bims-proteo 2020-05-24 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–05–24
forty-two papers selected by
Eric Chevet, INSERM

Discovery of a potent SCAP degrader that ameliorates HFD-induced obesity, hyperlipidemia and insulin resistance via an autophagy-independent lysosomal pathway.
The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.
EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.
Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.
Split-TurboID enables contact-dependent proximity labeling in cells.
Molecular Mechanisms of ER Stress and UPR in the Pathogenesis of Alzheimer's Disease.
NADPH and Glutathione Redox Link TCA Cycle Activity to Endoplasmic Reticulum Homeostasis.
Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p.
ERp29 as a regulator of Insulin biosynthesis.
IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.
The Roles of Endoplasmic Reticulum in NLRP3 Inflammasome Activation.
The mystery of mitochondria-ER contact sites in physiology and pathology: A cancer perspective.
ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila.
Structural basis for membrane insertion by the human ER membrane protein complex.
Membrane Curvature Catalyzes Lipid Droplet Assembly.
Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function.
KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity.
Synergies in exosomes and autophagy pathways for cellular homeostasis and metastasis of tumor cells.
Mitochondrial Protein Quality Control Mechanisms.
p62/SQSTM1 accumulation due to degradation inhibition and transcriptional activation plays a critical role in silica nanoparticle-induced airway inflammation via NF-κB activation.
Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders.
Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids.
The plant endoplasmic reticulum: An organized chaos of tubules and sheets with multiple functions.
A selective role for a component of the autophagy pathway in coupling the Golgi apparatus to dendrite polarity in pyramidal neurons.
The pepper RING-type E3 ligase, CaATIR1, positively regulates ABA signaling and drought response by modulating the stability of CaATBZ1.
Calcineurin-dependent regulation of endocytosis by a plasma membrane ubiquitin ligase adaptor, Rcr1.
Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.
Chaperone-Mediated Autophagy in the light of evolution: insight from fish.
The proteasome activator REGγ accelerates cardiac hypertrophy by declining PP2Acα-SOD2 pathway.
ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.
Autophagy Participates in Lysosomal Vacuolation-Mediated Cell Death in RGNNV-Infected Cells.
Mechanisms Regulating the UPS-ALS Crosstalk: The Role of Proteaphagy.
The hypothetical role of Phosphatidic Acid in subverting ER membranes during SARS-CoV infection.
Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling.
Adaptor protein RapZ activates endoribonuclease RNase E by protein-protein interaction to cleave a small regulatory RNA.
Mitochondrial translation, dynamics, and lysosomes combine to extend lifespan.
Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13.
UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner.
Small heat shock proteins determine synapse number and neuronal activity during development.
Honokiol bis-dichloroacetate is a selective allosteric inhibitor of the mitochondrial chaperone TRAP1.
USP9X prevents AGEs-induced upregulation of FN and TGF-β1 through activating Nrf2-ARE pathway in rat glomerular mesangial cells.
Editorial: Stem cell secretome.

Autophagy. 2020 May 20. 1-22

Discovery of a potent SCAP degrader that ameliorates HFD-induced obesity, hyperlipidemia and insulin resistance via an autophagy-independent lysosomal pathway.

Zu-Guo Zheng, Si-Tong Zhu, Hui-Min Cheng, Xin Zhang, Gang Cheng, Pyone Myat Thu, Supeng Perry Wang, Hui-Jun Li, Ming Ding, Lei Qiang, Xiao-Wei Chen, Qing Zhong, Ping Li, Xiaojun Xu.

  SCAP (SREBF chaperone) regulates SREBFs (sterol regulatory element binding transcription factors) processing and stability, and, thus, becomes an emerging drug target to treat dyslipidemia and fatty liver disease. However, the current known SCAP inhibitors, such as oxysterols, induce endoplasmic reticulum (ER) stress and NR1H3/LXRα (nuclear receptor subfamily 1 group H member 3)-SREBF1/SREBP-1 c-mediated hepatic steatosis, which severely limited the clinical application of this inhibitor. In this study, we identified a small molecule, lycorine, which binds to SCAP, which suppressed the SREBF pathway without inducing ER stress or activating NR1H3. Mechanistically, lycorine promotes SCAP lysosomal degradation in a macroautophagy/autophagy-independent pathway, a mechanism completely distinct from current SCAP inhibitors. Furthermore, we determined that SQSTM1 captured SCAP after its exit from the ER. The interaction of SCAP and SQSTM1 requires the WD40 domain of SCAP and the TB domain of SQSTM1. Interestingly, lycorine triggers the lysosome translocation of SCAP independent of autophagy. We termed this novel protein degradation pathway as the SQSTM1-mediated autophagy-independent lysosomal degradation (SMAILD) pathway. In vivo, lycorine ameliorates high-fat diet-induced hyperlipidemia, hepatic steatosis, and insulin resistance in mice. Our study demonstrated that the inhibition of SCAP through the SMAILD pathway could be employed as a useful therapeutic strategy for treating metabolic diseases.
ABBREVIATION: 25-OHD: 25-hydroxyvitamin D; 3-MA: 3-methyladenine; ABCG5: ATP binding cassette subfamily G member 5; ABCG8: ATP binding cassette subfamily G member 8; ACACA: acetyl-CoA carboxylase alpha; AEBSF: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; AHI: anhydroicaritin; AKT/protein kinase B: AKT serine/threonine kinase; APOE: apolipoprotein E; ATF6: activating transcription factor 6; ATG: autophagy-related; BAT: brown adipose tissue; CD274/PD-L1: CD274 molecule; CETSA: cellular thermal shift assay; CMA: chaperone-mediated autophagy; COPII: cytoplasmic coat protein complex-II; CQ: chloroquine; DDIT3/CHOP: DNA damage inducible transcript 3; DNL: de novo lipogenesis; EE: energy expenditure; EGFR: epithelial growth factor receptor; eMI: endosomal microautophagy; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FADS2: fatty acid desaturase 2; FASN: fatty acid synthase; GOT1/AST: glutamic-oxaloacetic transaminase 1; GPT/ALT: glutamic-pyruvate transaminase; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; HMGCS1: 3-hydroxy-3-methylglutaryl-CoA synthase 1; HSP90B1/GRP94: heat shock protein 90 beta family member 1; HSPA5/GRP78: heat hock protein family A (Hsp70) member 5; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INSIG1: insulin induced gene 1; LAMP2A: lysosomal associated membrane protein 2A; LDLR: low density lipoprotein receptor; LyTACs: lysosome targeting chimeras; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MBTPS1: membrane bound transcription factor peptidase, site 1; MEF: mouse embryonic fibroblast; MST: microscale thermophoresis; MTOR: mechanistic target of rapamycin kinase; MVK: mevalonate kinase; PROTAC: proteolysis targeting chimera; RQ: respiratory quotient; SCAP: SREBF chaperone; SCD1: stearoyl-coenzemy A desaturase 1; SMAILD: sequestosome 1 mediated autophagy-independent lysosomal degradation; SQSTM1: sequestosome 1; SREBF: sterol regulatory element binding transcription factor; TNFRSF10B/DR5: TNF receptor superfamily member 10b; TRAF6: TNF receptor associated factor 6; UPR: unfolded protein response; WAT: white adipose tissue; XBP1: X-box binding protein 1.

Keywords:  Autophagy; ER stress; SCAP; SQSTM1; SREBFs; lycorine

DOI:  https://doi.org/10.1080/15548627.2020.1757955
J Biol Chem. 2020 May 19. pii: jbc.RA120.012979. [Epub ahead of print]

The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis.

Xin Jin, Jianling Xie, Michael Zabolocki, Xuemin Wang, Tao Jiang, Dong Wang, Laurent Desaubry, Cedric Bardy, Christopher G Proud.

  Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca2+-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca2+ levels, likely explaining the effects on eEF2. Disruption of normal Ca2+ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely due to activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca2+ levels, cellular protein synthesis, and cell survival.

Keywords:  calcium; cancer; cell death; eEF2; endoplasmic reticulum stress (ER stress); eukaryotic initiation factor 2 (eIF2); eukaryotic translation initiation; prohibitin; protein synthesis; translation elongation factor

DOI:  https://doi.org/10.1074/jbc.RA120.012979
Int J Mol Sci. 2020 May 14. pii: E3468. [Epub ahead of print]21(10):

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired.

