bims-proteo 2020-08-30 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–08–30
thirty-six papers selected by
Eric Chevet, INSERM

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.
Beyond the cell factory: Homeostatic regulation of and by the UPRER.
N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle.
BAG5 Promotes Alpha-Synuclein Oligomer Formation and Functionally Interacts With the Autophagy Adaptor Protein p62.
Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.
Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.
Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response.
A Quantitative Analysis of Cellular Lipid Compositions During Acute Proteotoxic ER Stress Reveals Specificity in the Production of Asymmetric Lipids.
Synaptic Protein Degradation Controls Sexually Dimorphic Circuits through Regulation of DCC/UNC-40.
Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.
GRP78 targeting: Hitting two birds with a stone.
GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate.
Glycan dependent refolding activity of ER glucosyltransferase (UGGT).
Mechanisms of sensing and response to proteotoxic stress.
A Cell Cycle Checkpoint for the Endoplasmic Reticulum.
Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone.
Proteotoxic Stress and Cell Death in Cancer Cells.
Characterization of aminobenzylphenols as protein disulfide isomerase inhibitors in glioblastoma cell lines.
Hsp90 Co-chaperones Form Plastic Genetic Networks Adapted to Client Maturation.
Role of the Sec22b/E-Syt complex in neurite growth and ramification.
PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB.
The role and therapeutic potential of Hsp90, Hsp70, and smaller heat shock proteins in peripheral and central neuropathies.
ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis.
Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity.
Cyclosporin A but not FK506 activates the integrated stress response in human cells.
Mitophagy is induced by short ubiquitin chains on mitochondria.
Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation.
DDHD1, but Not DDHD2, Suppresses Neurite Outgrowth in SH-SY5Y and PC12 Cells by Regulating Protein Transport From Recycling Endosomes.
Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter.
Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.
The Autophagy-RNA Interplay: Degradation and Beyond.
Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress.
The ubiquitin ligase UBE4B regulates amyloid precursor protein ubiquitination, endosomal trafficking, and amyloid β42 generation and secretion.
ATG16L1 autophagy pathway regulates BAX protein levels and programmed cell death.
Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.
The Transcription Factor bZIP60 Links the Unfolded Protein Response (UPR) to the Heat Stress Response (HSR) in Maize.

Elife. 2020 Aug 27. pii: e58396. [Epub ahead of print]9

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.

Madlen Stephani, Lorenzo Picchianti, Alexander Gajic, Rebecca Beveridge, Emilio Skarwan, Victor Sanchez de Medina Hernandez, Azadeh Mohseni, Marion Clavel, Yonglun Zeng, Christin Naumann, Mateusz Matuszkiewicz, Eleonora Turco, Christian Loefke, Baiying Li, Gerhard Dürnberger, Michael Schutzbier, Hsiao Tieh Chen, Alibek Abdrakhmanov, Adriana Savova, Khong-Sam Chia, Armin Djamei, Irene Schaffner, Steffen Abel, Liwen Jiang, Karl Mechtler, Fumiyo Ikeda, Sascha Martens, Tim Clausen, Yasin Dagdas.

  Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

Keywords:  A. thaliana; cell biology; human; plant biology; viruses

DOI:  https://doi.org/10.7554/eLife.58396
Sci Adv. 2020 Jul;6(29): eabb9614

Beyond the cell factory: Homeostatic regulation of and by the UPRER.

Melissa G Metcalf, Ryo Higuchi-Sanabria, Gilberto Garcia, C Kimberly Tsui, Andrew Dillin.

The endoplasmic reticulum (ER) is commonly referred to as the factory of the cell, as it is responsible for a large amount of protein and lipid synthesis. As a membrane-bound organelle, the ER has a distinct environment that is ideal for its functions in synthesizing these primary cellular components. Many different quality control machineries exist to maintain ER stability under the stresses associated with synthesizing, folding, and modifying complex proteins and lipids. The best understood of these mechanisms is the unfolded protein response of the ER (UPRER), in which transmembrane proteins serve as sensors, which trigger a coordinated transcriptional response of genes dedicated for mitigating the stress. As the name suggests, the UPRER is most well described as a functional response to protein misfolding stress. Here, we focus on recent findings and emerging themes in additional roles of the UPRER outside of protein homeostasis, including lipid homeostasis, autophagy, apoptosis, and immunity.

DOI: https://doi.org/10.1126/sciadv.abb9614
Viruses. 2020 Aug 23. pii: E925. [Epub ahead of print]12(9):

N-glycosylation in the Pre-Membrane Protein Is Essential for the Zika Virus Life Cycle.

Yong-Dae Gwon, Eva Zusinaite, Andres Merits, Anna K Överby, Magnus Evander.

  Asparagine (N)-linked protein glycosylation plays an important role in protein synthesis and modification. Two Zika virus (ZIKV) structural proteins, the pre-membrane (prM) and envelope (E) protein are N-glycosylated. The prM protein of all ZIKV strains contains a single N-linked glycosylation site, while not all strains contain an N-linked site in the E protein. Our aim was to examine the impact of prM and E N-linked glycosylation on ZIKV infectivity and cell trafficking. Using a ZIKV infectious clone, we found that when the N-glycan sites were removed, the prM- and the prM/E-double mutants did not produce an infectious virus in the supernatant. Further, by using ZIKV prME constructs, we found that N-glycosylation was necessary for effective secretion of ZIKV virions. The absence of the N-glycan on prM or E caused protein aggregation in the rough endoplasmatic reticulum (ER) compartment. The aggregation was more pronounced for the prM-mutation, and the mutant virus lost the ER-Golgi intermediate compartment (ERGIC) localization. In addition, lack of the N-glycan on prM induced nuclear translocation of CCAAT-enhancer-binding protein homologous protein (CHOP), an ER stress marker. To conclude, we show that the prM N-glycan is essential for the ZIKV infectious cycle, and plays an important role in viral protein trafficking, protein folding, and virion assembly.

Keywords:  N-glycosylation; Zika virus; envelope; pre-membrane; virus life cycle

DOI:  https://doi.org/10.3390/v12090925
Front Cell Dev Biol. 2020 ;8 716

BAG5 Promotes Alpha-Synuclein Oligomer Formation and Functionally Interacts With the Autophagy Adaptor Protein p62.

