bims-proteo Biomed News
on Proteostasis
Issue of 2021–10–10
twenty papers selected by
Eric Chevet, INSERM
  1. Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex.
  2. The deubiquitinase USP11 is a versatile and conserved regulator of autophagy.
  3. Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion.
  4. OsFes1C, a potential nucleotide exchange factor for OsBiP1, is involved in the ER and salt stress responses.
  5. The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila.
  6. Two ubiquitin-associated ER proteins interact with COPT copper transporters and modulate their accumulation.
  7. Mechanism of lipid droplet formation by the yeast Sei1/Ldb16 Seipin complex.
  8. TFG: a novel regulator of ULK1-dependent autophagy.
  9. Sirt1 promotes tissue regeneration in zebrafish through regulating the mitochondrial unfolded protein response.
  10. Autophagy deficiency activates rDNA transcription.
  11. Oncogenic activity of glucocorticoid receptor β is controlled by ubiquitination-dependent interaction with USP49 in glioblastoma cells.
  12. Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound.
  13. Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein.
  14. Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly.
  15. E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor.
  16. Single-cell analysis reveals androgen receptor regulates the ER-to-Golgi trafficking pathway with CREB3L2 to drive prostate cancer progression.
  17. OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.
  18. USP29 coordinates MYC and HIF1α stabilization to promote tumor metabolism and progression.
  19. A preferred sequence for organelle inheritance during polarized cell growth.
  20. DDX3 interacts with USP9X and participates in deubiquitination of the anti-apoptotic protein MCL1.
  1. EMBO J. 2021 Oct 06. e107958
    Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex.
    Dimitrios Papagiannidis, Peter W Bircham, Christian Lüchtenborg, Oliver Pajonk, Giulia Ruffini, Britta Brügger, Sebastian Schuck.
      Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the underlying regulatory mechanisms are largely unclear. Here, we describe a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, called lipin in metazoa. Specifically, Ice2 inhibits the conserved Nem1-Spo7 complex and thus suppresses the dephosphorylation and activation of Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain cell homeostasis during ER stress. These findings establish the control of the lipin phosphatase complex as an important mechanism for regulating ER membrane biogenesis.
    Keywords:  Opi1; endoplasmic reticulum; lipid droplets; lipin; organelle biogenesis
    DOI:  https://doi.org/10.15252/embj.2021107958
  2. J Biol Chem. 2021 Sep 30. pii: S0021-9258(21)01066-8. [Epub ahead of print] 101263
    The deubiquitinase USP11 is a versatile and conserved regulator of autophagy.
    Mila Basic, Alexandra Hertel, Justyna Bajdzienko, Florian Bonn, Mariana Tellechea, Alexandra Stolz, Andreas Kern, Christian Behl, Anja Bremm.
      Autophagy is a major cellular quality control system responsible for degradation of proteins and organelles in response to stress and damage in order to maintain homeostasis. Ubiquitination of autophagy-related proteins or regulatory components is important for the precise control of autophagy pathways. Here, we show that the deubiquitinase USP11 restricts autophagy and that knockout (KO) of USP11 in mammalian cells results in elevated autophagic flux. We also demonstrate that depletion of the USP11 homolog H34C03.2 in Caenorhabditis elegans triggers hyperactivation of autophagy and protects the animals against human β-amyloid peptide 42 aggregation-induced paralysis. USP11 co-precipitated with the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) and limited its interaction with nuclear receptor-binding factor 2 (NRBF2), thus decreasing lipid kinase activity of the PI3KC3-C1 and subsequent recruitment of effectors like WIPI proteins to the autophagosomal membrane. Accordingly, more WIPI2-puncta accumulated in USP11 KO cells. In addition, USP11 interacts with and stabilizes the serine/threonine kinase mTOR, thereby further contributing to the regulation of autophagy induction. Taken together, our data suggested that USP11 impinges on the autophagy pathway at multiple sites and that inhibiting USP11 alleviates symptoms of proteotoxicity, which is a major hallmark of neurodegenerative diseases.
