bims-proteo Biomed News
on Proteostasis
Issue of 2023–03–05
25 papers selected by
Eric Chevet, INSERM
  1. SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum.
  2. VMP1 affects endoplasmic reticulum stress sensitivity via differential modulation of the three unfolded protein response arms.
  3. GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.
  4. K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants.
  5. CARPs regulate STUB1 and its pathogenic mutants aggregation kinetics by mono-ubiquitination.
  6. HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies.
  7. UFL1, a UFMylation E3 ligase, plays a crucial role in multiple cellular stress responses.
  8. A Novel Ubiquitin Complex Regulates Aneuploid Epithelial Tumors by Moderating an Integrated Stress Response.
  9. Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis.
  10. ATG16L1 adopts a dual-binding site mode to interact with WIPI2b in autophagy.
  11. AlphaFold-multimer predicts ATG8 protein binding motifs crucial for autophagy research.
  12. Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis.
  13. mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria.
  14. A tell tail sign: A conserved C-terminal tail-anchor domain targets a subset of pathogen effectors to the plant endoplasmic reticulum.
  15. Tumor suppressor p53 regulates heat shock factor 1 protein degradation in Huntington's disease.
  16. Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.
  17. Lysosomal control of senescence and inflammation through cholesterol partitioning.
  18. Preparation of chaperone-loaded neural stem cell-derived extracellular vesicles to reduce protein aggregation in Huntington's disease cellular models.
  19. ATF6β Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.
  20. Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.
  21. Ubiquitin-like Conjugation by Bacterial cGAS Enhances Anti-phage Defence.
  22. Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells.
  23. A covalent BTK ternary complex compatible with targeted protein degradation.
  24. A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells.
  25. Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells.
  1. Cell Rep. 2023 Jan 31. pii: S2211-1247(23)00039-6. [Epub ahead of print]42(1): 112028
    SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum.
    Lihui Wang, Yue Xu, Sijung Yun, Quan Yuan, Prasanna Satpute-Krishnan, Yihong Ye.
      Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating translocation-associated quality control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR-Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.
    Keywords:  CP: Molecular biology; Drosophila; SAYSD1; Sec61; UFM1/UFMylation; co-translational protein translocation; collagen biogenesis; ribosome stalling/translation arrest; translocation-associated quality control/TAQC; translocon clogging
    DOI:  https://doi.org/10.1016/j.celrep.2023.112028
  2. Cell Rep. 2023 Mar 03. pii: S2211-1247(23)00220-6. [Epub ahead of print]42(3): 112209
    VMP1 affects endoplasmic reticulum stress sensitivity via differential modulation of the three unfolded protein response arms.
    Tao Li, Hongyu Zhao, Gaofeng Guo, Shuwei Xia, Likun Wang.
      Consisting of three signaling pathways, the unfolded protein response (UPR) can be either protective or detrimental to cells that undergo ER stress. Elaborate regulation of the UPR is key to the cell-fate decision, but how it is achieved remains vague. Here, by studying cells deficient in vacuole membrane protein 1 (VMP1), a UPR regulator, we report a model of UPR regulation in which the three pathways are divergently controlled. Under basal conditions, calcium binding specifically activates PERK. Under ER stress, ER-mitochondria interaction-induced mitochondrial stress cooperates with PERK to suppress IRE1α and ATF6 by decelerating global protein synthesis. Such sophisticated regulation commits limited activation of the UPR yet refrains from UPR hyperactivation, protecting cells from chronic ER stress despite decreasing cell proliferation. Therefore, our study reveals interorganelle-interaction-dependent and calcium-dependent regulation of the UPR that dictates cell fate.
    Keywords:  CP: Cell biology; ER stress; ER stress resistance; ER-mitochondria contact; VMP1; calcium; integrated stress response; mitochondrial stress; unfolded protein response
    DOI:  https://doi.org/10.1016/j.celrep.2023.112209
  3. Biochim Biophys Acta Gene Regul Mech. 2023 Feb 24. pii: S1874-9399(23)00019-6. [Epub ahead of print] 194924
    GLI1, a novel target of the ER stress regulator p97/VCP, promotes ATF6f-mediated activation of XBP1.
    Luciana L Almada, Kim Barroso, Sandhya Sen, Murat Toruner, Ashley N Sigafoos, Glancis L Raja Arul, David R Pease, Renzo E Vera, Rachel L O Olson, Holger W Auner, Rémy Pedeux, Juan L Iovanna, Eric Chevet, Martin E Fernandez-Zapico.
