bims-proteo 2022-02-27 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–02–27
39 papers selected by
Eric Chevet, INSERM

ER-phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.
Targeting EDEM protects against ER stress and improves development and survival in C. elegans.
Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.
ATase inhibition rescues age-associated proteotoxicity of the secretory pathway.
Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses.
Covalent fragment-based ligand screening approaches for identification of novel ubiquitin proteasome system modulators.
Histone ubiquitination controls organ size in cotton (Gossypium hirsutum).
Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.
Emerging roles of the HECT-type E3 ubiquitin ligases in hematological malignancies.
The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation.
Oligomannose-Type Glycan Processing in the Endoplasmic Reticulum and Its Importance in Misfolding Diseases.
K48/K63-linked polyubiquitination of ATG9A by TRAF6 E3 ligase regulates oxidative stress-induced autophagy.
Stress-responsive regulation of extracellular proteostasis.
Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting.
ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.
How do protein aggregates escape quality control in neurodegeneration?
USP33 deubiquitinates and stabilizes HIF-2alpha to promote hypoxia response in glioma stem cells.
Chemical biology tools to study Deubiquitinases and Ubl proteases.
Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates.
The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress.
Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer's disease.
Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.
Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles.
Cryo-EM structures of human p97 double hexamer capture potentiated ATPase-competent state.
The role of endoplasmic reticulum stress in the MHC class I antigen presentation pathway of dendritic cells.
Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.
Vault RNA1-1 riboregulates the autophagic function of p62 by binding to K7/R21 that are critical for p62 oligomerisation.
Deciphering the molecular organization of GET pathway chaperones through native intact dissociation of multiprotein complexes.
Defective autophagy contributes to endometrial epithelial-mesenchymal transition in intrauterine adhesions.
PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2.
Inhibition of SRP-dependent protein secretion by the bacterial alarmone (p)ppGpp.
Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E).
Recent advances in the pharmacological targeting of ubiquitin-regulating enzymes in cancer.
Targeting the ubiquitin-proteasome system using the UBA1 inhibitor TAK-243 is a potential therapeutic strategy for small cell lung cancer.
Deubiquitinase-targeting chimeras for targeted protein stabilization.
Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance.
A dominant-negative regulatory mechanism of SQSTM1 droplets-based autophagy.
Prostate-specific oncogene OTUD6A promotes prostatic tumorigenesis via deubiquitinating and stabilizing c-Myc.
Organellar homeostasis and innate immune sensing.

Physiol Rev. 2022 Feb 21.

ER-phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum.

Fulvio Reggiori, Maurizio Molinari.

  ER-phagy (reticulo-phagy) defines the degradation of portions of the endoplasmic reticulum (ER) within lysosomes or vacuoles. It is part of the self-digestion (i.e., auto-phagic) programs recycling cytoplasmic material and organelles, which rapidly mobilize metabolites in cells confronted with nutrient shortage. Moreover, selective clearance of ER subdomains participates to the control of ER size and activity during ER stress, the re-establishment of ER homeostasis after ER stress resolution and the removal of ER parts, in which aberrant and potentially cytotoxic material has been segregated. ER-phagy relies on the individual and/or concerted activation of the ER-phagy receptors, ER peripheral or integral membrane proteins that share the presence of LC3/Atg8-binding motifs in their cytosolic domains. ER-phagy involves the physical separation of portions of the ER from the bulk ER network, and their delivery to the endolysosomal/vacuolar catabolic district. This last step is accomplished by a variety of mechanisms including macro-ER-phagy (in which ER fragments are sequestered by double-membrane autophagosomes that eventually fuse with lysosomes/vacuoles), micro-ER-phagy (in which ER fragments are directly engulfed by endosomes/lysosomes/vacuoles), or direct fusion of ER-derived vesicles with lysosomes/vacuoles. ER-phagy is dysfunctional in specific human diseases and its regulators are subverted by pathogens, highlighting its crucial role for cell and organism life.

Keywords:  Autophagy; Disease; ER-phagy; Endoplasmic Reticulum; Lysosomal degradation

DOI:  https://doi.org/10.1152/physrev.00038.2021
PLoS Genet. 2022 Feb 22. 18(2): e1010069

Targeting EDEM protects against ER stress and improves development and survival in C. elegans.

Simona Ghenea, Marioara Chiritoiu, Robi Tacutu, Antonio Miranda-Vizuete, Stefana Maria Petrescu.

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival.

DOI: https://doi.org/10.1371/journal.pgen.1010069
Life Sci Alliance. 2022 May;pii: e202201379. [Epub ahead of print]5(5):

Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs.

Alexandra Papaioannou, Federica Centonze, Alice Metais, Marion Maurel, Luc Negroni, Matías Gonzalez-Quiroz, Sayyed Jalil Mahdizadeh, Gabriella Svensson, Ensieh Zare, Alice Blondel, Albert C Koong, Claudio Hetz, Rémy Pedeux, Michel L Tremblay, Leif A Eriksson, Eric Chevet.

ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs.

DOI: https://doi.org/10.26508/lsa.202201379
Commun Biol. 2022 Feb 25. 5(1): 173

ATase inhibition rescues age-associated proteotoxicity of the secretory pathway.

Maeghan Murie, Yajing Peng, Michael J Rigby, Inca A Dieterich, Mark A Farrugia, Andreas Endresen, Anita Bhattacharyya, Luigi Puglielli.