    Keywords:  Autophagy; PI3KC3-C1; deubiquitinase (DUB); mTORC1; proteostasis; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2021.101263
  3. J Cell Biol. 2021 Nov 01. pii: e202103079. [Epub ahead of print]220(11):
    Subunit cooperation in the Get1/2 receptor promotes tail-anchored membrane protein insertion.
    Un Seng Chio, Yumeng Liu, SangYoon Chung, Woo Jun Shim, Sowmya Chandrasekar, Shimon Weiss, Shu-Ou Shan.
      The guided entry of tail-anchored protein (GET) pathway, in which the Get3 ATPase delivers an essential class of tail-anchored membrane proteins (TAs) to the Get1/2 receptor at the endoplasmic reticulum, provides a conserved mechanism for TA biogenesis in eukaryotic cells. The membrane-associated events of this pathway remain poorly understood. Here we show that complex assembly between the cytosolic domains (CDs) of Get1 and Get2 strongly enhances the affinity of the individual subunits for Get3•TA, thus enabling efficient capture of the targeting complex. In addition to the known role of Get1CD in remodeling Get3 conformation, two molecular recognition features (MoRFs) in Get2CD induce Get3 opening, and both subunits are required for optimal TA release from Get3. Mutation of the MoRFs attenuates TA insertion into the ER in vivo. Our results demonstrate extensive cooperation between the Get1/2 receptor subunits in the capture and remodeling of the targeting complex, and emphasize the role of MoRFs in receptor function during membrane protein biogenesis.
    DOI:  https://doi.org/10.1083/jcb.202103079
  4. Plant Physiol. 2021 Sep 04. 187(1): 396-408
    OsFes1C, a potential nucleotide exchange factor for OsBiP1, is involved in the ER and salt stress responses.
    Dandan Qian, Shuo Xiong, Mei Li, Lihong Tian, Le Qing Qu.
      The endoplasmic reticulum (ER) quality control system monitors protein homeostasis and relies on the activity of many molecular chaperones. Binding immunoglobulin protein (BiP) is a major ER luminal chaperone that is involved in most functions of the organelle. BiP activity is tightly regulated by nucleotide exchange factors (NEFs). However, information about NEFs in plants is limited. We obtained a Fes1-like protein (OsFes1C) through isobaric tags for relative and absolute quantitation-based proteomics analysis of ER-stressed rice (Oryza sativa) seeds. Unlike its homologs in yeast and mammals, which are located in the cytosol and respond to heat stress, OsFes1C is an ER membrane protein and responds to ER and salt stresses. OsFes1C interacts directly with OsBiP1 and the interaction is inhibited by ATP but promoted by ADP, suggesting that OsFes1C acts as a potential NEF of OsBiP1 in vivo. Overexpression or suppression of OsFes1C led to hypersensitivity to ER stress and affected the growth of rice. Furthermore, we established that OsFes1C directly interacts with a putative salt response protein and is involved in the salt response. Taken together, our study marks an important step toward elucidating the functional mechanisms of an identified ER stress response factor in rice.
    DOI:  https://doi.org/10.1093/plphys/kiab263
  5. Elife. 2021 Oct 04. pii: e74047. [Epub ahead of print]10
    The transcription factor Xrp1 is required for PERK-mediated antioxidant gene induction in Drosophila.
    Brian Brown, Sahana Mitra, Finnegan D Roach, Deepika Vasudevan, Hyung Don Ryoo.
      PERK is an endoplasmic reticulum (ER) transmembrane sensor that phosphorylates eIF2a to initiate the Unfolded Protein Response (UPR). eIF2a phosphorylation promotes stress-responsive gene expression most notably through the transcription factor ATF4 that contains a regulatory 5' leader. Possible PERK effectors other than ATF4 remain poorly understood. Here, we report that the bZIP transcription factor Xrp1 is required for ATF4-independent PERK signaling. Cell type-specific gene expression profiling in Drosophila indicated that delta-family glutathione-S-transferases (gstD) are prominently induced by the UPR-activating transgene Rh1G69D. Perk was necessary and sufficient for such gstD induction, but ATF4 was not required. Instead, Perk and other regulators of eIF2a phosphorylation regulated Xrp1 protein levels to induce gstDs. The Xrp1 5' leader has a conserved upstream Open Reading Frame (uORF) analogous to those that regulate ATF4 translation. The gstD-GFP reporter induction required putative Xrp1 binding sites. These results indicate that antioxidant genes are highly induced by a previously unrecognized UPR signaling axis consisting of PERK and Xrp1.
