bims-proteo Biomed News
on Proteostasis
Issue of 2020‒10‒25
forty-three papers selected by
Eric Chevet
INSERM


  1. Elife. 2020 Oct 19. pii: e58155. [Epub ahead of print]9
    Hepowit NL, Pereira KN, Tumolo JM, Chazin WJ, MacGurn JA.
      Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.
    Keywords:  S. cerevisiae; cell biology; human
    DOI:  https://doi.org/10.7554/eLife.58155
  2. Nat Cell Biol. 2020 Oct 19.
    Ros M, Nguyen AT, Chia J, Le Tran S, Le Guezennec X, McDowall R, Vakhrushev S, Clausen H, Humphries MJ, Saltel F, Bard FA.
      Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.
    DOI:  https://doi.org/10.1038/s41556-020-00590-w
  3. Cells. 2020 Oct 18. pii: E2314. [Epub ahead of print]9(10):
    Roscoe JM, Sevier CS.
      The endoplasmic reticulum (ER) has emerged as a source of hydrogen peroxide (H2O2) and a hub for peroxide-based signaling events. Here we outline cellular sources of ER-localized peroxide, including sources within and near the ER. Focusing on three ER-localized proteins-the molecular chaperone BiP, the transmembrane stress-sensor IRE1, and the calcium pump SERCA2-we discuss how post-translational modification of protein cysteines by H2O2 can alter ER activities. We review how changed activities for these three proteins upon oxidation can modulate signaling events, and also how cysteine oxidation can serve to limit the cellular damage that is most often associated with elevated peroxide levels.
    Keywords:  BiP; IRE1; SERCA2; cysteine oxidation; endoplasmic reticulum (ER); hydrogen peroxide; reactive oxygen species (ROS); redox signaling; unfolded protein response (UPR)
    DOI:  https://doi.org/10.3390/cells9102314
  4. Trends Biochem Sci. 2020 Oct 17. pii: S0968-0004(20)30249-8. [Epub ahead of print]
    Pathinayake PS, Hsu AC, Wark PAB.
      Integral membrane proteins (IMPs) have crucial roles in many cellular processes. A novel intramembrane chaperone complex, recently elucidated by Chitwood and Hedge, provides mechanistic insight of IMP biogenesis and folding, illustrating how IMPs with multiple transmembrane domains (TMDs) are assembled within the endoplasmic reticulum (ER) membrane.
    Keywords:  PAT complex; chaperone; endoplasmic reticulum; membrane proteins
    DOI:  https://doi.org/10.1016/j.tibs.2020.10.001
  5. Cell Death Differ. 2020 Oct 21.
    Zhang Y, Xu X, Hu M, Wang X, Cheng H, Zhou R.
      Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.
    DOI:  https://doi.org/10.1038/s41418-020-00638-2
  6. Cell Mol Gastroenterol Hepatol. 2020 Oct 17. pii: S2352-345X(20)30171-5. [Epub ahead of print]
    Cooley MM, Thomas DDH, Deans K, Peng Y, Lugea A, Pandol SJ, Puglielli L, Groblewski GE.
      BACKGROUND & AIMS: Maintaining endoplasmic reticulum (ER) proteostasis is essential for pancreatic acinar cell function. Under conditions of severe ER stress, activation of pathogenic unfolded protein response pathways plays a central role in the development and progression of pancreatitis. Less is known, however, of the consequence of perturbing ER-associated post-translational protein modifications on pancreatic outcomes. Here, we examined the role of the ER acetyl-CoA transporter AT-1 on pancreatic homeostasis.METHODS: We utilized an AT-1S113R/+ hypomorphic mouse model, and generated an inducible, acinar-specific AT-1 knockout mouse model, and performed histological and biochemical analyses to probe the effect of AT-1 loss on acinar cell physiology.
    RESULTS: We found that AT-1 expression is significantly downregulated during both acute and chronic pancreatitis. Furthermore, acinar-specific deletion of AT-1 in acinar cells induces chronic ER stress marked by activation of both the XBP1s and PERK pathways, leading to spontaneous mild/moderate chronic pancreatitis evidenced by accumulation of intracellular trypsin, immune cell infiltration, and fibrosis. Induction of acute on chronic pancreatitis in the AT-1 model led to acinar cell loss and glad atrophy.
    CONCLUSIONS: These results indicate a key role for AT-1 in pancreatic acinar cell homeostasis, the unfolded protein response (UPR), and that perturbations in AT-1 function leads to pancreatic disease.
    Keywords:  AT-1; ER stress; unfolded protein response
    DOI:  https://doi.org/10.1016/j.jcmgh.2020.10.008
  7. mSphere. 2020 Oct 21. pii: e00879-20. [Epub ahead of print]5(5):
    Guirao-Abad JP, Weichert M, Albee A, Deck K, Askew DS.
      The unfolded protein response (UPR) is a signaling network that maintains homeostasis of the endoplasmic reticulum (ER). In the human-pathogenic fungus Aspergillus fumigatus, the UPR is initiated by activation of an endoribonuclease (RNase) domain in the ER transmembrane stress sensor IreA, which splices the downstream mRNA hacAu into its active form, hacAi, encoding the master transcriptional regulator of the pathway. Small-molecule inhibitors against IRE1, the human ortholog of IreA, have been developed for anticancer therapy, but their effects on the fungal UPR are unexplored. Here, we demonstrate that the IRE1 RNase inhibitor 4μ8C prevented A. fumigatus from increasing the levels of hacAi mRNA, thereby blocking induction of downstream UPR target gene expression. Treatment with 4μ8C had minimal effects on growth in minimal medium but severely impaired growth on a collagen substrate that requires high levels of hydrolytic enzyme secretion, mirroring the phenotype of other fungal UPR mutants. 4μ8C also increased sensitivity to carvacrol, a natural compound that disrupts ER integrity in fungi, and hygromycin B, which correlated with reduced expression of glycosylation-related genes. Interestingly, treatment with 4μ8C was unable to induce all of the phenotypes attributed to the loss of the canonical UPR in a ΔhacA mutant but showed remarkable similarity to the phenotype of an RNase-deficient IreA mutant that is also unable to generate the hacAi mRNA. These results establish proof of principle that pharmacological inhibition of the canonical UPR pathway is feasible in A. fumigatus and support a noncanonical role for the hacAu mRNA in ER stress response.IMPORTANCE The unfolded protein response (UPR) is a signaling pathway that maintains endoplasmic reticulum (ER) homeostasis, with functions that overlap virulence mechanisms in the human-pathogenic mold Aspergillus fumigatus The canonical pathway centers on HacA, its master transcriptional regulator. Translation of this protein requires the removal of an unconventional intron from the cytoplasmic mRNA of the hacA gene, which is achieved by an RNase domain located in the ER-transmembrane stress sensor IreA. Here, we show that targeting this RNase activity with a small-molecule inhibitor effectively blocked UPR activation, resulting in effects that mirror the consequences of genetic deletion of the RNase domain. However, these phenotypes were surprisingly narrow in scope relative to those associated with a complete deletion of the hacA gene. These findings expand the understanding of UPR signaling in this species by supporting the existence of noncanonical functions for the unspliced hacA mRNA in ER stress response.
