bims-proteo Biomed News
on Proteostasis
Issue of 2022–10–30
25 papers selected by
Eric Chevet, INSERM
  1. ATP13A1 prevents ERAD of folding-competent mislocalized and misoriented proteins.
  2. A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation.
  3. LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles.
  4. The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection.
  5. Digest it all: the lysosomal turnover of cytoplasmic aggregates.
  6. An expanded lexicon for the ubiquitin code.
  7. Termination of the unfolded protein response is guided by ER stress-induced HAC1 mRNA nuclear retention.
  8. The mechanisms and roles of selective autophagy in mammals.
  9. A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.
  10. Phospholipid imbalance impairs autophagosome completion.
  11. PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805.
  12. USP7 substrates identified by proteomics analysis reveal the specificity of USP7.
  13. The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.
  14. PROTAC-DB 2.0: an updated database of PROTACs.
  15. Yeast Smy2 and its human homologs GIGYF1 and -2 regulate Cdc48/VCP function during transcription stress.
  16. Small-molecule inhibition of the archetypal UbiB protein COQ8.
  17. Atypical nuclear envelope condensates linked to neurological disorders reveal nucleoporin-directed chaperone activities.
  18. HOIP modulates the stability of GPx4 by linear ubiquitination.
  19. Autophagic perturbation caused by reduced lysosomal activity positively regulates cell competition.
  20. UBE2O promotes lipid metabolic reprogramming and liver cancer progression by mediating HADHA ubiquitination.
  21. Calreticulin surface presentation can promote quality control of hematopoietic stem cells.
  22. Formation of toxic oligomers of polyQ-expanded Huntingtin by prion-mediated cross-seeding.
  23. Many roads lead to CASM: Diverse stimuli of noncanonical autophagy share a unifying molecular mechanism.
  24. Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.
  25. PROTACting the kinome with covalent warheads.
  1. Mol Cell. 2022 Oct 15. pii: S1097-2765(22)00956-X. [Epub ahead of print]
    ATP13A1 prevents ERAD of folding-competent mislocalized and misoriented proteins.
    Michael J McKenna, Benjamin M Adams, Vincent Chu, Joao A Paulo, Sichen Shao.
      The biosynthesis of thousands of proteins requires targeting a signal sequence or transmembrane segment (TM) to the endoplasmic reticulum (ER). These hydrophobic ɑ helices must localize to the appropriate cellular membrane and integrate in the correct topology to maintain a high-fidelity proteome. Here, we show that the P5A-ATPase ATP13A1 prevents the accumulation of mislocalized and misoriented proteins, which are eliminated by different ER-associated degradation (ERAD) pathways in mammalian cells. Without ATP13A1, mitochondrial tail-anchored proteins mislocalize to the ER through the ER membrane protein complex and are cleaved by signal peptide peptidase for ERAD. ATP13A1 also facilitates the topogenesis of a subset of proteins with an N-terminal TM or signal sequence that should insert into the ER membrane with a cytosolic N terminus. Without ATP13A1, such proteins accumulate in the wrong orientation and are targeted for ERAD by distinct ubiquitin ligases. Thus, ATP13A1 prevents ERAD of diverse proteins capable of proper folding.
    Keywords:  ER-associated degradation; protein localization; protein topology; quality control; signal sequence; transmembrane proteins
    DOI:  https://doi.org/10.1016/j.molcel.2022.09.035
  2. Nat Commun. 2022 Oct 27. 13(1): 6411
    A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation.
    Momoko Narita, Timo Denk, Yoshitaka Matsuo, Takato Sugiyama, Chisato Kikuguchi, Sota Ito, Nichika Sato, Toru Suzuki, Satoshi Hashimoto, Iva Machová, Petr Tesina, Roland Beckmann, Toshifumi Inada.
      Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.
    DOI:  https://doi.org/10.1038/s41467-022-34097-9
  3. Autophagy. 2022 Oct 26.
    LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles.
    Jackson O Gardner, Andrew M Leidal, Tan A Nguyen, Jayanta Debnath.