      Upon accumulation of improperly folded proteins in the Endoplasmic Reticulum (ER), the Unfolded Protein Response (UPR) is triggered to restore ER homeostasis. The induction of stress genes is a sine qua non condition for effective adaptive UPR. Although this requirement has been extensively described, the mechanisms underlying this process remain in part uncharacterized. Here, we show that p97/VCP, an AAA+ ATPase known to contribute to ER stress-induced gene expression, regulates the transcription factor GLI1, a primary effector of Hedgehog (Hh) signaling. Under basal (non-ER stress) conditions, GLI1 is repressed by a p97/VCP-HDAC1 complex while upon ER stress GLI1 is induced through a mechanism requiring both USF2 binding and increase histone acetylation at its promoter. Interestingly, the induction of GLI1 was independent of ligand-regulated Hh signaling. Further analysis showed that GLI1 cooperates with ATF6f to induce promoter activity and expression of XBP1, a key transcription factor driving UPR. Overall, our work demonstrates a novel role for GLI1 in the regulation of ER stress gene expression and defines the interplay between p97/VCP, HDAC1 and USF2 as essential players in this process.
    Keywords:  ER stress; GLI1; HDAC1; Hedgehog; UPR; USF2; p97/VCP
    DOI:  https://doi.org/10.1016/j.bbagrm.2023.194924
  4. Curr Biol. 2023 Feb 22. pii: S0960-9822(23)00164-1. [Epub ahead of print]
    K63-linked ubiquitin chains are a global signal for endocytosis and contribute to selective autophagy in plants.
    Bushra Saeed, Florian Deligne, Carla Brillada, Kai Dünser, Franck Aniset Ditengou, Ilona Turek, Alaa Allahham, Nenad Grujic, Yasin Dagdas, Thomas Ott, Jürgen Kleine-Vehn, Grégory Vert, Marco Trujillo.
      In contrast to other eukaryotic model organisms, the closely related ubiquitin (Ub)-conjugating enzymes UBC35 and UBC36 are the main sources of K63-linked Ub chains in Arabidopsis.1 Although K63-linked chains have been associated with the regulation of vesicle trafficking, definitive proof for their role in endocytosis was missing. We show that the ubc35 ubc36 mutant has pleiotropic phenotypes related to hormone and immune signaling. Specifically, we reveal that ubc35-1 ubc36-1 plants have altered turnover of integral membrane proteins including FLS2, BRI1, and PIN1 at the plasma membrane. Our data indicates that K63-Ub chains are generally required for endocytic trafficking in plants. In addition, we show that in plants K63-Ub chains are involved in selective autophagy through NBR1, the second major pathway delivering cargoes to the vacuole for degradation. Similar to autophagy-defective mutants, ubc35-1 ubc36-1 plants display an accumulation of autophagy markers. Moreover, autophagy receptor NBR1 interacts with K63-Ub chains, which are required for its delivery to the lytic vacuole.2 Together, we show that K63-Ub chains act as a general signal required for the two main pathways delivering cargo to the vacuole and thus, to maintain proteostasis.
    Keywords:  E2; endocytosis; signaling; ubiquitin; ubiquitin-conjugating enzymes; vesicle trafficking
    DOI:  https://doi.org/10.1016/j.cub.2023.02.024
  5. FEBS J. 2023 Feb 28.
    CARPs regulate STUB1 and its pathogenic mutants aggregation kinetics by mono-ubiquitination.
    Rahul Sharma, Prema Mondal, Srinivasa M Srinivasula.
      The development of neurological pathologies is linked to the accumulation of protein aggregates like alpha-synuclein in Parkinson's disease and tau protein in Alzheimer's disease. Mono or di ubiquitination of these molecules has been reported to stabilize aggregates and contribute to the disorders. STUB1(STIP1 Homologous and U-Box containing protein 1) is a multifunctional protein that maintains proteostasis and insulin signaling. In spinocerebellar Ataxia 16 (SCA16), an autosomal recessive neurodegenerative disease, mutations in and aggregation of STUB1 are reported. Despite the well-accepted neuroprotective role for STUB1, very little is known of regulatory mechanisms that control the dynamics of STUB1 aggregate assembly. Here we report that CARP2, a ubiquitin ligase, is a novel regulator of STUB1. CARP2 interacts and mono-ubiquitinates STUB1. Furthermore, we found that CARP2 regulates STUB1 through its TPR motif, a domain that is also associated with HSP70. Modification of STUB1 by CARP2 leads to detergent-insoluble aggregate formation. Importantly, pathogenic mutants of STUB1 are more prone than the wild type to CARP2-mediate aggregate assembly. Hence our findings revealed CARPs as novel regulators of STUB1 and control its cytosolic vs. aggregate dynamics.