    Keywords:  D. melanogaster; genetics; genomics
    DOI:  https://doi.org/10.7554/eLife.74047
  6. Plant Physiol. 2021 Aug 09. pii: kiab381. [Epub ahead of print]
    Two ubiquitin-associated ER proteins interact with COPT copper transporters and modulate their accumulation.
    Xifeng Li, Zhe Wang, Yunting Fu, Xi Cheng, Yan Zhang, Baofang Fan, Cheng Zhu, Zhixiang Chen.
      The endoplasmic reticulum (ER) contains an elaborate protein quality control network that promotes protein folding and prevents accumulation of misfolded proteins. Evolutionarily conserved UBIQUITIN-ASSOCIATED DOMAIN-CONTAINING PROTEIN 2 (UBAC2) is involved in ER-associated protein degradation in metazoans. We have previously reported that two close UBAC2 homologs from Arabidopsis (Arabidopsis thaliana) not only participate in selective autophagy of ER components but also interact with plant-specific PATHOGEN-ASSOCIATED MOLECULAR PATTERN (PAMP)-INDUCED COILED COIL (PICC) protein to increase the accumulation of POWDERY MILDEW-RESISTANT 4 callose synthase. Here, we report that UBAC2s also interacted with COPPER (Cu) TRANSPORTER 1 (COPT1) and plasma membrane-targeted members of the Cu transporter family. The ubac2 mutants were significantly reduced in both the accumulation of COPT proteins and Cu content, and also displayed increased sensitivity to a Cu chelator. Therefore, UBAC2s positively regulate the accumulation of COPT transporters, thereby increasing Cu uptake by plant cells. Unlike with POWDERY MILDEW RESISTANCE 4, however, the positive role of UBAC2s in the accumulation of COPT1 is not dependent on PICC or the UBA domain of UBAC2s. When COPT1 was overexpressed under the CaMV 35S promoter, the increased accumulation of COPT1 was strongly UBAC2-dependent, particularly when a signal peptide was added to the N-terminus of COPT1. Further analysis using inhibitors of protein synthesis and degradation strongly suggested that UBAC2s stabilize newly synthesized COPT proteins against degradation by the proteasome system. These results indicate that plant UBAC2s are multifunctional proteins that regulate the degradation and accumulation of specific ER-synthesized proteins.
    DOI:  https://doi.org/10.1093/plphys/kiab381
  7. Nat Commun. 2021 Oct 08. 12(1): 5892
    Mechanism of lipid droplet formation by the yeast Sei1/Ldb16 Seipin complex.
    Yoel A Klug, Justin C Deme, Robin A Corey, Mike F Renne, Phillip J Stansfeld, Susan M Lea, Pedro Carvalho.
      Lipid droplets (LDs) are universal lipid storage organelles with a core of neutral lipids, such as triacylglycerols, surrounded by a phospholipid monolayer. This unique architecture is generated during LD biogenesis at endoplasmic reticulum (ER) sites marked by Seipin, a conserved membrane protein mutated in lipodystrophy. Here structural, biochemical and molecular dynamics simulation approaches reveal the mechanism of LD formation by the yeast Seipin Sei1 and its membrane partner Ldb16. We show that Sei1 luminal domain assembles a homooligomeric ring, which, in contrast to other Seipins, is unable to concentrate triacylglycerol. Instead, Sei1 positions Ldb16, which concentrates triacylglycerol within the Sei1 ring through critical hydroxyl residues. Triacylglycerol recruitment to the complex is further promoted by Sei1 transmembrane segments, which also control Ldb16 stability. Thus, we propose that LD assembly by the Sei1/Ldb16 complex, and likely other Seipins, requires sequential triacylglycerol-concentrating steps via distinct elements in the ER membrane and lumen.
