bims-proteo 2021-04-18 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2021‒04‒18
forty papers selected by
Eric Chevet
INSERM

SLFN11 inactivation induces proteotoxic stress and sensitizes cancer cells to ubiquitin activating enzyme inhibitor TAK-243.
WLS/wntless is essential in controlling dendritic cell homeostasis via a WNT signaling-independent mechanism.
Membrane protein folding and quality control.
The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.
The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.
A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage.
Cellular senescence and tumor promotion: Role of the Unfolded Protein Response.
A model for a partnership of lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis.
Structural and Functional Significance of the Endoplasmic Reticulum Unfolded Protein Response Transducers and Chaperones at the Mitochondria-ER Contacts: A Cancer Perspective.
Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism.
Major Advances in Targeted Protein Degradation: PROTACs, LYTACs, and MADTACs.
ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.
Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation.
The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Proteostasis Factors.
Phosphorylation of the LIR domain of SCOC modulates ATG8 binding affinity and specificity.
The Dose-Dependent Pleiotropic Effects of the UBB+1 Ubiquitin Mutant.
COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers.
Inhibitors of heat shock protein 70 (Hsp70) with enhanced metabolic stability reduce tau levels.
Acetylated Ubiquitin Modulates the Catalytic Activity of the E1 Enzyme Uba1.
Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING.
Novel Insight into the Role of Endoplasmic Reticulum Stress in the Pathogenesis of Myocardial Ischemia-Reperfusion Injury.
Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.
Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells.
Human SND2 mediates ER targeting of GPI-anchored proteins with low hydrophobic GPI-attachment signals.
Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.
Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.
p62/SQSTM1 droplets initiate autophagosome biogenesis and oxidative stress control.
Development of MDM2 degraders based on ligands derived from Ugi reactions: Lessons and discoveries.
The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.
Subtle genomic DNA damage induces intraneuronal production of amyloid-β (1-42) by increasing β-secretase activity.
FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins.
Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases.
A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization.
DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network.
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.
NUCB1 Suppresses Growth and Shows Additive Effects With Gemcitabine in Pancreatic Ductal Adenocarcinoma via the Unfolded Protein Response.
The E3 ligase MREL57 modulates microtubule stability and stomatal closure in response to ABA.
Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease.
Roles of FAM134B in diseases from the perspectives of organelle membrane morphogenesis and cellular homeostasis.
UBC13 is an RNF213-associated E2 ubiquitin-conjugating enzyme, and Lysine 63-linked ubiquitination by the RNF213-UBC13 axis is responsible for angiogenic activity.

Cancer Res. 2021 Apr 16. pii: canres.2694.2020. [Epub ahead of print]

SLFN11 inactivation induces proteotoxic stress and sensitizes cancer cells to ubiquitin activating enzyme inhibitor TAK-243.

Yasuhisa Murai, Ukhyun Jo, Junko Murai, Lisa Jenkins, Shar-Yin N Huang, Sirisha Chakka, Lu Chen, Ken Cheng, Shinsaku Fukuda, Naoko Takebe, Yves Pommier.

Schlafen11 (SLFN11) inactivation occurs in approximately 50% of cancer cell lines and in a large fraction of patient tumor samples, which leads to chemoresistance. Therefore, new therapeutic approaches are needed to target SLFN11-deficient cancers. To that effect, we conducted a drug screen with the NCATS mechanistic drug library of 1978 compounds in isogenic SLFN11-knockout (KO) and wild-type (WT) leukemia cell lines. Here we report that TAK-243, a first-in-class ubiquitin activating enzyme UBA1 inhibitor in clinical development, causes preferential cytotoxicity in SLFN11-KO cells; this effect is associated with claspin-mediated DNA replication inhibition by CHK1 independently of ATR. Additional analyses showed that SLFN11-KO cells exhibit consistently enhanced global protein ubiquitylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and protein aggregation. TAK-243 suppressed global protein ubiquitylation and activated the UPR transducers PERK, phosphorylated eIF2alpha, phosphorylated IRE1, and ATF6 more effectively in SLFN11-KO cells than WT cells. Proteomic analysis using biotinylated mass spectrometry and RNAi screening also showed physical and functional interactions of SLFN11 with translation initiation complexes and protein folding machinery. These findings uncover a previously unknown function of SLFN11 as a regulator of protein quality control and attenuator of ER stress and UPR. Moreover, they suggest the potential value of TAK-243 in SLFN11-deficient tumors.

DOI: https://doi.org/10.1158/0008-5472.CAN-20-2694
Autophagy. 2021 Apr 14. 1-16

WLS/wntless is essential in controlling dendritic cell homeostasis via a WNT signaling-independent mechanism.

Li-Ting Wang, Ming-Hong Lin, Kwei-Yan Liu, Shyh-Shin Chiou, Shen-Nien Wang, Chee-Yin Chai, Li-Wen Tseng, Hsin-Ying Clair Chiou, Hsueh-Chun Wang, Kazunari K Yokoyama, Shih-Hsien Hsu, Shau-Ku Huang.

  We propose that beyond its role in WNT secretion, WLS/GPR177 (wntless, WNT ligand secretion mediator) acts as an essential regulator controlling protein glycosylation, endoplasmic reticulum (ER) homeostasis, and dendritic cell (DC)-mediated immunity. WLS deficiency in bone marrow-derived DCs (BMDCs) resulted in poor growth and an inability to mount cytokine and T-cell responses in vitro, phenotypes that were irreversible by the addition of exogenous WNTs. In fact, WLS was discovered to integrate a protein complex in N-glycan-dependent and WLS domain-selective manners, comprising ER stress sensors and lectin chaperones. WLS deficiency in BMDCs led to increased ER stress response and macroautophagy/autophagy, decreased calcium efflux from the ER, and the loss of CALR (calreticulin)-CANX (calnexin) cycle, and hence protein hypo-glycosylation. Consequently, DC-specific wls-null mice were unable to develop both Th1-, Th2- and Th17-associated responses in the respective autoimmune and allergic disease models. These results suggest that WLS is a critical chaperone in maintaining ER homeostasis, glycoprotein quality control and calcium dynamics in DCs.Abbreviations: ATF6: activating transcription factor 6; ATG5: autophagy related 5; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; ATP2A1/SERCA1: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1; BALF: bronchoalveolar lavage fluid; BFA: brefeldin A; BMDC: bone marrow-derived dendritic cell; CALR: calreticulin; CANX: calnexin; CCL2/MCP-1: C-C motif chemokine ligand 2; CNS: central nervous system; CT: C-terminal domain; DTT: dithiothreitol; DNAJB9/ERDJ4: DnaJ heat shock protein family (Hsp40) member B9; EAE: experimental autoimmune encephalomyelitis; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1/IRE1: endoplasmic reticulum (ER) to nucleus signaling 1; GFP: green fluorescent protein; HSPA5/GRP78/BiP: heat shock protein A5; IFNA: interferon alpha; IFNAR1: interferon alpha and beta receptor subunit 1; IFNB: interferon beta; IFNG/INFγ: interferon gamma; IFNGR2: interferon gamma receptor 2; IL6: interleukin 6; IL10: interleukin 10; IL12A: interleukin 12A; IL23A: interleukin 23 subunit alpha; ITGAX/CD11c: integrin subunit alpha X; ITPR1/InsP3R1: inositol 1,4,5-trisphosphate receptor type 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; OVA: ovalbumin; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PLF: predicted lipocalin fold; PPP1R15A/GADD34: protein phosphatase 1 regulatory subunit 15A; RYR1/RyanR1: ryanodine receptor 1, skeletal muscle; SD: signal domain; TGFB/TGF-β: transforming growth factor beta family; Th1: T helper cell type 1; Th17: T helper cell type 17; TM: tunicamycin; TNF/TNF-α: tumor necrosis factor; UPR: unfolded protein response; WLS/wntless: WNT ligand secretion mediator.

