicin.
    Kai Hou, Jianliang Shen, Junrong Yan, Chuannan Zhai, Jingxia Zhang, Ji-An Pan, Ye Zhang, Yaping Jiang, Yongbo Wang, Richard Z Lin, Hongliang Cong, Shenglan Gao, Wei-Xing Zong.
       BACKGROUND: Doxorubicin, an anthracycline chemotherapeutic agent, is widely used in the treatment of many cancers. However, doxorubicin posts a great risk of adverse cardiovascular events, which are thought to be caused by oxidative stress. We recently reported that the ubiquitin E3 ligase TRIM21 interacts and ubiquitylates p62 and negatively regulates the p62-Keap1-Nrf2 antioxidant pathway. Therefore, we sought to determine the role TRIM21 in cardiotoxicity induced by oxidative damage.
    METHODS: Using TRIM21 knockout mice, we examined the effects of TRIM21 on cardiotoxicity induced by two oxidative damage models: the doxorubicin treatment model and the Left Anterior Descending (LAD) model. We also explored the underlying mechanism by RNA-sequencing of the heart tissues, and by treating the mouse embryonic fibroblasts (MEFs), immortalized rat cardiomyocyte line H9c2, and immortalized human cardiomyocyte line AC16 with doxorubicin.
    FINDINGS: TRIM21 knockout mice are protected from heart failure and fatality in both the doxorubicin and LAD models. Hearts of doxorubicin-treated wild-type mice exhibit deformed mitochondria and elevated level of lipid peroxidation reminiscent of ferroptosis, which is alleviated in TRIM21 knockout hearts. Mechanistically, TRIM21-deficient heart tissues and cultured MEFs and H9c2 cells display enhanced p62 sequestration of Keap1 and are protected from doxorubicin-induced ferroptosis. Reconstitution of wild-type but not the E3 ligase-dead and the p62 binding-deficient TRIM21 mutants impedes the protection from doxorubicin-induced cell death.
    INTERPRETATION: Our study demonstrates that TRIM21 ablation protects doxorubicin-induced cardiotoxicity and illustrates a new function of TRIM21 in ferroptosis, and suggests TRIM21 as a therapeutic target for reducing chemotherapy-related cardiotoxicity.
    Keywords:  Antioxidant; Cardiotoxicity; Doxorubicin; Ferroptosis; TRIM21
    DOI:  https://doi.org/10.1016/j.ebiom.2021.103456
  32. Cell Death Dis. 2021 Jul 03. 12(7): 671
    Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes.
    Sentiljana Gumeni, Eleni-Dimitra Papanagnou, Maria S Manola, Ioannis P Trougakos.
      The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies' tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.
    DOI:  https://doi.org/10.1038/s41419-021-03952-w
  33. Cell Mol Gastroenterol Hepatol. 2021 Jul 05. pii: S2352-345X(21)00142-9. [Epub ahead of print]
    Human AGR2 Deficiency Causes Mucus Barrier Dysfunction and Infantile Inflammatory Bowel Disease.
    COLORS in IBD-Qatar study group
       BACKGROUND: S AND AIM: The gastrointestinal epithelium plays a crucial role in maintaining homeostasis with the gut microbiome. Mucins are essential for intestinal barrier function and serve as a scaffold for antimicrobial factors. MUC2 is the major intestinal gel-forming mucin produced predominantly by goblet cells. Goblet cells express AGR2, a protein disulfide isomerase (PDI) that is crucial for proper processing of gel-forming mucins. Here, we investigated two siblings who presented with severe infantile onset inflammatory bowel disease.
    METHODS: We performed whole genome sequencing to identify candidate variants. We quantified goblet cell numbers using H&E histology and investigated the expression of gel-forming mucins, stress markers, and goblet cell markers using immunohistochemistry. AGR2-MUC2 binding was evaluated using co-immunoprecipitation. Endoplasmic reticulum (ER) stress regulatory function of mutant AGR2 was examined by expression studies in HEK293T using tunicamycin to induce ER stress.