    Keywords:  Protein quality control; protein aggregates; spinocerebellar ataxia; ubiquitination
    DOI:  https://doi.org/10.1111/febs.16766
  6. Autophagy. 2023 Feb 28. 1-23
    HSPB8 frameshift mutant aggregates weaken chaperone-assisted selective autophagy in neuromyopathies.
    Barbara Tedesco, Leen Vendredy, Elias Adriaenssens, Marta Cozzi, Bob Asselbergh, Valeria Crippa, Riccardo Cristofani, Paola Rusmini, Veronica Ferrari, Elena Casarotto, Marta Chierichetti, Francesco Mina, Paola Pramaggiore, Mariarita Galbiati, Margherita Piccolella, Jonathan Baets, Femke Baeke, Riet De Rycke, Vincent Mouly, Tommaso Laurenzi, Ivano Eberini, Anna Vihola, Bjarne Udd, Lan Weiss, Virginia Kimonis, Vincent Timmerman, Angelo Poletti.
      Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.
    Keywords:  BAG3; CASA; HSPA; HSPB8; misfolding; myopathy; neuromuscular disorders; neuropathy; protein quality control
    DOI:  https://doi.org/10.1080/15548627.2023.2179780
  7. Front Endocrinol (Lausanne). 2023 ;14 1123124
    UFL1, a UFMylation E3 ligase, plays a crucial role in multiple cellular stress responses.
    Qiang Jiang, Yongsheng Wang, Minghui Xiang, Jiamin Hua, Tianci Zhou, Fanghui Chen, Xiaoyang Lv, Jinming Huang, Yafei Cai.
      The UFM1 conjugation system(UFMylation)is a novel type of ubiquitin-like system that plays an indispensable role in maintaining cell homeostasis under various cellular stress. Similar to ubiquitination, UFMylation consists of a three-step enzymatic reaction with E1-like enzymes ubiquitin-like modifier activating enzyme5 (UBA5), E2-like enzymes ubiquitin-fold modifier-conjugating enzyme 1(UFC1), and E3-like ligase UFM1-specific ligase 1 (UFL1). As the only identified E3 ligase, UFL1 is responsible for specific binding and modification of the substrates to mediate numerous hormone signaling pathways and endocrine regulation under different physiological or pathological stress, such as ER stress, genotoxic stress, oncogenic stress, and inflammation. Further elucidation of the UFL1 working mechanism in multiple cellular stress responses is essential for revealing the disease pathogenesis and providing novel potential therapeutic targets. In this short review, we summarize the recent advances in novel UFL1 functions and shed light on the potential challenges ahead, thus hopefully providing a better understanding of UFMylation-mediated cellular stress.
    Keywords:  ER stress; UFL1; Ufmylation modification; genotoxic stress; inflammation; oncogenic stress
    DOI:  https://doi.org/10.3389/fendo.2023.1123124
  8. Cancer Discov. 2023 Mar 01. 13(3): 535-537
    A Novel Ubiquitin Complex Regulates Aneuploid Epithelial Tumors by Moderating an Integrated Stress Response.
    Nektaria Maria Leli, Constantinos Koumenis.
       SUMMARY: Tumor fitness coessentiality gene analysis that aims to expand the repertoire of druggable targets reveals a novel ubiquitin ligase complex, the BICR6 module. Along with the other complex members (UBA6, KCMF1, and UBR4), BIRC6 selectively contributes to the survival of a subset of epithelial tumors with a high degree of aneuploidy by ubiquitinating and suppressing HRI, a component of the integrated stress response adaptive pathway. See related article by Cervia et al., p. 766 (2).
    DOI:  https://doi.org/10.1158/2159-8290.CD-22-1440
  9. Nat Commun. 2023 Feb 28. 14(1): 947
    Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis.
    David Paul, Omer Stern, Yvonne Vallis, Jatinder Dhillon, Andrew Buchanan, Harvey McMahon.
