bims-proteo Biomed News
on Proteostasis
Issue of 2021–05–09
forty-two papers selected by
Eric Chevet, INSERM
  1. Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
  2. Membrane protein biogenesis at the ER: the highways and byways.
  3. Unconventional p97/VCP-Mediated ER to Endosomal Trafficking of a Retroviral Protein.
  4. Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.
  5. The PDB and protein homeostasis: from chaperones to degradation and disaggregase machines.
  6. Characterization of the Structural Determinants of the Ubiquitin-Dependent Proteasomal Degradation of Human Hepatic Tryptophan 2,3-Dioxygenase.
  7. The Ca2+ -binding protein sorcin stimulates transcriptional activity of the unfolded protein response mediator ATF6.
  8. NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway.
  9. Organelle-Specific Autophagy in Cellular Aging and Rejuvenation.
  10. The Unfolded Protein Response: An Overview.
  11. Glucose -regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers and neurological disorders.
  12. Ub and Dub of RNF43/ZNRF3 in the WNT signalling pathway.
  13. GRAMD1-mediated accessible cholesterol sensing and transport.
  14. Distinct fission signatures predict mitochondrial degradation or biogenesis.
  15. NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4.
  16. Metabolic flexibility via mitochondrial BCAA carrier SLC25A44 is required for optimal fever.
  17. Expanding the arsenal of E3 ubiquitin ligases for proximity-induced protein degradation.
  18. E3 ligase TRIM25 ubiquitinates RIP3 to inhibit TNF induced cell necrosis.
  19. The Role of Endoplasmic Reticulum Stress Response in Pollen Development and Heat Stress Tolerance.
  20. Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.
  21. Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.
  22. Insights into the Role of P-Bodies and Stress Granules in Protein Quality Control.
  23. Insights into the Role of P-Bodies and Stress Granules in Protein Quality Control.
  24. Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids.
  25. Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.
  26. Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans.
  27. The role of autophagy in cardiovascular pathology.
  28. Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70-Hsp40 Substrates.
  29. Integration of temporal single cell cellular stress response activity with Logic-ODE modeling reveals activation of ATF4-CHOP axis as a critical predictor of drug-induced liver injury.
  30. Fic and non-Fic AMPylases: protein AMPylation in metazoans.
  31. Giantin is required for intracellular N-terminal processing of type I procollagen.
  32. Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.
  33. Chemical Chaperone PBA Attenuates ER Stress and Upregulates SOCS3 Expression as a Regulator of Leptin Signaling.
  34. O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA.
  35. Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.
  36. Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461.
  37. MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells.
  38. The ribosome modulates folding inside the ribosomal exit tunnel.
  39. The E3 ubiquitin ligase Itch limits the progression of post-traumatic osteoarthritis in mice by inhibiting macrophage polarization.
  40. Human Ubiquilin 2 and TDP-43 co-pathology drives neurodegeneration in transgenic C. elegans.
  41. Ufmylation regulates granulosa cell apoptosis via ER stress but not oxidative stress during goat follicular atresia.
  42. The surface of lipid droplets constitutes a barrier for endoplasmic reticulum residential integral membrane proteins.
  1. Cell Stress Chaperones. 2021 May 03.
    Kinetic and structural parameters governing Fic-mediated adenylylation/AMPylation of the Hsp70 chaperone, BiP/GRP78.
    Anwesha Sanyal, Erica A Zbornik, Ben G Watson, Charles Christoffer, Jia Ma, Daisuke Kihara, Seema Mattoo.
      Fic (filamentation induced by cAMP) proteins regulate diverse cell signaling events by post-translationally modifying their protein targets, predominantly by the addition of an AMP (adenosine monophosphate). This modification is called Fic-mediated adenylylation or AMPylation. We previously reported that the human Fic protein, HYPE/FicD, is a novel regulator of the unfolded protein response (UPR) that maintains homeostasis in the endoplasmic reticulum (ER) in response to stress from misfolded proteins. Specifically, HYPE regulates UPR by adenylylating the ER chaperone, BiP/GRP78, which serves as a sentinel for UPR activation. Maintaining ER homeostasis is critical for determining cell fate, thus highlighting the importance of the HYPE-BiP interaction. Here, we study the kinetic and structural parameters that determine the HYPE-BiP interaction. By measuring the binding and kinetic efficiencies of HYPE in its activated (Adenylylation-competent) and wild type (de-AMPylation-competent) forms for BiP in its wild type and ATP-bound conformations, we determine that HYPE displays a nearly identical preference for the wild type and ATP-bound forms of BiP in vitro and preferentially de-AMPylates the wild type form of adenylylated BiP. We also show that AMPylation at BiP's Thr366 versus Thr518 sites differentially affect its ATPase activity, and that HYPE does not adenylylate UPR accessory proteins like J-protein ERdJ6. Using molecular docking models, we explain how HYPE is able to adenylylate Thr366 and Thr518 sites in vitro. While a physiological role for AMPylation at both the Thr366 and Thr518 sites has been reported, our molecular docking model supports Thr518 as the structurally preferred modification site. This is the first such analysis of the HYPE-BiP interaction and offers critical insights into substrate specificity and target recognition.
    Keywords:  AMPylation; Adenylylation; BiP/GRP78; ER stress; FicD/HYPE; Hsp70; Post-translational modification; Unfolded protein response
    DOI:  https://doi.org/10.1007/s12192-021-01208-2
  2. FEBS J. 2021 May 07.
    Membrane protein biogenesis at the ER: the highways and byways.
    Sarah O'Keefe, Martin R Pool, Stephen High.
      The Sec61 complex is the major protein translocation channel of the endoplasmic reticulum (ER), where it plays a central role in the biogenesis of membrane and secretory proteins. Whilst Sec61-mediated protein translocation is typically coupled to polypeptide synthesis, suggestive of significant complexity, an obvious characteristic of this core translocation machinery is its surprising simplicity. Over thirty years after its initial discovery, we now understand that the Sec61 complex is in fact the central piece of an elaborate jigsaw puzzle which can be partly solved using new research findings. We propose that the Sec61 complex acts as a dynamic hub for co-translational protein translocation at the ER, proactively recruiting a range of accessory complexes that enhance and regulate its function in response to different protein clients. It is now clear that the Sec61 complex does not have a monopoly on co-translational insertion, with some transmembrane proteins preferentially utilising the ER membrane complex instead. We also have a better understanding of post-insertion events, where at least one membrane-embedded chaperone complex can capture the newly inserted transmembrane domains of multi-span proteins and co-ordinate their assembly into a native structure. Having discovered this array of Sec61-associated components and competitors, our next challenge is to understand how they act together in order to expand the range and complexity of the membrane proteins that can be synthesised at the ER. Furthermore, this diversity of components and pathways may open up new opportunities for targeted therapeutic interventions designed to selectively modulate protein biogenesis at the ER.
    Keywords:  Co-translational translocation; EMC; PAT complex; SRP; Sec61; Sec62/Sec63; TMCO1 translocon; TRAP complex; membrane protein insertion
    DOI:  https://doi.org/10.1111/febs.15905
  3. J Virol. 2021 May 05. pii: JVI.00531-21. [Epub ahead of print]
    Unconventional p97/VCP-Mediated ER to Endosomal Trafficking of a Retroviral Protein.
    Wendy Kaichun Xu, Yongqiang Gou, Mary M Lozano, Jaquelin P Dudley.
      Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with Endoglycosidase H indicated that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was co-localized with Rab5+ early endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes.IMPORTANCEMouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.
    DOI:  https://doi.org/10.1128/JVI.00531-21
  4. Sci Adv. 2021 May;pii: eabe8349. [Epub ahead of print]7(19):
    Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells.
    Despoina Kerselidou, Bushra Saeed Dohai, David R Nelson, Sarah Daakour, Nicolas De Cock, Zahra Al Oula Hassoun, Dae-Kyum Kim, Julien Olivet, Diana C El Assal, Ashish Jaiswal, Amnah Alzahmi, Deeya Saha, Charlotte Pain, Filip Matthijssens, Pierre Lemaitre, Michael Herfs, Julien Chapuis, Bart Ghesquiere, Didier Vertommen, Verena Kriechbaumer, Kèvin Knoops, Carmen Lopez-Iglesias, Marc van Zandvoort, Jean-Charles Lambert, Julien Hanson, Christophe Desmet, Marc Thiry, Kyle J Lauersen, Marc Vidal, Pieter Van Vlierberghe, Franck Dequiedt, Kourosh Salehi-Ashtiani, Jean-Claude Twizere.
