bims-proteo Biomed News
on Proteostasis
Issue of 2023–04–30
twenty-six papers selected by
Eric Chevet, INSERM
  1. Mathematical Model for Covalent Proteolysis Targeting Chimeras: Thermodynamics and Kinetics Underlying Catalytic Efficiency.
  2. Chaperone-directed ribosome repair after oxidative damage.
  3. FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels.
  4. The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein.
  5. Autophagy of the ER requires actin assembly driven by the interaction of ER with endocytic pits.
  6. Parallel phospholipid transfer by Vps13 and Atg2 determines autophagosome biogenesis dynamics.
  7. The ASC-1 complex promotes translation initiation by scanning ribosomes.
  8. DTX3L E3 ligase targets p53 for degradation at poly ADP-ribose polymerase-associated DNA damage sites.
  9. Human atlastin-3 is a constitutive ER membrane fusion catalyst.
  10. Modeling the Effect of Cooperativity in Ternary Complex Formation and Targeted Protein Degradation Mediated by Heterobifunctional Degraders.
  11. Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.
  12. Small-Molecule Ferritin Degrader as a Pyroptosis Inducer.
  13. A tubule-sheet continuum model for the mechanism of nuclear envelope assembly.
  14. Autophagy Inhibition as a Potential Therapeutic Strategy for Breast Cancer with Mitochondrial Translation Defect Caused by CBFB-Deficiency.
  15. The role of crinophagy in quality control of the regulated secretory pathway.
  16. Chaperone-Dependent Degradation of Cdc42 Promotes Cell Polarity and Shields the Protein from Aggregation.
  17. Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.
  18. Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases.
  19. The mechanism of STING autoinhibition and activation.
  20. MicroRNAs in Age-Related Proteostasis and Stress Responses.
  21. Mapping the in vivo fitness landscape of a tethered ribosome.
  22. ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.
  23. Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.
  24. USP7 controls NGN3 stability and pancreatic endocrine lineage development.
  25. Nucleophagy delays aging and preserves germline immortality.
  26. A new kid in the folding funnel: Molecular chaperone activities of the BRICHOS domain.
  1. J Med Chem. 2023 Apr 26.
    Mathematical Model for Covalent Proteolysis Targeting Chimeras: Thermodynamics and Kinetics Underlying Catalytic Efficiency.
    Charu Chaudhry.
      Proteolysis targeting chimeras (PROTACs), heterobifunctional protein degraders, have emerged as an exciting and transformative technology in chemical biology and drug discovery to degrade disease-causing proteins through co-opting of the ubiquitin-proteasome system (UPS). Here, we develop a mechanistic mathematical model for the use of irreversible covalent chemistry in targeted protein degradation (TPD) either to a target protein of interest (POI) or an E3 ligase ligand, considering the thermodynamic and kinetic factors governing ternary complex formation, ubiquitination, and degradation through the UPS. We highlight key advantages of covalency to the POI and E3 ligase and the underlying theoretical basis in the TPD reaction framework. We further identify regimes where covalency can serve to overcome weak binary binding affinities and improve the kinetics of ternary complex formation and degradation. Our results highlight the enhanced catalytic efficiency of covalent E3 PROTACs and thus their potential to improve the degradation of fast turnover targets.
    DOI:  https://doi.org/10.1021/acs.jmedchem.2c02076
  2. Mol Cell. 2023 Apr 18. pii: S1097-2765(23)00244-7. [Epub ahead of print]
    Chaperone-directed ribosome repair after oxidative damage.
    Yoon-Mo Yang, Youngeun Jung, Daniel Abegg, Alexander Adibekian, Kate S Carroll, Katrin Karbstein.
      Because of the central role ribosomes play for protein translation and ribosome-mediated mRNA and protein quality control (RQC), the ribosome pool is surveyed and dysfunctional ribosomes degraded both during assembly, as well as the functional cycle. Oxidative stress downregulates translation and damages mRNAs and ribosomal proteins (RPs). Although damaged mRNAs are detected and degraded via RQC, how cells mitigate damage to RPs is not known. Here, we show that cysteines in Rps26 and Rpl10 are readily oxidized, rendering the proteins non-functional. Oxidized Rps26 and Rpl10 are released from ribosomes by their chaperones, Tsr2 and Sqt1, and the damaged ribosomes are subsequently repaired with newly made proteins. Ablation of this pathway impairs growth, which is exacerbated under oxidative stress. These findings reveal an unanticipated mechanism for chaperone-mediated ribosome repair, augment our understanding of ribosome quality control, and explain previous observations of protein exchange in ribosomes from dendrites, with broad implications for aging and health.