    RESULTS: Both affected siblings were homozygous for a missense variant in AGR2. Patient biopsies showed reduced goblet cells, depletion of MUC2, MUC5AC, and MUC6, upregulation of AGR2, and elevated ER stress. The mutant AGR2 showed reduced capacity to bind MUC2 and alleviate tunicamycin-induced ER stress.
    CONCLUSIONS: Phenotype-genotype segregation, functional experiments, and the striking similarity of the human phenotype to the AGR2-/- mouse models suggest that the AGR2 missense variant is pathogenic. The Mendelian deficiency of AGR2, termed "Enteropathy caused by AGR2 deficiency, Goblet cell Loss, and ER Stress" (EAGLES), results in a mucus barrier defect, the inability to mitigate ER stress, and causes infantile onset IBD.
    Keywords:  AGR2; ER stress; Goblet cells,; MUC2; intestinal metaplasia
    DOI:  https://doi.org/10.1016/j.jcmgh.2021.07.001
  34. Mol Biol Cell. 2021 Jul 07. mbcE21010031
    SUMO Paralog Specific Functions Revealed through Systematic Analysis of Human Knockout Cell Lines and Gene Expression Data.
    Danielle Bouchard, Wei Wang, Wei-Chih Yang, Shuying He, Anthony Garcia, Michael J Matunis.
      The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogs with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogs, and also the best studied. To what extent SUMO1, SUMO2 and the other paralogs impart unique and non-redundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogs during development and studies in cell culture have relied largely on transient paralog overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralog gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralog-specific functions in adult human tissues. To systematically identify and characterize unique and non-redundant functions of the SUMO paralogs in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, PML nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal non-redundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.
    DOI:  https://doi.org/10.1091/mbc.E21-01-0031
  35. Cancer Discov. 2021 Apr 23.
    Bortezomib Induces Anti-Multiple Myeloma Immune Response Mediated by cGAS/STING Pathway Activation.
    Annamaria Gulla, Eugenio Morelli, Mehmet K Samur, Cirino Botta, Teru Hideshima, Giada Bianchi, Mariateresa Fulciniti, Stefano Malvestiti, Rao H Prabhala, Srikanth Talluri, Kenneth Wen, Yu-Tzu Tai, Paul G Richardson, Dharminder Chauhan, Tomasz Sewastianik, Ruben D Carrasco, Nikhil C Munshi, Kenneth C Anderson.
      The proteasome inhibitor bortezomib induces apoptosis in multiple myeloma cells and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine multiple myeloma models, we here show that bortezomib also triggers immunogenic cell death (ICD), characterized by exposure of calreticulin on dying multiple myeloma cells, phagocytosis of tumor cells by dendritic cells, and induction of multiple myeloma-specific immunity. We identify a bortezomib-triggered specific ICD gene signature associated with better outcome in two independent cohorts of patients with multiple myeloma. Importantly, bortezomib stimulates multiple myeloma cell immunogenicity via activation of the cGAS/STING pathway and production of type I IFNs, and STING agonists significantly potentiate bortezomib-induced ICD. Our study therefore delineates mechanisms whereby bortezomib exerts immunotherapeutic activity and provides the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in multiple myeloma. SIGNIFICANCE: Our study demonstrates that cGAS/STING-dependent immunostimulatory activity mediates bortezomib anti-myeloma activity in experimental models and associates with clinical response to bortezomib in patients with multiple myeloma. These findings provide the rationale for clinical evaluation of STING agonists to further potentiate anti-multiple myeloma immune response.See related commentary by Zitvogel and Kroemer.
    DOI:  https://doi.org/10.1158/2643-3230.BCD-21-0047
  36. J Biol Chem. 2021 Jul 02. pii: S0021-9258(21)00735-3. [Epub ahead of print] 100935
    Molecular basis for RASSF10/NPM/RNF2 feedback cascade-mediated regulation of gastric cancer cell proliferation.
    Naga Padma Lakshmi Ch, Ananthi Sivagnanam, Sebastian Raja, Sundarasamy Mahalingam.
