bims-proteo 2020-09-13 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2020‒09‒13
thirty-six papers selected by
Eric Chevet
INSERM

Atf-6 Regulates Lifespan through ER-Mitochondrial Calcium Homeostasis.
FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis.
The intrinsic proteostasis network of stem cells.
Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.
Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor.
The E3 Ubiquitin Ligase SYVN1 Ubiquitinates Atlastins to Remodel the Endoplasmic Reticulum Network.
O-GlcNAcylation modulates HBV replication through regulating cellular autophagy at multiple levels.
The eIF2α kinase HRI in innate immunity, proteostasis and mitochondrial stress.
HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation.
DEPTOR Prevents Osteoarthritis Development Via Interplay with TRC8 To Reduce Endoplasmic Reticulum Stress in Chondrocytes.
Mechanisms Targeting the Unfolded Protein Response in Asthma.
Increasing the chemical space of proteins in living cells via genetic code expansion.
The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement.
Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase.
Structural-functional diversity of malaria parasite's PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding.
Aneuploidy-induced proteotoxic stress can be effectively tolerated without dosage compensation, genetic mutations, or stress responses.
Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.
BBS4 Is Essential for Nuclear Transport of Transcription Factors Mediating Neuronal ER Stress Response.
SUMOylation enhances the activity of IDH2 under oxidative stress.
Sigma-1 (σ1) receptor activity is necessary for physiological brain plasticity in mice.
A Novel Mechanism For NF-κB Activation Via IκB Aggregation: Implications For Hepatic Mallory-Denk-Body Induced Inflammation.
Records of RNA locations in living yeast revealed through covalent marks.
The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.
Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.
The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug Resistance.
UBE2O targets Mxi1 for ubiquitination and degradation to promote lung cancer progression and radioresistance.
TRIM37 controls cancer-specific vulnerability to PLK4 inhibition.
RNF216 mediates neuronal injury following experimental subarachnoid hemorrhage through the Arc/Arg3.1-AMPAR pathway.
CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage.
tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast.
Chimera induced protein degradation: PROTACs and beyond.
Selective Lysosome Membrane Turnover Is Induced by Nutrient Starvation.
Small ubiquitin-like modifier 2 (SUMO2) is critical for memory processes in mice.
Suppression of DRR1 results in the accumulation of insoluble ubiquitinated proteins, which impairs drought stress tolerance.
p24 family proteins are involved in transport to the plasma membrane of GPI-anchored proteins in plants.
TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks.

Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31114-1. [Epub ahead of print]32(10): 108125

Atf-6 Regulates Lifespan through ER-Mitochondrial Calcium Homeostasis.

Kristopher Burkewitz, Gaomin Feng, Sneha Dutta, Charlotte A Kelley, Michael Steinbaugh, Erin J Cram, William B Mair.

  Individually, dysfunction of both the endoplasmic reticulum (ER) and mitochondria has been linked to aging, but how communication between these organelles might be targeted to promote longevity is unclear. Here, we provide evidence that, in Caenorhabditis elegans, inhibition of the conserved unfolded protein response (UPRER) mediator, activating transcription factor (atf)-6, increases lifespan by modulating calcium homeostasis and signaling to mitochondria. Atf-6 loss confers longevity via downregulation of the ER calcium buffer, calreticulin. ER calcium release via the inositol triphosphate receptor (IP3R/itr-1) is required for longevity, while IP3R/itr-1 gain of function is sufficient to extend lifespan. Highlighting coordination between organelles, the mitochondrial calcium import channel mcu-1 is also required for atf-6 longevity. IP3R inhibition leads to impaired mitochondrial bioenergetics and hyperfusion, which is sufficient to suppress long life in atf-6 mutants. This study reveals the importance of organellar calcium handling as a critical output for the UPRER in determining the quality of aging.

Keywords:  InsP3R; UPR; aging; calreticulin; interorganelle communication; longevity

DOI:  https://doi.org/10.1016/j.celrep.2020.108125
J Cell Biol. 2020 Oct 05. pii: e202006111. [Epub ahead of print]219(10):

FIT2 is an acyl-coenzyme A diphosphatase crucial for endoplasmic reticulum homeostasis.

Michel Becuwe, Laura M Bond, Antonio F M Pinto, Sebastian Boland, Niklas Mejhert, Shane D Elliott, Marcelo Cicconet, Morven M Graham, Xinran N Liu, Olga Ilkayeva, Alan Saghatelian, Tobias C Walther, Robert V Farese.

The endoplasmic reticulum is a cellular hub of lipid metabolism, coordinating lipid synthesis with continuous changes in metabolic flux. Maintaining ER lipid homeostasis despite these fluctuations is crucial to cell function and viability. Here, we identify a novel mechanism that is crucial for normal ER lipid metabolism and protects the ER from dysfunction. We identify the molecular function of the evolutionarily conserved ER protein FIT2 as a fatty acyl-coenzyme A (CoA) diphosphatase that hydrolyzes fatty acyl-CoA to yield acyl 4'-phosphopantetheine. This activity of FIT2, which is predicted to be active in the ER lumen, is required in yeast and mammalian cells for maintaining ER structure, protecting against ER stress, and enabling normal lipid storage in lipid droplets. Our findings thus solve the long-standing mystery of the molecular function of FIT2 and highlight the maintenance of optimal fatty acyl-CoA levels as key to ER homeostasis.

DOI: https://doi.org/10.1083/jcb.202006111
Curr Opin Cell Biol. 2020 Sep 02. pii: S0955-0674(20)30101-0. [Epub ahead of print]67 46-55

The intrinsic proteostasis network of stem cells.

Ernesto Llamas, Hafiza Alirzayeva, Rute Loureiro, David Vilchez.

  The proteostasis network adjusts protein composition and maintains protein integrity, which are essential processes for cell function and viability. Current efforts, given their intrinsic characteristics, regenerative potential and fundamental biological functions, have been directed to define proteostasis of stem cells. These insights demonstrate that embryonic stem cells and induced pluripotent stem cells exhibit an endogenous proteostasis network that not only modulates their pluripotency and differentiation but also provides a striking ability to suppress aggregation of disease-related proteins. Moreover, recent findings establish a central role of enhanced proteostasis to prevent the aging of somatic stem cells in adult organisms. Notably, proteostasis is also required for the biological purpose of adult germline stem cells, that is to be passed from one generation to the next. Beyond these links between proteostasis and stem cell function, we also discuss the implications of these findings for disease, aging, and reproduction.

Keywords:  Adult somatic stem cells; Aging; Autophagy; Cell reprogramming; Differentiation; Embryonic stem cells; Germline stem cells; Hematopoietic stem cells; Induced pluripotent stem cells; Muscle stem cells; Neural stem cells; Protein chaperones; Protein synthesis; Proteostasis; RNA-binding proteins; Ubiquitin proteasome system

DOI:  https://doi.org/10.1016/j.ceb.2020.08.005
Mol Cell. 2020 Aug 31. pii: S1097-2765(20)30575-X. [Epub ahead of print]

Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.