Keywords:  Dendritic cells; er stress; gpr177; protein glycosylation; unfold protein response

DOI:  https://doi.org/10.1080/15548627.2021.1907516
Curr Opin Struct Biol. 2021 Apr 12. pii: S0959-440X(21)00035-X. [Epub ahead of print]69 50-54

Membrane protein folding and quality control.

Ben P Phillips, Elizabeth A Miller.

Membrane proteins account for a quarter of cellular proteins, and most are synthesised at the endoplasmic reticulum (ER). Insertion and folding of polypeptides in the membrane environment is prone to error, necessitating diverse quality control systems. Recent discoveries have demonstrated how forces act on the nascent chain during insertion, and revealed new translocon components and accessories that facilitate the correct biogenesis of substrates. Our understanding of one of the best studied quality control systems-ER-associated degradation-has been advanced through new structural and functional studies of the core Hrd1 complex, and through the discovery of a new branch of this degradative pathway. New data also reveal how cells resolve clogged translocons, which would otherwise be unable to function. Finally, new work elucidates how mitochondrial tail-anchored proteins that have been mistargeted to the ER are identified and destroyed. Overall, we describe an emerging picture of an increasingly complex quality control network.

DOI: https://doi.org/10.1016/j.sbi.2021.03.003
J Biol Chem. 2021 Jan 08. pii: S0021-9258(21)00011-9. [Epub ahead of print]296 100246

The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.

Lee D Harris, Janic Le Pen, Nico Scholz, Juliusz Mieszczanek, Natalie Vaughan, Simon Davis, Georgina Berridge, Benedikt M Kessler, Mariann Bienz, Julien D F Licchesi.

  Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

Keywords:  E3 ubiquitin ligase; HECTD1; K29/K48-linked polyubiquitin chain; TRABID; deubiquitination; polyubiquitin chain; protein degradation; ubiquitin; ubiquitin thioesterase

DOI:  https://doi.org/10.1074/jbc.RA120.015162
EMBO Rep. 2021 Apr 14. e50684

The deubiquitinase USP36 promotes snoRNP group SUMOylation and is essential for ribosome biogenesis.

Hyunju Ryu, Xiao-Xin Sun, Yingxiao Chen, Yanping Li, Xiaoyan Wang, Roselyn S Dai, Hong-Ming Zhu, John Klimek, Larry David, Lev M Fedorov, Yoshiaki Azuma, Rosalie C Sears, Mu-Shui Dai.

  SUMOylation plays a crucial role in regulating diverse cellular processes including ribosome biogenesis. Proteomic analyses and experimental evidence showed that a number of nucleolar proteins involved in ribosome biogenesis are modified by SUMO. However, how these proteins are SUMOylated in cells is less understood. Here, we report that USP36, a nucleolar deubiquitinating enzyme (DUB), promotes nucleolar SUMOylation. Overexpression of USP36 enhances nucleolar SUMOylation, whereas its knockdown or genetic deletion reduces the levels of SUMOylation. USP36 interacts with SUMO2 and Ubc9 and directly mediates SUMOylation in cells and in vitro. We show that USP36 promotes the SUMOylation of the small nucleolar ribonucleoprotein (snoRNP) components Nop58 and Nhp2 in cells and in vitro and their binding to snoRNAs. It also promotes the SUMOylation of snoRNP components Nop56 and DKC1. Functionally, we show that knockdown of USP36 markedly impairs rRNA processing and translation. Thus, USP36 promotes snoRNP group SUMOylation and is critical for ribosome biogenesis and protein translation.

Keywords:  SUMOylation; USP36; deubiquitinating enzyme; ribosome biogenesis; snoRNP

DOI:  https://doi.org/10.15252/embr.202050684
Elife. 2021 04 13. pii: e62653. [Epub ahead of print]10

A genetic screen identifies new steps in oocyte maturation that enhance proteostasis in the immortal germ lineage.

Madhuja Samaddar, Jérôme Goudeau, Melissa Sanchez, David H Hall, K Adam Bohnert, Maria Ingaramo, Cynthia Kenyon.

  Somatic cells age and die, but the germ-cell lineage is immortal. In Caenorhabditis elegans, germline immortality involves proteostasis renewal at the beginning of each new generation, when oocyte maturation signals from sperm trigger the clearance of carbonylated proteins and protein aggregates. Here, we explore the cell biology of this proteostasis renewal in the context of a whole-genome RNAi screen. Oocyte maturation signals are known to trigger protein-aggregate removal via lysosome acidification. Our findings suggest that lysosomes are acidified as a consequence of changes in endoplasmic reticulum activity that permit assembly of the lysosomal V-ATPase, which in turn allows lysosomes to clear the aggregates via microautophagy. We define two functions for mitochondria, both of which appear to be independent of ATP generation. Many genes from the screen also regulate lysosome acidification and age-dependent protein aggregation in the soma, suggesting a fundamental mechanistic link between proteostasis renewal in the germline and somatic longevity.

Keywords:  C. elegans; cell biology; genetic screen; germ lineage; lysosome acidification; oocyte maturation; proteostasis

DOI:  https://doi.org/10.7554/eLife.62653
Adv Cancer Res. 2021 ;pii: S0065-230X(21)00001-4. [Epub ahead of print]150 285-334

Cellular senescence and tumor promotion: Role of the Unfolded Protein Response.