    DOI:  https://doi.org/10.1038/s41467-021-26162-6
  8. Mol Cell Oncol. 2021 ;8(4): 1945895
    TFG: a novel regulator of ULK1-dependent autophagy.
    Francesco Cecconi, Francesca Nazio.
      TRK-fused gene (TFG) is a protein implicated in multiple neurodegenerative diseases and oncogenesis. We have recently shown that, under starvation conditions, TFG contributes to spatial control of autophagy by facilitating Unc-51 like autophagy activating kinase 1 (ULK1)-microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C) interaction to modulate omegasome and autophagosome formation. Defective TFG-mediated autophagy could thus be postulated as a possible contributor to ontogenesis or progression of TFG-related diseases.
    Keywords:  ERGIC; TFG; ULK1; neurological disorders; omegasome
    DOI:  https://doi.org/10.1080/23723556.2021.1945895
  9. iScience. 2021 Oct 22. 24(10): 103118
    Sirt1 promotes tissue regeneration in zebrafish through regulating the mitochondrial unfolded protein response.
    Yi-Fan Lin, Jessica Sam, Todd Evans.
      The mitochondrial unfolded protein response (UPRmt) is an organellar stress signaling pathway that functions to detect and restore disruption of mitochondrial proteostasis. The UPRmt is involved in a wide range of physiological and disease conditions, including aging, stem cell maintenance, innate immunity, neurodegeneration, and cancer. Here we report that the UPRmt is integral to zebrafish fin regeneration. Taking advantage of a novel zebrafish UPRmt reporter, we observed that UPRmt activation occurs in regenerating fin tissue shortly after injury. Through chemical and genetic approaches, we discovered that the Sirt1-UPRmt pathway, best known for its role in promoting lifespan extension, is crucial for fin regeneration. The metabolism of NAD+ is an important contributor to Sirt1 activity in this context. We propose that Sirt1 activation induces mitochondrial biogenesis in injured fin tissue, which leads to UPRmt activation and promotes tissue regeneration.
    Keywords:  Cell biology; Developmental biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2021.103118
  10. Autophagy. 2021 Oct 06. 1-12
    Autophagy deficiency activates rDNA transcription.
    Yinfeng Xu, Yaosen Wu, Lei Wang, Zhuo Ren, Lijiang Song, Hui Zhang, Chuying Qian, Qian Wang, Zhengfu He, Wei Wan.
      Macroautophagy/autophagy, a highly conserved lysosome-dependent degradation pathway, has been intensively studied in regulating cell metabolism by degradation of intracellular components. In this study, we link autophagy to RNA metabolism by uncovering a regulatory role of autophagy in ribosomal RNA (rRNA) synthesis. Autophagy-deficient cells exhibit much higher 47S precursor rRNA level, which is caused by the accumulation of SQSTM1/p62 (sequestosome 1) but not other autophagy receptors. Mechanistically, SQSTM1 accumulation potentiates the activation of MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) signaling and promotes the assembly of RNA polymerase I pre-initiation complex at ribosomal DNA (rDNA) promoters, which leads to an increase of 47S rRNA transcribed from rDNA. Functionally, autophagy deficiency promotes protein synthesis, cell growth and cell proliferation, both of which are dependent on SQSTM1 accumulation. Taken together, our findings suggest that autophagy deficiency is involved in RNA metabolism by activating rDNA transcription and provide novel mechanisms for the reprogramming of cell metabolism in autophagy-related diseases including multiple types of cancers.Abbreviations: 5-FUrd: 5-fluorouridine; AMPK: AMP-activated protein kinase; ATG: autophagy related; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; ChIP: chromatin immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK/ERK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NFKB/NF-κB: nuclear factor kappa B; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; OPTN: optineurin; PIC: pre-initiation complex; POLR1: RNA polymerase I; POLR1A/RPA194: RNA polymerase I subunit A; POLR2A: RNA polymerase II subunit A; rDNA: ribosomal DNA; RPS6KB1/S6K1: ribosomal protein S6 kinase B1; rRNA: ribosomal RNA; RUBCN/Rubicon: rubicon autophagy regulator; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; SUnSET: surface sensing of translation; TAX1BP1: Tax1 binding protein 1; UBTF/UBF1: upstream binding transcription factor; WIPI2: WD repeat domain, phosphoinositide interacting 2; WT: wild-type.