Melanie A McDowell, Michael Heimes, Francesco Fiorentino, Shahid Mehmood, Ákos Farkas, Javier Coy-Vergara, Di Wu, Jani Reddy Bolla, Volker Schmid, Roger Heinze, Klemens Wild, Dirk Flemming, Stefan Pfeffer, Blanche Schwappach, Carol V Robinson, Irmgard Sinning.

  Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.

Keywords:  EMC; GET/TRC pathway; YidC; cryo-EM; lipid binding; membrane proteins; native mass spectrometry; protein transport; tail anchor

DOI:  https://doi.org/10.1016/j.molcel.2020.08.012
Sci Adv. 2020 May;pii: eaba5412. [Epub ahead of print]6(22):

Oncoprotein SND1 hijacks nascent MHC-I heavy chain to ER-associated degradation, leading to impaired CD8+ T cell response in tumor.

Yuan Wang, Xinting Wang, Xiaoteng Cui, Yue Zhuo, Hongshuai Li, Chuanbo Ha, Lingbiao Xin, Yuanyuan Ren, Wei Zhang, Xiaoming Sun, Lin Ge, Xin Liu, Jinyan He, Tao Zhang, Kai Zhang, Zhi Yao, Xi Yang, Jie Yang.

SND1 is highly expressed in various cancers. Here, we identify oncoprotein SND1 as a previously unidentified endoplasmic reticulum (ER) membrane-associated protein. The amino-terminal peptide of SND1 predominantly associates with SEC61A, which anchors on ER membrane. The SN domain of SND1 catches and guides the nascent synthesized heavy chain (HC) of MHC-I to ER-associated degradation (ERAD), hindering the normal assembly of MHC-I in the ER lumen. In mice model bearing tumors, especially in transgenic OT-I mice, deletion of SND1 promotes the presentation of MHC-I in both B16F10 and MC38 cells, and the infiltration of CD8+ T cells is notably increased in tumor tissue. It was further confirmed that SND1 impaired tumor antigen presentation to cytotoxic CD8+ T cells both in vivo and in vitro. These findings reveal SND1 as a novel ER-associated protein facilitating immune evasion of tumor cells through redirecting HC to ERAD pathway that consequently interrupts antigen presentation.

DOI: https://doi.org/10.1126/sciadv.aba5412
iScience. 2020 Aug 24. pii: S2589-0042(20)30686-6. [Epub ahead of print]23(9): 101494

The E3 Ubiquitin Ligase SYVN1 Ubiquitinates Atlastins to Remodel the Endoplasmic Reticulum Network.

Yupeng Zhao, Zhigang Feng, Yan Zou, Yanfen Liu.

  Atlastin (ATL) is a class of dynamin-like GTPases shaping endoplasmic reticulum (ER) by mediating homotypic membrane fusion. Defect of ATLs leads to abnormal ER structure and hereditary spastic paraplegia (HSP), a neurodegenerative disease with progressive spasticity. How ATLs are regulated to maintain the ER dynamics is not clear. Here, we found that SYVN1, an E3 ubiquitin ligase on the ER membrane, regulates ER shape and COPII exporting by mediating ubiquitination on ATLs, especially ATL1. ATL1 is ubiquitinated by SYVN1 strongly on K285 and mildly on K287. Ubiquitination on ATL1 does not result in protein degradation but inhibits ATL1 GTPase activity. SYVN1 overexpression compensates the excessive ER network fusion caused by ATL1 overexpression. Accordingly, the role of SYVN1 and ATL1 in regulating ER morphology is also recapitulated in Caenorhabditis elegans. Taken together, our study reveals a different role of SYVN1 in ER remodeling through mediating ubiquitination on ATLs.

Keywords:  Biological Sciences; Cell Biology; Molecular Biology

DOI:  https://doi.org/10.1016/j.isci.2020.101494
FASEB J. 2020 Sep 06.

O-GlcNAcylation modulates HBV replication through regulating cellular autophagy at multiple levels.

Xueyu Wang, Yong Lin, Shi Liu, Ying Zhu, Kefeng Lu, Ruth Broering, Mengji Lu.

  O-GlcNAcylation is a form of posttranslational modification, and serves various functions, including modulation of location, stability, and activity for the modified proteins. O-linked-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is an essential cellular enzyme that posttranslationally modifies the cellular proteins with O-GlcNAc moiety. Early studies reported that the decreased O-GlcNAcylation regulates cellular autophagy, a process relevant for hepatitis B virus replication (HBV) and assembly. Therefore, we addressed the question how O-GlcNAcylation regulates cellular autophagy and HBV replication. Inhibition of OGT activity with a small molecule inhibitor OSMI-1 or silencing OGT significantly enhanced HBV replication and HBsAg production in hepatoma cells and primary human hepatocytes (PHHs). Western blotting analysis showed that inhibition of O-GlcNAcylation-induced endoplasmic reticulum (ER) stress and cellular autophagy, two processes subsequently leading to enhanced HBV replication. Importantly, the numbers of autophagosomes and the levels of autophagic markers LC3-II and SQSTM1/p62 in hepatoma cells were elevated after inhibition of O-GlcNAcylation. Further analysis revealed that inhibition of O-GlcNAcylation blocked autophagosome-lysosome fusion and thereby prevented autophagic degradation of HBV virions and proteins. Moreover, OSMI-1 further promoted HBV replication by inducing autophagosome formation via inhibiting the O-GlcNAcylation of Akt and mTOR. In conclusion, decreased O-GlcNAcylation enhanced HBV replication through increasing autophagosome formation at multiple levels, including triggering ER-stress, Akt/mTOR inhibition, and blockade of autophagosome-lysosome fusion.

Keywords:   O-GlcNAcylation ; Akt/mTOR signaling; ER stress; HBV; autophagosome-lysosome fusion; autophagy

DOI:  https://doi.org/10.1096/fj.202001168RR
FEBS J. 2020 Sep 05.

The eIF2α kinase HRI in innate immunity, proteostasis and mitochondrial stress.

Stephen E Girardin, Camille Cuziol, Dana J Philpott, Damien Arnoult.

  The integrated stress response (ISR) is an evolutionary conserved stress response pathway that leads to a global arrest in translation as well as to the expression of specific genes, such as the transcription factor ATF4, to promote cellular recovery. The central nexus of this pathway is the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) by one of the four eIF2α kinases that sense specific cellular stressors. The heme-regulated inhibitor (HRI) is one of these kinases and it was initially reported to be activated in response to heme deprivation. Nevertheless, further studies have established that cytosolic proteotoxicity, resulting from oxidative or osmotic stress, heat shock and proteasome inhibition, is the predominant trigger for HRI to induce the ISR. In this review, we present newly identified functions of HRI in innate immunity, proteostasis and mitochondrial stress. Indeed, HRI-mediated signaling defines a novel cytosolic unfolded protein response (cUPR) required for the proper formation of some innate immune signalosomes and the control of toxic protein aggregates, and this eIF2α kinase also serves as a relay for mitonuclear communication after a mitochondrial stress.

Keywords:  HRI; ISR; innate immunity; mitochondrial stress; proteostasis

DOI:  https://doi.org/10.1111/febs.15553
iScience. 2020 Aug 21. pii: S2589-0042(20)30685-4. [Epub ahead of print]23(9): 101493

HRD Complex Self-Remodeling Enables a Novel Route of Membrane Protein Retrotranslocation.