      LC3-dependent EV loading and secretion (LDELS) is a secretory autophagy pathway in which the macroautophagy/autophagy machinery facilitates the packaging of cytosolic cargos, such as RNA-binding proteins, into extracellular vesicles (EVs) for secretion outside of the cell. Here, we identify TFRC (transferrin receptor), one of the first proteins found to be secreted via EVs, as a transmembrane cargo of the LDELS pathway. Similar to other LDELS targets, TFRC secretion via EVs genetically requires components of the MAP1LC3/LC3-conjugation machinery but is independent of other ATGs involved in classical autophagosome formation. Furthermore, the packaging and secretion of this transmembrane protein into EVs depends on multiple ESCRT pathway components and the small GTPase RAB27A. Based on these results, we propose that the LDELS pathway promotes TFRC incorporation into EVs and its secretion outside the cell.
    Keywords:  ATG7; ATG8; LC3-conjugation; extracellular vesicles; secretory autophagy; transferrin receptor
    DOI:  https://doi.org/10.1080/15548627.2022.2140557
  4. EMBO J. 2022 Oct 25. e108970
    The E3 ubiquitin ligase RNF115 regulates phagosome maturation and host response to bacterial infection.
    Orsolya Bilkei-Gorzo, Tiaan Heunis, José Luis Marín-Rubio, Francesca Romana Cianfanelli, Benjamin Bernard Armando Raymond, Joseph Inns, Daniela Fabrikova, Julien Peltier, Fiona Oakley, Ralf Schmid, Anetta Härtlova, Matthias Trost.
      Phagocytosis is a key process in innate immunity and homeostasis. After particle uptake, newly formed phagosomes mature by acquisition of endolysosomal enzymes. Macrophage activation by interferon gamma (IFN-γ) increases microbicidal activity, but delays phagosomal maturation by an unknown mechanism. Using quantitative proteomics, we show that phagosomal proteins harbour high levels of typical and atypical ubiquitin chain types. Moreover, phagosomal ubiquitylation of vesicle trafficking proteins is substantially enhanced upon IFN-γ activation of macrophages, suggesting a role in regulating phagosomal functions. We identified the E3 ubiquitin ligase RNF115, which is enriched on phagosomes of IFN-γ activated macrophages, as an important regulator of phagosomal maturation. Loss of RNF115 protein or ligase activity enhanced phagosomal maturation and increased cytokine responses to bacterial infection, suggesting that both innate immune signalling from the phagosome and phagolysosomal trafficking are controlled through ubiquitylation. RNF115 knock-out mice show less tissue damage in response to S. aureus infection, indicating a role of RNF115 in inflammatory responses in vivo. In conclusion, RNF115 and phagosomal ubiquitylation are important regulators of innate immune functions during bacterial infections.
    Keywords:  E3 ligase; RNF115; macrophage; phagosome; ubiquitin
    DOI:  https://doi.org/10.15252/embj.2021108970
  5. Trends Biochem Sci. 2022 Oct 21. pii: S0968-0004(22)00271-7. [Epub ahead of print]
    Digest it all: the lysosomal turnover of cytoplasmic aggregates.
    Mario Mauthe, Harm H Kampinga, Mark S Hipp, Fulvio Reggiori.
      Aggrephagy describes the selective lysosomal transport and turnover of cytoplasmic protein aggregates by macro-autophagy. In this process, protein aggregates and conglomerates are polyubiquitinated and then sequestered by autophagosomes. Soluble selective autophagy receptors (SARs) are central to aggrephagy and physically bind to both ubiquitin and the autophagy machinery, thus linking the cargo to the forming autophagosomal membrane. Because the accumulation of protein aggregates is associated with cytotoxicity in several diseases, a better molecular understanding of aggrephagy might provide a conceptual framework to develop therapeutic strategies aimed at delaying the onset of these pathologies by preventing the buildup of potentially toxic aggregates. We review recent advances in our knowledge about the mechanism of aggrephagy.
    Keywords:  cellular protein quality control; chaperone-mediated autophagy; macro-autophagy; micro-autophagy; p62 bodies; selective autophagy receptors
    DOI:  https://doi.org/10.1016/j.tibs.2022.09.012
  6. Nat Rev Mol Cell Biol. 2022 Oct 25.
    An expanded lexicon for the ubiquitin code.
    Ivan Dikic, Brenda A Schulman.