Olivier Pluquet, Corinne Abbadie.

  Senescence is a cellular state which can be viewed as a stress response phenotype implicated in various physiological and pathological processes, including cancer. Therefore, it is of fundamental importance to understand why and how a cell acquires and maintains a senescent phenotype. Direct evidence has pointed to the homeostasis of the endoplasmic reticulum whose control appears strikingly affected during senescence. The endoplasmic reticulum is one of the sensing organelles that transduce signals between different pathways in order to adapt a functional proteome upon intrinsic or extrinsic challenges. One of these signaling pathways is the Unfolded Protein Response (UPR), which has been shown to be activated during senescence. Its exact contribution to senescence onset, maintenance, and escape, however, is still poorly understood. In this article, we review the mechanisms through which the UPR contributes to the appearance and maintenance of characteristic senescent features. We also discuss whether the perturbation of the endoplasmic reticulum proteostasis or accumulation of misfolded proteins could be possible causes of senescence, and-as a consequence-to what extent the UPR components could be considered as therapeutic targets allowing for the elimination of senescent cells or altering their secretome to prevent neoplastic transformation.

Keywords:  Aging; Endoplasmic reticulum; Homeostasis; Secretome; Senescence; Unfolded protein response

DOI:  https://doi.org/10.1016/bs.acr.2021.01.001
Proc Natl Acad Sci U S A. 2021 Apr 20. pii: e2101562118. [Epub ahead of print]118(16):

A model for a partnership of lipid transfer proteins and scramblases in membrane expansion and organelle biogenesis.

Alireza Ghanbarpour, Diana P Valverde, Thomas J Melia, Karin M Reinisch.

  The autophagy protein ATG2, proposed to transfer bulk lipid from the endoplasmic reticulum (ER) during autophagosome biogenesis, interacts with ER residents TMEM41B and VMP1 and with ATG9, in Golgi-derived vesicles that initiate autophagosome formation. In vitro assays reveal TMEM41B, VMP1, and ATG9 as scramblases. We propose a model wherein membrane expansion results from the partnership of a lipid transfer protein, moving lipids between the cytosolic leaflets of apposed organelles, and scramblases that reequilibrate the leaflets of donor and acceptor organelle membranes as lipids are depleted or augmented. TMEM41B and VMP1 are implicated broadly in lipid homeostasis and membrane dynamics processes in which their scrambling activities likely are key.

Keywords:  ATG9A; TMEM41B; VMP1; scramblase

DOI:  https://doi.org/10.1073/pnas.2101562118
Front Cell Dev Biol. 2021 ;9 641194

Structural and Functional Significance of the Endoplasmic Reticulum Unfolded Protein Response Transducers and Chaperones at the Mitochondria-ER Contacts: A Cancer Perspective.

Giuseppina Amodio, Valentina Pagliara, Ornella Moltedo, Paolo Remondelli.

  In the last decades, the endoplasmic reticulum (ER) has emerged as a key coordinator of cellular homeostasis, thanks to its physical interconnection to almost all intracellular organelles. In particular, an intense and mutual crosstalk between the ER and mitochondria occurs at the mitochondria-ER contacts (MERCs). MERCs ensure a fine-tuned regulation of fundamental cellular processes, involving cell fate decision, mitochondria dynamics, metabolism, and proteostasis, which plays a pivotal role in the tumorigenesis and therapeutic response of cancer cells. Intriguingly, recent studies have shown that different components of the unfolded protein response (UPR) machinery, including PERK, IRE1α, and ER chaperones, localize at MERCs. These proteins appear to exhibit multifaceted roles that expand beyond protein folding and UPR transduction and are often related to the control of calcium fluxes to the mitochondria, thus acquiring relevance to cell survival and death. In this review, we highlight the novel functions played by PERK, IRE1α, and ER chaperones at MERCs focusing on their impact on tumor development.

Keywords:  cancer; endoplasmic reticulum; mitochondria–ER contacts; molecular chaperones; unfolded protein response

DOI:  https://doi.org/10.3389/fcell.2021.641194
Commun Biol. 2021 Apr 12. 4(1): 454

Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism.

Michael J Rigby, Alexis J Lawton, Gulpreet Kaur, Varuna C Banduseela, William E Kamm, Aparna Lakkaraju, John M Denu, Luigi Puglielli.

Nε-lysine acetylation in the ER lumen is a recently discovered quality control mechanism that ensures proteostasis within the secretory pathway. The acetyltransferase reaction is carried out by two type-II membrane proteins, ATase1/NAT8B and ATase2/NAT8. Prior studies have shown that reducing ER acetylation can induce reticulophagy, increase ER turnover, and alleviate proteotoxic states. Here, we report the generation of Atase1-/- and Atase2-/- mice and show that these two ER-based acetyltransferases play different roles in the regulation of reticulophagy and macroautophagy. Importantly, knockout of Atase1 alone results in activation of reticulophagy and rescue of the proteotoxic state associated with Alzheimer's disease. Furthermore, loss of Atase1 or Atase2 results in widespread adaptive changes in the cell acetylome and acetyl-CoA metabolism. Overall, our study supports a divergent role of Atase1 and Atase2 in cellular biology, emphasizing ATase1 as a valid translational target for diseases characterized by toxic protein aggregation in the secretory pathway.

DOI: https://doi.org/10.1038/s42003-021-01992-8
J Biol Chem. 2021 Apr 08. pii: S0021-9258(21)00433-6. [Epub ahead of print] 100647

Major Advances in Targeted Protein Degradation: PROTACs, LYTACs, and MADTACs.

Shanique Alabi, Craig Crews.

  Of late, targeted protein degradation (TPD) has surfaced as a novel and innovative chemical tool and therapeutic modality. By co-opting protein degradation pathways, TPD facilitates complete removal of the protein molecules from within or outside the cell. While the pioneering Proteolysis Targeting Chimera (PROTAC) technology and molecular glues hijack the ubiquitin-proteasome system, newer modalities co-opt autophagy or the endo-lysosomal pathway. Using this mechanism, TPD is posited to largely expand the druggable space far beyond small molecule inhibitors. In this review, we discuss the major advances in TPD, highlight our current understanding, and explore outstanding questions in the field.

Keywords:  AUTACs; LYTACs; Molecular Glues; PROTACs; chemical biology; drug action; lysosome; protein degradation; ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2021.100647
Cell. 2021 Apr 13. pii: S0092-8674(21)00366-4. [Epub ahead of print]

ER-to-Golgi protein delivery through an interwoven, tubular network extending from ER.