    Keywords:  4μ8C; A. fumigatus ; ER stress; Hac1; HacA; IRE1; Ire1; IreA; RNase; STF-083010; UPR; XBP1; Xbp1; secretion
    DOI:  https://doi.org/10.1128/mSphere.00879-20
  8. Oncogenesis. 2020 Oct 18. 9(10): 93
    Giuli MV, Diluvio G, Giuliani E, Franciosa G, Di Magno L, Pignataro MG, Tottone L, Nicoletti C, Besharat ZM, Peruzzi G, Pelullo M, Palermo R, Canettieri G, Talora C, d'Amati G, Bellavia D, Screpanti I, Checquolo S.
      Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets.
    DOI:  https://doi.org/10.1038/s41389-020-00279-7
  9. Cell Chem Biol. 2020 Oct 20. pii: S2451-9456(20)30382-2. [Epub ahead of print]
    Esquejo RM, Roqueta-Rivera M, Shao W, Phelan PE, Seneviratne U, Am Ende CW, Hershberger PM, Machamer CE, Espenshade PJ, Osborne TF.
      Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.
    Keywords:  Insig; SCAP inhibitor; SREBP; cancer; cholesterol; gene expression; lipogenesis; obesity; repurposing dipyridamole; triglycerides
    DOI:  https://doi.org/10.1016/j.chembiol.2020.10.003
  10. Proc Natl Acad Sci U S A. 2020 Oct 19. pii: 202007366. [Epub ahead of print]
    Pan W, Lin B, Yang X, Liu L, Xia R, Li J, Wu Y, Xie Q.
      Abscisic acid (ABA) is the key phytohormone in plant drought tolerance and stress adaptation. The clade A protein phosphatase 2Cs (PP2Cs) like ABI1 (ABA-INSENSITIVE 1) work as coreceptors of ABA and regulate multiple ABA responses. Ubiquitination of ABI1 has been proven to play important regulatory roles in ABA signaling. However, the specific ubiquitin conjugating enzyme (E2) involved is unknown. Here, we report that UBC27 is an active E2 that positively regulates ABA signaling and drought tolerance. UBC27 forms the E2-E3 pair with the drought regulator RING E3 ligase AIRP3. Both UBC27 and AIRP3 interact with ABI1 and affect the ubiquitination and degradation of ABI1. ABA activates the expression of UBC27, inhibits the proteasome degradation of UBC27, and enhances the interaction between UBC27 and ABI1 to increase its activity. These findings uncover a regulatory mechanism in ABA signaling and drought response and provide a further understanding of the plant ubiquitination system and ABA signaling pathway.
    Keywords:  ABA; ABI1; drought response; ubiquitin conjugating enzyme; ubiquitination
    DOI:  https://doi.org/10.1073/pnas.2007366117
  11. Nat Rev Mol Cell Biol. 2020 Oct 22.
    Song J, Herrmann JM, Becker T.
      Mitochondria contain about 1,000-1,500 proteins that fulfil multiple functions. Mitochondrial proteins originate from two genomes: mitochondrial and nuclear. Hence, proper mitochondrial function requires synchronization of gene expression in the nucleus and in mitochondria and necessitates efficient import of mitochondrial proteins into the organelle from the cytosol. Furthermore, the mitochondrial proteome displays high plasticity to allow the adaptation of mitochondrial function to cellular requirements. Maintenance of this complex and adaptable mitochondrial proteome is challenging, but is of crucial importance to cell function. Defects in mitochondrial proteostasis lead to proteotoxic insults and eventually cell death. Different quality control systems monitor the mitochondrial proteome. The cytosolic ubiquitin-proteasome system controls protein transport across the mitochondrial outer membrane and removes damaged or mislocalized proteins. Concomitantly, a number of mitochondrial chaperones and proteases govern protein folding and degrade damaged proteins inside mitochondria. The quality control factors also regulate processing and turnover of native proteins to control protein import, mitochondrial metabolism, signalling cascades, mitochondrial dynamics and lipid biogenesis, further ensuring proper function of mitochondria. Thus, mitochondrial protein quality control mechanisms are of pivotal importance to integrate mitochondria into the cellular environment.
    DOI:  https://doi.org/10.1038/s41580-020-00300-2
  12. Thorax. 2020 Oct 19. pii: thoraxjnl-2019-213738. [Epub ahead of print]
    Bradley KL, Stokes CA, Marciniak SJ, Parker LC, Condliffe AM.