      The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of guanosine triphosphate nucleotides. Among posttranslational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of ER morphology and dynamics. We have integrated multiomics and superresolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Last, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome structure axis, with implications in biotechnology and medicine.
    DOI:  https://doi.org/10.1126/sciadv.abe8349
  5. J Biol Chem. 2021 May 03. pii: S0021-9258(21)00534-2. [Epub ahead of print] 100744
    The PDB and protein homeostasis: from chaperones to degradation and disaggregase machines.
    Helen R Saibil.
      This review contains a personal account of the role played by the PDB in the development of the field of molecular chaperones and protein homeostasis, from the viewpoint of someone who experienced the concurrent advances in the structural biology, electron microscopy and chaperone fields. The emphasis is on some key structures, including those of Hsp70, GroEL, Hsp90 and small heat shock proteins, that were determined as the molecular chaperone concept and systems for protein quality control were emerging. These structures were pivotal in demonstrating how seemingly non-specific chaperones could assist the specific folding pathways of a variety of substrates. Moreover, they have provided mechanistic insights into the ATPase machinery of complexes such as GroEL/GroES that promote unfolding and folding, and the disaggregases that extract polypeptides from large aggregates and disassemble amyloid fibers. The PDB has provided a framework for the current success in curating, evaluating and distributing structural biology data, both through the PDB and the EMDB.
    Keywords:  ATPases associated with diverse cellular activities (AAA); Chaperone; DnaK; GroEL; heat shock protein 90 (Hsp90); protein aggregation; protein folding; protein misfolding; proteostasis
    DOI:  https://doi.org/10.1016/j.jbc.2021.100744
  6. Biochem J. 2021 May 07. pii: BCJ20210213. [Epub ahead of print]
    Characterization of the Structural Determinants of the Ubiquitin-Dependent Proteasomal Degradation of Human Hepatic Tryptophan 2,3-Dioxygenase.
    Yi Liu, Sung Mi Kim, YongQiang Wang, Shay Karkashon, Ariel Lewis-Ballester, Syun Ru Yeh, Maria Almira Correia.
      Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ~2.3 h, relative to an average t1/2 of ~2-3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation (UPD) pathway. Additionally, we identified 15 ubiquitination K sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here we further established autophagic lysosomal degradation (ALD) as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.
    Keywords:  Human tryptophan 2,3-dioxygenase; Ub-dependent proteasomal degradation; a-methyltryptophan; disordered termini; exosite; surface DEpSpT-clusters
    DOI:  https://doi.org/10.1042/BCJ20210213
  7. FEBS Lett. 2021 May 07.
    The Ca2+ -binding protein sorcin stimulates transcriptional activity of the unfolded protein response mediator ATF6.
    Steven Z Parks, Tian Gao, Natalia Jimenez Awuapura, Joseph Ayathamattam, Pauline L Chabosseau, Dhananjaya V Kalvakolanu, Héctor H Valdivia, Guy A Rutter, Isabelle Leclerc.
      Sorcin is a calcium-binding protein involved in maintaining endoplasmic reticulum (ER) Ca2+ stores. We have previously shown that overexpressing sorcin under the rat insulin promoter was protective against high fat diet-induced pancreatic beta cell dysfunction in vivo. Activating Transcription Factor 6 (ATF6) is a key mediator of the unfolded protein response (UPR) that provides cellular protection during the progression of ER stress. Here, using non-excitable HEK293 cells, we show that sorcin overexpression increased ATF6 signalling, whereas sorcin knock out caused a reduction in ATF6 transcriptional activity and increased ER stress. Altogether, our data suggest that sorcin downregulation during lipotoxic stress may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance.
    Keywords:  ATF6; ER stress; Sorcin; lipotoxicity
    DOI:  https://doi.org/10.1002/1873-3468.14101
  8. J Biomed Sci. 2021 May 07. 28(1): 35
    NEDD4 regulates ubiquitination and stability of the cell adhesion molecule IGPR-1 via lysosomal pathway.
    Linzi Sun, Razie Amraei, Nader Rahimi.
       BACKGROUND: The cell adhesion molecule IGPR-1 regulates various critical cellular processes including, cell-cell adhesion, mechanosensing and autophagy and plays important roles in angiogenesis and tumor growth; however, the molecular mechanism governing the cell surface levels of IGPR-1 remains unknown.
    RESULTS: In the present study, we used an in vitro ubiquitination assay and identified ubiquitin E3 ligase NEDD4 and the ubiquitin conjugating enzyme UbcH6 involved in the ubiquitination of IGPR-1. In vitro GST-pulldown and in vivo co-immunoprecipitation assays demonstrated that NEDD4 binds to IGPR-1. Over-expression of wild-type NEDD4 downregulated IGPR-1 and deletion of WW domains (1-4) of NEDD4 revoked its effects on IGPR-1. Knockdown of NEDD4 increased IGPR-1 levels in A375 melanoma cells. Deletion of 57 amino acids encompassing the polyproline rich (PPR) motifs on the C-terminus of IGPR-1 nullified its binding with NEDD4. Furthermore, we demonstrate that NEDD4 promotes K48- and K63-dependent polyubiquitination of IGPR-1. The NEDD4-mediated polyubiquitination of IGPR-1 stimulates lysosomal-dependent degradation of IGPR-1 as the treatment of cells with the lysosomal inhibitors, bafilomycine or ammonium chloride increased IGPR-1 levels ectopically expressed in HEK-293 cells and in multiple endogenously IGPR-1 expressing human skin melanoma cell lines.
    CONCLUSIONS: NEDD4 ubiquitin E3 ligase binds to and mediates polyubiquitination of IGPR-1 leading to its lysosomal-dependent degradation. NEDD4 is a key regulator of IGPR-1 expression with implication in the therapeutic targeting of IGPR-1 in human cancers.
    Keywords:  CD28H; IGPR-1; K48-linked ubiquitination; K63-linked ubiquitination; Lysosomal degradation; Melanoma; NEDD4; TMIGD2; UbcH6; Ubiquitination
    DOI:  https://doi.org/10.1186/s12929-021-00731-9
  9. Adv Geriatr Med Res. 2021 ;pii: e210010. [Epub ahead of print]3(2):
    Organelle-Specific Autophagy in Cellular Aging and Rejuvenation.
    Tyler J Butsch, Bhaswati Ghosh, K Adam Bohnert.
      The health of a cell requires proper functioning, regulation, and quality control of its organelles, the membrane-enclosed compartments inside the cell that carry out its essential biochemical tasks. Aging commonly perturbs organelle homeostasis, causing problems to cellular health that can spur the initiation and progression of degenerative diseases and related pathologies. Here, we discuss emerging evidence indicating that age-related defects in organelle homeostasis stem in part from dysfunction of the autophagy-lysosome system, a pivotal player in cellular quality control and damage clearance. We also highlight natural examples from biology where enhanced activity of the autophagy-lysosome system might be harnessed to erase age-related organelle damage, raising potential implications for cellular rejuvenation.
    Keywords:  aging; autophagy; cell biology; organelles; rejuvenation
    DOI:  https://doi.org/10.20900/agmr20210010
  10. Biology (Basel). 2021 Apr 29. pii: 384. [Epub ahead of print]10(5):
    The Unfolded Protein Response: An Overview.
    Adam Read, Martin Schröder.
      The unfolded protein response is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. Under normal conditions, the UPR is not activated; however, under certain stresses, such as hypoxia or altered glycosylation, the UPR can be activated due to an accumulation of unfolded proteins. The activation of the UPR involves three signaling pathways, IRE1, PERK and ATF6, which all play vital roles in returning protein homeostasis to levels seen in non-stressed cells. IRE1 is the best studied of the three pathways, as it is the only pathway present in Saccharomyces cerevisiae. This pathway involves spliceosome independent splicing of HAC1 or XBP1 in yeast and mammalians cells, respectively. PERK limits protein synthesis, therefore reducing the number of new proteins requiring folding. ATF6 is translocated and proteolytically cleaved, releasing a NH2 domain fragment which is transported to the nucleus and which affects gene expression. If the UPR is unsuccessful at reducing the load of unfolded proteins in the ER and the UPR signals remain activated, this can lead to programmed cell death.