Marioara Chiritoiu, Gabriela N Chiritoiu, Cristian V A Munteanu, Florin Pastrama, N Erwin Ivessa, Stefana M Petrescu.

  Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein-protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Keywords:  EDEM1; EDEM1 interaction network; ER-phagy; ERAD; autophagy; endoplasmic reticulum; intrinsically disordered region; mass spectrometry; protein degradation; protein quality control

DOI:  https://doi.org/10.3390/ijms21103468
Cell Rep. 2020 May 19. pii: S2211-1247(20)30617-3. [Epub ahead of print]31(7): 107664

Ubiquitylation of the ER-Shaping Protein Lunapark via the CRL3KLHL12 Ubiquitin Ligase Complex.

Laurensia Yuniati, Angela Lauriola, Manouk Gerritsen, Susana Abreu, Eric Ni, Chiara Tesoriero, Jacob O Onireti, Teck Yew Low, Albert J R Heck, Andrea Vettori, Timothy Cardozo, Daniele Guardavaccaro.

  Cullin-RING ligases (CRLs) control key cellular processes by promoting ubiquitylation of a multitude of soluble cytosolic and nuclear proteins. Subsets of CRL complexes are recruited and activated locally at cellular membranes; however, few CRL functions and substrates at these distinct cellular compartments are known. Here, we use a proteomic screen to identify proteins that are ubiquitylated at cellular membranes and found that Lunapark, an endoplasmic reticulum (ER)-shaping protein localized to ER three-way junctions, is ubiquitylated by the CRL3KLHL12 ubiquitin ligase. We demonstrate that Lunapark interacts with mechanistic target of rapamycin complex-1 (mTORC1), a central cellular regulator that coordinates growth and metabolism with environmental conditions. We show that mTORC1 binds Lunapark specifically at three-way junctions, and lysosomes, where mTORC1 is activated, make contact with three-way junctions where Lunapark resides. Inhibition of Lunapark ubiquitylation results in neurodevelopmental defects indicating that KLHL12-dependent ubiquitylation of Lunapark is required for normal growth and development.

Keywords:  ER three-way junction; Lunapark; cullin-RING ligases; endoplasmic reticulum; lysosome; mTORC1; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2020.107664
Proc Natl Acad Sci U S A. 2020 May 18. pii: 201919528. [Epub ahead of print]

Split-TurboID enables contact-dependent proximity labeling in cells.

Kelvin F Cho, Tess C Branon, Sanjana Rajeev, Tanya Svinkina, Namrata D Udeshi, Themis Thoudam, Chulhwan Kwak, Hyun-Woo Rhee, In-Kyu Lee, Steven A Carr, Alice Y Ting.

  Proximity labeling catalyzed by promiscuous enzymes, such as TurboID, have enabled the proteomic analysis of subcellular regions difficult or impossible to access by conventional fractionation-based approaches. Yet some cellular regions, such as organelle contact sites, remain out of reach for current PL methods. To address this limitation, we split the enzyme TurboID into two inactive fragments that recombine when driven together by a protein-protein interaction or membrane-membrane apposition. At endoplasmic reticulum-mitochondria contact sites, reconstituted TurboID catalyzed spatially restricted biotinylation, enabling the enrichment and identification of >100 endogenous proteins, including many not previously linked to endoplasmic reticulum-mitochondria contacts. We validated eight candidates by biochemical fractionation and overexpression imaging. Overall, split-TurboID is a versatile tool for conditional and spatially specific proximity labeling in cells.

Keywords:  ER–mitochondria contacts; proximity labeling; split-TurboID

DOI:  https://doi.org/10.1073/pnas.1919528117
Mol Neurobiol. 2020 May 19.

Molecular Mechanisms of ER Stress and UPR in the Pathogenesis of Alzheimer's Disease.

Md Sahab Uddin, Devesh Tewari, Gaurav Sharma, Md Tanvir Kabir, George E Barreto, May N Bin-Jumah, Asma Perveen, Mohamed M Abdel-Daim, Ghulam Md Ashraf.

  Alzheimer's disease (AD) is a progressive neurodegenerative disease involving aggregation of misfolded proteins inside the neuron causing prolonged cellular stress. The neuropathological hallmarks of AD include the formation of senile plaques and neurofibrillary tangles in specific brain regions that lead to synaptic loss and neuronal death. The exact mechanism of neuron dysfunction in AD remains obscure. In recent years, endoplasmic reticulum (ER) dysfunction has been implicated in neuronal degeneration seen in AD. Apart from AD, many other diseases also involve misfolded proteins aggregations in the ER, a condition referred to as ER stress. The response of the cell to ER stress is to activate a group of signaling pathways called unfolded protein response (UPR) that stimulates a particular transcriptional program to restore ER function and ensure cell survival. ER stress also involves the generation of reactive oxygen species (ROS) that, together with mitochondrial ROS and decreased effectiveness of antioxidant mechanisms, producing a condition of chronic oxidative stress. The unfolded proteins may not always produce a response that leads to the restoration of cellular functions, but they may also lead to inflammation by a set of different pathways with deleterious consequences. In this review, we extensively discuss the role of ER stress and how to target it using different pharmacological approaches in AD development and onset.

Keywords:  Alzheimer’s disease; Amyloid β; Endoplasmic reticulum; Tau; Unfolded protein response

DOI:  https://doi.org/10.1007/s12035-020-01929-y
iScience. 2020 Apr 29. pii: S2589-0042(20)30301-1. [Epub ahead of print]23(5): 101116

NADPH and Glutathione Redox Link TCA Cycle Activity to Endoplasmic Reticulum Homeostasis.

Erica R Gansemer, Kyle S McCommis, Michael Martino, Abdul Qaadir King-McAlpin, Matthew J Potthoff, Brian N Finck, Eric B Taylor, D Thomas Rutkowski.

  Many metabolic diseases disrupt endoplasmic reticulum (ER) homeostasis, but little is known about how metabolic activity is communicated to the ER. Here, we show in hepatocytes and other metabolically active cells that decreasing the availability of substrate for the tricarboxylic acid (TCA) cycle diminished NADPH production, elevated glutathione oxidation, led to altered oxidative maturation of ER client proteins, and attenuated ER stress. This attenuation was prevented when glutathione oxidation was disfavored. ER stress was also alleviated by inhibiting either TCA-dependent NADPH production or Glutathione Reductase. Conversely, stimulating TCA activity increased NADPH production, glutathione reduction, and ER stress. Validating these findings, deletion of the Mitochondrial Pyruvate Carrier-which is known to decrease TCA cycle activity and protect the liver from steatohepatitis-also diminished NADPH, elevated glutathione oxidation, and alleviated ER stress. Together, our results demonstrate a novel pathway by which mitochondrial metabolic activity is communicated to the ER through the relay of redox metabolites.

Keywords:  biological sciences; cell biology; functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2020.101116
Biochim Biophys Acta Biomembr. 2020 May 13. pii: S0005-2736(20)30182-6. [Epub ahead of print] 183342

Proteasome-dependent protein quality control of the peroxisomal membrane protein Pxa1p.

S Devarajan, M Meurer, C W T van Roermund, X Chen, E H Hettema, S Kemp, M Knop, C Williams.

  Peroxisomes are eukaryotic organelles that function in numerous metabolic pathways and defects in peroxisome function can cause serious developmental brain disorders such as adrenoleukodystrophy (ALD). Peroxisomal membrane proteins (PMPs) play a crucial role in regulating peroxisome function. Therefore, PMP homeostasis is vital for peroxisome function. Recently, we established that certain PMPs are degraded by the Ubiquitin Proteasome System yet little is known about how faulty/non-functional PMPs undergo quality control. Here we have investigated the degradation of Pxa1p, a fatty acid transporter in the yeast Saccharomyces cerevisiae. Pxa1p is a homologue of the human protein ALDP and mutations in ALDP result in the severe disorder ALD. By introducing two corresponding ALDP mutations into Pxa1p (Pxa1MUT), fused to mGFP, we show that Pxa1MUT-mGFP is rapidly degraded from peroxisomes in a proteasome-dependent manner, while wild type Pxa1-mGFP remains relatively stable. Furthermore, we identify a role for the ubiquitin ligase Ufd4p in Pxa1MUT-mGFP degradation. Finally, we establish that inhibiting Pxa1MUT-mGFP degradation results in a partial rescue of Pxa1p activity in cells. Together, our data demonstrate that faulty PMPs can undergo proteasome-dependent quality control. Furthermore, our observations may provide new insights into the role of ALDP degradation in ALD.