Sonya Neal, Della Syau, Anahita Nejatfard, Samantha Nadeau, Randolph Y Hampton.

  ER-associated degradation (ERAD) targets misfolded ER proteins for degradation. Retrotranslocation, a key feature of ERAD, entails removal of ubiquitinated substrates into the cytosol for proteasomal destruction. Recently, it has been shown that the Hrd1 E3 ligase forms a retrotranslocation channel for luminal (ERAD-L) substrates. Conversely, our studies found that integral membrane (ERAD-M) substrates exit the ER through a distinct pathway mediated by the Dfm1 rhomboid protein. Those studies also revealed a second, Hrd1-dependent pathway of ERAD-M retrotranslocation can arise in dfm1Δ null. Here we show that, in the dfm1Δ null, the HRD complex undergoes remodeling to a form that mediates ERAD-M retrotranslocation. Specifically, Hrd1's normally present stochiometric partner Hrd3 is efficiently removed during suppressive remodeling, allowing Hrd1 to function in this novel capacity. Neither Hrd1 autoubiquitination nor its cytosolic domain is required for suppressive ERAD-M retrotranslocation. Thus, the HRD complex displays remarkable functional flexibility in response to ER stress.

Keywords:  Biological Sciences; Cell Biology; Molecular Biology

DOI:  https://doi.org/10.1016/j.isci.2020.101493
J Bone Miner Res. 2020 Sep 11.

DEPTOR Prevents Osteoarthritis Development Via Interplay with TRC8 To Reduce Endoplasmic Reticulum Stress in Chondrocytes.

Kai Li, Panpan Yang, Yuwei Zhang, Yue Zhang, He Cao, Peilin Liu, Bin Huang, Song Xu, Pinglin Lai, Guanghua Lei, Jia Liu, Yujin Tang, Xiaochun Bai, Zhipeng Zou.

  Endoplasmic reticulum (ER) stress has been shown to promote chondrocyte apoptosis and osteoarthritis (OA) progression, but the precise mechanisms via which ER stress is modulated in OA remain unclear. Here we report that DEPTOR (DEP domain-containing mTOR-interacting protein) negatively regulated ER stress and OA development independent of mTOR signaling. DEPTOR is ubiquitinated in articular chondrocytes and its expression is markedly reduced along with OA progression. Deletion of DEPTOR in chondrocytes significantly promoted destabilized medial meniscus (DMM) surgery-induced OA development, whereas intra-articular injection of lentivirus expressing DEPTOR delayed OA progression in mice. Proteomics analysis revealed that DEPTOR interplayed with TRC8, which promoted TRC8 auto-ubiquitination and degraded by the ubiquitin-proteasome system (UPS) in chondrocytes. Loss of DEPTOR leaded to TRC8 accumulation and excessive ER stress, with subsequent chondrocytes apoptosis and OA progression. Importantly, an inhibitor of ER stress eliminated chondrocyte DEPTOR deletion-exacerbated OA in mice. Together, these findings establish a novel mechanism essential for OA pathogenesis, where decreasing DEPTOR in chondrocytes during OA progression relieves the auto-ubiquitination of TRC8, resulting in TRC8 accumulation, excessive ER stress and OA progression. Targeting this pathway has promising therapeutic potential for OA treatment. This article is protected by copyright. All rights reserved.

Keywords:  DEPTOR; ER stress; TRC8; osteoarthritis; ubiquitination

DOI:  https://doi.org/10.1002/jbmr.4176
Am J Respir Cell Mol Biol. 2020 Sep 11.

Mechanisms Targeting the Unfolded Protein Response in Asthma.

Sanaz Dastghaib, P Sravan Kumar, Sajjad Aftabi, Gautam Damera, Azadeh Dalvand, Adel Sepanjnia, Mohammad Kiumarsi, Mohamad-Reza Aghanoori, Sukhwinder Singh Sohal, Sudharsan R Ande, Javad Alizadeh, Pooneh Mokarram, Saeid Ghavami, Pawan Sharma, Amir A Zeki.

  Lung cells are constantly exposed to various internal and external stressors that disrupt protein homeostasis. To cope with these stimuli, cells evoke a highly conserved adaptive mechanism called the unfolded protein response (UPR). UPR stressors can impose greater protein secretory demands on the endoplasmic reticulum (ER) resulting in the development, differentiation, and survival of these cell types to meet these increasing functional needs. Dysregulation of the UPR leads to the development of the disease. The UPR and ER stress are involved in several human conditions such as chronic inflammation, neurodegeneration, metabolic syndrome, and cancer. Further, potent and specific compounds that target the UPR pathway are under development as future therapies. The focus of this review is to thoroughly describe the effects of both internal and external stressors on the ER in asthma. Further, we discuss how the UPR signaling pathway is activated in the lungs to overcome cellular damage. We also present an overview of the pathogenic mechanisms with a brief focus on potential strategies for pharmacological interventions.

Keywords:  Asthma; ER stress; Endoplasmic Reticulum (ER); Unfolded Protein Response

DOI:  https://doi.org/10.1165/rcmb.2019-0235TR
Curr Opin Chem Biol. 2020 Sep 07. pii: S1367-5931(20)30108-3. [Epub ahead of print]58 112-120

Increasing the chemical space of proteins in living cells via genetic code expansion.

Kristina Krauskopf, Kathrin Lang.

  In recent years it has become possible to genetically encode an expanded set of designer amino acids with tailored chemical and physical properties (dubbed unnatural amino acids, UAAs) into proteins in living cells by expanding the genetic code. Together with developments in chemistries that are amenable to and selective within physiological settings, these strategies have started to have a big impact on biological studies, as they enable exciting in cellulo applications. Here we highlight recent advances to covalently stabilize transient protein-protein interactions and capture enzyme substrate-complexes in living cells using proximity-triggered and residue-selective photo-induced crosslinking approaches. Furthermore, we describe recent efforts in controlling enzyme activity with photocaged UAAs and in extending their application to a variety of enzymatic scaffolds. In addition, we discuss the site-specific incorporation of UAAs mimicking post-translational modifications (PTMs) and approaches to generate natively-linked ubiquitin-protein conjugates to probe the role of PTMs in modulating complex cellular networks.

Keywords:  Genetic code expansion; Photocaging; Photocrosslinking; Post-translational modifications; Proximity-triggered crosslinking; Unnatural amino acids

DOI:  https://doi.org/10.1016/j.cbpa.2020.07.012
Int J Mol Sci. 2020 Sep 03. pii: E6429. [Epub ahead of print]21(17):

The Ubiquitin Proteasome System in Neuromuscular Disorders: Moving Beyond Movement.

Sara Bachiller, Isabel M Alonso-Bellido, Luis Miguel Real, Eva María Pérez-Villegas, José Luis Venero, Tomas Deierborg, José Ángel Armengol, Rocío Ruiz.