    Keywords:  ATF6; ERAD; IRE1; PERK; RIDD; UPR; inactivation
    DOI:  https://doi.org/10.3390/biology10050384
  11. IUBMB Life. 2021 May 07.
    Glucose -regulated protein (GRP78) is an important cell surface receptor for viral invasion, cancers and neurological disorders.
    Mario Gonzalez-Gronow, Udhayakumar Gopal, Richard C Austin, Salvatore V Pizzo.
      The 78 kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone. GRP78 is a member of the 70 kDa heat shock family of proteins involved in correcting and clearing misfolded proteins in the ER. In response to cellular stress, GRP78 escapes from the ER and moves to the plasma membrane where it (i) functions as a receptor for many ligands, and (ii) behaves as an autoantigen for autoantibodies that contribute to human disease and cancer. Cell surface GRP78 (csGRP78) associates with the major histocompatibility complex class I (MHC-I), and is the port of entry for several viruses, including the predictive binding of the novel SARS-CoV-2. Furthermore, csGRP78 is found in association with partners as diverse as the teratocarcinoma-derived growth factor 1 (Cripto), the melanocortin-4 receptor (MC4R) and the DnaJ-like protein MTJ-1. CsGRP78 also serves as a receptor for a large variety of ligands including activated α2 -macroglobulin (α2 M*), plasminogen kringle 5 (K5), microplasminogen, the voltage-dependent anion channel (VDAC), tissue factor (TF) and the prostate apoptosis response-4 protein (Par-4). In this review, we discuss the mechanisms involved in the translocation of GRP78 from the ER to the cell surface, and the role of secreted GRP78 and its autoantibodies in cancer and neurological disorders. This article is protected by copyright. All rights reserved.
    Keywords:  GRP78; GRP78 autoantibodies; GRP78 signaling; cancer; heat shock protein; neurological disorders
    DOI:  https://doi.org/10.1002/iub.2502
  12. EMBO Rep. 2021 May 05. 22(5): e52970
    Ub and Dub of RNF43/ZNRF3 in the WNT signalling pathway.
    Gabriele Colozza, Bon-Kyoung Koo.
      The E3 ubiquitin ligases RING finger protein 43 (RNF43) and zinc and RING finger 3 (ZNRF3) have received great attention for their critical role in regulating WNT signalling during adult stem cell homeostasis. By promoting the turnover of WNT receptors, Frizzled and LRP5/6, RNF43 and ZNRF3 ensure that proper levels of WNT activity are maintained in stem cells. The molecular mechanism of RNF43/ZNRF3 activity is beginning to emerge from several recent studies, yet little is known about the regulation of RNF43/ZNRF3 at the post-translational level. A study in this issue of EMBO Reports identifies the deubiquitinating enzyme USP42 as a key regulator of WNT signalling, which acts by antagonizing the ubiquitin-dependent clearance of RNF43/ZNRF3 induced by R-spondins (Giebel et al, 2021).
    DOI:  https://doi.org/10.15252/embr.202152970
  13. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Apr 28. pii: S1388-1981(21)00085-8. [Epub ahead of print]1866(8): 158957
    GRAMD1-mediated accessible cholesterol sensing and transport.
    Tomoki Naito, Yasunori Saheki.
      Cholesterol, an essential lipid for cell signaling and structural integrity of cellular membranes, is highly enriched in the plasma membrane (PM). However, the regulatory mechanisms that control its biosynthesis and uptake both reside in the endoplasmic reticulum (ER). Thus, the ER needs to constantly monitor the levels of PM cholesterol. This is in part mediated by regulated transport of a biochemically defined pool of cholesterol, termed "accessible" cholesterol, from the PM to the ER via evolutionarily conserved ER-anchored lipid transfer proteins, the GRAMD1s/Asters (GRAMD1a/1b/1c) (Lam/Ltc proteins in yeast). GRAMD1s possess cytosolically exposed GRAM domain and StART-like domain followed by a transmembrane ER anchor. They form homo- and hetero-meric complexes and move to the contacts formed between the ER and the PM by sensing a transient expansion of the accessible pool of cholesterol in the PM via the GRAM domain and facilitate its extraction and transport to the ER via the StART-like domain. The GRAMD1b GRAM domain possesses distinct, but synergistic sites, for recognizing accessible cholesterol and anionic lipids, including phosphatidylserine, within the PM. This property of the GRAM domain contributes to regulated tethering of the PM to ER membrane where GRAMD1s are anchored and fine-tunes StART-like domain-dependent accessible cholesterol transport. Thus, cells use GRAMD1s to sense the levels of cholesterol in the PM and regulate transport of accessible PM cholesterol to the ER in order to maintain cholesterol homeostasis.
    Keywords:  Cholesterol; GRAM domain; Membrane contact sites; Non-vesicular lipid transport; Phosphatidylserine; StART-like domain
    DOI:  https://doi.org/10.1016/j.bbalip.2021.158957
  14. Nature. 2021 May 05.
    Distinct fission signatures predict mitochondrial degradation or biogenesis.
    Tatjana Kleele, Timo Rey, Julius Winter, Sofia Zaganelli, Dora Mahecic, Hélène Perreten Lambert, Francesco Paolo Ruberto, Mohamed Nemir, Timothy Wai, Thierry Pedrazzini, Suliana Manley.
      Mitochondrial fission is a highly regulated process that, when disrupted, can alter metabolism, proliferation and apoptosis1-3. Dysregulation has been linked to neurodegeneration3,4, cardiovascular disease3 and cancer5. Key components of the fission machinery include the endoplasmic reticulum6 and actin7, which initiate constriction before dynamin-related protein 1 (DRP1)8 binds to the outer mitochondrial membrane via adaptor proteins9-11, to drive scission12. In the mitochondrial life cycle, fission enables both biogenesis of new mitochondria and clearance of dysfunctional mitochondria through mitophagy1,13. Current models of fission regulation cannot explain how those dual fates are decided. However, uncovering fate determinants is challenging, as fission is unpredictable, and mitochondrial morphology is heterogeneous, with ultrastructural features that are below the diffraction limit. Here, we used live-cell structured illumination microscopy to capture mitochondrial dynamics. By analysing hundreds of fissions in African green monkey Cos-7 cells and mouse cardiomyocytes, we discovered two functionally and mechanistically distinct types of fission. Division at the periphery enables damaged material to be shed into smaller mitochondria destined for mitophagy, whereas division at the midzone leads to the proliferation of mitochondria. Both types are mediated by DRP1, but endoplasmic reticulum- and actin-mediated pre-constriction and the adaptor MFF govern only midzone fission. Peripheral fission is preceded by lysosomal contact and is regulated by the mitochondrial outer membrane protein FIS1. These distinct molecular mechanisms explain how cells independently regulate fission, leading to distinct mitochondrial fates.
    DOI:  https://doi.org/10.1038/s41586-021-03510-6
  15. Sci Adv. 2021 May;pii: eabf7114. [Epub ahead of print]7(19):
    NOX4 links metabolic regulation in pancreatic cancer to endoplasmic reticulum redox vulnerability and dependence on PRDX4.
    Pallavi Jain, Anna Dvorkin-Gheva, Erik Mollen, Lucie Malbeteau, Michael Xie, Fatima Jessa, Piriththiv Dhavarasa, Stephen Chung, Kevin R Brown, Gun Ho Jang, Parth Vora, Faiyaz Notta, Jason Moffat, David Hedley, Paul C Boutros, Bradly G Wouters, Marianne Koritzinsky.
      There is an urgent need to identify vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). PDAC cells acquire metabolic changes that augment NADPH production and cytosolic redox homeostasis. Here, we show that high NADPH levels drive activity of NADPH oxidase 4 (NOX4) expressed in the endoplasmic reticulum (ER) membrane. NOX4 produces H2O2 metabolized by peroxiredoxin 4 (PRDX4) in the ER lumen. Using functional genomics and subsequent in vitro and in vivo validations, we find that PDAC cell lines with high NADPH levels are dependent on PRDX4 for their growth and survival. PRDX4 addiction is associated with increased reactive oxygen species, a DNA-PKcs-governed DNA damage response and radiosensitivity, which can be rescued by depletion of NOX4 or NADPH. Hence, this study has identified NOX4 as a protein that paradoxically converts the reducing power of the cytosol to an ER-specific oxidative stress vulnerability in PDAC that may be therapeutically exploited by targeting PRDX4.