Erik L Friesen, Yu Tong Zhang, Rebecca Earnshaw, Mitch L De Snoo, Darren M O'Hara, Victoria Agapova, Hien Chau, Sophie Ngana, Kevin S Chen, Lorraine V Kalia, Suneil K Kalia.

  Molecular chaperones are critical to maintaining intracellular proteostasis and have been shown to have a protective role against alpha-synuclein-mediated toxicity. Co-chaperone proteins regulate the activity of molecular chaperones and connect the chaperone network to protein degradation and cell death pathways. Bcl-2 associated athanogene 5 (BAG5) is a co-chaperone that modulates proteostasis by inhibiting the activity of Heat shock protein 70 (Hsp70) and several E3 ubiquitin ligases, resulting in enhanced neurodegeneration in models of Parkinson's disease (PD). Here we identify a novel interaction between BAG5 and p62/sequestosome-1 (SQSTM1), suggesting that BAG5 may bridge the chaperone network to autophagy-mediated protein degradation. We found that BAG5 enhanced the formation of pathogenic alpha-synuclein oligomers and regulated the levels and subcellular distribution of p62. These results extend the role of BAG5 in alpha-synuclein processing and intracellular proteostasis.

Keywords:  BAG5; alpha-synuclein; bcl-2 associated athanogene; chaperones; p62; proteostasis; sequestosome-1

DOI:  https://doi.org/10.3389/fcell.2020.00716
Proc Natl Acad Sci U S A. 2020 Aug 27. pii: 202005301. [Epub ahead of print]

Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.

Ye Zhou, Panagiotis L Kastritis, Shannon E Dougherty, Jonathan Bouvette, Allen L Hsu, Laura Burbaum, Shyamal Mosalaganti, Stefan Pfeffer, Wim J H Hagen, Friedrich Förster, Mario J Borgnia, Christine Vogel, Martin Beck, Alberto Bartesaghi, Gustavo M Silva.

  Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Keywords:  K63 ubiquitin; cryo-EM; oxidative stress; ribosome; translation

DOI:  https://doi.org/10.1073/pnas.2005301117
BMC Biol. 2020 Aug 28. 18(1): 107

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy.

Dan Li, Shu-Gao Yang, Cheng-Wen He, Zheng-Tan Zhang, Yongheng Liang, Hui Li, Jing Zhu, Xiong Su, Qingqiu Gong, Zhiping Xie.

   BACKGROUND: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive.
RESULTS: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ER-accumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response.
CONCLUSIONS: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.

Keywords:  Autophagy; Glycerolipid; Intracellular trafficking; Organelle; Phospholipid

DOI:  https://doi.org/10.1186/s12915-020-00837-w
Int J Mol Sci. 2020 Aug 24. pii: E6088. [Epub ahead of print]21(17):

Sephin1 Protects Neurons against Excitotoxicity Independently of the Integrated Stress Response.

Asier Ruiz, Jone Zuazo, Carolina Ortiz-Sanz, Celia Luchena, Carlos Matute, Elena Alberdi.

  Sephin1 is a derivative of guanabenz that inhibits the dephosphorylation of the eukaryotic initiation factor 2 alpha (eIF2α) and therefore may enhance the integrated stress response (ISR), an adaptive mechanism against different cellular stresses, such as accumulation of misfolded proteins. Unlike guanabenz, Sephin1 provides neuroprotection without adverse effects on the α2-adrenergic system and therefore it is considered a promising pharmacological therapeutic tool. Here, we have studied the effects of Sephin1 on N-methyl-D-aspartic acid (NMDA) receptor signaling which may modulate the ISR and contribute to excitotoxic neuronal loss in several neurodegenerative conditions. Time-course analysis of peIF2α levels after NMDA receptor overactivation showed a delayed dephosphorylation that occurred in the absence of activating transcription factor 4 (ATF4) and therefore independently of the ISR, in contrast to that observed during endoplasmic reticulum (ER) stress induced by tunicamycin and thapsigargin. Similar to guanabenz, Sephin1 completely blocked NMDA-induced neuronal death and was ineffective against AMPA-induced excitotoxicity, whereas it did not protect from experimental ER stress. Interestingly, both guanabenz and Sephin1 partially but significantly reduced NMDA-induced cytosolic Ca2+ increase, leading to a complete inhibition of subsequent calpain activation. We conclude that Sephin1 and guanabenz share common strong anti-excitotoxic properties with therapeutic potential unrelated to the ISR.

Keywords:  NMDA; Sephin1; calcium; calpain; excitotoxicity; guanabenz; integrated stress response

DOI:  https://doi.org/10.3390/ijms21176088
Front Cell Dev Biol. 2020 ;8 756

A Quantitative Analysis of Cellular Lipid Compositions During Acute Proteotoxic ER Stress Reveals Specificity in the Production of Asymmetric Lipids.

John Reinhard, Carsten Mattes, Kristina Väth, Toni Radanović, Michal A Surma, Christian Klose, Robert Ernst.

  The unfolded protein response (UPR) is central to endoplasmic reticulum (ER) homeostasis by controlling its size and protein folding capacity. When activated by unfolded proteins in the ER-lumen or aberrant lipid compositions, the UPR adjusts the expression of hundreds of target genes to counteract ER stress. The proteotoxic drugs dithiothreitol (DTT) and tunicamycin (TM) are commonly used to induce misfolding of proteins in the ER and to study the UPR. However, their potential impact on the cellular lipid composition has never been systematically addressed. Here, we report the quantitative, cellular lipid composition of Saccharomyces cerevisiae during acute, proteotoxic stress in both rich and synthetic media. We show that DTT causes rapid remodeling of the lipidome when used in rich medium at growth-inhibitory concentrations, while TM has only a marginal impact on the lipidome under our conditions of cultivation. We formulate recommendations on how to study UPR activation by proteotoxic stress without interferences from a perturbed lipid metabolism. Furthermore, our data suggest an intricate connection between the cellular growth rate, the abundance of the ER, and the metabolism of fatty acids. We show that Saccharomyces cerevisiae can produce asymmetric lipids with two saturated fatty acyl chains differing substantially in length. These observations indicate that the pairing of saturated fatty acyl chains is tightly controlled and suggest an evolutionary conservation of asymmetric lipids and their biosynthetic machineries.

Keywords:  DTT; Ire1; UPR; asymmetric lipids; lipid bilayer stress; lipidomics; proteotoxic stress; tunicamycin

DOI:  https://doi.org/10.3389/fcell.2020.00756
Curr Biol. 2020 Aug 18. pii: S0960-9822(20)31156-8. [Epub ahead of print]

Synaptic Protein Degradation Controls Sexually Dimorphic Circuits through Regulation of DCC/UNC-40.