  Neuromuscular disorders (NMDs) affect 1 in 3000 people worldwide. There are more than 150 different types of NMDs, where the common feature is the loss of muscle strength. These disorders are classified according to their neuroanatomical location, as motor neuron diseases, peripheral nerve diseases, neuromuscular junction diseases, and muscle diseases. Over the years, numerous studies have pointed to protein homeostasis as a crucial factor in the development of these fatal diseases. The ubiquitin-proteasome system (UPS) plays a fundamental role in maintaining protein homeostasis, being involved in protein degradation, among other cellular functions. Through a cascade of enzymatic reactions, proteins are ubiquitinated, tagged, and translocated to the proteasome to be degraded. Within the ubiquitin system, we can find three main groups of enzymes: E1 (ubiquitin-activating enzymes), E2 (ubiquitin-conjugating enzymes), and E3 (ubiquitin-protein ligases). Only the ubiquitinated proteins with specific chain linkages (such as K48) will be degraded by the UPS. In this review, we describe the relevance of this system in NMDs, summarizing the UPS proteins that have been involved in pathological conditions and neuromuscular disorders, such as Spinal Muscular Atrophy (SMA), Charcot-Marie-Tooth disease (CMT), or Duchenne Muscular Dystrophy (DMD), among others. A better knowledge of the processes involved in the maintenance of proteostasis may pave the way for future progress in neuromuscular disorder studies and treatments.

Keywords:  UPS; neuromuscular disorder; neuromuscular junction; proteasome; synapse; ubiquitin

DOI:  https://doi.org/10.3390/ijms21176429
Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31096-2. [Epub ahead of print]32(10): 108107

Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase.

Tomoe Kitao, Kyoichiro Taguchi, Shintaro Seto, Kohei Arasaki, Hiroki Ando, Hiroki Nagai, Tomoko Kubori.

  The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.

Keywords:  COPI; Legionella; SNARE; Sec22b; deubiquitinase; early secretory pathway; effector protein; syntaxin; type IV secretion system; ubiquitin

DOI:  https://doi.org/10.1016/j.celrep.2020.108107
Biochem J. 2020 Sep 07. pii: BCJ20200434. [Epub ahead of print]

Structural-functional diversity of malaria parasite's PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding.

Mohammad Anas, Ankita Shukla, Aradhya Tripathi, Varsha Kumari, Chetan Prakash, Priyabrata Nag, L Sathish Kumar, Sandeep Sharma, Ravishankar Ramachandran, Niti Kumar.

  Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite's chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition and oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate folding of client-substrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain substrate in folding-competent state to minimize its misfolding. Structural investigation through SAXS gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggests that parasite has evolved functionally diverged and efficient chaperone machinery which allows human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for exploration of small-molecule based antimalarial interventions.

Keywords:  HSP; Plasmodium; aggregation; malaria; protein conformation

DOI:  https://doi.org/10.1042/BCJ20200434
BMC Biol. 2020 Sep 08. 18(1): 117

Aneuploidy-induced proteotoxic stress can be effectively tolerated without dosage compensation, genetic mutations, or stress responses.

Katherine E Larrimore, Natalia S Barattin-Voynova, David W Reid, Davis T W Ng.

  BACKGROUND: The protein homeostasis (proteostasis) network maintains balanced protein synthesis, folding, transport, and degradation within a cell. Failure to maintain proteostasis is associated with aging and disease, leading to concerted efforts to study how the network responds to various proteotoxic stresses. This is often accomplished using ectopic overexpression of well-characterized, model misfolded protein substrates. However, how cells tolerate large-scale, diverse burden to the proteostasis network is not understood. Aneuploidy, the state of imbalanced chromosome content, adversely affects the proteostasis network by dysregulating the expression of hundreds of proteins simultaneously. Using aneuploid haploid yeast cells as a model, we address whether cells can tolerate large-scale, diverse challenges to the proteostasis network.RESULTS: Here we characterize several aneuploid Saccharomyces cerevisiae strains isolated from a collection of stable, randomly generated yeast aneuploid cells. These strains exhibit robust growth and resistance to multiple drugs which induce various forms of proteotoxic stress. Whole genome re-sequencing of the strains revealed this was not the result of genetic mutations, and transcriptome profiling combined with ribosome footprinting showed that genes are expressed and translated in accordance to chromosome copy number. In some strains, various facets of the proteostasis network are mildly upregulated without chronic activation of environmental stress response or heat shock response pathways. No severe defects were observed in the degradation of misfolded proteins, using model misfolded substrates of endoplasmic reticulum-associated degradation or cytosolic quality control pathways, and protein biosynthesis capacity was not impaired.
CONCLUSIONS: We show that yeast strains of some karyotypes in the genetic background studied here can tolerate the large aneuploidy-associated burden to the proteostasis machinery without genetic changes, dosage compensation, or activation of canonical stress response pathways. We suggest that proteotoxic stress, while common, is not always an obligate consequence of aneuploidy, but rather certain karyotypes and genetic backgrounds may be able to tolerate the excess protein burden placed on the protein homeostasis machinery. This may help clarify how cancer cells are paradoxically both highly aneuploid and highly proliferative at the same time.

Keywords:  Aneuploidy; Protein homeostasis; Protein quality control; Proteostasis; Proteotoxic stress

DOI:  https://doi.org/10.1186/s12915-020-00852-x
EMBO J. 2020 Sep 09. e105087

Molecular basis for the bifunctional Uba4-Urm1 sulfur-relay system in tRNA thiolation and ubiquitin-like conjugation.

Marta Pabis, Martin Termathe, Keerthiraju E Ravichandran, Sandra D Kienast, Rościsław Krutyhołowa, Mikołaj Sokołowski, Urszula Jankowska, Przemysław Grudnik, Sebastian A Leidel, Sebastian Glatt.

  The chemical modification of tRNA bases by sulfur is crucial to tune translation and to optimize protein synthesis. In eukaryotes, the ubiquitin-related modifier 1 (Urm1) pathway is responsible for the synthesis of 2-thiolated wobble uridine (U34 ). During the key step of the modification cascade, the E1-like activating enzyme ubiquitin-like protein activator 4 (Uba4) first adenylates and thiocarboxylates the C-terminus of its substrate Urm1. Subsequently, activated thiocarboxylated Urm1 (Urm1-COSH) can serve as a sulfur donor for specific tRNA thiolases or participate in ubiquitin-like conjugation reactions. Structural and mechanistic details of Uba4 and Urm1 have remained elusive but are key to understand the evolutionary branch point between ubiquitin-like proteins (UBL) and sulfur-relay systems. Here, we report the crystal structures of full-length Uba4 and its heterodimeric complex with its substrate Urm1. We show how the two domains of Uba4 orchestrate recognition, binding, and thiocarboxylation of the C-terminus of Urm1. Finally, we uncover how the catalytic domains of Uba4 communicate efficiently during the reaction cycle and identify a mechanism that enables Uba4 to protect itself against self-conjugation with its own product, namely activated Urm1-COSH.

Keywords:  adenylation; tRNA modification; thioester; thiolation; ubiquitin-like proteins

DOI:  https://doi.org/10.15252/embj.2020105087
Mol Neurobiol. 2020 Sep 07.

BBS4 Is Essential for Nuclear Transport of Transcription Factors Mediating Neuronal ER Stress Response.

Avital Horwitz, Ruth Birk.