      Our understanding of the ubiquitin code has greatly evolved from conventional E1, E2 and E3 enzymes that modify Lys residues on specific substrates with a single type of ubiquitin chain to more complex processes that regulate and mediate ubiquitylation. In this Review, we discuss recently discovered endogenous mechanisms and unprecedented pathways by which pathogens rewrite the ubiquitin code to promote infection. These processes include unconventional ubiquitin modifications involving ester linkages with proteins, lipids and sugars, or ubiquitylation through a phosphoribosyl bridge involving Arg42 of ubiquitin. We also introduce the enzymatic pathways that write and reverse these modifications, such as the papain-like proteases of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Furthermore, structural studies have revealed that the ultimate functions of ubiquitin are mediated not simply by straightforward recognition by ubiquitin-binding domains. Instead, elaborate multivalent interactions between ubiquitylated targets or ubiquitin chains and their readers (for example, the proteasome, the MLL1 complex or DOT1L) can elicit conformational changes that regulate protein degradation or transcription. The newly discovered mechanisms provide opportunities for innovative therapeutic interventions for diseases such as cancer and infectious diseases.
    DOI:  https://doi.org/10.1038/s41580-022-00543-1
  7. Nat Commun. 2022 Oct 25. 13(1): 6331
    Termination of the unfolded protein response is guided by ER stress-induced HAC1 mRNA nuclear retention.
    Laura Matabishi-Bibi, Drice Challal, Mara Barucco, Domenico Libri, Anna Babour.
      Cellular homeostasis is maintained by surveillance mechanisms that intervene at virtually every step of gene expression. In the nucleus, the yeast chromatin remodeler Isw1 holds back maturing mRNA ribonucleoparticles to prevent their untimely export, but whether this activity operates beyond quality control of mRNA biogenesis to regulate gene expression is unknown. Here, we identify the mRNA encoding the central effector of the unfolded protein response (UPR) HAC1, as an Isw1 RNA target. The direct binding of Isw1 to the 3' untranslated region of HAC1 mRNA restricts its nuclear export and is required for accurate UPR abatement. Accordingly, ISW1 inactivation sensitizes cells to endoplasmic reticulum (ER) stress while its overexpression reduces UPR induction. Our results reveal an unsuspected mechanism, in which binding of ER-stress induced Isw1 to HAC1 mRNA limits its nuclear export, providing a feedback loop that fine-tunes UPR attenuation to guarantee homeostatic adaptation to ER stress.
    DOI:  https://doi.org/10.1038/s41467-022-34133-8
  8. Nat Rev Mol Cell Biol. 2022 Oct 27.
    The mechanisms and roles of selective autophagy in mammals.
    Jose Norberto S Vargas, Maho Hamasaki, Tsuyoshi Kawabata, Richard J Youle, Tamotsu Yoshimori.
      Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.
    DOI:  https://doi.org/10.1038/s41580-022-00542-2
  9. J Biol Chem. 2022 Oct 20. pii: S0021-9258(22)01072-9. [Epub ahead of print] 102629
    A cell-based chemical-genetic screen for amino acid stress response inhibitors reveals torins reverse stress kinase GCN2 signaling.
    Johanna B Brüggenthies, Alessandra Fiore, Marion Russier, Christina Bitsina, Julian Brötzmann, Susanne Kordes, Sascha Menninger, Alexander Wolf, Elena Conti, Jan E Eickhoff, Peter J Murray.
      mTORC1 and GCN2 are serine/threonine kinases that control how cells adapt to amino acid availability. mTORC1 responds to amino acids to promote translation and cell growth while GCN2 senses limiting amino acids to hinder translation via eIF2α phosphorylation. GCN2 is an appealing target for cancer therapies because malignant cells can harness the GCN2 pathway to temper the rate of translation during rapid amino acid consumption. To isolate new GCN2 inhibitors, we created cell-based, amino acid limitation reporters via genetic manipulation of Ddit3 (encoding the transcription factor CHOP). CHOP is strongly induced by limiting amino acids and in this context, GCN2-dependent. Using leucine starvation as a model for essential amino acid sensing, we unexpectedly discovered ATP-competitive PI3 kinase-related kinase inhibitors, including ATR and mTOR inhibitors like torins, completely reversed GCN2 activation in a time-dependent way. Mechanistically, via inhibiting mTORC1-dependent translation, torins increased intracellular leucine, which was sufficient to reverse GCN2 activation and the downstream integrated stress response including stress-induced transcriptional factor ATF4 expression. Strikingly, we found that general translation inhibitors mirrored the effects of torins. Therefore, we propose that mTOR kinase inhibitors concurrently inhibit different branches of amino acid sensing by a dual mechanism involving direct inhibition of mTOR and indirect suppression of GCN2 that are connected by effects on the translation machinery. Collectively, our results highlight distinct ways of regulating GCN2 activity.
