bims-proteo 2022-04-03 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022‒04‒03
forty papers selected by
Eric Chevet
INSERM

Protein quality control at the Golgi.
Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation.
K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation.
Update on the Protein Homeostasis Network in Bacillus subtilis.
The Ubiquitin Ligase RNF34 Participates in the Peripheral Quality Control of CFTR (RNF34 Role in CFTR PeriQC).
Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle.
Linear ubiquitination in immune and neurodegenerative diseases, and beyond.
Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.
A second chance for protein targeting/folding: Ubiquitination and deubiquitination of nascent proteins.
Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis.
Upstream open reading frames mediate autophagy-related protein translation.
Mechanisms underlying ubiquitin-driven selective mitochondrial and bacterial autophagy.
Naturally-derived endoplasmic reticulum stress inhibitors for osteoarthritis?
Ubiquitination of the ubiquitin-binding machinery: how early ESCRT components are controlled.
FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway.
Structural and biochemical insights into lipid transport by VPS13 proteins.
Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.
Organelle transporters and inter-organelle communication as drivers of metabolic regulation and cellular homeostasis.
The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.
Transmembrane protein ATG-9 links presynaptic autophagy with the synaptic vesicle cycle.
Ubiquitinomics: history, methods and applications in basic research and drug discovery.
Glypican-1 drives unconventional secretion of Fibroblast Growth Factor 2.
eIF4A2 targets developmental potency and histone H3.3 transcripts for translational control of stem cell pluripotency.
Protective role of chaperone-mediated autophagy against atherosclerosis.
RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation.
Membrane trafficking: ESCRTs act here, there, and everywhere.
RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules.
Development of a High-Throughput Assay to Identify Inhibitors of the Ubiquitin-Conjugating Enzyme UBCH10.
Liver X receptor-agonist treatment rescues degeneration in a Drosophila model of hereditary spastic paraplegia.
TMED2 binding restricts SMO to the ER and Golgi compartments.
FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9/CYLD/NF-κB axis.
NFATc1 signaling drives chronic ER stress responses to promote NAFLD progression.
K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.
Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation.
Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.
Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.
Discovery of 3-(2-aminoethyl)-thiazolidine-2,4-diones as a Novel Chemotype of Sigma-1 Receptor Ligands.
Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression.
ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.
USP28 enables oncogenic transformation of respiratory cells and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

Curr Opin Cell Biol. 2022 Mar 29. pii: S0955-0674(22)00020-5. [Epub ahead of print]75 102074

Protein quality control at the Golgi.

Sinead Schwabl, David Teis.

The majority of the proteome in eukaryotic cells is targeted to organelles. To maintain protein homeostasis (proteostasis), distinct protein quality control (PQC) machineries operate on organelles, where they detect misfolded proteins, orphaned and mis-localized proteins and selectively target these proteins into different ubiquitin-dependent or -independent degradation pathways. Thereby, PQC prevents proteotoxic effects that would disrupt organelle integrity and cause cellular damage that leads to diseases. Here, we will discuss emerging mechanisms for PQC machineries at the Golgi apparatus, the central station for the sorting and the modification of proteins that traffic to the endo-lysosomal system, or along the secretory pathway to the PM and to the extracellular space. We will focus on Golgi PQC pathways that (1) retrieve misfolded and orphaned proteins from the Golgi back to the endoplasmic reticulum, (2) extract these proteins from Golgi membranes for proteasomal degradation, (3) or selectively target these proteins to lysosomes for degradation.

DOI: https://doi.org/10.1016/j.ceb.2022.02.008
J Biol Chem. 2022 Mar 28. pii: S0021-9258(22)00315-5. [Epub ahead of print] 101875

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation.

Galen Andrew Collins, Zhe Sha, Chueh-Ling Kuo, Beyza Erbil, Alfred L Goldberg.

  Although several proteasome subunits have been shown to bind ubiquitin chains, many ubiquitylated substrates also associate with 26S proteasomes via "shuttling factors." Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a ubiquitin-associating (UBA) domain; therefore, it is unclear how they bind ubiquitin. Here, we show that deletion of Ddi2 leads to the accumulation of ubiquitin conjugates with K11/K48 branched chains. We found using affinity co-purifications that Ddi2 binds ubiquitin conjugates through its ubiquitin-like (UBL) domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding ubiquitin conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of ubiquitin conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking Ddi2's endoprotease activity either genetically or with the HIV protease inhibitor nelfinavir increased its binding to ubiquitin conjugates, but decreased its binding to proteasomes, and reduced subsequent protein degradation by proteasomes leading to further accumulation of ubiquitin conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum-associated protein degradation (ERAD) and to deliver K11/K48-ubiquitylated proteins to the proteasome. We conclude that Ddi2's protease activity influences this shuttling factor activity, promotes protein turnover, and helps prevent ER stress, which may explain nelfinavir's ability to enhance cell killing by proteasome inhibitors.

Keywords:  26S Proteasome; Aspartic Protease; Ddi2; HIV Protease Inhibitor; Nelfinavir; Polyubiquitin chain; Protein Degradation; Shuttling Factor; Ubiquitin-Like Domain (UBL); Unfolded Protein Response (UPR)

DOI:  https://doi.org/10.1016/j.jbc.2022.101875
EMBO J. 2022 Mar 29. e110145

K27-linked ubiquitylation promotes p97 substrate processing and is essential for cell proliferation.

Robert F Shearer, Dimitris Typas, Fabian Coscia, Sofie Schovsbo, Thomas Kruse, Andreas Mund, Niels Mailand.

  Conjugation of ubiquitin (Ub) to numerous substrate proteins regulates virtually all cellular processes. Eight distinct ubiquitin polymer linkages specifying different functional outcomes are generated in cells. However, the roles of some atypical poly-ubiquitin topologies, in particular linkages via lysine 27 (K27), remain poorly understood due to a lack of tools for their specific detection and manipulation. Here, we adapted a cell-based ubiquitin replacement strategy to enable selective and conditional abrogation of K27-linked ubiquitylation, revealing that this ubiquitin linkage type is essential for proliferation of human cells. We demonstrate that K27-linked ubiquitylation is predominantly a nuclear modification whose ablation deregulates nuclear ubiquitylation dynamics and impairs cell cycle progression in an epistatic manner with inactivation of the ATPase p97/VCP. Moreover, we show that a p97-proteasome pathway model substrate (Ub(G76V)-GFP) is directly modified by K27-linked ubiquitylation, and that disabling the formation of K27-linked ubiquitin signals or blocking their decoding via overexpression of the K27 linkage-specific binder UCHL3 impedes Ub(G76V)-GFP turnover at the level of p97 function. Our findings suggest a critical role of K27-linked ubiquitylation in supporting cell fitness by facilitating p97-dependent processing of ubiquitylated nuclear proteins.

Keywords:  K27-linked ubiquitylation; cell cycle; p97/VCP; ubiquitin; ubiquitin-proteasome system

DOI:  https://doi.org/10.15252/embj.2021110145
Front Microbiol. 2022 ;13 865141

Update on the Protein Homeostasis Network in Bacillus subtilis.

Judith Matavacas, Claes von Wachenfeldt.

  Protein homeostasis is fundamental to cell function and survival. It relies on an interconnected network of processes involving protein synthesis, folding, post-translational modification and degradation as well as regulators of these processes. Here we provide an update on the roles, regulation and subcellular localization of the protein homeostasis machinery in the Gram-positive model organism Bacillus subtilis. We discuss emerging ideas and current research gaps in the field that, if tackled, increase our understanding of how Gram-positive bacteria, including several human pathogens, maintain protein homeostasis and cope with stressful conditions that challenge their survival.

