bims-proteo Biomed News
on Proteostasis
Issue of 2023–02–19
24 papers selected by
Eric Chevet, INSERM
  1. Interference with the HNF4-dependent gene regulatory network diminishes ER stress in hepatocytes.
  2. Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.
  3. Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2.
  4. Neuronal endoplasmic reticulum architecture and roles in axonal physiology.
  5. Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation.
  6. The impact of endoplasmic reticulum morphology on IRE1 protein clustering.
  7. The outer mitochondrial membrane protein TMEM11 demarcates spatially restricted BNIP3/BNIP3L-mediated mitophagy.
  8. SQSTM1 facilitates oocyte maturation through inducing ACLY degradation mediated by selective autophagy.
  9. Quality control ensures fidelity in ribosome assembly and cellular health.
  10. Network of hotspot interactions cluster tau amyloid folds.
  11. A highly efficient human cell-free translation system.
  12. A Year at the Forefront of Proteostasis and Aging.
  13. Dynamic mapping of proteome trafficking within and between living cells by TransitID.
  14. Chemoproteomic discovery of a human RNA ligase.
  15. Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.
  16. Linear ubiquitination improves NFAT1 protein stability and facilitates NFAT1 signaling in Kawasaki disease.
  17. AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in a neurodevelopmental disorder.
  18. Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase.
  19. BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.
  20. Transcellular chaperone signaling is an intercellular stress-response distinct from the HSF-1-mediated heat shock response.
  21. Intestinal SEC16B modulates obesity by regulating chylomicron metabolism.
  22. SUMOylation of NaV1.2 channels regulates the velocity of backpropagating action potentials in cortical pyramidal neurons.
  23. Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline.
  24. A NPAS4-NuA4 complex couples synaptic activity to DNA repair.
  1. bioRxiv. 2023 Feb 09. pii: 2023.02.09.527889. [Epub ahead of print]
    Interference with the HNF4-dependent gene regulatory network diminishes ER stress in hepatocytes.
    Anit Shah, Ian Huck, Kaylia Duncan, Erica R Gansemer, Udayan Apte, Mark A Stamnes, D Thomas Rutkowski.
      In all eukaryotic cell types, the unfolded protein response (UPR) upregulates factors that promote protein folding and misfolded protein clearance to help alleviate endoplasmic reticulum (ER) stress. Yet ER stress in the liver is uniquely accompanied by the suppression of metabolic genes, the coordination and purpose of which is largely unknown. Here, we used unsupervised machine learning to identify a cluster of correlated genes that were profoundly suppressed by persistent ER stress in the liver. These genes, which encode diverse functions including metabolism, coagulation, drug detoxification, and bile synthesis, are likely targets of the master regulator of hepatocyte differentiation HNF4α. The response of these genes to ER stress was phenocopied by liver-specific deletion of HNF4α. Strikingly, while deletion of HNF4α exacerbated liver injury in response to an ER stress challenge, it also diminished UPR activation and partially preserved ER ultrastructure, suggesting attenuated ER stress. Conversely, pharmacological maintenance of hepatocyte identity in vitro enhanced sensitivity to stress. Several pathways potentially link HNF4α to ER stress sensitivity, including control of expression of the tunicamycin transporter MFSD2A; modulation of IRE1/XBP1 signaling; and regulation of Pyruvate Dehydrogenase. Together, these findings suggest that HNF4α activity is linked to hepatic ER homeostasis through multiple mechanisms.
    DOI:  https://doi.org/10.1101/2023.02.09.527889
  2. Chem Commun (Camb). 2023 Feb 15.
    Squaryl group-modified UDP analogs as inhibitors of the endoplasmic reticulum-resident folding sensor enzyme UGGT.
    Junpei Abe, Yoichi Takeda, Takashi Kikuma, Yasuhiko Kizuka, Hiroyuki Kajiura, Yasuhiro Kajihara, Yukishige Ito.
      UDP-Glc:glycoprotein glucosyltransferase (UGGT) has a central role to retain quality control of correctly folded N-glycoprotein in the endoplasmic reticulum (ER). A selective and potent inhibitor against UGGT could lead to elucidation of UGGT-related events, but such a molecule has not been identified so far. Examples of small molecules with UGGT inhibitory activity are scarce. Here, we report squaryl group-modified UDP analogs as a promising UGGT inhibitor. Among these, the compound possessing a 2'-amino group of the uridine moiety and hydroxyethyl-substituted squaramide exhibited the highest potency, suggesting its relevance as a molecule for further optimization.
