bims-proteo Biomed News
on Proteostasis
Issue of 2020‒05‒31
forty-nine papers selected by
Eric Chevet

  1. Cell Death Dis. 2020 May 26. 11(5): 397
    Bugallo R, Marlin E, Baltanás A, Toledo E, Ferrero R, Vinueza-Gavilanes R, Larrea L, Arrasate M, Aragón T.
      Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.
  2. Nat Rev Mol Cell Biol. 2020 May 26.
    Hetz C, Zhang K, Kaufman RJ.
      Cellular stress induced by the abnormal accumulation of unfolded or misfolded proteins at the endoplasmic reticulum (ER) is emerging as a possible driver of human diseases, including cancer, diabetes, obesity and neurodegeneration. ER proteostasis surveillance is mediated by the unfolded protein response (UPR), a signal transduction pathway that senses the fidelity of protein folding in the ER lumen. The UPR transmits information about protein folding status to the nucleus and cytosol to adjust the protein folding capacity of the cell or, in the event of chronic damage, induce apoptotic cell death. Recent advances in the understanding of the regulation of UPR signalling and its implications in the pathophysiology of disease might open new therapeutic avenues.
  3. FEBS J. 2020 May 23.
    Doultsinos D, Carlesso A, Chintha C, Paton JC, Paton AW, Samali A, Chevet E, Eriksson LA.
      Inositol Requiring Enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the Unfolded Protein Response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here we report the discovery of the FDA approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimization and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard of care chemotherapy temozolomide (TMZ).
    Keywords:  Endoplasmic Reticulum; IRE1; Inhibitors; Unfolded Protein Response; glioblastoma
  4. EMBO J. 2020 May 25. e103954
    Tittelmeier J, Sandhof CA, Ries HM, Druffel-Augustin S, Mogk A, Bukau B, Nussbaum-Krammer C.
      The accumulation and prion-like propagation of α-synuclein and other amyloidogenic proteins are associated with devastating neurodegenerative diseases. Metazoan heat shock protein HSP70 and its co-chaperones DNAJB1 and HSP110 constitute a disaggregation machinery that is able to disassemble α-synuclein fibrils in vitro, but its physiological effects on α-synuclein toxicity are unknown. Here, we depleted Caenorhabditis elegans HSP-110 and monitored the consequences on α-synuclein-related pathological phenotypes such as misfolding, intercellular spreading, and toxicity in C. elegans in vivo models. Depletion of HSP-110 impaired HSP70 disaggregation activity, prevented resolubilization of amorphous aggregates, and compromised the overall cellular folding capacity. At the same time, HSP-110 depletion reduced α-synuclein foci formation, cell-to-cell transmission, and toxicity. These data demonstrate that the HSP70 disaggregation activity constitutes a double-edged sword, as it is essential for maintaining cellular proteostasis but also involved in the generation of toxic amyloid-type protein species.
    Keywords:  chaperones; neurodegenerative diseases; prion-like propagation; protein disaggregation; proteostasis
  5. Amino Acids. 2020 May 24.
    Adacan K, Obakan-Yerlikaya P, Arisan ED, Coker-Gurkan A, Kaya RI, Palavan-Unsal N.
      Epibrassinolide (EBR), a polyhydroxysteroid belongs to plant growth regulator family, brassinosteroids and has been shown to have a similar chemical structure to mammalian steroid hormones. Our findings indicated that EBR could trigger apoptosis in cancer cells via induction of endoplasmic reticulum (ER) stress, caused by protein folding disturbance in the ER. Normal cells exhibited a remarkable resistance to EBR treatment and avoid from apoptotic cell death. The unfolded protein response clears un/misfolded proteins and restore ER functions. When stress is chronic, cells tend to die due to improper cellular functions. To understand the effect of EBR in non-malign cells, mouse embryonic fibroblast (MEF) cells were investigated in detail for ER stress biomarkers, autophagy, and polyamine metabolism in this study. Evolutionary conserved autophagy mechanism is a crucial cellular process to clean damaged organelles and protein aggregates through lysosome under the control of autophagy-related genes (ATGs). Cells tend to activate autophagy to promote cell survival under stress conditions. Polyamines are polycationic molecules playing a role in the homeostasis of important cellular events such as cell survival, growth, and, proliferation. The administration of PAs has been markedly extended the lifespan of various organisms via inducing autophagy and inhibiting oxidative stress. Our data indicated that ER stress is induced following EBR treatment in MEF cells as well as MEF Atg5-/- cells. In addition, autophagy is activated following EBR treatment by targeting PI3K/Akt/mTOR in wildtype (wt) cells. However, EBR-induced autophagy targets ULK1 in MEF cells lacking Atg5 expression. Besides, EBR treatment depleted the PA pool in MEF cells through the alterations of metabolic enzymes. The administration of Spd with EBR further increased autophagic vacuole formation. In conclusion, EBR is an anticancer drug candidate with selective cytotoxicity for cancer cells, in addition the induction of autophagy and PA metabolism are critical for responses of normal cells against EBR.
    Keywords:  Apoptosis; Autophagy; ER stress; Epibrassinolide; Polyamines
  6. Cells. 2020 May 22. pii: E1295. [Epub ahead of print]9(5):
    Lopez S, Perez-Linero AM, Manzano-Lopez J, Sabido-Bozo S, Cortes-Gomez A, Rodriguez-Gallardo S, Aguilera-Romero A, Goder V, Muñiz M.
      The cellular mechanisms that ensure the selectivity and fidelity of secretory cargo protein transport from the endoplasmic reticulum (ER) to the Golgi are still not well understood. The p24 protein complex acts as a specific cargo receptor for GPI-anchored proteins by facilitating their ER exit through a specialized export pathway in yeast. In parallel, the p24 complex can also exit the ER using the general pathway that exports the rest of secretory proteins with their respective cargo receptors. Here, we show biochemically that the p24 complex associates at the ER with other cargo receptors in a COPII-dependent manner, forming high-molecular weight multireceptor complexes. Furthermore, live cell imaging analysis reveals that the p24 complex is required to retain in the ER secretory cargos when their specific receptors are absent. This requirement does not involve neither the unfolded protein response nor the retrograde transport from the Golgi. Our results suggest that, in addition to its role as a cargo receptor in the specialized GPI-anchored protein pathway, the p24 complex also plays an independent role in secretory cargo selectivity during its exit through the general ER export pathway, preventing the non-selective bulk flow of native secretory cargos. This mechanism would ensure receptor-regulated cargo transport, providing an additional layer of regulation of secretory cargo selectivity during ER export.
    Keywords:  bulk flow; cargo receptor; endoplasmic reticulum; p24 complex; secretory cargo
  7. Elife. 2020 May 26. pii: e57822. [Epub ahead of print]9
    Raote I, Ernst AM, Campelo F, Rothman JE, Pincet F, Malhotra V.
      We have previously shown TANGO1 organises membranes at the interface of the endoplasmic reticulum (ER) and ERGIC/Golgi (Raote et al., 2018). TANGO1 corrals retrograde membranes at ER exit sites to create an export conduit. Here the retrograde membrane is, in itself, an anterograde carrier. This mode of forward transport necessitates a mechanism to prevent membrane mixing between ER and the retrograde membrane. TANGO1 has an unusual membrane helix organisation, composed of one membrane-spanning helix (TM) and another that penetrates the inner leaflet (IM). We have reconstituted these membrane helices in model membranes and shown that TM and IM together reduce the flow of lipids at a region of defined shape. We have also shown that the helices align TANGO1 around an ER exit site. We suggest this is a mechanism to prevent membrane mixing during TANGO1-mediated transfer of bulky secretory cargos from the ER to the ERGIC/Golgi via a tunnel.