Keywords:  chaperone; degradation tags; protease; protein aggregation; protein quality control; proteotoxic stress

DOI:  https://doi.org/10.3389/fmicb.2022.865141
Front Mol Biosci. 2022 ;9 840649

The Ubiquitin Ligase RNF34 Participates in the Peripheral Quality Control of CFTR (RNF34 Role in CFTR PeriQC).

Shogo Taniguchi, Yukiko Ito, Hibiki Kiritani, Asuka Maruo, Ryohei Sakai, Yuji Ono, Ryosuke Fukuda, Tsukasa Okiyoneda.

  The peripheral protein quality control (periQC) system eliminates the conformationally defective cystic fibrosis transmembrane conductance regulator (CFTR), including ∆F508-CFTR, from the plasma membrane (PM) and limits the efficacy of pharmacological therapy for cystic fibrosis (CF). The ubiquitin (Ub) ligase RFFL is responsible for the chaperone-independent ubiquitination and lysosomal degradation of CFTR in the periQC. Here, we report that the Ub ligase RNF34 participates in the CFTR periQC in parallel to RFFL. An in vitro study reveals that RNF34 directly recognizes the CFTR NBD1 and selectively promotes the ubiquitination of unfolded proteins. RNF34 was localized in the cytoplasm and endosomes, where RFFL was equally colocalized. RNF34 ablation increased the PM density as well as the mature form of ∆F508-CFTR rescued at low temperatures. RFFL ablation, with the exception of RNF34 ablation, increased the functional PM expression of ∆F508-CFTR upon a triple combination of CFTR modulators (Trikafta) treatment by inhibiting the K63-linked polyubiquitination. Interestingly, simultaneous ablation of RNF34 and RFFL dramatically increased the functional PM ∆F508-CFTR by inhibiting the ubiquitination in the post-Golgi compartments. The CFTR-NLuc assay demonstrates that simultaneous ablation of RNF34 and RFFL dramatically inhibits the degradation of mature ∆F508-CFTR after Trikafta treatment. Therefore, these results suggest that RNF34 plays a crucial role in the CFTR periQC, especially when there is insufficient RFFL. We propose that simultaneous inhibition of RFFL and RNF34 may improve the efficacy of CFTR modulators.

Keywords:  CFTR; RFFL; RNF34; Trikafta; cystic fibrosis; peripheral quality control; ubiquitin ligase

DOI:  https://doi.org/10.3389/fmolb.2022.840649
Nat Commun. 2022 Apr 01. 13(1): 1748

Long-chain ceramides are cell non-autonomous signals linking lipotoxicity to endoplasmic reticulum stress in skeletal muscle.

Ben D McNally, Dean F Ashley, Lea Hänschke, Hélène N Daou, Nicole T Watt, Steven A Murfitt, Amanda D V MacCannell, Anna Whitehead, T Scott Bowen, Francis W B Sanders, Michele Vacca, Klaus K Witte, Graeme R Davies, Reinhard Bauer, Julian L Griffin, Lee D Roberts.

The endoplasmic reticulum (ER) regulates cellular protein and lipid biosynthesis. ER dysfunction leads to protein misfolding and the unfolded protein response (UPR), which limits protein synthesis to prevent cytotoxicity. Chronic ER stress in skeletal muscle is a unifying mechanism linking lipotoxicity to metabolic disease. Unidentified signals from cells undergoing ER stress propagate paracrine and systemic UPR activation. Here, we induce ER stress and lipotoxicity in myotubes. We observe ER stress-inducing lipid cell non-autonomous signal(s). Lipidomics identifies that palmitate-induced cell stress induces long-chain ceramide 40:1 and 42:1 secretion. Ceramide synthesis through the ceramide synthase 2 de novo pathway is regulated by UPR kinase Perk. Inactivation of CerS2 in mice reduces systemic and muscle ceramide signals and muscle UPR activation. The ceramides are packaged into extracellular vesicles, secreted and induce UPR activation in naïve myotubes through dihydroceramide accumulation. This study furthers our understanding of ER stress by identifying UPR-inducing cell non-autonomous signals.

DOI: https://doi.org/10.1038/s41467-022-29363-9
Biochem Soc Trans. 2022 Mar 28. pii: BST20211078. [Epub ahead of print]

Linear ubiquitination in immune and neurodegenerative diseases, and beyond.

Fuminori Tokunaga, Fumiyo Ikeda.

  Ubiquitin regulates numerous aspects of biology via a complex ubiquitin code. The linear ubiquitin chain is an atypical code that forms a unique structure, with the C-terminal tail of the distal ubiquitin linked to the N-terminal Met1 of the proximal ubiquitin. Thus far, LUBAC is the only known ubiquitin ligase complex that specifically generates linear ubiquitin chains. LUBAC-induced linear ubiquitin chains regulate inflammatory responses, cell death and immunity. Genetically modified mouse models and cellular assays have revealed that LUBAC is also involved in embryonic development in mice. LUBAC dysfunction is associated with autoimmune diseases, myopathy, and neurodegenerative diseases in humans, but the underlying mechanisms are poorly understood. In this review, we focus on the roles of linear ubiquitin chains and LUBAC in immune and neurodegenerative diseases. We further discuss LUBAC inhibitors and their potential as therapeutics for these diseases.

Keywords:  LUBAC; LUBAC inhibitors; inflammation; linear ubiquitination; neurodegeneration

DOI:  https://doi.org/10.1042/BST20211078
Proc Natl Acad Sci U S A. 2022 Apr 05. 119(14): e2115083119

Genome-wide CRISPR screen reveals CLPTM1L as a lipid scramblase required for efficient glycosylphosphatidylinositol biosynthesis.

Yicheng Wang, Anant K Menon, Yuta Maki, Yi-Shi Liu, Yugo Iwasaki, Morihisa Fujita, Paula A Guerrero, Daniel Varó'n Silva, Peter H Seeberger, Yoshiko Murakami, Taroh Kinoshita.

  SignificanceScramblases translocate lipids across the lipid bilayer without consumption of ATP, thereby regulating lipid distributions in cellular membranes. Cytosol-to-lumen translocation across the endoplasmic reticulum (ER) membrane is a common process among lipid glycoconjugates involved in posttranslational protein modifications in eukaryotes. These translocations are thought to be mediated by specific ER-resident scramblases, but the identity of these proteins and the underlying molecular mechanisms have been elusive. Here, we show that CLPTM1L, an integral membrane protein with eight putative transmembrane domains, is the major lipid scramblase involved in efficient glycosylphosphatidylinositol biosynthesis in the ER membrane. Our results validate the long-standing hypothesis that lipid scramblases ensure the efficient translocations of lipid glycoconjugates across the ER membrane for protein glycosylation pathways.

Keywords:  CLPTM1L; endoplasmic reticulum; glycobiology; glycosylphosphatidylinositol; scramblase

DOI:  https://doi.org/10.1073/pnas.2115083119
Bioessays. 2022 Mar 31. e2200014

A second chance for protein targeting/folding: Ubiquitination and deubiquitination of nascent proteins.

Jacob A Culver, Xia Li, Matthew Jordan, Malaiyalam Mariappan.

  Molecular chaperones in cells constantly monitor and bind to exposed hydrophobicity in newly synthesized proteins and assist them in folding or targeting to cellular membranes for insertion. However, proteins can be misfolded or mistargeted, which often causes hydrophobic amino acids to be exposed to the aqueous cytosol. Again, chaperones recognize exposed hydrophobicity in these proteins to prevent nonspecific interactions and aggregation, which are harmful to cells. The chaperone-bound misfolded proteins are then decorated with ubiquitin chains denoting them for proteasomal degradation. It remains enigmatic how molecular chaperones can mediate both maturation of nascent proteins and ubiquitination of misfolded proteins solely based on their exposed hydrophobic signals. In this review, we propose a dynamic ubiquitination and deubiquitination model in which ubiquitination of newly synthesized proteins serves as a "fix me" signal for either refolding of soluble proteins or retargeting of membrane proteins with the help of chaperones and deubiquitinases. Such a model would provide additional time for aberrant nascent proteins to fold or route for membrane insertion, thus avoiding excessive protein degradation and saving cellular energy spent on protein synthesis.