    DOI:  https://doi.org/10.1039/d2cc06634c
  3. Nat Commun. 2023 Feb 16. 14(1): 870
    Spatial proteomics reveals secretory pathway disturbances caused by neuropathy-associated TECPR2.
    Karsten Nalbach, Martina Schifferer, Debjani Bhattacharya, Hung Ho-Xuan, Wei Tseng, Luis A Williams, Alexandra Stolz, Stefan F Lichtenthaler, Zvulun Elazar, Christian Behrends.
      Hereditary sensory and autonomic neuropathy 9 (HSAN9) is a rare fatal neurological disease caused by mis- and nonsense mutations in the gene encoding for Tectonin β-propeller repeat containing protein 2 (TECPR2). While TECPR2 is required for lysosomal consumption of autophagosomes and ER-to-Golgi transport, it remains elusive how exactly TECPR2 is involved in autophagy and secretion and what downstream sequels arise from defective TECPR2 due to its involvement in these processes. To address these questions, we determine molecular consequences of TECPR2 deficiency along the secretory pathway. By employing spatial proteomics, we describe pronounced changes with numerous proteins important for neuronal function being affected in their intracellular transport. Moreover, we provide evidence that TECPR2's interaction with the early secretory pathway is not restricted to COPII carriers. Collectively, our systematic profiling of a HSAN9 cell model points to specific trafficking and sorting defects which might precede autophagy dysfunction upon TECPR2 deficiency.
    DOI:  https://doi.org/10.1038/s41467-023-36553-6
  4. Mol Cell Neurosci. 2023 Feb 11. pii: S1044-7431(23)00016-7. [Epub ahead of print] 103822
    Neuronal endoplasmic reticulum architecture and roles in axonal physiology.
    Klaas Yperman, Marijn Kuijpers.
      The endoplasmic reticulum (ER) is the largest membrane compartment within eukaryotic cells and is emerging as a key coordinator of many cellular processes. The ER can modulate local calcium fluxes and communicate with other organelles like the plasma membrane. The importance of ER in neuronal processes such as neurite growth, axon repair and neurotransmission has recently gained much attention. In this review, we highlight the importance of the ER tubular network in axonal homeostasis and discuss how the generation and maintenance of the thin tubular ER network in axons and synapses, requires a cooperative effort of ER-shaping proteins, cytoskeleton and autophagy processes.
    Keywords:  ER-phagy; Endoplasmic reticulum; Neuron; Neurotransmission; Synapse
    DOI:  https://doi.org/10.1016/j.mcn.2023.103822
  5. Sci Adv. 2023 Feb 15. 9(7): eadg2997
    Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation.
    Xuefeng Ren, Thanh N Nguyen, Wai Kit Lam, Cosmo Z Buffalo, Michael Lazarou, Adam L Yokom, James H Hurley.
      The assembly of the autophagy initiation machinery nucleates autophagosome biogenesis, including in the PINK1- and Parkin-dependent mitophagy pathway implicated in Parkinson's disease. The structural interaction between the sole transmembrane autophagy protein, autophagy-related protein 9A (ATG9A), and components of the Unc-51-like autophagy activating kinase (ULK1) complex is one of the major missing links needed to complete a structural map of autophagy initiation. We determined the 2.4-Å x-ray crystallographic structure of the ternary structure of ATG9A carboxyl-terminal tail bound to the ATG13:ATG101 Hop1/Rev7/Mad2 (HORMA) dimer, which is part of the ULK1 complex. We term the interacting portion of the extreme carboxyl-terminal part of the ATG9A tail the "HORMA dimer-interacting region" (HDIR). This structure shows that the HDIR binds to the HORMA domain of ATG101 by β sheet complementation such that the ATG9A tail resides in a deep cleft at the ATG13:ATG101 interface. Disruption of this complex in cells impairs damage-induced PINK1/Parkin mitophagy mediated by the cargo receptor NDP52.
    DOI:  https://doi.org/10.1126/sciadv.adg2997
  6. Biophys J. 2023 Feb 10. pii: S0006-3495(22)03632-3. [Epub ahead of print]122(3S1): 510a
    The impact of endoplasmic reticulum morphology on IRE1 protein clustering.
    Liam Kischuck, Aidan Brown.