    Keywords:  GCN2; amino acid starvation; integrated stress response; mTORC1; torins
    DOI:  https://doi.org/10.1016/j.jbc.2022.102629
  10. EMBO J. 2022 Oct 27. e110771
    Phospholipid imbalance impairs autophagosome completion.
    Alexandra Polyansky, Oren Shatz, Milana Fraiberg, Eyal Shimoni, Tali Dadosh, Muriel Mari, Fulvio M Reggiori, Chao Qin, Xianlin Han, Zvulun Elazar.
      Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.
    Keywords:  autophagosome biogenesis; autophagy; phagophore; phospholipids
    DOI:  https://doi.org/10.15252/embj.2022110771
  11. Autophagy. 2022 Oct 27.
    PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805.
    Yunpeng Bai, Guimei Yu, Hong-Ming Zhou, Ovini Amarasinghe, Yuan Zhou, Peipei Zhu, Qinglin Li, Lujuan Zhang, Frederick Nguele Meke, Yiming Miao, Eli Chapman, W Andy Tao, Zhong-Yin Zhang.
      Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about their physiological substrates. VCP is implicated in lysophagy via collaboration with specific cofactors in the ELDR complex. However, how the ELDR complex assembly is regulated has not been determined. Moreover, the functional significance of the penultimate and conserved Tyr805 phosphorylation in VCP has not been established. Here, we use an unbiased substrate trapping and mass spectrometry approach and identify VCP/p97 as a bona fide substrate of PTP4A2. Biochemical studies show that PTP4A2 dephosphorylates VCP at Tyr805, enabling the association of VCP with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy, the autophagic clearance of damaged lysosomes. Functionally, PTP4A2 is required for cellular homeostasis by promoting lysophagy through facilitating ELDR-mediated K48-linked ubiquitin conjugate removal and autophagosome formation on the damaged lysosomes. Deletion of Ptp4a2 in vivo compromises the recovery of glycerol-injection induced acute kidney injury due to impaired lysophagy and sustained lysosomal damage. Taken together, our data establish PTP4A2 as a critical regulator of VCP and uncover an important role for PTP4A2 in maintaining lysosomal homeostasis through dephosphorylation of VCP at Tyr805. Our study suggests that PTP4A2 targeting could be a potential therapeutic approach to treat cancers and other degenerative diseases by modulating lysosomal homeostasis and macroautophagy/autophagy.
    Keywords:  ELDR complex; PLAA; PRL phosphatase; PTP4A2; UBXN6; VCP; autophagy; dephosphorylation; lysosome
    DOI:  https://doi.org/10.1080/15548627.2022.2140558
  12. Genes Dev. 2022 Oct 27.
    USP7 substrates identified by proteomics analysis reveal the specificity of USP7.
    Litong Nie, Chao Wang, Xiaoguang Liu, Hongqi Teng, Siting Li, Min Huang, Xu Feng, Guangsheng Pei, Qinglei Hang, Zhongming Zhao, Boyi Gan, Li Ma, Junjie Chen.
      Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.
    Keywords:  MGA; USP7; proteomics; ubiquitination
    DOI:  https://doi.org/10.1101/gad.349848.122
  13. Nat Cell Biol. 2022 Oct 27.
    The chaperone DNAJB6 surveils FG-nucleoporins and is required for interphase nuclear pore complex biogenesis.
    E F Elsiena Kuiper, Paola Gallardo, Tessa Bergsma, Muriel Mari, Maiara Kolbe Musskopf, Jeroen Kuipers, Ben N G Giepmans, Anton Steen, Harm H Kampinga, Liesbeth M Veenhoff, Steven Bergink.
      Biogenesis of nuclear pore complexes (NPCs) includes the formation of the permeability barrier composed of phenylalanine-glycine-rich nucleoporins (FG-Nups) that regulate the selective passage of biomolecules across the nuclear envelope. The FG-Nups are intrinsically disordered and prone to liquid-liquid phase separation and aggregation when isolated. How FG-Nups are protected from making inappropriate interactions during NPC biogenesis is not fully understood. Here we find that DNAJB6, a molecular chaperone of the heat shock protein network, forms foci in close proximity to NPCs. The number of these foci decreases upon removal of proteins involved in the early steps of interphase NPC biogenesis. Conversely, when this process is stalled in the last steps, the number of DNAJB6-containing foci increases and these foci are identified as herniations at the nuclear envelope. Immunoelectron tomography shows that DNAJB6 localizes inside the lumen of the herniations arising at NPC biogenesis intermediates. Loss of DNAJB6 results in the accumulation of cytosolic annulate lamellae, which are structures containing partly assembled NPCs, a feature associated with disturbances in NPC biogenesis. We find that DNAJB6 binds to FG-Nups and can prevent the aggregation of the FG region of several FG-Nups in cells and in vitro. Together, our data show that the molecular chaperone DNAJB6 provides quality control during NPC biogenesis and is involved in the surveillance of native intrinsically disordered FG-Nups.