Keywords:  ALD; Peroxisome; Proteasome; Protein degradation; ufd4

DOI:  https://doi.org/10.1016/j.bbamem.2020.183342
PLoS One. 2020 ;15(5): e0233502

ERp29 as a regulator of Insulin biosynthesis.

Jeffrey Viviano, Margaret Brecker, Christine Ferrara-Cook, Laurence Suaud, Ronald C Rubenstein.

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.

DOI: https://doi.org/10.1371/journal.pone.0233502
Sci Rep. 2020 May 20. 10(1): 8348

IRE1α and IGF signaling predict resistance to an endoplasmic reticulum stress-inducing drug in glioblastoma cells.

Jeffrey J Rodvold, Su Xian, Julia Nussbacher, Brian Tsui, T Cameron Waller, Stephen C Searles, Alyssa Lew, Pengfei Jiang, Ivan Babic, Natsuko Nomura, Jonathan H Lin, Santosh Kesari, Hannah Carter, Maurizio Zanetti.

To date current therapies of glioblastoma multiforme (GBM) are largely ineffective. The induction of apoptosis by an unresolvable unfolded protein response (UPR) represents a potential new therapeutic strategy. Here we tested 12ADT, a sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor, on a panel of unselected patient-derived neurosphere-forming cells and found that GBM cells can be distinguished into "responder" and "non-responder". By RNASeq analysis we found that the non-responder phenotype is significantly linked with the expression of UPR genes, and in particular ERN1 (IRE1) and ATF4. We also identified two additional genes selectively overexpressed among non-responders, IGFBP3 and IGFBP5. CRISPR-mediated deletion of the ERN1, IGFBP3, IGFBP5 signature genes in the U251 human GBM cell line increased responsiveness to 12ADT. Remarkably, >65% of GBM cases in The Cancer Genome Atlas express the non-responder (ERN1, IGFBP3, IGFBP5) gene signature. Thus, elevated levels of IRE1α and IGFBPs predict a poor response to drugs inducing unresolvable UPR and possibly other forms of chemotherapy helping in a better stratification GBM patients.

DOI: https://doi.org/10.1038/s41598-020-65320-6
Cells. 2020 May 14. pii: E1219. [Epub ahead of print]9(5):

The Roles of Endoplasmic Reticulum in NLRP3 Inflammasome Activation.

Yang Zhou, Zhizi Tong, Songhong Jiang, Wenyan Zheng, Jianjun Zhao, Xiangmei Zhou.

  The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome senses pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), and activates caspase-1, which provokes release of proinflammatory cytokines such as interleukin-1β (IL-1β) and IL-18 as well as pyroptosis to engage in innate immune defense. The endoplasmic reticulum (ER) is a large and dynamic endomembrane compartment, critical to cellular function of organelle networks. Recent studies have unveiled the pivotal roles of the ER in NLRP3 inflammasome activation. ER-mitochondria contact sites provide a location for NLRP3 activation, its association with ligands released from or residing in mitochondria, and rapid Ca2+ mobilization from ER stores to mitochondria. ER-stress signaling plays a critical role in NLRP3 inflammasome activation. Lipid perturbation and cholesterol trafficking to the ER activate the NLRP3 inflammasome. These findings emphasize the importance of the ER in initiation and regulation of the NLRP3 inflammasome.

Keywords:  IRE1α; NLRP3; endoplasmic reticulum; inflammasome

DOI:  https://doi.org/10.3390/cells9051219
Biochim Biophys Acta Mol Basis Dis. 2020 May 10. pii: S0925-4439(20)30179-4. [Epub ahead of print] 165834

The mystery of mitochondria-ER contact sites in physiology and pathology: A cancer perspective.

Ines C M Simoes, Giampaolo Morciano, Magdalena Lebiedzinska-Arciszewska, Gianluca Aguiari, Paolo Pinton, Yaiza Potes, Mariusz R Wieckowski.

  Mitochondria-associated membranes (MAM), physical platforms that enable communication between mitochondria and the endoplasmic reticulum (ER), are enriched with many proteins and enzymes involved in several crucial cellular processes, such as calcium (Ca2+) homeostasis, lipid synthesis and trafficking, autophagy and reactive oxygen species (ROS) production. Accumulating studies indicate that tumor suppressors and oncogenes are present at these intimate contacts between mitochondria and the ER, where they influence Ca2+ flux between mitochondria and the ER or affect lipid homeostasis at MAM, consequently impacting cell metabolism and cell fate. Understanding these fundamental roles of mitochondria-ER contact sites as important domains for tumor suppressors and oncogenes can support the search for new and more precise anticancer therapies. In the present review, we summarize the current understanding of basic MAM biology, composition and function and discuss the possible role of MAM-resident oncogenes and tumor suppressors.

Keywords:  Cancer; Endoplasmic reticulum; Mitochondria; Mitochondria-associated membranes (MAM); Oncogenes; Oncosuppressors

DOI:  https://doi.org/10.1016/j.bbadis.2020.165834
Cell Rep. 2020 May 19. pii: S2211-1247(20)30612-4. [Epub ahead of print]31(7): 107659

ATF4-Induced Warburg Metabolism Drives Over-Proliferation in Drosophila.

Sebastian Sorge, Jonas Theelke, Kerem Yildirim, Helen Hertenstein, Ellen McMullen, Stephan Müller, Christian Altbürger, Stefanie Schirmeier, Ingrid Lohmann.

  The mitochondrial electron transport chain (ETC) enables essential metabolic reactions; nonetheless, the cellular responses to defects in mitochondria and the modulation of signaling pathway outputs are not understood. We show that Notch signaling and ETC attenuation via knockdown of COX7a induces massive over-proliferation. The tumor-like growth is caused by a transcriptional response through the eIF2α-kinase PERK and ATF4, which activates the expression of metabolic enzymes, nutrient transporters, and mitochondrial chaperones. We find this stress adaptation to be beneficial for progenitor cell fitness, as it renders cells sensitive to proliferation induced by the Notch signaling pathway. Intriguingly, over-proliferation is not caused by transcriptional cooperation of Notch and ATF4, but it is mediated in part by pH changes resulting from the Warburg metabolism induced by ETC attenuation. Our results suggest that ETC function is monitored by the PERK-ATF4 pathway, which can be hijacked by growth-promoting signaling pathways, leading to oncogenic pathway activity.

Keywords:  ATF4; Drosophila; ETC; ETC impairment; ISR; LDH; Notch pathway; PERK; UPR; lactate; mitochondrial electron transport chain; pH; proliferation

DOI:  https://doi.org/10.1016/j.celrep.2020.107659
Science. 2020 May 21. pii: eabb5008. [Epub ahead of print]

Structural basis for membrane insertion by the human ER membrane protein complex.

Tino Pleiner, Giovani Pinton Tomaleri, Kurt Januszyk, Alison J Inglis, Masami Hazu, Rebecca M Voorhees.

A defining step in the biogenesis of a membrane protein is the insertion of its hydrophobic transmembrane helices into the lipid bilayer. The nine-subunit ER membrane protein complex (EMC) is a conserved co- and post-translational insertase at the endoplasmic reticulum. We determined the structure of the human EMC in a lipid nanodisc to an overall resolution of 3.4 Å by cryo-electron microscopy, permitting building of a nearly complete atomic model. We used structure-guided mutagenesis to demonstrate that substrate insertion requires a methionine-rich cytosolic loop and occurs via an enclosed hydrophilic vestibule within the membrane formed by the subunits EMC3 and EMC6. We propose that the EMC uses local membrane thinning and a positively charged patch to decrease the energetic barrier for insertion into the bilayer.

DOI: https://doi.org/10.1126/science.abb5008
Curr Biol. 2020 May 15. pii: S0960-9822(20)30582-0. [Epub ahead of print]

Membrane Curvature Catalyzes Lipid Droplet Assembly.