      The lungs are exposed to a range of environmental toxins (including cigarette smoke, air pollution, asbestos) and pathogens (bacterial, viral and fungal), and most respiratory diseases are associated with local or systemic hypoxia. All of these adverse factors can trigger endoplasmic reticulum (ER) stress. The ER is a key intracellular site for synthesis of secretory and membrane proteins, regulating their folding, assembly into complexes, transport and degradation. Accumulation of misfolded proteins within the lumen results in ER stress, which activates the unfolded protein response (UPR). Effectors of the UPR temporarily reduce protein synthesis, while enhancing degradation of misfolded proteins and increasing the folding capacity of the ER. If successful, homeostasis is restored and protein synthesis resumes, but if ER stress persists, cell death pathways are activated. ER stress and the resulting UPR occur in a range of pulmonary insults and the outcome plays an important role in many respiratory diseases. The UPR is triggered in the airway of patients with several respiratory diseases and in corresponding experimental models. ER stress has been implicated in the initiation and progression of pulmonary fibrosis, and evidence is accumulating suggesting that ER stress occurs in obstructive lung diseases (particularly in asthma), in pulmonary infections (some viral infections and in the setting of the cystic fibrosis airway) and in lung cancer. While a number of small molecule inhibitors have been used to interrogate the role of the UPR in disease models, many of these tools have complex and off-target effects, hence additional evidence (eg, from genetic manipulation) may be required to support conclusions based on the impact of such pharmacological agents. Aberrant activation of the UPR may be linked to disease pathogenesis and progression, but at present, our understanding of the context-specific and disease-specific mechanisms linking these processes is incomplete. Despite this, the ability of the UPR to defend against ER stress and influence a range of respiratory diseases is becoming increasingly evident, and the UPR is therefore attracting attention as a prospective target for therapeutic intervention strategies.
    Keywords:  airway epithelium; asthma; innate immunity; respiratory infection; viral infection
    DOI:  https://doi.org/10.1136/thoraxjnl-2019-213738
  13. Autophagy. 2020 Oct 23. 1-2
    Metur SP, Klionsky DJ.
      Macroautophagy/autophagy is a complex process that involves over 40 proteins in Saccharomyces cerevisiae. How these proteins are organized, and their activities orchestrated to facilitate an efficient autophagic mechanism remain elusive. Sawa-Makarsha et al. reconstitute the initial steps of autophagosome biogenesis during selective autophagy using autophagy factors purified from yeast. Their results show that Atg9 vesicles serve as platforms for the recruitment of the autophagy machinery, and establish membrane contact sites to initiate lipid transfer for autophagosome biogenesis. Abbreviations: GUV, giant unilamellar vesicles; PAS, phagophore assembly site; PL, proteolipisomes.
    Keywords:  Membrane; phagophore; stress; vacuole; yeast
    DOI:  https://doi.org/10.1080/15548627.2020.1835231
  14. Plant J. 2020 Oct 20.
    Li Q, Serio RJ, Schofield A, Liu H, Rasmussen SR, Hofius D, Stone SL.
      Plants employ multiple mechanisms to cope with a constantly changing and challenging environment including utilizing the ubiquitin proteasome system (UPS) to alter their proteome to assist in initiating, modulating and terminating responses to stress. We previously reported that the ubiquitin ligase XBAT35.2 mediates the proteasome-dependent degradation of Accelerated Cell Death 11 (ACD11) to promote pathogen defense. Here, we demonstrate roles for XBAT35.2 and ACD11 in abiotic stress tolerance. Similar to pathogen infection, abiotic stress stabilizes XBAT35.2 and protein abundance rose consistently with increasing concentrations of abscisic acid (ABA) and salt. Surprisingly, exposure to ABA and salt increased ACD11 stability and overexpression of ACD11 improves survival of salt and drought stress, suggesting a role for ACD11 in promoting tolerance. However, prolonged exposure to high concentrations of ABA/salt resulted in ubiquitination and proteasome-dependent degradation of ACD11. The stress-induced turnover of ACD11 requires XBAT35.2 as degradation is slowed in the absence of the E3 ubiquitin ligase. Consistent with XBAT35.2 mediating the proteasome-dependent degradation of ACD11, loss of E3 ubiquitin ligase function enhances tolerance of salt and drought stress, while overexpression increases sensitivity. A model is presented where upon abiotic stress, ACD11 abundance increases to promote tolerance. Meanwhile, XBAT35.2 accumulates and in turn promotes the degradation of ACD11 to attenuate the stress response. The results characterize XBAT35.2 as an E3 ubiquitin ligase with opposing roles in abiotic and biotic stress. Supporting Information.
    Keywords:  Abiotic stress tolerance; Abscisic acid (ABA); Arabidopsis; RING-type E3 ubiquitin ligase; Regulated protein degradation; Ubiquitin Proteasome System (UPS); Ubiquitination; XBAT35 (for XB3 ortholog 5 in Arabidopsis thaliana)
    DOI:  https://doi.org/10.1111/tpj.15032
  15. Dis Model Mech. 2020 Oct 12. pii: dmm045096. [Epub ahead of print]13(10):
    Schuster S, Heuten E, Velic A, Admard J, Synofzik M, Ossowski S, Macek B, Hauser S, Schöls L.
      C-terminus of HSC70-interacting protein (CHIP) encoded by the gene STUB1 is a co-chaperone and E3 ligase that acts as a key regulator of cellular protein homeostasis. Mutations in STUB1 cause autosomal recessive spinocerebellar ataxia type 16 (SCAR16) with widespread neurodegeneration manifesting as spastic-ataxic gait disorder, dementia and epilepsy. CHIP-/- mice display severe cerebellar atrophy, show high perinatal lethality and impaired heat stress tolerance. To decipher the pathomechanism underlying SCAR16, we investigated the heat shock response (HSR) in primary fibroblasts of three SCAR16 patients. We found impaired HSR induction and recovery compared to healthy controls. HSPA1A/B transcript levels (coding for HSP70) were reduced upon heat shock but HSP70 remained higher upon recovery in patient- compared to control-fibroblasts. As SCAR16 primarily affects the central nervous system we next investigated the HSR in cortical neurons (CNs) derived from induced pluripotent stem cells of SCAR16 patients. We found CNs of patients and controls to be surprisingly resistant to heat stress with high basal levels of HSP70 compared to fibroblasts. Although heat stress resulted in strong transcript level increases of many HSPs, this did not translate into higher HSP70 protein levels upon heat shock, independent of STUB1 mutations. Furthermore, STUB1(-/-) neurons generated by CRISPR/Cas9-mediated genome editing from an isogenic healthy control line showed a similar HSR to patients. Proteomic analysis of CNs showed dysfunctional protein (re)folding and higher basal oxidative stress levels in patients. Our results question the role of impaired HSR in SCAR16 neuropathology and highlight the need for careful selection of proper cell types for modeling human diseases.