      The ability of cells to manage consequences of exogenous proteotoxicity is key to cellular homeostasis. While a plethora of well-characterised machinery aids intracellular proteostasis, mechanisms involved in the response to denaturation of extracellular proteins remain elusive. Here we show that aggregation of protein ectodomains triggers their endocytosis via a macroendocytic route, and subsequent lysosomal degradation. Using ERBB2/HER2-specific antibodies we reveal that their cross-linking ability triggers specific and fast endocytosis of the receptor, independent of clathrin and dynamin. Upon aggregation, canonical clathrin-dependent cargoes are redirected into the aggregation-dependent endocytosis (ADE) pathway. ADE is an actin-driven process, which morphologically resembles macropinocytosis. Physical and chemical stress-induced aggregation of surface proteins also triggers ADE, facilitating their degradation in the lysosome. This study pinpoints aggregation of extracellular domains as a trigger for rapid uptake and lysosomal clearance which besides its proteostatic function has potential implications for the uptake of pathological protein aggregates and antibody-based therapies.
    DOI:  https://doi.org/10.1038/s41467-023-36496-y
  10. Sci Adv. 2023 Mar;9(9): eadf0824
    ATG16L1 adopts a dual-binding site mode to interact with WIPI2b in autophagy.
    Xinyu Gong, Yingli Wang, Yubin Tang, Yaru Wang, Mingfang Zhang, Miao Li, Yuchao Zhang, Lifeng Pan.
      Macroautophagy plays crucial roles in the regulation of cellular physiology and requires de novo synthesis of double-membrane autophagosomes, which relies on a specific interaction between autophagy-related 16L1 (ATG16L1) and WD repeat domain phosphoinositide-interacting protein 2b (WIPI2b). However, the molecular mechanism governing the interaction of ATG16L1 with WIPI2b remains elusive. Here, we find that ATG16L1 has two distinct binding sites for interacting with WIPI2b, the previously reported WIPI2b-binding site (WBS1) and the previously unidentified site (WBS2). We determine the crystal structures of WIPI2b with ATG16L1 WBS1 and WBS2, respectively, and elucidate the molecular mechanism underpinning the recruitment of ATG16L1 by WIPI2b. Moreover, we uncover that ATG16L1 WBS2 and its binding mode with WIPI2b is well conserved from yeast to mammals, unlike ATG16L1 WBS1. Last, our cell-based functional assays demonstrate that both ATG16L1 WBS1 and WBS2 are required for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the key ATG16L1/WIPI2b interaction in autophagy.
    DOI:  https://doi.org/10.1126/sciadv.adf0824
  11. PLoS Biol. 2023 Feb;21(2): e3002002
    AlphaFold-multimer predicts ATG8 protein binding motifs crucial for autophagy research.
    Hallvard Lauritz Olsvik, Terje Johansen.
      In this issue of PLOS Biology, Ibrahim and colleagues demonstrate how AlphaFold-multimer, an artificial intelligence-based structure prediction tool, can be used to identify sequence motifs binding to the ATG8 family of proteins central to autophagy.
    DOI:  https://doi.org/10.1371/journal.pbio.3002002
  12. Autophagy. 2023 Feb 27. 1-17
    Non-muscle MYH10/myosin IIB recruits ESCRT-III to participate in autophagosome closure to maintain neuronal homeostasis.
    Yong-Woo Jun, Soojin Lee, Byung-Kwan Ban, Jin-A Lee, Fen-Biao Gao.
      Dysfunction of the endosomal sorting complex required for transport (ESCRT) has been linked to frontotemporal dementia (FTD) due in part to the accumulation of unsealed autophagosomes. However, the mechanisms of ESCRT-mediated membrane closure events on phagophores remain largely unknown. In this study, we found that partial knockdown of non-muscle MYH10/myosin IIB/zip rescues neurodegeneration in both Drosophila and human iPSC-derived cortical neurons expressing FTD-associated mutant CHMP2B, a subunit of ESCRT-III. We also found that MYH10 binds and recruits several autophagy receptor proteins during autophagosome formation induced by mutant CHMP2B or nutrient starvation. Moreover, MYH10 interacted with ESCRT-III to regulate phagophore closure by recruiting ESCRT-III to damaged mitochondria during PRKN/parkin-mediated mitophagy. Evidently, MYH10 is involved in the initiation of induced but not basal autophagy and also links ESCRT-III to mitophagosome sealing, revealing novel roles of MYH10 in the autophagy pathway and in ESCRT-related FTD pathogenesis.Abbreviations: ALS: amyotrophic lateral sclerosis; AP: autophagosome; Atg: autophagy-related; ESCRT: endosomal sorting complex required for transport; FTD: frontotemporal dementia.