Alexandre Santinho, Veijo T Salo, Aymeric Chorlay, Shiqian Li, Xin Zhou, Mohyeddine Omrane, Elina Ikonen, Abdou Rachid Thiam.

  Lipid droplet (LD) biogenesis begins in the endoplasmic reticulum (ER) bilayer, but how the ER topology impacts this process is unclear. An early step in LD formation is nucleation, wherein free neutral lipids, mainly triacylglycerols (TGs) and sterol esters (SEs), condense into a nascent LD. How this transition occurs is poorly known. Here, we found that LDs preferably assemble at ER tubules, with higher curvature than ER sheets, independently of the LD assembly protein seipin. Indeed, the critical TG concentration required for initiating LD assembly is lower at curved versus flat membrane regions. In agreement with this finding, flat ER regions bear higher amounts of free TGs than tubular ones and present less LDs. By using an in vitro approach, we discovered that the presence of free TGs in tubules is energetically unfavorable, leading to outflow of TGs to flat membrane regions or condensation into LDs. Accordingly, in vitro LD nucleation can be achieved by the sole increase of membrane curvature. In contrast to TGs, the presence of free SEs is favored at tubules and increasing SE levels is inhibitory to the curvature-induced nucleation of TG LDs. Finally, we found that seipin is enriched at ER tubules and controls the condensation process, preventing excessive tubule-induced nucleation. The absence of seipin provokes erratic LD nucleation events determined by the abundance of ER tubules. In summary, our data indicate that membrane curvature catalyzes LD assembly.

Keywords:  ER shaping proteins; ER tubules; endoplasmic reticulum; lipid droplet; lipid droplet biogenesis; membrane curvature; nucleation; seipin; sterol esters; triglycerides

DOI:  https://doi.org/10.1016/j.cub.2020.04.066
Biochim Biophys Acta Mol Cell Res. 2020 May 15. pii: S0167-4889(20)30099-9. [Epub ahead of print] 118741

Loss of tumor susceptibility gene 101 (TSG101) perturbs endoplasmic reticulum structure and function.

Zenia Kaul, Debdatto Mookherjee, Subhrangshu Das, Debmita Chatterjee, Saikat Chakrabarti, Oishee Chakrabarti.

  Tumor susceptibility gene 101 (TSG101), an ESCRT-I protein, is implicated in multiple cellular processes and its functional depletion can lead to blocked lysosomal degradation, cell cycle arrest, demyelination and neurodegeneration. Here, we show that loss of TSG101 results in endoplasmic reticulum (ER) stress and this causes ER membrane remodelling (EMR). This correlates with an expansion of ER, increased vacuolation, altered relative distribution of the rough and smooth ER and disruption of three-way junctions. Blocked lysosomal degradation due to TSG101 depletion leads to ER stress and Ca2+ leakage from ER stores, causing destabilization of actin cytoskeleton. Inhibiting Ca2+ release from the ER by blocking ryanodine receptors (RYRs) with Dantrolene partially rescues the ER stress phenotypes. Hence, in this study we have identified the involvement of TSG101 in modulating ER stress mediated remodelling by engaging the actin cytoskeleton. This is significant because functional depletion of TSG101 effectuates ER-stress, perturbs the structure, mobility and function of the ER, all aspects closely associated with neurodegenerative diseases. SUMMARY STATEMENT: We show that tumor susceptibility gene (TSG) 101 regulates endoplasmic reticulum (ER) stress and its membrane remodelling. Loss of TSG101 perturbs structure, mobility and function of the ER as a consequence of actin destabilization.

Keywords:  ER sheets; ER stress; ER translocon proteins; ER tubules; Reticulons; TSG101

DOI:  https://doi.org/10.1016/j.bbamcr.2020.118741
Proc Natl Acad Sci U S A. 2020 May 19. pii: 202005156. [Epub ahead of print]

KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity.

Romi Gupta, Parmanand Malvi, Keshab Raj Parajuli, Radoslav Janostiak, Suresh Bugide, Guoping Cai, Lihua Julie Zhu, Michael R Green, Narendra Wajapeyee.

  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.

Keywords:  Golgi complex; IFN-stimulated genes; KLF7; metastasis; pancreatic cancer

DOI:  https://doi.org/10.1073/pnas.2005156117
Cell Biosci. 2020 ;10 64

Synergies in exosomes and autophagy pathways for cellular homeostasis and metastasis of tumor cells.

Leila Salimi, Ali Akbari, Nassrollah Jabbari, Behnam Mojarad, Ali Vahhabi, Sławomir Szafert, Sadegh Asghari Kalashani, Hamid Soraya, Muhammad Nawaz, Jafar Rezaie.

   Background: Eukaryotic cells demonstrate two tightly linked vesicular transport systems, comprising intracellular vesicle transport and extracellular vesicle transport system. Intracellular transport vesicles can translocate biomolecules between compartments inside the cell, for example, proteins from the rough endoplasmic reticulum to the Golgi apparatus. Whereas, the secreted vesicles so-called extracellular vesicles facilitate the transport of biomolecules, for example, nucleic acids, proteins and lipids between cells. Vesicles can be formed during the process of endocytosis or/and autophagy and not only act as mediators of intra- and inter-cellular communication but also represent pathological conditions of cells or tissues.
Methods: In this review, we searched articles in PubMed, published between 2000 and 2020, with following terms: autophagy, autophagocytosis, transport vesicles, lysosomes, endosomes, exocytosis, exosomes, alone or in different combinations. The biological functions that were selected based on relevancy to our topic include cellular homeostasis and tumorigenesis.
Results: The searched literature shows that there is a high degree of synergies between exosome biogenesis and autophagy, which encompass endocytosis and endosomes, lysosomes, exocytosis and exosomes, autophagocytosis, autophagosomes and amphisomes. These transport systems not only maintain cellular homeostasis but also operate synergically against fluctuations in the external and internal environment such as during tumorigenesis and metastasis. Additionally, exosomal and autophagic proteins may serve as cancer diagnosis approaches.
Conclusion: Exosomal and autophagy pathways play pivotal roles in homeostasis and metastasis of tumor cells. Understanding the crosstalk between endomembrane organelles and vesicular trafficking may expand our insight into cooperative functions of exosomal and autophagy pathways during disease progression and may help to develop effective therapies against lysosomal diseases including cancers and beyond.

Keywords:  Autophagosomes; Autophagy; Autophagy associated tumorigenesis; Autophagy-mediated exosomes; Cancer cell metastasis; Endosomes; Extracellular vesicles

DOI:  https://doi.org/10.1186/s13578-020-00426-y
Genes (Basel). 2020 May 18. pii: E563. [Epub ahead of print]11(5):

Mitochondrial Protein Quality Control Mechanisms.

Pooja Jadiya, Dhanendra Tomar.

  Mitochondria serve as a hub for many cellular processes, including bioenergetics, metabolism, cellular signaling, redox balance, calcium homeostasis, and cell death. The mitochondrial proteome includes over a thousand proteins, encoded by both the mitochondrial and nuclear genomes. The majority (~99%) of proteins are nuclear encoded that are synthesized in the cytosol and subsequently imported into the mitochondria. Within the mitochondria, polypeptides fold and assemble into their native functional form. Mitochondria health and integrity depend on correct protein import, folding, and regulated turnover termed as mitochondrial protein quality control (MPQC). Failure to maintain these processes can cause mitochondrial dysfunction that leads to various pathophysiological outcomes and the commencement of diseases. Here, we summarize the current knowledge about the role of different MPQC regulatory systems such as mitochondrial chaperones, proteases, the ubiquitin-proteasome system, mitochondrial unfolded protein response, mitophagy, and mitochondria-derived vesicles in the maintenance of mitochondrial proteome and health. The proper understanding of mitochondrial protein quality control mechanisms will provide relevant insights to treat multiple human diseases.

Keywords:  chaperones; mitochondria; mitochondria-associated degradation; mitochondrial protein quality control; mitochondrial unfolded protein response; mitophagy; protease; proteasome; proteome; ubiquitin

DOI:  https://doi.org/10.3390/genes11050563
J Nanobiotechnology. 2020 May 19. 18(1): 77

p62/SQSTM1 accumulation due to degradation inhibition and transcriptional activation plays a critical role in silica nanoparticle-induced airway inflammation via NF-κB activation.