    DOI:  https://doi.org/10.1126/sciadv.abf7114
  16. Elife. 2021 May 04. pii: e66865. [Epub ahead of print]10
    Metabolic flexibility via mitochondrial BCAA carrier SLC25A44 is required for optimal fever.
    Takeshi Yoneshiro, Naoya Kataoka, Jacquelyn M Walejko, Kenji Ikeda, Zachary Brown, Momoko Yoneshiro, Scott B Crown, Tsuyoshi Osawa, Juro Sakai, Robert W McGarrah, Phillip J White, Kazuhiro Nakamura, Shingo Kajimura.
      Importing necessary metabolites into the mitochondrial matrix is a crucial step of fuel choice during stress adaptation. Branched chain-amino acids (BCAA) are essential amino acids needed for anabolic processes, but they are also imported into the mitochondria for catabolic reactions. What controls the distinct subcellular BCAA utilization during stress adaptation is insufficiently understood. The present study reports the role of SLC25A44, a recently identified mitochondrial BCAA carrier (MBC), in the regulation of mitochondrial BCAA catabolism and adaptive response to fever in rodents. We found that mitochondrial BCAA oxidation in brown adipose tissue (BAT) is significantly enhanced during fever in response to the pyrogenic mediator prostaglandin E2 (PGE2) and psychological stress in mice and rats. Genetic deletion of MBC in a BAT-specific manner blunts mitochondrial BCAA oxidation and non-shivering thermogenesis following intracerebroventricular PGE2 administration. At a cellular level, MBC is required for mitochondrial BCAA deamination as well as the synthesis of mitochondrial amino acids and TCA intermediates. Together, these results illuminate the role of MBC as a determinant of metabolic flexibility to mitochondrial BCAA catabolism and optimal febrile responses. This study also offers an opportunity to control fever by rewiring the subcellular BCAA fate.
    Keywords:  cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.66865
  17. Cell Chem Biol. 2021 Apr 27. pii: S2451-9456(21)00162-8. [Epub ahead of print]
    Expanding the arsenal of E3 ubiquitin ligases for proximity-induced protein degradation.
    Aimo Kannt, Ivan Đikić.
      Efficacy and selectivity of molecules inducing protein degradation depend on their affinity to the target protein but also on the type of E3 ubiquitin ligase that is recruited to trigger proteasomal degradation. While tremendous progress has been made on the former, the latter-the arsenal of E3 ligases that can be hijacked for targeted protein degradation-is still largely unexplored. Only about 2% of the more than 600 E3 ligases have been utilized to date. Exploiting additional E3 ligases that are, for example, selectively expressed in specific tissues or cells, or regulated under certain conditions, can considerably broaden the applicability of molecular degraders as a therapeutic modality. Here, we provide an overview of major classes of E3 ligases, review the enzymes that have been exploited for induced protein degradation and approaches used to identify or design E3 ligands, and highlight challenges and opportunities for targeting new E3 ligases.
    Keywords:  E3 ubiquitin ligase; PROTAC; molecular glue; targeted protein degradation; ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.chembiol.2021.04.007
  18. Cell Death Differ. 2021 May 05.
    E3 ligase TRIM25 ubiquitinates RIP3 to inhibit TNF induced cell necrosis.
    Pucheng Mei, Feiyan Xie, Jiasong Pan, Sen Wang, Wenqing Gao, Rui Ge, Baocai Gao, Siqi Gao, Xiangjun Chen, Yongming Wang, Jiaxue Wu, Chen Ding, Jixi Li.
      Receptor interacting protein kinase 3 (RIP3 or RIPK3), the critical executor of cell programmed necrosis, plays essential roles in maintaining immune responses and appropriate tissue homeostasis. Although the E3 ligases CHIP and PELI1 are reported to promote RIP3 degradation, however, how post-translational modification regulates RIP3 activity and stability is poorly understood. Here, we identify the tripartite motif protein TRIM25 as a negative regulator of RIP3-dependent necrosis. TRIM25 directly interacts with RIP3 through its SPRY domain and mediates the K48-linked polyubiquitination of RIP3 on residue K501. The RING domain of TRIM25 facilitates the polyubiquitination chain on RIP3, thereby promoting proteasomal degradation of RIP3. Also, TRIM25 deficiency inhibited the ubiquitination of RIP3, thus promoting TNF-induced cell necrosis. Our current finding reveals the regulating mechanism of polyubiquitination on RIP3, which might be a potential therapeutic target for the intervention of RIP3-dependent necrosis-related diseases.
    DOI:  https://doi.org/10.1038/s41418-021-00790-3
  19. Front Plant Sci. 2021 ;12 661062
    The Role of Endoplasmic Reticulum Stress Response in Pollen Development and Heat Stress Tolerance.
    Mohan B Singh, Neeta Lohani, Prem L Bhalla.
      Endoplasmic reticulum (ER) stress is defined by a protracted disruption in protein folding and accumulation of unfolded or misfolded proteins in the ER. This accumulation of unfolded proteins can result from excessive demands on the protein folding machinery triggered by environmental and cellular stresses such as nutrient deficiencies, oxidative stress, pathogens, and heat. The cell responds to ER stress by activating a protective pathway termed unfolded protein response (UPR), which comprises cellular mechanisms targeted to maintain cellular homeostasis by increasing the ER's protein folding capacity. The UPR is especially significant for plants as being sessile requires them to adapt to multiple environmental stresses. While multiple stresses trigger the UPR at the vegetative stage, it appears to be active constitutively in the anthers of unstressed plants. Transcriptome analysis reveals significant upregulation of ER stress-related transcripts in diploid meiocytes and haploid microspores. Interestingly, several ER stress-related genes are specifically upregulated in the sperm cells. The analysis of gene knockout mutants in Arabidopsis has revealed that defects in ER stress response lead to the failure of normal pollen development and enhanced susceptibility of male gametophyte to heat stress conditions. In this mini-review, we provide an overview of the role of ER stress and UPR in pollen development and its protective roles in maintaining male fertility under heat stress conditions.
    Keywords:  endoplasmic reticulum stress; heat stress; male gametophyte; plant reproduction; pollen; pollen development; sperm cell; unfolded protein response
    DOI:  https://doi.org/10.3389/fpls.2021.661062
  20. Genetics. 2019 Jul 01. 212(3): 757-771
    Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.
    Rebecca L Howie, Lina Manuela Jay-Garcia, Denis A Kiktev, Quincy L Faber, Margaret Murphy, Katherine A Rees, Numera Sachwani, Yury O Chernoff.
      Self-perpetuating protein aggregates (prions) cause diseases in mammals. Yeast prions are heritable in cell divisions. Howie et al. demonstrate that the cellular apparatus responsible for the asymmetry of cell division controls destabilization of a yeast prion... Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae. Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [PSI+], a prion isoform of the yeast translation termination factor Sup35. This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [PSI+] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [PSI+], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104-GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [PSI+], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.
    Keywords:  Hsp104; Sir2; Ssb; [PSI+]; stress
    DOI:  https://doi.org/10.1534/genetics.119.302237
  21. Genetics. 2019 Jul 01. 212(3): 757-771
    Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.
    Rebecca L Howie, Lina Manuela Jay-Garcia, Denis A Kiktev, Quincy L Faber, Margaret Murphy, Katherine A Rees, Numera Sachwani, Yury O Chernoff.
      Self-perpetuating protein aggregates (prions) cause diseases in mammals. Yeast prions are heritable in cell divisions. Howie et al. demonstrate that the cellular apparatus responsible for the asymmetry of cell division controls destabilization of a yeast prion... Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae. Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [PSI+], a prion isoform of the yeast translation termination factor Sup35. This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [PSI+] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [PSI+], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104-GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [PSI+], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.
    Keywords:  Hsp104; Sir2; Ssb; [PSI+]; stress
    DOI:  https://doi.org/10.1534/genetics.119.302237
  22. Genetics. 2019 Sep 01. 213(1): 251-265
    Insights into the Role of P-Bodies and Stress Granules in Protein Quality Control.
    Regina Nostramo, Siyuan Xing, Bo Zhang, Paul K Herman.