  Bardet-Biedl syndrome (BBS) is an autosomal recessive syndrome presenting with retinal dystrophy, cognitive impairment, and obesity. BBS is characterized by elevated endoplasmic reticulum (ER) stress in the early stages of adipocyte and retinal development. BBS expression in the CNS and indications of hippocampal dysgenesis suggest neural development abnormalities. However, the role of BBS in ER stress in neuronal cells has not yet been studied. Therefore, we aimed at studying the role of BBS4 in neuronal development under normal and ER stress conditions. ER stress and unfolded protein response (UPR) were studied in BBS4-silenced (SiBBS4) SH-SY5Y cells during differentiation under normal and stress states, using molecular and biochemical markers. ER stress was demonstrated at early neural differentiation, with significantly augmented expression of UPR markers corresponding to BBS4 expression. In the undifferentiated state, BBS4 silencing resulted in significantly reduced ER-stress markers' expression under normal and ER-stress states. Independent of ER stress, SiBBS4 cells demonstrated significant reduction in activated phospho-IRE1α. Under BBS4 silencing, both sXBP-1 and activated ATF6α p50 failed to translocate to the nucleus. Transcript levels of apoptosis markers were upregulated under BBS4 depletion and ER-stress induction, corresponding to decreased viability. BBS4 depletion in neuronal cells results in reduced sensitivity to ER stress during differentiation and under ER-stress induction, partly due to failure in translocation of ER-transcription factors (TF) sXBP-1 and ATF6α p50 to the nucleus. Hence, BBS4 is essential for nuclear transport under ER-stress response in neuronal cells during early differentiation. Our studies shed light on molecular mechanisms through which BBS4 malfunction alters neuronal ER stress response.

Keywords:  ATF6α; BBS4; Endoplasmic reticulum (ER); Unfolded protein response (UPR); XBP-1

DOI:  https://doi.org/10.1007/s12035-020-02104-z
Biochem Biophys Res Commun. 2020 Sep 05. pii: S0006-291X(20)31688-0. [Epub ahead of print]

SUMOylation enhances the activity of IDH2 under oxidative stress.

Yun Yu, Yalan Chen, Kexin Liu, Jinke Cheng, Jun Tu.

  Mitochondria play a central role in biological oxidation that inevitably generates reactive oxygen species (ROS) as by-products. Maintenance of mitochondrial redox balance status requires NADPH, which is primarily generated by the mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2). The activity of IDH2 is regulated by post-translational modifications (PTMs). In this study, we found IDH2 is modified by small ubiquitin-like modifier 1 (SUMO1) at lysine 45. SUMO specific protease 1 (SENP1) is responsible for deSUMOylation of IDH2. SUMOylation of IDH2 is induced by oxidants and enhances the antioxidant activity of IDH2 to protect cells against oxidative stress. Mutation of the SUMOylation site impairs the enzymatic activity of IDH2 and hence decreases levels of α-ketoglutarate (α-KG), NADPH and GSH. Cells with SUMOylation deficient IDH2 suffer more apoptosis than that with wild type IDH2 under oxidative stress. These results indicate that SUMOylation is an important way to regulate IDH2 activity to maintain mitochondrial redox balance.

Keywords:  IDH2; Mitochondria; Oxidative stress; SUMOylation

DOI:  https://doi.org/10.1016/j.bbrc.2020.08.089
Eur Neuropsychopharmacol. 2020 Sep 03. pii: S0924-977X(20)30260-1. [Epub ahead of print]

Sigma-1 (σ1) receptor activity is necessary for physiological brain plasticity in mice.

Lucie Crouzier, Simon Couly, Chloé Roques, Coralie Peter, Rislen Belkhiter, Maëva Arguel Jacquemin, Anna Bonetto, Benjamin Delprat, Tangui Maurice.

  The sigma-1 receptor (S1R) is a membrane-associated protein expressed in neurons and glia at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs). S1R interacts with different partners to regulate cellular responses, including ER stress, mitochondrial physiology and Ca2+ fluxes. S1R shapes cellular plasticity by directly modulating signaling pathways involved in inflammatory responses, cell survival and death. We here analyzed its impact on brain plasticity in vivo, in mice trained in a complex maze, the Hamlet test. The device, providing strong enriched environment (EE) conditions, mimics a small village. It has a central agora and streets expanding from it, leading to functionalized houses where animals can Drink, Eat, Hide, Run, or Interact. Animals were trained in groups, 4 h/day for two weeks, and their maze exploration and topographic memory could be analyzed. Several groups of mice were considered: non-trained vs. trained; repeatedly administered with saline vs. NE-100, a selective S1R antagonist; and wildtype vs. S1R KO mice. S1R inactivation altered maze exploration and prevented topographic learning. EE induced a strong plasticity measured through resilience to behavioral despair or to the amnesic effects of scopolamine, and increases in S1R expression and bdnf mRNA levels in the hippocampus; increases in neurogenesis (proliferation and maturation); and increases of histone acetylation in the hippocampus and cortex. S1R inactivation altered all these parameters significantly, showing that S1R activity plays a major role in physiological brain plasticity. As S1R is a major resident protein in MAMs, modulating ER responses and mitochondrial homeostasy, MAM physiology appeared impacted by enriched environment.

Keywords:  Brain plasticity; Enriched environment; Hamlet test; Histone acetylation; Neurogenesis; Sigma-1 receptor

DOI:  https://doi.org/10.1016/j.euroneuro.2020.08.010
Mol Cell Proteomics. 2020 Sep 10. pii: mcp.RA120.002316. [Epub ahead of print]

A Novel Mechanism For NF-κB Activation Via IκB Aggregation: Implications For Hepatic Mallory-Denk-Body Induced Inflammation.

Yi Liu, Michael J Trnka, Shenheng Guan, Doyoung Kwon, Do-Hyung Kim, Jane-Jane Chen, Peter A Greer, Alma L Burlingame, Maria Almira Correia.

  Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-kB activation. However, the precise mechanism that links protein aggregation to NF-kB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IkBa-loss with consequent NF-kB activation. Four known mechanisms of IkBa-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IkBa-loss was due to its sequestration along with IkBb into insoluble aggregates, thereby releasing NF-kB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-kB subunit p65, which stably interacts with IkBa under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IkBa under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IkBa after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IkBa-nuclear import. The concurrent aggregation of IkBa, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IkBa and its consequent binding and termination of NF-kB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

Keywords:  Affinity proteomics; Hepatotoxicity; IkBa; Immunoaffinity; Inflammation; Inflammatory response; Knockouts*; Label-free quantification; Liver disease; Mallory-Denk-bodies; Mass Spectrometry; NF-kB; Protein aggregation; Protein-Protein Interactions*; ZnPPIX

DOI:  https://doi.org/10.1074/mcp.RA120.002316
Proc Natl Acad Sci U S A. 2020 Sep 09. pii: 201921408. [Epub ahead of print]

Records of RNA locations in living yeast revealed through covalent marks.

Hugo C Medina-Munoz, Christopher P Lapointe, Douglas F Porter, Marvin Wickens.

  RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.