    Keywords:  chaperone; oxidative damage; repair; ribosome
    DOI:  https://doi.org/10.1016/j.molcel.2023.03.030
  3. EMBO J. 2023 Apr 27. e112799
    FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels.
    Hannah Elcocks, Ailbhe J Brazel, Katy R McCarron, Manuel Kaulich, Koraljka Husnjak, Heather Mortiboys, Michael J Clague, Sylvie Urbé.
      Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin-RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α-mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early-onset mitochondrial encephalomyopathy that is supported by analysis of a disease-associated mutation. We further show that the compound MLN4924, which globally interferes with cullin-RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.
    Keywords:  BNIP3; FBXL4; NIX; VHL; mitophagy
    DOI:  https://doi.org/10.15252/embj.2022112799
  4. iScience. 2023 May 19. 26(5): 106601
    The human E3 ligase RNF185 is a regulator of the SARS-CoV-2 envelope protein.
    Charles Zou, Hojong Yoon, Paul M C Park, J J Patten, Jesse Pellman, Jeannie Carreiro, Jonathan M Tsai, Yen-Der Li, Shourya S Roy Burman, Katherine A Donovan, Jessica Gasser, Adam S Sperling, Radosław P Nowak, Eric S Fischer, Robert A Davey, Benjamin L Ebert, Mikołaj Słabicki.
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.
    Keywords:  Cell biology; Virology
    DOI:  https://doi.org/10.1016/j.isci.2023.106601
  5. Contact (Thousand Oaks). 2022 Jan-Dec;5:5
    Autophagy of the ER requires actin assembly driven by the interaction of ER with endocytic pits.
    Peter Novick, Dongmei Liu, Susan Ferro-Novick.
      Autophagy of the cortical ER in budding yeast was unexpectedly found to require End3, a component of the endocytic machinery that promotes the assembly of actin at endocytic pits on the plasma membrane. The cortical ER transiently interacts with invaginating endocytic pits through a linkage consisting of VAP proteins, oxysterol binding proteins and type I myosins. These proteins are required for actin assembly and for autophagy of the ER. Assembly of actin at these contact sites may direct the movement of ER away from the cortex towards sites of autophagosome assembly.
    Keywords:  VAP; actin assembly; autophagy; autophagy receptor; endocytosis; endoplasmic reticulum
    DOI:  https://doi.org/10.1177/25152564221093215
  6. J Cell Biol. 2023 Jul 03. pii: e202211039. [Epub ahead of print]222(7):
    Parallel phospholipid transfer by Vps13 and Atg2 determines autophagosome biogenesis dynamics.
    Rahel Dabrowski, Susanna Tulli, Martin Graef.
      During autophagy, rapid membrane assembly expands small phagophores into large double-membrane autophagosomes. Theoretical modeling predicts that the majority of autophagosomal phospholipids are derived from highly efficient non-vesicular phospholipid transfer (PLT) across phagophore-ER contacts (PERCS). Currently, the phagophore-ER tether Atg2 is the only PLT protein known to drive phagophore expansion in vivo. Here, our quantitative live-cell imaging analysis reveals a poor correlation between the duration and size of forming autophagosomes and the number of Atg2 molecules at PERCS of starving yeast cells. Strikingly, we find that Atg2-mediated PLT is non-rate limiting for autophagosome biogenesis because membrane tether and the PLT protein Vps13 localizes to the rim and promotes the expansion of phagophores in parallel with Atg2. In the absence of Vps13, the number of Atg2 molecules at PERCS determines the duration and size of forming autophagosomes with an apparent in vivo transfer rate of ∼200 phospholipids per Atg2 molecule and second. We propose that conserved PLT proteins cooperate in channeling phospholipids across organelle contact sites for non-rate-limiting membrane assembly during autophagosome biogenesis.
    DOI:  https://doi.org/10.1083/jcb.202211039
  7. EMBO J. 2023 Apr 24. e112869
    The ASC-1 complex promotes translation initiation by scanning ribosomes.
    Yuki Kito, Akinobu Matsumoto, Kazuya Ichihara, Chisa Shiraishi, Ronghao Tang, Atsushi Hatano, Masaki Matsumoto, Peixun Han, Shintaro Iwasaki, Keiichi I Nakayama.
      Translation initiates when the eIF4F complex binds the 5' mRNA cap, followed by 5' untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domain-containing subunit of ASCC, localizes predominantly to the 5' untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.
    Keywords:  ASCC; Sel-TCP-MS; eIF4A; scanning ribosome; translation initiation
    DOI:  https://doi.org/10.15252/embj.2022112869
  8. iScience. 2023 Apr 21. 26(4): 106444
    DTX3L E3 ligase targets p53 for degradation at poly ADP-ribose polymerase-associated DNA damage sites.
