bims-proteo 2020-07-19 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020–07–19
thirty-one papers selected by
Eric Chevet, INSERM

Endoplasmic reticulum stress and liver diseases.
Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation.
Mitochondria-Associated Degradation Pathway (MAD) Function beyond the Outer Membrane.
Phosphorylation within the bipartite NLS alters the localization and toxicity of the ER stress response factor DDIT3/CHOP.
ATM-associated signalling triggers the unfolded protein response and cell death in response to stress.
The integrated stress response induces R-loops and hinders replication fork progression.
POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation.
A paralog-specific role of COPI vesicles in the neuronal differentiation of mouse pluripotent cells.
Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance.
SUMO: From Bench to Bedside.
Off and On Again: De- and Reubiquitination during Membrane Protein Degradation.
Endoplasmic reticulum stress-induced exosomal miR-27a-3p promotes immune escape in breast cancer via regulating PD-L1 expression in macrophages.
The Zscan4-Tet2 Transcription Nexus Regulates Metabolic Rewiring and Enhances Proteostasis to Promote Reprogramming.
The Cdc48 Complex Alleviates the Cytotoxicity of Misfolded Proteins by Regulating Ubiquitin Homeostasis.
Mitophagy, Mitochondrial Homeostasis, and Cell Fate.
Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.
Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.
Endoplasmic Reticulum Stress in Macrophages: The Vicious Circle of Lipid Accumulation and Pro-Inflammatory Response.
eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.
Autophagy regulates the localization and degradation of p16INK4a.
Uev1A amino terminus stimulates poly-ubiquitin chain assembly and is required for NF-κB activation.
Adenovirus-mediated ubiquitination alters protein-RNA binding and aids viral RNA processing.
Modeling UBQLN2-mediated neurodegenerative disease in mice: Shared and divergent properties of wild type and mutant UBQLN2 in phase separation, subcellular localization, altered proteostasis pathways, and selective cytotoxicity.
Necdin regulates BMAL1 stability and circadian clock through SGT1-HSP90 chaperone machinery.
Aging is associated with increased TRB3, ER stress, and hepatic glucose production in the liver of rats.
Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum-to-mitochondria Ca2+ transfer for survival.
Ribosome collisions trigger cis-acting feedback inhibition of translation initiation.
The deubiquitinase USP22 regulates PD-L1 degradation in human cancer cells.
MIB1-mediated degradation of WRN promotes cellular senescence in response to camptothecin treatment.
Ribosome profiling reveals a functional role for autophagy in mRNA translational control.
Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Liver Res. 2019 Mar;3(1): 55-64

Endoplasmic reticulum stress and liver diseases.

Xiaoying Liu, Richard M Green.

  Endoplasmic reticulum (ER) stress occurs when ER homeostasis is perturbed with accumulation of unfolded/misfolded protein or calcium depletion. The unfolded protein response (UPR), comprising of inositol-requiring enzyme 1α (IRE1α), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6) signaling pathways, is a protective cellular response activated by ER stress. However, UPR activation can also induce cell death upon persistent ER stress. The liver is susceptible to ER stress given its synthetic and other biological functions. Numerous studies from human liver samples and animal disease models have indicated a crucial role of ER stress and UPR signaling pathways in the pathogenesis of liver diseases, including non-alcoholic fatty liver disease, alcoholic liver disease, alpha-1 antitrypsin deficiency, cholestatic liver disease, drug-induced liver injury, ischemia/reperfusion injury, viral hepatitis and hepatocellular carcinoma. Extensive investigations have demonstrated the potential underlying mechanisms of the induction of ER stress and the contribution of UPR pathways during the development of the diseases. Moreover ER stress and the UPR proteins and genes have become emerging therapeutic targets to treat liver diseases.

Keywords:  ATF6; ER stress; IRE1α; PERK; UPR; liver diseases

DOI:  https://doi.org/10.1016/j.livres.2019.01.002
J Cell Sci. 2020 Jul 14. pii: jcs.246983. [Epub ahead of print]

Pex24 and Pex32 are required to tether peroxisomes to the ER for organelle biogenesis, positioning and segregation.

Fei Wu, Rinse de Boer, Arjen M Krikken, Arman Akşit, Nicola Bordin, Damien P Devos, Ida J van der Klei.

  The yeast Hansenula polymorpha contains four members of the Pex23 family of peroxins, which characteristically contain a DysF domain. Here we show that all four H. polymorpha Pex23 family proteins localize to the ER. Pex24 and Pex32, but not Pex23 and Pex29, predominantly accumulate at peroxisome-ER contacts. Upon deletion of PEX24 or PEX32 - and to a much lesser extent of PEX23 or PEX29 - peroxisome-ER contacts are lost, concomitant with defects in peroxisomal matrix protein import, membrane growth, organelle proliferation, positioning and segregation. These defects are suppressed by the introduction of an artificial peroxisome-ER tether, indicating that Pex24 and Pex32 contribute to tethering peroxisomes to the ER. Accumulation of Pex32 at these contact sites is lost in cells lacking the peroxisomal membrane protein Pex11 in conjunction with disruption of the contacts. This indicates that Pex11 contributes to Pex32-dependent peroxisome-ER contact formation. The absence of Pex32 has no major effect on pre-peroxisomal vesicles that occur in pex3 atg1 cells.

Keywords:  Endoplasmic reticulum; Membrane contact; Peroxisome; Pex24; Pex32; Yeast

DOI:  https://doi.org/10.1242/jcs.246983
Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30883-4. [Epub ahead of print]32(2): 107902

Mitochondria-Associated Degradation Pathway (MAD) Function beyond the Outer Membrane.

Pin-Chao Liao, Dana M Alessi Wolken, Edith Serrano, Pallavi Srivastava, Liza A Pon.

  The mitochondria-associated degradation pathway (MAD) mediates ubiquitination and degradation of mitochondrial outer membrane (MOM) proteins by the proteasome. We find that the MAD, but not other quality-control pathways including macroautophagy, mitophagy, or mitochondrial chaperones and proteases, is critical for yeast cellular fitness under conditions of paraquat (PQ)-induced oxidative stress in mitochondria. Specifically, inhibition of the MAD increases PQ-induced defects in growth and mitochondrial quality and decreases chronological lifespan. We use mass spectrometry analysis to identify possible MAD substrates as mitochondrial proteins that exhibit increased ubiquitination in response to PQ treatment and inhibition of the MAD. We identify candidate substrates in the mitochondrial matrix and inner membrane and confirm that two matrix proteins are MAD substrates. Our studies reveal a broader function for the MAD in mitochondrial protein surveillance beyond the MOM and a major role for the MAD in cellular and mitochondrial fitness in response to chronic, low-level oxidative stress in mitochondria.