    Keywords:  CHIP/STUB1; CRISPR/Cas9; Cortical neurons; Heat shock response; Induced pluripotent stem cells; SCAR16
    DOI:  https://doi.org/10.1242/dmm.045096
  16. J Am Chem Soc. 2020 Oct 21.
    Yan R, Chen K, Xu K.
      Diffusion properties notably determine the behavior of biomembranes. Here we report the concurrent nanoscale fine-mapping of membrane topography, diffusivity, and packing order in live mammalian cells through a synergy of single-molecule and super-resolution methods. By identifying a bright, lipophilic fluorescence turn-on probe that enables sustained single-molecule imaging of cellular membranes under stroboscopic excitation, we accumulate the positions and transient displacements of >106 probe molecules to achieve super-resolution topography and diffusivity mapping. We thus determine a trend that the membrane diffusivity drops with increased lipid packing order when comparing the endoplasmic reticulum (ER) membrane, plasma membrane, and nanodomains induced by cholera toxin B. Utilizing our nanoscale mapping capability, we further unveil reduced diffusivity in the ER membrane at ER-plasma membrane contact sites. By next integrating spectrally resolved single-molecule imaging, we show that this localized diffusion slowdown is not due to altered lipid packing order but may instead be attributed to local protein crowding. Our integrated multidimensional single-molecule approach thus unveils and differentiates between nanoscale diffusional heterogeneities of different origins in live-cell membranes.
    DOI:  https://doi.org/10.1021/jacs.0c08426
  17. J Pharmacokinet Pharmacodyn. 2020 Oct 22.
    Bartlett DW, Gilbert AM.
      Bispecific protein degraders (BPDs) engage the ubiquitin-proteasome system (UPS) to catalytically degrade intracellular proteins through the formation of ternary complexes with the target protein and E3 ubiquitin ligases. Here, we describe the development of a mechanistic modeling framework for BPDs that includes the reaction network governing ternary complex formation and degradation via the UPS. A critical element of the model framework is a multi-step process that results in a time delay between ternary complex formation and protein degradation, thereby balancing ternary complex stability against UPS degradation rates akin to the kinetic proofreading concept that has been proposed to explain the accuracy and specificity of biological processes including protein translation and T cell receptor signal transduction. Kinetic proofreading likely plays a central role in the cell's ability to regulate substrate recognition and degradation by the UPS, and the model presented here applies this concept in the context of a quantitative pharmacokinetic (PK)-pharmacodynamic (PD) framework to inform the design of potent and selective BPDs.
    Keywords:  Kinetic proofreading; Mechanistic modeling; Targeted protein degradation; Translational pharmacology
    DOI:  https://doi.org/10.1007/s10928-020-09722-z
  18. EMBO Rep. 2020 Oct 19. e49183
    Tiwari SK, Dang JW, Lin N, Qin Y, Wang S, Rana TM.
      Zika virus (ZIKV) is an emerging flavivirus, which when passed through vertical transmission from mother to developing fetus can lead to developmental abnormalities, including microcephaly. While there is mounting evidence that suggests a causal relationship between ZIKV infection and microcephaly, the mechanisms by which ZIKV induces these changes remain to be elucidated. Here, we demonstrate that ZIKV infection of neural stems cells, both in vitro and in vivo, induces macroautophagy to enhance viral replication. At the same time, ZIKV downregulates a number of essential selective autophagy genes, including the Fanconi anemia (FA) pathway genes. Bioinformatics analyses indicate that the transcription factor E2F4 promotes FANCC expression and is downregulated upon ZIKV infection. Gain and loss of function assays indicate that FANCC is essential for selective autophagy and acts as a negative regulator of ZIKV replication. Finally, we show that Fancc KO mice have increased ZIKV infection and autophagy protein levels in various brain regions. Taken together, ZIKV downregulates FANCC to modulate the host antiviral response and simultaneously attenuate neuronal growth.
    Keywords:  Fanconi anemia protein C; ZIKA virus replication; neural stem cells; selective autophagy; transcription factor E2F4
    DOI:  https://doi.org/10.15252/embr.201949183
  19. Cells. 2020 Oct 18. pii: E2315. [Epub ahead of print]9(10):
    Baeken MW, Weckmann K, Diefenthäler P, Schulte J, Yusifli K, Moosmann B, Behl C, Hajieva P.
      Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.
    Keywords:  ATG8; GABARAP; LC3A; LC3B; LC3C; autophagy; sequestosome 1 (p62); sirtuin 1
    DOI:  https://doi.org/10.3390/cells9102315
  20. J Biomed Sci. 2020 Oct 22. 27(1): 97
    Yamamoto YH, Noda T.
      Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.
    Keywords:  ATG9; Atg2; Autophagosome; Autophagy; CDIPT; COPII; ERdj8/DNAJC16; Endoplasmic reticulum; PIS; TMEM41b; VMP1
    DOI:  https://doi.org/10.1186/s12929-020-00691-6
  21. Sci Signal. 2020 Oct 20. pii: eaba8208. [Epub ahead of print]13(654):
    Liess AKL, Kucerova A, Schweimer K, Schlesinger D, Dybkov O, Urlaub H, Mansfeld J, Lorenz S.
      At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.
    DOI:  https://doi.org/10.1126/scisignal.aba8208
  22. Cancer Lett. 2020 Oct 15. pii: S0304-3835(20)30545-0. [Epub ahead of print]
    Zhang N, Sun P, Xu Y, Li H, Liu H, Wang L, Cao Y, Zhou K, TinghuaiWang .
      G protein-coupled oestrogen receptor 1 (GPER1), predicted to be a novel oestrogen receptor, has been linked to the development and progression of breast cancer. However, the molecular mechanisms underlying its functions remain elusive. Here, we show that the protein levels of GPER1 are negatively associated with those of ERα and that higher expression of GPER1 correlated with a better clinical outcome in oestrogen receptor-positive (ER+) breast cancer patients. Activation of GPER1 decreases ERα protein levels, which subsequently suppresses ERα-mediated transcription and target gene expression but does not affect its mRNA expression in ER + breast cancer cells. A mechanistic study revealed that GPER1 mediates ubiquitin (Ub)-proteasome-dependent degradation of ERα via upregulation of the Cullin3-based E3 ubiquitin ligase adaptor protein speckle-type POZ protein (SPOP), and depletion of SPOP abrogates GPER1-induced ERα ubiquitination and degradation. Functionally, GPER1 activation inhibits 17β-oestradiol (E2)-induced ER + breast cancer cell proliferation, migration, and invasion in vitro and tumour growth in vivo. Our findings reveal a novel mechanism by which GPER1 negatively modulates the ERα signalling pathway via promoting its ubiquitin-proteasome-dependent degradation, which may contribute to its inhibition of breast cancer progression.