Aubrey V Weigel, Chi-Lun Chang, Gleb Shtengel, C Shan Xu, David P Hoffman, Melanie Freeman, Nirmala Iyer, Jesse Aaron, Satya Khuon, John Bogovic, Wei Qiu, Harald F Hess, Jennifer Lippincott-Schwartz.

  Cellular versatility depends on accurate trafficking of diverse proteins to their organellar destinations. For the secretory pathway (followed by approximately 30% of all proteins), the physical nature of the vessel conducting the first portage (endoplasmic reticulum [ER] to Golgi apparatus) is unclear. We provide a dynamic 3D view of early secretory compartments in mammalian cells with isotropic resolution and precise protein localization using whole-cell, focused ion beam scanning electron microscopy with cryo-structured illumination microscopy and live-cell synchronized cargo release approaches. Rather than vesicles alone, the ER spawns an elaborate, interwoven tubular network of contiguous lipid bilayers (ER exit site) for protein export. This receptacle is capable of extending microns along microtubules while still connected to the ER by a thin neck. COPII localizes to this neck region and dynamically regulates cargo entry from the ER, while COPI acts more distally, escorting the detached, accelerating tubular entity on its way to joining the Golgi apparatus through microtubule-directed movement.

Keywords:  COPI; COPII; cholesterol; correlative light and electron microscopy; endoplasmic reticulum exit sites; endoplasmic reticulum to Golgi transport intermediate; focused ion beam-scanning electron microscopy; membrane trafficking; retention using selective hook system; secretory pathway

DOI:  https://doi.org/10.1016/j.cell.2021.03.035
iScience. 2021 Apr 23. 24(4): 102296

Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation.

Chihiro Hirayama, Kodai Machida, Kentaro Noi, Tadayoshi Murakawa, Masaki Okumura, Teru Ogura, Hiroaki Imataka, Kenji Inaba.

  The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.

Keywords:  Cell Biology; Molecular Biology; Structural Biology

DOI:  https://doi.org/10.1016/j.isci.2021.102296
J Neuropathol Exp Neurol. 2021 Apr 16. pii: nlab029. [Epub ahead of print]

The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Proteostasis Factors.

Benjamin C Creekmore, Yi-Wei Chang, Edward B Lee.

  Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.

Keywords:  Alzheimer disease; Amyotrophic lateral sclerosis; ClpX; Fibrils; Frontotemporal degeneration; Multisystem proteinopathy; Vacuolar tauopathy

DOI:  https://doi.org/10.1093/jnen/nlab029
J Mol Biol. 2021 Apr 09. pii: S0022-2836(21)00188-1. [Epub ahead of print] 166987

Phosphorylation of the LIR domain of SCOC modulates ATG8 binding affinity and specificity.

Martina Wirth, Stephane Mouilleron, Wenxin Zhang, Eva Sjottem, Yakubu Princely Abudu, Ashish Jain, Hallvard Lauritz Olsvik, Jack-Ansgar Bruun, Minoo Razi, Harold B J Jefferies, Rebecca Lee, Dhira Joshi, Nicola O'Reilly, Terje Johansen, Sharon A Tooze.

  Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors.

Keywords:  Autophagy; Bio-layer interferometry; LIR motif; crystal structure; phosphorylation

DOI:  https://doi.org/10.1016/j.jmb.2021.166987
Front Mol Biosci. 2021 ;8 650730

The Dose-Dependent Pleiotropic Effects of the UBB+1 Ubiquitin Mutant.

Katarzyna Banasiak, Natalia A Szulc, Wojciech Pokrzywa.

  The proteolytic machinery activity diminishes with age, leading to abnormal accumulation of aberrant proteins; furthermore, a decline in protein degradation capacity is associated with multiple age-related proteinopathies. Cellular proteostasis can be maintained via the removal of ubiquitin (Ub)-tagged damaged and redundant proteins by the ubiquitin-proteasome system (UPS). However, during aging, central nervous system (CNS) cells begin to express a frameshift-mutated Ub, UBB+1. Its accumulation is a neuropathological hallmark of tauopathy, including Alzheimer's disease and polyglutamine diseases. Mechanistically, in cell-free and cell-based systems, an increase in the UBB+1 concentration disrupts proteasome processivity, leading to increased aggregation of toxic proteins. On the other hand, a low level of UBB+1 improves stress resistance and extends lifespan. Here we summarize recent findings regarding the impact of UBB+1 on Ub signaling and neurodegeneration. We also review the molecular basis of how UBB+1 affects UPS components as well as its dose-dependent switch between cytoprotective and cytotoxic roles.

Keywords:  ROS generation and cytotoxicity; UBB+1; cellular viability; molecular misreading; neurodegeneration; proteotoxic stress response; ubiquitin proteasomal system

DOI:  https://doi.org/10.3389/fmolb.2021.650730
J Cell Biol. 2021 Jun 07. pii: e201907224. [Epub ahead of print]220(6):

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers.

Olga Shomron, Inbar Nevo-Yassaf, Tamar Aviad, Yakey Yaffe, Eitan Erez Zahavi, Anna Dukhovny, Eran Perlson, Ilya Brodsky, Adva Yeheskel, Metsada Pasmanik-Chor, Anna Mironov, Galina V Beznoussenko, Alexander A Mironov, Ella H Sklan, George H Patterson, Yoji Yonemura, Mara Sannai, Christoph Kaether, Koret Hirschberg.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.

DOI: https://doi.org/10.1083/jcb.201907224
Bioorg Med Chem Lett. 2021 Apr 08. pii: S0960-894X(21)00251-1. [Epub ahead of print] 128025

Inhibitors of heat shock protein 70 (Hsp70) with enhanced metabolic stability reduce tau levels.

Hao Shao, Xiaokai Li, Shigenari Hayashi, Jeanette L Bertron, Daniel M C Schwarz, Benjamin C Tang, Jason E Gestwicki.

  The molecular chaperone, Heat Shock Protein 70 (Hsp70), is an emerging drug target for neurodegenerative diseases, because of its ability to promote degradation of microtubule-associated protein tau (MAPT/tau). Recently, we reported YM-08 as a brain penetrant, allosteric Hsp70 inhibitor, which reduced tau levels. However, the benzothiazole moiety of YM-08 is vulnerable to metabolism by CYP3A4, limiting its further application as a chemical probe. In this manuscript, we designed and synthesized seventeen YM-08 derivatives by systematically introducing halogen atoms to the benzothiazole ring and shifting the position of the heteroatom in a distal pyridine. In microsome assays, we found that compound JG-23 has 12-fold better metabolic stability and it retained the ability to reduce tau levels in two cell-based models. These chemical probes of Hsp70 are expected to be useful tools for studying tau homeostasis.