    Keywords:  Autophagy; MTORC1; SQSTM1/p62; rDNA; rRNA
    DOI:  https://doi.org/10.1080/15548627.2021.1974178
  11. Mol Cancer Res. 2021 Oct 05. pii: molcanres.1068.2020. [Epub ahead of print]
    Oncogenic activity of glucocorticoid receptor β is controlled by ubiquitination-dependent interaction with USP49 in glioblastoma cells.
    Yaling Hu, Yingdi Jiang, Zhenhao Zhang, Jingjing Wang, Bo Zhang, Lingli Gong, Li Ji, Zhening Pu, Xusheng Yang, Jian Zou, Ying Yin.
      Previous studies have demonstrated that glucocorticoid receptor β (GRβ) functions as an oncoprotein, regulating the malignant phenotypes and stem-like cells maintaining in human glioblastoma (GBM). Of the GR isoforms, GRβ and GRα are highly homologous, though the mechanism underlying the distinct functions of these two isoforms in GBM has not been clarified. Here by establishing a C-terminal deletion mutant, we determined that GRβ can be ubiquitinated. We also found that its C-terminal is essential for this ubiquitination. The mutation of a lysine to arginine at residue 733 (K733R) blocked the ubiquitination of GRβ, indicating that K733 is a key site for ubiquitination. Using K733R to establish non-ubiquitinated GRβ, we demonstrated that ubiquitination not only regulates the stability and nuclear translocation of GRβ, but is also a vital mechanism for its oncogenic functions in vitro and in vivo. Protein interaction assay further indicated that ubiquitin-specific protease 49 (USP49) is a GRβ-binding protein and the interaction depends on GRβ ubiquitination. USP49 knockdown resulted in a decrease of cell proliferation, invasion, and an increase of cell apoptosis. More importantly, USP49 knockdown increased ubiquitination and amplified the oncogenic effects of GRβ, confirming the decisive role of ubiquitination on GRβ carcinogenicity. Taken together, these findings established that ubiquitination is a vial process for GRβ the execution of oncogenic functions in GBM and that the K733 site is crucial for ubiquitination of GRβ. Implications: This work is the first identify of the activation GRβ by a single lysine point-mediated ubiquitination and proteasome degradation, which determines its oncogenic functions in GBM.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-1068
  12. Cell Death Dis. 2021 Oct 06. 12(10): 914
    Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound.
    Tatiana A Giovannucci, Florian A Salomons, Martin Haraldsson, Lotta H M Elfman, Malin Wickström, Patrick Young, Thomas Lundbäck, Jürgen Eirich, Mikael Altun, Rozbeh Jafari, Anna-Lena Gustavsson, John Inge Johnsen, Nico P Dantuma.
      Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.
    DOI:  https://doi.org/10.1038/s41419-021-04191-9
  13. Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2016962118. [Epub ahead of print]118(41):
    Selective promiscuity in the binding of E. coli Hsp70 to an unfolded protein.
    Eugenia M Clerico, Alexandra K Pozhidaeva, Rachel M Jansen, Can Özden, Joseph M Tilitsky, Lila M Gierasch.