      Ras association domain family (RASSF) proteins are encoded by numerous tumor suppressor genes that frequently become silenced in human cancers. RASSF10 is downregulated by promoter hypermethylation in cancers and has been shown to inhibit cell proliferation; however the molecular mechanism(s) remains poorly understood. Here, we demonstrate for the first time that RASSF10 inhibits Cdk1/cyclin-B kinase complex formation to maintain stable levels of cyclin-B for inducing mitotic arrest during cell cycle. Using LC-MS/MS, live cell imaging and biochemical approaches, we identify Nucleophosmin (NPM) as a novel functional target of RASSF10 and revealed that RASSF10 expression promoted the nuclear accumulation of GADD45a and knockdown of either NPM or GADD45a, resulting in impairment of RASSF10 mediated G2/M phase arrest. Furthermore, we demonstrate that RASSF10 is a substrate for the E3 ligase RNF2 and show that an NPM dependent downregulation of RNF2 expression is critical to maintain stable RASSF10 levels in cells for efficient mitotic arrest. Interestingly, Kaplan-Meier plot analysis show a positive correlation of RASSF10 and NPM expression with greater gastric cancer patient survival and shows the reverse with expression of RNF2, suggesting that they may have a role in cancer progression. Finally, our findings provide insights into the mode of action of the RASSF10/NPM/RNF2 signaling cascade on controlling cell proliferation and may represent a novel therapeutic avenue for the prevention of gastric cancer metastasis.
    Keywords:  Cell Proliferation; GADD45a; Gastric cancer; Mass spectrometry; Mitosis; Nucleophosmin; Proteomics; RASSF10; RNF2; Ubiquitination
    DOI:  https://doi.org/10.1016/j.jbc.2021.100935
  37. Mol Plant. 2021 Jun 30. pii: S1674-2052(21)00262-8. [Epub ahead of print]
    The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.
    Wensi Yang, Kun Wu, Bo Wang, Huanhuan Liu, Siyi Guo, Xiaoyu Guo, Wei Luo, Shengyuan Sun, Yidan Ouyang, Xiangdong Fu, Kang Chong, Qifa Zhang, Yunyuan Xu.
      G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. CLG1 overexpression led to increased grain length, and overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. CLG1 physically interacted with and ubiquitinated GS3; the latter was subsequently degraded through the endosome degradation pathway, leading to increased grain size. We also identified a critical SNP in exon 3 of CLG1 that was significantly associated with grain-size variation in a core collection of cultivated rice. This SNP resulted in an amino acid substitution from Arg to Ser at position 163, which enhancing the E3 ligase activity of CLG1, thus increasing grain size. Both the CLG1 expression level and the SNP may be useful targets for manipulating grain size.
    Keywords:  CLG1; E3 ligase; GS3; endosome; grain size
    DOI:  https://doi.org/10.1016/j.molp.2021.06.027
  38. Geroscience. 2021 Jul 09.
    A quantitative yeast aging proteomics analysis reveals novel aging regulators.
    Yu Sun, Ruofan Yu, Hao-Bo Guo, Hong Qin, Weiwei Dang.
      Calorie restriction (CR) is the most robust longevity intervention, extending lifespan from yeast to mammals. Numerous conserved pathways regulating aging and mediating CR have been identified; however, the overall proteomic changes during these conditions remain largely unexplored. We compared proteomes between young and replicatively aged yeast cells under normal and CR conditions using the Stable-Isotope Labeling by Amino acids in Cell culture (SILAC) quantitative proteomics and discovered distinct signatures in the aging proteome. We found remarkable proteomic similarities between aged and CR cells, including induction of stress response pathways, providing evidence that CR pathways are engaged in aged cells. These observations also uncovered aberrant changes in mitochondria membrane proteins as well as a proteolytic cellular state in old cells. These proteomics analyses help identify potential genes and pathways that have causal effects on longevity.
    Keywords:  Aging; Calorie restriction; Proteome; SILAC; Yeast
    DOI:  https://doi.org/10.1007/s11357-021-00412-3
  39. Nat Commun. 2021 Jul 07. 12(1): 4194
    Chaperone-like protein DAY plays critical roles in photomorphogenesis.