    DOI:  https://doi.org/10.1038/s41556-022-01010-x
  14. Nucleic Acids Res. 2022 Oct 27. pii: gkac946. [Epub ahead of print]
    PROTAC-DB 2.0: an updated database of PROTACs.
    Gaoqi Weng, Xuanyan Cai, Dongsheng Cao, Hongyan Du, Chao Shen, Yafeng Deng, Qiaojun He, Bo Yang, Dan Li, Tingjun Hou.
      Proteolysis targeting chimeras (PROTACs), which harness the ubiquitin-proteasome system to selectively induce targeted protein degradation, represent an emerging therapeutic technology with the potential to modulate traditional undruggable targets. Over the past few years, this technology has moved from academia to industry and more than 10 PROTACs have been advanced into clinical trials. However, designing potent PROTACs with desirable drug-like properties still remains a great challenge. Here, we report an updated online database, PROTAC-DB 2.0, which is a repository of structural and experimental data about PROTACs. In this 2nd release, we expanded the number of PROTACs to 3270, which corresponds to a 96% expansion over the first version. Meanwhile, the numbers of warheads (small molecules targeting the proteins of interest), linkers, and E3 ligands (small molecules recruiting E3 ligases) have increased to over 360, 1500 and 80, respectively. In addition, given the importance and the limited number of the crystal target-PROTAC-E3 ternary complex structures, we provide the predicted ternary complex structures for PROTACs with good degradation capability using our PROTAC-Model method. To further facilitate the analysis of PROTAC data, a new filtering strategy based on the E3 ligases is also added. PROTAC-DB 2.0 is available online at http://cadd.zju.edu.cn/protacdb/.
    DOI:  https://doi.org/10.1093/nar/gkac946
  15. Cell Rep. 2022 Oct 25. pii: S2211-1247(22)01392-4. [Epub ahead of print]41(4): 111536
    Yeast Smy2 and its human homologs GIGYF1 and -2 regulate Cdc48/VCP function during transcription stress.
    Michelle Harreman Lehner, Jane Walker, Kotryna Temcinaite, Anna Herlihy, Michael Taschner, Adam C Berger, Anita H Corbett, A Barbara Dirac Svejstrup, Jesper Q Svejstrup.
      The "last resort" pathway results in ubiquitylation and degradation of RNA polymerase II in response to transcription stress and is governed by factors such as Def1 in yeast. Here, we show that the SMY2 gene acts as a multi-copy suppressor of DEF1 deletion and functions at multiple steps of the last resort pathway. We also provide genetic and biochemical evidence from disparate cellular processes that Smy2 works more broadly as a hitherto overlooked regulator of Cdc48 function. Similarly, the Smy2 homologs GIGYF1 and -2 affect the transcription stress response in human cells and regulate the function of the Cdc48 homolog VCP/p97, presently being explored as a target for cancer therapy. Indeed, we show that the apoptosis-inducing effect of VCP inhibitors NMS-873 and CB-5083 is GIGYF1/2 dependent.
    Keywords:  CP: Molecular biology; DEF1; GIGYF1; GIGYF2; RNA polymerase II; SMY2; VCP; cdc48; last resort pathway; p97; proteasome
    DOI:  https://doi.org/10.1016/j.celrep.2022.111536
  16. Nat Chem Biol. 2022 Oct 27.
    Small-molecule inhibition of the archetypal UbiB protein COQ8.
    Nathan H Murray, Christopher R M Asquith, Zixiang Fang, Michael P East, Naomi Ptak, Robert W Smith, James D Vasta, Chad A Zimprich, Cesear R Corona, Matthew B Robers, Gary L Johnson, Craig A Bingman, David J Pagliarini.