Yehuda Salzberg, Vladyslava Pechuk, Asaf Gat, Hagar Setty, Sapir Sela, Meital Oren-Suissa.

  Sexually dimorphic circuits underlie behavioral differences between the sexes, yet the molecular mechanisms involved in their formation are poorly understood. We show here that sexually dimorphic connectivity patterns arise in C. elegans through local ubiquitin-mediated protein degradation in selected synapses of one sex but not the other. Specifically, synaptic degradation occurs via binding of the evolutionary conserved E3 ligase SEL-10/FBW7 to a phosphodegron binding site of the netrin receptor UNC-40/DCC (Deleted in Colorectal Cancer), resulting in degradation of UNC-40. In animals carrying an undegradable unc-40 gain-of-function allele, synapses were retained in both sexes, compromising the activity of the circuit without affecting neurite guidance. Thus, by decoupling the synaptic and guidance functions of the netrin pathway, we reveal a critical role for dimorphic protein degradation in controlling neuronal connectivity and activity. Additionally, the interaction between SEL-10 and UNC-40 is necessary not only for sex-specific synapse pruning, but also for other synaptic functions. These findings provide insight into the mechanisms that generate sex-specific differences in neuronal connectivity, activity, and function.

Keywords:  C. elegans; DCC/UNC-40; FBW7/SEL-10; protein degradation; sexual dimorphism; synapse pruning; synaptic connectivity; ubiquitin-proteasome pathway

DOI:  https://doi.org/10.1016/j.cub.2020.08.002
Nat Commun. 2020 Aug 27. 11(1): 4286

Lumenal Galectin-9-Lamp2 interaction regulates lysosome and autophagy to prevent pathogenesis in the intestine and pancreas.

Janaki N Sudhakar, Hsueh-Han Lu, Hung-Yu Chiang, Ching-Shu Suen, Ming-Jing Hwang, Sung-Yu Wu, Chia-Ning Shen, Yao-Ming Chang, Fu-An Li, Fu-Tong Liu, Jr-Wen Shui.

Intracellular galectins are carbohydrate-binding proteins capable of sensing and repairing damaged lysosomes. As in the physiological conditions glycosylated moieties are mostly in the lysosomal lumen but not cytosol, it is unclear whether galectins reside in lysosomes, bind to glycosylated proteins, and regulate lysosome functions. Here, we show in gut epithelial cells, galectin-9 is enriched in lysosomes and predominantly binds to lysosome-associated membrane protein 2 (Lamp2) in a Asn(N)-glycan dependent manner. At the steady state, galectin-9 binding to glycosylated Asn175 of Lamp2 is essential for functionality of lysosomes and autophagy. Loss of N-glycan-binding capability of galectin-9 causes its complete depletion from lysosomes and defective autophagy, leading to increased endoplasmic reticulum (ER) stress preferentially in autophagy-active Paneth cells and acinar cells. Unresolved ER stress consequently causes cell degeneration or apoptosis that associates with colitis and pancreatic disorders in mice. Therefore, lysosomal galectins maintain homeostatic function of lysosomes to prevent organ pathogenesis.

DOI: https://doi.org/10.1038/s41467-020-18102-7
Life Sci. 2020 Aug 22. pii: S0024-3205(20)31069-9. [Epub ahead of print] 118317

GRP78 targeting: Hitting two birds with a stone.

Abdo A Elfiky, Ahmed M Baghdady, Shehab A Ali, Marwan I Ahmed.

   BACKGROUND: Glucose regulating protein 78 (GRP78) is one member of the Heat Shock Protein family of chaperone proteins (HSPA5) found in eukaryotes. It acts as the master of the Unfolded Protein Response (UPR) process in the lumen of the Endoplasmic Reticulum (ER).
SCOPE: Under the stress of unfolded proteins, GRP78 binds to the unfolded proteins to prevent misfolding, while under the load of the unfolded protein, it drives the cell to autophagy or apoptosis. Several attempts reported the overexpression of GRP78 on the cell membrane of cancer cells and cells infected with viruses or fungi.
MAJOR CONCLUSIONS: Cell-surface GRP78 is used as a cancer cell target in previous studies. Additionally, GRP78 is used as a drug target to stop the progression of cancer cells by different compounds, including peptides, antibodies, and some natural compounds. Additionally, it can be used as a protein target to reduce the infectivity of different viruses, including the pandemic SARS-CoV-2. Besides, GRP78 targeting is used in diagnosis and imaging modalities using radionuclides.
GENERAL SIGNIFICANCE: This review summarizes the various attempts that used GRP78 both in therapy (fighting cancer, viral and fungal infections) and diagnosis (imaging).

Keywords:  BiP; Cancer-targeting; GRP78; Natural compounds; Peptide inhibitors; SARS-CoV-2

DOI:  https://doi.org/10.1016/j.lfs.2020.118317
J Mol Biol. 2020 Aug 21. pii: S0022-2836(20)30509-X. [Epub ahead of print]

GROEL/ES Buffers Entropic Traps in Folding Pathway during Evolution of a Model Substrate.

Anwar Sadat, Satyam Tiwari, Kanika Verma, Arjun Ray, Mudassar Ali, Vaibhav Upadhyay, Anupam Singh, Aseem Chaphalkar, Asmita Ghosh, Rahul Chakraborty, Kausik Chakraborty, Koyeli Mapa.

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

DOI: https://doi.org/10.1016/j.jmb.2020.08.015
Biochim Biophys Acta Gen Subj. 2020 Aug 25. pii: S0304-4165(20)30221-X. [Epub ahead of print] 129709

Glycan dependent refolding activity of ER glucosyltransferase (UGGT).

Ning Wang, Akira Seko, Yoichi Takeda, Yukishige Ito.