Keywords:  Saccharomyces cerevisiae; chronological lifespan; mitochondrial quality control; mitophagy; oxidative stress; paraquat; proteasome; proteostasis; reactive oxygen species; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2020.107902
Cell Signal. 2020 Jul 13. pii: S0898-6568(20)30190-X. [Epub ahead of print] 109713

Phosphorylation within the bipartite NLS alters the localization and toxicity of the ER stress response factor DDIT3/CHOP.

Jonathan C Bartko, Yinghui Li, Zheng Sun, Marc W Halterman.

  Regulated nuclear-cytoplasmic trafficking is a well-established mechanism utilized by cells to regulate adaptive and maladaptive responses to acute oxidant stress. Commonly associated with endoplasmic reticulum stress, the bZIP transcription factor CCAAT/enhancer-binding protein homologous protein (CHOP/DDIT3) mediates the cellular response to redox stress with effects on cellular growth, differentiation, and survival. We show through functional analyses that CHOP contains a conserved, compound pat4/bipartite nuclear localization signal within the basic DNA-binding domain. Using phylogenetic analyses and mass spectrometry, we now show that Ser107 located within the linker region of the bipartite NLS domain is a substrate for phosphorylation under standard culture conditions. Studies using the S107E phospho-mimic of CHOP indicate that changes in the charge properties at this residue regulate CHOP's nuclear-to-cytoplasmic ratio. And while co-stimulation with the SERCA inhibitor thapsigargin induced injury in cells expressing wild-type CHOP, the S107A point-mutant blocked this response. These findings indicate that phosphorylation within the bipartite NLS exerts regulatory effects on both the subcellular localization and toxic potential of DDIT3/CHOP. Future studies geared towards defining the relevant kinase/phosphatase networks that converge on the phosphorylation-regulated NLS (prNLS) phosphoepitope may provide an opportunity to constrain cellular damage in the context of acute ER stress.

Keywords:  Apoptosis; DDIT3/CHOP; Growth arrest; Nuclear localization signal; Nuclear-cytoplasmic transport; Post-translational modification

DOI:  https://doi.org/10.1016/j.cellsig.2020.109713
Commun Biol. 2020 Jul 14. 3(1): 378

ATM-associated signalling triggers the unfolded protein response and cell death in response to stress.

Yuka Hotokezaka, Ikuo Katayama, Takashi Nakamura.

Endoplasmic reticulum (ER) stress can be caused by perturbations in ER function resulting from the accumulation of unfolded/misfolded proteins in the ER lumen. Accumulating unfolded proteins trigger unfolded protein responses (UPRs) through activating three transmembrane sensors on the ER: IRE1α, PERK, and ATF6. The orchestrated action of these molecules upregulates genes encoding proteins involved in the downregulation of protein synthesis and acceleration of protein secretion. Ineffectiveness of these fail-safe mechanisms may lead to apoptosis. However, the molecular mechanisms upstream of the UPR are not fully understood. Here we show participation of ataxia telangiectasia mutated (ATM) in stress-induced apoptosis. Cytoplasmic ATM serves as a platform on which protein phosphatase 2A-dependent dephosphorylation of AKT activates glycogen synthase kinase 3β, thereby downregulating nascent polypeptide-associated complex α subunit and γ-taxilin, triggering UPRs and leading to mitochondria-dependent apoptosis. These results suggest an ATM/AKT-dependent cell death pathway triggered by various forms of stress.

DOI: https://doi.org/10.1038/s42003-020-1102-2
Cell Death Dis. 2020 Jul 16. 11(7): 538

The integrated stress response induces R-loops and hinders replication fork progression.

Josephine Ann Mun Yee Choo, Denise Schlösser, Valentina Manzini, Anna Magerhans, Matthias Dobbelstein.

The integrated stress response (ISR) allows cells to rapidly shutdown most of their protein synthesis in response to protein misfolding, amino acid deficiency, or virus infection. These stresses trigger the phosphorylation of the translation initiation factor eIF2alpha, which prevents the initiation of translation. Here we show that triggering the ISR drastically reduces the progression of DNA replication forks within 1 h, thus flanking the shutdown of protein synthesis with immediate inhibition of DNA synthesis. DNA replication is restored by compounds that inhibit eIF2alpha kinases or re-activate eIF2alpha. Mechanistically, the translational shutdown blocks histone synthesis, promoting the formation of DNA:RNA hybrids (R-loops), which interfere with DNA replication. R-loops accumulate upon histone depletion. Conversely, histone overexpression or R-loop removal by RNaseH1 each restores DNA replication in the context of ISR and histone depletion. In conclusion, the ISR rapidly stalls DNA synthesis through histone deficiency and R-loop formation. We propose that this shutdown mechanism prevents potentially detrimental DNA replication in the face of cellular stresses.

DOI: https://doi.org/10.1038/s41419-020-2727-2
Protein Cell. 2020 Jul 14.

POST1/C12ORF49 regulates the SREBP pathway by promoting site-1 protease maturation.

Jian Xiao, Yanni Xiong, Liu-Ting Yang, Ju-Qiong Wang, Zi-Mu Zhou, Le-Wei Dong, Xiong-Jie Shi, Xiaolu Zhao, Jie Luo, Bao-Liang Song.

  Sterol-regulatory element binding proteins (SREBPs) are the key transcriptional regulators of lipid metabolism. The activation of SREBP requires translocation of the SREBP precursor from the endoplasmic reticulum to the Golgi, where it is sequentially cleaved by site-1 protease (S1P) and site-2 protease and releases a nuclear form to modulate gene expression. To search for new genes regulating cholesterol metabolism, we perform a genome-wide CRISPR/Cas9 knockout screen and find that partner of site-1 protease (POST1), encoded by C12ORF49, is critically involved in the SREBP signaling. Ablation of POST1 decreases the generation of nuclear SREBP and reduces the expression of SREBP target genes. POST1 binds S1P, which is synthesized as an inactive protease (form A) and becomes fully mature via a two-step autocatalytic process involving forms B'/B and C'/C. POST1 promotes the generation of the functional S1P-C'/C from S1P-B'/B (canonical cleavage) and, notably, from S1P-A directly (non-canonical cleavage) as well. This POST1-mediated S1P activation is also essential for the cleavages of other S1P substrates including ATF6, CREB3 family members and the α/β-subunit precursor of N-acetylglucosamine-1-phosphotransferase. Together, we demonstrate that POST1 is a cofactor controlling S1P maturation and plays important roles in lipid homeostasis, unfolded protein response, lipoprotein metabolism and lysosome biogenesis.