    Keywords:  cell biology; human; molecular biophysics; structural biology
  8. Biochimie. 2020 May 25. pii: S0300-9084(20)30127-9. [Epub ahead of print]
    Turpin J, Frumence E, Harrabi W, Haddad JG, El Kalamouni C, Desprès P, Krejbich-Trotot P, Viranaïcken W.
      Flaviviruses replicate in membranous factories associated with the endoplasmic reticulum (ER). Significant levels of flavivirus polyprotein integration contribute to ER stress and the host cell may exhibit an Unfolded Protein Response (UPR) to this protein accumulation, stimulating appropriate cellular responses such as adaptation, autophagy or cell death. These different stress responses support other antiviral strategies initiated by infected cells and can help to overcome viral infection. In epithelial A549 cells, a model currently used to study the flavivirus infection cycle and the host cell responses, all three pathways leading to UPR are activated during infection by Dengue virus (DENV), Yellow Fever virus (YFV) or West Nile virus (WNV). In the present study, we investigated the capacity of ZIKA virus (ZIKV) to induce ER stress in A549 cells. We observed that the cells respond to ZIKV infection by implementing an UPR through activation of the IRE1 and PERK pathway without activation of the ATF6 branch. By modulating the ER stress response, we found that UPR inducers significantly inhibit ZIKV replication. Interestingly, our findings provide evidence that ZIKV could manipulate the UPR to escape this host cell defence system by downregulating GRP78/BiP expression. This subversion of GRP78 expression could lead to unresolved and persistent ER stress which can be a benefit for virus growth.
    Keywords:  Persistent ER stress; Unfolded protein response; Viral growth; Zika virus
  9. Elife. 2020 May 27. pii: e57887. [Epub ahead of print]9
    O'Donnell JP, Phillips BP, Yagita Y, Juszkiewicz S, Wagner A, Malinverni D, Keenan RJ, Miller EA, Hegde RS.
      Approximately 25% of eukaryotic genes code for integral membrane proteins that are assembled at the endoplasmic reticulum. An abundant and widely conserved multi-protein complex termed EMC has been implicated in membrane protein biogenesis, but its mechanism of action is poorly understood. Here, we define the composition and architecture of human EMC using biochemical assays, crystallography of individual subunits, site-specific photocrosslinking, and cryo-EM reconstruction. Our results suggest that EMC's cytosolic domain contains a large, moderately hydrophobic vestibule that can bind a substrate's transmembrane domain (TMD). The cytosolic vestibule leads into a lumenally-sealed, lipid-exposed intramembrane groove large enough to accommodate a single substrate TMD. A gap between the cytosolic vestibule and intramembrane groove provides a potential path for substrate egress from EMC. These findings suggest how EMC facilitates energy-independent membrane insertion of TMDs, explain why only short lumenal domains are translocated by EMC, and constrain models of EMC's proposed chaperone function.
    Keywords:  biochemistry; chemical biology; molecular biophysics; none; structural biology
  10. Int J Mol Sci. 2020 May 23. pii: E3683. [Epub ahead of print]21(10):
    Hamazaki J, Murata S.
      Protein folding is a substantively error prone process, especially when it occurs in the endoplasmic reticulum (ER). The highly exquisite machinery in the ER controls secretory protein folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol; these misfolded proteins are then degraded by the ubiquitin-proteasome system termed as the ER-associated degradation (ERAD). The 26S proteasome is a multisubunit protease complex that recognizes and degrades ubiquitinated proteins in an ATP-dependent manner. The complex structure of the 26S proteasome requires exquisite regulation at the transcription, translation, and molecular assembly levels. Nuclear factor erythroid-derived 2-related factor 1 (Nrf1; NFE2L1), an ER-resident transcription factor, has recently been shown to be responsible for the coordinated expression of all the proteasome subunit genes upon proteasome impairment in mammalian cells. In this review, we summarize the current knowledge regarding the transcriptional regulation of the proteasome, as well as recent findings concerning the regulation of Nrf1 transcription activity in ER homeostasis and metabolic processes.
    Keywords:  DDI2; Nrf1; bounce back; proteasome; proteostasis
  11. Appl Microbiol Biotechnol. 2020 May 24.
    Li W, Cao T, Luo C, Cai J, Zhou X, Xiao X, Liu S.
      Endoplasmic reticulum stress (ERS) is a protective response to restore protein homeostasis by activating the unfolded protein response (UPR). However, UPR can trigger cell death under severe and/or persistently high ERS. The NLRP3 inflammasome is a complex of multiple proteins that activates the secretion of the proinflammatory cytokine IL-1β in a caspase-1-dependent manner to participate in the regulation of inflammation. The NLRP3 inflammasome involvement in ERS-induced inflammation has not been completely described. The intersection of ERS with multiple inflammatory pathways can initiate and aggravate chronic diseases. Accumulating evidence suggests that ERS-induced activation of NLRP3 inflammasome is the pathological basis of various inflammatory diseases. In this review, we have discussed the networks between ERS and NLRP3 inflammasome, with the view to identifying novel therapeutic targets in inflammatory diseases. KEY POINTS: • Endoplasmic reticulum stress (ERS) is an important factor for the activation of the NLRP3 inflammasomes that results in pathological processes. • ERS can activate the NLRP3 inflammasome to induce inflammatory responses via oxidative stress, calcium homeostasis, and NF-κB activation. • The interactions between ERS and NLRP3 inflammasome are associated with inflammation, which represent a potential therapeutic opportunity of inflammatory diseases.
    Keywords:  Endoplasmic reticulum stress; Inflammation; NF-κB; NLRP3 inflammasome; Reactive oxygen species
  12. Adv Exp Med Biol. 2020 ;1194 351-358
    Theocharopoulou G, Vlamos P.
      Protein homeostasis is a dynamic network that plays a pivotal role in systems' maintenance within a cell. This quality control system involves a number of mechanisms regarding the process of protein folding. Chaperones play a critical role in the folding, refolding, and unfolding of proteins. Aggregation of misfolded proteins is a common characteristic of neurodegenerative diseases. Chaperones act in a variety of pathways in this critical interplay between protein homeostasis network and misfolded protein's load. Moreover, ER stress-induced cell death mechanisms (such as the unfolded protein response) are activated as a response. Therefore, there is a critical balance in the accumulation of misfolded proteins and ER stress response mechanisms which can lead to cell death. Our focus is in understanding the different mechanisms that govern ER stress signaling in health and disease in order to represent the regulation of protein homeostasis and balance of protein synthesis and degradation in the ER. Our proposed model describes, using hybrid modeling, the function of chaperones' machinery for protein folding.
    Keywords:  Chaperones; Misfolded proteins; Neurodegenerative diseases; Protein homeostasis; Quality control
  13. Mol Neurobiol. 2020 May 25.
    Wang WX, Prajapati P, Nelson PT, Springer JE.
      The mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are specific ER domains that contact the mitochondria and function to facilitate communication between ER and mitochondria. Disruption of contact between the mitochondria and ER is associated with a variety of pathophysiological conditions including neurodegenerative diseases. Considering the many cellular functions of MAMs, we hypothesized that MAMs play an important role in regulating microRNA (miRNA) activity linked to its unique location between mitochondria and ER. Here we present new findings from human and rat brains indicating that the MAMs are subcellular sites enriched for specific miRNAs. We employed subcellular fractionation and TaqMan® RT-qPCR miRNA analysis to quantify miRNA levels in subcellular fractions isolated from male rat brains and six human brain samples. We found that MAMs contain a substantial number of miRNAs and the profile differs significantly from that of cytosolic, mitochondria, or ER. Interestingly, MAMs are particularly enriched in inflammatory-responsive miRNAs, including miR-146a, miR-142-3p, and miR-142-5p in both human and rat brains; miR-223 MAM enrichment was observed only in human brain samples. Further, mitochondrial uncoupling or traumatic brain injury in male rats resulted in the alteration of inflammatory miRNA enrichment in the isolated subcellular fractions. These observations demonstrate that miRNAs are distributed differentially in organelles and may re-distribute between organelles and the cytosol in response to cellular stress and metabolic demands.