    Qingsheng Yan, Jingyi Ding, Sumbul Jawed Khan, Lee N Lawton, Margaret A Shipp.
      P53 is a master transcriptional regulator and effector of the DNA damage response (DDR) that localizes to DNA damage sites, in part, via an interaction with PARP1. However, the mechanisms that regulate p53 abundance and activity at PARP1-decorated DNA damage sites remain undefined. The PARP9 (BAL1) macrodomain-containing protein and its partner DTX3L (BBAP) E3 ligase are rapidly recruited to PARP1-PARylated DNA damage sites. During an initial DDR, we found that DTX3L rapidly colocalized with p53, polyubiquitylated its lysine-rich C-terminal domain, and targeted p53 for proteasomal degradation. DTX3L knockout significantly increased and prolonged p53 retention at PARP-decorated DNA damage sites. These findings reveal a non-redundant, PARP- and PARylation-dependent role for DTX3L in the spatiotemporal regulation of p53 during an initial DDR. Our studies suggest that targeted inhibition of DTX3L may augment the efficacy of certain DNA-damaging agents by increasing p53 abundance and activity.
    Keywords:  Biochemical mechanism; Cell biology; Molecular mechanism of gene regulation
    DOI:  https://doi.org/10.1016/j.isci.2023.106444
  9. J Cell Biol. 2023 Jul 03. pii: e202211021. [Epub ahead of print]222(7):
    Human atlastin-3 is a constitutive ER membrane fusion catalyst.
    Samantha Bryce, Maureen Stolzer, Daniel Crosby, Ruijin Yang, Dannie Durand, Tina H Lee.
      Homotypic membrane fusion catalyzed by the atlastin (ATL) GTPase sustains the branched endoplasmic reticulum (ER) network in metazoans. Our recent discovery that two of the three human ATL paralogs (ATL1/2) are C-terminally autoinhibited implied that relief of autoinhibition would be integral to the ATL fusion mechanism. An alternative hypothesis is that the third paralog ATL3 promotes constitutive ER fusion with relief of ATL1/2 autoinhibition used conditionally. However, published studies suggest ATL3 is a weak fusogen at best. Contrary to expectations, we demonstrate here that purified human ATL3 catalyzes efficient membrane fusion in vitro and is sufficient to sustain the ER network in triple knockout cells. Strikingly, ATL3 lacks any detectable C-terminal autoinhibition, like the invertebrate Drosophila ATL ortholog. Phylogenetic analysis of ATL C-termini indicates that C-terminal autoinhibition is a recent evolutionary innovation. We suggest that ATL3 is a constitutive ER fusion catalyst and that ATL1/2 autoinhibition likely evolved in vertebrates as a means of upregulating ER fusion activity on demand.
    DOI:  https://doi.org/10.1083/jcb.202211021
  10. ACS Bio Med Chem Au. 2023 Feb 15. 3(1): 74-86
    Modeling the Effect of Cooperativity in Ternary Complex Formation and Targeted Protein Degradation Mediated by Heterobifunctional Degraders.
    Daniel Park, Jesus Izaguirre, Rory Coffey, Huafeng Xu.
      Chemically induced proximity between certain endogenous enzymes and a protein of interest (POI) inside cells may cause post-translational modifications to the POI with biological consequences and potential therapeutic effects. Heterobifunctional (HBF) molecules that bind with one functional part to a target POI and with the other to an E3 ligase induce the formation of a target-HBF-E3 ternary complex, which can lead to ubiquitination and proteasomal degradation of the POI. Targeted protein degradation (TPD) by HBFs offers a promising approach to modulate disease-associated proteins, especially those that are intractable using other therapeutic approaches, such as enzymatic inhibition. The three-way interactions among the HBF, the target POI, and the ligase-including the protein-protein interaction between the POI and the ligase-contribute to the stability of the ternary complex, manifested as positive or negative binding cooperativity in its formation. How such cooperativity affects HBF-mediated degradation is an open question. In this work, we develop a pharmacodynamic model that describes the kinetics of the key reactions in the TPD process, and we use this model to investigate the role of cooperativity in the ternary complex formation and in the target POI degradation. Our model establishes the quantitative connection between the ternary complex stability and the degradation efficiency through the former's effect on the rate of catalytic turnover. We also develop a statistical inference model for determining cooperativity in intracellular ternary complex formation from cellular assay data and demonstrate it by quantifying the change in cooperativity due to site-directed mutagenesis at the POI-ligase interface of the SMARCA2-ACBI1-VHL ternary complex. Our pharmacodynamic model provides a quantitative framework to dissect the complex HBF-mediated TPD process and may inform the rational design of effective HBF degraders.