Keywords:  SREBP; activating transcription factor 6; mannose-6-phosphate; proteolytic activation; site-1 protease; unfolded protein response

DOI:  https://doi.org/10.1007/s13238-020-00753-3
Life Sci Alliance. 2020 Sep;pii: e202000714. [Epub ahead of print]3(9):

A paralog-specific role of COPI vesicles in the neuronal differentiation of mouse pluripotent cells.

Manu Jain Goyal, Xiyan Zhao, Mariya Bozhinova, Karla Andrade-López, Cecilia de Heus, Sandra Schulze-Dramac, Michaela Müller-McNicoll, Judith Klumperman, Julien Béthune.

Coat protein complex I (COPI)-coated vesicles mediate membrane trafficking between Golgi cisternae as well as retrieval of proteins from the Golgi to the endoplasmic reticulum. There are several flavors of the COPI coat defined by paralogous subunits of the protein complex coatomer. However, whether paralogous COPI proteins have specific functions is currently unknown. Here, we show that the paralogous coatomer subunits γ1-COP and γ2-COP are differentially expressed during the neuronal differentiation of mouse pluripotent cells. Moreover, through a combination of genome editing experiments, we demonstrate that whereas γ-COP paralogs are largely functionally redundant, γ1-COP specifically promotes neurite outgrowth. Our work stresses a role of the COPI pathway in neuronal polarization and provides evidence for distinct functions for coatomer paralogous subunits in this process.

DOI: https://doi.org/10.26508/lsa.202000714
J Biol Chem. 2020 Jul 15. pii: jbc.RA120.013519. [Epub ahead of print]

Ubiquitin ligase SMURF2 enhances epidermal growth factor receptor stability and tyrosine-kinase inhibitor resistance.

Paramita Ray, Krishnan Raghunathan, Aarif Ahsan, Uday Sankar Allam, Shirish Shukla, Venkatesha Basrur, Sarah Veatch, Theodore S Lawrence, Mukesh K Nyati, Dipankar Ray.

  The discovery of activating epidermal growth factor receptor (EGFR) mutations spurred the use of EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib, as the first-line treatment of lung cancers. We previously reported that differential degradation of TKI-sensitive (e.g. L858R) and resistant (T790M) EGFR mutants upon erlotinib treatment correlates with drug sensitivity. We also reported that SMAD ubiquitination regulatory factor 2 (SMURF2) ligase activity is important in stabilizing EGFR. However, the molecular mechanisms involved remain unclear. Here, using in vitro and in vivo ubiquitination assays, mass spectrometry, and super-resolution microscopy, we show SMURF2-EGFR functional interaction is important for EGFR stability and response to TKI. We demonstrate that L858R/T790M EGFR is preferentially stabilized by SMURF2-UBCH5 (an E3-E2) mediated polyubiquitination. We identified four lysine residues as the sites of ubiquitination, and showed that replacement of one of them with acetylation-mimicking glutamine increases the sensitivity of mutant EGFR to erlotinib-induced degradation. We show that SMURF2 extends membrane retention of EGF bound EGFR, whereas SMURF2 knockdown increases receptor sorting to lysosomes. In lung cancer cell lines, SMURF2 overexpression increased EGFR levels, improving TKI tolerance, while SMURF2 knockdown decreased EGFR steady state levels and sensitized lung cancer cells. Overall, we propose that SMURF2-mediated polyubiquitination of L858R/T790M EGFR may be competing with acetylation-mediated receptor internalization that correlates with enhanced receptor stability, therefore, disruption of the E3-E2 complex may be an attractive target to overcome TKI resistance.

Keywords:  E3 ubiquitin ligase; Epidermal growth factor receptor (EGFR); Smad ubiquitination regulatory factor 2 (SMURF2); Ubiquitin conjugating enzyme H5 (UBCH5); epidermal growth factor receptor (EGFR); protective ubiquitination; receptor regulation; tyrosine kinase inhibitor (TKI) resistance; tyrosine-protein kinase (tyrosine kinase); ubiquitylation (ubiquitination)

DOI:  https://doi.org/10.1074/jbc.RA120.013519
Physiol Rev. 2020 Oct 01. 100(4): 1599-1619

SUMO: From Bench to Bedside.

Hui-Ming Chang, Edward T H Yeh.

  Sentrin/small ubiquitin-like modifier (SUMO) is protein modification pathway that regulates multiple biological processes, including cell division, DNA replication/repair, signal transduction, and cellular metabolism. In this review, we will focus on recent advances in the mechanisms of disease pathogenesis, such as cancer, diabetes, seizure, and heart failure, which have been linked to the SUMO pathway. SUMO is conjugated to lysine residues in target proteins through an isopeptide linkage catalyzed by SUMO-specific activating (E1), conjugating (E2), and ligating (E3) enzymes. In steady state, the quantity of SUMO-modified substrates is usually a small fraction of unmodified substrates due to the deconjugation activity of the family Sentrin/SUMO-specific proteases (SENPs). In contrast to the complexity of the ubiquitination/deubiquitination machinery, the biochemistry of SUMOylation and de-SUMOylation is relatively modest. Specificity of the SUMO pathway is achieved through redox regulation, acetylation, phosphorylation, or other posttranslational protein modification of the SUMOylation and de-SUMOylation enzymes. There are three major SUMOs. SUMO-1 usually modifies a substrate as a monomer; however, SUMO-2/3 can form poly-SUMO chains. The monomeric SUMO-1 or poly-SUMO chains can interact with other proteins through SUMO-interactive motif (SIM). Thus SUMO modification provides a platform to enhance protein-protein interaction. The consequence of SUMOylation includes changes in cellular localization, protein activity, or protein stability. Furthermore, SUMO may join force with ubiquitin to degrade proteins through SUMO-targeted ubiquitin ligases (STUbL). After 20 yr of research, SUMO has been shown to play critical roles in most, if not all, biological pathways. Thus the SUMO enzymes could be targets for drug development to treat human diseases.

Keywords:  SUMO; Sentrin; biochemistry; cancer; heart; physiology

DOI:  https://doi.org/10.1152/physrev.00025.2019
Mol Cell. 2020 Jul 16. pii: S1097-2765(20)30465-2. [Epub ahead of print]79(2): 203-204

Off and On Again: De- and Reubiquitination during Membrane Protein Degradation.

Claudia C Schmidt, Alexander Stein.

In this issue of Molecular Cell, Hu et al. (2020) show that the cytosolic E3 ligase RNF126 reubiquitinates membrane proteins after their extraction from the membrane of the endoplasmic reticulum to target them for proteasomal degradation.