    Keywords:  Mitochondria-associated ER membrane; Neurodegeneration; Subcellular; Traumatic brain injury; miR-146a; microRNA
  14. Cells. 2020 May 24. pii: E1308. [Epub ahead of print]9(5):
    Margulis B, Tsimokha A, Zubova S, Guzhova I.
      Throughout their life cycles, cells are subject to a variety of stresses that lead to a compromise between cell death and survival. Survival is partially provided by the cell proteostasis network, which consists of molecular chaperones, a ubiquitin-proteasome system of degradation and autophagy. The cooperation of these systems impacts the correct function of protein synthesis/modification/transport machinery starting from the adaption of nascent polypeptides to cellular overcrowding until the utilization of damaged or needless proteins. Eventually, aging cells, in parallel to the accumulation of flawed proteins, gradually lose their proteostasis mechanisms, and this loss leads to the degeneration of large cellular masses and to number of age-associated pathologies and ultimately death. In this review, we describe the function of proteostasis mechanisms with an emphasis on the possible associations between them.
    Keywords:  aging; autophagy; molecular chaperones; ubiquitin-proteasomal system
  15. FEBS Lett. 2020 May 23.
    Balchin D, Hayer-Hartl M, Hartl FU.
      Molecular chaperones are highly conserved proteins that promote proper folding of other proteins in vivo. Diverse chaperone systems assist de novo protein folding and trafficking, the assembly of oligomeric complexes, and recovery from stress-induced unfolding. A fundamental function of molecular chaperones is to inhibit unproductive protein interactions by recognizing and protecting hydrophobic surfaces that are exposed during folding or following proteotoxic stress. Beyond this basic principle, it is now clear that chaperones can also actively and specifically accelerate folding reactions in an ATP-dependent manner. We focus on the bacterial Hsp70 and chaperonin systems as paradigms, and review recent work that has advanced our understanding of how these chaperones act as catalysts of protein folding.
    Keywords:  DnaK; GroEL; Hsp40; Hsp60; Hsp70; Protein folding; chaperonin; confinement; molecular chaperones; protein misfolding
  16. Elife. 2020 May 29. pii: e57127. [Epub ahead of print]9
    Langer LM, Gat Y, Bonneau F, Conti E.
      PI3K-related kinases (PIKKs) are large Serine/Threonine (Ser/Thr)-protein kinases central to the regulation of many fundamental cellular processes. PIKK family member SMG1 orchestrates progression of an RNA quality control pathway, termed nonsense-mediated mRNA decay (NMD), by phosphorylating the NMD factor UPF1. Phosphorylation of UPF1 occurs in its unstructured N- and C-terminal regions at Serine/Threonine-Glutamine (SQ) motifs. How SMG1 and other PIKKs specifically recognize SQ motifs has remained unclear. Here, we present a cryo-electron microscopy (cryo-EM) reconstruction of a human SMG1-8-9 kinase complex bound to a UPF1 phosphorylation site at an overall resolution of 2.9 Å. This structure provides the first snapshot of a human PIKK with a substrate-bound active site. Together with biochemical assays, it rationalizes how SMG1 and perhaps other PIKKs specifically phosphorylate Ser/Thr-containing motifs with a glutamine residue at position +1 and a hydrophobic residue at position -1, thus elucidating the molecular basis for phosphorylation site recognition.
    Keywords:  human; molecular biophysics; structural biology
  17. Trends Cancer. 2020 May 22. pii: S2405-8033(20)30139-4. [Epub ahead of print]
    O'Malley J, Kumar R, Inigo J, Yadava N, Chandra D.
      Cancer cells survive and adapt to many types of stress including hypoxia, nutrient deprivation, metabolic, and oxidative stress. These stresses are sensed by diverse cellular signaling processes, leading to either degradation of mitochondria or alleviation of mitochondrial stress. This review discusses signaling during sensing and mitigation of stress involving mitochondrial communication with the endoplasmic reticulum, and how retrograde signaling upregulates the mitochondrial stress response to maintain mitochondrial integrity. The importance of the mitochondrial unfolded protein response, an emerging pathway that alleviates cellular stress, will be elaborated with respect to cancer. Detailed understanding of cellular pathways will establish mitochondrial stress response as a key mechanism for cancer cell survival leading to cancer progression and resistance, and provide a potential therapeutic target in cancer.
    Keywords:  cancer cell survival; cancer progression; heat shock protein 60; mitochondrial stress response; mitochondrial unfolded protein response; therapeutic resistance
  18. J Biochem. 2020 May 27. pii: mvaa058. [Epub ahead of print]
    Okuda A, Matsusaki M, Masuda T, Morishima K, Sato N, Inoue R, Sugiyama M, Urade R.
      Secretory and membrane proteins synthesized in the endoplasmic reticulum (ER) are folded with intramolecular disulfide bonds, viz. oxidative folding, catalyzed by the protein disulfide isomerase (PDI) family proteins. Here, we identified a novel soybean PDI family protein, GmPDIL6. GmPDIL6 has a single thioredoxin-domain with a putative N-terminal signal peptide and an active center (CKHC). Recombinant GmPDIL6 forms various oligomers binding iron. Oligomers with or without iron binding, and monomers exhibited a dithiol oxidase activity level comparable to those of other soybean PDI family proteins. However, they displayed no disulfide reductase and extremely low oxidative refolding activity. Interestingly, GmPDIL6 was mainly expressed in the cotyledon during synthesis of seed storage proteins and GmPDIL6 mRNA was upregulated under ER stress. GmPDIL6 may play a role in the formation of disulfide bonds in nascent proteins for oxidative folding in the ER.
    Keywords:  Endoplasmic Reticulum; Protein Disulfide Isomerase; Protein Folding; Soybean; Unfolded Protein Response
  19. Open Biol. 2020 May;10(5): 190244
    Russo A.
      In eukaryotic cells, about one-third of the synthesized proteins are translocated into the endoplasmic reticulum; they are membrane or lumen resident proteins and proteins direct to the Golgi apparatus. The co-translational translocation takes place through the heterotrimeric protein-conducting channel Sec61 which is associated with the ribosome and many accessory components, such as the heterotetrameric translocon-associated protein (TRAP) complex. Recently, microscopic techniques, such as cryo-electron microscopy and cryo-electron tomography, have enabled the determination of the translocation machinery structure. However, at present, there is a lack of understanding regarding the roles of some of its components; indeed, the TRAP complex function during co-translational translocation needs to be established. In addition, TRAP may play a role during unfolded protein response, endoplasmic-reticulum-associated protein degradation and congenital disorder of glycosylation (ssr4 CDG). In this article, I describe the current understanding of the TRAP complex in the light of its possible function(s).
    Keywords:  calcium-binding domain; endoplasmic reticulum protein translocation; endoplasmic-reticulum-associated protein degradation; glycosylation; translocon-associated protein complex; unfolded protein response
  20. Antioxidants (Basel). 2020 May 24. pii: E452. [Epub ahead of print]9(5):
    Jiang B, Adams Z, Moonah S, Shi H, Maupin-Furlow J, Moskovitz J.
      Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1's deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.