      
    DOI:  https://doi.org/10.1016/j.bpj.2022.11.2716
  7. J Cell Biol. 2023 Apr 03. pii: e202204021. [Epub ahead of print]222(4):
    The outer mitochondrial membrane protein TMEM11 demarcates spatially restricted BNIP3/BNIP3L-mediated mitophagy.
    Mehmet Oguz Gok, Olivia M Connor, Xun Wang, Cameron J Menezes, Claire B Llamas, Prashant Mishra, Jonathan R Friedman.
      Mitochondria play critical roles in cellular metabolism and to maintain their integrity, they are regulated by several quality control pathways, including mitophagy. During BNIP3/BNIP3L-dependent receptor-mediated mitophagy, mitochondria are selectively targeted for degradation by the direct recruitment of the autophagy protein LC3. BNIP3 and/or BNIP3L are upregulated situationally, for example during hypoxia and developmentally during erythrocyte maturation. However, it is not well understood how they are spatially regulated within the mitochondrial network to locally trigger mitophagy. Here, we find that the poorly characterized mitochondrial protein TMEM11 forms a complex with BNIP3 and BNIP3L and co-enriches at sites of mitophagosome formation. We find that mitophagy is hyper-active in the absence of TMEM11 during both normoxia and hypoxia-mimetic conditions due to an increase in BNIP3/BNIP3L mitophagy sites, supporting a model that TMEM11 spatially restricts mitophagosome formation.
    DOI:  https://doi.org/10.1083/jcb.202204021
  8. Cell Cycle. 2023 Feb 14. 1-3
    SQSTM1 facilitates oocyte maturation through inducing ACLY degradation mediated by selective autophagy.
    Heide Schatten.
      Selective autophagy specifically eliminates certain intracellular substrates through the autophagy pathway. Organelles and aggregation-prone proteins can be degraded through the autophagy receptor protein SQSTM1/p62, which renders them a promising therapeutic approach against infertility. He et al. demonstrate that blocking of autophagy in cumulus granulosa cells can directly attenuate citrate levels and in turn affect oocyte maturation quality. Further findings show that SQSTM1 connects K63-polyubiquitinated ACLY (ATP citrate lyase) during the process of selective autophagic degradation, which further compromises the homeostasis of citrate. Therefore, the quality of oocyte meiotic maturation can be evaluated by the levels of selective autophagy in cumulus granulosa cells.
    Keywords:  ACLY; Sqstm1/P62; citrate; meiotic resumption; selective autophagy
    DOI:  https://doi.org/10.1080/15384101.2023.2176673
  9. J Cell Biol. 2023 Apr 03. pii: e202209115. [Epub ahead of print]222(4):
    Quality control ensures fidelity in ribosome assembly and cellular health.
    Melissa D Parker, Katrin Karbstein.
      The coordinated integration of ribosomal RNA and protein into two functional ribosomal subunits is safeguarded by quality control checkpoints that ensure ribosomes are correctly assembled and functional before they engage in translation. Quality control is critical in maintaining the integrity of ribosomes and necessary to support healthy cell growth and prevent diseases associated with mistakes in ribosome assembly. Its importance is demonstrated by the finding that bypassing quality control leads to misassembled, malfunctioning ribosomes with altered translation fidelity, which change gene expression and disrupt protein homeostasis. In this review, we outline our understanding of quality control within ribosome synthesis and how failure to enforce quality control contributes to human disease. We first provide a definition of quality control to guide our investigation, briefly present the main assembly steps, and then examine stages of assembly that test ribosome function, establish a pass-fail system to evaluate these functions, and contribute to altered ribosome performance when bypassed, and are thus considered "quality control."
    DOI:  https://doi.org/10.1083/jcb.202209115
  10. Nat Commun. 2023 Feb 16. 14(1): 895
    Network of hotspot interactions cluster tau amyloid folds.
    Vishruth Mullapudi, Jaime Vaquer-Alicea, Vaibhav Bommareddy, Anthony R Vega, Bryan D Ryder, Charles L White, Marc I Diamond, Lukasz A Joachimiak.
      Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformations of β-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We evaluate our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.
    DOI:  https://doi.org/10.1038/s41467-023-36572-3
  11. bioRxiv. 2023 Feb 10. pii: 2023.02.09.527910. [Epub ahead of print]
    A highly efficient human cell-free translation system.