    Ho-Seok Lee, Ilyeong Choi, Young Jeon, Hee-Kyung Ahn, Huikyong Cho, JiWoo Kim, Jae-Hee Kim, Jung-Min Lee, SungHee Lee, Julian Bünting, Dong Hye Seo, Tak Lee, Du-Hwa Lee, Insuk Lee, Man-Ho Oh, Tae-Wuk Kim, Youssef Belkhadir, Hyun-Sook Pai.
      Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.
    DOI:  https://doi.org/10.1038/s41467-021-24446-5
  40. Mol Cell. 2021 Jun 29. pii: S1097-2765(21)00493-7. [Epub ahead of print]
    Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer.
    Jaclyn M Einstein, Mark Perelis, Isaac A Chaim, Jitendra K Meena, Julia K Nussbacher, Alexandra T Tankka, Brian A Yee, Heyuan Li, Assael A Madrigal, Nicholas J Neill, Archana Shankar, Siddhartha Tyagi, Thomas F Westbrook, Gene W Yeo.
      RNA-binding proteins (RBPs) are critical regulators of post-transcriptional gene expression, and aberrant RBP-RNA interactions can promote cancer progression. Here, we interrogate the function of RBPs in cancer using pooled CRISPR-Cas9 screening and identify 57 RBP candidates with distinct roles in supporting MYC-driven oncogenic pathways. We find that disrupting YTHDF2-dependent mRNA degradation triggers apoptosis in triple-negative breast cancer (TNBC) cells and tumors. eCLIP and m6A sequencing reveal that YTHDF2 interacts with mRNAs encoding proteins in the MAPK pathway that, when stabilized, induce epithelial-to-mesenchymal transition and increase global translation rates. scRibo-STAMP profiling of translating mRNAs reveals unique alterations in the translatome of single cells within YTHDF2-depleted solid tumors, which selectively contribute to endoplasmic reticulum stress-induced apoptosis in TNBC cells. Thus, our work highlights the therapeutic potential of RBPs by uncovering a critical role for YTHDF2 in counteracting the global increase of mRNA synthesis in MYC-driven breast cancers.
    Keywords:  CRISPR screening; MYC-driven cancer; N6-methyladenosine; RNA-binding protein; STAMP; YTHDF2; scRNA-seq
    DOI:  https://doi.org/10.1016/j.molcel.2021.06.014
  41. J Cell Sci. 2021 Jul 07. pii: jcs.258325. [Epub ahead of print]
    Large organellar changes occur during mild heat shock in yeast.
    Katharina S Keuenhof, Lisa Larsson Berglund, Sandra Malmgren Hill, Kara L Schneider, Per O Widlund, Thomas Nyström, Johanna L Höög.
      When the temperature is increased, the heat shock response is activated to protect the cellular environment. The transcriptomics and proteomics of this process are intensively studied, while information about how the cell responds structurally to heat stress is mostly lacking. Here, Saccharomyces cerevisiae were subjected to a mild continuous heat shock (38°C) and intermittently cryo-immobilized for electron microscopy. Through measuring changes in all distinguishable organelle numbers, sizes, and morphologies in over 2100 electron micrographs a major restructuring of the cell's internal architecture during the progressive heat shock was revealed. The cell grew larger but most organelles within it expanded even more, shrinking the volume of the cytoplasm. Organelles responded to heat shock at different times, both in terms of size and number, and adaptations of certain organelles' morphology (such as the vacuole), were observed. Multivesicular bodies grew to almost 170% in size, indicating a previously unknown involvement in the heat shock response. A previously undescribed electron translucent structure accumulated close to the plasma membrane. This all-encompassing approach provides a detailed chronological progression of organelle adaptation throughout the cellular heat-stress response.
    Keywords:  Budding yeast; Electron microscopy; Heat shock; Organelles; Ultrastructure
    DOI:  https://doi.org/10.1242/jcs.258325
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