    Keywords:  brain; methionine oxidation; neddylation; oxidative stress; posttranslational modification; ubiquitin
  21. J Biol Chem. 2020 May 28. pii: jbc.RA120.013478. [Epub ahead of print]
    Nachman E, Wentink AS, Madiona K, Bousset L, Katsinelos T, Allinson K, Kampinga H, McEwan WA, Jahn TR, Melki R, Mogk A, Bukau B, Nussbaum-Krammer C.
      The accumulation of amyloid Tau aggregates is implicated in Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are known to maintain protein homeostasis. Here we show that an ATP-dependent human chaperone system disassembles Tau fibrils in vitro We found that this function is mediated by the core chaperone HSC70, assisted by specific co-chaperones, in particular class B J‑domain proteins and a heat shock protein 110 (Hsp110)-type nucleotide exchange factor (NEF). The Hsp70 disaggregation machinery processed recombinant fibrils assembled from all six Tau isoforms as well as sarkosyl-resistant Tau aggregates extracted from cell cultures and human AD brain tissues, demonstrating the ability of the Hsp70 machinery to recognize a broad range of Tau aggregates. However, the chaperone activity released monomeric and small oligomeric Tau species, which induced the aggregation of self-propagating Tau conformers in a Tau cell culture model. We conclude that the activity of the Hsp70 disaggregation machinery is a double-sided sword, as it eliminates Tau amyloids at the cost of generating new seeds.
    Keywords:  70 kilodalton heat shock protein (Hsp70); Tau protein (Tau); amyloid; chaperone DnaJ (DnaJ); molecular chaperone; neurodegenerative disease; prion; protein aggregation; proteostasis; tauopathy
  22. Autophagy. 2020 May 26. 1-12
    Tian S, Jin S, Wu Y, Liu T, Luo M, Ou J, Xie W, Cui J.
      Macroautophagy/autophagy, a eukaryotic homeostatic process that sequesters cytoplasmic constituents for lysosomal degradation, is orchestrated by a number of autophagy-related (ATG) proteins tightly controlled by post-translational modifications. However, the involvement of reversible ubiquitination in the regulation of autophagy remains largely unclear. Here, we performed a single-guide RNA-based screening assay to investigate the functions of deubiquitinating enzymes (DUBs) in regulating autophagy. We identified previously unrecognized roles of several DUBs in modulating autophagy at multiple levels by targeting various ATG proteins. Mechanistically, we demonstrated that STAMBP/AMSH (STAM-binding protein) promotes the stabilization of ULK1 by removing its lysine 48 (K48)-linked ubiquitination, whereas OTUD7B mediates the degradation of PIK3 C3 by enhancing its K48-linked ubiquitination, thus positively or negatively affects autophagy flux, respectively. Together, our study elaborated on the broad involvement of DUBs in regulating autophagy and uncovered the critical roles of the reversible ubiquitination in the modification of ATG proteins.ABBREVIATIONS: ATG: autophagy-related; Baf A1: bafilomycin A1; DUB: deubiquitinating enzyme; EBSS: Earle's balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OTUD7B: OTU domain-containing protein 7B; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; sgRNA: single-guide RNA; SQSTM1/p62: sequestosome 1; STAMBP/AMSH: STAM-binding protein; ULK1: unc-51 like autophagy activating kinase 1; USP: ubiquitin specific peptidase.
    Keywords:  Autophagy; OTUD7B; PIK3C3; STAMBP; ULK1; deubiquitinating enzymes; ubiquitination
  23. PLoS Pathog. 2020 May 26. 16(5): e1008618
    Zhi H, Guo X, Ho YK, Pasupala N, Engstrom HAA, Semmes OJ, Giam CZ.
      The genomic instability associated with adult T cell leukemia/lymphoma (ATL) is causally linked to Tax, the HTLV-1 viral oncoprotein, but the underlying mechanism is not fully understood. We have previously shown that Tax hijacks and aberrantly activates ring finger protein 8 (RNF8) - a lysine 63 (K63)-specific ubiquitin E3 ligase critical for DNA double-strand break (DSB) repair signaling - to assemble K63-linked polyubiquitin chains (K63-pUbs) in the cytosol. Tax and the cytosolic K63-pUbs, in turn, initiate additional recruitment of linear ubiquitin assembly complex (LUBAC) to produce hybrid K63-M1 pUbs, which trigger a kinase cascade that leads to canonical IKK:NF-κB activation. Here we demonstrate that HTLV-1-infected cells are impaired in DNA damage response (DDR). This impairment correlates with the induction of microscopically visible nuclear speckles by Tax known as the Tax-speckle structures (TSS), which act as pseudo DNA damage signaling scaffolds that sequester DDR factors such as BRCA1, DNA-PK, and MDC1. We show that TSS co-localize with Tax, RNF8 and K63-pUbs, and their formation depends on RNF8. Tax mutants defective or attenuated in inducing K63-pUb assembly are deficient or tempered in TSS induction and DDR impairment. Finally, our results indicate that loss of RNF8 expression reduces HTLV-1 viral gene expression and frequently occurs in ATL cells. Thus, during HTLV-1 infection, Tax activates RNF8 to assemble nuclear K63-pUbs that sequester DDR factors in Tax speckles, disrupting DDR signaling and DSB repair. Down-regulation of RNF8 expression is positively selected during infection and progression to disease, and further exacerbates the genomic instability of ATL.
  24. Curr Opin Hematol. 2020 May 20.
    Chua BA, Signer RAJ.
      PURPOSE OF REVIEW: Protein homeostasis (proteostasis) is maintained by an integrated network of physiological mechanisms and stress response pathways that regulate the content and quality of the proteome. Maintenance of cellular proteostasis is key to ensuring normal development, resistance to environmental stress, coping with infection, and promoting healthy aging and lifespan. Recent studies have revealed that several proteostasis mechanisms can function in a cell-type-specific manner within hematopoietic stem cells (HSCs). Here, we review recent studies demonstrating that the proteostasis network functions uniquely in HSCs to promote their maintenance and regenerative function.RECENT FINDINGS: The proteostasis network is regulated differently in HSCs as compared with restricted hematopoietic progenitors. Disruptions in proteostasis are particularly detrimental to HSC maintenance and function. These findings suggest that multiple aspects of cellular physiology are uniquely regulated in HSCs to maintain proteostasis, and that precise control of proteostasis is particularly important to support life-long HSC maintenance and regenerative function.
    SUMMARY: The proteostasis network is uniquely configured within HSCs to promote their longevity and hematopoietic function. Future work uncovering cell-type-specific differences in proteostasis network configuration, integration, and function will be essential for understanding how HSCs function during homeostasis, in response to stress, and in disease.
  25. Cells. 2020 May 26. pii: E1332. [Epub ahead of print]9(6):
    Fu SJ, Hu MC, Peng YJ, Fang HY, Hsiao CT, Chen TY, Jeng CJ, Tang CY.
      Voltage-gated ClC-2 channels are essential for chloride homeostasis. Complete knockout of mouse ClC-2 leads to testicular degeneration and neuronal myelin vacuolation. Gain-of-function and loss-of-function mutations in the ClC-2-encoding human CLCN2 gene are linked to the genetic diseases aldosteronism and leukodystrophy, respectively. The protein homeostasis (proteostasis) mechanism of ClC-2 is currently unclear. Here, we aimed to identify the molecular mechanism of endoplasmic reticulum-associated degradation of ClC-2, and to explore the pathophysiological significance of disease-associated anomalous ClC-2 proteostasis. In both heterologous expression system and native neuronal and testicular cells, ClC-2 is subject to significant regulation by cullin-RING E3 ligase-mediated polyubiquitination and proteasomal degradation. The cullin 4 (CUL4)-damage-specific DNA binding protein 1 (DDB1)-cereblon (CRBN) E3 ubiquitin ligase co-exists in the same complex with and promotes the degradation of ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide and the cullin E3 ligase inhibitor MLN4924 promotes and attenuates, respectively, proteasomal degradation of ClC-2. Analyses of disease-related ClC-2 mutants reveal that aldosteronism and leukodystrophy are associated with opposite alterations in ClC-2 proteostasis. Modifying CUL4 E3 ligase activity with lenalidomide and MLN4924 ameliorates disease-associated ClC-2 proteostasis abnormality. Our results highlight the significant role and therapeutic potential of CUL4 E3 ubiquitin ligase in regulating ClC-2 proteostasis.