Keywords:  chaperones; deubiquitination; protein folding; protein misfolding; protein quality control; protein unfolding; tail-anchored proteins; ubiquitination

DOI:  https://doi.org/10.1002/bies.202200014
Elife. 2022 Mar 31. pii: e74255. [Epub ahead of print]11

Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis.

Benjamin Pillet, Alfonso Méndez-Godoy, Guillaume Murat, Sébastien Favre, Michael Stumpe, Laurent Falquet, Dieter Kressler.

  The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, reduces the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide-associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production.

Keywords:  Ccr4-Not complex; S. cerevisiae; cell biology; chromosomes; gene expression; mRNA degradation; protein aggregation; protein homeostasis; ribosome biogenesis; yeast genetics

DOI:  https://doi.org/10.7554/eLife.74255
Autophagy. 2022 Apr 01.

Upstream open reading frames mediate autophagy-related protein translation.

Ying Yang, Damian Gatica Mizala, Xu Liu, Runliu Wu, Rui Kang, Daolin Tang, Daniel J Klionsky.

  Macroautophagy/autophagy, a highly conserved catabolic pathway that maintains proper cellular homeostasis is stringently regulated by numerous autophagy-related (Atg) proteins. Many studies have investigated autophagy regulation at the transcriptional level; however, relatively little is known about translational control. Here, we report the upstream open reading frames (uORFs)-mediated translational control of multiple Atg proteins in Saccharomyces cerevisiae and in human cells. The translation of several essential autophagy regulators in yeast, including Atg13, is suppressed by canonical uORFs under nutrient-rich conditions, and is activated during nitrogen-starvation conditions. We also found that the predicted human ATG4B and ATG12 non-canonical uORFs suppress downstream coding sequence translation. These results demonstrate that uORF-mediated translational control is a widely used mechanism among ATG genes from yeast to human and suggest a model for how some ATG genes bypass the general translational suppression that occurs under stress conditions to maintain a proper level of autophagy.

Keywords:  Autophagy; human; lysosome; stress; translational regulation; vacuole; yeast

DOI:  https://doi.org/10.1080/15548627.2022.2059744
Mol Cell. 2022 Mar 29. pii: S1097-2765(22)00221-0. [Epub ahead of print]

Mechanisms underlying ubiquitin-driven selective mitochondrial and bacterial autophagy.

Ellen A Goodall, Felix Kraus, J Wade Harper.

  Selective autophagy specifically eliminates damaged or superfluous organelles, maintaining cellular health. In this process, a double membrane structure termed an autophagosome captures target organelles or proteins and delivers this cargo to the lysosome for degradation. The attachment of the small protein ubiquitin to cargo has emerged as a common mechanism for initiating organelle or protein capture by the autophagy machinery. In this process, a suite of ubiquitin-binding cargo receptors function to initiate autophagosome assembly in situ on the target cargo, thereby providing selectivity in cargo capture. Here, we review recent efforts to understand the biochemical mechanisms and principles by which cargo are marked with ubiquitin and how ubiquitin-binding cargo receptors use conserved structural modules to recruit the autophagosome initiation machinery, with a particular focus on mitochondria and intracellular bacteria as cargo. These emerging mechanisms provide answers to long-standing questions in the field concerning how selectivity in cargo degradation is achieved.

Keywords:  cargo receptor; mitophagy; selective autophagy; ubiquitin; xenophagy

DOI:  https://doi.org/10.1016/j.molcel.2022.03.012
Eur J Pharmacol. 2022 Mar 26. pii: S0014-2999(22)00164-9. [Epub ahead of print]922 174903

Naturally-derived endoplasmic reticulum stress inhibitors for osteoarthritis?

Siam-Yee Lee, Pooi-Fong Wong, Juliana Jamal, Margaret M Roebuck.

  Osteoarthritis (OA) is a chronic degenerative disease that affects the whole synovial joint. OA causes severe pain and disability that significantly affects the livelihood of an individual and incurs a huge socioeconomic burden. Current management strategies are limited to supporting functional improvement with physiotherapy and pain reduction as there are no drugs available that can reverse the progression of OA with only joint replacement surgery for late stage OA. OA is associated with advancing age and obesity, both of which compromise the functions of key endoplasmic reticulum (ER) molecular chaperones leading to improper protein folding and ER stress. Failure to restore protein homeostasis leads to increased cellular stress and eventually apoptotic cell death. Cartilage is avascular and is dependent on its constituent cells, chondrocytes, for extracellular matrix maintenance. Chondrocytes have limited proliferative capacity and their apoptosis eventually leads to extracellular matrix loss and cartilage degeneration. Recent studies on attenuating ER stress and chondrocytes apoptosis offer a credible strategy for reducing OA progression. The established roles of ER stress responses in OA have paved the way for targeted drug discovery studies aiming to mitigate ER stress and OA progression. In this review, in vitro, pre-clinical and clinical evidence of naturally-derived ER stress inhibitors for OA, the prospect and challenges in bringing these compounds to clinics are discussed.

Keywords:  Arthritis; Cartilage; Chondrocytes; ER stress; ER stress Inhibitors; Therapeutics

DOI:  https://doi.org/10.1016/j.ejphar.2022.174903
Essays Biochem. 2022 Mar 30. pii: EBC20210042. [Epub ahead of print]

Ubiquitination of the ubiquitin-binding machinery: how early ESCRT components are controlled.

Barbara Korbei.

  To be able to quickly and accurately respond to the environment, cells need to tightly control the amount and localization of plasma membrane proteins. The post-translation modification by the protein modifier ubiquitin is the key signal for guiding membrane-associated cargo to the lysosome/vacuole for their degradation. The machinery responsible for such sorting contains several subunits that function as ubiquitin receptors, many of which are themselves subjected to ubiquitination. This review will focus on what is currently known about the modulation of the machinery itself by ubiquitination and how this might affect its function with a special emphasis on current findings from the plant field.

Keywords:  Endosomal Sorting Complexes Required for Transport; ubiquitin; ubiquitin binding domains

DOI:  https://doi.org/10.1042/EBC20210042
J Clin Invest. 2022 Mar 29. pii: e151591. [Epub ahead of print]

FOXK2 promotes ovarian cancer stemness by regulating the unfolded protein response pathway.

Yaqi Zhang, Yinu Wang, Guangyuan Zhao, Edward J Tanner, Mazhar Adli, Daniela Matei.

  Understanding the regulatory programs enabling cancer stem cells (CSCs) to self-renew and drive tumorigenicity could identify new treatments. Through comparative chromatin state and gene expression analyses in ovarian CSCs vs. non-CSCs, we identified FOXK2 as a highly expressed stemness-specific transcription factor in ovarian cancer. Its genetic depletion diminished stemness features and reduced tumor initiation capacity. Our mechanistic studies highlight that FOXK2 directly regulated IRE1α (ERN1 gene) expression, a key sensor for the unfolded protein response (UPR). Chromatin immunoprecipitation-sequencing revealed that FOXK2 bound to an intronic regulatory element of ERN1. Blocking FOXK2 from binding to this enhancer by using a catalytically inactive CRISPR/Cas9 (dCas9) diminished IRE1α transcription. At the molecular level, FOXK2-driven upregulation of IRE1α led to alternative XBP1 splicing and activation of stemness pathways, while genetic or pharmacological blockade of this sensor of the UPR inhibited ovarian CSCs. Collectively, these data establish a new function for FOXK2 as a key transcriptional regulator of CSCs and a mediator of the UPR, providing insight into potentially targetable new pathways in CSCs.