   BACKGROUND: In the endoplasmic reticulum (ER), folding of glycoproteins is assisted by a combined action of enzymes and chaperones that leads them to biologically functional structures. In this system, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) plays an essential role as the "folding sensor" by virtue of its ability to discriminate folding states of client glycoproteins. However, besides its transferase activity, whether UGGT1 possesses any chaperone activity that facilitates protein folding is yet to be addressed.
METHODS: We prepared oligomannose-type glycan modified RNase (M9GN2-RNase) by chemoenzymatic means using M9GN-oxazoline and glycan truncated RNase B and analyzed the effect of human UGGT1 (HUGT1) for refolding of the denatured M9GN2-RNase. Refolding was evaluated based on the RNase activity which was measured by the cleavage of the RNA substrate.
RESULTS: HUGT1 slightly accelerated the folding of M9GN2-RNase and non-glycosylated RNase A as the same extent. However, HUGT1 remarkably accelerated the folding of M9GN2-RNase in the presence of UDP-Glc. In contrast, neither UDP nor UDP-Gal was effective in enhancing the folding. Additionally, an HUGT1 mutant which lacks the glucosyltransferase activity did not accelerate the protein folding of M9GN2-RNase.
CONCLUSIONS: HUGT1has the ability to promote the refolding of denatured protein and the effect would be enhanced when HUGT1 tightly interacts with the client protein via glycan recognition.
GENERAL SIGNIFICANCE: Our study provides a possibility that HUGT1 play a role not only in sensing the misfolded glycoprotein but also in promoting folding of glycoproteins in the endoplasmic reticulum glycoprotein quality control.

Keywords:  Endoplasmic reticulum quality control; Glycoprotein folding; Glycoprotein glucosyltransferase; Oligomannose-type glycan; Ribonuclease; UDP-glucose

DOI:  https://doi.org/10.1016/j.bbagen.2020.129709
Exp Cell Res. 2020 Aug 20. pii: S0014-4827(20)30489-4. [Epub ahead of print]395(2): 112240

Mechanisms of sensing and response to proteotoxic stress.

Alec M Santiago, Davi L Gonçalves, Kevin A Morano.

  Cells are continuously subject to various stresses, battling both exogenous insults as well as toxic by-products of normal cellular metabolism and nutrient deprivation. Throughout the millennia, cells developed a core set of general stress responses that promote survival and reproduction under adverse circumstances. Past and current research efforts have been devoted to understanding how cells sense stressors and how that input is deciphered and transduced, resulting in stimulation of stress management pathways. A prime element of cellular stress responses is the increased transcription and translation of proteins specialized in managing and mitigating distinct types of stress. In this review, we focus on recent developments in our understanding of cellular sensing of proteotoxic stressors that impact protein synthesis, folding, and maturation provided by the model eukaryote the budding yeast, Saccharomyces cerevisiae, with reference to similarities and differences with other model organisms and humans.

Keywords:  Heat shock; Human; Oxidative stress; Proteostasis; Stress sensing; Yeast

DOI:  https://doi.org/10.1016/j.yexcr.2020.112240
Biochim Biophys Acta Mol Cell Res. 2020 Aug 20. pii: S0167-4889(20)30183-X. [Epub ahead of print] 118825

A Cell Cycle Checkpoint for the Endoplasmic Reticulum.

Maho Niwa.

  The generation of new cells is one of the most fundamental aspects of cell biology. Proper regulation of the cell cycle is critical for human health, as underscored by many diseases associated with errors in cell cycle regulation, including both cancer and hereditary diseases. A large body of work has identified regulatory mechanisms and checkpoints that ensure accurate and timely replication and segregation of chromosomal DNA. However, few studies have evaluated the extent to which similar checkpoints exist for the division of cytoplasmic components, including organelles. Such checkpoint mechanisms might be crucial for compartments that cannot be generated de novo, such as the endoplasmic reticulum (ER). In this review, we highlight recent work in the model organism Saccharomyces cerevisiae that led to the discovery of such a checkpoint that ensures that cells inherit functional ER into the daughter cell.

Keywords:  ER Cell Cycle Surveillance pathway or ERSU; ER stress; Endoplasmic reticulum; Reticulon; cell cycle; cell cycle checkpoint

DOI:  https://doi.org/10.1016/j.bbamcr.2020.118825
mBio. 2020 Aug 25. pii: e01545-20. [Epub ahead of print]11(4):

Mycobacterium tuberculosis Rv0991c Is a Redox-Regulated Molecular Chaperone.

Samuel H Becker, Kathrin Ulrich, Avantika Dhabaria, Beatrix Ueberheide, William Beavers, Eric P Skaar, Lakshminarayan M Iyer, L Aravind, Ursula Jakob, K Heran Darwin.

  The bacterial pathogen Mycobacterium tuberculosis is the leading cause of death by an infectious disease among humans. Here, we describe a previously uncharacterized M. tuberculosis protein, Rv0991c, as a molecular chaperone that is activated by oxidation. Rv0991c has homologs in most bacterial lineages and appears to function analogously to the well-characterized Escherichia coli redox-regulated chaperone Hsp33, despite a dissimilar protein sequence. Rv0991c is transcriptionally coregulated with hsp60 and hsp70 chaperone genes in M. tuberculosis, suggesting that Rv0991c functions with these chaperones in maintaining protein quality control. Supporting this hypothesis, we found that, like oxidized Hsp33, oxidized Rv0991c prevents the aggregation of a model unfolded protein in vitro and promotes its refolding by the M. tuberculosis Hsp70 chaperone system. Furthermore, Rv0991c interacts with DnaK and can associate with many other M. tuberculosis proteins. We therefore propose that Rv0991c, which we named "Ruc" (redox-regulated protein with unstructured C terminus), represents a founding member of a new chaperone family that protects M. tuberculosis and other species from proteotoxicity during oxidative stress.IMPORTANCE M. tuberculosis infections are responsible for more than 1 million deaths per year. Developing effective strategies to combat this disease requires a greater understanding of M. tuberculosis biology. As in all cells, protein quality control is essential for the viability of M. tuberculosis, which likely faces proteotoxic stress within a host. Here, we identify an M. tuberculosis protein, Ruc, that gains chaperone activity upon oxidation. Ruc represents a previously unrecognized family of redox-regulated chaperones found throughout the bacterial superkingdom. Additionally, we found that oxidized Ruc promotes the protein-folding activity of the essential M. tuberculosis Hsp70 chaperone system. This work contributes to a growing body of evidence that oxidative stress provides a particular strain on cellular protein stability.

Keywords:  Hsp70; Mycobacterium; chaperone; protein; proteostasis; tuberculosis

DOI:  https://doi.org/10.1128/mBio.01545-20
Cancers (Basel). 2020 Aug 23. pii: E2385. [Epub ahead of print]12(9):

Proteotoxic Stress and Cell Death in Cancer Cells.