    Keywords:  MG132; MLN4924; channelopathy; cullin E3 ubiquitin ligase; lenalidomide; polyubiquitination; proteasomal degradation
  26. J Phys Chem B. 2020 May 26.
    Addabbo RM, Dalphin MD, Mecha MF, Liu Y, Staikos A, Guzman-Luna V, Cavagnero S.
      The relation between co- and post-translational protein folding and aggregation in the cell is poorly understood. Here, we employ a combination of fluorescence anisotropy decays in the frequency domain, fluorescence-detected solubility assays and NMR spectroscopy to explore the role of the ribosome in protein folding within a biologically relevant context. First, we find that a primary function of the ribosome is to promote cotranslational nascent-protein solubility, thus supporting cotranslational folding even in the absence of molecular chaperones. Under these conditions, however, only a fraction of the soluble expressed protein is folded and freely tumbling in solution. Hence the ribosome alone is insufficient to guarantee quantitative formation of the native state of the apomyoglobin (apoMb) model protein. Right after biosynthesis, nascent chains encoding apoMb emerge from the ribosomal exit tunnel and undergo a crucial irreversible post-translational kinetic partitioning between further folding and aggregation. Mutational analysis in combination with protein-expression kinetics and NMR show that nascent proteins can attain their native state only when the relative rates of soluble and insoluble product formation immediately upon release from the ribosome are tilted in favor of soluble species. Finally, the outcome of the above immediately post-translational kinetic partitioning is much more sensitive to amino-acid sequence perturbations than the native fold, which is rather mutation-insensitive. Hence, kinetic channeling of nascent-protein conformation upon release from the ribosome may be a major determinant of evolutionary pressure.
  27. Cancer Lett. 2020 May 21. pii: S0304-3835(20)30283-4. [Epub ahead of print]
    Barua D, Gupta A, Gupta S.
      Estrogen receptor 1 (ESR1, which encodes estrogen receptor-alpha) is a key driver gene for the initiation and progression of hormone receptor-positive breast cancer. Estrogen receptor-alpha (ER) is expressed in up to 70% of cases, and patients are routinely treated with endocrine therapies. However, the development of resistance over time is common and occurs in one-third of ER-positive breast tumors, leading to disease progression and death. X-box binding protein 1 (XBP1), a key component of the unfolded protein response (UPR) and ER signaling pathway, generates a positive feedback regulatory loop that leads to increased expression of XBP1 and ER in luminal breast cancer. In this review, we highlight new insights into the mechanisms of crosstalk between XBP1 and ER signaling and its clinical implications. Next, we describe the key signaling nodes that play an important role in XBP1-mediated endocrine resistance in breast cancer. Further, we discuss XBP1 gene mutations in breast cancer and the role of these mutations in the emergence of endocrine resistance and response to treatment. Finally, we discuss the current state and future directions for targeting XBP1 in combination with standard endocrine therapy to improve clinical outcomes in endocrine-resistant breast cancer patients.
    Keywords:  Breast cancer; ESR1 mutations; Endocrine resistance; Unfolded protein response; XBP1
  28. Development. 2020 May 28. pii: dev.189886. [Epub ahead of print]
    Zheng C, Atlas E, Lee HMT, Jao SLJ, Nguyen KCQ, Hall DH, Chalfie M.
      Molecular chaperones often work collaboratively with the ubiquitination-proteasome system (UPS) to facilitate the degradation of misfolded proteins, which typically safeguards cellular differentiation and protects cells from stress. In this study, however, we report that the Hsp70/Hsp90 chaperone machinery and an F-box protein, MEC-15, have opposing effects on neuronal differentiation and that the chaperones negatively regulate neuronal morphogenesis and functions. Using the touch receptor neurons (TRNs) of Caenorhabditis elegans, we find that mec-15(-) mutants display defects in microtubule formation, neurite growth, synaptic development, and neuronal functions, and these defects can be rescued by the loss of Hsp70/Hsp90 chaperones and cochaperones. MEC-15 likely functions in a SCF complex to degrade DLK-1, which is an Hsp90 client protein stabilized by the chaperones. The abundance of DLK-1, and likely other Hsp90 substrates, is fine-tuned by the antagonism between MEC-15 and chaperones; this antagonism regulates TRN development as well as synaptic functions of GABAergic motor neurons. Therefore, a balance between UPS and chaperones tightly controls neuronal differentiation.
    Keywords:  Microtubules; Molecular chaperones; Neurite growth; Protein homeostasis; Touch receptor neurons; Ubiquitination-proteasome system
  29. Cell Rep. 2020 May 26. pii: S2211-1247(20)30648-3. [Epub ahead of print]31(8): 107695
    Jacomin AC, Petridi S, Di Monaco M, Bhujabal Z, Jain A, Mulakkal NC, Palara A, Powell EL, Chung B, Zampronio C, Jones A, Cameron A, Johansen T, Nezis IP.
      Autophagy is the degradation of cytoplasmic material through the lysosomal pathway. One of the most studied autophagy-related proteins is LC3. Despite growing evidence that LC3 is enriched in the nucleus, its nuclear role is poorly understood. Here, we show that Drosophila Atg8a protein, homologous to mammalian LC3, interacts with the transcription factor Sequoia in a LIR motif-dependent manner. We show that Sequoia depletion induces autophagy in nutrient-rich conditions through the enhanced expression of autophagy genes. We show that Atg8a interacts with YL-1, a component of a nuclear acetyltransferase complex, and that it is acetylated in nutrient-rich conditions. We also show that Atg8a interacts with the deacetylase Sir2, which deacetylates Atg8a during starvation to activate autophagy. Our results suggest a mechanism of regulation of the expression of autophagy genes by Atg8a, which is linked to its acetylation status and its interaction with Sequoia, YL-1, and Sir2.
    Keywords:  LC3/Atg8; LIR motif; acetylation; autophagy; nucleus; transcription
  30. Cell Mol Life Sci. 2020 May 23.
    Lingelem ABD, Kavaliauskiene S, Halsne R, Klokk TI, Surma MA, Klose C, Skotland T, Sandvig K.
      The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.
    Keywords:  CAY10594; Intracellular transport; PLD3; R59022; SNX2; Vps35
  31. Plant Physiol. 2020 May 26. pii: pp.00225.2020. [Epub ahead of print]
    Neubauer M, Innes RW.
      Stress signaling in plants is carefully regulated to ensure proper development and reproductive fitness. Overactive defense signaling can result in dwarfism as well as developmental defects. In addition to requiring a substantial amount of energy, plant stress responses place a burden upon the cellular machinery, which can result in the accumulation of misfolded proteins and endoplasmic reticulum (ER) stress. Negative regulators of stress signaling, such as ENHANCED DISEASE RESISTANCE 1 (EDR1), ensure that stress responses are properly suspended when they are not needed, thereby conserving energy for growth and development. Here, we describe the role of an uncharacterized N-terminal acetyltransferase, NAA50, in the regulation of plant development and stress responses in Arabidopsis thaliana. Our results demonstrate that NAA50, an interactor of EDR1, plays an important role in regulating the tradeoff between plant growth and defense. Plants lacking NAA50 display severe developmental defects as well as induced stress responses. Reduction of NAA50 expression results in arrested stem and root growth as well as senescence. Furthermore, our results demonstrate that the loss of NAA50 results in constitutive ER stress signaling, indicating that NAA50 may be required for the suppression of ER stress. This work establishes NAA50 as essential for plant development and the suppression of stress responses, potentially through the regulation of ER stress.