    DOI:  https://doi.org/10.1021/acsbiomedchemau.2c00037
  11. J Cell Biol. 2023 Jul 03. pii: e202210078. [Epub ahead of print]222(7):
    Quantitative analysis of autophagy reveals the role of ATG9 and ATG2 in autophagosome formation.
    David G Broadbent, Carlo Barnaba, Gloria I Perez, Jens C Schmidt.
      Autophagy is a catabolic pathway required for the recycling of cytoplasmic materials. To define the mechanisms underlying autophagy it is critical to quantitatively characterize the dynamic behavior of autophagy factors in living cells. Using a panel of cell lines expressing HaloTagged autophagy factors from their endogenous loci, we analyzed the abundance, single-molecule dynamics, and autophagosome association kinetics of autophagy proteins involved in autophagosome biogenesis. We demonstrate that autophagosome formation is inefficient and ATG2-mediated tethering to donor membranes is a key commitment step in autophagosome formation. Furthermore, our observations support the model that phagophores are initiated by the accumulation of autophagy factors on mobile ATG9 vesicles, and that the ULK1 complex and PI3-kinase form a positive feedback loop required for autophagosome formation. Finally, we demonstrate that the duration of autophagosome biogenesis is ∼110 s. In total, our work provides quantitative insight into autophagosome biogenesis and establishes an experimental framework to analyze autophagy in human cells.
    DOI:  https://doi.org/10.1083/jcb.202210078
  12. J Am Chem Soc. 2023 Apr 24.
    Small-Molecule Ferritin Degrader as a Pyroptosis Inducer.
    Yu Chen, Wen Li, Song Kwon, Yixin Wang, Zhaoting Li, Quanyin Hu.
      Exploring the response of malignant cells to intracellular metabolic stress is critical for understanding pathologic processes and developing anticancer therapies. Herein, we developed ferritin-targeting proteolysis targeting chimeras (PROTACs) to establish the iron excess stress inside cancer cells and investigated subsequent cellular behaviors. We conjugated oleic acid that binds to the ferritin dimer to the ligand of von Hippel-Lindau (VHL) E3 ligase through an alkyl linker. The screened chimera, DeFer-2, degraded ferritin and then rapidly elevated the free iron content, thereby initiating the caspase 3-GSDME-mediated pyroptosis in cancer cells rather than typical ferroptosis that is always associated with iron ion overload. According to its structural and physicochemical characteristics, DeFer-2 was loaded into a tailored albumin-based nano-formulation, which substantially inhibited tumor growth and prolonged the survival time of mice bearing B16F10 subcutaneous tumors with negligible adverse effects. This study developed a ferritin-targeting PROTAC for iron overload stress, revealed iron metabolic dysregulation-mediated pyroptosis, and provided a PROTAC-based pyroptosis inducer for anticancer treatment.
    DOI:  https://doi.org/10.1021/jacs.3c01852
  13. Dev Cell. 2023 Apr 24. pii: S1534-5807(23)00156-9. [Epub ahead of print]
    A tubule-sheet continuum model for the mechanism of nuclear envelope assembly.
    Gengjing Zhao, Shiwei Liu, Sanjana Arun, Fioranna Renda, Alexey Khodjakov, David Pellman.
      Nuclear envelope (NE) assembly defects cause chromosome fragmentation, cancer, and aging. However, major questions about the mechanism of NE assembly and its relationship to nuclear pathology are unresolved. In particular, how cells efficiently assemble the NE starting from vastly different, cell type-specific endoplasmic reticulum (ER) morphologies is unclear. Here, we identify a NE assembly mechanism, "membrane infiltration," that defines one end of a continuum with another NE assembly mechanism, "lateral sheet expansion," in human cells. Membrane infiltration involves the recruitment of ER tubules or small sheets to the chromatin surface by mitotic actin filaments. Lateral sheet expansion involves actin-independent envelopment of peripheral chromatin by large ER sheets that then extend over chromatin within the spindle. We propose a "tubule-sheet continuum" model that explains the efficient NE assembly from any starting ER morphology, the cell type-specific patterns of nuclear pore complex (NPC) assembly, and the obligatory NPC assembly defect of micronuclei.
    Keywords:  endoplasmic reticulum; micronuclei; mitosis; mitotic actin; myosin V; nuclear envelope; nuclear pore complexes
    DOI:  https://doi.org/10.1016/j.devcel.2023.04.003
  14. Autophagy. 2023 Apr 28.
    Autophagy Inhibition as a Potential Therapeutic Strategy for Breast Cancer with Mitochondrial Translation Defect Caused by CBFB-Deficiency.