Keywords:  Hsc70; Hsp72; Metabolism; Molecular chaperone; Neurodegeneration; Protein folding; Tauopathy

DOI:  https://doi.org/10.1016/j.bmcl.2021.128025
Biochemistry. 2021 Apr 13.

Acetylated Ubiquitin Modulates the Catalytic Activity of the E1 Enzyme Uba1.

Rachel E Lacoursiere, Gary S Shaw.

Ubiquitin (Ub) signaling requires the covalent passage of Ub among E1, E2, and E3 enzymes. The choice of E2 and E3 enzymes combined with multiple rounds of the cascade leads to the formation of polyubiquitin chains linked through any one of the seven lysines on Ub. The linkage type and length act as a signal to trigger important cellular processes such as protein degradation or the DNA damage response. Recently, proteomics studies have identified that Ub can be acetylated at six of its seven lysine residues under various cell stress conditions. To understand the potential differences in Ub signaling caused by acetylation, we synthesized all possible acetylated ubiquitin (acUb) variants and examined the E1-mediated formation of the corresponding E2∼acUb conjugates in vitro using kinetic methods. A Förster resonance energy transfer assay was optimized in which the Ub constructs were labeled with a CyPet fluorophore and the E2 UBE2D1 was labeled with a YPet fluorophore to monitor the formation of E2∼Ub conjugates. Our methods enable the detection of small differences that may otherwise be concealed in steady-state ubiquitination experiments. We determined that Ub, acetylated at K11, K27, K33, K48, or K63, has altered turnover numbers for E2∼Ub conjugate formation by the E1 enzyme Uba1. This work provides evidence that acetylation of Ub can alter the catalysis of ubiquitination early on in the pathway.

DOI: https://doi.org/10.1021/acs.biochem.1c00145
Immunity. 2021 Apr 07. pii: S1074-7613(21)00123-0. [Epub ahead of print]

Golgi apparatus-synthesized sulfated glycosaminoglycans mediate polymerization and activation of the cGAMP sensor STING.

Run Fang, Qifei Jiang, Yukun Guan, Pengfei Gao, Rui Zhang, Zhen Zhao, Zhengfan Jiang.

  Activation of the cyclic guanosine monophosphate (GMP)-AMP (cGAMP) sensor STING requires its translocation from the endoplasmic reticulum to the Golgi apparatus and subsequent polymerization. Using a genome-wide CRISPR-Cas9 screen to define factors critical for STING activation in cells, we identified proteins critical for biosynthesis of sulfated glycosaminoglycans (sGAGs) in the Golgi apparatus. Binding of sGAGs promoted STING polymerization through luminal, positively charged, polar residues. These residues are evolutionarily conserved, and selective mutation of specific residues inhibited STING activation. Purified or chemically synthesized sGAGs induced STING polymerization and activation of the kinase TBK1. The chain length and O-linked sulfation of sGAGs directly affected the level of STING polymerization and, therefore, its activation. Reducing the expression of Slc35b2 to inhibit GAG sulfation in mice impaired responses to vaccinia virus infection. Thus, sGAGs in the Golgi apparatus are necessary and sufficient to drive STING polymerization, providing a mechanistic understanding of the requirement for endoplasmic reticulum (ER)-to-Golgi apparatus translocation for STING activation.

Keywords:  STING-associated vasculopathy with onset in infancy (SAVI); anti-viral innate immunity; cGAS-STING; sulfated glycosaminoglycans (sGAGs)

DOI:  https://doi.org/10.1016/j.immuni.2021.03.011
Oxid Med Cell Longev. 2021 ;2021 5529810

Novel Insight into the Role of Endoplasmic Reticulum Stress in the Pathogenesis of Myocardial Ischemia-Reperfusion Injury.

Hang Zhu, Hao Zhou.

Impaired function of the endoplasmic reticulum (ER) is followed by evolutionarily conserved cell stress responses, which are employed by cells, including cardiomyocytes, to maintain and/or restore ER homeostasis. ER stress activates the unfolded protein response (UPR) to degrade and remove abnormal proteins from the ER lumen. Although the UPR is an intracellular defense mechanism to sustain cardiomyocyte viability and heart function, excessive activation initiates ER-dependent cardiomyocyte apoptosis. Myocardial ischemia/reperfusion (I/R) injury is a pathological process occurring during or after revascularization of ischemic myocardium. Several molecular mechanisms contribute to the pathogenesis of cardiac I/R injury. Due to the dual protective/degradative effects of ER stress on cardiomyocyte viability and function, it is of interest to understand the basic concepts, regulatory signals, and molecular processes involved in ER stress following myocardial I/R injury. In this review, therefore, we present recent findings related to the novel components of ER stress activation. The complex effects of ER stress and whether they mitigate or exacerbate myocardial I/R injury are summarized to serve as the basis for research into potential therapies for cardioprotection through control of ER homeostasis.

DOI: https://doi.org/10.1155/2021/5529810
Nat Commun. 2021 04 12. 12(1): 2176

Protein kinase A controls the hexosamine pathway by tuning the feedback inhibition of GFAT-1.

Sabine Ruegenberg, Felix A M C Mayr, Ilian Atanassov, Ulrich Baumann, Martin S Denzel.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

DOI: https://doi.org/10.1038/s41467-021-22320-y
Bio Protoc. 2021 Mar 20. 11(6): e3958

Retention Using Selective Hooks (RUSH) Cargo Sorting Assay for Live-cell Vesicle Tracking in the Secretory Pathway Using HeLa Cells.

Mehrshad Pakdel, Natalia Pacheco-Fernandez, Julia von Blume.

  More than 30% of the total amount of proteins synthesized in mammalian cells follow the secretory pathway in order to mature and be properly sorted to their final destinations. Among several methodologies that describe live-cell monitoring of vesicles, the Retention Using Selective Hooks (RUSH) system is a powerful one that allows to visualize cargo trafficking under physiological conditions. The present protocol describes a method to use the RUSH system in live-cell microscopy and a subsequent quantitative analysis of cargo vesicles to dissect protein trafficking. In brief, HeLa cells are transiently transfected with an MMP2-RUSH construct and vesicle trafficking is evaluated by wide-field microscopy, recording videos in 1-min time frames for 45 min. We also present a quantitative approach that can be used to identify kinetics of uncharacterized protein cargo, as well as to evaluate with more detail processes such as ER-to-Golgi vesicle trafficking. Graphic abstract: Live-cell RUSH: a tool to monitor real-time protein trafficking in the secretory pathway.