      Nostramo et al. identify a potential role for two novel RNA-protein granules, the P-body and stress granule, in the maintenance of normal protein homeostasis. These cytoplasmic granules are members of an ever-growing family of membraneless organelles in eukaryotic cells... The eukaryotic cell is highly compartmentalized, and contains a variety of both membrane-bound and membraneless organelles. The latter include the cytoplasmic ribonucleoprotein (RNP) granules, known as the processing body (P-body) and the stress granule. These RNP structures are thought to be involved in the storage of particular mRNAs during periods of stress. Here, we find that a mutant lacking both P-bodies and stress granules exhibits phenotypes suggesting that these structures also have a role in the maintenance of protein homeostasis. In particular, there was an increased occurrence of specific protein quality control (PQC) compartments in this mutant, an observation that is consistent with there being an elevated level of protein misfolding. These compartments normally house soluble misfolded proteins and allow the cell to sequester these polypeptides away from the remaining cellular milieu. Moreover, specific proteins that are normally targeted to both P-bodies and stress granules were found to instead associate with these PQC compartments in this granuleless mutant. This observation is interesting as our data indicate that this association occurs specifically in cells that have been subjected to an elevated level of proteotoxic stress. Altogether, the results here are consistent with P-bodies and stress granules having a role in normal protein homeostasis in eukaryotic cells.
    Keywords:  processing bodies; protein homeostasis; protein quality control compartments; stress granules
    DOI:  https://doi.org/10.1534/genetics.119.302376
  23. Genetics. 2019 Sep 01. 213(1): 251-265
    Insights into the Role of P-Bodies and Stress Granules in Protein Quality Control.
    Regina Nostramo, Siyuan Xing, Bo Zhang, Paul K Herman.
      Nostramo et al. identify a potential role for two novel RNA-protein granules, the P-body and stress granule, in the maintenance of normal protein homeostasis. These cytoplasmic granules are members of an ever-growing family of membraneless organelles in eukaryotic cells... The eukaryotic cell is highly compartmentalized, and contains a variety of both membrane-bound and membraneless organelles. The latter include the cytoplasmic ribonucleoprotein (RNP) granules, known as the processing body (P-body) and the stress granule. These RNP structures are thought to be involved in the storage of particular mRNAs during periods of stress. Here, we find that a mutant lacking both P-bodies and stress granules exhibits phenotypes suggesting that these structures also have a role in the maintenance of protein homeostasis. In particular, there was an increased occurrence of specific protein quality control (PQC) compartments in this mutant, an observation that is consistent with there being an elevated level of protein misfolding. These compartments normally house soluble misfolded proteins and allow the cell to sequester these polypeptides away from the remaining cellular milieu. Moreover, specific proteins that are normally targeted to both P-bodies and stress granules were found to instead associate with these PQC compartments in this granuleless mutant. This observation is interesting as our data indicate that this association occurs specifically in cells that have been subjected to an elevated level of proteotoxic stress. Altogether, the results here are consistent with P-bodies and stress granules having a role in normal protein homeostasis in eukaryotic cells.
    Keywords:  processing bodies; protein homeostasis; protein quality control compartments; stress granules
    DOI:  https://doi.org/10.1534/genetics.119.302376
  24. mBio. 2021 05 04. pii: e00167-21. [Epub ahead of print]12(3):
    Human Cytomegalovirus Uses a Host Stress Response To Balance the Elongation of Saturated/Monounsaturated and Polyunsaturated Very-Long-Chain Fatty Acids.
    Yuecheng Xi, Lena Lindenmayer, Ian Kline, Jens von Einem, John G Purdy.
      Stress and virus infection regulate lipid metabolism. Human cytomegalovirus (HCMV) infection induces fatty acid (FA) elongation and increases the abundance of lipids with very-long-chain FA (VLCFA) tails. While reprogramming of metabolism can be stress related, the role of stress in HCMV reprogramming of lipid metabolism is poorly understood. In this study, we engineered cells to knock out protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) in the ER stress pathway and measured lipid changes using lipidomics to determine if PERK is needed for lipid changes associated with HCMV infection. In HCMV-infected cells, PERK promotes increases in the levels of phospholipids with saturated FA (SFA) and monounsaturated FA (MUFA) VLCFA tails. Further, PERK enhances FA elongase 7 (ELOVL7) protein levels, which elongates SFA and MUFA VLCFAs. Additionally, we found that increases in the elongation of polyunsaturated fatty acids (PUFAs) associated with HCMV infection were independent of PERK and that lipids with PUFA tails accumulated in HCMV-infected PERK knockout cells. Additionally, the protein levels of ELOVL5, which elongates PUFAs, are increased by HCMV infection through a PERK-independent mechanism. These observations show that PERK differentially regulates ELOVL7 and ELOVL5, creating a balance between the synthesis of lipids with SFA/MUFA tails and PUFA tails. Additionally, we found that PERK was necessary for virus replication and the infectivity of released viral progeny. Overall, our findings indicate that PERK-and, more broadly, ER stress-may be necessary for the membrane biogenesis needed to generate infectious HCMV virions.IMPORTANCE HCMV is a common herpesvirus that establishes lifelong persistent infections. While infection is asymptomatic in most people, HCMV causes life-threatening illnesses in immunocompromised people, including transplant recipients and cancer patients. Additionally, HCMV infection is a leading cause of congenital disabilities. HCMV replication relies on lipid synthesis. Here, we demonstrated that the ER stress mediator PERK controls FA elongation and the cellular abundance of several types of lipids following HCMV infection. Specifically, PERK promotes FA elongase 7 synthesis and phospholipids with saturated/monounsaturated very-long-chain FA tails. Overall, our study shows that PERK is an essential host factor that supports HCMV replication and promotes lipidome changes caused by HCMV infection.
    Keywords:  ER stress; PERK; fatty acid elongases; herpesviruses; human cytomegalovirus; lipidomics; lipids; very-long-chain fatty acids
    DOI:  https://doi.org/10.1128/mBio.00167-21
  25. Mol Cell. 2021 Apr 27. pii: S1097-2765(21)00317-8. [Epub ahead of print]
    Comprehensive interactome profiling of the human Hsp70 network highlights functional differentiation of J domains.
    Benjamin L Piette, Nader Alerasool, Zhen-Yuan Lin, Jessica Lacoste, Mandy Hiu Yi Lam, Wesley Wei Qian, Stephanie Tran, Brett Larsen, Eric Campos, Jian Peng, Anne-Claude Gingras, Mikko Taipale.
      Hsp70s comprise a deeply conserved chaperone family that has a central role in maintaining protein homeostasis. In humans, Hsp70 client specificity is provided by 49 different co-factors known as J domain proteins (JDPs). However, the cellular function and client specificity of JDPs have largely remained elusive. We have combined affinity purification-mass spectrometry (AP-MS) and proximity-dependent biotinylation (BioID) to characterize the interactome of all human JDPs and Hsp70s. The resulting network suggests specific functions for many uncharacterized JDPs, and we establish a role of conserved JDPs DNAJC9 and DNAJC27 in histone chaperoning and ciliogenesis, respectively. Unexpectedly, we find that the J domain of DNAJC27 but not of other JDPs can fully replace the function of endogenous DNAJC27, suggesting a previously unappreciated role for J domains themselves in JDP specificity. More broadly, our work expands the role of the Hsp70-regulated proteostasis network and provides a platform for further discovery of JDP-dependent functions.
    DOI:  https://doi.org/10.1016/j.molcel.2021.04.012
  26. Genetics. 2019 Aug 01. 212(4): 1259-1278
    Restoration of Proteostasis in the Endoplasmic Reticulum Reverses an Inflammation-Like Response to Cytoplasmic DNA in Caenorhabditis elegans.
    Ashley B Williams, Felix Heider, Jan-Erik Messling, Matthias Rieckher, Wilhelm Bloch, Björn Schumacher.
      Caenorhabditis elegans possesses a rudimentary innate immune response that serves as a model for various aspects of the human innate immune response. To date, a nematode response to pathogenic cytoplasmic DNA has not been identified... Innate immune responses protect organisms against various insults, but may lead to tissue damage when aberrantly activated. In higher organisms, cytoplasmic DNA can trigger inflammatory responses that can lead to tissue degeneration. Simpler metazoan models could shed new mechanistic light on how inflammatory responses to cytoplasmic DNA lead to pathologies. Here, we show that in a DNase II-defective Caenorhabditis elegans strain, persistent cytoplasmic DNA leads to systemic tissue degeneration and loss of tissue functionality due to impaired proteostasis. These pathological outcomes can be therapeutically alleviated by restoring protein homeostasis, either via ectopic induction of the ER unfolded protein response or N-acetylglucosamine treatment. Our results establish C. elegans as an ancestral metazoan model for studying the outcomes of inflammation-like conditions caused by persistent cytoplasmic DNA and provide insight into potential therapies for human conditions involving chronic inflammation.