      Small-molecule tools have enabled mechanistic investigations and therapeutic targeting of the protein kinase-like (PKL) superfamily. However, such tools are still lacking for many PKL members, including the highly conserved and disease-related UbiB family. Here, we sought to develop and characterize an inhibitor for the archetypal UbiB member COQ8, whose function is essential for coenzyme Q (CoQ) biosynthesis. Guided by crystallography, activity assays and cellular CoQ measurements, we repurposed the 4-anilinoquinoline scaffold to selectively inhibit human COQ8A in cells. Our chemical tool promises to lend mechanistic insights into the activities of these widespread and understudied proteins and to offer potential therapeutic strategies for human diseases connected to their dysfunction.
    DOI:  https://doi.org/10.1038/s41589-022-01168-3
  17. Nat Cell Biol. 2022 Oct 27.
    Atypical nuclear envelope condensates linked to neurological disorders reveal nucleoporin-directed chaperone activities.
    Sarah M Prophet, Anthony J Rampello, Robert F Niescier, Juliana E Gentile, Sunanda Mallik, Anthony J Koleske, Christian Schlieker.
      DYT1 dystonia is a debilitating neurological movement disorder arising from mutation in the AAA+ ATPase TorsinA. The hallmark of Torsin dysfunction is nuclear envelope blebbing resulting from defects in nuclear pore complex biogenesis. Whether blebs actively contribute to disease manifestation is unknown. We report that FG-nucleoporins in the bleb lumen form aberrant condensates and contribute to DYT1 dystonia by provoking two proteotoxic insults. Short-lived ubiquitylated proteins that are normally rapidly degraded partition into the bleb lumen and become stabilized. In addition, blebs selectively sequester a specific HSP40-HSP70 chaperone network that is modulated by the bleb component MLF2. MLF2 suppresses the ectopic accumulation of FG-nucleoporins and modulates the selective properties and size of condensates in vitro. Our study identifies dual mechanisms of proteotoxicity in the context of condensate formation and establishes FG-nucleoporin-directed activities for a nuclear chaperone network.
    DOI:  https://doi.org/10.1038/s41556-022-01001-y
  18. Proc Natl Acad Sci U S A. 2022 Nov;119(44): e2214227119
    HOIP modulates the stability of GPx4 by linear ubiquitination.
    Kangyun Dong, Ran Wei, Taijie Jin, Mengmeng Zhang, Jiali Shen, Huaijiang Xiang, Bing Shan, Junying Yuan, Ying Li.
      LUBAC-mediated linear ubiquitination plays a pivotal role in regulation of cell death and inflammatory pathways. Genetic deficiency in LUBAC components leads to severe immune dysfunction or embryonic lethality. LUBAC has been extensively studied for its role in mediating TNF signaling. However, Tnfr1 knockout is not able to fully rescue the embryonic lethality of LUBAC deficiency, suggesting that LUBAC may modify additional key cellular substrates in promoting cell survival. GPx4 is an important selenoprotein involved in regulating cellular redox homeostasis in defense against lipid peroxidation-mediated cell death known as ferroptosis. Here we demonstrate that LUBAC deficiency sensitizes to ferroptosis by promoting GPx4 degradation and downstream lipid peroxidation. LUBAC binds and stabilizes GPx4 by modulating its linear ubiquitination both in normal condition and under oxidative stress. Our findings identify GPx4 as a key substrate of LUBAC and a previously unrecognized role of LUBAC-mediated linear ubiquitination in regulating cellular redox status and cell death.
    Keywords:  GPx4; LUBAC; TNF; ferroptosis
    DOI:  https://doi.org/10.1073/pnas.2214227119
  19. Autophagy. 2022 Oct 26.
    Autophagic perturbation caused by reduced lysosomal activity positively regulates cell competition.
    Eilma Akter, Shunsuke Kon.
      Newly emerging transformed epithelial cells are recognized and apically removed by surrounding normal cells through a biological event termed "cell competition". However, little is known about the mechanisms underlying this process. In a recent study, we describe that RASG12V/RasV12-transformed cells surrounded by normal cells exhibit decreased lysosomal activity accompanied with accumulation of autophagosomes. Restoration of low lysosomal activity or inhibition of autophagosome formation significantly antagonizes apical extrusion of RASG12V cells, suggesting that non-degradable autophagosomes are required for cell competition. Notably, analysis of a cell competition mouse model demonstrates that macroautophagy/autophagy-ablated RASG12V cells are less readily eliminated by cell competition, and remaining transformed cells destroy ductal integrity, leading to chronic pancreatitis. Thus, our findings illuminate a critical role for non-degradable autophagosomes in cell competition and reveal a homeostasis-preserving role of autophagy upon emergence of transformed cells.