Yifan Wu, Yang Jin, Tianyu Sun, Piaoyu Zhu, Jinlong Li, Qinglin Zhang, Xiaoke Wang, Junkang Jiang, Gang Chen, Xinyuan Zhao.

   BACKGROUND: Most nanoparticles (NPs) reportedly block autophagic flux, thereby upregulating p62/SQSTM1 through degradation inhibition. p62 also acts as a multifunctional scaffold protein with multiple domains, and is involved in various cellular processes. However, the autophagy substrate-independent role of p62 and its regulation at the transcriptional level upon NPs exposure remain unclear.
RESULTS: In this work, we exposed BEAS-2b cells and mice to silica nanoparticles (SiNPs), and found that SiNPs increased p62 protein levels in vivo and vitro. Then, we further explored the role and mechanism of SiNPs-stimulated p62 in vitro, and found that p62 degradation was inhibited due to autophagic flux blockade. Mechanistically, SiNPs blocked autophagic flux through impairment of lysosomal capacity rather than defective autophagosome fusion with lysosomes. Moreover, SiNPs stimulated translocation of NF-E2-related factor 2 (Nrf2) to the nucleus from the cytoplasm, which upregulated p62 transcriptional activation through direct binding of Nrf2 to the p62 promoter. Nrf2 siRNA dramatically reduced both the mRNA and protein levels of p62. These two mechanisms led to p62 protein accumulation, thus increasing interleukin (IL)-1 and IL-6 expression. SiNPs activated nuclear factor kappa B (NF-κB), and this effect could be alleviated by p62 knockdown.
CONCLUSION: SiNPs caused accumulation of p62 through both pre- and post-translational mechanisms, resulting in airway inflammation. These findings improve our understanding of SiNP-induced pulmonary damage and the molecular targets available to mitigate it.

Keywords:  Airway inflammation; Autophagic flux blockade; Nrf2; Silica nanoparticle; Transcriptional activation; p62 accumulation

DOI:  https://doi.org/10.1186/s12951-020-00634-1
Proc Natl Acad Sci U S A. 2020 May 18. pii: 202000848. [Epub ahead of print]

Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders.

Yao-Cheng Li, Nikki K Lytle, Seth T Gammon, Luke Wang, Tikvah K Hayes, Margie N Sutton, Robert C Bast, Channing J Der, David Piwnica-Worms, Frank McCormick, Geoffrey M Wahl.

  HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.

Keywords:  HRAS; KRAS; NRAS; protein–protein interaction; split-luciferase complementation

DOI:  https://doi.org/10.1073/pnas.2000848117
Cell Death Differ. 2020 May 20.

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids.

Daniela Stöhr, Jens O Schmid, Tobias B Beigl, Alexandra Mack, Daniela S Maichl, Kai Cao, Beate Budai, Gavin Fullstone, Roland E Kontermann, Thomas E Mürdter, Stephen W G Tait, Cathrin Hagenlocher, Nadine Pollak, Peter Scheurich, Markus Rehm.

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

DOI: https://doi.org/10.1038/s41418-020-0559-3
J Microsc. 2020 May 19.

The plant endoplasmic reticulum: An organized chaos of tubules and sheets with multiple functions.

V Kriechbaumer, F Brandizzi.

The endoplasmic reticulum (ER) is a fascinating organelle at the core of the secretory pathway. It is responsible for the synthesis of one third of the cellular proteome and, in plant cells, it produces receptors and transporters of hormones as well as the proteins responsible for the biosynthesis of critical components of a cellulosic cell wall. The ER structure resembles a spider-web network of interconnected tubules and cisternae that pervades the cell. The study of the dynamics and interaction of this organelles with other cellular structures such as the plasma membrane, the Golgi apparatus and the cytoskeleton, have been permitted by the implementation of fluorescent protein and advanced confocal imaging. In this review, we report on the findings that contributed toward the understanding of the ER morphology and function with the aid of fluorescent proteins, focusing on the contributions provided by pioneering work from the lab of the late Professor Chris Hawes. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1111/jmi.12909
Neurosci Lett. 2020 May 18. pii: S0304-3940(20)30318-9. [Epub ahead of print] 135048

A selective role for a component of the autophagy pathway in coupling the Golgi apparatus to dendrite polarity in pyramidal neurons.

Cheryl Ligon, Yu Cai, Shilpa Buch, Jyothi Arikkath.

  Pyramidal neurons have a characteristic morphology that is critical to their ability to integrate into functional neural circuits. In addition to axon dendrite polarity, pyramidal neurons also exhibit dendritic polarity such that apical and basolateral dendrites differ in size, structure and inputs. Dendrite polarity in pyramidal neurons coincides with polarity of the Golgi apparatus, a key feature relevant to directed secretory trafficking, both in vitro and in vivo. We identify a novel autophagy based mechanism that uncouples the polarity of the Golgi apparatus from dendrite polarity. Autophagy is a universal cellular pathway that promotes cellular homeostasis via degradation of cellular components. Our data indicate that knockdown of ATG7, a key component of the autophagy mechanism, disrupts the polarity of the Golgi apparatus without impacting dendritic polarity in primary pyramidal neurons, providing the first evidence that dendrite polarity can be uncoupled from Golgi polarity. Interestingly, these effects are restricted to ATG7 knockdown and are not replicated by the knockdown of ATG16L1, another component of the autophagy mechanism. We propose that cellular mechanisms exist to couple Golgi polarity to dendrite polarity. Components of the autophagy mechanism are leveraged to actively couple Golgi polarity to dendrite polarity, thus impacting secretory trafficking into individual dendrites in pyramidal neurons.

Keywords:  Golgi polarity; dendrite polarity; pyramidal neurons

DOI:  https://doi.org/10.1016/j.neulet.2020.135048
Plant Cell Environ. 2020 May 18.

The pepper RING-type E3 ligase, CaATIR1, positively regulates ABA signaling and drought response by modulating the stability of CaATBZ1.

Hyunhee Joo, Chae Woo Lim, Sung Chul Lee.

  Protein degradation by the ubiquitin/26S proteasome system is a critical process that modulates many eukaryotic cellular processes. E3 ligase usually modulates stress response by adjusting the stability of transcription factors. Previous studies have shown that a RING-type E3 ligase, CaASRF1, positively modulates abscisic acid (ABA) signaling and ABA-mediated drought response by modulating the stability of CaAIBZ1 and CaATBZ1. In this study, we conducted yeast two-hybrid (Y2H) screening with CaATBZ1 to isolate an additional modulator, identified as CaATIR1 (Capsicum annuum ATBZ1 Interacting RING finger protein 1). CaATIR1 has E3 ligase activity and promoted CaATBZ1 degradation using the 26S proteasome system. We investigated the loss-of and gain-of functions of this E3 ligase by using silencing pepper and overexpressing Arabidopsis plants, respectively. In response to ABA and drought treatments, CaATIR1-silenced pepper plants showed ABA insensitive and drought sensitive phenotypes, while CaATIR1-overexpressing plants showed the opposite phenotypes. Additionally, CaATBZ1-silencing rescued the ABA insensitive and drought sensitive phenotypes of CaATIR1-silencing pepper plants. Taken together, these data demonstrate that the stability of CaATBZ1 mediated by CaATIR1 has a crucial role in drought stress signaling in pepper plants. This article is protected by copyright. All rights reserved.

Keywords:  E3 ligase; abscisic acid; bZIP transcription factor; drought; protein degradation; ubiquitination

DOI:  https://doi.org/10.1111/pce.13789
J Cell Biol. 2020 Aug 03. pii: e201909158. [Epub ahead of print]219(8):

Calcineurin-dependent regulation of endocytosis by a plasma membrane ubiquitin ligase adaptor, Rcr1.

Lu Zhu, Richa Sardana, Daniel K Jin, Scott D Emr.