    Nikolay A Aleksashin, Stacey Tsai-Lan Chang, Jamie H D Cate.
      Cell-free protein synthesis (CFPS) systems enable easy in vitro expression of proteins with many scientific, industrial, and therapeutic applications. Here we present an optimized, highly efficient human cell-free translation system that bypasses many limitations of currently used in vitro systems. This CFPS system is based on extracts from human HEK293T cells engineered to endogenously express GADD34 and K3L proteins, which suppress phosphorylation of translation initiation factor eIF2α. Overexpression of GADD34 and K3L proteins in human cells before cell lysate preparation rather than adding them after the fact significantly simplifies lysate preparation. The new CFPS system improves the translation of 5' cap-dependent mRNAs as well as those that use internal ribosome entry site (IRES) mediated translation initiation. We find that expression of the GADD34 and K3L accessory proteins before cell lysis maintains low levels of phosphorylation of eIF2α in the extracts. During in vitro translation reactions, eIF2α phosphorylation increases moderately in a GCN2-dependent fashion that can be inhibited by GCN2 kinase inhibitors. We also find evidence for activation of regulatory pathways related to eukaryotic translation elongation factor 2 (eEF2) phosphorylation and ribosome quality control in the extracts. This new CFPS system should be useful for exploring human translation mechanisms in more physiological conditions outside the cell.
    DOI:  https://doi.org/10.1101/2023.02.09.527910
  12. Biol Open. 2023 Feb 15. pii: bio059750. [Epub ahead of print]12(2):
    A Year at the Forefront of Proteostasis and Aging.
    Maximilian A Thompson, Evandro A De-Souza.
      During aging, animals experience a decline in proteostasis activity, including loss of stress-response activation, culminating in the accumulation of misfolded proteins and toxic aggregates, which are causal in the onset of some chronic diseases. Finding genetic and pharmaceutical treatments that can increase organismal proteostasis and lengthen life is an ongoing goal of current research. The regulation of stress responses by cell non-autonomous mechanisms appears to be a potent way to impact organismal healthspan. In this Review, we cover recent findings in the intersection of proteostasis and aging, with a special focus on articles and preprints published between November 2021 and October 2022. A significant number of papers published during this time increased our understanding of how cells communicate with each other during proteotoxic stress. Finally, we also draw attention to emerging datasets that can be explored to generate new hypotheses that explain age-related proteostasis collapse.
    Keywords:  Cell non-autonomous response; Heat shock response; Longevity; Protein homeostasis; UPR
    DOI:  https://doi.org/10.1242/bio.059750
  13. bioRxiv. 2023 Feb 08. pii: 2023.02.07.527548. [Epub ahead of print]
    Dynamic mapping of proteome trafficking within and between living cells by TransitID.
    Wei Qin Xu, Joleen S Cheah, Charles Xu, James Messing, Brian D Freibaum, Steven Boeynaems, J Paul Taylor, Namrata D Udeshi, Steven A Carr, Alice Y Ting.
      The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules, uncovering a role for stress granules in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID introduces a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
    DOI:  https://doi.org/10.1101/2023.02.07.527548
  14. Nat Commun. 2023 Feb 15. 14(1): 842
    Chemoproteomic discovery of a human RNA ligase.
    Yizhi Yuan, Florian M Stumpf, Lisa A Schlor, Olivia P Schmidt, Philip Saumer, Luisa B Huber, Matthias Frese, Eva Höllmüller, Martin Scheffner, Florian Stengel, Kay Diederichs, Andreas Marx.
      RNA ligases are present across all forms of life. While enzymatic RNA ligation between 5'-PO4 and 3'-OH termini is prevalent in viruses, fungi, and plants, such RNA ligases are yet to be identified in vertebrates. Here, using a nucleotide-based chemical probe targeting human AMPylated proteome, we have enriched and identified the hitherto uncharacterised human protein chromosome 12 open reading frame 29 (C12orf29) as a human enzyme promoting RNA ligation between 5'-PO4 and 3'-OH termini. C12orf29 catalyses ATP-dependent RNA ligation via a three-step mechanism, involving tandem auto- and RNA AMPylation. Knock-out of C12ORF29 gene impedes the cellular resilience to oxidative stress featuring concurrent RNA degradation, which suggests a role of C12orf29 in maintaining RNA integrity. These data provide the groundwork for establishing a human RNA repair pathway.