Keywords:  Cancer; Cell Biology; Cell stress; Oncology; Transcription

DOI:  https://doi.org/10.1172/JCI151591
J Cell Biol. 2022 Jun 06. pii: e202202030. [Epub ahead of print]221(5):

Structural and biochemical insights into lipid transport by VPS13 proteins.

Jyoti Adlakha, Zhouping Hong, PeiQi Li, Karin M Reinisch.

VPS13 proteins are proposed to function at contact sites between organelles as bridges for lipids to move directionally and in bulk between organellar membranes. VPS13s are anchored between membranes via interactions with receptors, including both peripheral and integral membrane proteins. Here we present the crystal structure of VPS13s adaptor binding domain (VAB) complexed with a Pro-X-Pro peptide recognition motif present in one such receptor, the integral membrane protein Mcp1p, and show biochemically that other Pro-X-Pro motifs bind the VAB in the same site. We further demonstrate that Mcp1p and another integral membrane protein that interacts directly with human VPS13A, XK, are scramblases. This finding supports an emerging paradigm of a partnership between bulk lipid transport proteins and scramblases. Scramblases can re-equilibrate lipids between membrane leaflets as lipids are removed from or inserted into the cytosolic leaflet of donor and acceptor organelles, respectively, in the course of protein-mediated transport.

DOI: https://doi.org/10.1083/jcb.202202030
Hum Mol Genet. 2022 Mar 26. pii: ddac072. [Epub ahead of print]

Transverse endoplasmic reticulum expansion in hereditary spastic paraplegia corticospinal axons.

Peng-Peng Zhu, Hui-Fang Hung, Natalia Batchenkova, Jonathon Nixon-Abell, James Henderson, Pengli Zheng, Benoit Renvoisé, Song Pang, C Shan Xu, Stephan Saalfeld, Jan Funke, Yuxiang Xie, Fabian Svara, Harald F Hess, Craig Blackstone.

Hereditary spastic paraplegias (HSPs) comprise a large group of inherited neurologic disorders affecting the longest corticospinal axons (SPG1-86 plus others), with shared manifestations of lower extremity spasticity and gait impairment. Common autosomal dominant HSPs are caused by mutations in genes encoding the microtubule-severing ATPase spastin (SPAST; SPG4), the membrane-bound GTPase atlastin-1 (ATL1; SPG3A), and the reticulon-like, microtubule-binding protein REEP1 (REEP1; SPG31). These proteins bind one another and function in shaping the tubular endoplasmic reticulum (ER) network. Typically, mouse models of HSPs have mild, later-onset phenotypes, possibly reflecting far shorter lengths of their corticospinal axons relative to humans. Here, we have generated a robust, double mutant mouse model of HSP in which atlastin-1 is genetically modified with a K80A knock-in (KI) missense change that abolishes its GTPase activity, while its binding partner Reep1 is knocked out. Atl1KI/KI/Reep1-/- mice exhibit early-onset and rapidly progressive declines in several motor function tests. Also, ER in mutant corticospinal axons dramatically expands transversely and periodically in a mutation dosage-dependent manner to create a ladder-like appearance, based on reconstructions of focused ion beam-scanning electron microscopy datasets using machine learning-based auto-segmentation. In lockstep with changes in ER morphology, axonal mitochondria are fragmented and proportions of hypophosphorylated neurofilament H and M subunits are dramatically increased in Atl1KI/KI/Reep1-/- spinal cord. Co-occurrence of these findings links ER morphology changes to alterations in mitochondrial morphology and cytoskeletal organization. Atl1KI/KI/Reep1-/- mice represent an early-onset rodent HSP model with robust behavioral and cellular readouts for testing novel therapies.

DOI: https://doi.org/10.1093/hmg/ddac072
Mol Metab. 2022 Mar 25. pii: S2212-8778(22)00050-3. [Epub ahead of print] 101481

Organelle transporters and inter-organelle communication as drivers of metabolic regulation and cellular homeostasis.

Aakriti Jain, Roberto Zoncu.

  Spatial compartmentalization of metabolic pathways within membrane-separated organelles is key to the ability of eukaryotic cells to precisely regulate their biochemical functions. Membrane-bound organelles such as mitochondria, endoplasmic reticulum (ER) and lysosomes enable the concentration of metabolic precursors within optimized chemical environments, greatly accelerating the efficiency of both anabolic and catabolic reactions, enabling division of labor and optimal utilization of resources. However, metabolic compartmentalization also poses a challenge to cells because it creates spatial discontinuities that must be bridged for reaction cascades to be connected and completed. To do so, cells employ different methods to coordinate metabolic fluxes occurring in different organelles, such as membrane-localized transporters to facilitate regulated metabolite exchange between mitochondria and lysosomes, non-vesicular transport pathways via physical contact sites connecting the ER with both mitochondria and lysosomes, as well as localized regulatory signaling processes that coordinately regulate the activity of all these organelles. Effective communication among these systems is essential to cellular health and function, whereas disruption of inter-organelle communication is an emerging driver in a multitude of diseases, from cancer to neurodegeneration.

Keywords:  Contact sites; Lysosome; Metabolism; Mitochondria; Transporters; mTORC1

DOI:  https://doi.org/10.1016/j.molmet.2022.101481
J Cell Sci. 2022 Mar 28. pii: jcs.259421. [Epub ahead of print]

The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.

Prado Vargas Duarte, Ralph Hardenberg, Muriel Mari, Stefan Walter, Fulvio Reggiori, Florian Fröhlich, Ayelén González Montoro, Christian Ungermann.

  Lysosomes mediate degradation of macromolecules to their precursors for their cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. The Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein, and an effector of the minor Rab5 isoform Ypt52. Strikingly, the bph1▵ mutant has lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy inducing conditions. In agreement, proteomics of bph1▵ vacuoles reveal an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1▵ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.

Keywords:  Atg8; Autophagy; Endosome; LYST; Rab5; Ypt52

DOI:  https://doi.org/10.1242/jcs.259421
Autophagy. 2022 Mar 29. 1-2

Transmembrane protein ATG-9 links presynaptic autophagy with the synaptic vesicle cycle.

Sisi Yang, Daniel A Colón-Ramos.

  Macroautophagy/autophagy occurs preferentially at synapses and responds to increased neuronal activity states. How synaptic autophagy is coupled to the neuronal activity state is largely unknown. Through genetic approaches we find that ATG-9, the only transmembrane protein in the core autophagy pathway, is transported from the trans-Golgi network to synapses in C. elegans via the AP-3 complex. At synapses ATG-9 undergoes exo-endocytosis in an activity-dependent manner. Mutations that disrupt the endocytosis pathway, including a mutation associated with early onset Parkinsonism (EOP), lead to abnormal ATG-9 accumulation into subsynaptic clathrin-rich foci, and defects in activity-induced synaptic autophagy. We propose that ATG-9 exo-endocytosis links the activity-dependent synaptic vesicle cycle with autophagosome formation at synapses.

Keywords:  AP-3; ATG-9; Golgi apparatus; Parkinson disease; autophagy; clathrin; endocytosis; neuronal activity state; synaptic vesicle cycle; synaptojanin 1/UNC-26

DOI:  https://doi.org/10.1080/15548627.2022.2049151
Proteomics. 2022 Mar 30. e2200074

Ubiquitinomics: history, methods and applications in basic research and drug discovery.

Martin Steger, Özge Karayel, Vadim Demichev.