Keywords:  Cargo sorting; Confocal microscopy; Protein trafficking; RUSH; Vesicle trafficking

DOI:  https://doi.org/10.21769/BioProtoc.3958
FEBS Lett. 2021 Apr 10.

Human SND2 mediates ER targeting of GPI-anchored proteins with low hydrophobic GPI-attachment signals.

Jing Yang, Tetsuya Hirata, Yi-Shi Liu, Xin-Yu Guo, Xiao-Dong Gao, Taroh Kinoshita, Morihisa Fujita.

  Over 100 glycosylphosphatidylinositol-anchored proteins (GPI-APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI-APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)-dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI-APs to the ER. Both the N-terminal signal sequence and C-terminal GPI-attachment signal of GPI-APs contribute to ER targeting via the hSND2-dependent pathway. Particularly, the hydrophobicity of the C-terminal GPI-attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI-APs in mammalian cells.

Keywords:  SND2; endoplasmic reticulum; glycosylphosphatidylinositol; protein targeting; signal recognition particle

DOI:  https://doi.org/10.1002/1873-3468.14083
Nat Commun. 2021 04 15. 12(1): 2170

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate.

Francesca Rapino, Zhaoli Zhou, Ana Maria Roncero Sanchez, Marc Joiret, Christian Seca, Najla El Hachem, Gianluca Valenti, Sara Latini, Kateryna Shostak, Liesbet Geris, Ping Li, Gang Huang, Gabriel Mazzucchelli, Dominique Baiwir, Christophe J Desmet, Alain Chariot, Michel Georges, Pierre Close.

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

DOI: https://doi.org/10.1038/s41467-021-22254-5
J Clin Invest. 2021 Apr 15. pii: 137689. [Epub ahead of print]

Desmosomal COP9 regulates proteome degradation in arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Yan Liang, Robert C Lyon, Jason Pellman, William H Bradford, Stephan Lange, Julius Bogomolovas, Nancy D Dalton, Yusu Gu, Marcus Bobar, Mong-Hong Lee, Tomoo Iwakuma, Vishal Nigam, Angeliki Asimaki, Melvin Scheinman, Kirk L Peterson, Farah Sheikh.

  Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease, arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we uncovered a cardiac COP9 desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels and function were impacted in hearts of classic mouse and human models of ARVD/C impacted by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to CSN6 loss and human desmosomal mutations destabilizing CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

Keywords:  Cardiology; Muscle Biology; Ubiquitin-proteosome system

DOI:  https://doi.org/10.1172/JCI137689
Mol Cell Oncol. 2021 Mar 09. 8(2): 1890990

p62/SQSTM1 droplets initiate autophagosome biogenesis and oxidative stress control.

Eeva-Liisa Eskelinen, Shun Kageyama, Masaaki Komatsu.

  Selective autophagy contributes to the degradation of condensates, such as sequestosome 1-bodies, also called p62/SQSTM1-bodies. We showed that endogenous p62 forms gel-like structures, which serve as platforms for autophagosome formation and nuclear factor erythroid 2-related factor 2 (NRF2) activation. Further, p62-mediated NRF2 activation is not cytotoxic, but combination of NRF2 activation with impaired bulk and selective autophagy causes liver injury.

Keywords:  GABARAP; KEAP1; LC3; NRF2; autophagy; liquid-liquid phase separation; oxidative stress; p62/SQSTM1

DOI:  https://doi.org/10.1080/23723556.2021.1890990
Eur J Med Chem. 2021 Apr 04. pii: S0223-5234(21)00274-9. [Epub ahead of print]219 113425

Development of MDM2 degraders based on ligands derived from Ugi reactions: Lessons and discoveries.

Bo Wang, Jin Liu, Ira Tandon, Suzhen Wu, Peng Teng, Jianhua Liao, Weiping Tang.

  Proteolysis targeting chimeras (PROTACs) have gained tremendous interest in both the academic and pharmaceutical communities. This opens a new way to regulate the cellular protein homeostasis, especially for disease-related proteins. In this work, we designed and synthesized a series of MDM2 degraders based on ligands that were readily prepared by a four-component Ugi reaction. After extensive optimization based on anti-proliferation and MDM2 degradation, WB214 was identified as the most potent anti-proliferative agent in various leukemia cell lines. Surprisingly, our mechanistic investigations indicated that WB214 not only effectively induced the degradation of MDM2, but also led to the degradation of p53. Further studies revealed that WB214 degraded MDM2 as a molecular glue. WB214 and its related analogues did not bind to MDM2 in the p53 binding region and MDM2 was discovered as a novel neo-substrate of the E3 ligase cereblon. Finally, we found that WB214 could potently degrade GSPT1, which could rationalize the inhibition of cell growth. A selective degrader for GSPT1 over MDM2 was then developed through systematically varying different motifs.

Keywords:  GSPT1; MDM2 degraders; Molecular glue; PROTAC

DOI:  https://doi.org/10.1016/j.ejmech.2021.113425
Nature. 2021 Apr 14.

The AMBRA1 E3 ligase adaptor regulates the stability of cyclin D.

Andrea C Chaikovsky, Chuan Li, Edwin E Jeng, Samuel Loebell, Myung Chang Lee, Christopher W Murray, Ran Cheng, Janos Demeter, Danielle L Swaney, Si-Han Chen, Billy W Newton, Jeffrey R Johnson, Alexandros P Drainas, Yan Ting Shue, Jose A Seoane, Preethi Srinivasan, Andy He, Akihiro Yoshida, Susan Q Hipkins, Edel McCrea, Carson D Poltorack, Nevan J Krogan, J Alan Diehl, Christina Kong, Peter K Jackson, Christina Curtis, Dmitri A Petrov, Michael C Bassik, Monte M Winslow, Julien Sage.

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication1. Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer2,3. However, the mechanisms that regulate levels of cyclin D are incompletely understood4,5. Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

DOI: https://doi.org/10.1038/s41586-021-03474-7
FASEB J. 2021 May;35(5): e21569

Subtle genomic DNA damage induces intraneuronal production of amyloid-β (1-42) by increasing β-secretase activity.

Hrishita Das, Sukanya Sarkar, Ramesh K Paidi, Subhas C Biswas.