    DOI:  https://doi.org/10.1038/s41467-023-36451-x
  15. Cell Chem Biol. 2023 Feb 08. pii: S2451-9456(23)00030-2. [Epub ahead of print]
    Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.
    Scott C Rosenberg, Frances Shanahan, Sayumi Yamazoe, Marc Kschonsak, Yi J Zeng, James Lee, Emile Plise, Ivana Yen, Christopher M Rose, John G Quinn, Lewis J Gazzard, Benjamin T Walters, Donald S Kirkpatrick, Steven T Staben, Scott A Foster, Shiva Malek.
      Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.
    Keywords:  EGFR; PROTAC; VHL; chemical biology; degrader; inhibitor
    DOI:  https://doi.org/10.1016/j.chembiol.2023.01.007
  16. FEBS J. 2023 Feb 13.
    Linear ubiquitination improves NFAT1 protein stability and facilitates NFAT1 signaling in Kawasaki disease.
    Ying Miao, Guanghui Qian, Renxia Zhang, Yukang Yuan, Yibo Zuo, Yueyue Ding, Xuan Li, Yunjia Tang, Hui Zheng, Haitao Lv.
      NFAT1 is known for its roles in T cell development and activation. So far, phosphorylation of NFAT1 has been extensively studied, but the other post-translational modifications of NFAT1 remain largely unknown. In this study, we reported that NFAT1 is a linearly ubiquitinated substrate of linear ubiquitin chain assembly complex (LUBAC). LUBAC promoted NFAT1 linear ubiquitination, which in turn inhibited K48-linked polyubiquitination of NFAT1 and therefore increased NFAT1 protein stability. Interestingly, the linear ubiquitination levels of NFAT1 in patients with the Kawasaki disease were upregulated. Further studies demonstrated that the patients with the Kawasaki disease had increased mRNA levels of HOIL-1L. These findings revealed a linearly ubiquitinated substrate of LUBAC and an important biological function of NFAT1 linear ubiquitination in the Kawasaki disease, and therefore may provide a novel strategy for treatment of the Kawasaki disease.
    Keywords:  Kawasaki disease; LUBAC; Linear ubiquitination; NFAT1; Protein stability
    DOI:  https://doi.org/10.1111/febs.16749
  17. bioRxiv. 2023 Jan 31. pii: 2023.01.31.526497. [Epub ahead of print]
    AGAP1-associated endolysosomal trafficking abnormalities link gene-environment interactions in a neurodevelopmental disorder.
    Sara A Lewis, Somayeh Bakhtiari, Jacob Forstrom, Allan Bayat, Frédéric Bilan, Gwenaël Le Guyader, Ebba Alkhunaizi, Hilary Vernon, Sergio R Padilla-Lopez, Michael C Kruer.
      AGAP1 is an Arf1 GAP that regulates endolysosomal trafficking. Damaging variants have been linked to cerebral palsy and autism. We report 3 new individuals with microdeletion variants in AGAP1 . Affected individuals have intellectual disability (3/3), autism (3/3), dystonia with axial hypotonia (1/3), abnormalities of brain maturation (1/3), growth impairment (2/3) and facial dysmorphism (2/3). We investigated mechanisms potentially underlying AGAP1 neurodevelopmental impairments using the Drosophila ortholog, CenG1a . We discovered reduced axon terminal size, increased neuronal endosome abundance, and elevated autophagy at baseline. Given potential incomplete penetrance, we assessed gene-environment interactions. We found basal elevation in phosphorylation of the integrated stress-response protein eIF2α and inability to further increase eIF2α-P with subsequent cytotoxic stressors. CenG1a -mutant flies have increased lethality from exposure to environmental insults. We propose a model wherein disruption of AGAP1 function impairs endolysosomal trafficking, chronically activating the integrated stress response, and leaving AGAP1-deficient cells susceptible to a variety of second hit cytotoxic stressors. This model may have broader applicability beyond AGAP1 in instances where both genetic and environmental insults co-occur in individuals with neurodevelopmental disorders.
    Summary statement: We describe 3 additional patients with heterozygous AGAP1 deletion variants and use a loss of function Drosophila model to identify defects in synaptic morphology with increased endosomal sequestration, chronic autophagy induction, basal activation of eIF2α-P, and sensitivity to environmental stressors.