Claudio Brancolini, Luca Iuliano.

  To maintain proteostasis, cells must integrate information and activities that supervise protein synthesis, protein folding, conformational stability, and also protein degradation. Extrinsic and intrinsic conditions can both impact normal proteostasis, causing the appearance of proteotoxic stress. Initially, proteotoxic stress elicits adaptive responses aimed at restoring proteostasis, allowing cells to survive the stress condition. However, if the proteostasis restoration fails, a permanent and sustained proteotoxic stress can be deleterious, and cell death ensues. Many cancer cells convive with high levels of proteotoxic stress, and this condition could be exploited from a therapeutic perspective. Understanding the cell death pathways engaged by proteotoxic stress is instrumental to better hijack the proliferative fate of cancer cells.

Keywords:  BCL2; DR5; NOXA; UPR; apoptosis; ferroptosis; necroptosis; proteotoxic stress

DOI:  https://doi.org/10.3390/cancers12092385
J Med Chem. 2020 Aug 24.

Characterization of aminobenzylphenols as protein disulfide isomerase inhibitors in glioblastoma cell lines.

Andrea Shergalis, Ding Xue, Fatma Z Gharbia, Hannah Driks, Binita Shrestha, Amina Tanweer, Kirin Cromer, Mats Ljungman, Nouri Neamati.

Disulfide bond formation is a critical post-translational modification of newly synthesized polypeptides in the oxidizing environment of the endoplasmic reticulum and is mediated by protein disulfide isomerase (PDIA1). In this study, we report a series of α-aminobenzylphenol analogues as potent PDI inhibitors. The lead compound, AS15, is a covalent nanomolar inhibitor of PDI and the combination of AS15 analogues with glutathione synthesis inhibitor BSO leads to synergistic cell growth inhibition. Using a nascent RNA sequencing we show that an AS15 analogue triggers the unfolded protein response in glioblastoma cells. A BODIPY-labeled analogue binds proteins including PDIA1, suggesting the compounds are cell-permeable and reach the intended target. Taken together, these findings demonstrate an extensive biochemical characterization of a novel series of highly potent reactive small molecules that covalently bind to PDI.

DOI: https://doi.org/10.1021/acs.jmedchem.0c00728
Cell Rep. 2020 Aug 25. pii: S2211-1247(20)31048-2. [Epub ahead of print]32(8): 108063

Hsp90 Co-chaperones Form Plastic Genetic Networks Adapted to Client Maturation.

Maximilian M Biebl, Maximilian Riedl, Johannes Buchner.

  Heat shock protein 90 (Hsp90) is a molecular chaperone regulating the activity of diverse client proteins together with a plethora of different co-chaperones. Whether these functionally cooperate has remained enigmatic. We analyze all double mutants of 11 Saccharomyces cerevisiae Hsp90 co-chaperones in vivo concerning effects on cell physiology and the activation of specific client proteins. We find that client activation is supported by a genetic network with weak epistasis between most co-chaperones and a few modules with strong genetic interactions. These include an epistatic module regulating protein translation and dedicated epistatic networks for specific clients. For kinases, the bridging of Hsp70 and Hsp90 by Sti1/Hop is essential for activation, whereas for steroid hormone receptors, an epistatic module regulating their dwell time on Hsp90 is crucial, highlighting the specific needs of different clients. Thus, the Hsp90 system is characterized by plastic co-chaperone networks fine-tuning the conformational processing in a client-specific manner.

Keywords:  Hsp90; Molecular chaperones; Src kinase; client maturation; co-chaperones; epistasis; steroid hormone receptors

DOI:  https://doi.org/10.1016/j.celrep.2020.108063
J Cell Sci. 2020 Aug 25. pii: jcs.247148. [Epub ahead of print]

Role of the Sec22b/E-Syt complex in neurite growth and ramification.

Alessandra Gallo, Lydia Danglot, Francesca Giordano, Bailey Hewlett, Thomas Binz, Christian Vannier, Thierry Galli.

  Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here we show that Sec22b interacts with the endoplasmic reticulum lipid transfer proteins (LTPs) Extended-Synaptotagmins (E-Syts) and this interaction depends on the Longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, expression of Sec22b Longin domain and a Sec22b mutant with extended linker between SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with LTPs such as E-Syts.

Keywords:  Axonal growth; Filopodia; Lipid-transfer protein; Membrane contact site; Membrane trafficking; SNARE

DOI:  https://doi.org/10.1242/jcs.247148
Front Immunol. 2020 ;11 1559

PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB.

Aya Jodo, Azusa Shibazaki, Asuka Onuma, Tsuneyasu Kaisho, Takashi Tanaka.

  Activation of NF-κB transcription factors is critical for innate immune cells to induce inflammation and fight against microbial pathogens. On the other hand, the excessive and prolonged activation of NF-κB causes massive inflammatory damage to the host, suggesting that regulatory mechanisms to promptly terminate NF-κB activation are important to prevent immunopathology. We have previously reported that PDLIM2, a PDZ-LIM domain-containing protein, is a nuclear ubiquitin E3 ligase that targets the p65 subunit of NF-κB for degradation, thereby suppressing NF-κB activation. Here we show that PDLIM7, another member of LIM protein family, is also a ubiquitin E3 ligase that inhibits NF-κB-mediated inflammatory responses. PDLIM7 directly polyubiquitinates p65 and promotes its proteasomal degradation. Moreover, PDLIM7 heterodimerizes with PDLIM2 to promote synergistic PDLIM2-mediated degradation of p65. Mechanistically, PDLIM7 promotes K63-linked ubiquitination of PDLIM2 and then the proteasome/autophagosome cargo protein p62/Sqstm1 binds to both polyubiquitinated PDLIM2 and the proteasome, thereby facilitating the delivery of the NF-κB-PDLIM2 complex to the proteasome and subsequent p65 degradation. Consistently, double knockdown of PDLIM7 and either PDLIM2 or p62/Sqstm1 results in augmented proinflammatory cytokine production compared to control cells or single knockdown cells. These data delineate a new role for PDLIM7 and p62/Sqstm1 in the regulation of NF-κB signaling by bridging a ubiquitin E3 ligase and the proteasome.

Keywords:  LIM protein; NF-κB; inflammation; p62/Sqstm1; ubiquitin E3 ligase

DOI:  https://doi.org/10.3389/fimmu.2020.01559
Med Res Rev. 2020 Aug 25.