    Keywords:  Autophagy; Drosophila; ESCRT; mitophagy; myosin II; neurodegeneration; neuron
    DOI:  https://doi.org/10.1080/15548627.2023.2169309
  13. Cell Rep. 2023 Feb 24. pii: S2211-1247(23)00151-1. [Epub ahead of print]42(3): 112140
    mRNA targeting eliminates the need for the signal recognition particle during membrane protein insertion in bacteria.
    Pinku Sarmah, Wenkang Shang, Andrea Origi, Mariya Licheva, Claudine Kraft, Maximilian Ulbrich, Elisabeth Lichtenberg, Annegret Wilde, Hans-Georg Koch.
      Signal-sequence-dependent protein targeting is essential for the spatiotemporal organization of eukaryotic and prokaryotic cells and is facilitated by dedicated protein targeting factors such as the signal recognition particle (SRP). However, targeting signals are not exclusively contained within proteins but can also be present within mRNAs. By in vivo and in vitro assays, we show that mRNA targeting is controlled by the nucleotide content and by secondary structures within mRNAs. mRNA binding to bacterial membranes occurs independently of soluble targeting factors but is dependent on the SecYEG translocon and YidC. Importantly, membrane insertion of proteins translated from membrane-bound mRNAs occurs independently of the SRP pathway, while the latter is strictly required for proteins translated from cytosolic mRNAs. In summary, our data indicate that mRNA targeting acts in parallel to the canonical SRP-dependent protein targeting and serves as an alternative strategy for safeguarding membrane protein insertion when the SRP pathway is compromised.
    Keywords:  (p)ppGpp; CP: Microbiology; FtsY; SecYEG translocon; YidC; alarmones; mRNA targeting; signal recognition particle; small membrane proteins; stringent response; translation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112140
  14. J Exp Bot. 2023 Mar 01. pii: erad075. [Epub ahead of print]
    A tell tail sign: A conserved C-terminal tail-anchor domain targets a subset of pathogen effectors to the plant endoplasmic reticulum.
    Emily Breeze, Victoria Vale, Hazel McLellan, Yann Pecrix, Laurence Godiard, Murray Grant, Lorenzo Frigerio.
      The endoplasmic reticulum (ER) is the entry point to the secretory pathway and, as such, is critical for adaptive responses to biotic stress, when the demand for de novo synthesis of immunity-related proteins and signalling components increases significantly. Successful phytopathogens have evolved an arsenal of small effector proteins which collectively reconfigure multiple host components and signalling pathways to promote virulence; a small, but important, subset of which are targeted to the endomembrane system including the ER. We identified and validated a conserved C-terminal tail-anchor motif in a set of pathogen effectors known to localize to the ER from the oomycetes Hyaloperonospora arabidopsidis and Plasmopara halstedii (downy mildew of Arabidopsis and sunflower, respectively) and used this protein topology to develop a bioinformatic pipeline to identify putative ER-localised effectors within the effectorome of the related oomycete, Phytophthora infestans, the causal agent of potato late blight. Many of the identified P. infestans tail-anchor effectors converged on ER-localised NAC transcription factors, suggesting this family is a critical host target for multiple pathogens.
    Keywords:   Phytophthora infestans ; Endomembrane; NAC with Transmembrane Motif1-like (NTL); endoplasmic reticulum (ER); oomycete effectors; tail-anchor
    DOI:  https://doi.org/10.1093/jxb/erad075
  15. Cell Rep. 2023 Mar 02. pii: S2211-1247(23)00209-7. [Epub ahead of print]42(3): 112198
    Tumor suppressor p53 regulates heat shock factor 1 protein degradation in Huntington's disease.
    Rachel H Mansky, Erin A Greguske, Dahyun Yu, Nicole Zarate, Taylor A Intihar, Wei Tsai, Taylor G Brown, Mackenzie N Thayer, Kompal Kumar, Rocio Gomez-Pastor.
      p53 and HSF1 are two major transcription factors involved in cell proliferation and apoptosis, whose dysregulation contributes to cancer and neurodegeneration. Contrary to most cancers, p53 is increased in Huntington's disease (HD) and other neurodegenerative diseases, while HSF1 is decreased. p53 and HSF1 reciprocal regulation has been shown in different contexts, but their connection in neurodegeneration remains understudied. Using cellular and animal models of HD, we show that mutant HTT stabilized p53 by abrogating the interaction between p53 and E3 ligase MDM2. Stabilized p53 promotes protein kinase CK2 alpha prime and E3 ligase FBXW7 transcription, both of which are responsible for HSF1 degradation. Consequently, p53 deletion in striatal neurons of zQ175 HD mice restores HSF1 abundance and decrease HTT aggregation and striatal pathology. Our work shows the mechanism connecting p53 stabilization with HSF1 degradation and pathophysiology in HD and sheds light on the broader molecular differences and commonalities between cancer and neurodegeneration.