    Keywords:  GPCR; Hormone receptor; Nuclear receptor; Protein interaction; Ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.1016/j.canlet.2020.10.019
  23. Autophagy. 2020 Oct 19.
    Kumar S, Jain A, Choi SW, Peixoto Duarte da Silva G, Allers L, Mudd MH, Peters RS, Anonsen JH, Rusten TE, Lazarou M, Deretic V.
      Macroautophagy/autophagy delivers cytoplasmic cargo to lysosomes for degradation. In yeast, the single Atg8 protein plays a role in the formation of autophagosomes whereas in mammalian cells there are five to seven paralogs, referred to as mammalian Atg8s (mAtg8s: GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, LC3B2 and LC3C) with incompletely defined functions. Here we show that a subset of mAtg8s directly control lysosomal biogenesis. This occurs at the level of TFEB, the principal regulator of the lysosomal transcriptional program. mAtg8s promote TFEB's nuclear translocation in response to stimuli such as starvation. GABARAP interacts directly with TFEB, whereas RNA-Seq analyses reveal that knockout of six genes encoding mAtg8s, or a triple knockout of the genes encoding all GABARAPs, diminishes the TFEB transcriptional program. We furthermore show that GABARAPs in cooperation with other proteins, IRGM, a factor implicated in tuberculosis and Crohn disease, and STX17, are required during starvation for optimal inhibition of MTOR, an upstream kinase of TFEB, and activation of the PPP3/calcineurin phosphatase that dephosphorylates TFEB, thus promoting its nuclear translocation. In conclusion, mAtg8s, IRGM and STX17 control lysosomal biogenesis by their combined or individual effects on MTOR, TFEB, and PPP3/calcineurin, independently of their roles in the formation of autophagosomal membranes.
    Keywords:  Crohn’s disease; GABARAP; HIV; LC3; MTOR; Mycobacterium tuberculosis; TFEB; autophagy; lysosome; metabolism
    DOI:  https://doi.org/10.1080/15548627.2020.1837423
  24. Biosci Rep. 2020 Oct 30. pii: BSR20193271. [Epub ahead of print]40(10):
    Young HS, McGowan LM, Jepson KA, Adams JC.
      Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.
    Keywords:  Cell attachment; Cell migration; Erp57; PDIA3; Secretome; breast cancer
    DOI:  https://doi.org/10.1042/BSR20193271
  25. Cell Death Differ. 2020 Oct 20.
    Krassikova L, Zhang B, Nagarajan D, Queiroz AL, Kacal M, Samakidis E, Vakifahmetoglu-Norberg H, Norberg E.
      Cancer cells undergo complex metabolic alterations. The mechanisms underlying the tuning of cancer metabolism are under active investigation. Here, we identify the uncharacterized deubiquitinase JOSD2 as a positive regulator of cancer cell proliferation by displaying comprehensive effects on glucose catabolism. We found that JOSD2 directly controls a metabolic enzyme complex that includes Aldolase A, Phosphofructokinase-1 and Phosphoglycerate dehydrogenase, in vitro and in vivo. Further, JOSD2 expression, but not a catalytically inactive mutant, deubiquitinates and stabilizes the enzyme complex, thereby enhancing their activities and the glycolytic rate. This represents a selective JOSD2 feature that is not shared among other Machado-Joseph disease DUBs or observed in nontransformed cells. JOSD2 deficiency displays cytostatic effects and reduces glycolysis in a broad spectrum of tumor cells of distinct origin and its expression correlates with poor prognosis in non-small cell lung cancer. Overall, our study provides evidence for a previously unknown biological mechanism in which JOSD2 integrates glucose and serine metabolism with potential therapeutic implications.
    DOI:  https://doi.org/10.1038/s41418-020-00639-1
  26. Commun Biol. 2020 Oct 20. 3(1): 592
    Watanabe M, Saeki Y, Takahashi H, Ohtake F, Yoshida Y, Kasuga Y, Kondo T, Yaguchi H, Suzuki M, Ishida H, Tanaka K, Hatakeyama S.
      The identification of true substrates of an E3 ligase is biologically important but biochemically difficult. In recent years, several techniques for identifying substrates have been developed, but these approaches cannot exclude indirect ubiquitination or have other limitations. Here we develop an E3 ligase substrate-trapping strategy by fusing a tandem ubiquitin-binding entity (TUBE) with an anti-ubiquitin remnant antibody to effectively identify ubiquitinated substrates. We apply this method to one of the RBR-type ligases, Parkin, and to one of the RING-type ligases, TRIM28, and identify previously unknown substrates for TRIM28 including cyclin A2 and TFIIB. Furthermore, we find that TRIM28 promotes cyclin A2 ubiquitination and degradation at the G1/S phase and suppresses premature entry into S phase. Taken together, the results indicate that this method is a powerful tool for comprehensively identifying substrates of E3 ligases.
    DOI:  https://doi.org/10.1038/s42003-020-01328-y
  27. Cell Rep. 2020 Oct 20. pii: S2211-1247(20)31275-4. [Epub ahead of print]33(3): 108286
    Balakrishnan I, Danis E, Pierce A, Madhavan K, Wang D, Dahl N, Sanford B, Birks DK, Davidson N, Metselaar DS, Meel MH, Lemma R, Donson A, Vijmasi T, Katagi H, Sola I, Fosmire S, Alimova I, Steiner J, Gilani A, Hulleman E, Serkova NJ, Hashizume R, Hawkins C, Carcaboso AM, Gupta N, Monje M, Jabado N, Jones K, Foreman N, Green A, Vibhakar R, Venkataraman S.
      Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.
    Keywords:  BH3 mimetics; BMI1; DIPG; H3K27M mutant; H3WT; PTC 028; RNAi screen; SASP; senescence
    DOI:  https://doi.org/10.1016/j.celrep.2020.108286
  28. Open Biol. 2020 Oct;10(10): 200279
    Suresh HG, Pascoe N, Andrews B.
      Protein ubiquitination is a key post-translational modification that regulates diverse cellular processes in eukaryotic cells. The specificity of ubiquitin (Ub) signalling for different bioprocesses and pathways is dictated by the large variety of mono-ubiquitination and polyubiquitination events, including many possible chain architectures. Deubiquitinases (DUBs) reverse or edit Ub signals with high sophistication and specificity, forming an integral arm of the Ub signalling machinery, thus impinging on fundamental cellular processes including DNA damage repair, gene expression, protein quality control and organellar integrity. In this review, we discuss the many layers of DUB function and regulation, with a focus on insights gained from budding yeast. Our review provides a framework to understand key aspects of DUB biology.
    Keywords:  deubiquitinases; protein degradation; ubiquitin signalling
    DOI:  https://doi.org/10.1098/rsob.200279
  29. FASEB J. 2020 Oct 23.
    Han Y, Yun CC.
      Na+ /H+ exchanger NHE3 of human or primates differs from NHE3 of other animals by having a PY motif, which mediates interaction with the E3 ubiquitin (Ub) ligase Nedd4-2. Ub-conjugation of human NHE3 by Nedd4-2 regulates endocytosis of NHE3 but does not affect its cellular expression. Because Ub-conjugation is a reversible process, the aim of this study is to identify deubiquitinating enzyme (DUB) regulating the post-endosomal fate of human NHE3. Using an activity-based chemical screening, we identified ubiquitin specific protease-7 (USP7) and USP10 that bind NHE3. The roles of DUBs in regulation of NHE3 were studied by determining the effects of silencing of USP7 and USP10. Knockdown of USP7 or USP10 resulted in increased NHE3 ubiquitination and decreased NHE3 expression at the surface membrane and cellular level. The endocytic retrieval of NHE3 was promoted by depletion of USP7 or USP10, with increased association of NHE3 with Rab5a and Rab7. Inhibition of USP7 and USP10 by chemical inhibitors or knockdown had an additive effect on NHE3. In addition, NHE3 half-life was reduced accounting for decreased NHE3 protein abundance. NHE3 is inhibited by protein kinase A. Activation of PKA by forskolin decreased the binding of USP7 and USP10 to NHE3, while increasing ubiquitination of NHE3. Knockdown of USP10 had an additive effect on PKA-dependent inhibition of NHE3. These findings demonstrate that USP7 and USP10 are DUBs that regulate NHE3 ubiquitination and expression, and reveal a new mechanism of NHE3 inhibition involving DUBs.
    Keywords:  Na+/H+ exchange; deubiquitinating enzyme; diarrhea; ubiquitination
    DOI:  https://doi.org/10.1096/fj.202001875R
  30. Neoplasia. 2020 Nov;pii: S1476-5586(20)30132-9. [Epub ahead of print]22(11): 644-658
    Meyer AE, Furumo Q, Stelloh C, Minella AC, Rao S.
      Fbw7 is a tumor suppressor that regulates the degradation of oncogenic substrates such as c-Jun, c-Myc, Notch1 intracellular domain (ICD), and cyclin E by functioning as the substrate recognition protein in the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex. Consequently, low expression or loss of FBXW7 in breast cancer has been hypothesized to result in the accumulation of oncogenic transcription factors that are master regulators of proliferation, apoptosis, and ultimately transformation. Despite this, the direct effect of Fbw7 loss on mammary gland morphology and tumorigenesis has not been examined. Here, we demonstrate that conditional deletion of Fbxw7 in murine mammary tissue initiates breast tumor development and also results in lactation and involution defects. Further, while Fbxw7 loss results in the overexpression of Notch1-ICD, c-Jun, cyclin E, and c-Myc, the downstream transcription factor pathways associated with c-Myc and cyclin E are the most dysregulated, including at the single-cell level. These pathways are dysregulated early after Fbxw7 loss, and their sustained loss results in tumorigenesis and reinforced c-Myc and cyclin E-E2F pathway disruption. We also find that loss of Fbxw7 is linked to the acquisition of Trp53 mutations, similar to the mutational spectrum observed in patients. Our results demonstrate that the loss of Fbxw7 promotes the acquisition of Trp53 mutations and that the two cooperate in breast tumor development. Targeting c-Myc, E2F, or p53 may therefore be a beneficial treatment strategy for FBXW7-altered breast cancer patients.
    Keywords:  Breast cancer; Cell cycle; Fbw7; P53; Tumor cell biology
    DOI:  https://doi.org/10.1016/j.neo.2020.07.001
  31. Expert Opin Drug Discov. 2020 Oct 17.
    Maurice T.
      INTRODUCTION: The sigma-1 receptor (S1R) is attracting much attention as a target for disease-modifying therapies in neurodegenerative diseases. It is a highly conserved protein, present in plasma and endoplasmic reticulum (ER) membranes and enriched in mitochondria-associated ER membranes (MAMs). It modulates ER-mitochondria Ca2+ transfer and activation of the ER stress pathways. Mitochondrial and MAM dysfunctions contribute to neurodegenerative processes in pathologies including Alzheimer's disease and Parkinson's disease. Interestingly, the S1R can be activated by small druggable molecules and accumulating preclinical data suggest that S1R agonists are effective protectants in these neurodegenerative diseases.AREA COVERED: In this review, the author presents the data showing the high therapeutic potential of S1R drugs for the treatment of neurodegenerative diseases, focusing on pridopidine as a potent and selective S1R agonist under clinical development. Of particular interest is the bi-phasic (bell-shaped) dose-response effect described in numerous preclinical models in vitro, in vivo and in clinical trials.
    EXPERT OPINION: S1R agonists modulate essential inter-organelles communication altered in all neurodegenerative diseases and activate numerous intracellular survival pathways. Research in the field will continue growing in the near future. The particular cellular nature of this unique chaperone protein must be better understood to facilitate the development of promising molecules at the clinical stage.