The role and therapeutic potential of Hsp90, Hsp70, and smaller heat shock proteins in peripheral and central neuropathies.

Subhabrata Chaudhury, Bradley M Keegan, Brian S J Blagg.

  Heat shock proteins (Hsps) are molecular chaperones that also play important roles in the activation of the heat shock response (HSR). The HSR is an evolutionary conserved and protective mechanism that is used to counter abnormal physiological conditions, stressors, and disease states, such as those exemplified in cancer and/or neurodegeneration. In normal cells, heat shock factor-1 (HSF-1), the transcription factor that regulates the HSR, remains in a dormant multiprotein complex that is formed upon association with chaperones (Hsp90, Hsp70, etc.), co-chaperones, and client proteins. However, under cellular stress, HSF-1 dissociates from Hsp90 and induces the transcriptional upregulation of Hsp70 to afford protection against the encountered cellular stress. As a consequence of both peripheral and central neuropathies, cellular stress occurs and results in the accumulation of unfolded and/or misfolded proteins, which can be counterbalanced by activation of the HSR. Since Hsp90 is the primary regulator of the HSR, modulation of Hsp90 by small molecules represents an attractive therapeutic approach against both peripheral and central neuropathies.

Keywords:  HSF-1; Hsp90; chaperones; diabetic peripheral neuropathy; heat shock response; neurodegeneration; neuropathy

DOI:  https://doi.org/10.1002/med.21729
J Cell Sci. 2020 Aug 25. pii: jcs.243477. [Epub ahead of print]

ACSL3 is a novel GABARAPL2 interactor that links ufmylation and lipid droplet biogenesis.

Franziska Eck, Santosh Phuyal, Matthew D Smith, Manuel Kaulich, Simon Wilkinson, Hesso Farhan, Christian Behrends.

  While studies of ATG genes in knockout models led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP proteins are still poorly understood. A major drawback for their understanding is that the available interactome data was largely acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was exemplarily employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Further, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together, we describe ACSL3 as novel regulator of the enigmatic UFM1 conjugation pathway.

Keywords:  ACSL3; ER-phagy; GABARAPL2; Lipid droplets; UBA5; Ufmylation

DOI:  https://doi.org/10.1242/jcs.243477
Proc Natl Acad Sci U S A. 2020 Aug 24. pii: 202013543. [Epub ahead of print]

Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity.

Hong Zhang, Zhihui Lyu, Yongqiang Fan, Christopher R Evans, Karl W Barber, Kinshuk Banerjee, Oleg A Igoshin, Jesse Rinehart, Jiqiang Ling.

Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.

DOI: https://doi.org/10.1073/pnas.2013543117
J Biol Chem. 2020 Aug 24. pii: jbc.RA120.014531. [Epub ahead of print]

Cyclosporin A but not FK506 activates the integrated stress response in human cells.

Anthony O Fedele, Valérie Carraro, Jianling Xie, Julien Averous, Christopher G Proud.

  Cyclosporin A (CsA) and tacrolimus (FK506) are valuable immunosuppressants for a range of clinical settings, including (but not limited to) organ transplantation and the treatment of autoimmune diseases. They function by inhibiting the activity of the Ca2+/calmodulin-dependent phosphatase calcineurin towards nuclear factor of activated T-cells (NF-AT) in T-lymphocytes. However, use of CsA is associated with more serious side-effects and worse clinical outcomes than FK506. Here we show that CsA, but not FK506, causes activation of the integrated stress response (ISR), an event which is normally an acute reaction to various types of intracellular insults, such as nutrient deficiency or endoplasmic reticulum stress. These effects of CsA involve at least two of the stress-activated protein kinases (GCN2 and PERK) that act on the translational machinery to slow down protein synthesis via phosphorylation of the eukaryotic initiation factor (eIF) 2α and thereby induce the ISR. These actions of CsA likely contribute to the adverse effects associated with its clinical application.

Keywords:  FK506; cyclosporin; immunosuppressor; protein kinase; protein synthesis; stress response; translation initiation factor

DOI:  https://doi.org/10.1074/jbc.RA120.014531
J Cell Biol. 2020 Sep 07. pii: e202008031. [Epub ahead of print]219(9):

Mitophagy is induced by short ubiquitin chains on mitochondria.

Fumiyo Ikeda.

Mitophagy has a critical role in maintaining cellular homeostasis by removing damaged mitochondria. In this issue, Yamano et al. (2020. J. Cell Biol. https://doi.org/10.1083/jcb.201912144) uncover that a novel complex of the autophagy adaptor optineurin and the membrane protein ATG9A specifically regulate ubiquitin-induced mitophagy.

DOI: https://doi.org/10.1083/jcb.202008031
Endocr Regul. 2020 Jul 01. pii: /j/enr.2020.54.issue-3/enr-2020-0021/enr-2020-0021.xml. [Epub ahead of print]54(3): 183-195

Expression of IDE and PITRM1 genes in ERN1 knockdown U87 glioma cells: effect of hypoxia and glucose deprivation.

Dmytro O Minchenko, Olena O Khita, Dariia O Tsymbal, Serhij V Danilovskyi, Olha V Rudnytska, Oleh V Halkin, Iryna V Kryvdiuk, Maria V Smeshkova, Mykhailo M Yakymchuk, Borys H Bezrodnyi, Oleksandr H Minchenko.