    Navdeep Malik, Hualong Yan, Young-Im Kim, Gamze Ayaz, Shasha Wang, Payel Mondal, Ji Luo, Jing Huang.
      Autophagy plays a crucial role in tumor initiation and progression. However, targeting autophagy in cancer has proven challenging due to genetic or epigenetic factors that may affect the efficacy of autophagy inhibition. Therefore, identifying biomarkers is crucial for selecting patients who are likely to benefit from this treatment modality. We show that dysregulation of mitochondrial translation caused by CBFB (core-binding factor subunit beta) deficiency can sensitize the tumors to autophagy inhibition. CBFB and its binding partner HNRNPK (heterogeneous nuclear ribonucleoprotein K) interact with mRNAs encoded by the mitochondrial genome (mt-mRNAs) and maintain their translation. Specifically, CBFB enhances the binding of TUFM (Tu translation elongation factor, mitochondrial), an elongation factor for mitochondrial translation, to mt-mRNAs. CBFB deficiency, which often occurs in estrogen receptor-positive breast tumors, results in elevated autophagy and mitophagy that promote cancer cell survival. Consequently, these cells are hypersensitive to autophagy inhibition, creating a targetable vulnerability. Studies using in vivo models have shown that inhibiting autophagy selectively eliminates breast tumor cells with mitochondrial translation defects resulting from CBFB deficiency. Our results suggest that autophagy inhibition may be an effective treatment option for breast tumors carrying CBFB alterations.
    Keywords:  Autophagy; CBFB; PIK3CA; autophagy in cancer; autophagy targeting; breast cancer; mitochondria; mitochondrial translation; mitophagy
    DOI:  https://doi.org/10.1080/15548627.2023.2208481
  15. J Cell Sci. 2023 Apr 15. pii: jcs260741. [Epub ahead of print]136(8):
    The role of crinophagy in quality control of the regulated secretory pathway.
    Győző Szenci, Tamás Csizmadia, Gábor Juhász.
      In specialized secretory cells that produce and release biologically active substances in a regulated fashion, tight control of both the quantity and quality of secretory material is of paramount importance. During crinophagy, abnormal, excess or obsolete secretory granules directly fuse with lysosomes to yield crinosomes, in which the delivered secretory material is degraded. Crinophagy maintains the proper intracellular pool of secretory granules, and it is enhanced when secretory material accumulates because of compromised secretion. Recent studies highlight that it can even degrade newly formed, nascent secretory granules that shed from the trans-Golgi network. This implies that crinophagy provides a quality control checkpoint acting at the formation of secretory vesicles, and this degradation mechanism might survey secretory granules throughout their maturation. Of note, a plethora of human disorders is associated with defective lysosomal clearance of secretory material via crinophagy or similar pathways, including macro- or micro-autophagic degradation of secretory granules (referred to here as macro- and micro-secretophagy, respectively). In our Review, we summarize key recent advances in this field and discuss potential links with disease.
    Keywords:  Autophagy; Crinophagy; Secretion
    DOI:  https://doi.org/10.1242/jcs.260741
  16. Mol Cell Biol. 2023 Apr 28. 1-23
    Chaperone-Dependent Degradation of Cdc42 Promotes Cell Polarity and Shields the Protein from Aggregation.
    Beatriz González, Martí Aldea, Paul J Cullen.
      Rho GTPases are global regulators of cell polarity and signaling. By exploring the turnover regulation of the yeast Rho GTPase Cdc42p, we identified new regulatory features surrounding the stability of the protein. We specifically show that Cdc42p is degraded at 37 °C by chaperones through lysine residues located in the C-terminus of the protein. Cdc42p turnover at 37 °C occurred by the 26S proteasome in an ESCRT-dependent manner in the lysosome/vacuole. By analyzing versions of Cdc42p that were defective for turnover, we show that turnover at 37 °C promoted cell polarity but was defective for sensitivity to mating pheromone, presumably mediated through a Cdc42p-dependent MAP kinase pathway. We also identified one residue (K16) in the P-loop of the protein that was critical for Cdc42p stability. Accumulation of Cdc42pK16R in some contexts led to the formation of protein aggregates, which were enriched in aging mother cells and cells undergoing proteostatic stress. Our study uncovers new aspects of protein turnover regulation of a Rho-type GTPase that may extend to other systems. Moreover, residues identified here that mediate Cdc42p turnover correlate with several human diseases, which may suggest that turnover regulation of Cdc42p is important to aspects of human health.