  Aberrant accumulation of amyloid-β (Aβ) in brain is the major trigger for pathogenesis in Alzheimer's disease (AD). It is imperative to understand how Aβ attains such toxic levels in the brain parenchyma. We detected that a subtle and tolerable amount of DNA damage, related to aging, increased intraneuronal Aβ1-42 production both in cultured neuron and in cortex of rodent brain. Strikingly, we also observed elevated levels of mitochondrial fusion and of its major driver protein, MFN2. Hyperfusion of mitochondria may be seen as an adaptive stress response resulting from the induction of ER stress since we detected the activation of both PERK and IRE1α arms of unfolded protein response of ER stress. We found increased phosphorylation of PERK substrate eukaryotic initiation factor 2 α (eIF2α), and upregulation of the downstream effector proteins, ATF4 and CHOP. Concomitantly, increased XBP1 level, the direct effecter protein of IRE-1α, was observed. Reports suggest that eIF2α phosphorylation can increase BACE1 activity, the rate limiting enzyme in Aβ production. Here, we show that inhibiting PERK, decreased Aβ1-42 level while direct BACE1 inhibition, reduced the mitochondrial fusion. We found increased MFN2 expression in young 5xFAD mice when Aβ plaques and neurodegeneration were absent. Thus, our study indicates that mild DNA damage leads to increased Aβ1-42 production almost as a consequence of an initial ER stress-directed protective mitochondrial fusion in brain. We propose that an age-related subtle genomic DNA damage may trigger enhanced intraneuronal Aβ1-42 production in an apparently healthy neuron way before the appearance of clinical symptoms in AD.

Keywords:  5xFAD; BACE1; DNA damage; MFN2; PERK; amyloid-β; eIF2α

DOI:  https://doi.org/10.1096/fj.202001676RR
J Cell Biol. 2021 May 03. pii: e201907183. [Epub ahead of print]220(5):

FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins.

Fang Chen, Bing Yan, Jie Ren, Rui Lyu, Yanfang Wu, Yuting Guo, Dong Li, Hong Zhang, Junjie Hu.

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage-inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins.

DOI: https://doi.org/10.1083/jcb.201907183
Nat Commun. 2021 04 12. 12(1): 2155

Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases.

Yadi Chen, Xiaohua Hu, Siyuan Liu, Tiantian Su, Hsiaochi Huang, Huibo Ren, Zhensheng Gao, Xu Wang, Deshu Lin, James A Wohlschlegel, Qin Wang, Chentao Lin.

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

DOI: https://doi.org/10.1038/s41467-021-22410-x
Structure. 2021 Apr 09. pii: S0969-2126(21)00114-3. [Epub ahead of print]

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization.

Masaki Okumura, Shingo Kanemura, Motonori Matsusaki, Misaki Kinoshita, Tomohide Saio, Dai Ito, Chihiro Hirayama, Hiroyuki Kumeta, Mai Watabe, Yuta Amagai, Young-Ho Lee, Shuji Akiyama, Kenji Inaba.

  P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.

Keywords:  ER quality control; NMR; P5; SAXS; calcium binding; dimerization motif; oxidative protein folding; protein disulfide isomerase

DOI:  https://doi.org/10.1016/j.str.2021.03.016
Mol Cell. 2021 Apr 08. pii: S1097-2765(21)00262-8. [Epub ahead of print]

DNAJC9 integrates heat shock molecular chaperones into the histone chaperone network.

Colin M Hammond, Hongyu Bao, Ivo A Hendriks, Massimo Carraro, Alberto García-Nieto, Yanhong Liu, Nazaret Reverón-Gómez, Christos Spanos, Liu Chen, Juri Rappsilber, Michael L Nielsen, Dinshaw J Patel, Hongda Huang, Anja Groth.

  From biosynthesis to assembly into nucleosomes, histones are handed through a cascade of histone chaperones, which shield histones from non-specific interactions. Whether mechanisms exist to safeguard the histone fold during histone chaperone handover events or to release trapped intermediates is unclear. Using structure-guided and functional proteomics, we identify and characterize a histone chaperone function of DNAJC9, a heat shock co-chaperone that promotes HSP70-mediated catalysis. We elucidate the structure of DNAJC9, in a histone H3-H4 co-chaperone complex with MCM2, revealing how this dual histone and heat shock co-chaperone binds histone substrates. We show that DNAJC9 recruits HSP70-type enzymes via its J domain to fold histone H3-H4 substrates: upstream in the histone supply chain, during replication- and transcription-coupled nucleosome assembly, and to clean up spurious interactions. With its dual functionality, DNAJC9 integrates ATP-resourced protein folding into the histone supply pathway to resolve aberrant intermediates throughout the dynamic lives of histones.

Keywords:  DNAJC9; HSP40; HSP70; MCM2; TONSL; chromatin replication; heat shock co-chaperone; histone chaperone; nucleosome assembly; transcription

DOI:  https://doi.org/10.1016/j.molcel.2021.03.041
EMBO Mol Med. 2021 Apr 14. e13258

Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation.

Reini E N van der Welle, Rebekah Jobling, Christian Burns, Paolo Sanza, Jan A van der Beek, Alfonso Fasano, Lan Chen, Fried J Zwartkruis, Susan Zwakenberg, Edward F Griffin, Corlinda Ten Brink, Tineke Veenendaal, Nalan Liv, Conny M A van Ravenswaaij-Arts, Henny H Lemmink, Rolph Pfundt, Susan Blaser, Carolina Sepulveda, Andres M Lozano, Grace Yoon, Teresa Santiago-Sim, Cedric S Asensio, Guy A Caldwell, Kim A Caldwell, David Chitayat, Judith Klumperman.

  Vacuolar protein sorting 41 (VPS41) is as part of the Homotypic fusion and Protein Sorting (HOPS) complex required for lysosomal fusion events and, independent of HOPS, for regulated secretion. Here, we report three patients with compound heterozygous mutations in VPS41 (VPS41S285P and VPS41R662 * ; VPS41c.1423-2A>G and VPS41R662 * ) displaying neurodegeneration with ataxia and dystonia. Cellular consequences were investigated in patient fibroblasts and VPS41-depleted HeLa cells. All mutants prevented formation of a functional HOPS complex, causing delayed lysosomal delivery of endocytic and autophagic cargo. By contrast, VPS41S285P enabled regulated secretion. Strikingly, loss of VPS41 function caused a cytosolic redistribution of mTORC1, continuous nuclear localization of Transcription Factor E3 (TFE3), enhanced levels of LC3II, and a reduced autophagic response to nutrient starvation. Phosphorylation of mTORC1 substrates S6K1 and 4EBP1 was not affected. In a C. elegans model of Parkinson's disease, co-expression of VPS41S285P /VPS41R662 * abolished the neuroprotective function of VPS41 against α-synuclein aggregates. We conclude that the VPS41 variants specifically abrogate HOPS function, which interferes with the TFEB/TFE3 axis of mTORC1 signaling, and cause a neurodegenerative disease.