DOI: https://doi.org/10.1016/j.molcel.2020.06.036
J Cell Mol Med. 2020 Jul 16.

Endoplasmic reticulum stress-induced exosomal miR-27a-3p promotes immune escape in breast cancer via regulating PD-L1 expression in macrophages.

Xiaoli Yao, Yi Tu, Yulin Xu, Yueyue Guo, Feng Yao, Xinghua Zhang.

  Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA-mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)-27a-3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR-27a-3p and MAGI2 was predicted using bioinformatic analysis and verified by dual-luciferase reporter assay. Ectopic expression and inhibition of miR-27a-3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co-cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD-L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR-27a-3p expression. Elevation of miR-27a-3p and PD-L1 levels in macrophages was observed in response to exosomes-overexpressing miR-27a-3p in vivo and in vitro. miR-27a-3p could target and negatively regulate MAGI2, while MAGI2 down-regulated PD-L1 by up-regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4+ , CD8+ T cells and IL-2, and T cells apoptosis were observed in response to co-culture of macrophages and CD3+ T cells. Conjointly, exosomal miR-27a-3p promotes immune evasion by up-regulating PD-L1 via MAGI2/PTEN/PI3K axis in breast cancer.

Keywords:  MicroRNA-27a-3p; breast cancer; endoplasmic reticulum; exosomes; macrophages; programmed cell death-Ligand 1; tumour immune evasion of breast cancer cells

DOI:  https://doi.org/10.1111/jcmm.15367
Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30858-5. [Epub ahead of print]32(2): 107877

The Zscan4-Tet2 Transcription Nexus Regulates Metabolic Rewiring and Enhances Proteostasis to Promote Reprogramming.

Zhou-Li Cheng, Meng-Li Zhang, Huai-Peng Lin, Chao Gao, Jun-Bin Song, Zhihong Zheng, Linpeng Li, Yanan Zhang, Xiaoqi Shen, Hao Zhang, Zhenghui Huang, Wuqiang Zhan, Cheng Zhang, Xu Hu, Yi-Ping Sun, Lubing Jiang, Lei Sun, Yanhui Xu, Chen Yang, Yuanlong Ge, Yong Zhao, Xingguo Liu, Hui Yang, Pengyuan Liu, Xing Guo, Kun-Liang Guan, Yue Xiong, Mingliang Zhang, Dan Ye.

  Evolutionarily conserved SCAN (named after SRE-ZBP, CTfin51, AW-1, and Number 18 cDNA)-domain-containing zinc finger transcription factors (ZSCAN) have been found in both mouse and human genomes. Zscan4 is transiently expressed during zygotic genome activation (ZGA) in preimplantation embryos and induced pluripotent stem cell (iPSC) reprogramming. However, little is known about the mechanism of Zscan4 underlying these processes of cell fate control. Here, we show that Zscan4f, a representative of ZSCAN proteins, is able to recruit Tet2 through its SCAN domain. The Zscan4f-Tet2 interaction promotes DNA demethylation and regulates the expression of target genes, particularly those encoding glycolytic enzymes and proteasome subunits. Zscan4f regulates metabolic rewiring, enhances proteasome function, and ultimately promotes iPSC generation. These results identify Zscan4f as an important partner of Tet2 in regulating target genes and promoting iPSC generation and suggest a possible and common mechanism shared by SCAN family transcription factors to recruit ten-eleven translocation (TET) DNA dioxygenases to regulate diverse cellular processes, including reprogramming.

Keywords:  SCAN domain; TET2; ZSCAN4; iPSCs; induced pluripotent stem cells; metabolic rewiring; proteasome function; stem cell potency

DOI:  https://doi.org/10.1016/j.celrep.2020.107877
Cell Rep. 2020 Jul 14. pii: S2211-1247(20)30879-2. [Epub ahead of print]32(2): 107898

The Cdc48 Complex Alleviates the Cytotoxicity of Misfolded Proteins by Regulating Ubiquitin Homeostasis.

Ryan Higgins, Marie-Helene Kabbaj, Delaney Sherwin, Lauren A Howell, Alexa Hatcher, Robert J Tomko, Yanchang Wang.

  The accumulation of misfolded proteins is associated with multiple neurodegenerative disorders, but it remains poorly defined how this accumulation causes cytotoxicity. Here, we demonstrate that the Cdc48/p97 segregase machinery drives the clearance of ubiquitinated model misfolded protein Huntingtin (Htt103QP) and limits its aggregation. Nuclear ubiquitin ligase San1 acts upstream of Cdc48 to ubiquitinate Htt103QP. Unexpectedly, deletion of SAN1 and/or its cytosolic counterpart UBR1 rescues the toxicity associated with Cdc48 deficiency, suggesting that ubiquitin depletion, rather than compromised proteolysis of misfolded proteins, causes the growth defect in cells with Cdc48 deficiency. Indeed, Cdc48 deficiency leads to elevated protein ubiquitination levels and decreased free ubiquitin, which depends on San1/Ubr1. Furthermore, enhancing free ubiquitin levels rescues the toxicity in various Cdc48 pathway mutants and restores normal turnover of a known Cdc48-independent substrate. Our work highlights a previously unappreciated function for Cdc48 in ensuring the regeneration of monoubiquitin that is critical for normal cellular function.

Keywords:  Cdc48; San1/Ubr1 E3 ligases; mutated Huntingtin; proteotoxicity; ubiquitin homeostasis

DOI:  https://doi.org/10.1016/j.celrep.2020.107898
Front Cell Dev Biol. 2020 ;8 467

Mitophagy, Mitochondrial Homeostasis, and Cell Fate.

Kaili Ma, Guo Chen, Wenhui Li, Oliver Kepp, Yushan Zhu, Quan Chen.

  Mitochondria are highly plastic and dynamic organelles that have graded responses to the changing cellular, environmental, and developmental cues. Mitochondria undergo constant mitochondrial fission and fusion, mitochondrial biogenesis, and mitophagy, which coordinately control mitochondrial morphology, quantity, quality, turnover, and inheritance. Mitophagy is a cellular process that selectively removes the aged and damaged mitochondria via the specific sequestration and engulfment of mitochondria for subsequent lysosomal degradation. It plays a pivotal role in reinstating cellular homeostasis in normal physiology and conditions of stress. Damaged mitochondria may either instigate innate immunity through the overproduction of ROS or the release of mtDNA, or trigger cell death through the release of cytochrome c and other apoptogenic factors when mitochondria damage is beyond repair. Distinct molecular machineries and signaling pathways are found to regulate these mitochondrial dynamics and behaviors. It is less clear how mitochondrial behaviors are coordinated at molecular levels. BCL2 family proteins interact within family members to regulate mitochondrial outer membrane permeabilization and apoptosis. They were also described as global regulators of mitochondrial homeostasis and mitochondrial fate through their interaction with distinct partners including Drp1, mitofusins, PGAM5, and even LC3 that involved mitochondrial dynamics and behaviors. In this review, we summarize recent findings on molecular pathways governing mitophagy and its coordination with other mitochondrial behaviors, which together determine cellular fate.