Rsp5, the Nedd4 family member in yeast, is an E3 ubiquitin ligase involved in numerous cellular processes, many of which require Rsp5 to interact with PY-motif containing adaptor proteins. Here, we show that two paralogous transmembrane Rsp5 adaptors, Rcr1 and Rcr2, are sorted to distinct cellular locations: Rcr1 is a plasma membrane (PM) protein, whereas Rcr2 is sorted to the vacuole. Rcr2 is delivered to the vacuole using ubiquitin as a sorting signal. Rcr1 is delivered to the PM by the exomer complex using a newly uncovered PM sorting motif. Further, we show that Rcr1, but not Rcr2, is up-regulated via the calcineurin/Crz1 signaling pathway. Upon exogenous calcium treatment, Rcr1 ubiquitinates and down-regulates the chitin synthase Chs3. We propose that the PM-anchored Rsp5/Rcr1 ubiquitin ligase-adaptor complex can provide an acute response to degrade unwanted proteins under stress conditions, thereby maintaining cell integrity.

DOI: https://doi.org/10.1083/jcb.201909158
J Clin Invest. 2020 May 19. pii: 136167. [Epub ahead of print]

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy.

Rocio Bengoechea, Andrew R Findlay, Ankan K Bhadra, Hao Shao, Kevin C Stein, Sara K Pittman, Jill Daw, Jason E Gestwicki, Heather L True, Conrad C Weihl.

  Dominant mutations in the HSP70 co-chaperone DNAJB6 cause a late onset muscle disease termed limb girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70, and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of HSP70's diffusion in LGMDD1 mouse muscle was diminished likely because it has an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small molecule inhibitor of the DNAJ-HSP70 complex re-mobilized HSP70, improved strength and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counter-intuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70:DNAJB6 may be effective in treating this inherited muscular dystrophy.

Keywords:  Cell Biology; Chaperones; Muscle Biology; Protein misfolding; Skeletal muscle

DOI:  https://doi.org/10.1172/JCI136167
Mol Biol Evol. 2020 May 21. pii: msaa127. [Epub ahead of print]

Chaperone-Mediated Autophagy in the light of evolution: insight from fish.

Laury Lescat, Vincent Véron, Brigitte Mourot, Sandrine Péron, Nathalie Chenais, Karine Dias, Natàlia Riera, Florian Beaumatin, Karine Pinel, Muriel Priault, Stéphane Panserat, Bénédicte Salin, Yann Guiguen, Julien Bobe, Amaury Herpin, Iban Seiliez.

  Chaperone-Mediated-Autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in non-tetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.

Keywords:  Autophagy; CMA; Lamp2a; chaperone-mediated autophagy; evolution; fish; medaka

DOI:  https://doi.org/10.1093/molbev/msaa127
Cell Death Differ. 2020 May 18.

The proteasome activator REGγ accelerates cardiac hypertrophy by declining PP2Acα-SOD2 pathway.

Yifan Xie, Yang Gao, Rifeng Gao, Wenlong Yang, Zheng Dong, Robb E Moses, Aijun Sun, Xiaotao Li, Junbo Ge.

Pathological cardiac hypertrophy eventually leads to heart failure without adequate treatment. REGγ is emerging as 11S proteasome activator of 20S proteasome to promote the degradation of cellular proteins in a ubiquitin- and ATP-independent manner. Here, we found that REGγ was significantly upregulated in the transverse aortic constriction (TAC)-induced hypertrophic hearts and angiotensin II (Ang II)-treated cardiomyocytes. REGγ deficiency ameliorated pressure overload-induced cardiac hypertrophy were associated with inhibition of cardiac reactive oxygen species (ROS) accumulation and suppression of protein phosphatase 2A catalytic subunit α (PP2Acα) decay. Mechanistically, REGγ interacted with and targeted PP2Acα for degradation directly, thereby leading to increase of phosphorylation levels and nuclear export of Forkhead box protein O (FoxO) 3a and subsequent of SOD2 decline, ROS accumulation, and cardiac hypertrophy. Introducing exogenous PP2Acα or SOD2 to human cardiomyocytes significantly rescued the REGγ-mediated ROS accumulation of Ang II stimulation in vitro. Furthermore, treatment with superoxide dismutase mimetic, MnTBAP prevented cardiac ROS production and hypertrophy features that REGγ caused in vivo, thereby establishing a REGγ-PP2Acα-FoxO3a-SOD2 pathway in cardiac oxidative stress and hypertrophy, indicates modulating the REGγ-proteasome activity may be a potential therapeutic approach in cardiac hypertrophy-associated disorders.

DOI: https://doi.org/10.1038/s41418-020-0554-8
Elife. 2020 May 18. pii: e53531. [Epub ahead of print]9

ER-luminal [Ca2+] regulation of InsP3 receptor gating mediated by an ER-luminal peripheral Ca2+-binding protein.

Horia Vais, Min Wang, Karthik Mallilankaraman, Riley Payne, Chris McKennan, Jeffrey T Lock, Lynn A Spruce, Carly Fiest, Matthew Y Chan, Ian Parker, Steven H Seeholzer, J Kevin Foskett, Don-On Daniel Mak.

  Modulating cytoplasmic Ca2+ concentration ([Ca2+]i) by endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate receptor (InsP3R) Ca2+-release channels is a universal signaling pathway that regulates numerous cell-physiological processes. Whereas much is known regarding regulation of InsP3R activity by cytoplasmic ligands and processes, its regulation by ER-luminal Ca2+ concentration ([Ca2+]ER) is poorly understood and controversial. We discovered that the InsP3R is regulated by a peripheral membrane-associated ER-luminal protein that strongly inhibits the channel in the presence of high, physiological [Ca2+]ER. The widely-expressed Ca2+-binding protein annexin A1 (ANXA1) is present in the nuclear envelope lumen and, through interaction with a luminal region of the channel, can modify high-[Ca2+]ER inhibition of InsP3R activity. Genetic knockdown of ANXA1 expression enhanced global and local elementary InsP3-mediated Ca2+ signaling events. Thus, [Ca2+]ER is a major regulator of InsP3R channel activity and InsP3R-mediated [Ca2+]i signaling in cells by controlling an interaction of the channel with a peripheral membrane-associated Ca2+-binding protein, likely ANXA1.

Keywords:  molecular biophysics; none; structural biology

DOI:  https://doi.org/10.7554/eLife.53531
Front Microbiol. 2020 ;11 790

Autophagy Participates in Lysosomal Vacuolation-Mediated Cell Death in RGNNV-Infected Cells.

Youhua Huang, Ya Zhang, Zetian Liu, Chuanhe Liu, Jiaying Zheng, Qiwei Qin, Xiaohong Huang.

  Nervous necrosis virus (NNV) is the etiological agent of viral nervous necrosis (VNN), also known as viral encephalopathy and retinopathy (VER), which results in heavy economic losses to the aquaculture industry worldwide. Dramatic cytoplasmic vacuoles were observed during NNV infection both in vitro and in vivo; however, the origin and mechanism of cytoplasmic vacuolization remains unknown. In this report, we found that the cytoplasmic vacuole morphology became fused and enlarged during infection with red spotted grouper nervous necrosis virus (RGNNV), which was accompanied by increased cell death. Notably, Lyso-Tracker, but not Mito-Tracker or ER-Tracker, was accumulated in the vacuoles, and abnormal lysosome swelling was observed in RGNNV-infected cells, suggesting that the cytoplasmic vacuoles originated from lysosomal organelles. Cytoplasmic vacuolization and cell death in RGNNV-infected cells was completely blocked by the vacuolar H+-ATPase inhibitor (bafilomycin A1), and was significantly weakened by chloroquine (CQ), a lysosomotropic agent that induces the acidification of the lysosomes. This suggests that lysosome acidification was essential for vacuole formation. Significant inhibitory effects on vacuolization and cell death were also observed in the RGNNV-infected cells following treatment with nigericin and monensin (ionophores that uncouple the proton gradient present in lysosomes). This indicated that lysosome function was tightly associated with RGNNV infection-induced cell death. In addition, vacuoles were found to be partially co-localized with GFP-LC3II punctate dots during RGNNV infection. Moreover, the severity of vacuolization and cell death were both significantly decreased after treatment with the autophagy inhibitor, 3-MA, suggesting that autophagy was involved in lysosomal vacuolization and cell death evoked by RGNNV infection. Thus, our results demonstrate that autophagy participates in lysosomal vacuolation-mediated cell death during RGNNV infection, and provides new insight into our understanding of the potential mechanisms underlying nodavirus pathogenesis in vitro.