    Keywords:  CP: Molecular biology; CP: Neuroscience; HSF1; Huntington’s disease; MDM2; p53; striatal pathology
    DOI:  https://doi.org/10.1016/j.celrep.2023.112198
  16. Sci Adv. 2023 Mar 03. 9(9): eadf0797
    Structural insights into TRAP association with ribosome-Sec61 complex and translocon inhibition by a CADA derivative.
    Eva Pauwels, Neesha R Shewakramani, Brent De Wijngaert, Anita Camps, Becky Provinciael, Joren Stroobants, Kai-Uwe Kalies, Enno Hartmann, Piet Maes, Kurt Vermeire, Kalyan Das.
      During cotranslational translocation, the signal peptide of a nascent chain binds Sec61 translocon to initiate protein transport through the endoplasmic reticulum (ER) membrane. Our cryo-electron microscopy structure of ribosome-Sec61 shows binding of an ordered heterotetrameric translocon-associated protein (TRAP) complex, in which TRAP-γ is anchored at two adjacent positions of 28S ribosomal RNA and interacts with ribosomal protein L38 and Sec61α/γ. Four transmembrane helices (TMHs) of TRAP-γ cluster with one C-terminal helix of each α, β, and δ subunits. The seven TMH bundle helps position a crescent-shaped trimeric TRAP-α/β/δ core in the ER lumen, facing the Sec61 channel. Further, our in vitro assay establishes the cyclotriazadisulfonamide derivative CK147 as a translocon inhibitor. A structure of ribosome-Sec61-CK147 reveals CK147 binding the channel and interacting with the plug helix from the lumenal side. The CK147 resistance mutations surround the inhibitor. These structures help in understanding the TRAP functions and provide a new Sec61 site for designing translocon inhibitors.
    DOI:  https://doi.org/10.1126/sciadv.adf0797
  17. Nat Metab. 2023 Mar 02.
    Lysosomal control of senescence and inflammation through cholesterol partitioning.
    Kyeonghwan Roh, Jeonghwan Noh, Yeonju Kim, Yeji Jang, Jaejin Kim, Haebeen Choi, Yeonghyeon Lee, Moongi Ji, Donghyun Kang, Mi-Sung Kim, Man-Jeong Paik, Jongkyeong Chung, Jin-Hong Kim, Chanhee Kang.
      Whereas cholesterol is vital for cell growth, proliferation, and remodeling, dysregulation of cholesterol metabolism is associated with multiple age-related pathologies. Here we show that senescent cells accumulate cholesterol in lysosomes to maintain the senescence-associated secretory phenotype (SASP). We find that induction of cellular senescence by diverse triggers enhances cellular cholesterol metabolism. Senescence is associated with the upregulation of the cholesterol exporter ABCA1, which is rerouted to the lysosome, where it moonlights as a cholesterol importer. Lysosomal cholesterol accumulation results in the formation of cholesterol-rich microdomains on the lysosomal limiting membrane enriched with the mammalian target of rapamycin complex 1 (mTORC1) scaffolding complex, thereby sustaining mTORC1 activity to support the SASP. We further show that pharmacological modulation of lysosomal cholesterol partitioning alters senescence-associated inflammation and in vivo senescence during osteoarthritis progression in male mice. Our study reveals a potential unifying theme for the role of cholesterol in the aging process through the regulation of senescence-associated inflammation.
    DOI:  https://doi.org/10.1038/s42255-023-00747-5
  18. STAR Protoc. 2023 Feb 24. pii: S2666-1667(23)00092-8. [Epub ahead of print]4(1): 102134
    Preparation of chaperone-loaded neural stem cell-derived extracellular vesicles to reduce protein aggregation in Huntington's disease cellular models.
    Bhagyashree S Joshi, Inge S Zuhorn.
      Here, we present a protocol using genetic engineering techniques to prepare small extracellular vesicles (sEVs) enriched in the chaperone protein DNAJB6. We describe steps to prepare cell lines overexpressing DNAJB6, followed by the isolation and characterization of sEVs from cell conditioned media. Further, we describe assays to examine effects of DNAJB6-loaded sEVs on protein aggregation in Huntington's disease cellular models. The protocol can be readily repurposed to study protein aggregation in other neurodegenerative disorders or extended to other therapeutic proteins. For complete details on the use and execution of this protocol, please refer to Joshi et al. (2021).1.