      Heat shock protein 70 (Hsp70) chaperones bind many different sequences and discriminate between incompletely folded and folded clients. Most research into the origins of this "selective promiscuity" has relied on short peptides as substrates to dissect the binding, but much less is known about how Hsp70s bind full-length client proteins. Here, we connect detailed structural analyses of complexes between the Escherichia coli Hsp70 (DnaK) substrate-binding domain (SBD) and peptides encompassing five potential binding sites in the precursor to E. coli alkaline phosphatase (proPhoA) with SBD binding to full-length unfolded proPhoA. Analysis of SBD complexes with proPhoA peptides by a combination of X-ray crystallography, methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), and paramagnetic relaxation enhancement (PRE) NMR and chemical cross-linking experiments provided detailed descriptions of their binding modes. Importantly, many sequences populate multiple SBD binding modes, including both the canonical N to C orientation and a C to N orientation. The favored peptide binding mode optimizes substrate residue side-chain compatibility with the SBD binding pockets independent of backbone orientation. Relating these results to the binding of the SBD to full-length proPhoA, we observe that multiple chaperones may bind to the protein substrate, and the binding sites, well separated in the proPhoA sequence, behave independently. The hierarchy of chaperone binding to sites on the protein was generally consistent with the apparent binding affinities observed for the peptides corresponding to these sites. Functionally, these results reveal that Hsp70s "read" sequences without regard to the backbone direction and that both binding orientations must be considered in current predictive algorithms.
    Keywords:  DnaK; Hsp70 molecular chaperone; NMR; crystallography; substrate binding
    DOI:  https://doi.org/10.1073/pnas.2016962118
  14. Nat Cell Biol. 2021 Oct 06.
    Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly.
    Gea Cereghetti, Caroline Wilson-Zbinden, Vera M Kissling, Maren Diether, Alexandra Arm, Haneul Yoo, Ilaria Piazza, Shady Saad, Paola Picotti, D Allan Drummond, Uwe Sauer, Reinhard Dechant, Matthias Peter.
      Cells respond to stress by blocking translation, rewiring metabolism and forming transient messenger ribonucleoprotein assemblies called stress granules (SGs). After stress release, re-establishing homeostasis and disassembling SGs requires ATP-consuming processes. However, the molecular mechanisms whereby cells restore ATP production and disassemble SGs after stress remain poorly understood. Here we show that upon stress, the ATP-producing enzyme Cdc19 forms inactive amyloids, and that their rapid re-solubilization is essential to restore ATP production and disassemble SGs in glucose-containing media. Cdc19 re-solubilization is initiated by the glycolytic metabolite fructose-1,6-bisphosphate, which directly binds Cdc19 amyloids, allowing Hsp104 and Ssa2 chaperone recruitment and aggregate re-solubilization. Fructose-1,6-bisphosphate then promotes Cdc19 tetramerization, which boosts its activity to further enhance ATP production and SG disassembly. Together, these results describe a molecular mechanism that is critical for stress recovery and directly couples cellular metabolism with SG dynamics via the regulation of reversible Cdc19 amyloids.
    DOI:  https://doi.org/10.1038/s41556-021-00760-4
  15. Oncogene. 2021 Oct 07.
    E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor.
    Alfonso García-Valverde, Jordi Rosell, Sergi Sayols, David Gómez-Peregrina, Daniel F Pilco-Janeta, Iván Olivares-Rivas, Enrique de Álava, Joan Maurel, Jordi Rubió-Casadevall, Anna Esteve, Marta Gut, Claudia Valverde, Jordi Barretina, Joan Carles, George D Demetri, Jonathan A Fletcher, Joaquín Arribas, César Serrano.