Keywords:  RNA localization; RNA regulation; localized RNA records

DOI:  https://doi.org/10.1073/pnas.1921408117
Am J Cancer Res. 2020 ;10(8): 2621-2634

The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

Cheng-Wei Chou, Ri-Yao Yang, Li-Chuan Chan, Ching-Fei Li, Linlin Sun, Heng-Huan Lee, Pei-Chih Lee, Yuh-Pyng Sher, Haoqiang Ying, Mien-Chie Hung.

The immune checkpoint blockade therapy has emerged as encouraging treatment strategies in various cancer types. Anti-PD-L1 (programmed death-ligand 1) antibodies have been approved for triple-negative breast cancer, however the response rate yet to be optimized. It would be imperative to further understand and investigate the molecular mechanisms of PD-L1 regulation. Here, we identified glucose regulatory protein 78 (GRP78), a major endoplasmic reticulum (ER) stress responding protein, as a novel binding partner of PD-L1. GRP78 interacts with PD-L1 at the ER region and increases PD-L1 levels via regulating its stability. ER stress, triggered by different stimuli such as conventional chemotherapy, leads to the induction of PD-L1 in a GRP78-dependent manner. We showed that GRP78 modulates the response to chemotherapy, and dual-high levels of GRP78 and PD-L1 correlates with poor relapse-free survival in triple-negative breast cancer. Altogether, our study provides novel molecular insights into the regulatory mechanism of PD-L1 by revealing its interaction with GRP78, and offers a rationale to target GRP78 as a potential therapeutic strategy to enhance anti-tumor immunity.

Keywords: ER stress; GRP78; PD-L1; Triple-negative breast cancer
Autophagy. 2020 Sep 07. 1-3

Two different axes CALCOCO2-RB1CC1 and OPTN-ATG9A initiate PRKN-mediated mitophagy.

Koji Yamano, Richard J Youle.

  PINK1 and PRKN, proteins mutated in Parkinson disease, selectively amplify ubiquitin signals on damaged mitochondria for elimination via mitophagy. Because all five macroautophagy/autophagy receptors in mammals possess domains binding to ubiquitin and Atg8-family proteins, they were thought to recruit Atg8-family protein labeled phagophores from a cytosolic pool. However, our recent findings show that, in addition to Atg8-family protein binding, two of the receptors CALCOCO2 and OPTN interact with RB1CC1 and ATG9A, respectively, indicating that two different axes, CALCOCO2-RB1CC1 and OPTN-ATG9A, can initiate de novo biogenesis of autophagic membranes on ubiquitin-coated damaged mitochondria. These results explain the critical roles of the autophagy receptors CALCOCO2 and OPTN in mitochondrial degradation, and their abilities to simultaneously bind multiple autophagy core proteins propose a new function, i.e. a scaffold to build multivalent interactions for the orchestrated assembly of autophagy proteins near the ubiquitinated cargo.ABBREVIATIONS: ATG: autophagy-related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CRABP2: cellular retinoic acid binding protein 2; LIR: MAP1LC3/LC3-interacting region; MAP1LC3: microtubule associated protein 1 light chain 3; NBR1: NBR1 autophagy cargo receptor; OPTN: optineurin; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SNIPER: specific and nongenetic IAP-dependent protein eraser; SQSTM1/p62: sequestosome 1; ULK: unc-51 like autophagy activating kinase.

Keywords:  Mitochondria; PINK1; Parkin; Parkinson’s disease; ubiquitin

DOI:  https://doi.org/10.1080/15548627.2020.1815457
Cell Rep. 2020 Sep 08. pii: S2211-1247(20)31091-3. [Epub ahead of print]32(10): 108102

The Negative Cross-Talk between SAG/RBX2/ROC2 and APC/C E3 Ligases in Regulation of Cell Cycle Progression and Drug Resistance.

Shizhen Zhang, Yanwen Shen, Hua Li, Chao Bi, Yilun Sun, Xiufang Xiong, Wenyi Wei, Yi Sun.

  Anaphase-promoting complex/cyclosome (APC/C) is a well-characterized E3 ligase that couples with UBE2C and UBE2S E2s for substrate ubiquitylation by the K11 linkage. Our recent data show that SAG/RBX2/ROC2, a RING component of Cullin-RING E3 ligase, also complexes with these E2s for K11-linked substrate polyubiquitylation. Whether these two E3s cross-talk with each other was previously unknown. Here, we report that SAG competes with APC2 for UBE2C/UBE2S binding to act as a potential endogenous inhibitor of APC/C, thereby regulating the G2-to-M progression. As such, SAG knockdown triggers premature activation of APC/C, leading to mitotic slippage and resistance to anti-microtubule drugs. On the other hand, SAG itself is a substrate of APC/CCDH1 for targeted degradation at the G1 phase. The degradation-resistant mutant of SAG-R98A/L101A accelerates the G1-to-S progression. Our study reveals that the negative cross-talk between SAG and APC/C is likely a mechanism to ensure the fidelity of cell cycle progression.

Keywords:  APC/C; APC2; SAG; UBE2C; UBE2S; cell cycle; mitotic slippage; ubiquitylation

DOI:  https://doi.org/10.1016/j.celrep.2020.108102
Cell Death Differ. 2020 Sep 08.

UBE2O targets Mxi1 for ubiquitination and degradation to promote lung cancer progression and radioresistance.

Yumei Huang, Xijie Yang, Yanwei Lu, Ye Zhao, Rui Meng, Sheng Zhang, Xiaorong Dong, Shuangbing Xu, Gang Wu.

UBE2O, an E2/E3 hybrid ubiquitin-protein ligase, has been implicated in the regulation of adipogenesis, erythroid differentiation, and tumor proliferation. However, its role in cancer radioresistance remains completely unknown. Here, we uncover that UBE2O interacts and targets Mxi1 for ubiquitination and degradation at the K46 residue. Furthermore, we show that genetical or pharmacological blockade of UBE2O impairs tumor progression and radioresistance in lung cancer in vitro and in vivo, and these effects can be restored by Mxi1 inhibition. Moreover, we demonstrate that UBE2O is overexpressed and negatively correlated with Mxi1 protein levels in lung cancer tissues. Collectively, our work reveals that UBE2O facilitates tumorigenesis and radioresistance by promoting Mxi1 ubiquitination and degradation, suggesting that UBE2O is an attractive radiosensitization target for the treatment of lung cancer.

DOI: https://doi.org/10.1038/s41418-020-00616-8
Nature. 2020 Sep 09.

TRIM37 controls cancer-specific vulnerability to PLK4 inhibition.

Franz Meitinger, Midori Ohta, Kian-Yong Lee, Sadanori Watanabe, Robert L Davis, John V Anzola, Ruth Kabeche, David A Jenkins, Andrew K Shiau, Arshad Desai, Karen Oegema.

Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus1. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. 2,3). When PLK4 is chemically inhibited, cell division proceeds without centrosome duplication, generating centrosome-less cells that exhibit delayed, acentrosomal spindle assembly4. Whether PLK4 inhibitors can be leveraged as a treatment for cancer is not yet clear. Here we show that acentrosomal spindle assembly following PLK4 inhibition depends on levels of the centrosomal ubiquitin ligase TRIM37. Low TRIM37 levels accelerate acentrosomal spindle assembly and improve proliferation following PLK4 inhibition, whereas high TRIM37 levels inhibit acentrosomal spindle assembly, leading to mitotic failure and cessation of proliferation. The Chr17q region containing the TRIM37 gene is frequently amplified in neuroblastoma and in breast cancer5-8, rendering these cancer types highly sensitive to PLK4 inhibition. We find that inactivating TRIM37 improves acentrosomal mitosis because TRIM37 prevents PLK4 from self-assembling into centrosome-independent condensates that serve as ectopic microtubule-organizing centres. By contrast, elevated TRIM37 expression inhibits acentrosomal spindle assembly through a distinct mechanism that involves degradation of the centrosomal component CEP192. Thus, TRIM37 is an essential determinant of mitotic vulnerability to PLK4 inhibition. Linkage of TRIM37 to prevalent cancer-associated genomic changes-including 17q gain in neuroblastoma and 17q23 amplification in breast cancer-may offer an opportunity to use PLK4 inhibition to trigger selective mitotic failure and provide new avenues to treatments for these cancers.

DOI: https://doi.org/10.1038/s41586-020-2710-1
FASEB J. 2020 Sep 12.

RNF216 mediates neuronal injury following experimental subarachnoid hemorrhage through the Arc/Arg3.1-AMPAR pathway.

Tao Chen, Jie Zhu, Yu-Hai Wang.

  Subarachnoid hemorrhage (SAH), mostly caused by aneurysm rupture, is a pathological condition associated with oxidative stress and neuroinflammation. Toll-like receptors (TLRs) are a family of key regulators of neuroinflammation, and RNF216 is an E3 ubiquitin-protein ligase that regulates TLRs via ubiquitination and proteolytic degradation. However, the role of RNF216 in SAH has not been determined. In this study, we investigated the biological function of RNF216 in experimental SAH models both in vitro and in vivo. The expression of RNF216 was found to be upregulated in cortical neurons after oxyhemoglobin (OxyHb) treatment, and increased RNF216 expression was also observed in brain tissues in the single-hemorrhage model of SAH. Downregulation of RNF216 expression by short interfering RNA (siRNA) transfection significantly reduced cytotoxicity and apoptosis after OxyHb exposure. The results of western blot showed that the RNF216-mediated neuronal injury in vitro was associated with the regulation of the Arc-AMPAR pathway, which was related to intracellular Ca2+ dysfunction, as evidenced by Ca2+ imaging. In addition, knockdown of RNF216 in vivo using intraventricular injection of siRNA was found to attenuate brain injury and neuroinflammation via the Arc-AMPAR pathway after SAH in the animal model. In summary, we demonstrated that silence of RNF216 expression protects against neuronal injury and neurological dysfunction in experimental SAH models. These data support for the first time that RNF216 may represent a novel candidate for therapies against SAH.

Keywords:  AMPA receptors; Arc/Arg3.1; RNF216; subarachnoid hemorrhage

DOI:  https://doi.org/10.1096/fj.201903151RRRR
Cell Death Dis. 2020 Sep 10. 11(9): 740

CHIP ubiquitylates NOXA and induces its lysosomal degradation in response to DNA damage.

Marie-Christine Albert, Kerstin Brinkmann, Wojciech Pokrzywa, Saskia Diana Günther, Martin Krönke, Thorsten Hoppe, Hamid Kashkar.

The BH3-only protein NOXA is a regulator of mitochondrial apoptosis by specifically antagonizing the anti-apoptotic protein MCL-1. Here we show that the E3 ubiquitin ligase CHIP controls NOXA stability after DNA damage. Our findings reveal that CHIP and MCL-1 are binding partners of NOXA and differentially define the fate of NOXA. Whereas NOXA is initially targeted to mitochondria upon MCL-1-binding, CHIP mediates ubiquitylation of cytosolic NOXA and promotes lysosomal degradation of NOXA, which is not bound by MCL-1. Our data indicate that MCL-1 defines NOXA abundance and its pro-apoptotic activity. Increased NOXA levels beyond this threshold are effectively removed by lysosomal protein degradation triggered via CHIP-mediated ubiquitylation. Together, these results shed new light on regulatory circuits controlling DNA damage response and identified the E3 ligase CHIP as a new molecular guardian, which restricts the cytosolic accumulation of NOXA upon genotoxic stress.

DOI: https://doi.org/10.1038/s41419-020-02923-x
RNA Biol. 2020 Sep 06.

tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast.

Joana F Tavares, Nick K Davis, Ana Poim, Andreia Reis, Stefanie Kellner, Inês Sousa, Ana R Soares, Gabriela M R Moura, Peter C Dedon, Manuel A S Santos.

  /Summary Protein synthesis rate and accuracy are tightly controlled by the cell and are essential for proteome homeostasis for bona fide protein synthesis (proteostasis), however the full picture of how mRNA translational factors maintain protein synthesis accuracy and co-translational protein folding are far from being fully understood. To address this question, we evaluated the role of 70 yeast tRNA modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated proteins. We show that modification of uridine at anticodon position 34 (U34) by the tRNA modifying enzymes Elp1, Elp3, Sml3 and Trm9 is critical for proteostasis, the mitochondrial tRNA-modifying enzyme Slm3 plays a fundamental role in general proteostasis and that stress response proteins whose genes are enriched in codons decoded by tRNAs lacking mcm5U34, mcm5s2U34, ncm5U34, ncm5Um34, modifications are overrepresented in protein aggregates of the ELP1, SLM3 and TRM9 KO strains. Increased rates of amino acid misincorporation were also detected in these strains at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U34 tRNA modifications safeguard the proteome from translational errors, protein misfolding and proteotoxic stress.

Keywords:  (5-10) tRNA modifying enzymes; protein aggregation; proteome; tRNA; yeast

DOI:  https://doi.org/10.1080/15476286.2020.1819671
Eur J Med Chem. 2020 Jul 19. pii: S0223-5234(20)30466-9. [Epub ahead of print]206 112494

Chimera induced protein degradation: PROTACs and beyond.

Lina Yin, Qingzhong Hu.

  Ubiquitin-proteasome system, autophagy-lysosome pathway and N-end rule pathway are crucial protein quality control mechanisms in human body. Hijacking these endogenous protein degrading measures by chimera degraders could be a revolutionary strategy for the discovery of small-molecule drugs. As the most advanced chimera degraders, PROTACs have demonstrated the potential by delivering two drug candidates into clinical trials. The development of chimera degraders exploiting these three pathways are reviewed, a focus is given on the chemical structures and their influences on biological effects from a viewpoint of medicinal chemistry.

Keywords:  Autophagy-lysosome pathway; Chimera degraders; Drug discovery; N-end rule; PROTACs; Ubiquitin–proteasome system

DOI:  https://doi.org/10.1016/j.ejmech.2020.112494
Dev Cell. 2020 Aug 31. pii: S1534-5807(20)30666-3. [Epub ahead of print]

Selective Lysosome Membrane Turnover Is Induced by Nutrient Starvation.

Chan Lee, Lilian Lamech, Eleanor Johns, Michael Overholtzer.