    Keywords:   Caenorhabditis elegans ; DNA sensing; DNase II; inflammatory responses; innate immunity
    DOI:  https://doi.org/10.1534/genetics.119.302422
  27. Cardiovasc Res. 2021 May 06. pii: cvab158. [Epub ahead of print]
    The role of autophagy in cardiovascular pathology.
    Damián Gatica, Mario Chiong, Sergio Lavandero, Daniel J Klionsky.
      Macroautophagy/autophagy is a conserved catabolic recycling pathway in which cytoplasmic components are sequestered, degraded, and recycled to survive various stress conditions. Autophagy dysregulation has been observed and linked with the development and progression of several pathologies, including cardiovascular diseases, the leading cause of death in the developed world. In this review, we aim to provide a broad understanding of the different molecular factors that govern autophagy regulation and how these mechanisms are involved in the development of specific cardiovascular pathologies, including ischemic and reperfusion injury, myocardial infarction, cardiac hypertrophy, cardiac remodeling, and heart failure.
    Keywords:  Autophagosome; cardiomyocyte; heart; lysosome; vascular
    DOI:  https://doi.org/10.1093/cvr/cvab158
  28. Front Mol Biosci. 2021 ;8 653073
    Bacterial Hsp90 Facilitates the Degradation of Aggregation-Prone Hsp70-Hsp40 Substrates.
    Bruno Fauvet, Andrija Finka, Marie-Pierre Castanié-Cornet, Anne-Marie Cirinesi, Pierre Genevaux, Manfredo Quadroni, Pierre Goloubinoff.
      In eukaryotes, the 90-kDa heat shock proteins (Hsp90s) are profusely studied chaperones that, together with 70-kDa heat shock proteins (Hsp70s), control protein homeostasis. In bacteria, however, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains poorly characterized. To uncover physiological processes that depend on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild-type (WT) Escherichia coli and from knockout mutants ΔdnaKdnaJ (ΔKJ), ΔhtpG (ΔG), and ΔdnaKdnaJΔhtpG (ΔKJG). Whereas the ΔG mutant showed no detectable proteomic differences with wild-type, ΔKJ expressed more chaperones, proteases and ribosomes and expressed dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that the triple mutant ΔKJG showed higher levels of metabolic and respiratory enzymes than ΔKJ, suggesting that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates. Further in vivo experiments suggest that such Hsp90-mediated degradation possibly occurs through the HslUV protease.
    Keywords:  DnaJ; DnaK; HslV; HtpG; chaperones; proteostasis
    DOI:  https://doi.org/10.3389/fmolb.2021.653073
  29. Biochem Pharmacol. 2021 May 03. pii: S0006-2952(21)00197-0. [Epub ahead of print] 114591
    Integration of temporal single cell cellular stress response activity with Logic-ODE modeling reveals activation of ATF4-CHOP axis as a critical predictor of drug-induced liver injury.
    Lukas Surya Wijaya, Panuwat Trairatphisan, Attila Gabor, Marije Niemeijer, Jason Keet, Ariadna Alcalà Morera, Kirsten E Snijders, Steven Wink, Huan Yang, Stefan Schildknecht, James L Stevens, Peter Bouwman, Hennicke Kamp, Jan Hengstler, Joost Beltman, Marcel Leist, Sylvia Le Dévédec, Julio Saez-Rodriguez, Bob van de Water.
      Drug-induced liver injury (DILI) is the most prevalent adversity encountered in drug development and clinical settings leading to urgent needs to understand the underlying mechanisms. In this study, we have systematically investigated the dynamics of the activation of cellular stress response pathways and cell death outcomes upon exposure of a panel of liver toxicants using live cell imaging of fluorescent reporter cell lines. We established a comprehensive temporal dynamic response profile of a large set of BAC-GFP HepG2 cell lines representing the following components of stress signaling: i) unfolded protein response (UPR) [ATF4, XBP1, BIP and CHOP]; ii) oxidative stress [NRF2, SRXN1, HMOX1]; iii) DNA damage [P53, P21, BTG2, MDM2]; and iv) NF-κB pathway [A20, ICAM1]. We quantified the single cell GFP expression as a surrogate for endogenous protein expression using live cell imaging over >60 hours upon exposure to 14 DILI compounds at multiple concentrations. Using logic-based ordinary differential equation (Logic-ODE), we modelled the dynamic profiles of the different stress responses and extracted specific descriptors potentially predicting the progressive outcomes.We identified the activation of ATF4-CHOP axis of the UPR as the key pathway showing the highest correlation with cell death upon DILI compound perturbation. Knocking down main components of the UPR provided partial protection from compound-induced cytotoxicity, indicating a complex interplay among UPR components as well as other stress pathways. Our results suggest that a systematic analysis of the temporal dynamics of ATF4-CHOP axis activation can support the identification of DILI risk for new candidate drugs.
    Keywords:  Drug-induced liver injury (DILI); GFP-reporter; Logic-ODE; Stress response pathway
    DOI:  https://doi.org/10.1016/j.bcp.2021.114591
  30. Open Biol. 2021 May;11(5): 210009
    Fic and non-Fic AMPylases: protein AMPylation in metazoans.
    Bhaskar K Chatterjee, Matthias C Truttmann.
      Protein AMPylation refers to the covalent attachment of an AMP moiety to the amino acid side chains of target proteins using ATP as nucleotide donor. This process is catalysed by dedicated AMP transferases, called AMPylases. Since this initial discovery, several research groups have identified AMPylation as a critical post-translational modification relevant to normal and pathological cell signalling in both bacteria and metazoans. Bacterial AMPylases are abundant enzymes that either regulate the function of endogenous bacterial proteins or are translocated into host cells to hijack host cell signalling processes. By contrast, only two classes of metazoan AMPylases have been identified so far: enzymes containing a conserved filamentation induced by cAMP (Fic) domain (Fic AMPylases), which primarily modify the ER-resident chaperone BiP, and SelO, a mitochondrial AMPylase involved in redox signalling. In this review, we compare and contrast bacterial and metazoan Fic and non-Fic AMPylases, and summarize recent technological and conceptual developments in the emerging field of AMPylation.
    Keywords:  ampylases; chaperone and neurodegeneration; filamentation induced by cAMP; non-Fic; post-translational modification
    DOI:  https://doi.org/10.1098/rsob.210009
  31. J Cell Biol. 2021 Jun 07. pii: e202005166. [Epub ahead of print]220(6):
    Giantin is required for intracellular N-terminal processing of type I procollagen.
    Nicola L Stevenson, Dylan J M Bergen, Yinhui Lu, M Esther Prada-Sanchez, Karl E Kadler, Chrissy L Hammond, David J Stephens.
      Knockout of the golgin giantin leads to skeletal and craniofacial defects driven by poorly studied changes in glycosylation and extracellular matrix deposition. Here, we sought to determine how giantin impacts the production of healthy bone tissue by focusing on the main protein component of the osteoid, type I collagen. Giantin mutant zebrafish accumulate multiple spontaneous fractures in their caudal fin, suggesting their bones may be more brittle. Inducing new experimental fractures revealed defects in the mineralization of newly deposited collagen as well as diminished procollagen reporter expression in mutant fish. Analysis of a human giantin knockout cell line expressing a GFP-tagged procollagen showed that procollagen trafficking is independent of giantin. However, our data show that intracellular N-propeptide processing of pro-α1(I) is defective in the absence of giantin. These data demonstrate a conserved role for giantin in collagen biosynthesis and extracellular matrix assembly. Our work also provides evidence of a giantin-dependent pathway for intracellular procollagen processing.
    DOI:  https://doi.org/10.1083/jcb.202005166
  32. Angiogenesis. 2021 May 06.
    Notch regulates vascular collagen IV basement membrane through modulation of lysyl hydroxylase 3 trafficking.