Keywords:  cell fate; mitochondrial apoptosis; mitochondrial dynamics; mitophagy; mitophagy receptors

DOI:  https://doi.org/10.3389/fcell.2020.00467
Proc Natl Acad Sci U S A. 2020 Jul 13. pii: 202004410. [Epub ahead of print]

Peptidylglycine α-amidating monooxygenase is required for atrial secretory granule formation.

Nils Bäck, Raj Luxmi, Kathryn G Powers, Richard E Mains, Betty A Eipper.

  The discovery of atrial secretory granules and the natriuretic peptides stored in them identified the atrium as an endocrine organ. Although neither atrial nor brain natriuretic peptide (ANP, BNP) is amidated, the major membrane protein in atrial granules is peptidylglycine α-amidating monooxygenase (PAM), an enzyme essential for amidated peptide biosynthesis. Mice lacking cardiomyocyte PAM (Pam Myh6-cKO/cKO) are viable, but a gene dosage-dependent drop in atrial ANP and BNP content occurred. Ultrastructural analysis of adult Pam Myh6-cKO/cKO atria revealed a 13-fold drop in the number of secretory granules. When primary cultures of Pam 0-Cre-cKO/cKO atrial myocytes (no Cre recombinase, PAM floxed) were transduced with Cre-GFP lentivirus, PAM protein levels dropped, followed by a decline in ANP precursor (proANP) levels. Expression of exogenous PAM in Pam Myh6-cKO/cKO atrial myocytes produced a dose-dependent rescue of proANP content; strikingly, this response did not require the monooxygenase activity of PAM. Unlike many prohormones, atrial proANP is stored intact. A threefold increase in the basal rate of proANP secretion by Pam Myh6-cKO/cKO myocytes was a major contributor to its reduced levels. While proANP secretion was increased following treatment of control cultures with drugs that block the activation of Golgi-localized Arf proteins and COPI vesicle formation, proANP secretion by Pam Myh6-cKO/cKO myocytes was unaffected. In cells lacking secretory granules, expression of exogenous PAM led to the accumulation of fluorescently tagged proANP in the cis-Golgi region. Our data indicate that COPI vesicle-mediated recycling of PAM from the cis-Golgi to the endoplasmic reticulum plays an essential role in the biogenesis of proANP containing atrial granules.

Keywords:  PAM; atrial natriuretic peptide; golgicide A; secretion; ultrastructure

DOI:  https://doi.org/10.1073/pnas.2004410117
Cell Death Differ. 2020 Jul 13.

Defective endoplasmic reticulum-mitochondria contacts and bioenergetics in SEPN1-related myopathy.

Anne Filipe, Alexander Chernorudskiy, Sandrine Arbogast, Ersilia Varone, Rocío-Nur Villar-Quiles, Diego Pozzer, Maryline Moulin, Stefano Fumagalli, Eva Cabet, Swati Dudhal, Maria-Grazia De Simoni, Raphaël Denis, Nathalie Vadrot, Corinne Dill, Matteo Giovarelli, Luke Szweda, Clara De Palma, Paolo Pinton, Carlotta Giorgi, Carlo Viscomi, Emilio Clementi, Sonia Missiroli, Simona Boncompagni, Ester Zito, Ana Ferreiro.

SEPN1-related myopathy (SEPN1-RM) is a muscle disorder due to mutations of the SEPN1 gene, which is characterized by muscle weakness and fatigue leading to scoliosis and life-threatening respiratory failure. Core lesions, focal areas of mitochondria depletion in skeletal muscle fibers, are the most common histopathological lesion. SEPN1-RM underlying mechanisms and the precise role of SEPN1 in muscle remained incompletely understood, hindering the development of biomarkers and therapies for this untreatable disease. To investigate the pathophysiological pathways in SEPN1-RM, we performed metabolic studies, calcium and ATP measurements, super-resolution and electron microscopy on in vivo and in vitro models of SEPN1 deficiency as well as muscle biopsies from SEPN1-RM patients. Mouse models of SEPN1 deficiency showed marked alterations in mitochondrial physiology and energy metabolism, suggesting that SEPN1 controls mitochondrial bioenergetics. Moreover, we found that SEPN1 was enriched at the mitochondria-associated membranes (MAM), and was needed for calcium transients between ER and mitochondria, as well as for the integrity of ER-mitochondria contacts. Consistently, loss of SEPN1 in patients was associated with alterations in body composition which correlated with the severity of muscle weakness, and with impaired ER-mitochondria contacts and low ATP levels. Our results indicate a role of SEPN1 as a novel MAM protein involved in mitochondrial bioenergetics. They also identify a systemic bioenergetic component in SEPN1-RM and establish mitochondria as a novel therapeutic target. This role of SEPN1 contributes to explain the fatigue and core lesions in skeletal muscle as well as the body composition abnormalities identified as part of the SEPN1-RM phenotype. Finally, these results point out to an unrecognized interplay between mitochondrial bioenergetics and ER homeostasis in skeletal muscle. They could therefore pave the way to the identification of biomarkers and therapeutic drugs for SEPN1-RM and for other disorders in which muscle ER-mitochondria cross-talk are impaired.

DOI: https://doi.org/10.1038/s41418-020-0587-z
Biomedicines. 2020 Jul 13. pii: E210. [Epub ahead of print]8(7):

Endoplasmic Reticulum Stress in Macrophages: The Vicious Circle of Lipid Accumulation and Pro-Inflammatory Response.

Vasily N Sukhorukov, Victoria A Khotina, Mariam Bagheri Ekta, Ekaterina A Ivanova, Igor A Sobenin, Alexander N Orekhov.