Keywords:  RGNNV; autophagy; cell death; lysosome; vacuolization

DOI:  https://doi.org/10.3389/fmicb.2020.00790
Molecules. 2020 May 18. pii: E2352. [Epub ahead of print]25(10):

Mechanisms Regulating the UPS-ALS Crosstalk: The Role of Proteaphagy.

Grégoire Quinet, Maria Gonzalez-Santamarta, Clara Louche, Manuel S Rodriguez.

  Protein degradation is tightly regulated inside cells because of its utmost importance for protein homeostasis (proteostasis). The two major intracellular proteolytic pathways are the ubiquitin-proteasome and the autophagy-lysosome systems which ensure the fate of proteins when modified by various members of the ubiquitin family. These pathways are tightly interconnected by receptors and cofactors that recognize distinct chain architectures to connect with either the proteasome or autophagy under distinct physiologic and pathologic situations. The degradation of proteasome by autophagy, known as proteaphagy, plays an important role in this crosstalk since it favours the activity of autophagy in the absence of fully active proteasomes. Recently described in several biological models, proteaphagy appears to help the cell to survive when proteostasis is broken by the absence of nutrients or the excess of proteins accumulated under various stress conditions. Emerging evidence indicates that proteaphagy could be permanently activated in some types of cancer or when chemoresistance is observed in patients.

Keywords:  autophagy; pathology; proteaphagy; ubiquitin proteasome system; ubiquitin-like

DOI:  https://doi.org/10.3390/molecules25102352
Traffic. 2020 May 18.

The hypothetical role of Phosphatidic Acid in subverting ER membranes during SARS-CoV infection.

María Moriel-Carretero.

  Positive sense (+) RNA viruses exploit membranes from a variety of cellular organelles to support the amplification of their genomes. This association concurs with the formation of vesicles whose main morphological feature is that of being wrapped by a double membrane. In the case of the SARS-CoV virus, the outer membrane is not discrete for each vesicle, but seems to be continuous and shared between many individual vesicles, a difference with other +RNA viruses whose nature has remained elusive. I present morphological, biochemical and pharmacological arguments defending the striking analogy of this arrangement and that of entangled, nascent Lipid Droplets whose birth has been aborted by an excess of Phosphatidic Acid. Since Phosphatidic Acid can be targeted with therapeutical purposes, considering this working hypothesis may prove important in tackling SARS-CoV infection. This article is protected by copyright. All rights reserved.

Keywords:  Endoplasmic Reticulum; Lipid Droplets; Phosphatidic Acid; SARS-CoV; membrane subversion

DOI:  https://doi.org/10.1111/tra.12738
J Cell Biol. 2020 Jul 06. pii: e201912104. [Epub ahead of print]219(7):

Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling.

Paurav B Desai, Michael W Stuck, Bo Lv, Gregory J Pazour.

In the absence of Hedgehog ligand, patched-1 (Ptch1) localizes to cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon ligand binding, Ptch1 is removed from cilia, and Smo is derepressed and accumulates in cilia where it activates signaling. The mechanisms regulating these dynamic movements are not well understood, but defects in intraflagellar transport components, including Ift27 and the BBSome, cause Smo to accumulate in cilia without pathway activation. We find that in the absence of ligand-induced pathway activation, Smo is ubiquitinated and removed from cilia, and this process is dependent on Ift27 and BBSome components. Activation of Hedgehog signaling decreases Smo ubiquitination and ciliary removal, resulting in its accumulation. Blocking ubiquitination of Smo by an E1 ligase inhibitor or by mutating two lysine residues in intracellular loop three causes Smo to aberrantly accumulate in cilia without pathway activation. These data provide a mechanism to control Smo's ciliary level during Hedgehog signaling by regulating the ubiquitination state of the receptor.

DOI: https://doi.org/10.1083/jcb.201912104
RNA. 2020 May 18. pii: rna.074047.119. [Epub ahead of print]

Adaptor protein RapZ activates endoribonuclease RNase E by protein-protein interaction to cleave a small regulatory RNA.

Svetlana Durica-Mitic, Yvonne Göpel, Fabian Amman, Boris Görke.

  In Escherichia coli, endoribonuclease RNase E initiates degradation of many RNAs and represents a hub for post-transcriptional regulation. The tetrameric adaptor protein RapZ targets the small regulatory RNA GlmZ to degradation by RNase E. RapZ binds GlmZ through a domain located at the C-terminus and interacts with RNase E, promoting GlmZ cleavage in the base-pairing region. When necessary, cleavage of GlmZ is counteracted by the homologous small RNA GlmY, which sequesters RapZ through molecular mimicry. In the current study, we addressed the molecular mechanism employed by RapZ. We show that RapZ mutants impaired in RNA-binding but proficient in binding RNase E are able to stimulate GlmZ cleavage in vivo and in vitro when provided at increased concentrations. In contrast, a truncated RapZ variant retaining RNA-binding activity but incapable of contacting RNase E lacks this activity. In agreement, we find that tetrameric RapZ binds the likewise tetrameric RNase E through direct interaction with its large globular domain within the catalytic N-terminus, independent of RNA. Although RapZ stimulates cleavage of at least one non-cognate RNA by RNase E in vitro, its activity is restricted to GlmZ in vivo as revealed by RNA sequencing, suggesting that certain features within the RNA substrate are also required for cleavage. In conclusion, RapZ boosts RNase E activity through interaction with its catalytic domain, which represents a novel mechanism of RNase E activation. In contrast, RNA-binding has a recruiting role, increasing the likelihood that productive RapZ/GlmZ/RNase E complexes form.

Keywords:  RNA-binding protein; adaptor protein RapZ; endoribonuclease RNase E; regulated RNA degradation; small RNA GlmZ

DOI:  https://doi.org/10.1261/rna.074047.119
J Cell Biol. 2020 Jun 01. pii: e202005084. [Epub ahead of print]219(6):

Mitochondrial translation, dynamics, and lysosomes combine to extend lifespan.

Levi Ali, Cole M Haynes.

In this issue, Liu et al. (2019. J. Cell. Biol.https://doi.org/10.1083/jcb.201907067) find that the inhibition of mitochondrial ribosomes in combination with impaired mitochondrial fission or fusion increases C. elegans lifespan by activating the transcription factor HLH-30, which promotes lysosomal biogenesis.

DOI: https://doi.org/10.1083/jcb.202005084
Leukemia. 2020 May 18.

Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13.

Ting Du, Yan Song, Arghya Ray, Dharminder Chauhan, Kenneth C Anderson.

Our prior study showed that inhibition of 19S proteasome-associated ubiquitin receptor Rpn13 can overcome bortezomib resistance in MM cells. Here, we performed proteomic analysis of Rpn13 inhibitor (RA190)-treated MM cells and identified an antioxidant enzyme superoxide dismutase (SOD1) as a mediator of Rpn13 signaling. SOD1 levels are higher in MM patient cells versus normal PBMCs; and importantly, SOD1 expression correlates with the progression of disease and shorter survival. Functional validation studies show that RA190-induced cytotoxicity in bortezomib-sensitive and -resistant MM cells is associated with decrease in SOD1 levels; conversely, forced expression of SOD1 inhibits RA190-induced cell death. Genetic knockdown and biochemical blockade of SOD1 with LCS-1 sensitizes bortezomib-resistant MM cells to bortezomib. SOD1 inhibitor LCS-1 decreases viability in MM cell lines and patient cells. LCS-1-induced cell death is associated with: (1) increase in superoxide and ROS levels; (2) activation of caspases, and p53/p21 signaling; (3) decrease in MCL-1, BCLxL, CDC2, cyclin-B1, and c-Myc; (4) ER stress response; and (5) inhibition of proteasome function. In animal model studies, LCS-1 inhibits xenografted bortezomib-resistant human MM cell growth and prolongs host survival. Our studies therefore show that targeting Rpn13 overcomes bortezomib resistance by decreasing cellular SOD1 levels, and provide the rationale for novel therapeutics targeting SOD1 to improve patient outcome in MM.

DOI: https://doi.org/10.1038/s41375-020-0865-2
Biochem Biophys Res Commun. 2020 May 15. pii: S0006-291X(20)30903-7. [Epub ahead of print]

UGGT1 retains proinsulin in the endoplasmic reticulum in an arginine dependent manner.