    Keywords:  cell competition; hindered autophagic flux; lysosomal dysfunction; non-degradable autophagosomes; pancreatic cancer
    DOI:  https://doi.org/10.1080/15548627.2022.2140559
  20. Oncogene. 2022 Oct 22.
    UBE2O promotes lipid metabolic reprogramming and liver cancer progression by mediating HADHA ubiquitination.
    Meilin Ma, Changhui Zhang, Rong Cao, Dongmei Tang, Xiongbo Sang, Sailan Zou, Xiuxuan Wang, Haixia Xu, Geng Liu, Lunzhi Dai, Yan Tian, Xiang Gao, Xianghui Fu.
      Cancer cells rely on heightened protein quality control mechanisms, including the ubiquitin-proteosome system that is predominantly driven by ubiquitination comprising E1, E2, and E3 trienzyme cascades. Although E3s have been extensively studied, the implication of E2s in tumorigenesis is poorly defined. Here we reveal a critical E2 in the pathogenesis of hepatocellular carcinoma (HCC). Among all of E2s, UBE2O shows the strongest association with HCC survival prognosis, and its expression is increased in HCC tumors. Accordingly, UBE2O deficiency inhibits HCC growth and metastasis both in vitro and in vivo, while its overexpression has opposite effects. Depending on both E2 and E3 enzymatic activities, UBE2O can interact with and mediate the ubiquitination and degradation of HADHA, a mitochondrial β-oxidation enzyme, thereby modulating lipid metabolic reprogramming. HADHA is reduced in HCC tumors and inversely correlated with UBE2O levels. Importantly, HADHA acts as a tumor suppressor and primarily mediates UBE2O's function on HCC. Moreover, liver-specific deletion of Ube2o in mice are resistant to DEN-induced hepatocarcinogenesis, along with HADHA upregulation and reduced hepatic lipid accumulation. These data reveal UBE2O as a novel oncogenic driver for metabolic reprogramming and HCC development, highlighting the potential of targeting UBE2O/HADHA axis for HCC therapy.
    DOI:  https://doi.org/10.1038/s41388-022-02509-1
  21. Trends Immunol. 2022 Oct 25. pii: S1471-4906(22)00229-0. [Epub ahead of print]
    Calreticulin surface presentation can promote quality control of hematopoietic stem cells.
    Lorenzo Galluzzi, Guido Kroemer.
      Endoplasmic reticulum stress can stimulate calreticulin (CALR) presentation on the cell surface, promoting the phagocytic uptake of stressed cells by myeloid cells. Recent findings from Wattrus et al. demonstrate that zebrafish and mouse embryonic macrophages engulf CALR-exposing nascent hematopoietic stem cells to ensure the selective survival of stem cells apt for adult hematopoiesis.
    Keywords:  autophagy; cancer immunosurveillance; hematopoiesis; immunogenic cell death; sterile inflammation
    DOI:  https://doi.org/10.1016/j.it.2022.10.008
  22. Mol Cell. 2022 Oct 13. pii: S1097-2765(22)00952-2. [Epub ahead of print]
    Formation of toxic oligomers of polyQ-expanded Huntingtin by prion-mediated cross-seeding.
    Michael H M Gropp, Courtney L Klaips, F Ulrich Hartl.
      Manifestation of aggregate pathology in Huntington's disease is thought to be facilitated by a preferential vulnerability of affected brain cells to age-dependent proteostatic decline. To understand how specific cellular backgrounds may facilitate pathologic aggregation, we utilized the yeast model in which polyQ-expanded Huntingtin forms aggregates only when the endogenous prion-forming protein Rnq1 is in its amyloid-like prion [PIN+] conformation. We employed optogenetic clustering of polyQ protein as an orthogonal method to induce polyQ aggregation in prion-free [pin-] cells. Optogenetic aggregation circumvented the prion requirement for the formation of detergent-resistant polyQ inclusions but bypassed the formation of toxic polyQ oligomers, which accumulated specifically in [PIN+] cells. Reconstitution of aggregation in vitro suggested that these polyQ oligomers formed through direct templating on Rnq1 prions. These findings shed light on the mechanism of prion-mediated formation of oligomers, which may play a role in triggering polyQ pathology in the patient brain.