   OBJECTIVE: The aim of the present investigation was to study the expression of genes encoding polyfunctional proteins insulinase (insulin degrading enzyme, IDE) and pitrilysin metallopeptidase 1 (PITRM1) in U87 glioma cells in response to inhibition of endoplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of metabolism through ERN1 signaling as well as hypoxia, glucose and glutamine deprivations.
METHODS: The expression level of IDE and PITRM1 genes was studied in control and ERN1 knockdown U87 glioma cells under glucose and glutamine deprivations as well as hypoxia by quantitative polymerase chain reaction.
RESULTS: It was found that the expression level of IDE and PITRM1 genes was down-regulated in ERN1 knockdown (without ERN1 protein kinase and endoribonuclease activity) glioma cells in comparison with the control glioma cells, being more significant for PITRM1 gene. We also found up-regulation of microRNA MIR7-2 and MIRLET7A2, which have specific binding sites in 3'-untranslated region of IDE and PITRM1 mRNAs, correspondingly, and can participate in posttranscriptional regulation of these mRNA expressions. Only inhibition of ERN1 endoribonuclease did not change significantly the expression of IDE and PITRM1 genes in glioma cells. The expression of IDE and PITRM1 genes is preferentially regulated by ERN1 protein kinase. We also showed that hypoxia down-regulated the expression of IDE and PITRM1 genes and that knockdown of ERN1 signaling enzyme function modified the response of these gene expressions to hypoxia. Glucose deprivation increased the expression level of IDE and PITRM1 genes, but ERN1 knockdown enhanced only the effect of glucose deprivation on PITRM1 gene expression. Glutamine deprivation did not affect the expression of IDE gene in both types of glioma cells, but up-regulated PITRM1 gene and this up-regulation was stronger in ERN1 knockdown cells.
CONCLUSIONS: Results of this investigation demonstrate that ERN1 knockdown significantly decreases the expression of IDE and PITRM1 genes by ERN1 protein kinase mediated mechanism. The expression of both studied genes was sensitive to hypoxia as well as glucose deprivation and dependent on ERN1 signaling in gene-specific manner. It is possible that the level of these genes expression under hypoxia and glucose deprivation is a result of complex interaction of variable endoplasmic reticulum stress related and unrelated regulatory factors and contributed to the control of the cell metabolism.

Keywords:  ERN1 knockdown; IDE; PITRM1; U87 glioma cells; glucose deprivation; hypoxia; mRNA expression; microRNA

DOI:  https://doi.org/10.2478/enr-2020-0021
Front Cell Dev Biol. 2020 ;8 670

DDHD1, but Not DDHD2, Suppresses Neurite Outgrowth in SH-SY5Y and PC12 Cells by Regulating Protein Transport From Recycling Endosomes.

Yuki Maemoto, Tomohiro Maruyama, Kazuaki Nemoto, Takashi Baba, Manae Motohashi, Akihiro Ito, Mitsuo Tagaya, Katsuko Tani.

  DDHD1 and DDHD2 are both intracellular phospholipases A1 and hydrolyze phosphatidic acid in vitro. Given that phosphatidic acid participates in neurite outgrowth, we examined whether DDHD1 and DDHD2 regulate neurite outgrowth. Depletion of DDHD1 from SH-SY5Y and PC12 cells caused elongation of neurites, whereas DDHD2 depletion prevented neurite elongation. Rescue experiments demonstrated that the enzymatic activity of DDHD1 is necessary for the prevention of neurite elongation. Depletion of DDHD1 caused enlargement of early endosomes and stimulated tubulation of recycling endosomes positive for phosphatidic acid-binding proteins syndapin2 and MICAL-L1. Knockout of DDHD1 enhanced transferrin recycling from recycling endosomes to the cell surface. Our results suggest that DDHD1 negatively controls the formation of a local phosphatidic acid-rich domain in recycling endosomes that serves as a membrane source for neurite outgrowth.

Keywords:  endosome; neurite; phosphatidic acid; phospholipase; recycling

DOI:  https://doi.org/10.3389/fcell.2020.00670
Biochem Eng J. 2019 Nov 15. pii: 107320. [Epub ahead of print]151

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter.

Nora Safa, Jacob H Pettigrew, Ted J Gauthier, Adam T Melvin.

  Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal β-hairpin motif that acts as both a 'protectide' to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The β-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Keywords:  Cell penetrating peptides; Deubiquitinating enzymes (DUBs); Fluorometry; Peptide-based biosensors; Protease-resilient peptides; Single cell analysis

DOI:  https://doi.org/10.1016/j.bej.2019.107320
FASEB J. 2020 Aug 26.

Deficiency of ULK1/ATG1 in the follicle cells disturbs ER homeostasis and causes defective chorion deposition in the vector Rhodnius prolixus.

Larissa Bomfim, Isabela Ramos.

  In insects, synthesis and deposition of the chorion (eggshell) are performed by the professional secretory follicle cells (FCs) that surround the oocytes in the course of oogenesis. Here, we found that ULK1/ATG1, an autophagy-related protein, is highly expressed in the FCs of the Chagas-Disease vector Rhodnius prolixus, and that parental RNAi silencing of ULK1/ATG1 results in oocytes with abnormal chorion ultrastructure and FCs presenting expanded rough ER membranes as well as increased expression of the ER chaperone BiP3, both indicatives of ER stress. Silencing of LC3/ATG8, another essential autophagy protein, did not replicate the ULK1/ATG1 phenotypes, whereas silencing of SEC16A, a known partner of the noncanonical ULK1/ATG1 function in the ER exit sites phenocopied the silencing of ULK1/ATG1. Our findings point to a cooperated function of ULK1/ATG1 and SEC16A in the FCs to complete choriogenesis and provide additional in vivo phenotype-based evidence to the literature of the role of ULK1/ATG1 in the ER in a professional secretory cell.

Keywords:  ULK1/ATG1; choriogenesis; follicle cells; vector biology

DOI:  https://doi.org/10.1096/fj.202001396R
Trends Biochem Sci. 2020 Aug 19. pii: S0968-0004(20)30192-4. [Epub ahead of print]

The Autophagy-RNA Interplay: Degradation and Beyond.

Marie H Abildgaard, Sólveig H Brynjólfsdóttir, Lisa B Frankel.

  Autophagy is a highly conserved degradation pathway that ensures nutrient recycling and removal of unwanted substrates. This process has a fundamental role in stress adaptation and maintenance of cellular homeostasis. Here, we discuss emerging aspects of the autophagy-RNA interplay, including autophagy-mediated degradation of RNA, RNA-binding proteins (RBPs), and ribonucleoprotein (RNP) complexes. Beyond degradation, we review new roles for autophagy players in the secretion and intracellular transport of RNA and related complexes. We discuss the physiological importance of these events for RNA homeostasis and gene expression programs, as well as their implications for disease, including cancer and neurodegeneration. Lastly, we examine how post-transcriptional regulation of autophagy, through specialized processing and selective translation of key transcripts, challenges and updates our current view of autophagy complexity.

Keywords:  RNA homeostasis; RNA-binding proteins; autophagy; degradation; post-transcriptional regulation; translation

DOI:  https://doi.org/10.1016/j.tibs.2020.07.007
Int J Mol Sci. 2020 Aug 26. pii: E6146. [Epub ahead of print]21(17):

Derlin-3 Is Required for Changes in ERAD Complex Formation under ER Stress.