    DOI:  https://doi.org/10.1080/17460441.2021.1838483
  32. Cell. 2020 Oct 08. pii: S0092-8674(20)31310-6. [Epub ahead of print]
    Banerjee AK, Blanco MR, Bruce EA, Honson DD, Chen LM, Chow A, Bhat P, Ollikainen N, Quinodoz SA, Loney C, Thai J, Miller ZD, Lin AE, Schmidt MM, Stewart DG, Goldfarb D, De Lorenzo G, Rihn SJ, Voorhees RM, Botten JW, Majumdar D, Guttman M.
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.
    Keywords:  NSP1; NSP16; NSP8; NSP9; RNA-protein interactions; SARS-CoV-2; interferon; mRNA splicing; protein trafficking; translation
    DOI:  https://doi.org/10.1016/j.cell.2020.10.004
  33. EMBO Rep. 2020 Oct 19. e49684
    Lu X, Huang J, Wu S, Zheng Q, Liu P, Feng H, Su X, Fu H, Xi Q, Wang G.
      mascRNA is a small cytoplasmic RNA derived from the lncRNA MALAT1. After being processed by the tRNA processing enzymes RNase P and RNase Z, mascRNA undergoes CCA addition like tRNAs and folds into a tRNA-like cloverleaf structure. While MALAT1 functions in multiple cellular processes, the role of mascRNA was largely unknown. Here, we show that mascRNA binds directly to the multi-tRNA synthetase complex (MSC) component glutaminyl-tRNA synthetase (QARS). mascRNA promotes global protein translation and cell proliferation by positively regulating QARS protein levels. Our results uncover a role of mascRNA that is independent of MALAT1, but could be part of the molecular mechanism of MALAT1's function in cancer, and provide a paradigm for understanding tRNA-like structures in mammalian cells.
    Keywords:   MALAT1 ; mascRNA; multi-tRNA synthetase complex; tRNA-like structure; translation
    DOI:  https://doi.org/10.15252/embr.201949684
  34. J Cell Sci. 2020 Oct 19. pii: jcs.248336. [Epub ahead of print]
    Murakawa T, Kiger AA, Sakamaki Y, Fukuda M, Fujita N.
      Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.
    Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17
    DOI:  https://doi.org/10.1242/jcs.248336
  35. Nat Commun. 2020 10 19. 11(1): 5267
    Chen BH, Chang YJ, Lin S, Yang WY.
      Peroxisomes perform beta-oxidation of branched and very-long chain fatty acids, which leads to the formation of reactive oxygen species (ROS) within the peroxisomal lumen. Peroxisomes are therefore prone to ROS-mediated damages. Here, using light to specifically and acutely induce ROS formation within the peroxisomal lumen, we find that cells individually remove ROS-stressed peroxisomes through ubiquitin-dependent pexophagy. Heat shock protein 70 s mediates the translocation of the ubiquitin E3 ligase Stub1 (STIP1 Homology and U-Box Containing Protein 1) onto oxidatively-stressed peroxisomes to promote their selective ubiquitination and autophagic degradation. Artificially targeting Stub1 to healthy peroxisomes is sufficient to trigger pexophagy, suggesting a key role Stub1 plays in regulating peroxisome quality. We further determine that Stub1 mutants found in Ataxia patients are defective in pexophagy induction. Dysfunctional peroxisomal quality control may therefore contribute to the development of Ataxia.
    DOI:  https://doi.org/10.1038/s41467-020-18942-3
  36. Biochem J. 2020 Oct 23. pii: BCJ20200536. [Epub ahead of print]
    Xue W, Zhang SX, He WT, Hong JY, Jiang LL, Hu HY.
      Ubiquitin-specific protease 19 (USP19) is a member of the deubiquitinating (DUB) enzymes that catalyze removing the ubiquitin signals from target proteins. Our previous research has demonstrated that USP19 up-regulates the protein level and aggregation of polyQ-expanded huntingtin through the involvement of heat shock protein 90 (HSP90). Here, we present solution structures of the CS1, CS2 and UbL domains of USP19 and structural insights into their domain interactions. We found that the tandem CS domains fold back to interact with the C-terminal USP domain (USPD) intra-molecularly that leads to inhibition of the catalytic core of USP19, especially CS1 interacts with the embedded UbL domain and CS2 does with the CH2 catalytic core. Moreover, CS2 specifically interacts with the NBD domain of HSP90, which can activate the deubiquitinating enzyme. A mechanism of auto-inhibition of USP19 and activation by HSP90 is proposed, on which USP19 modulates the protein level of polyQ-expanded huntingtin in cells. This study provides structural and mechanistic insights into the modulation of protein level and aggregation by USP19 with the assistance of HSP90.
    Keywords:  HSP90; USP19; auto-inhibition; deubiquitination; domain interaction
    DOI:  https://doi.org/10.1042/BCJ20200536
  37. Nat Commun. 2020 10 20. 11(1): 5237
    Ranek MJ, Oeing C, Sanchez-Hodge R, Kokkonen-Simon KM, Dillard D, Aslam MI, Rainer PP, Mishra S, Dunkerly-Eyring B, Holewinski RJ, Virus C, Zhang H, Mannion MM, Agrawal V, Hahn V, Lee DI, Sasaki M, Van Eyk JE, Willis MS, Page RC, Schisler JC, Kass DA.
      Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.
    DOI:  https://doi.org/10.1038/s41467-020-18980-x
  38. Nat Microbiol. 2020 Oct 19.
    Zhou B, Liu J, Zeng L, Zhu S, Wang H, Billiar TR, Kroemer G, Klionsky DJ, Zeh HJ, Jiang J, Tang D, Kang R.