    DOI:  https://doi.org/10.1101/2023.01.31.526497
  18. Commun Biol. 2023 Feb 15. 6(1): 177
    Cooperation of N- and C-terminal substrate transmembrane domain segments in intramembrane proteolysis by γ-secretase.
    Nadine T Werner, Philipp Högel, Gökhan Güner, Walter Stelzer, Manfred Wozny, Marlene Aßfalg, Stefan F Lichtenthaler, Harald Steiner, Dieter Langosch.
      Intramembrane proteases play a pivotal role in biology and medicine, but how these proteases decode cleavability of a substrate transmembrane (TM) domain remains unclear. Here, we study the role of conformational flexibility of a TM domain, as determined by deuterium/hydrogen exchange, on substrate cleavability by γ-secretase in vitro and in cellulo. By comparing hybrid TMDs based on the natural amyloid precursor protein TM domain and an artificial poly-Leu non-substrate, we find that substrate cleavage requires conformational flexibility within the N-terminal half of the TMD helix (TM-N). Robust cleavability also requires the C-terminal TM sequence (TM-C) containing substrate cleavage sites. Since flexibility of TM-C does not correlate with cleavage efficiency, the role of the TM-C may be defined mainly by its ability to form a cleavage-competent state near the active site, together with parts of presenilin, the enzymatic component of γ-secretase. In sum, cleavability of a γ-secretase substrate appears to depend on cooperating TM domain segments, which deepens our mechanistic understanding of intramembrane proteolysis.
    DOI:  https://doi.org/10.1038/s42003-023-04470-5
  19. bioRxiv. 2023 Feb 09. pii: 2023.02.08.527546. [Epub ahead of print]
    BAG3 regulates the specificity of the recognition of specific MAPT species by NBR1 and SQSTM1.
    Heng Lin, Sarah Sandkuhler, Colleen Dunlea, Darron H King, Gail V W Johnson.
      Autophagy receptors are essential for the recognition and clearance of specific cargos by selective autophagy, which is essential for maintaining MAPT proteostasis. Previous studies have implicated different autophagy receptors in directing distinct species of MAPT to autophagy, but the underlying mechanisms have not been fully investigated. Here we examine how the autophagy receptors NBR1 and SQSTM1 differentially engage specific forms of MAPT and facilitate their clearance. In primary neurons depletion of NBR1, unlike depletion of SQSTM1, significantly increased phosphorylated MAPT levels. The specificity of the interactions were confirmed using in vitro binding assays with purified proteins. We provide direct evidence that NBR1 preferentially binds to monomeric MAPT, while SQSTM1 interacts predominantly with oligomeric MAPT, and that the co-chaperone BAG3 regulates the specificity of these interactions. Using an in vitro pulldown assay, we show that SQSTM1 only binds to monomeric MAPT when BAG3 is absent and fails to bind when BAG3 is present. The opposite is true of NBR1; its binding to monomeric MAPT was dependent on the presence of BAG3. Interestingly, in Alzheimer’s disease brain the association of NBR1 with BAG3 was significantly decreased. In a mouse model, ablation of BAG3 in neural cells disrupted the association of NBR1 with phosphorylated MAPT and lead to increased levels of phosphorylated and oligomeric MAPT. Overall, our results uncover a novel role for BAG3 in regulating the specificity of selective autophagy receptors in targeting different species of MAPT and provide compelling evidence that BAG3 plays a key role in maintaining MAPT proteostasis.
    Highlights: First direct evidence of the district role of NBR1 and SQSTM1 in binding with monomeric and oligomeric MAPT, respectively.Demonstration of a novel mechanism by which BAG3 regulates the specificity of the recognition of monomeric MAPT by NBR1 and oligomeric MAPT by SQSTM1.Conditional knockout of BAG3 in the brain disrupted the association of NBR1 with phosphorylated MAPT and lead to increased levels of phosphorylated and oligomeric MAPT.
    DOI:  https://doi.org/10.1101/2023.02.08.527546
  20. PLoS Biol. 2023 Feb 13. 21(2): e3001605
    Transcellular chaperone signaling is an intercellular stress-response distinct from the HSF-1-mediated heat shock response.
    Jay Miles, Sarah Townend, Dovilė Milonaitytė, William Smith, Francesca Hodge, David R Westhead, Patricija van Oosten-Hawle.