    Stephen J Gross, Amelia M Webb, Alek D Peterlin, Jessica R Durrant, Rachel J Judson, Qanber Raza, Jan K Kitajewski, Erich J Kushner.
      Collagen type IV (Col IV) is a basement membrane protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. Defects in vascular Col IV deposition are the basis of heritable disorders, such as small vessel disease, marked by cerebral hemorrhage and drastically shorten lifespan. To date, little is known about how endothelial cells regulate the intracellular transport and selective secretion of Col IV in response to angiogenic cues, leaving a void in our understanding of this critical process. Our aim was to identify trafficking pathways that regulate Col IV deposition during angiogenic blood vessel development. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development using both in vitro imaging and biochemistry as well as in vivo models. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 results in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrate that Rab10 is Notch responsive, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune basement membrane secretion during blood vessel development.
    Keywords:  Angiogenesis; Blood vessels; Collagen IV; Development; Lysyl hydroxlyase 3; Notch1; Rab10; Rab25; Secretion; Small vessel disease; Trafficking; Zebrafish
    DOI:  https://doi.org/10.1007/s10456-021-09791-9
  33. Biochemistry (Mosc). 2021 Apr;86(4): 480-488
    Chemical Chaperone PBA Attenuates ER Stress and Upregulates SOCS3 Expression as a Regulator of Leptin Signaling.
    Burcu Baba, Mursel Caliskan, Gulbahar Boyuk, Aysun Hacisevki.
      Endoplasmic reticulum (ER) is very sensitive to the nutritional and energy states of the cells. Disruption of ER homeostasis leads to the accumulation of unfolded/misfolded proteins in the ER lumen, which is defined as ER stress. ER stress triggers the unfolded protein response (UPR). It is suggested that chronic ER stress is associated with obesity and leptin resistance. We investigated the role of ER stress and the effect of the ER stress inhibitor phenylbutyric acid (PBA) of ER stress, in obesity, as well as their impact on leptin signaling. This study involved twenty-four lean and twenty-four leptin-deficient (ob/ob) mice divided into PBA- and vehicle-treated groups. Pancreatic islets were isolated, incubated with leptin for 48 h, and assayed for the expression of CHOP and XBP1s (UPR signaling indicators) and SOCS3 (regulator of leptin signaling) by RT-qPCR. The expression levels of XBP1s and CHOP were markedly increased in the ob/ob controls compared to other groups with and without leptin treatment. No significant differences in the XBP1s and CHOP expression levels were found between the PBA-treated ob/ob and lean mice. SOCS3 expression was significantly upregulated in the PBA-treated ob/ob mice compared to the ob/ob controls after leptin treatment; but no significant difference in the SOCS3 expression was found between the PBA-treated ob/ob and lean mice with and without leptin treatment. Our findings suggested that ER stress plays an important role in the pathology of obesity, while PBA reduces ER stress and may potentially ameliorate leptin signaling.
    Keywords:  chemical chaperone; endoplasmic reticulum stress; leptin signaling; obesity; phenylbutyric acid; unfolded protein response
    DOI:  https://doi.org/10.1134/S0006297921040088
  34. Curr Protoc. 2021 May;1(5): e117
    O-GlcNAc Engineering on a Target Protein in Cells with Nanobody-OGT and Nanobody-splitOGA.
    Daniel H Ramirez, Yun Ge, Christina M Woo.
      The monosaccharide O-linked N-acetyl glucosamine (O-GlcNAc) is an essential and dynamic post-translational modification (PTM) that decorates thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a target protein is crucial yet challenging to perform in cells. We recently reported a pair of methods to selectively install or remove O-GlcNAc on a target protein in cells using an engineered O-GlcNAc transferase (OGT) or split O-GlcNAcase (OGA) fused to a nanobody. Target protein O-GlcNAcylation and de-O-GlcNAcylation complements methods to interrogate the role of O-GlcNAc on a global scale or at individual glycosites. Herein, we describe a protocol for utilizing the nanobody-OGT and nanobody-splitOGA systems to screen for O-GlcNAc functionality on a target protein. We additionally include associated protocols for the detection of O-GlcNAc and cloning procedures to adapt the method for the user's target protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Target protein O-GlcNAcylation of JunB using nanobody-OGT Basic Protocol 2: Target protein deglycosylation of Nup62 using nanobody-splitOGA Alternate Protocol: Verification of the O-GlcNAc state of a tagged target protein through chemoenzymatic labeling Support Protocol: Cloning of new nanobody-OGT/nanobody-splitOGA and target protein pairs.
    Keywords:  O-GlcNAc; deglycosylation; glycosylation; nanobodies; post-translational modification; proximity-direction
    DOI:  https://doi.org/10.1002/cpz1.117
  35. PLoS One. 2021 ;16(5): e0251184
    Genetic analysis of the Drosophila ESCRT-III complex protein, VPS24, reveals a novel function in lysosome homeostasis.
    Jonathan R Florian, Samuel J DeMatte, Devon M Sweeder, Richard W Ordway, Fumiko Kawasaki.
      The ESCRT pathway is evolutionarily conserved across eukaryotes and plays key roles in a variety of membrane remodeling processes. A new Drosophila mutant recovered in our forward genetic screens for synaptic transmission mutants mapped to the vps24 gene encoding a subunit of the ESCRT-III complex. Molecular characterization indicated a loss of VPS24 function, however the mutant is viable and thus loss of VPS24 may be studied in a developed multicellular organism. The mutant exhibits deficits in locomotion and lifespan and, notably, these phenotypes are rescued by neuronal expression of wild-type VPS24. At the cellular level, neuronal and muscle cells exhibit marked expansion of a ubiquitin-positive lysosomal compartment, as well as accumulation of autophagic intermediates, and these phenotypes are rescued cell-autonomously. Moreover, VPS24 expression in glia suppressed the mutant phenotype in muscle, indicating a cell-nonautonomous function for VPS24 in protective intercellular signaling. Ultrastructural analysis of neurons and muscle indicated marked accumulation of the lysosomal compartment in the vps24 mutant. In the neuronal cell body, this included characteristic lysosomal structures associated with an expansive membrane compartment with a striking tubular network morphology. These findings further define the in vivo roles of VPS24 and the ESCRT pathway in lysosome homeostasis and their potential contributions to neurodegenerative diseases characterized by defective ESCRT or lysosome function.
    DOI:  https://doi.org/10.1371/journal.pone.0251184
  36. Sci Rep. 2021 May 07. 11(1): 9812
    Ubiquitin-mediated DNA damage response is synthetic lethal with G-quadruplex stabilizer CX-5461.
    Tehmina Masud, Charles Soong, Hong Xu, Justina Biele, Saelin Bjornson, Steven McKinney, Samuel Aparicio.
      CX-5461 is a G-quadruplex (G4) ligand currently in trials with initial indications of clinical activity in cancers with defects in homologous recombination repair. To identify more genetic defects that could sensitize tumors to CX-5461, we tested synthetic lethality for 480 DNA repair and genome maintenance genes to CX-5461, pyridostatin (PDS), a structurally unrelated G4-specific stabilizer, and BMH-21, which binds GC-rich DNA but not G4 structures. We identified multiple members of HRD, Fanconi Anemia pathways, and POLQ, a polymerase with a helicase domain important for G4 structure resolution. Significant synthetic lethality was observed with UBE2N and RNF168, key members of the DNA damage response associated ubiquitin signaling pathway. Loss-of-function of RNF168 and UBE2N resulted in significantly lower cell survival in the presence of CX-5461 and PDS but not BMH-21. RNF168 recruitment and histone ubiquitination increased with CX-5461 treatment, and nuclear ubiquitination response frequently co-localized with G4 structures. Pharmacological inhibition of UBE2N acted synergistically with CX-5461. In conclusion, we have uncovered novel genetic vulnerabilities to CX-5461 with potential significance for patient selection in future clinical trials.
    DOI:  https://doi.org/10.1038/s41598-021-88988-w
  37. Nat Commun. 2021 05 04. 12(1): 2522
    MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells.
    Qiaozhi Wei, Sandra Pinho, Shuxian Dong, Halley Pierce, Huihui Li, Fumio Nakahara, Jianing Xu, Chunliang Xu, Philip E Boulais, Dachuan Zhang, Maria Maryanovich, Ana Maria Cuervo, Paul S Frenette.
      Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.