    Keywords:  Huntington’s disease; Rnq1; cross-seeding; neurodegeneration; oligomers; optogenetics; polyQ; prion; protein aggregation; proteostasis
    DOI:  https://doi.org/10.1016/j.molcel.2022.09.031
  23. Sci Adv. 2022 Oct 28. 8(43): eabo1274
    Many roads lead to CASM: Diverse stimuli of noncanonical autophagy share a unifying molecular mechanism.
    Joanne Durgan, Oliver Florey.
      Autophagy is a fundamental catabolic process coordinated by a network of autophagy-related (ATG) proteins. These ATG proteins also perform an important parallel role in "noncanonical" autophagy, a lysosome-associated signaling pathway with key functions in immunity, inflammation, cancer, and neurodegeneration. While the noncanonical autophagy pathway shares the common ATG machinery, it bears key mechanistic and functional distinctions, and is characterized by conjugation of ATG8 to single membranes (CASM). Here, we review the diverse, and still expanding, collection of stimuli and processes now known to harness the noncanonical autophagy pathway, including engulfment processes, drug treatments, TRPML1 and STING signaling, viral infection, and other pathogenic factors. We discuss the multiple associated routes to CASM and assess their shared and distinctive molecular features. By integrating these findings, we propose an updated and unifying mechanism for noncanonical autophagy, centered on ATG16L1 and V-ATPase.
    DOI:  https://doi.org/10.1126/sciadv.abo1274
  24. Sci Adv. 2022 Oct 28. 8(43): eabo1304
    Ribosome changes reprogram translation for chemosurvival in G0 leukemic cells.
    Chandreyee Datta, Samuel S Truesdell, Keith Q Wu, Syed I A Bukhari, Harrison Ngue, Brienna Buchanan, Olivier Le Tonqueze, Sooncheol Lee, Swapna Kollu, Madeleine A Granovetter, Myriam Boukhali, Johannes Kreuzer, Maheen S Batool, Leonora Balaj, Wilhelm Haas, Shobha Vasudevan.
      Quiescent leukemic cells survive chemotherapy, with translation changes. Our data reveal that FXR1, a protein amplified in several aggressive cancers, is elevated in quiescent and chemo-treated leukemic cells and promotes chemosurvival. This suggests undiscovered roles for this RNA- and ribosome-associated protein in chemosurvival. We find that FXR1 depletion reduces translation, with altered rRNAs, snoRNAs, and ribosomal proteins (RPs). FXR1 regulates factors that promote transcription and processing of ribosomal genes and snoRNAs. Ribosome changes in FXR1-overexpressing cells, including RPLP0/uL10 levels, activate eIF2α kinases. Accordingly, phospho-eIF2α increases, enabling selective translation of survival and immune regulators in FXR1-overexpressing cells. Overriding these genes or phospho-eIF2α with inhibitors reduces chemosurvival. Thus, elevated FXR1 in quiescent or chemo-treated leukemic cells alters ribosomes that trigger stress signals to redirect translation for chemosurvival.
    DOI:  https://doi.org/10.1126/sciadv.abo1304
  25. Drug Discov Today. 2022 Oct 25. pii: S1359-6446(22)00410-X. [Epub ahead of print] 103417
    PROTACting the kinome with covalent warheads.
    Deep Rohan Chatterjee, Saumya Kapoor, Meenakshi Jain, Rudradip Das, Moumita Ghosh Chowdhury, Amit Shard.
      The dawn of targeted degradation using proteolysis-targeting chimeras (PROTACs) against recalcitrant proteins has prompted numerous efforts to develop complementary drugs. Although many of these are specifically directed against undruggable proteins, there is increasing interest in small molecule-based PROTACs that target intracellular pathways, and some have recently entered clinical trials. Concurrently, small molecule-based PROTACs that target protumorigenic pathways in cancer cells, the tumor microenvironment (TME), and angiogenesis have been found to have potent effects that synergize with the action of antibodies. This has led to the augmentation of PROTACs with variable substitution patterns. Several combinations with small molecules targeting undruggable proteins are now under clinical investigation. In this review, we discuss the recent milestones achieved as well as challenges encountered in this area of drug development, as well as our opinion on the best path forward.
    Keywords:  Covalent inhibitors; E3 ligase; Kinase; PROTACs; Targeted kinase degradation
    DOI:  https://doi.org/10.1016/j.drudis.2022.103417
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