      Organismal proteostasis is maintained by intercellular signaling processes including cell nonautonomous stress responses such as transcellular chaperone signaling (TCS). When TCS is activated upon tissue-specific knockdown of hsp-90 in the Caenorhabditis elegans intestine, heat-inducible hsp-70 is induced in muscle cells at the permissive temperature resulting in increased heat stress resistance and lifespan extension. However, our understanding of the molecular mechanism and signaling factors mediating transcellular activation of hsp-70 expression from one tissue to another is still in its infancy. Here, we conducted a combinatorial approach using transcriptome RNA-Seq profiling and a forward genetic mutagenesis screen to elucidate how stress signaling from the intestine to the muscle is regulated. We find that the TCS-mediated "gut-to-muscle" induction of hsp-70 expression is suppressed by HSF-1 and instead relies on transcellular-X-cross-tissue (txt) genes. We identify a key role for the PDZ-domain guanylate cyclase txt-1 and the homeobox transcription factor ceh-58 as signaling hubs in the stress receiving muscle cells to initiate hsp-70 expression and facilitate TCS-mediated heat stress resistance and lifespan extension. Our results provide a new view on cell-nonautonomous regulation of "inter-tissue" stress responses in an organism that highlight a key role for the gut. Our data suggest that the HSF-1-mediated heat shock response is switched off upon TCS activation, in favor of an intercellular stress-signaling route to safeguard survival.
    DOI:  https://doi.org/10.1371/journal.pbio.3001605
  21. Mol Metab. 2023 Feb 14. pii: S2212-8778(23)00027-3. [Epub ahead of print] 101693
    Intestinal SEC16B modulates obesity by regulating chylomicron metabolism.
    Ruicheng Shi, Wei Lu, Ye Tian, Bo Wang.
       OBJECTIVE: Genome-wide association studies (GWAS) have identified genetic variants in SEC16 homolog B (SEC16B) locus to be associated with obesity and body mass index (BMI) in various populations. SEC16B encodes a scaffold protein located at endoplasmic reticulum (ER) exit sites that is implicated to participate in the trafficking of COPII vesicles in mammalian cells. However, the function of SEC16B in vivo, especially in lipid metabolism, has not been investigated.
    METHODS: We generated Sec16b intestinal knockout (IKO) mice and assessed the impact of its deficiency on high-fat diet (HFD) induced obesity and lipid absorption in both male and female mice. We examined lipid absorption in vivo by acute oil challenge and fasting/HFD refeeding. Biochemical analyses and imaging studies were performed to understand the underlying mechanisms.
    RESULTS: Our results showed that Sec16b intestinal knockout (IKO) mice, especially female mice, were protected from HFD-induced obesity. Loss of Sec16b in intestine dramatically reduced postprandial serum triglyceride output upon intragastric lipid load or during overnight fasting and HFD refeeding. Further studies showed that intestinal Sec16b deficiency impaired apoB lipidation and chylomicron secretion.
    CONCLUSIONS: Our studies demonstrated that intestinal SEC16B is required for dietary lipid absorption in mice. These results revealed that SEC16B plays important roles in chylomicron metabolism, which may shed light on the association between variants in SEC16B and obesity in human.
    Keywords:  Obesity; SEC16B; chylomicron metabolism; lipid absorption
    DOI:  https://doi.org/10.1016/j.molmet.2023.101693
  22. Elife. 2023 Feb 16. pii: e81463. [Epub ahead of print]12
    SUMOylation of NaV1.2 channels regulates the velocity of backpropagating action potentials in cortical pyramidal neurons.
    Oron Kotler, Yana Khrapunsky, Arik Shvartsman, Hui Dai, Leigh D Plant, Steven A N Goldstein, Ilya Fleidervish.
      Voltage-gated sodium channels located in axon initial segments (AIS) trigger action potentials (AP) and play pivotal roles in the excitability of cortical pyramidal neurons. The differential electrophysiological properties and distributions of NaV1.2 and NaV1.6 channels lead to distinct contributions to AP initiation and propagation. While NaV1.6 at the distal AIS promotes AP initiation and forward propagation, NaV1.2 at the proximal AIS promotes the backpropagation of APs to the soma. Here, we show the Small Ubiquitin-like Modifier (SUMO) pathway modulates Na+ channels at the AIS to increase neuronal gain and the speed of backpropagation. Since SUMO does not affect NaV1.6, these effects were attributed to SUMOylation of NaV1.2. Moreover, SUMO effects were absent in a mouse engineered to express NaV1.2-Lys38Gln channels that lack the site for SUMO linkage. Thus, SUMOylation of NaV1.2 exclusively controls INaP generation and AP backpropagation, thereby playing a prominent role in synaptic integration and plasticity.