Jaeyong Cho, Masaki Hiramoto, Yuka Masaike, Satoshi Sakamoto, Yoichi Imai, Yumi Imai, Hiroshi Handa, Takeshi Imai.

  We sought to clarify a pathway by which L- and dD-arginine simulate insulin secretion in mice and cell lines and obtained the following novel two findings. (1) Using affinity magnetic nanobeads technology, we identified that proinsulin is retained in the endoplasmic reticulum (ER) through UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) when arginine availability is limited. (2) L- and d-arginine release proinsulin from UGGT1 through competition with proinsulin and promote exit of proinsulin from the ER to Golgi apparatus. The ability of arginine to release proinsulin from UGGT1 closely correlates with arginine-induced insulin secretion in several models of β cells indicating that UGGT1-proinsulin interaction regulates arginine-induced insulin secretion.

Keywords:  Arginine; Endoplasmic; Insulin; Reticulum; UGGT1

DOI:  https://doi.org/10.1016/j.bbrc.2020.04.158
PLoS One. 2020 ;15(5): e0233231

Small heat shock proteins determine synapse number and neuronal activity during development.

Elena Santana, Teresa de Los Reyes, Sergio Casas-Tintó.

Environmental changes cause stress, Reactive Oxygen Species and unfolded protein accumulation which hamper synaptic activity and trigger cell death. Heat shock proteins (HSPs) assist protein refolding to maintain proteostasis and cellular integrity. Mechanisms regulating the activity of HSPs include transcription factors and posttranslational modifications that ensure a rapid response. HSPs preserve synaptic function in the nervous system upon environmental insults or pathological factors and contribute to the coupling between environmental cues and neuron control of development. We have performed a biased screening in Drosophila melanogaster searching for synaptogenic modulators among HSPs during development. We explore the role of two small-HSPs (sHSPs), sHSP23 and sHSP26 in synaptogenesis and neuronal activity. Both sHSPs immunoprecipitate together and the equilibrium between both chaperones is required for neuronal development and activity. The molecular mechanism controlling HSP23 and HSP26 accumulation in neurons relies on a novel gene (CG1561), which we name Pinkman (pkm). We propose that sHSPs and Pkm are targets to modulate the impact of stress in neurons and to prevent synapse loss.

DOI: https://doi.org/10.1371/journal.pone.0233231
Antioxid Redox Signal. 2020 May 22.

Honokiol bis-dichloroacetate is a selective allosteric inhibitor of the mitochondrial chaperone TRAP1.

Carlos Sanchez-Martin, Daniela Menon, Elisabetta Moroni, Mariarosaria Ferraro, Ionica Masgras, Justin Elsey, Jack L Arbiser, Giorgio Colombo, Andrea Rasola.

INTRODUCTION: TRAP1, the mitochondrial paralog of the Hsp90 family of molecular chaperones, is required for neoplastic growth in several tumor cell models, where it inhibits succinate dehydrogenase (SDH) activity, thus favoring bioenergetic rewiring, maintenance of redox homeostasis and orchestration of a HIF1α-mediated pseudohypoxic program. Development of selective TRAP1 inhibitors is instrumental for targeted development of anti-neoplastic drugs, but was hampered up to now by the high degree of homology among catalytic pockets of Hsp90 family members. The vegetal derivative honokiol and its lipophilic bis-dichloroacetate ester, honokiol DCA (HDCA), are small molecule compounds with antineoplastic activity. HDCA leads to oxidative stress and apoptosis in in vivo tumor models and displays an action that is functionally opposed to that of TRAP1, as it induces both SDH and the mitochondrial deacetylase SIRT3, which further enhances SDH activity.
AIMS: We investigated whether HDCA could interact with TRAP1 inhibiting its chaperone function, and the effects of HDCA on tumor cells harboring TRAP1.
RESULTS: An allosteric binding site in TRAP1 is able to host HDCA, which inhibits TRAP1 but not Hsp90 ATPase activity. In neoplastic cells, HDCA reverts TRAP1-dependent downregulation of SDH, decreases proliferation rate, increases mitochondrial superoxide levels and abolishes tumorigenic growth.
INNOVATION: HDCA is a potential lead compound for the generation of anti-neoplastic approaches based on the allosteric inhibition of TRAP1 chaperone activity.
CONCLUSIONS: We have identified a selective TRAP1 inhibitor that can be used to better dissect TRAP1 biochemical functions and to tailor novel tumor-targeting strategies.

DOI: https://doi.org/10.1089/ars.2019.7972
Exp Cell Res. 2020 May 19. pii: S0014-4827(20)30344-X. [Epub ahead of print] 112100

USP9X prevents AGEs-induced upregulation of FN and TGF-β1 through activating Nrf2-ARE pathway in rat glomerular mesangial cells.

Kaipeng Huang, Xilin Zhao.

  Oxidative stress is a key pathological factor for diabetic renal fibrosis by activating TGF-β/Smad pathway in glomerular mesangial cells (GMCs) to promote the synthesis of extracellular matrix such as fibronectin (FN). Nuclear factor-E2-related factor (Nrf2)- anti-oxidant response element (ARE) anti-oxidative pathway has crucial renoprotective effects, and inhibiting ubiquitin-mediated degradation of Nrf2 delays diabetic renal fibrosis development. Ubiquitin-specific protease 9X (USP9X) has close relationship with oxidative stress and TGF-β/Smad pathway, but whether it regulate diabetic renal fibrosis remains unclarified. Here, we found that advanced glycation-end products (AGEs) dose- and time-dependently reduced the protein expression and deubiquitinase activity of USP9X in GMCs. USP9X overexpression attenuated AGEs-induced upregulation of FN, TGF-β1, and Collagen Ⅳ, three fibrosis-related marker proteins, in a deubiquitinase activity-dependent manner. While USP9X depletion with siRNAs further promoted the expressions of those proteins in AGEs-treated GMCs. Under AGEs treatment conditions, USP9X overexpression markedly increased the total and nuclear levels, ARE-binding ability, and transcriptional activity of Nrf2, upregulated the protein expressions of Nrf2 downstream genes HO-1 and NQO1, and eventually reduced the excessive production of ROS. Overexpression of the deubiquitinase catalytically inactive USP9X-C1556S mutant failed to exert such effects. Silencing Nrf2 abolished the renoprotective effects of USP9X. Further study showed that upon AGEs stimulation, Nrf2 transferred into the nucleus and the interaction between USP9X and Nrf2 was weakened. AGEs also increased Nrf2 ubiquitination level, and overexpression of USP9X, instead of USP9X-C1556S, significantly reduced the ubiquitination level of Nrf2. Taken together, USP9X reduced Nrf2 ubiquitination level and promoted Nrf2-ARE pathway activation to prevent the accumulation of extracellular matrix, eventually alleviated the pathological process of diabetic renal fibrosis.

Keywords:  Diabetic renal fibrosis; Nrf2-ARE pathway; Oxidative stress; USP9X

DOI:  https://doi.org/10.1016/j.yexcr.2020.112100
Brain Res. 2020 May 19. pii: S0006-8993(20)30255-9. [Epub ahead of print] 146899

Editorial: Stem cell secretome.

Joshua D Rieskamp, Elizabeth D Kirby.

Stem cells have the potential to advance therapy for many neurological diseases that are currently refractive to treatment. They are also key cellular players in homeostasis within several adult brain regions that host endogenous populations of neural stem cells. Investigations of the functions of stem cells in the adult CNS have historically approached these cells as sources of differentiated progeny, whether it be new neurons or new glial cells. Yet, as both basic research and pre-clinical efforts centered on stem cells in the brain push forward, it has become evident that this initial framework is incomplete. Emerging evidence indicates that stem and progenitor cells from a variety of tissues can regulate their microenvironment through production of secreted factors. This special issue highlights work investigating the role of the neural and non-neural stem cell secretome in regulating CNS function. These studies represent efforts both to more fully delineate the suite of factors secreted by stem cells and to evaluate its impact on CNS health and disease. Together, they demonstrate a broad potential for stem cell function through secreted proteins that urges continued basic and translational research in the years to come.

DOI: https://doi.org/10.1016/j.brainres.2020.146899