Keywords:  Autophagy; HOPS complex; TFEB/TFE3; lysosome-associated disorder; mTORC1

DOI:  https://doi.org/10.15252/emmm.202013258
Front Cell Dev Biol. 2021 ;9 641836

NUCB1 Suppresses Growth and Shows Additive Effects With Gemcitabine in Pancreatic Ductal Adenocarcinoma via the Unfolded Protein Response.

Yong-Qiang Hua, Ke Zhang, Jie Sheng, Zhou-Yu Ning, Ye Li, Wei-Dong Shi, Lu-Ming Liu.

  Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor patient prognosis. A cellular stress response mechanism called the unfolded protein response (UPR) has been implicated in PDAC progression. More recently, nucleobindin 1 (NUCB1), a calcium-binding protein, has been shown to control the UPR but its precise role in PDAC has not been explored. Here, we found that downregulation of NUCB1 was associated with poor prognosis in patients with PDAC. Functionally, NUCB1 overexpression suppressed pancreatic cancer cell proliferation and showed additive effects with gemcitabine (GEM) in vitro and in vivo. Moreover, by controlling ATF6 activity, NUCB1 overexpression suppressed GEM-induced UPR and autophagy. Last but not least, we uncovered METTL3-mediated m6A modification on NUCB1 5'UTR via the reader YTHDF2 as a mechanism for NUCB1 downregulation in PDAC. Taken together, our study revealed crucial functions of NUCB1 in suppressing proliferation and enhancing the effects of gemcitabine in pancreatic cancer cells and identified METTL3-mediated m6A modification as a mechanism for NUCB1 downregulation in PDAC.

Keywords:  NUCB1; autophagy; m6A modification; pancreatic ductal adenocarcinoma; unfolded protein response

DOI:  https://doi.org/10.3389/fcell.2021.641836
Nat Commun. 2021 04 12. 12(1): 2181

The E3 ligase MREL57 modulates microtubule stability and stomatal closure in response to ABA.

Liru Dou, Kaikai He, Jialin Peng, Xiangfeng Wang, Tonglin Mao.

Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA.

DOI: https://doi.org/10.1038/s41467-021-22455-y
Acta Neuropathol Commun. 2021 Apr 14. 9(1): 70

Correlative light and electron microscopy suggests that mutant huntingtin dysregulates the endolysosomal pathway in presymptomatic Huntington's disease.

Ya Zhou, Thomas R Peskett, Christian Landles, John B Warner, Kirupa Sathasivam, Edward J Smith, Shu Chen, Ronald Wetzel, Hilal A Lashuel, Gillian P Bates, Helen R Saibil.

  Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.

Keywords:  Amphisome; Autolysosome; Correlative light and electron microscopy; Electron tomography; Endolysosomal system; Exocytosis; Huntingtin aggregation; Huntington’s disease; Multivesicular body; Polyglutamine

DOI:  https://doi.org/10.1186/s40478-021-01172-z
J Cell Physiol. 2021 Apr 12.

Roles of FAM134B in diseases from the perspectives of organelle membrane morphogenesis and cellular homeostasis.

Luoyi Zhu, Xinxia Wang, Yizhen Wang.

  Family with sequence similarity 134 member B (FAM134B)/RETREG1/JK1 is a novel gene with recently reported roles in various diseases. Understanding the function and mechanism of action of FAM134B is necessary to develop disease therapies. Notably, emerging data are clarifying the molecular mechanisms of FAM134B function in organelle membrane morphogenesis and the regulation of signaling pathways, such as the Wnt and AKT signaling pathways. In addition, transcription factors, RNA N6 -methyladenosine-mediated epigenetic regulation, microRNA, and small molecules are involved in the regulation of FAM134B expression. This review comprehensively considers recent studies on the role of FAM134B and its potential mechanisms in neurodegenerative diseases, obesity, viral diseases, cancer, and other diseases. The functions of FAM134B in maintaining cell homeostasis by regulating Golgi morphology, endoplasmic reticulum autophagy, and mitophagy are also highlighted, which may be the underlying mechanism of FAM134B gene mutation-induced diseases. Moreover, the molecular mechanisms of the FAM134B function during numerous biological processes are discussed. This review provides novel insights into the functions and mechanisms of FAM134B in various diseases, which will inform the development of effective drugs to treat diseases.

Keywords:  ER-phagy; FAM134B; Golgi apparatus; HSAN ⅡB; cancer; mitophagy; obesity; signal pathways

DOI:  https://doi.org/10.1002/jcp.30377
FASEB Bioadv. 2021 Apr;3(4): 243-258

UBC13 is an RNF213-associated E2 ubiquitin-conjugating enzyme, and Lysine 63-linked ubiquitination by the RNF213-UBC13 axis is responsible for angiogenic activity.

Toshiyuki Habu, Kouji H Harada.

  Moyamoya disease (MMD) is a cryptogenic vascular disorder in the intracranial arteries. RING protein 213 (RNF213) is the susceptibility gene for MMD, and encodes a RING domain and a Walker motif. Herein, we identified UBC13 (UBE2N) as an E2 ubiquitin-conjugating enzyme for RNF213 E3 ubiquitin ligase by yeast two-hybrid screening with a fragment containing RNF213 RING domain as bait, and the immunocomplex of RNF213-UBC13 was detected in vivo. Analysis of the ubiquitin chain on RNF213 by monitoring autoubiquitination showed that RNF213 was autoubiquitinated in a K63 chain fashion, but not in a K48 chain fashion. Finally, this RNF213 ubiquitination in a UBC13-dependent manner was required for cell mobility and invasion activity for HUVEC cells in UBC13 knock-down and ubiquitination-dead RNF213 mutant expressing experiments. These findings demonstrated that RNF213 is a K63-linked E3 ubiquitin ligase, and UBC13 is responsible for RNF213 dependent ubiquitination. The RNF213-UBC13 axis may be associated with angiogenic activity and MMD.

Keywords:  Moyamoya disease; RNF213; angiogenic activity; lysine‐linked ubiquitination

DOI:  https://doi.org/10.1096/fba.2019-00092