      KIT/PDGFRA oncogenic tyrosine kinase signaling is the central oncogenic event in most gastrointestinal stromal tumors (GIST), which are human malignant mesenchymal neoplasms that often feature myogenic differentiation. Although targeted inhibition of KIT/PDGFRA provides substantial clinical benefit, GIST cells adapt to KIT/PDGFRA driver suppression and eventually develop resistance. The specific molecular events leading to adaptive resistance in GIST remain unclear. By using clinically representative in vitro and in vivo GIST models and GIST patients' samples, we found that the E3 ubiquitin ligase Atrogin-1 (FBXO32)-the main effector of muscular atrophy in cachexia-resulted in the most critical gene derepressed in response to KIT inhibition, regardless the type of KIT primary or secondary mutation. Atrogin-1 in GISTs is transcriptionally controlled by the KIT-FOXO3a axis, thus indicating overlap with Atrogin-1 regulation mechanisms in nonneoplastic muscle cells. Further, Atrogin-1 overexpression was a GIST-cell-specific pro-survival mechanism that enabled the adaptation to KIT-targeted inhibition by apoptosis evasion through cell quiescence. Buttressed on these findings, we established in vitro and in vivo the preclinical proof-of-concept for co-targeting KIT and the ubiquitin pathway to maximize the therapeutic response to first-line imatinib treatment.
    DOI:  https://doi.org/10.1038/s41388-021-02049-0
  16. Oncogene. 2021 Oct 05.
    Single-cell analysis reveals androgen receptor regulates the ER-to-Golgi trafficking pathway with CREB3L2 to drive prostate cancer progression.
    Lingling Hu, Xin Chen, Nitin Narwade, Michelle Gek Liang Lim, Zikai Chen, Chandana Tennakoon, Peiyong Guan, Un In Chan, Zuxianglan Zhao, Mokan Deng, Xiaoling Xu, Wing-Kin Sung, Edwin Cheung.
      Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.
    DOI:  https://doi.org/10.1038/s41388-021-02026-7
  17. Nat Commun. 2021 Oct 08. 12(1): 5913
    OTULIN maintains skin homeostasis by controlling keratinocyte death and stem cell identity.
    Esther Hoste, Kim Lecomte, Karl Annusver, Niels Vandamme, Jana Roels, Sophia Maschalidi, Lien Verboom, Hanna-Kaisa Vikkula, Mozes Sze, Lisette Van Hove, Kevin Verstaen, Arne Martens, Tino Hochepied, Yvan Saeys, Kodi Ravichandran, Maria Kasper, Geert van Loo.
      OTULIN is a deubiquitinase that specifically cleaves linear ubiquitin chains. Here we demonstrate that the ablation of Otulin selectively in keratinocytes causes inflammatory skin lesions that develop into verrucous carcinomas. Genetic deletion of Tnfr1, knockin expression of kinase-inactive Ripk1 or keratinocyte-specific deletion of Fadd and Mlkl completely rescues mice with OTULIN deficiency from dermatitis and tumorigenesis, thereby identifying keratinocyte cell death as the driving force for inflammation. Single-cell RNA-sequencing comparing non-lesional and lesional skin reveals changes in epidermal stem cell identity in OTULIN-deficient keratinocytes prior to substantial immune cell infiltration. Keratinocytes lacking OTULIN display a type-1 interferon and IL-1β response signature, and genetic or pharmacologic inhibition of these cytokines partially inhibits skin inflammation. Finally, expression of a hypomorphic mutant Otulin allele, previously shown to cause OTULIN-related autoinflammatory syndrome in humans, induces a similar inflammatory phenotype, thus supporting the importance of OTULIN for restraining skin inflammation and maintaining immune homeostasis.
    DOI:  https://doi.org/10.1038/s41467-021-25944-2
  18. Oncogene. 2021 Oct 02.
    USP29 coordinates MYC and HIF1α stabilization to promote tumor metabolism and progression.
    Rongfu Tu, Wenqian Kang, Mengjie Yang, Liyuan Wang, Qing Bao, Zhi Chen, Yang Dong, Jingchao Wang, Jue Jiang, Hudan Liu, Guoliang Qing.