    Keywords:  mouse; neuroscience
    DOI:  https://doi.org/10.7554/eLife.81463
  23. J Cell Biol. 2023 Apr 03. pii: e202204093. [Epub ahead of print]222(4):
    Quantifying organellar ultrastructure in cryo-electron tomography using a surface morphometrics pipeline.
    Benjamin A Barad, Michaela Medina, Daniel Fuentes, R Luke Wiseman, Danielle A Grotjahn.
      Cellular cryo-electron tomography (cryo-ET) enables three-dimensional reconstructions of organelles in their native cellular environment at subnanometer resolution. However, quantifying ultrastructural features of pleomorphic organelles in three dimensions is challenging, as is defining the significance of observed changes induced by specific cellular perturbations. To address this challenge, we established a semiautomated workflow to segment organellar membranes and reconstruct their underlying surface geometry in cryo-ET. To complement this workflow, we developed an open-source suite of ultrastructural quantifications, integrated into a single pipeline called the surface morphometrics pipeline. This pipeline enables rapid modeling of complex membrane structures and allows detailed mapping of inter- and intramembrane spacing, curvedness, and orientation onto reconstructed membrane meshes, highlighting subtle organellar features that are challenging to detect in three dimensions and allowing for statistical comparison across many organelles. To demonstrate the advantages of this approach, we combine cryo-ET with cryo-fluorescence microscopy to correlate bulk mitochondrial network morphology (i.e., elongated versus fragmented) with membrane ultrastructure of individual mitochondria in the presence and absence of endoplasmic reticulum (ER) stress. Using our pipeline, we demonstrate ER stress promotes adaptive remodeling of ultrastructural features of mitochondria including spacing between the inner and outer membranes, local curvedness of the inner membrane, and spacing between mitochondrial cristae. We show that differences in membrane ultrastructure correlate to mitochondrial network morphologies, suggesting that these two remodeling events are coupled. Our pipeline offers opportunities for quantifying changes in membrane ultrastructure on a single-cell level using cryo-ET, opening new opportunities to define changes in ultrastructural features induced by diverse types of cellular perturbations.
    DOI:  https://doi.org/10.1083/jcb.202204093
  24. Nature. 2023 Feb 15.
    A NPAS4-NuA4 complex couples synaptic activity to DNA repair.
    Elizabeth A Pollina, Daniel T Gilliam, Andrew T Landau, Cindy Lin, Naomi Pajarillo, Christopher P Davis, David A Harmin, Ee-Lynn Yap, Ian R Vogel, Eric C Griffith, M Aurel Nagy, Emi Ling, Erin E Duffy, Bernardo L Sabatini, Charles J Weitz, Michael E Greenberg.
      Neuronal activity is crucial for adaptive circuit remodelling but poses an inherent risk to the stability of the genome across the long lifespan of postmitotic neurons1-5. Whether neurons have acquired specialized genome protection mechanisms that enable them to withstand decades of potentially damaging stimuli during periods of heightened activity is unknown. Here we identify an activity-dependent DNA repair mechanism in which a new form of the NuA4-TIP60 chromatin modifier assembles in activated neurons around the inducible, neuronal-specific transcription factor NPAS4. We purify this complex from the brain and demonstrate its functions in eliciting activity-dependent changes to neuronal transcriptomes and circuitry. By characterizing the landscape of activity-induced DNA double-strand breaks in the brain, we show that NPAS4-NuA4 binds to recurrently damaged regulatory elements and recruits additional DNA repair machinery to stimulate their repair. Gene regulatory elements bound by NPAS4-NuA4 are partially protected against age-dependent accumulation of somatic mutations. Impaired NPAS4-NuA4 signalling leads to a cascade of cellular defects, including dysregulated activity-dependent transcriptional responses, loss of control over neuronal inhibition and genome instability, which all culminate to reduce organismal lifespan. In addition, mutations in several components of the NuA4 complex are reported to lead to neurodevelopmental and autism spectrum disorders. Together, these findings identify a neuronal-specific complex that couples neuronal activity directly to genome preservation, the disruption of which may contribute to developmental disorders, neurodegeneration and ageing.
    DOI:  https://doi.org/10.1038/s41586-023-05711-7
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