  32. Cells. 2020 May 22. pii: E1296. [Epub ahead of print]9(5):
    Dobner J, Simons IM, Rufinatscha K, Hänsch S, Schwarten M, Weiergräber OH, Abdollahzadeh I, Gensch T, Bode JG, Hoffmann S, Willbold D.
      The γ-aminobutyric acid type A receptor-associated protein (GABARAP) and its close paralogs GABARAPL1 and GABARAPL2 constitute a subfamily of the autophagy-related 8 (Atg8) protein family. Being associated with a variety of dynamic membranous structures of autophagic and non-autophagic origin, Atg8 proteins functionalize membranes by either serving as docking sites for other proteins or by acting as membrane tethers or adhesion factors. In this study, we describe that deficiency for GABARAP alone, but not for its close paralogs, is sufficient for accelerated EGF receptor (EGFR) degradation in response to EGF, which is accompanied by the downregulation of EGFR-mediated MAPK signaling, altered target gene expression, EGF uptake, and EGF vesicle composition over time. We further show that GABARAP and EGFR converge in the same distinct compartments at endogenous GABARAP expression levels in response to EGF stimulation. Furthermore, GABARAP associates with EGFR in living cells and binds to synthetic peptides that are derived from the EGFR cytoplasmic tail in vitro. Thus, our data strongly indicate a unique and novel role for GABARAP during EGFR trafficking.
    Keywords:  Atg8; EGFR; GABARAP; degradation; genome editing; receptor trafficking
  33. J Mol Biol. 2020 May 25. pii: S0022-2836(20)30355-7. [Epub ahead of print]
    Yücel SS, Lemberg MK.
      Intramembrane proteases catalyze the unusual cleavage of peptide bonds in the plane of biological membranes. They are categorized according to their active site. The GxGD aspartyl proteases comprise presenilin and the signal peptide peptidase (SPP), and SPP-like (SPPL) proteases. Here we focus on the functionally related SPP and SPPL proteases and review the current understanding of their substrate specificity and summarize known physiological functions in mammalian cells. We discuss how on one side regulated intramembrane proteolysis (RIP) generates signaling molecules and on the other side how processes, such as regulatory Endoplasmic Reticulum-associated degradation (ERAD-R) controls the quantity and activity of central regulators. While the enzymatic core of GxGD intramembrane proteases is conserved, association with regulatory factors and substrate adaptors may have tailored enzymes for various specific functions.
    Keywords:  Intramembrane proteolysis; Limited proteolysis; Presenilin fold; Protein degradation; signaling
  34. Clin Chim Acta. 2020 May 24. pii: S0009-8981(20)30246-1. [Epub ahead of print]
    Ouyang S, Mo Z, Sun S, Yin K, Lv Y.
      Growing evidence has demonstrated that Insig-1 is intricately involved in lipid metabolism regulation and the progression of lipid disorders. Our review summarizes updated information on the role and underlying mechanisms of Insig-1 in lipid metabolism dyshomeostasis and lipid disorders. As a member of the insulin-induced gene family, insulin-induced gene 1 (Insig-1) is a six-span transmembrane protein embedded in the endoplasmic reticulum (ER) membrane. Insig-1 is widely involved in the maintenance of intracellular lipid metabolism homeostasis by controlling the activation of sterol regulatory element-binding proteins (SREBPs) and the degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Growing experimental and clinical data have identified that Insig-1 reduces lipid accumulation in hepatocytes to relieve the development of nonalcoholic fatty liver disease (NAFLD), downregulates the plasma level of free cholesterol and protects β cells against lipotoxicity to alleviate diabetic dyslipidemia. In addition, Insig-1 suppresses adipogenesis and inhibits the differentiation of preadipocytes to prevent the occurrence of obesity. Insig-1 is a key regulatory factor that maintains intracellular lipid metabolism homeostasis and is a promising therapeutic target for lipid disorders.
    Keywords:  Diabetic dyslipidemia; Insig-1; Lipid metabolism; NAFLD; Obesity; SREBPs
  35. Life Sci. 2020 May 23. pii: S0024-3205(20)30592-0. [Epub ahead of print]255 117842
    Banerjee A, Czinn SJ, Reiter RJ, Blanchard TG.
      The outbreak of COVID-19 caused by 2019-nCov/SARS-CoV-2 has become a pandemic with an urgent need for understanding the mechanisms and identifying a treatment. Viral infections including SARS-CoV are associated with increased levels of reactive oxygen species, disturbances of Ca++ caused by unfolded protein response (UPR) mediated by endoplasmic reticulum (ER) stress and is due to the exploitation of virus's own protein i.e., viroporins into the host cells. Several clinical trials are on-going including testing Remdesivir (anti-viral), Chloroquine and Hydroxychloroquine derivatives (anti-malarial drugs) etc. Unfortunately, each drug has specific limitations. Herein, we review the viral protein involvement to activate ER stress transducers (IRE-1, PERK, ATF-6) and their downstream signals; and evaluate combination therapies for COVID-19 mediated ER stress alterations. Melatonin is an immunoregulator, anti-pyretic, antioxidant, anti-inflammatory and ER stress modulator during viral infections. It enhances protective mechanisms for respiratory tract disorders. Andrographolide, isolated from Andrographis paniculata, has versatile biological activities including immunomodulation and determining SARS-CoV-2 binding site. Considering the properties of both compounds in terms of anti-inflammatory, antioxidant, anti-pyrogenic, anti-viral and ER stress modulation and computational approaches revealing andrographolide docks with the SARS-CoV2 binding site, we predict that this combination therapy may have potential utility against COVID-19.
    Keywords:  2019-nCov/SARS-CoV-2; Andrographolide; COVID-19; Endoplasmic reticulum stress; Melatonin; Unfolded protein response
  36. Neurobiol Dis. 2020 May 23. pii: S0969-9961(20)30230-8. [Epub ahead of print] 104955
    Callender JA, Sevillano AM, Soldau K, Kurt TD, Schumann T, Pizzo DP, Altmeppen H, Glatzel M, Esko JD, Sigurdson CJ.
      Many aggregation-prone proteins linked to neurodegenerative disease are post-translationally modified during their biogenesis. In vivo pathogenesis studies have suggested that the presence of post-translational modifications can shift the aggregate assembly pathway and profoundly alter the disease phenotype. In prion disease, the N-linked glycans and GPI-anchor on the prion protein (PrP) impair fibril assembly. However, the relevance of the two glycans to aggregate structure and disease progression remains unclear. Here we show that prion-infected knockin mice expressing an additional PrP glycan (tri-glycosylated PrP) develop new plaque-like deposits on neuronal cell membranes, along the subarachnoid space, and periventricularly, suggestive of high prion mobility and transit through the interstitial fluid. The plaque-like deposits were largely non-congophilic and composed of full length, uncleaved PrP, indicating retention of the glycophosphatidylinositol (GPI) anchor. Prion aggregates sedimented in low density fractions following ultracentrifugation, consistent with oligomers, and bound low levels of heparan sulfate similar to other predominantly GPI-anchored prions. These results suggest that highly glycosylated PrP primarily converts as a GPI-anchored glycoform with low involvement of HS co-factors, limiting PrP assembly mainly to oligomers. Thus, these findings may explain the high frequency of diffuse, synaptic, and plaque-like deposits and rapid conversion commonly observed in human and animal prion disease.