Yuka Eura, Toshiyuki Miyata, Koichi Kokame.

  Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is a quality control system that induces the degradation of ER terminally misfolded proteins. The ERAD system consists of complexes of multiple ER membrane-associated and luminal proteins that function cooperatively. We aimed to reveal the role of Derlin-3 in the ERAD system using the liver, pancreas, and kidney obtained from different mouse genotypes. We performed coimmunoprecipitation and sucrose density gradient centrifugation to unravel the dynamic nature of ERAD complexes. We observed that Derlin-3 is exclusively expressed in the pancreas, and its deficiency leads to the destabilization of Herp and accumulation of ERAD substrates. Under normal conditions, Complex-1a predominantly contains Herp, Derlin-2, HRD1, and SEL1L, and under ER stress, Complex-1b contains Herp, Derlin-3 (instead of Derlin-2), HRD1, and SEL1L. Complex-2 is upregulated under ER stress and contains Derlin-1, Derlin-2, p97, and VIMP. Derlin-3 deficiency suppresses the transition of Derlin-2 from Complex-1a to Complex-2 under ER stress. In the pancreas, Derlin-3 deficiency blocks Derlin-2 transition. In conclusion, the composition of ERAD complexes is tissue-specific and changes in response to ER stress in a Derlin-3-dependent manner. Derlin-3 may play a key role in changing ERAD complex compositions to overcome ER stress.

Keywords:  Derlin-1; Derlin-2; Derlin-3; ER stress; ERAD; ERAD complex; HRD1; Herp

DOI:  https://doi.org/10.3390/ijms21176146
Mol Cell Neurosci. 2020 Aug 22. pii: S1044-7431(20)30165-2. [Epub ahead of print] 103542

The ubiquitin ligase UBE4B regulates amyloid precursor protein ubiquitination, endosomal trafficking, and amyloid β42 generation and secretion.

Monica Gireud-Goss, Sahily Reyes, Ritika Tewari, Anthony Patrizz, Matthew D Howe, Julia Kofler, M Neal Waxham, Louise D McCullough, Andrew J Bean.

  The extracellular accumulation of amyloid β (Aβ) fragments of amyloid precursor protein (APP) in brain parenchyma is a pathological hallmark of Alzheimer's disease (AD). APP can be cleaved into Aβ on late endosomes/multivesicular bodies (MVBs). E3 ubiquitin ligases have been linked to Aβ production, but specific E3 ligases associated with APP ubiquitination that may affect targeting of APP to endosomes have not yet been described. Using cultured cortical neurons isolated from rat pups, we reconstituted APP movement into the internal vesicles (ILVs) of MVBs. Loss of endosomal sorting complexes required for transport (ESCRT) components inhibited APP movement into ILVs and increased endosomal Aβ42 generation, implying a requirement for APP ubiquitination. We identified an ESCRT-binding and APP-interacting endosomal E3 ubiquitin ligase, ubiquitination factor E4B (UBE4B), that regulates APP ubiquitination. Depleting UBE4B in neurons inhibited APP ubiquitination and internalization into MVBs, resulting in increased endosomal Aβ42 levels and increased neuronal secretion of Aβ42. When we examined AD brains, we found levels of the UBE4B-interacting ESCRT component, hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), were significantly decreased in AD brains. These data suggest that ESCRT components critical for membrane protein sorting in the endocytic pathway are altered in AD. These results indicate that the molecular machinery underlying endosomal trafficking of APP, including the ubiquitin ligase UBE4B, regulates Aβ levels and may play an essential role in AD progression.

Keywords:  Alzheimer’s disease (AD); amyloid β (Aβ); endocytosis; membrane trafficking; multivesicular body (MVB); ubiquitination factor E4B (UBE4B)

DOI:  https://doi.org/10.1016/j.mcn.2020.103542
J Biol Chem. 2020 Aug 26. pii: jbc.RA120.013999. [Epub ahead of print]

ATG16L1 autophagy pathway regulates BAX protein levels and programmed cell death.

Fenfen Chen, Dulguun Amgalan, Richard N Kitsis, Jeffrey E Pessin, Daorong Feng.

  Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo. Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, while in the nutrient-deplete state BAX binds to SNAP23 to form a ternary BAX/SNAP23/ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.

Keywords:  ATG16L1; ATG9; Bax; SNAP23; SNARE proteins; adipocyte; apoptosis; autophagy

DOI:  https://doi.org/10.1074/jbc.RA120.013999
PLoS Genet. 2020 Aug 26. 16(8): e1008745

Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.

Bianca M Esch, Sergej Limar, André Bogdanowski, Christos Gournas, Tushar More, Celine Sundag, Stefan Walter, Jürgen J Heinisch, Christer S Ejsing, Bruno André, Florian Fröhlich.

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.

DOI: https://doi.org/10.1371/journal.pgen.1008745
Plant Cell. 2020 Aug 25. pii: tpc.00260.2020. [Epub ahead of print]

The Transcription Factor bZIP60 Links the Unfolded Protein Response (UPR) to the Heat Stress Response (HSR) in Maize.

Zhaoxia Li, Jie Tang, Renu Srivastava, Diane C Bassham, Stephen H Howell.

The unfolded protein response (UPR) and the heat shock response (HSR) are two evolutionarily conserved systems to protect plants from heat stress. The UPR and HSR occur in different cellular compartments and both responses are elicited by misfolded proteins which accumulate under adverse environmental conditions such as heat stress. While the UPR and HSR appear to operate independently, we have found a link between them in maize (Zea mays) involving the production of the bZIP60 transcription factor, a pivotal response of the UPR to heat stress. Surprisingly, a mutant (bzip60 2) knocking down bZIP60 expression blunted the HSR at elevated temperatures and prevented the normal upregulation of a group of heat shock protein (HSP) genes in response to elevated temperature. The expression of a key heat shock factor HSFTF13 (a HSFA6B family member) was compromised in bzip60-2, and the HSFTF13 promoter was shown to be a target of bZIP60 in maize protoplasts. In addition, the upregulation by heat of genes involved in chlorophyll catabolism and chloroplast protein turnover were subdued in bzip60-2, and these genes were also found to be targets of bZIP60. Thus, the UPR, an endoplasmic reticulum associated response, quite unexpectedly contributes to the nuclear/cytoplasmic heat shock response in maize.

DOI: https://doi.org/10.1105/tpc.20.00260