    Keywords:  Cell Biology; Cell Separation/Fractionation; Gene Expression; Health Sciences; Microscopy; Molecular Biology; Molecular/Chemical Probes; Neuroscience; Protein Biochemistry; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2023.102134
  19. Neurochem Res. 2023 Feb 28.
    ATF6β Deficiency Elicits Anxiety-like Behavior and Hyperactivity Under Stress Conditions.
    Takashi Tanaka, Dinh Thi Nguyen, Nichakarn Kwankaew, Megumi Sumizono, Reika Shinoda, Hiroshi Ishii, Mika Takarada-Iemata, Tsuyoshi Hattori, Seiichi Oyadomari, Nobuo Kato, Kazutoshi Mori, Osamu Hori.
      Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER) stress-regulated transcription factor that induces expression of major molecular chaperones in the ER. We recently reported that ATF6β, a subtype of ATF6, promoted survival of hippocampal neurons exposed to ER stress and excitotoxicity, at least in part by inducing expression of calreticulin, an ER molecular chaperone with high Ca2+-binding capacity. In the present study, we demonstrate that ATF6β deficiency in mice also decreases calreticulin expression and increases expression of glucose-regulated protein 78, another ER molecular chaperone, in emotional brain regions such as the prefrontal cortex (PFC), hypothalamus, hippocampus, and amygdala. Comprehensive behavioral analyses revealed that Atf6b-/- mice exhibit anxiety-like behavior in the light/dark transition test and hyperactivity in the forced swim test. Consistent with these results, PFC and hypothalamic corticotropin-releasing hormone (CRH) expression was increased in Atf6b-/- mice, as was circulating corticosterone. Moreover, CRH receptor 1 antagonism alleviated anxiety-like behavior in Atf6b-/- mice. These findings suggest that ATF6β deficiency produces anxiety-like behavior and hyperactivity via a CRH receptor 1-dependent mechanism. ATF6β could play a role in psychiatric conditions in the emotional centers of the brain.
    Keywords:  Activating transcription factor 6β; Anxiety-like behavior; Endoplasmic reticulum stress; Hyperactivity; Hypothalamic-pituitary-adrenal axis
    DOI:  https://doi.org/10.1007/s11064-023-03900-4
  20. Mol Biol Cell. 2023 Mar 01. mbcE23010037
    Exploring the consequences of redirecting an exocytic Rab onto endocytic vesicles.
    Xia Li, Dongmei Liu, Eric Griffis, Peter Novick.
      Bidirectional vesicular traffic links compartments along the exocytic and endocytic pathways. Rab GTPases have been implicated in specifying the direction of vesicular transport. To explore this proposal we sought to redirect an exocytic Rab, Sec4, onto endocytic vesicles by fusing the catalytic domain of the Sec4 GEF, Sec2, onto the CUE localization domain of Vps9, a GEF for the endocytic Rab Ypt51. The Sec2GEF-GFP-CUE construct localized to bright puncta predominantly near sites of polarized growth and this localization was dependent upon the ability of the CUE domain to bind to the ubiquitin moieties added to the cytoplasmic tails of proteins destined for endocytic internalization. Sec4 and Sec4 effectors were recruited to these puncta with varying efficiency. Cells expressing Sec2GEF-GFP-CUE grew surprisingly well and secreted protein at near normal efficiency, implying that Golgi derived secretory vesicles were delivered to polarized sites of cell growth despite the misdirection of Sec4 and its effectors. A low efficiency mechanism for localization of Sec2 to secretory vesicles that is independent of known cues might be responsible. In total, the results suggest that while Rabs may play a critical role in specifying the direction of vesicular transport cells are remarkably tolerant of Rab misdirection. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E23-01-0037
  21. Nature. 2023 Feb 27.
    Ubiquitin-like Conjugation by Bacterial cGAS Enhances Anti-phage Defence.