    Keywords:  ADAM10 cleavage; Amyloid; Glycans; Glycosaminoglycans; Glycosylation; Neurodegeneration; Prion strains; Protein misfolding
  37. iScience. 2020 May 22. pii: S2589-0042(20)30282-0. [Epub ahead of print]23(5): 101097
    Parakh S, Shadfar S, Perri ER, Ragagnin AMG, Piattoni CV, Fogolín MB, Yuan KC, Shahheydari H, Don EK, Thomas CJ, Hong Y, Comini MA, Laird AS, Spencer DM, Atkin JD.
      Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in almost all cases of amyotrophic lateral sclerosis (ALS), and 20% of familial ALS cases are due to mutations in superoxide dismutase 1 (SOD1). Redox regulation is critical to maintain cellular homeostasis, although how this relates to ALS is unclear. Here, we demonstrate that the redox function of protein disulfide isomerase (PDI) is protective against protein misfolding, cytoplasmic mislocalization of TDP-43, ER stress, ER-Golgi transport dysfunction, and apoptosis in neuronal cells expressing mutant TDP-43 or SOD1, and motor impairment in zebrafish expressing mutant SOD1. Moreover, previously described PDI mutants present in patients with ALS (D292N, R300H) lack redox activity and were not protective against ALS phenotypes. Hence, these findings implicate the redox activity of PDI centrally in ALS, linking it to multiple cellular processes. They also imply that therapeutics based on PDI's redox activity will be beneficial in ALS.
    Keywords:  Molecular Biology; Neurogenetics; Neuroscience
  38. Commun Biol. 2020 May 25. 3(1): 262
    Fatima A, Irmak D, Noormohammadi A, Rinschen MM, Das A, Leidecker O, Schindler C, Sánchez-Gaya V, Wagle P, Pokrzywa W, Hoppe T, Rada-Iglesias A, Vilchez D.
      Histones modulate gene expression by chromatin compaction, regulating numerous processes such as differentiation. However, the mechanisms underlying histone degradation remain elusive. Human embryonic stem cells (hESCs) have a unique chromatin architecture characterized by low levels of trimethylated histone H3 at lysine 9 (H3K9me3), a heterochromatin-associated modification. Here we assess the link between the intrinsic epigenetic landscape and ubiquitin-proteasome system of hESCs. We find that hESCs exhibit high expression of the ubiquitin-conjugating enzyme UBE2K. Loss of UBE2K upregulates the trimethyltransferase SETDB1, resulting in H3K9 trimethylation and repression of neurogenic genes during differentiation. Besides H3K9 trimethylation, UBE2K binds histone H3 to induce its polyubiquitination and degradation by the proteasome. Notably, ubc-20, the worm orthologue of UBE2K, also regulates histone H3 levels and H3K9 trimethylation in Caenorhabditis elegans germ cells. Thus, our results indicate that UBE2K crosses evolutionary boundaries to promote histone H3 degradation and reduce H3K9me3 repressive marks in immortal cells.
  39. Mol Cell Proteomics. 2020 May 28. pii: mcp.RA120.002078. [Epub ahead of print]
    Adams MK, Banks CAS, Thornton JL, Kempf C, Zhang Y, Miah S, Hao Y, Sardiu ME, Killer M, Hattem G, Murray A, Katt M, Florens LA, Washburn MP.
      Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical crosslinking mass spectrometry identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.
    Keywords:  Chromatin function or biology; Cross linking; DSSO; Epigenetics; Histone deacetylase; Nuclear Translocation; Pathway Analysis; Protein complex analysis; Protein-Protein Interactions*; SIN3; Subcellular analysis; Systems biology*
  40. Cell Death Differ. 2020 May 26.
    Seo J, Seong D, Nam YW, Hwang CH, Lee SR, Lee CS, Jin Y, Lee HW, Oh DB, Vandenabeele P, Song J.
      Necroptosis is a form of regulated cell death caused by formation of the necrosome complex. However, the factors modulating this process and the systemic pathophysiological effects of necroptosis are yet to be understood. Here, we identified that Beclin 1 functions as an anti-necroptosis factor by being recruited into the necrosome complex upon treatment with TNFα, Smac mimetic, and pan-caspase inhibitor and by repressing MLKL oligomerisation, thus preventing the disruption of the plasma membrane. Cells ablated or knocked-out for Beclin 1 become sensitised to necroptosis in an autophagy-independent manner without affecting the necrosome formation itself. Interestingly, the recruitment of Beclin 1 into the necrosome complex is dependent on the activation and phosphorylation of MLKL. Biochemically, the coiled-coil domain (CCD) of Beclin 1 binds to the CCD of MLKL, which restrains the oligomerisation of phosphorylated MLKL. Finally, Beclin 1 depletion was found to promote necroptosis in leukaemia cells and enhance regression of xenografted-tumour upon treatment with Smac mimetics and caspase inhibitors. These results suggest that Beclin 1 functions as a negative regulator in the execution of necroptosis by suppressing MLKL oligomerisation.
  41. Trends Biochem Sci. 2020 May 26. pii: S0968-0004(20)30119-5. [Epub ahead of print]
    Hayer-Hartl M, Hartl FU.
      A major challenge faced by human civilization is to ensure that agricultural productivity keeps pace with population growth and a changing climate. All food supply is generated, directly or indirectly, through the process of photosynthesis, with the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzing the assimilation of atmospheric CO2. Despite its pivotal role, Rubisco is a remarkably inefficient enzyme and must be made by plants in large quantities. However, efforts to enhance Rubisco performance by bioengineering have been hampered by its extensive reliance on molecular chaperones and auxiliary factors for biogenesis, metabolic repair, and packaging into membraneless microcompartments. Here, we review recent advances in understanding these complex machineries and discuss their implications for improving Rubisco carboxylase activity with the goal to increase crop yields.
    Keywords:  AAA+ protein; CO(2) concentration mechanism; Rubisco activase; carboxysomes; chaperonins; photosynthesis
  42. EMBO Rep. 2020 May 24. e50094
    Bar-Ziv R, Bolas T, Dillin A.
      Multicellular organisms are complex biological systems, composed of specialized tissues that require coordination of the metabolic and fitness state of each component. In the cells composing the tissues, one central organelle is the mitochondrion, a compartment essential for many energetic and fundamental biological processes. Beyond serving these functions, mitochondria have emerged as signaling hubs in biological systems, capable of inducing changes to the cell they are in, to cells in distal tissues through secreted factors, and to overall animal physiology. Here, we describe our current understanding of these communication mechanisms in the context of mitochondrial stress. We focus on cellular mechanisms that deal with perturbations to the mitochondrial proteome and outline recent advances in understanding how local perturbations can affect distal tissues and animal physiology in model organisms. Finally, we discuss recent findings of these responses associated with metabolic and age-associated diseases in mammalian systems, and how they may be employed as diagnostic and therapeutic tools.
    Keywords:  aging; mitochondria; stress
  43. Sci Rep. 2020 May 29. 10(1): 8755
    Adriaenssens E, Tedesco B, Mediani L, Asselbergh B, Crippa V, Antoniani F, Carra S, Poletti A, Timmerman V.