      Tumor cells must rewire cellular metabolism to satisfy the demands of unbridled growth and proliferation. How these metabolic processes are integrated to fuel cancer cell growth remains largely unknown. Deciphering the regulatory mechanisms is vital to develop targeted strategies for tumor-selective therapies. We herein performed an unbiased and functional siRNA screen against 96 deubiquitinases, which play indispensable roles in cancer and are emerging as therapeutic targets, and identified USP29 as a top candidate essential for metabolic reprogramming that support biosynthesis and survival in tumor cells. Integrated metabolic flux analysis and molecular investigation reveal that USP29 directly deubiquitinates and stabilizes MYC and HIF1α, two master regulators of metabolic reprogramming, enabling adaptive response of tumor cells in both normoxia and hypoxia. Systemic knockout of Usp29 depleted MYC and HIF1α in MYC-driven neuroblastoma and B cell lymphoma, inhibited critical metabolic targets and significantly prolonged survival of tumor-bearing mice. Strikingly, mice homozygous null for the Usp29 gene are viable, fertile, and display no gross phenotypic abnormalities. Altogether, these results demonstrate that USP29 selectively coordinates MYC and HIF1α to integrate metabolic processes critical for cancer cell growth, and therapeutic targeting of USP29, a potentially targetable enzyme, could create a unique vulnerability given deregulation of MYC and HIF1α frequently occurs in human cancers.
    DOI:  https://doi.org/10.1038/s41388-021-02031-w
  19. J Cell Sci. 2021 Oct 08. pii: jcs.258856. [Epub ahead of print]
    A preferred sequence for organelle inheritance during polarized cell growth.
    Kathryn W Li, Michelle S Lu, Yuichiro Iwamoto, David G Drubin, Ross T A Pedersen.
      Some organelles cannot be synthesized anew, so they are segregated into daughter cells during cell division. In Saccharomyces cerevisiae, daughter cells bud from mother cells and are populated by organelles inherited from the mothers. To determine whether this organelle inheritance occurs in a stereotyped manner, we tracked organelles using fluorescence microscopy. We describe a program for organelle inheritance in budding yeast. The cortical endoplasmic reticulum (ER) and peroxisomes are inherited concomitant with bud emergence. Next, vacuoles are inherited in small buds, followed closely by mitochondria. Finally, the nucleus and perinuclear ER are inherited when buds have nearly reached their maximal size. Because organelle inheritance timing correlates with bud morphology, which is coupled to the cell cycle, we tested whether disrupting the cell cycle alters organelle inheritance order. By arresting cell cycle progression but allowing continued bud growth, we determined that organelle inheritance still occurs when DNA replication is blocked, and that the general inheritance order is maintained. Thus, organelle inheritance follows a preferred order during polarized cell division and does not require completion of S-phase.
    Keywords:  Mitosis; Organelles; Polarity
    DOI:  https://doi.org/10.1242/jcs.258856
  20. FEBS J. 2021 Oct 04.
    DDX3 interacts with USP9X and participates in deubiquitination of the anti-apoptotic protein MCL1.
    Ming-Chih Lai, Yi-Pin Chen, Ding-An Li, Jau-Song Yu, Hsin-Yuan Hung, Woan-Yuh Tarn.
      Here, we describe a novel interaction between the RNA helicase DDX3 and the deubiquitinase USP9X in human cells. Domain mapping studies reveal that the C-terminal region of DDX3 interacted with the N-terminus of USP9X. USP9X was predominantly localized in the cytoplasm where the interaction between DDX3 and USP9X occurred. USP9X was not visibly enriched in cytoplasmic stress granules (SGs) under oxidative stress conditions, whereas overexpression of GFP-DDX3 induced SG formation and recruited USP9X to SGs in HeLa cells. Luciferase reporter assays showed that depletion of USP9X had no significant effect on DDX3-mediated translation. Given that DDX3 is not ubiquitinated upon ubiquitin overexpression, it is unlikely that DDX3 serves as a substrate of USP9X. Importantly, we found that ubiquitinated MCL1 was accumulated upon depletion of USP9X and/or DDX3 in MG132-treated cells, suggesting that USP9X and DDX3 play a role in regulating MCL1 protein stability and anti-apoptotic function. This study indicates that DDX3 exerts anti-apoptotic effects probably by coordinating with USP9X in promoting MCL1 deubiquitination.
    Keywords:  DDX3; MCL1; USP9X; anti-apoptosis; deubiquitination
    DOI:  https://doi.org/10.1111/febs.16219
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