  Lysosome function is essential for cellular homeostasis, but quality-control mechanisms that maintain healthy lysosomes remain poorly characterized. Here, we developed a method to measure lysosome turnover and use this to identify a selective mechanism of membrane degradation that involves lipidation of the autophagy protein LC3 onto lysosomal membranes and the formation of intraluminal vesicles through microautophagy. This mechanism is induced in response to metabolic stress resulting from glucose starvation or by treatment with pharmacological agents that induce osmotic stress on lysosomes. Cells lacking ATG5, an essential component of the LC3 lipidation machinery, show reduced ability to regulate lysosome size and degradative capacity in response to activation of this mechanism. These findings identify a selective mechanism of lysosome membrane turnover that is induced by stress and uncover a function for LC3 lipidation in regulating lysosome size and activity through microautophagy.

Keywords:  ATG5; LAP; LC3; ammonium; autophagy; glucose; glutamine; lysosome; metabolism; microautophagy

DOI:  https://doi.org/10.1016/j.devcel.2020.08.008
FASEB J. 2020 Sep 10.

Small ubiquitin-like modifier 2 (SUMO2) is critical for memory processes in mice.

Shu Yu, Francesca Galeffi, Ramona M Rodriguiz, Zhuoran Wang, Yuntian Shen, Jingjun Lyu, Ran Li, Joshua D Bernstock, Kory R Johnson, Shuai Liu, Huaxin Sheng, Dennis A Turner, William C Wetsel, Wulf Paschen, Wei Yang.

  Small ubiquitin-like modifier (SUMO1-3) conjugation (SUMOylation), a posttranslational modification, modulates almost all major cellular processes. Mounting evidence indicates that SUMOylation plays a crucial role in maintaining and regulating neural function, and importantly its dysfunction is implicated in cognitive impairment in humans. We have previously shown that simultaneously silencing SUMO1-3 expression in neurons negatively affects cognitive function. However, the roles of the individual SUMOs in modulating cognition and the mechanisms that link SUMOylation to cognitive processes remain unknown. To address these questions, in this study, we have focused on SUMO2 and generated a new conditional Sumo2 knockout mouse line. We found that conditional deletion of Sumo2 predominantly in forebrain neurons resulted in marked impairments in various cognitive tests, including episodic and fear memory. Our data further suggest that these abnormalities are attributable neither to constitutive changes in gene expression nor to alterations in neuronal morphology, but they involve impairment in dynamic SUMOylation processes associated with synaptic plasticity. Finally, we provide evidence that dysfunction on hippocampal-based cognitive tasks was associated with a significant deficit in the maintenance of hippocampal long-term potentiation in Sumo2 knockout mice. Collectively, these data demonstrate that protein conjugation by SUMO2 is critically involved in cognitive processes.

Keywords:  LTP; knockout; memory impairment; posttranslational modification

DOI:  https://doi.org/10.1096/fj.202000850RR
J Integr Plant Biol. 2020 Sep 10.

Suppression of DRR1 results in the accumulation of insoluble ubiquitinated proteins, which impairs drought stress tolerance.

Seong Gwan Yu, Na Hyun Cho, Jong Hum Kim, Tae Rin Oh, Woo Taek Kim.

Drought stress has detrimental effects on plants. Although the ABA-mediated drought response is well established, defensive mechanisms to cope with dehydration-induced proteotoxicity have been rarely studied. DRR1 was identified as an Arabidopsis drought-induced gene encoding an ER-localized RING-type E3 Ub ligase. Suppression of DRR1 markedly reduced tolerance to drought and proteotoxic stress without altering ABA-mediated germination and stomatal movement. Proteotoxicity- and dehydration-induced insoluble ubiquitinated protein accumulation was more obvious in DRR1 loss-of-function plants than in wild-type plants. These results suggest that DRR1 is involved in an ABA-independent drought stress response possibly through the mitigation of dehydration-induced proteotoxic stress. This article is protected by copyright. All rights reserved.

DOI: https://doi.org/10.1111/jipb.13014
Plant Physiol. 2020 Sep 08. pii: pp.00880.2020. [Epub ahead of print]

p24 family proteins are involved in transport to the plasma membrane of GPI-anchored proteins in plants.

César Bernat-Silvestre, Vanessa De Sousa Vieira, Judit Sánchez-Simarro, Noelia Pastor-Cantizano, Chris Hawes, María Jesús Marcote, Fernando Aniento.

p24 proteins are a family of type-I membrane proteins which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus via Coat Protein I (COPI)- and COPII-coated vesicles. They have been proposed to function as cargo receptors, but the identity of putative cargos in plants is still elusive. We previously generated an Arabidopsis thaliana quadruple loss-of-function mutant affecting p24 genes from the delta-1 subclass of the p24 delta subfamily (p24δ3δ4δ5δ6 mutant). This mutant also had reduced protein levels of other p24 family proteins and was found to be sensitive to salt stress. Here, we used this mutant to test the possible involvement of p24 proteins in the transport to the plasma membrane of glycosylphosphatidylinositol (GPI)-anchored proteins. We found that GPI-anchored proteins mostly localized to the ER in p24δ3δ4δ5δ6-mutant cells, in contrast to plasma-membrane proteins with other types of membrane attachment. The plasma-membrane localization of GPI-anchored proteins was restored in the p24δ3δ4δ5δ6 mutant upon transient expression of a single member of the p24delta-1 subclass, RFP-p24δ5, which was dependent on the coiled-coil domain in p24δ5. The coiled-coil domain was also important for a direct interaction between p24δ5 and the GPI-anchored protein arabinogalactan protein 4 (AGP4). These results suggest that Arabidopsis p24 proteins are involved in ER export and transport to the plasma membrane of GPI-anchored proteins.

DOI: https://doi.org/10.1104/pp.20.00880
Proc Natl Acad Sci U S A. 2020 Sep 08. pii: 202004122. [Epub ahead of print]

TRIM28 functions as the SUMO E3 ligase for PCNA in prevention of transcription induced DNA breaks.

Min Li, Xiaohua Xu, Chou-Wei Chang, Yilun Liu.

  In human cells, the DNA replication factor proliferating cell nuclear antigen (PCNA) can be conjugated to either the small ubiquitinlike modifier SUMO1 or SUMO2, but only SUMO2-conjugated PCNA is induced by transcription to facilitate resolution of transcription-replication conflict (TRC). To date, the SUMO E3 ligase that provides substrate specificity for SUMO2-PCNA conjugation in response to TRC remains unknown. Using a proteomic approach, we identified TRIM28 as the E3 ligase that catalyzes SUMO2-PCNA conjugation. In vitro, TRIM28, together with the RNA polymerase II (RNAPII)-interacting protein RECQ5, promotes SUMO2-PCNA conjugation but inhibits SUMO1-PCNA formation. This activity requires a PCNA-interacting protein (PIP) motif located within the bromodomain of TRIM28. In cells, TRIM28 interaction with PCNA on human chromatin is dependent on both transcription and RECQ5, and SUMO2-PCNA level correlates with TRIM28 expression. As a consequence, TRIM28 depletion led to RNAPII accumulation at TRC sites, and expression of a TRIM28 PIP mutant failed to suppress TRC-induced DNA breaks.

Keywords:  DNA replication; PCNA; RECQ5; SUMO2; TRIM28

DOI:  https://doi.org/10.1073/pnas.2004122117