      Sepsis is the most common cause of death for patients in intensive care worldwide due to a dysregulated host response to infection. Here, we investigate the role of sequestosome-1 (SQSTM1/p62), an autophagy receptor that functions as a regulator of innate immunity, in sepsis. We find that lipopolysaccharide elicits gasdermin D-dependent pyroptosis to enable passive SQSTM1 release from macrophages and monocytes, whereas transmembrane protein 173-dependent TANK-binding kinase 1 activation results in the phosphorylation of SQSTM1 at Ser403 and subsequent SQSTM1 secretion from macrophages and monocytes. Moreover, extracellular SQSTM1 binds to insulin receptor, which in turn activates a nuclear factor kappa B-dependent metabolic pathway, leading to aerobic glycolysis and polarization of macrophages. Intraperitoneal injection of anti-SQSTM1-neutralizing monoclonal antibodies or conditional depletion of Insr in myeloid cells using the Cre-loxP system protects mice from lethal sepsis (caecal ligation and puncture or infection by Escherichia coli or Streptococcus pneumoniae) and endotoxaemia. We also report that circulating SQSTM1 and the messenger RNA expression levels of SQSTM1 and INSR in peripheral blood mononuclear cells are related to the severity of sepsis in 40 patients. Thus, extracellular SQSTM1 has a pathological role in sepsis and could be targeted to develop therapies for sepsis.
    DOI:  https://doi.org/10.1038/s41564-020-00795-7
  39. J Biol Chem. 2020 Oct 22. pii: jbc.RA120.015216. [Epub ahead of print]
    Turan NN, Moshal KS, Roder K, Baggett BC, Kabakov AY, Dhakal S, Teramoto R, Chiang DY, Zhong M, Xie A, Lu Y, Dudley SC, MacRae CA, Karma A, Koren G.
      The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies (GWAS) identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-alpha factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, while co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggests LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression downregulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I Na We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a non-significant 15% prolongation of action potential duration (APD). Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca2+ and Na+ homeostasis are responsible for the surprisingly modest APD shortening. These computational data thus corroborate findings from several GWAS that associated LITAF with QT interval variation.
    Keywords:  Action potential duration; Computer modelling; E3 ubiquitin ligase; LITAF; NEDD4-2; Nav1.5; cardiomyocyte; sodium channel; ubiquitin; zebrafish
    DOI:  https://doi.org/10.1074/jbc.RA120.015216
  40. Immunohorizons. 2020 Oct 22. 4(10): 670-678
    Xu W, Rush JS, Graham DB, Cao Z, Xavier RJ.
      Posttranslational modifications are efficient means to rapidly regulate protein function in response to a stimulus. Although ubiquitination events and the E3 ubiquitin ligases involved are increasingly characterized in many signaling pathways, their regulation by deubiquitinating enzymes remains less understood. The C-type lectin receptor (CLR) signaling adaptor CARD9 was previously reported to be activated via TRIM62-mediated ubiquitination. In this study, we identify the deubiquitinase USP15 as a novel regulator of CARD9, demonstrating that USP15 constitutively associates with CARD9 and removes TRIM62-deposited ubiquitin marks. Furthermore, USP15 knockdown and knockout specifically enhance CARD9-dependent CLR signaling in both mouse and human immune cells. Altogether, our study identifies a novel regulator of innate immune signaling and provides a blueprint for the identification of additional deubiquitinases that are likely to control these processes.
    DOI:  https://doi.org/10.4049/immunohorizons.2000036
  41. Nat Commun. 2020 Oct 23. 11(1): 5362
    Gao M, Guo G, Huang J, Kloeber JA, Zhao F, Deng M, Tu X, Kim W, Zhou Q, Zhang C, Yin P, Luo K, Lou Z.
      Human C-terminal binding protein (CtBP)-interacting protein (CtIP) is a central regulator to initiate DNA end resection and homologous recombination (HR). Several studies have shown that post-translational modifications control the activity or expression of CtIP. However, it remains unclear whether and how cells restrain CtIP activity in unstressed cells and activate CtIP when needed. Here, we identify that USP52 directly interacts with and deubiquitinates CtIP, thereby promoting DNA end resection and HR. Mechanistically, USP52 removes the ubiquitination of CtIP to facilitate the phosphorylation and activation of CtIP at Thr-847. In addition, USP52 is phosphorylated by ATM at Ser-1003 after DNA damage, which enhances the catalytic activity of USP52. Furthermore, depletion of USP52 sensitizes cells to PARP inhibition in a CtIP-dependent manner in vitro and in vivo. Collectively, our findings reveal the key role of USP52 and the regulatory complexity of CtIP deubiquitination in DNA repair.
    DOI:  https://doi.org/10.1038/s41467-020-19202-0
  42. Dev Cell. 2020 Oct 10. pii: S1534-5807(20)30757-7. [Epub ahead of print]
    Bustos F, Segarra-Fas A, Nardocci G, Cassidy A, Antico O, Davidson L, Brandenburg L, Macartney TJ, Toth R, Hastie CJ, Moran J, Gourlay R, Varghese J, Soares RF, Montecino M, Findlay GM.
      Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.
    Keywords:  development; metazoan evolution; neural development; neurodevelopmental disorders; protein kinase; protein phosphorylation; signal transduction; stem cells; transcriptomics; ubiquitin signaling
    DOI:  https://doi.org/10.1016/j.devcel.2020.09.025
  43. Cell Mol Neurobiol. 2020 Oct 23.
    Couly S, Goguadze N, Yasui Y, Kimura Y, Wang SM, Sharikadze N, Wu HE, Su TP.
      Sigma-1 receptor (Sig-1R) is a protein present in several organs such as brain, lung, and heart. In a cell, Sig-1R is mainly located across the membranes of the endoplasmic reticulum and more specifically at the mitochondria-associated membranes. Despite numerous studies showing that Sig-1R could be targeted to rescue several cellular mechanisms in different pathological conditions, less is known about its fundamental relevance. In this review, we report results from various studies and focus on the importance of Sig-1R in physiological conditions by comparing Sig-1R KO mice to wild-type mice in order to investigate the fundamental functions of Sig-1R. We note that the Sig-1R deletion induces cognitive, psychiatric, and motor dysfunctions, but also alters metabolism of heart. Finally, taken together, observations from different experiments demonstrate that those dysfunctions are correlated to poor regulation of ER and mitochondria metabolism altered by stress, which could occur with aging.
    Keywords:  Endoplasmic reticulum; Mitochondria; Neurodegenerative disorders; Sigma-1 receptor
    DOI:  https://doi.org/10.1007/s10571-020-00983-3