  The endoplasmic reticulum (ER) stress is an important event in the pathogenesis of different human disorders, including atherosclerosis. ER stress leads to disturbance of cellular homeostasis, apoptosis, and in the case of macrophages, to foam cell formation and pro-inflammatory cytokines production. In atherosclerosis, several cell types can be affected by ER stress, including endothelial cells, vascular smooth muscular cells, and macrophages. Modified low-density lipoproteins (LDL) and cytokines, in turn, can provoke ER stress through different processes. The signaling cascades involved in ER stress initiation are complex and linked to other cellular processes, such as lysosomal biogenesis and functioning, autophagy, mitochondrial homeostasis, and energy production. In this review, we discuss the underlying mechanisms of ER stress formation and the interplay of lipid accumulation and pro-inflammatory response. We will specifically focus on macrophages, which are the key players in maintaining chronic inflammatory milieu in atherosclerotic lesions, and also a major source of lipid-accumulating foam cells.

Keywords:  atherosclerosis; endoplasmic reticulum stress; foam cells; macrophages; pro-inflammatory response

DOI:  https://doi.org/10.3390/biomedicines8070210
mBio. 2020 Jul 14. pii: e00976-20. [Epub ahead of print]11(4):

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

Jennifer Deborah Wuerth, Matthias Habjan, Markus Kainulainen, Besim Berisha, Damien Bertheloot, Giulio Superti-Furga, Andreas Pichlmair, Friedemann Weber.

  RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αβγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

Keywords:  NSs protein; PKR; eIF2B; integrated stress response; phospho-eIF2α; sandfly fever Sicilian phlebovirus; translation inhibition; viral PKR antagonist

DOI:  https://doi.org/10.1128/mBio.00976-20
Aging Cell. 2020 Jul 13. e13171

Autophagy regulates the localization and degradation of p16INK4a.

Philip R Coryell, Supreet K Goraya, Katherine A Griffin, Margaret A Redick, Samuel R Sisk, Jeremy E Purvis.

  The tumor suppressor protein p16INK4a (p16) is a well-established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy-lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress-response proteins and has also been shown to attenuate age-related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time-lapse fluorescence microscopy using an endogenous p16-mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3-II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age-related diseases.

Keywords:  Ink4a; autophagy; lysosomes; p16

DOI:  https://doi.org/10.1111/acel.13171
Cell Signal. 2020 Jul 10. pii: S0898-6568(20)30189-3. [Epub ahead of print] 109712

Uev1A amino terminus stimulates poly-ubiquitin chain assembly and is required for NF-κB activation.

Zhaojia Wu, Parker L Andersen, Trevor Moraes, Sean A McKenna, Yiran Zhang, Weiwei Zhang, Michael J Ellison, Wei Xiao.

  The ubiquitin (Ub)-conjugating enzyme variants (Uev) Uev1A and Mms2 interact with Ubc13 to form heterodimeric complexes with different biological functions. Uev1A-Ubc13 is involved in NF-κB activation while Mms2-Ubc13 is required for the DNA-damage response. The structural comparison of the core domains of these two Uevs reveals no obvious difference, suggesting that the amino terminal extension of Uev1A plays a critical role in the functional determination. Indeed, truncated Uev1A lacking the N-terminal extension behaves like Mms2, while a chimeric protein containing the N-terminal Uev1A fused to Mms2 functionally resembles Uev1A. Interestingly, the N-terminal extension of Uev1A also dictates whether to assemble di- or poly-Ub chains in an in vitro reaction. Both thermodynamic measurements and enzymatic assays revealed that the Uev1A N-terminal extension weakens the Uev-Ubc13 interaction; however, other means capable of causing a reduced Uev1A-Ubc13 affinity and poly-Ub chain assembly do not necessarily promote NF-κB activation, indicating that the poly-Ub chain formation is not the only component contributed by the N-terminal extension of Uev1A. The physiological relevance of the Uev1A N-terminal truncation is presented and discussed.

Keywords:  DNA-damage response; Mms2; NF-κB signaling; Poly-ubiquitination; Ubc13; Uev1A

DOI:  https://doi.org/10.1016/j.cellsig.2020.109712
Nat Microbiol. 2020 Jul 13.

Adenovirus-mediated ubiquitination alters protein-RNA binding and aids viral RNA processing.

Christin Herrmann, Joseph M Dybas, Jennifer C Liddle, Alexander M Price, Katharina E Hayer, Richard Lauman, Caitlin E Purman, Matthew Charman, Eui Tae Kim, Benjamin A Garcia, Matthew D Weitzman.

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

DOI: https://doi.org/10.1038/s41564-020-0750-9
Neurobiol Dis. 2020 Jul 09. pii: S0969-9961(20)30291-6. [Epub ahead of print] 105016

Modeling UBQLN2-mediated neurodegenerative disease in mice: Shared and divergent properties of wild type and mutant UBQLN2 in phase separation, subcellular localization, altered proteostasis pathways, and selective cytotoxicity.

Lisa M Sharkey, Stephanie S Sandoval-Pistorius, Shannon J Moore, Julia E Gerson, Robert Komlo, Svetlana Fischer, Keyshla Y Negron-Rios, Emily V Crowley, Francisco Padron, Ronak Patel, Geoffrey G Murphy, Henry L Paulson.

  The ubiquitin-binding proteasomal shuttle protein UBQLN2 is implicated in common neurodegenerative disorders due to its accumulation in disease-specific aggregates and, when mutated, directly causes familial frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). Like other proteins linked to FTD/ALS, UBQLN2 undergoes phase separation to form condensates. The relationship of UBQLN2 phase separation and accumulation to neurodegeneration, however, remains uncertain. Employing biochemical, neuropathological and behavioral assays, we studied the impact of overexpressing WT or mutant UBQLN2 in the CNS of transgenic mice. Expression of UBQLN2 harboring a pathogenic mutation (P506T) elicited profound and widespread intraneuronal inclusion formation and aggregation without prominent neurodegenerative or behavioral changes. Both WT and mutant UBQLN2 formed ubiquitin- and P62-positive inclusions in neurons, supporting the view that UBQLN2 is intrinsically prone to phase separate, with the size, shape and frequency of inclusions depending on expression level and the presence or absence of a pathogenic mutation. Overexpression of WT or mutant UBQLN2 resulted in a dose-dependent decrease in levels of a key interacting chaperone, HSP70, as well as dose-dependent profound degeneration of the retina. We conclude that, at least in mice, robust aggregation of a pathogenic form of UBQLN2 is insufficient to cause neuronal loss recapitulating that of human FTD/ALS. Our results nevertheless support the view that altering the normal cellular balance of UBQLN2, whether wild type or mutant protein, has deleterious effects on cells of the CNS and retina that likely reflect perturbations in ubiquitin-dependent protein homeostasis.

Keywords:  Amyotrophic lateral sclerosis; Frontotemporal dementia; Neurodegeneration; Protein aggregation; Proteostasis; Ubiquilin; Ubiquitin proteasome system

DOI:  https://doi.org/10.1016/j.nbd.2020.105016
Nucleic Acids Res. 2020 Jul 15. pii: gkaa601. [Epub ahead of print]

Necdin regulates BMAL1 stability and circadian clock through SGT1-HSP90 chaperone machinery.