  The ubiquitin-proteasome system (UPS) was discovered about 40 years ago and is known to regulate a multitude of cellular processes including protein homeostasis. ubiquitylated proteins are recognized by downstream effectors, resulting in alterations of protein abundance, activity, or localization. Not surprisingly, the ubiquitylation machinery is dysregulated in numerous diseases, including cancers and neurodegeneration. Mass spectrometry (MS)-based proteomics has emerged as a transformative technology for characterizing protein ubiquitylation in an unbiased fashion. Here, we provide an overview of the different MS-based approaches for studying protein ubiquitylation. We review various methods for enriching and quantifying ubiquitin modifications at the peptide or protein level, outline MS acquisition and data processing approaches and discuss key challenges. Finally, we examine how MS-based ubiquitinomics can aid both basic biology and drug discovery research. This article is protected by copyright. All rights reserved.

Keywords:  Ubiquitin-proteasome system; Ubiquitinomics; drug discovery; mass spectrometry

DOI:  https://doi.org/10.1002/pmic.202200074
Elife. 2022 Mar 29. pii: e75545. [Epub ahead of print]11

Glypican-1 drives unconventional secretion of Fibroblast Growth Factor 2.

Carola Sparn, Eleni Dimou, Annalena Meyer, Roberto Saleppico, Sabine Wegehingel, Matthias Gerstner, Severina Klaus, Helge Ewers, Walter Nickel.

  Fibroblast Growth Factor 2 (FGF2) is a tumor cell survival factor that is transported into the extracellular space by an unconventional secretory mechanism. Cell surface heparan sulfate proteoglycans are known to play an essential role in this process. Unexpectedly, we found that among the diverse sub-classes consisting of syndecans, perlecans, glypicans and others, Glypican-1 (GPC1) is the principle and rate-limiting factor that drives unconventional secretion of FGF2. By contrast, we demonstrate GPC1 to be dispensable for FGF2 signaling into cells. We provide first insights into the structural basis for GPC1-dependent FGF2 secretion, identifying disaccharides with N-linked sulfate groups to be enriched in the heparan sulfate chains of GPC1 to which FGF2 binds with high affinity. Our findings have broad implications for the role of GPC1 as a key molecule in tumor progression.

Keywords:  biochemistry; cell biology; chemical biology; human

DOI:  https://doi.org/10.7554/eLife.75545
Sci Adv. 2022 Apr;8(13): eabm0478

eIF4A2 targets developmental potency and histone H3.3 transcripts for translational control of stem cell pluripotency.

Dan Li, Jihong Yang, Xin Huang, Hongwei Zhou, Jianlong Wang.

Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNA interference screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eukaryotic translation initiation factor 4A2 (eIF4A2) and defined its mechanistic action through ribosomal protein S26-independent and -dependent ribosomes in translation initiation activation of messenger RNAs (mRNAs) encoding pluripotency factors and the histone variant H3.3 with demonstrated roles in maintaining stem cell pluripotency. eIF4A2 also mediates translation initiation activation of Ddx6, which acts together with eIF4A2 to restrict the totipotent two-cell transcription program in ESCs through Zscan4 mRNA degradation and translation repression. Accordingly, knockdown of eIF4A2 disrupts ESC proteome, causing the loss of ESC identity. Collectively, we establish a translational paradigm of the protein synthesis of pluripotency transcription factors and epigenetic regulators imposed on their established roles in controlling pluripotency.

DOI: https://doi.org/10.1126/sciadv.abm0478
Proc Natl Acad Sci U S A. 2022 Apr 05. 119(14): e2121133119

Protective role of chaperone-mediated autophagy against atherosclerosis.

Julio Madrigal-Matute, Jenny de Bruijn, Kim van Kuijk, Dario F Riascos-Bernal, Antonio Diaz, Inmaculada Tasset, Adrián Martín-Segura, Marion J J Gijbels, Bianca Sander, Susmita Kaushik, Erik A L Biessen, Simoni Tiano, Mathieu Bourdenx, Gregory J Krause, Ian McCracken, Andrew H Baker, Han Jin, Nicholas E S Sibinga, Jose Javier Bravo-Cordero, Fernando Macian, Rajat Singh, Patrick C N Rensen, Jimmy F P Berbée, Gerard Pasterkamp, Judith C Sluimer, Ana Maria Cuervo.

  Significance Cardiovascular diseases remain the leading cause of death worldwide, with atherosclerosis being the most common source of clinical events. Metabolic changes with aging associate with concurrent increased risk of both type 2 diabetes and cardiovascular disease, with the former further raising the risk of the latter. The activity of a selective type of autophagy, chaperone-mediated autophagy (CMA), decreases with age or upon dietary excesses. Here we study whether reduced CMA activity increases risk of atherosclerosis in mouse models. We have identified that CMA is up-regulated early in response to proatherogenic challenges and demonstrate that reduced systemic CMA aggravates vascular pathology in these conditions. We also provide proof-of-concept support that CMA up-regulation is an effective intervention to reduce atherosclerosis severity and progression.

Keywords:  atherosclerotic plaques; lipid challenge; lysosomes; proteolysis; vascular disease

DOI:  https://doi.org/10.1073/pnas.2121133119
Exp Hematol Oncol. 2022 Mar 31. 11(1): 18

RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation.

Dalia Quwaider, Luis A Corchete, Marta Martín-Izquierdo, Jesús M Hernández-Sánchez, Elizabeta A Rojas, Ignacio J Cardona-Benavides, Ramón García-Sanz, Ana B Herrero, Norma C Gutiérrez.

  BACKGROUND: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells.METHODS: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot.
RESULTS: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found.
CONCLUSION: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.

Keywords:  ER stress; IRE1; Multiple myeloma; RIDD; UPR

DOI:  https://doi.org/10.1186/s40164-022-00271-4
Curr Biol. 2022 Mar 28. pii: S0960-9822(22)00265-2. [Epub ahead of print]32(6): R292-R294

Membrane trafficking: ESCRTs act here, there, and everywhere.

Leticia Lemus, Veit Goder.

Endosomal sorting complex required for transport (ESCRT) proteins can promote extreme membrane deformations, including scission and sealing. New work uncovers a link between these proteins and the early secretory pathway that is functionally important for programmed autophagy during Drosophila development.

DOI: https://doi.org/10.1016/j.cub.2022.02.043
EMBO J. 2022 Mar 31. e110137

RNA is required for the integrity of multiple nuclear and cytoplasmic membrane-less RNP granules.

Carolyn J Decker, James M Burke, Patrick K Mulvaney, Roy Parker.

  Numerous membrane-less organelles, composed of a combination of RNA and proteins, are observed in the nucleus and cytoplasm of eukaryotic cells. These RNP granules include stress granules (SGs), processing bodies (PBs), Cajal bodies, and nuclear speckles. An unresolved question is how frequently RNA molecules are required for the integrity of RNP granules in either the nucleus or cytosol. To address this issue, we degraded intracellular RNA in either the cytosol or the nucleus by the activation of RNase L and examined the impact of RNA loss on several RNP granules. We find the majority of RNP granules, including SGs, Cajal bodies, nuclear speckles, and the nucleolus, are altered by the degradation of their RNA components. In contrast, PBs and super-enhancer complexes were largely not affected by RNA degradation in their respective compartments. RNA degradation overall led to the apparent dissolution of some membrane-less organelles, whereas others reorganized into structures with altered morphology. These findings highlight a critical and widespread role of RNA in the organization of several RNP granules.

Keywords:  RNA granules; RNP granules; RNase L; condensates; membrane-less organelles

DOI:  https://doi.org/10.15252/embj.2021110137
SLAS Discov. 2022 Mar 24. pii: S2472-5552(22)12653-5. [Epub ahead of print]

Development of a High-Throughput Assay to Identify Inhibitors of the Ubiquitin-Conjugating Enzyme UBCH10.