    DOI:  https://doi.org/10.1038/s41467-021-22749-1
  38. Commun Biol. 2021 May 05. 4(1): 523
    The ribosome modulates folding inside the ribosomal exit tunnel.
    Florian Wruck, Pengfei Tian, Renuka Kudva, Robert B Best, Gunnar von Heijne, Sander J Tans, Alexandros Katranidis.
      Proteins commonly fold co-translationally at the ribosome, while the nascent chain emerges from the ribosomal exit tunnel. Protein domains that are sufficiently small can even fold while still located inside the tunnel. However, the effect of the tunnel on the folding dynamics of these domains is not well understood. Here, we combine optical tweezers with single-molecule FRET and molecular dynamics simulations to investigate folding of the small zinc-finger domain ADR1a inside and at the vestibule of the ribosomal tunnel. The tunnel is found to accelerate folding and stabilize the folded state, reminiscent of the effects of chaperonins. However, a simple mechanism involving stabilization by confinement does not explain the results. Instead, it appears that electrostatic interactions between the protein and ribosome contribute to the observed folding acceleration and stabilization of ADR1a.
    DOI:  https://doi.org/10.1038/s42003-021-02055-8
  39. Osteoarthritis Cartilage. 2021 Apr 30. pii: S1063-4584(21)00704-4. [Epub ahead of print]
    The E3 ubiquitin ligase Itch limits the progression of post-traumatic osteoarthritis in mice by inhibiting macrophage polarization.
    Xi Lin, Wensheng Wang, Andrew McDavid, Hao Xu, Brendan F Boyce, Lianping Xing.
       OBJECTIVE: Osteoarthritis (OA) is characterized by articular cartilage loss, associated with synovial inflammation. We recently reported increased pro-inflammatory macrophages in murine post-traumatic OA (PTOA) joints, and blockade of the ubiquitin-proteasome system alleviates PTOA progression. However, the mechanisms whereby protein ubiquitination influences PTOA pathology are not well studied. We hypothesized that loss of the negative regulator of inflammation, E3 ligase Itch, in macrophages contributes to joint OA tissue damage by promoting pro-inflammatory polarization of macrophages.
    METHODS: Mice deficient Itch in macrophages (MΔItch) were generated by crossing Itchfl/fl mice with LysM-Cre mice. PTOA surgery was performed on global Itch knockout, Itch-/-, mice and MΔItch mice. Joint tissue damage and synovial macrophages were examined. Itch-/- cells were treated with IL-1 and pro-inflammatory polarization was determined. Expression of Itch protein and mRNA in PTOA synovium were assessed at different time points post PTOA.
    RESULTS: Similar to Itch-/- mice, MΔItch mice developed more severe joint damage than control mice following PTOA surgery (mean difference of OARSI score: 1.17 (95% CI 0.31∼2.03) between MΔItch and Itchfl/fl mice), accompanied by increased the inflammatory macrophage infiltration in the synovium (mean difference of % F4/80+CD86+CD36-inflammatory macrophages: 14.81 (95% CI 8.90∼20.73) between MΔItch and Itchfl/fl mice). Itch-/- macrophages exerted pro-inflammatory phenotype in response to IL-1β treatment. Itch protein, but not mRNA levels decreased during PTOA progression.
    CONCLUSION: The negative regulator of inflammation, Itch, limits PTOA progression by inhibiting macrophage pro-inflammatory polarization. Itch protein degradation may contribute to PTOA pathology.
    Keywords:  Itch; Post-traumatic osteoarthritis; macrophage; pro-inflammatory cytokine
    DOI:  https://doi.org/10.1016/j.joca.2021.04.009
  40. G3 (Bethesda). 2021 May 08. pii: jkab158. [Epub ahead of print]
    Human Ubiquilin 2 and TDP-43 co-pathology drives neurodegeneration in transgenic C. elegans.
    Aleen D Saxton, Brian C Kraemer.
      Amyotrophic lateral sclerosis (ALS) is a debilitating, fatal neurodegenerative disease that causes rapid muscle wasting. It shares a spectrum of symptoms and pathology with frontotemporal lobar degeneration (FTLD). These diseases are caused by aberrant activity of a set of proteins including TDP-43 and UBIQUILIN-2 (UBQLN2). UBQLN2 encodes an ubiquitin-like adaptor protein involved in the ubiquitin-proteasome protein degradation pathway. Mutations in the PXX domain of UBQLN2 cause familial ALS. UBQLN2 aggregates in skein-like inclusions with other ALS and FTLD associated proteins including TDP-43 and ubiquitin. To facilitate further investigation of UBQLN2-mediated mechanisms of neurodegeneration, we made Caenorhabditis elegans transgenic lines pan-neuronally expressing human UBQLN2 cDNAs carrying either the wild-type UBQLN2 sequence or UBQLN2 with ALS causing mutations. Transgenic animals exhibit motor dysfunction accompanied by neurodegeneration of GABAergic motor neurons. At low levels of UBQLN2 expression, wild-type UBQLN2 causes significant motor impairment and neurodegeneration that is exacerbated by ALS associated mutations in UBQLN2. At higher levels of UBQLN2 expression, both wild-type and ALS mutated versions of UBQLN2 cause severe impairment. Molecular genetic investigation revealed that UBQLN2 dependent locomotor defects do not require the involvement of the endogenous homolog of TDP-43 in C. elegans (tdp-1). However, co-expression of wild-type human TDP-43 exacerbates UBQLN2 deficits. This model of UBQLN2-mediated neurodegeneration may be useful for further mechanistic investigation into the molecular cascades driving neurodegeneration in ALS and ALS-FTLD.
    Keywords:  Amyotrophic Lateral Sclerosis; C. elegans; TDP-43; UBQLN2; neurodegeneration; transgenic
    DOI:  https://doi.org/10.1093/g3journal/jkab158
  41. Theriogenology. 2021 Apr 22. pii: S0093-691X(21)00135-7. [Epub ahead of print]169 47-55
    Ufmylation regulates granulosa cell apoptosis via ER stress but not oxidative stress during goat follicular atresia.
    Xinyan Zhang, Tong Yu, Xinyan Guo, Ruixue Zhang, Yanni Jia, Chunmei Shang, Aihua Wang, Yaping Jin, Pengfei Lin.
      Follicular atresia is primarily caused by granulosa cell (GC) apoptosis, although the mechanisms are largely unknown. Ufmylation is a recently identified ubiquitin-like post-translational modifier that plays an important role in cell proliferation and apoptosis. The purpose of this study was to investigate the effects of Ufmylation on GC apoptosis during goat follicular atresia. Ubiquitin-fold modifier 1 (UFM1) and its target DDRGK domain containing 1 (DDRGK1) proteins were identified in granulosa cells (GCs) isolated from all stages of preantral follicles and from healthy (HF), early atretic (EF) and progressed atretic (PF) antral follicles. The expression levels were higher in GCs derived from antral atretic follicles than healthy follicles. Although the viability of GCs was not affected after overexpression of UFM1, siRNA-mediated UFM1 silencing significantly inhibited GC proliferation and induced apoptosis. Notably, components of the ufmylation pathway were significantly upregulated in GCs induced by the ER stress agent tunicamycin (Tm) and thapsigargin (Tg), but not affected by oxidative stress inducer H2O2. Furthermore, UFM1 silencing markedly increased the apoptosis of GCs upon Tg treatment by stimulating the ER stress-related gene expression. Our results provide evidence that UFM1 and its target DDRGK1 are expressed in the goat GCs during follicular development and atresia, and ufmylation may play an important role in the prevention of ER stress but not oxidative stress-induced GCs apoptosis.
    Keywords:  Apoptosis; ER stress; Follicular atresia; Goat; Ufmylation
    DOI:  https://doi.org/10.1016/j.theriogenology.2021.04.009
  42. J Cell Sci. 2021 May 05. pii: jcs.256206. [Epub ahead of print]
    The surface of lipid droplets constitutes a barrier for endoplasmic reticulum residential integral membrane proteins.
    Rasha Khaddaj, Muriel Mari, Stéphanie Cottier, Fulvio Reggiori, Roger Schneiter.
      Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER), and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER residential proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-residential integral membrane proteins.
    Keywords:  Endoplasmic reticulum; Lipid droplets; Perilipins; Saccharomyces cerevisiae; Seipin; Steryl esters; Triacylglycerols
    DOI:  https://doi.org/10.1242/jcs.256206
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