Renbin Lu, Yufan Dong, Jia-Da Li.

Circadian clocks are endogenous oscillators that control ∼24-hour physiology and behaviors in virtually all organisms. The circadian oscillator comprises interconnected transcriptional and translational feedback loops, but also requires finely coordinated protein homeostasis including protein degradation and maturation. However, the mechanisms underlying the mammalian clock protein maturation is largely unknown. In this study, we demonstrate that necdin, one of the Prader-Willi syndrome (PWS)-causative genes, is highly expressed in the suprachiasmatic nuclei (SCN), the pacemaker of circadian clocks in mammals. Mice deficient in necdin show abnormal behaviors during an 8-hour advance jet-lag paradigm and disrupted clock gene expression in the liver. By using yeast two hybrid screening, we identified BMAL1, the core component of the circadian clock, and co-chaperone SGT1 as two necdin-interactive proteins. BMAL1 and SGT1 associated with the N-terminal and C-terminal fragments of necdin, respectively. Mechanistically, necdin enables SGT1-HSP90 chaperone machinery to stabilize BMAL1. Depletion of necdin or SGT1/HSP90 leads to degradation of BMAL1 through the ubiquitin-proteasome system, resulting in alterations in both clock gene expression and circadian rhythms. Taken together, our data identify the PWS-associated protein necdin as a novel regulator of the circadian clock, and further emphasize the critical roles of chaperone machinery in circadian clock regulation.

DOI: https://doi.org/10.1093/nar/gkaa601
Exp Gerontol. 2020 Jul 10. pii: S0531-5565(20)30369-7. [Epub ahead of print] 111021

Aging is associated with increased TRB3, ER stress, and hepatic glucose production in the liver of rats.

Rafael Calais Gaspar, Vitor Rosetto Muñoz, Susana Castelo Branco Ramos Nakandakari, Renan Fudoli Lins Vieira, Luciana Renata da Conceição, Fellipe de Oliveira, Barbara Moreira Crisol, Adelino S R da Silva, Dennys Esper Cintra, Leandro Pereira de Moura, Eduardo Rochete Ropelle, Iman Zaghloul, Rania A Mekary, José Rodrigo Pauli.

  TRB3, a mammalian homolog of Drosophila tribbles, plays an important role in multiple tissues and it has been implicated in stress response regulation and metabolic control. However, the role of hepatic TRB3 and its relationship with endoplasmic reticulum stress (ER stress) during aging has not been elucidated. Thus, the present study aimed to explore the association of aging with TRB3 and ER stress on the hepatic glucose production in Wistar rats. We found the TRB3 protein content to be higher in livers of old rats (27 months) compared to young (3 months) and middle-aged (17 months) rats. The increased content of hepatic TRB3 of the old rats was associated with insulin resistance (decreased protein kinase B (Akt) and Forkhead Box O1 (FoxO1) phosphorylation) and increased enzymes of gluconeogenesis (phosphoenolpyruvate carboxykinase (PEPCK) and Glucose 6-phosphatase (G6Pase)). Moreover, aging was associated with activation of the endoplasmic reticulum stress pathway-related molecules, with an increase in phosphorylation of Inositol-requiring enzyme 1 (p-IRE1α), the protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), eukaryotic translation initiation factor-α (p-eIF2α), binding immunoglobulin protein (BiP), and the C/EBP homologous protein (CHOP) contents in rats. These molecular changes resulted in increased liver glucose production in response to the pyruvate challenge and hyperglycemia of the old rats. In conclusion, our results suggested that, by interfering with insulin signaling in the liver, TRB3 was associated with ER stress and increased hepatic glucose production in aging rats.

Keywords:  Aging; Er stress; Gluconeogenesis; Insulin resistance; TRB3

DOI:  https://doi.org/10.1016/j.exger.2020.111021
Sci Signal. 2020 Jul 14. pii: eaay1212. [Epub ahead of print]13(640):

Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum-to-mitochondria Ca2+ transfer for survival.

Cesar Cardenas, Alenka Lovy, Eduardo Silva-Pavez, Felix Urra, Craig Mizzoni, Ulises Ahumada-Castro, Galdo Bustos, Fabian Jaňa, Pablo Cruz, Paula Farias, Elizabeth Mendoza, Hernan Huerta, Paola Murgas, Martin Hunter, Melany Rios, Oscar Cerda, Irene Georgakoudi, Armen Zakarian, Jordi Molgó, J Kevin Foskett.

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

DOI: https://doi.org/10.1126/scisignal.aay1212
Elife. 2020 Jul 13. pii: e60038. [Epub ahead of print]9

Ribosome collisions trigger cis-acting feedback inhibition of translation initiation.

Szymon Juszkiewicz, Greg Slodkowicz, Zhewang Lin, Paula Freire-Pritchett, Sew-Yeu Peak-Chew, Ramanujan S Hegde.

  Translation of aberrant mRNAs can cause ribosomes to stall, leading to collisions with trailing ribosomes. Collided ribosomes are specifically recognized by ZNF598 to initiate protein and mRNA quality control pathways. Here we found using quantitative proteomics of collided ribosomes that EDF1 is a ZNF598-independent sensor of ribosome collisions. EDF1 stabilizes GIGYF2 at collisions to inhibit translation initiation in cis via 4EHP. The GIGYF2 axis acts independently of the ZNF598 axis, but each pathway's output is more pronounced without the other. We propose that the widely conserved and highly abundant EDF1 monitors the transcriptome for excessive ribosome density, then triggers a GIGYF2-mediated response to locally and temporarily reduce ribosome loading. Only when collisions persist is translation abandoned to initiate ZNF598-dependent quality control. This tiered response to ribosome collisions would allow cells to dynamically tune translation rates while ensuring fidelity of the resulting protein products.

Keywords:  cell biology; none

DOI:  https://doi.org/10.7554/eLife.60038
Cell Commun Signal. 2020 Jul 14. 18(1): 112

The deubiquitinase USP22 regulates PD-L1 degradation in human cancer cells.

Yu Wang, Qingguo Sun, Ning Mu, Xiaoyang Sun, Yingying Wang, Songqing Fan, Ling Su, Xiangguo Liu.