      Three missense mutations targeting the same proline 209 (Pro209) codon in the co-chaperone Bcl2-associated athanogene 3 (BAG3) have been reported to cause distal myopathy, dilated cardiomyopathy or Charcot-Marie-Tooth type 2 neuropathy. Yet, it is unclear whether distinct molecular mechanisms underlie the variable clinical spectrum of the rare patients carrying these three heterozygous Pro209 mutations in BAG3. Here, we studied all three variants and compared them to the BAG3_Glu455Lys mutant, which causes dilated cardiomyopathy. We found that all BAG3_Pro209 mutants have acquired a toxic gain-of-function, which causes these variants to accumulate in the form of insoluble HDAC6- and vimentin-positive aggresomes. The aggresomes formed by mutant BAG3 led to a relocation of other chaperones such as HSPB8 and Hsp70, which, together with BAG3, promote the so-called chaperone-assisted selective autophagy (CASA). As a consequence of their increased aggregation-proneness, mutant BAG3 trapped ubiquitinylated client proteins at the aggresome, preventing their efficient clearance. Combined, these data show that all BAG3_Pro209 mutants, irrespective of their different clinical phenotypes, are characterized by a gain-of-function that contributes to the gradual loss of protein homeostasis.
  44. Elife. 2020 May 26. pii: e54612. [Epub ahead of print]9
    Cook GM, Sousa C, Schaeffer J, Wiles K, Jareonsettasin P, Kalyanasundaram A, Walder E, Casper C, Patel S, Chua PW, Riboni-Verri G, Raza M, Swaddiwudhipong N, Hui A, Abdullah A, Wajed S, Keynes RJ.
      Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in chick half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.
    Keywords:  chicken; developmental biology; human; neuroscience; rat
  45. J Cell Physiol. 2020 May 26.
    Nouri-Vaskeh M, Alizadeh L, Hajiasgharzadeh K, Mokhtarzadeh A, Halimi M, Baradaran B.
      Misfolded proteins have enhanced formation of toxic oligomers and nonfunctional protein copies lead to recruiting wild-type protein types. Heat shock protein 90 (HSP90) is a molecular chaperone generated by cells that are involved in many cellular functions through regulation of folding and/or localization of large multi-protein complexes as well as client proteins. HSP90 can regulate a number of different cellular processes including cell proliferation, motility, angiogenesis, signal transduction, and adaptation to stress. HSP90 makes the mutated oncoproteins able to avoid misfolding and degradation and permits the malignant transformation. As a result, HSP90 is an important factor in several signaling pathways associated with tumorigenicity, therapy resistance, and inhibiting apoptosis. Clinically, the upregulation of HSP90 expression in hepatocellular carcinoma (HCC) is linked with advanced stages and inappropriate survival in cases suffering from this kind of cancer. The present review comprehensively assesses HSP90 functions and its possible usefulness as a potential diagnostic biomarker and therapeutic option for HCC.
    Keywords:  chaperone proteins; heat shock protein 90; hepatocellular carcinoma; liver cancer
  46. Trends Biochem Sci. 2020 May 21. pii: S0968-0004(20)30118-3. [Epub ahead of print]
    Dai X, Zhu M.
      Ribosome and protein synthesis lie at the core of cell growth and are major consumers of the cellular budget. Here we review recent progress in the coupling of ribosome synthesis and translational capacity with cell growth in bacteria. We elaborate on the different strategies of bacteria to modulate the protein synthesis rate at fast and slow growth rates. In particular, bacterial cells maintain translational potential at very slow growth as a strategy to keep fitness in fluctuating environments. We further discuss the important role of ribosome synthesis in rapidly proliferating eukaryotic cells such as yeast cells and cancer cells. The tight relation between ribosome and cell growth provides a broad research avenue for researchers from various disciplines.
    Keywords:  bacterial growth; cancer cell growth; nutrient availability; protein synthesis; yeast growth
  47. Cell Immunol. 2020 May 18. pii: S0008-8749(20)30190-8. [Epub ahead of print]353 104133
    Douanne T, Chapelier S, Rottapel R, Gavard J, Bidère N.
      The natural bioactive glycerophospholipid lysophosphatidic acid (LPA) binds to its cognate G protein-coupled receptors (GPCRs) on the cell surface to promote the activation of several transcription factors, including NF-κB. LPA-mediated activation of NF-κB relies on the formation of a signalosome that contains the scaffold CARMA3, the adaptor BCL10 and the paracaspase MALT1 (CBM complex). The CBM complex has been extensively studied in lymphocytes, where it links antigen receptors to NF-κB activation via the recruitment of the linear ubiquitin assembly complex (LUBAC), a tripartite complex of HOIP, HOIL1 and SHARPIN. Moreover, MALT1 cleaves the LUBAC subunit HOIL1 to further enhance NF-κB activation. However, the contribution of the LUBAC downstream of GPCRs has not been investigated. By using murine embryonic fibroblasts from mice deficient for HOIP, HOIL1 and SHARPIN, we report that the LUBAC is crucial for the activation of NF-κB in response to LPA. Further echoing the situation in lymphocytes, LPA unbridles the protease activity of MALT1, which cleaves HOIL1 at the Arginine 165. The expression of a MALT1-insensitive version of HOIL1 reveals that this processing is involved in the optimal production of the NF-κB target cytokine interleukin-6. Lastly, we provide evidence that the guanine exchange factor GEF-H1 favors MALT1-mediated cleavage of HOIL1 and NF-κB signaling in this context. Together, our results unveil a critical role for the LUBAC as a positive regulator of NF-κB signaling downstream of LPA receptors.
    Keywords:  CBM complex; GEF-H1; LPA; LUBAC; MALT1; NF-κB
  48. Proc Natl Acad Sci U S A. 2020 May 28. pii: 201918844. [Epub ahead of print]
    Meng B, Lan K, Xie J, Lerner RA, Wilson IA, Yang B.
      The existence of multiple serotypes renders vaccine development challenging for most viruses in the Enterovirus genus. An alternative and potentially more viable strategy for control of these viruses is to develop broad-spectrum antivirals by targeting highly conserved proteins that are indispensable for the virus life cycle, such as the 3C protease. Previously, two single-chain antibody fragments, YDF and GGVV, were reported to effectively inhibit human rhinovirus 14 proliferation. Here, we found that both single-chain antibody fragments target sites on the 3C protease that are distinct from its known drug site (peptidase active site) and possess different mechanisms of inhibition. YDF does not block the active site but instead noncompetitively inhibits 3C peptidase activity through an allosteric effect that is rarely seen for antibody protease inhibitors. Meanwhile, GGVV antagonizes the less-explored regulatory function of 3C in genome replication. The interaction between 3C and the viral genome 5' noncoding region has been reported to be important for enterovirus genome replication. Here, the interface between human rhinovirus 14 3C and its 5' noncoding region was probed by hydrogen-deuterium exchange coupled mass spectrometry and found to partially overlap with the interface between GGVV and 3C. Consistently, prebinding of GGVV completely abolishes interaction between human rhinovirus 14 3C and its 5' noncoding region. The epitopes of YDF and GGVV, therefore, represent two additional sites of therapeutic vulnerability in rhinovirus. Importantly, the GGVV epitope appears to be conserved across many enteroviruses, suggesting that it is a promising target for pan-enterovirus inhibitor screening and design.
    Keywords:  HDX-MS; X-ray crystallography; allosteric inhibitor; genome replication regulation; pan-enterovirus antiviral target
  49. Int J Mol Sci. 2020 May 27. pii: E3781. [Epub ahead of print]21(11):
    Shiiba I, Takeda K, Nagashima S, Yanagi S.
      The molecular pathology of diseases seen from the mitochondrial axis has become more complex with the progression of research. A variety of factors, including the failure of mitochondrial dynamics and quality control, have made it extremely difficult to narrow down drug discovery targets. We have identified MITOL (mitochondrial ubiquitin ligase: also known as MARCH5) localized on the mitochondrial outer membrane and previously reported that it is an important regulator of mitochondrial dynamics and mitochondrial quality control. In this review, we describe the pathological aspects of MITOL revealed through functional analysis and its potential as a drug discovery target.
    Keywords:  E3 ubiquitin ligase; MITOL/MARCH5; mitochondria