    Keywords:  ESCRT; aging; protein aggregation; protein trafficking; quality control; sexual selection; temperature; tradeoffs
    DOI:  https://doi.org/10.1080/10985549.2023.2198171
  17. J Cell Biol. 2023 Jul 03. pii: e202203060. [Epub ahead of print]222(7):
    Reticulons promote formation of ER-derived double-membrane vesicles that facilitate SARS-CoV-2 replication.
    Jeffrey M Williams, Yu-Jie Chen, Woo Jung Cho, Andrew W Tai, Billy Tsai.
      Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiologic agent for the global COVID-19 pandemic, triggers the formation of endoplasmic reticulum (ER)-derived replication organelles, including double-membrane vesicles (DMVs), in the host cell to support viral replication. Here, we clarify how SARS-CoV-2 hijacks host factors to construct the DMVs. We show that the ER morphogenic proteins reticulon-3 (RTN3) and RTN4 help drive DMV formation, enabling viral replication, which leads to productive infection. Different SARS-CoV-2 variants, including the delta variant, use the RTN-dependent pathway to promote infection. Mechanistically, our results reveal that the membrane-embedded reticulon homology domain (RHD) of the RTNs is sufficient to functionally support viral replication and physically engage NSP3 and NSP4, two viral non-structural membrane proteins known to induce DMV formation. Our findings thus identify the ER morphogenic RTN3 and RTN4 membrane proteins as host factors that help promote the biogenesis of SARS-CoV-2-induced DMVs, which can act as viral replication platforms.
    DOI:  https://doi.org/10.1083/jcb.202203060
  18. Life Sci Alliance. 2023 Jul;pii: e202201876. [Epub ahead of print]6(7):
    Structural insights into ubiquitin chain cleavage by Legionella ovarian tumor deubiquitinases.
    Sangwoo Kang, Gyuhee Kim, Minhyeong Choi, Minwoo Jeong, Gerbrand J van der Heden van Noort, Soung-Hun Roh, Donghyuk Shin.
      Although ubiquitin is found only in eukaryotes, several pathogenic bacteria and viruses possess proteins that hinder the host ubiquitin system. Legionella, a gram-negative intracellular bacterium, possesses an ovarian tumor (OTU) family of deubiquitinases (Lot DUBs). Herein, we describe the molecular characteristics of Lot DUBs. We elucidated the structure of the LotA OTU1 domain and revealed that entire Lot DUBs possess a characteristic extended helical lobe that is not found in other OTU-DUBs. The structural topology of an extended helical lobe is the same throughout the Lot family, and it provides an S1' ubiquitin-binding site. Moreover, the catalytic triads of Lot DUBs resemble those of the A20-type OTU-DUBs. Furthermore, we revealed a unique mechanism by which LotA OTU domains cooperate together to distinguish the length of the chain and preferentially cleave longer K48-linked polyubiquitin chains. The LotA OTU1 domain itself cleaves K6-linked ubiquitin chains, whereas it is also essential for assisting the cleavage of longer K48-linked polyubiquitin chains by the OTU2 domain. Thus, this study provides novel insights into the structure and mechanism of action of Lot DUBs.
    DOI:  https://doi.org/10.26508/lsa.202201876
  19. Mol Cell. 2023 Apr 14. pii: S1097-2765(23)00243-5. [Epub ahead of print]
    The mechanism of STING autoinhibition and activation.
    Sheng Liu, Bo Yang, Yingxiang Hou, Kaige Cui, Xiaozhu Yang, Xiaoxiong Li, Lianwan Chen, Shichao Liu, Zhichao Zhang, Yuanyuan Jia, Yufeng Xie, Ying Xue, Xiaomei Li, Bingxue Yan, Changxin Wu, Wen Deng, Jianxun Qi, Defen Lu, George F Gao, Peiyi Wang, Guijun Shang.
      2',3'-cGAMP, produced by the DNA sensor cGAS, activates stimulator of interferon genes (STING) and triggers immune response during infection. Tremendous effort has been placed on unraveling the mechanism of STING activation. However, little is known about STING inhibition. Here, we found that apo-STING exhibits a bilayer with head-to-head as well as side-by-side packing, mediated by its ligand-binding domain (LBD). This type of assembly holds two endoplasmic reticulum (ER) membranes together not only to prevent STING ER exit but also to eliminate the recruitment of TBK1, representing the autoinhibited state of STING. Additionally, we obtained the filament structure of the STING/2',3'-cGAMP complex, which adopts a bent monolayer assembly mediated by LBD and transmembrane domain (TMD). The active, curved STING polymer could deform ER membrane to support its ER exit and anterograde transportation. Our data together provide a panoramic vision regarding STING autoinhibition and activation, which adds substantially to current understanding of the cGAS-STING pathway.