   BACKGROUND: Many cancers evade immune surveillance by overexpressing PD-L1. PD-L1 interacted with its receptor PD-1, resulting in reduction of T cell proliferation and activation and thereafter cancer cell death mediated by T-lymphocyte. Understanding the mechanisms that regulate PD-L1 was of vital importance for immune checkpoint blockade therapy (ICBT).
METHODS: Human non-small cell lung cancer cells and 293FT cells were used to investigate the function of USP22 upon PD-L1 and CSN5 by WB, Immunoprecipitation, Immunofluorescence and Flow cytometry analysis. B16-F10 cells were used to explore the role of USP22 on tumorigenesis and T cell cytotoxicity. The relationship between USP22 and PD-L1 expression was investigated by Immunohistochemistry analysis in human non-small cell lung cancer samples.
RESULTS: Our data showed that USP22 interacted with PD-L1 and promoted its stability. USP22 deubiquitinated PD-L1 and inhibited its proteasome degradation. Moreover, USP22 also interacted with CSN5 and stabilized CSN5 through deubiquitination. Either USP22 or CSN5 could facilitate the interaction of PD-L1 with the other one. Furthermore, USP22 removed K6, K11, K27, K29, K33 and K63-linked ubiquitin chain of both CSN5 and PD-L1. In addition, USP22 depletion inhibited tumorigenesis and promoted T cell cytotoxicity. Besides, USP22 expression positively correlated with PD-L1 expression in human non-small cell lung cancer samples.
CONCLUSIONS: Here, we suggested that USP22 is a new regulator for PD-L1. On the one hand, USP22 could directly regulate PD-L1 stability through deubiquitination. On the other hand, USP22 regulated PD-L1 protein level through USP22-CSN5-PD-L1 axis. In addition, USP22 depletion inhibited tumorigenesis and promoted T cell cytotoxicity. Besides, USP22 expression positively correlated with PD-L1 expression in human non-small cell lung cancer samples. Together, we identified a new regulator of PD-L1 and characterized the important role of USP22 in PD-L1 mediated immune evasion. Targeting USP22 might be a new solution to ICBT. Video abstract.

Keywords:  CSN5; Deubiquitination; Immune checkpoint blockade therapy; PD-L1; USP22

DOI:  https://doi.org/10.1186/s12964-020-00612-y
FASEB J. 2020 Jul 11.

MIB1-mediated degradation of WRN promotes cellular senescence in response to camptothecin treatment.

Meiting Li, Boya Liu, Jingjie Yi, Yang Yang, Jiadong Wang, Wei-Guo Zhu, Jianyuan Luo.

  Werner syndrome protein (WRN) plays critical roles in DNA replication, recombination, and repair, as well as transcription and cellular senescence. Ubiquitination and degradation of WRN have been reported, however, the E3 ubiquitin ligase of WRN is little known. Here, we identify mindbomb E3 ubiquitin protein ligase 1 (MIB1) as a novel E3 ubiquitin ligase for WRN protein. MIB1 physically interacts with WRN in vitro and in vivo and induces ubiquitination and degradation of WRN in the ubiquitin-proteasome pathway. Camptothecin (CPT) enhances the interaction between MIB1 and WRN, and promotes WRN degradation in a MIB1-dependent manner. In addition, CPT-induced cellular senescence is facilitated by the expression of MIB1 and attenuated by WRN expression. Our results show that MIB1-mediated degradation of WRN promotes cellular senescence and reveal a novel model executed by MIB1 and WRN to regulate cellular senescence.

Keywords:  CPT; Mind bomb 1; Werner syndrome protein; aging; protein stability

DOI:  https://doi.org/10.1096/fj.202000268RRR
Commun Biol. 2020 Jul 17. 3(1): 388

Ribosome profiling reveals a functional role for autophagy in mRNA translational control.

Juliet Goldsmith, Timothy Marsh, Saurabh Asthana, Andrew M Leidal, Deepthisri Suresh, Adam Olshen, Jayanta Debnath.

Autophagy promotes protein degradation, and therefore has been proposed to maintain amino acid pools to sustain protein synthesis during metabolic stress. To date, how autophagy influences the protein synthesis landscape in mammalian cells remains unclear. Here, we utilize ribosome profiling to delineate the effects of genetic ablation of the autophagy regulator, ATG12, on translational control. In mammalian cells, genetic loss of autophagy does not impact global rates of cap dependent translation, even under starvation conditions. Instead, autophagy supports the translation of a subset of mRNAs enriched for cell cycle control and DNA damage repair. In particular, we demonstrate that autophagy enables the translation of the DNA damage repair protein BRCA2, which is functionally required to attenuate DNA damage and promote cell survival in response to PARP inhibition. Overall, our findings illuminate that autophagy impacts protein translation and shapes the protein landscape.

DOI: https://doi.org/10.1038/s42003-020-1090-2
EMBO Mol Med. 2020 Jul 15. e11908

Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity.

Claudia Koch, Andra Kuske, Simon A Joosse, Gökhan Yigit, George Sflomos, Sonja Thaler, Daniel J Smit, Stefan Werner, Kerstin Borgmann, Sebastian Gärtner, Parinaz Mossahebi Mohammadi, Laura Battista, Laure Cayrefourcq, Janine Altmüller, Gabriela Salinas-Riester, Kaamini Raithatha, Arne Zibat, Yvonne Goy, Leonie Ott, Kai Bartkowiak, Tuan Zea Tan, Qing Zhou, Michael R Speicher, Volkmar Müller, Tobias M Gorges, Manfred Jücker, Jean-Paul Thiery, Cathrin Brisken, Sabine Riethdorf, Catherine Alix-Panabières, Klaus Pantel.

  Functional studies giving insight into the biology of circulating tumor cells (CTCs) remain scarce due to the low frequency of CTCs and lack of appropriate models. Here, we describe the characterization of a novel CTC-derived breast cancer cell line, designated CTC-ITB-01, established from a patient with metastatic estrogen receptor-positive (ER+ ) breast cancer, resistant to endocrine therapy. CTC-ITB-01 remained ER+ in culture, and copy number alteration (CNA) profiling showed high concordance between CTC-ITB-01 and CTCs originally present in the patient with cancer at the time point of blood draw. RNA-sequencing data indicate that CTC-ITB-01 has a predominantly epithelial expression signature. Primary tumor and metastasis formation in an intraductal PDX mouse model mirrored the clinical progression of ER+ breast cancer. Downstream ER signaling was constitutively active in CTC-ITB-01 independent of ligand availability, and the CDK4/6 inhibitor Palbociclib strongly inhibited CTC-ITB-01 growth. Thus, we established a functional model that opens a new avenue to study CTC biology.

Keywords:  breast cancer; circulating tumor cells; functional studies; liquid biopsy; metastasis

DOI:  https://doi.org/10.15252/emmm.201911908