Patrick Cléroux, Laure Voisin, Sylvain Meloche.

  UBCH10 is an ubiquitin-conjugating enzyme (E2) of the anaphase-promoting complex E3 ligase, a key regulator of the cell cycle. The UBCH10 gene and protein are frequently upregulated in multiple solid tumors, associated with an unfavorable outcome. Accumulating evidence from studies of human cancer cell lines, mouse transgenic models, and analyses of clinical samples suggest that UBCH10 is a potential cancer drug target. No small molecule inhibitor of UBCH10 has been reported in the literature. Here, we described the development and optimization of a novel time-resolved fluorescence resonance energy transfer (TR-FRET) UBCH10 assay based on the self-polyubiquitination of the enzyme in the absence of E3. The homogenous assay is robust, sensitive, and scalable to different multi-well formats for high-throughput screening (HTS). We demonstrate the suitability of the TR-FRET assay to identify chemical inhibitors of UBCH10 in a pilot HTS campaign.

Keywords:  HTS; UBCH10; ubiquitin-conjugating enzymes; ubiquitin-proteasome system; ubiquitination assay

DOI:  https://doi.org/10.1016/j.slasd.2022.03.007
Acta Neuropathol Commun. 2022 Mar 28. 10(1): 40

Liver X receptor-agonist treatment rescues degeneration in a Drosophila model of hereditary spastic paraplegia.

Dwayne J Byrne, M Elena Garcia-Pardo, Nelson B Cole, Belguun Batnasan, Sophia Heneghan, Anood Sohail, Craig Blackstone, Niamh C O'Sullivan.

  Hereditary spastic paraplegias (HSPs) are a group of inherited, progressive neurodegenerative conditions characterised by prominent lower-limb spasticity and weakness, caused by a length-dependent degeneration of the longest corticospinal upper motor neurons. While more than 80 spastic paraplegia genes (SPGs) have been identified, many cases arise from mutations in genes encoding proteins which generate and maintain tubular endoplasmic reticulum (ER) membrane organisation. The ER-shaping proteins are essential for the health and survival of long motor neurons, however the mechanisms by which mutations in these genes cause the axonopathy observed in HSP have not been elucidated. To further develop our understanding of the ER-shaping proteins, this study outlines the generation of novel in vivo and in vitro models, using CRISPR/Cas9-mediated gene editing to knockout the ER-shaping protein ADP-ribosylation factor-like 6 interacting protein 1 (ARL6IP1), mutations in which give rise to the HSP subtype SPG61. Loss of Arl6IP1 in Drosophila results in progressive locomotor deficits, emulating a key aspect of HSP in patients. ARL6IP1 interacts with ER-shaping proteins and is required for regulating the organisation of ER tubules, particularly within long motor neuron axons. Unexpectedly, we identified physical and functional interactions between ARL6IP1 and the phospholipid transporter oxysterol-binding protein-related protein 8 in both human and Drosophila model systems, pointing to a conserved role for ARL6IP1 in lipid homeostasis. Furthermore, loss of Arl6IP1 from Drosophila neurons results in a cell non-autonomous accumulation of lipid droplets in axonal glia. Importantly, treatment with lipid regulating liver X receptor-agonists blocked lipid droplet accumulation, restored axonal ER organisation, and improved locomotor function in Arl6IP1 knockout Drosophila. Our findings indicate that disrupted lipid homeostasis contributes to neurodegeneration in HSP, identifying a potential novel therapeutic avenue for the treatment of this disorder.

Keywords:  Drosophila model; Endoplasmic reticulum; Lipid droplet; Neurodegeneration

DOI:  https://doi.org/10.1186/s40478-022-01343-6
PLoS Biol. 2022 Mar 30. 20(3): e3001596

TMED2 binding restricts SMO to the ER and Golgi compartments.

Giulio Di Minin, Markus Holzner, Alice Grison, Charles E Dumeau, Wesley Chan, Asun Monfort, Loydie A Jerome-Majewska, Henk Roelink, Anton Wutz.

Hedgehog (HH) signaling is important for embryonic pattering and stem cell differentiation. The G protein-coupled receptor (GPCR) Smoothened (SMO) is the key HH signal transducer modulating both transcription-dependent and transcription-independent responses. We show that SMO protects naive mouse embryonic stem cells (ESCs) from dissociation-induced cell death. We exploited this SMO dependency to perform a genetic screen in haploid ESCs where we identify the Golgi proteins TMED2 and TMED10 as factors for SMO regulation. Super-resolution microscopy shows that SMO is normally retained in the endoplasmic reticulum (ER) and Golgi compartments, and we demonstrate that TMED2 binds to SMO, preventing localization to the plasma membrane. Mutation of TMED2 allows SMO accumulation at the plasma membrane, recapitulating early events after HH stimulation. We demonstrate the physiologic relevance of this interaction in neural differentiation, where TMED2 functions to repress HH signal strength. Identification of TMED2 as a binder and upstream regulator of SMO opens the way for unraveling the events in the ER-Golgi leading to HH signaling activation.

DOI: https://doi.org/10.1371/journal.pbio.3001596
Mol Ther. 2022 Mar 26. pii: S1525-0016(22)00174-5. [Epub ahead of print]

FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9/CYLD/NF-κB axis.

Zhengxin Chen, Shuai Wang, Hai-Lin Li, Hui Luo, Xiaoting Wu, Jiacheng Lu, Hong-Wei Wang, Yuanyuan Chen, Dan Chen, Wen-Ting Wu, Shuyu Zhang, Qiongqiong He, Daru Lu, Ning Liu, Yongping You, Wei Wu, Huibo Wang.

Proneural (PN) to Mesenchymal (MES) Transition (PMT) is a crucial phenotypic shift in glioblastoma stem cells (GSCs). However, the mechanisms driving this process remain poorly understood. Here, we report that FOSL1, a component of AP-1 transcription factor complexes, is a key player in regulating PMT. FOSL1 is predominantly expressed in the MES subtype, but not PN subtype of GSCs. Knocking down FOSL1 expression in MES GSCs leads to the loss of MES features and tumor-initiating ability, whereas ectopic expression of FOSL1 in PN GSCs is able to induce PMT and maintain MES features. Moreover, FOSL1 facilitates IR-induced PMT and radioresistance of PN GSCs. Inhibition of FOSL1 could enhance the anti-tumor effects of IR by preventing IR-induced PMT. Mechanistically, we find that FOSL1 promotes UBC9-dependent CYLD SUMOylation, thereby inducing K63-linked polyubiquitination of major NF-κB intermediaries and subsequent NF-κB activation, which results in PMT induction in GSCs. Our study underscores the importance of FOSL1 in the regulation of PMT and suggests that therapeutic targeting of FOSL1 holds promise to attenuate the molecular subtype switching in patients with glioblastomas.

DOI: https://doi.org/10.1016/j.ymthe.2021.10.028
Gut. 2022 Apr 01. pii: gutjnl-2021-325013. [Epub ahead of print]

NFATc1 signaling drives chronic ER stress responses to promote NAFLD progression.

Muhammad Umair Latif, Geske Elisabeth Schmidt, Sercan Mercan, Raza Rahman, Christine Silvia Gibhardt, Ioana Stejerean-Todoran, Kristina Reutlinger, Elisabeth Hessmann, Shiv K Singh, Abdul Moeed, Abdul Rehman, Umer Javed Butt, Hanibal Bohnenberger, Philipp Stroebel, Sebastian Christopher Bremer, Albrecht Neesse, Ivan Bogeski, Volker Ellenrieder.

  OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) can persist in the stage of simple hepatic steatosis or progress to steatohepatitis (NASH) with an increased risk for cirrhosis and cancer. We examined the mechanisms controlling the progression to severe NASH in order to develop future treatment strategies for this disease.DESIGN: NFATc1 activation and regulation was examined in livers from patients with NAFLD, cultured and primary hepatocytes and in transgenic mice with differential hepatocyte-specific expression of the transcription factor (Alb-cre, NFATc1c.a . and NFATc1Δ/Δ ). Animals were fed with high-fat western diet (WD) alone or in combination with tauroursodeoxycholic acid (TUDCA), a candidate drug for NAFLD treatment. NFATc1-dependent ER stress-responses, NLRP3 inflammasome activation and disease progression were assessed both in vitro and in vivo.
RESULTS: NFATc1 expression was weak in healthy livers but strongly induced in advanced NAFLD stages, where it correlates with liver enzyme values as well as hepatic inflammation and fibrosis. Moreover, high-fat WD increased NFATc1 expression, nuclear localisation and activation to promote NAFLD progression, whereas hepatocyte-specific depletion of the transcription factor can prevent mice from disease acceleration. Mechanistically, NFATc1 drives liver cell damage and inflammation through ER stress sensing and activation of the PERK-CHOP unfolded protein response (UPR). Finally, NFATc1-induced disease progression towards NASH can be blocked by TUDCA administration.
CONCLUSION: NFATc1 stimulates NAFLD progression through chronic ER stress sensing and subsequent activation of terminal UPR signalling in hepatocytes. Interfering with ER stress-responses, for example, by TUDCA, protects fatty livers from progression towards manifest NASH.

Keywords:  fatty liver; hepatic fibrosis; inflammation; nonalcoholic steatohepatitis

DOI:  https://doi.org/10.1136/gutjnl-2021-325013
J Leukoc Biol. 2022 Mar 30.

K48- and K27-mutant ubiquitin regulates adaptive immune response by affecting cross-presentation in bone marrow precursor cells.

Jun Jun Jia, Xiao Yan Liao, Yi Yun Liang, Rui Ling Chen, Feng Guang Gao.

  K48-linked ubiquitination determines antigen degradation and plays vital roles in the process of cross-presentation of bone marrow precursor cell (BMPC)-derived dendritic cells (DCs). Although previous studies revealed that K48 and K27-linked ubiquitination regulates innate immunity, the exact roles of K48 and K27-linked ubiquitination in cross-presentation and BMPC-based adaptive immunity are still uncertain. In this study, we investigated the effects of K48- and K27-mutant ubiquitin (Ub) on BMPC-based adaptive immune response by observing the effects of MG132, Ub deficiency, and K48/K27-mutant Ub on cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and thereby the efficiency of cytolytic capacity of BMPC-activate T cells. We demonstrated that MG132, Ub deficiency, and K48-mutant Ub impair cross-presentation, T cell proliferation, IFN-γ secretion, BMPC-based CTL priming, and the cytolytic capacity of BMPC-activated T cells. Moreover, although K27-only Ub decreases cross-presentation, the replenishment of K27-mutant Ub restores Ub deficiency impaireds the abilities of T cell proliferation, IFN-γ secretion, CTL priming, and the cytolytic capacity of BMPC-activated T cells. Thus, these data suggest that K48- and K27-linked ubiquitination contributes to BMPC-mediated adaptive immune response by affecting BMPC cross-presentation and the cytolytic capacity by up-regulating both perforin and granzyme B in BMPC-activated T cells. K48- and K27-mutant Ub might have potential clinical therapeutic function in adaptive immune response-associated diseases.

Keywords:  CTL; Cross-presentation; Cytotoxicity; T cells; adaptive immunity; bone marrow; cross-presentation; ubiquitination

DOI:  https://doi.org/10.1002/JLB.4MA0222-419RR
Sci Adv. 2022 Apr;8(13): eabm5149

Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation.

Sudeep Banjade, Lu Zhu, Jeffrey R Jorgensen, Sho W Suzuki, Scott D Emr.

The general mechanisms by which ESCRTs (Endosomal Sorting Complexes Required for Transport) are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin are critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.

DOI: https://doi.org/10.1126/sciadv.abm5149
PLoS Biol. 2022 Mar 31. 20(3): e3001590

Identification of substrates of palmitoyl protein thioesterase 1 highlights roles of depalmitoylation in disulfide bond formation and synaptic function.

Erica L Gorenberg, Sofia Massaro Tieze, Betül Yücel, Helen R Zhao, Vicky Chou, Gregory S Wirak, Susumu Tomita, TuKiet T Lam, Sreeganga S Chandra.

Loss-of-function mutations in the depalmitoylating enzyme palmitoyl protein thioesterase 1 (PPT1) cause neuronal ceroid lipofuscinosis (NCL), a devastating neurodegenerative disease. The substrates of PPT1 are largely undescribed, posing a limitation on molecular dissection of disease mechanisms and therapeutic development. Here, we provide a resource identifying >100 novel PPT1 substrates. We utilized Acyl Resin-Assisted Capture (Acyl RAC) and mass spectrometry to identify proteins with increased in vivo palmitoylation in PPT1 knockout (KO) mouse brains. We then validated putative substrates through direct depalmitoylation with recombinant PPT1. This stringent screen elucidated diverse PPT1 substrates at the synapse, including channels and transporters, G-protein-associated molecules, endo/exocytic components, synaptic adhesion molecules, and mitochondrial proteins. Cysteine depalmitoylation sites in transmembrane PPT1 substrates frequently participate in disulfide bonds in the mature protein. We confirmed that depalmitoylation plays a role in disulfide bond formation in a tertiary screen analyzing posttranslational modifications (PTMs). Collectively, these data highlight the role of PPT1 in mediating synapse functions, implicate molecular pathways in the etiology of NCL and other neurodegenerative diseases, and advance our basic understanding of the purpose of depalmitoylation.

DOI: https://doi.org/10.1371/journal.pbio.3001590
J Hepatol. 2022 Mar 28. pii: S0168-8278(22)00185-4. [Epub ahead of print]

Endoplasmic reticulum-mitochondria miscommunication is an early and causal trigger of hepatic insulin resistance and steatosis.

Agathe Beaulant, Maya Dia, Bruno Pillot, Marie-Agnes Chauvin, Jingwei Ji-Cao, Christine Durand, Nadia Bendridi, Stephanie Chanon, Aurelie Vieille-Marchiset, Claire Crola Da Silva, Stéphanie Patouraux, Rodolphe Anty, Antonio Iannelli, Albert Tran, Philippe Gual, Hubert Vidal, Ludovic Gomez, Melanie Paillard, Jennifer Rieusset.

  BACKGROUND & AIMS: Hepatic insulin resistance in obesity and type 2 diabetes was recently associated with endoplasmic reticulum (ER)-mitochondria miscommunication. These contact sites (mitochondria-associated membranes: MAMs) are highly dynamic and involved in many functions. Up to now, it is not clear if MAM miscommunication could have a causal role in hepatic insulin resistance and steatosis. We therefore aimed to determine whether and how organelle miscommunication plays a role in the onset and progression of hepatic metabolic impairment.METHODS: We analyzed hepatic ER-mitochondria interactions and calcium exchange in diet-induced obese mice in a time-dependent and reversible manner, and investigated causality in hepatic metabolic alterations by expressing a specific organelle spacer or linker in mouse liver, using adenovirus.
RESULTS: Disruption of ER-mitochondria interactions and calcium exchange is an early event preceding hepatic insulin resistance and steatosis in diet-induced obese mice. Interestingly, an 8-week reversal diet concomitantly reversed hepatic organelle miscommunication and insulin resistance in obese mice. Mechanistically, disrupting structural and functional ER-mitochondria interactions through the hepatic overexpression of the organelle spacer FATE1 was sufficient to impair hepatic insulin action and glucose homeostasis. In addition, FATE1-mediated organelle miscommunication disrupted lipid-related mitochondrial oxidative metabolism and induced hepatic steatosis. Conversely, reinforcement of ER-mitochondria interactions through hepatic expression of a synthetic linker prevented diet-induced glucose intolerance after 4 weeks' overnutrition. Importantly, ER-mitochondria miscommunication was confirmed in the liver of obese patients with type-2 diabetes, and correlated with glycemia, HbA1c and HOMA-IR index.
CONCLUSIONS: ER-mitochondria miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be reversed by switching to a healthy diet. Thus, targeting MAMs could contribute to restoring metabolic homeostasis.
LAY SUMMARY: The literature suggests that interactions between endoplasmic reticulum (ER) and mitochondria could play a dual role in hepatic insulin resistance and steatosis during chronic obesity. The present study reappraised time-dependent regulation of hepatic ER-mitochondria interactions and calcium exchange in diet-induced obese mice and their causal role in hepatic insulin resistance and steatosis. We show that organelle miscommunication is an early causal trigger of hepatic insulin resistance and steatosis, and can be improved by nutritional strategies.