    Keywords:  STING; activation; autoinhibition; cGAMP; cGAS
    DOI:  https://doi.org/10.1016/j.molcel.2023.03.029
  20. Noncoding RNA. 2023 Apr 13. pii: 26. [Epub ahead of print]9(2):
    MicroRNAs in Age-Related Proteostasis and Stress Responses.
    Latika Matai, Frank J Slack.
      Aging is associated with the accumulation of damaged and misfolded proteins through a decline in the protein homeostasis (proteostasis) machinery, leading to various age-associated protein misfolding diseases such as Huntington's or Parkinson's. The efficiency of cellular stress response pathways also weakens with age, further contributing to the failure to maintain proteostasis. MicroRNAs (miRNAs or miRs) are a class of small, non-coding RNAs (ncRNAs) that bind target messenger RNAs at their 3'UTR, resulting in the post-transcriptional repression of gene expression. From the discovery of aging roles for lin-4 in C. elegans, the role of numerous miRNAs in controlling the aging process has been uncovered in different organisms. Recent studies have also shown that miRNAs regulate different components of proteostasis machinery as well as cellular response pathways to proteotoxic stress, some of which are very important during aging or in age-related pathologies. Here, we present a review of these findings, highlighting the role of individual miRNAs in age-associated protein folding and degradation across different organisms. We also broadly summarize the relationships between miRNAs and organelle-specific stress response pathways during aging and in various age-associated diseases.
    Keywords:  HSPs; UPR; aging; autophagy; health-span; heat-shock; lifespan; longevity; miR; miRNA; proteostasis; stress response
    DOI:  https://doi.org/10.3390/ncrna9020026
  21. Sci Adv. 2023 Apr 28. 9(17): eade8934
    Mapping the in vivo fitness landscape of a tethered ribosome.
    Felix Radford, Jesse Rinehart, Farren J Isaacs.
      Fitness landscapes are models of the sequence space of a genetic element that map how each sequence corresponds to its activity and can be used to guide laboratory evolution. The ribosome is a macromolecular machine that is essential for protein synthesis in all organisms. Because of the prevalence of dominant lethal mutations, a comprehensive fitness landscape of the ribosomal peptidyl transfer center (PTC) has not yet been attained. Here, we develop a method to functionally map an orthogonal tethered ribosome (oRiboT), which permits complete mutagenesis of nucleotides located in the PTC and the resulting epistatic interactions. We found that most nucleotides studied showed flexibility to mutation, and identified epistatic interactions between them, which compensate for deleterious mutations. This work provides a basis for a deeper understanding of ribosome function and malleability and could be used to inform design of engineered ribosomes with applications to synthesize next-generation biomaterials and therapeutics.
    DOI:  https://doi.org/10.1126/sciadv.ade8934
  22. J Cell Biol. 2023 Jul 03. pii: e202208088. [Epub ahead of print]222(7):
    ATG9 vesicles comprise the seed membrane of mammalian autophagosomes.
    Taryn J Olivas, Yumei Wu, Shenliang Yu, Lin Luan, Peter Choi, Emily D Guinn, Shanta Nag, Pietro V De Camilli, Kallol Gupta, Thomas J Melia.
      As the autophagosome forms, its membrane surface area expands rapidly, while its volume is kept low. Protein-mediated transfer of lipids from another organelle to the autophagosome likely drives this expansion, but as these lipids are only introduced into the cytoplasmic-facing leaflet of the organelle, full membrane growth also requires lipid scramblase activity. ATG9 harbors scramblase activity and is essential to autophagosome formation; however, whether ATG9 is integrated into mammalian autophagosomes remains unclear. Here we show that in the absence of lipid transport, ATG9 vesicles are already competent to collect proteins found on mature autophagosomes, including LC3-II. Further, we use styrene-maleic acid lipid particles to reveal the nanoscale organization of protein on LC3-II membranes; ATG9 and LC3-II are each fully integrated into expanding autophagosomes. The ratios of these two proteins at different stages of maturation demonstrate that ATG9 proteins are not continuously integrated, but rather are present on the seed vesicles only and become diluted in the expanding autophagosome membrane.
    DOI:  https://doi.org/10.1083/jcb.202208088
  23. ACS Chem Biol. 2023 Apr 25.
    Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.