    Justin M Jenson, Tuo Li, Fenghe Du, Chee-Kwee Ea, Zhijian J Chen.
      cGAS is an evolutionarily conserved enzyme that plays a pivotal role in immune defense against infection1-3. In vertebrate animals, cGAS is activated by DNA to produce cyclic GMP-AMP (cGAMP)4,5, which leads to the expression of antimicrobial genes6,7. In bacteria, cyclic dinucleotide (CDN)-based anti-phage signaling systems (CBASS) have been discovered8-11. These systems are composed of cGAS-like enzymes and various effector proteins that kill bacteria upon phage infection, thereby stopping phage spread. Of the CBASS systems reported, ~39% contain Cap2 and Cap3, which encode proteins with homology to ubiquitin conjugating (E1/E2) and deconjugating (DUB) enzymes, respectively8,12. Although these proteins are required to prevent infection of some bacteriophages8, the mechanism by which the enzymatic activities exert anti-phage effect is unknown. Here, we show that Cap2 forms a thioester bond with the C-terminal glycine of cGAS and promotes conjugation of cGAS to target proteins in a process that resembles ubiquitin conjugation. The covalent conjugation of cGAS increased the production of cGAMP. Through a genetic screen, we found that the phage protein Vs.4 antagonized cGAS signaling by binding tightly to cGAMP (Kd ~30 nM) and sequestering it. A crystal structure of Vs.4 bound to cGAMP showed that Vs.4 formed a hexamer that bound to three molecules of cGAMP. These results reveal a ubiquitin-like conjugation mechanism that regulates cGAS activity in bacteria and illustrate an arms race between bacteria and viruses through controlling CDN levels.
    DOI:  https://doi.org/10.1038/s41586-023-05862-7
  22. STAR Protoc. 2023 Jan 13. pii: S2666-1667(22)00892-9. [Epub ahead of print]4(1): 102012
    Tandem mass tag-based thermal proteome profiling for the discovery of drug-protein interactions in cancer cells.
    Fraser D Johnson, Christopher S Hughes, Alvin Liu, William W Lockwood, Gregg B Morin.
      Identification of effector targets is imperative to the characterization of the mechanisms of action of novel small molecules. Here, we describe steps to identify effector drug-protein interactions in lysates derived from cancer cell lines using a thermal proteome profiling (TPP) protocol. Building on existing TTP approaches, we detail the use of an in-solution trypsin digestion technique to streamline sample preparation, a nonparametric analysis to rank proteins for prioritization, and a follow-up strategy for identifying effector interactors. For complete details on the use and execution of this protocol, please refer to Johnson et al. (2022).1.
    Keywords:  Cancer; Cell Biology; Mass Spectrometry; Molecular Biology; Protein Biochemistry; Proteomics
    DOI:  https://doi.org/10.1016/j.xpro.2022.102012
  23. Nat Commun. 2023 Mar 02. 14(1): 1189
    A covalent BTK ternary complex compatible with targeted protein degradation.
    James Schiemer, Andrew Maxwell, Reto Horst, Shenping Liu, Daniel P Uccello, Kris Borzilleri, Nisha Rajamohan, Matthew F Brown, Matthew F Calabrese.
      Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.
    DOI:  https://doi.org/10.1038/s41467-023-36738-z
  24. Mol Syst Biol. 2023 Mar 01. e11127
    A circuit for secretion-coupled cellular autonomy in multicellular eukaryotic cells.
    Lingxia Qiao, Saptarshi Sinha, Amer Ali Abd El-Hafeez, I-Chung Lo, Krishna K Midde, Tony Ngo, Nicolas Aznar, Inmaculada Lopez-Sanchez, Vijay Gupta, Marilyn G Farquhar, Padmini Rangamani, Pradipta Ghosh.
      Cancers represent complex autonomous systems, displaying self-sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to "secrete-and-sense" growth factors (GFs): a poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback-coupled GTPase circuit within the secretory pathway that imparts secretion-coupled autonomy. The circuit is assembled when the Ras-superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαβγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed-loop control, allow the two GTPases to coregulate each other, and convert the expected switch-like behavior of Arf1-dependent secretion into an unexpected dose-response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self-sustained by stimulus-proportionate secretion. Proteomic studies and protein-protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self-sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion-coupled autonomy of growth factors.
    Keywords:  G proteins; Golgi secretion; cellular autonomy; dose-response alignment (DoRA); epidermal growth factor receptor (EGFR)
    DOI:  https://doi.org/10.15252/msb.202211127
  25. Redox Biol. 2023 Feb 24. pii: S2213-2317(23)00043-5. [Epub ahead of print]61 102642
    Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells.
    Ting Wu, Shang-Tong Li, Yu Ran, Yinuo Lin, Lu Liu, Xiajun Zhang, Lianqi Zhou, Long Zhang, Donghai Wu, Bing Yang, Shibing Tang.
      Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.
    DOI:  https://doi.org/10.1016/j.redox.2023.102642
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