Keywords:  calcium signaling; hepatic insulin resistance; hepatic steatosis; lipid oxidation; mitochondria oxidative metabolism; mitochondria-associated membranes

DOI:  https://doi.org/10.1016/j.jhep.2022.03.017
Chem Biol Drug Des. 2022 Mar 30.

Discovery of 3-(2-aminoethyl)-thiazolidine-2,4-diones as a Novel Chemotype of Sigma-1 Receptor Ligands.

Nada Elkholy, Ahmad Abdelwaly, Karim Mohamed, Emanuele Amata, Jessica Lombino, Giuseppe Cosentino, Sebastiano Intagliata, Mohamed A Helal.

  Sigma receptor is a transmembrane non-GPCR protein expressed mainly in the endoplasmic reticulum (ER) membrane associated with mitochondria. It is classified into two types; Sigma-1 (S1R) and Sigma-2 (S2R) based on their biological functions. S1R has been implicated in many neurological disorders such as anxiety, schizophrenia, and depression. Therefore, S1R ligands possess a variety of potential clinical applications with a great interest in the treatment of neuropathic pain. In this study, we report the discovery of a novel lead compound for S1R binding, based on the thiazolidine-2,4-dione nucleus. We have explored hydrophobic groups of different sizes on both sides of the five-membered ring scaffold guided by the crystal structure of S1R. Six compounds showed more than 50% displacement of the radioligand at 10 µM concentration with compound 6c resulting in 100% displacement and a Ki of 95.5 nM. Moreover, compounds 6c and 6e showed a significant selectivity over S2R. In addition, molecular docking predicted that all the compounds showed the critical salt bridge with Glu172 with variable degrees of π-stacking interaction with Tyr103. Upon optimization, this series of compounds could represent potential clinically useful S1R ligands for pain management.

Keywords:  Docking; Neuropathic Pain; Scaffold Hopping; Sigma Receptor; Thiazolidinedione

DOI:  https://doi.org/10.1111/cbdd.14047
Cell Death Dis. 2022 Apr 01. 13(4): 293

Class I HDAC overexpression promotes temozolomide resistance in glioma cells by regulating RAD18 expression.

Daniela Hanisch, Andrea Krumm, Tamara Diehl, Carla M Stork, Mario Dejung, Falk Butter, Ella Kim, Walburgis Brenner, Gerhard Fritz, Thomas G Hofmann, Wynand P Roos.

Overexpression of histone deacetylases (HDACs) in cancer commonly causes resistance to genotoxic-based therapies. Here, we report on the novel mechanism whereby overexpressed class I HDACs increase the resistance of glioblastoma cells to the SN1 methylating agent temozolomide (TMZ). The chemotherapeutic TMZ triggers the activation of the DNA damage response (DDR) in resistant glioma cells, leading to DNA lesion bypass and cellular survival. Mass spectrometry analysis revealed that the catalytic activity of class I HDACs stimulates the expression of the E3 ubiquitin ligase RAD18. Furthermore, the data showed that RAD18 is part of the O6-methylguanine-induced DDR as TMZ induces the formation of RAD18 foci at sites of DNA damage. Downregulation of RAD18 by HDAC inhibition prevented glioma cells from activating the DDR upon TMZ exposure. Lastly, RAD18 or O6-methylguanine-DNA methyltransferase (MGMT) overexpression abolished the sensitization effect of HDAC inhibition on TMZ-exposed glioma cells. Our study describes a mechanism whereby class I HDAC overexpression in glioma cells causes resistance to TMZ treatment. HDACs accomplish this by promoting the bypass of O6-methylguanine DNA lesions via enhancing RAD18 expression. It also provides a treatment option with HDAC inhibition to undermine this mechanism.

DOI: https://doi.org/10.1038/s41419-022-04751-7
Life Sci Alliance. 2022 Jul;pii: e202101239. [Epub ahead of print]5(7):

ESCRT-I fuels lysosomal degradation to restrict TFEB/TFE3 signaling via the Rag-mTORC1 pathway.

Marta Wróbel, Jarosław Cendrowski, Ewelina Szymańska, Malwina Grębowicz-Maciukiewicz, Noga Budick-Harmelin, Matylda Macias, Aleksandra Szybińska, Michał Mazur, Krzysztof Kolmus, Krzysztof Goryca, Michalina Dąbrowska, Agnieszka Paziewska, Michał Mikula, Marta Miączyńska.

Within the endolysosomal pathway in mammalian cells, ESCRT complexes facilitate degradation of proteins residing in endosomal membranes. Here, we show that mammalian ESCRT-I restricts the size of lysosomes and promotes degradation of proteins from lysosomal membranes, including MCOLN1, a Ca2+ channel protein. The altered lysosome morphology upon ESCRT-I depletion coincided with elevated expression of genes annotated to biogenesis of lysosomes due to prolonged activation of TFEB/TFE3 transcription factors. Lack of ESCRT-I also induced transcription of cholesterol biosynthesis genes, in response to inefficient delivery of cholesterol from endolysosomal compartments. Among factors that could possibly activate TFEB/TFE3 signaling upon ESCRT-I deficiency, we excluded lysosomal cholesterol accumulation and Ca2+-mediated dephosphorylation of TFEB/TFE3. However, we discovered that this activation occurs due to the inhibition of Rag GTPase-dependent mTORC1 pathway that specifically reduced phosphorylation of TFEB at S112. Constitutive activation of the Rag GTPase complex in cells lacking ESCRT-I restored S112 phosphorylation and prevented TFEB/TFE3 activation. Our results indicate that ESCRT-I deficiency evokes a homeostatic response to counteract lysosomal nutrient starvation, that is, improper supply of nutrients derived from lysosomal degradation.

DOI: https://doi.org/10.26508/lsa.202101239
Mol Oncol. 2022 Apr 01.

USP28 enables oncogenic transformation of respiratory cells and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K.

Cristian Prieto-Garcia, Oliver Hartmann, Michaela Reissland, Fabian Braun, Süleyman Bozkurt, Nikolett Pahor, Carmina Fuss, Andreas Schirbel, Christina Schülein-Völk, Alexander Buchberger, Marco A Calzado Canale, Mathias Rosenfeldt, Ivan Dikic, Christian Münch, Markus E Diefenbacher.

  Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared to in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a 'pre-malignant' state, and its inhibition synergises with clinically established compounds used to target EGFRL858R -, BRAFV600E - or PI3KH1047R -driven tumor cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumors.

Keywords:  BRAF; Buparlisib; EGFR; HRAS Gefitinib; PIK3CA; USP28; Vemurafenib; c-JUN; c-MYC; lung cancer

DOI:  https://doi.org/10.1002/1878-0261.13217