    Benoit Pelletier, Stéphanie Duhamel, Guillaume Tambutet, Scott Jarvis, Patrick Cléroux, Maud David, Pierre-Luc Tanguay, Laure Voisin, Clint James, Rico Lavoie, Yves Gareau, Joël Flynn-Robitaille, Thierry Lorca, Réjean Ruel, Anne Marinier, Sylvain Meloche.
      p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.
    DOI:  https://doi.org/10.1021/acschembio.2c00909
  24. Nat Commun. 2023 Apr 28. 14(1): 2457
    USP7 controls NGN3 stability and pancreatic endocrine lineage development.
    Teodora Manea, Jessica Kristine Nelson, Cristina Maria Garrone, Karin Hansson, Ian Evans, Axel Behrens, Rocio Sancho.
      Understanding the factors and mechanisms involved in beta-cell development will guide therapeutic efforts to generate fully functional beta cells for diabetes. Neurogenin 3 (NGN3) is the key transcription factor that marks endocrine progenitors and drives beta-cell differentiation. Here we screen for binding partners of NGN3 and identify the deubiquitylating enzyme USP7 as a key regulator of NGN3 stability. Mechanistically, USP7 interacts with, deubiquitinates and stabilizes NGN3. In vivo, conditional knockout of Usp7 in the mouse embryonic pancreas causes a dramatic reduction in islet formation and hyperglycemia in adult mice, due to impaired NGN3-mediated endocrine specification during pancreatic development. Furthermore, pharmacological inhibition of USP7 during endocrine specification in human iPSC models of beta-cell differentiation decreases NGN3 expressing progenitor cell numbers and impairs beta cell differentiation. Thus, the USP7-NGN3 axis is an essential mechanism for driving endocrine development and beta-cell differentiation, which can be therapeutically exploited.
    DOI:  https://doi.org/10.1038/s41467-023-38146-9
  25. Nat Aging. 2023 Jan;3(1): 34-46
    Nucleophagy delays aging and preserves germline immortality.
    Margarita-Elena Papandreou, Georgios Konstantinidis, Nektarios Tavernarakis.
      Marked alterations in nuclear ultrastructure are a universal hallmark of aging, progeroid syndromes and other age-related pathologies. Here we show that autophagy of nuclear proteins is an important determinant of fertility and aging. Impairment of nucleophagy diminishes stress resistance, germline immortality and longevity. We found that the nematode Caenorhabditis elegans nuclear envelope anchor protein, nuclear anchorage protein 1 (ANC-1) and its mammalian ortholog nesprin-2 are cleared out by autophagy and restrict nucleolar size, a biomarker of aging. We further uncovered a germline immortality assurance mechanism, which involves nucleolar degradation at the most proximal oocyte by ANC-1 and key autophagic components. Perturbation of this clearance pathway causes tumor-like structures in C. elegans, and genetic ablation of nesprin-2 causes ovarian carcinomas in mice. Thus, autophagic recycling of nuclear components is a conserved soma longevity and germline immortality mechanism that promotes youthfulness and delays aging under conditions of stress.
    DOI:  https://doi.org/10.1038/s43587-022-00327-4
  26. Protein Sci. 2023 Apr 25. e4645
    A new kid in the folding funnel: Molecular chaperone activities of the BRICHOS domain.
    Axel Leppert, Helen Poska, Michael Landreh, Axel Abelein, Gefei Chen, Jan Johansson.
      The BRICHOS protein superfamily is a diverse group of proteins associated with a wide variety of human diseases, including respiratory distress, covid-19, dementia, and cancer. A key characteristic of these proteins - besides their BRICHOS domain present in the ER lumen/extracellular part - is that they harbor an aggregation-prone region, which the BRICHOS domain is proposed to chaperone during biosynthesis. All so far studied BRICHOS domains modulate the aggregation pathway of various amyloid-forming substrates, but not all of them can keep denaturing proteins in a folding-competent state, in a similar manner as small heat shock proteins. Current evidence suggests that the ability to interfere with the aggregation pathways of substrates with entirely different end-point structures is dictated by BRICHOS quaternary-structure as well as specific surface motifs. This review aims to provide an overview of the BRICHOS protein family and a perspective of the diverse molecular chaperone-like functions of various BRICHOS domains in relation to their structure and conformational plasticity. Furthermore, we speculate about the physiological implication of the diverse molecular chaperone functions and discuss the possibility to use the BRICHOS domain as a blood brain barrier permeable molecular chaperone treatment of protein aggregation disorders. This article is protected by copyright. All rights reserved.
    Keywords:  Alzheimer disease treatment; amyloid; blood brain barrier; molecular chaperone; protein misfolding
    DOI:  https://doi.org/10.1002/pro.4645
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