Malfunction of autophagy contributes to the progression of many chronic age-associated diseases. As such, improving normal proteostatic mechanisms is an active target for biomedical research and a key focal area for aging research. Endoplasmic reticulum (ER)-based acetylation has emerged as a mechanism that ensures proteostasis within the ER by regulating the induction of ER specific autophagy. ER acetylation is ensured by two ER-membrane bound acetyltransferases, ATase1 and ATase2. Here, we show that ATase inhibitors can rescue ongoing disease manifestations associated with the segmental progeria-like phenotype of AT-1 sTg mice. We also describe a pipeline to reliably identify ATase inhibitors with promising druggability properties. Finally, we show that successful ATase inhibitors can rescue the proteopathy of a mouse model of Alzheimer's disease. In conclusion, our study proposes that ATase-targeting approaches might offer a translational pathway for many age-associated proteopathies affecting the ER/secretory pathway.

DOI: https://doi.org/10.1038/s42003-022-03118-0
Autophagy. 2022 Feb 20. 1-12

Loss of ubiquitinated protein autophagy is compensated by persistent cnc/NFE2L2/Nrf2 antioxidant responses.

Arindam Bhattacharjee, Adél Ürmösi, András Jipa, Levente Kovács, Péter Deák, Áron Szabó, Gábor Juhász.

  SQSTM1/p62-type selective macroautophagy/autophagy receptors cross-link poly-ubiquitinated cargo and autophagosomal LC3/Atg8 proteins to deliver them for lysosomal degradation. Consequently, loss of autophagy leads to accumulation of polyubiquitinated protein aggregates that are also frequently seen in various human diseases, but their physiological relevance is incompletely understood. Here, using a genetically non-redundant Drosophila model, we show that specific disruption of ubiquitinated protein autophagy and concomitant formation of polyubiquitinated aggregates has hardly any effect on bulk autophagy, proteasome activity and fly healthspan. We find that accumulation of ref(2)P/SQSTM1 due to a mutation that disrupts its binding to Atg8a results in the co-sequestering of Keap1 and thus activates the cnc/NFE2L2/Nrf2 antioxidant pathway. These mutant flies have increased tolerance to oxidative stress and reduced levels of aging-associated mitochondrial superoxide. Interestingly, ubiquitin overexpression in ref(2)P point mutants prevents the formation of large aggregates and restores the cargo recognition ability of ref(2)P, although it does not prevent the activation of antioxidant responses. Taken together, potential detrimental effects of impaired ubiquitinated protein autophagy are compensated by the aggregation-induced antioxidant response.

Keywords:  Autophagic receptor; Drosophila; autophagy; longevity; oxidative stress

DOI:  https://doi.org/10.1080/15548627.2022.2037852
Biol Chem. 2022 Feb 23.

Covalent fragment-based ligand screening approaches for identification of novel ubiquitin proteasome system modulators.

Elisabeth M Rothweiler, Paul E Brennan, Kilian V M Huber.

  Ubiquitination is a key regulatory mechanism vital for maintenance of cellular homeostasis. Protein degradation is induced by E3 ligases via attachment of ubiquitin chains to substrates. Pharmacological exploitation of this phenomenon via targeted protein degradation (TPD) can be achieved with molecular glues or bifunctional molecules facilitating the formation of ternary complexes between an E3 ligase and a given protein of interest (POI), resulting in ubiquitination of the substrate and subsequent proteolysis by the proteasome. Recently, the development of novel covalent fragment screening approaches has enabled the identification of first-in-class ligands for E3 ligases and deubiquitinases revealing so far unexplored binding sites which highlights the potential of these methods to uncover and expand druggable space for new target classes.

Keywords:  ABPP; E3 ligase; PROTAC; chemoproteomics; covalent fragments; ubiquitin

DOI:  https://doi.org/10.1515/hsz-2021-0396
Plant J. 2022 Feb 25.

Histone ubiquitination controls organ size in cotton (Gossypium hirsutum).

Pengcheng Wang, Kai Guo, Qian Su, Jinwu Deng, Xianlong Zhang, Lili Tu.

  Ubiquitination plays a vital role in modifying protein activity and destiny. Ubiquitin-conjugating enzyme E2 is one of the enzymes that participates in this precise process. There are at least 169 E2 proteins in the allotetraploid cotton Gossypium hirsutum, but their function remains unknown. Here we identify an E2 gene GhUBC2L and illustrate its positive role in cell proliferation and expansion. Complete knock-down of GhUBC2L in cotton resulted in retarded growth and reduced organ size. Conversely, overexpression of GhUBC2L promoted cotton growth, generating enlarged organs in size. Monoubiquitination of H2A and H2B was strongly impaired in GhUBC2L-suppressed cotton but slightly enhanced in GhUBC2L overexpressing plants. GhUbox8, a U-box type E3 ligase protein, was found to interact with GhUBC2L both in vivo and in vitro, indicating their synergistical function in protein ubiquitination. Furthermore, GhUbox8 was shown to interact with a series of histone proteins, including histone H2A and H2B, indicating its potential monoubiquitination on H2A and H2B. Expression of genes relating to cell cycle and organ development were altered when expression of GhUBC2L was changed. Our results show that GhUBC2L modulates histone monoubiquitination synergistically with GhUbox8 to regulate the expression of genes involved in organ development and cell cycle, thus controlling organ size in cotton. This research provides new insights into the role of protein ubiquitination in organ size control.

Keywords:  Cotton; Histone; Organ size; Ubiquitin ligase; Ubiquitin-conjugating enzyme; Ubiquitination

DOI:  https://doi.org/10.1111/tpj.15716
EMBO Mol Med. 2022 Feb 22. e15344

Intercepting IRE1 kinase-FMRP signaling prevents atherosclerosis progression.

Zehra Yildirim, Sabyasachi Baboo, Syed M Hamid, Asli E Dogan, Ozlem Tufanli, Sabrina Robichaud, Christina Emerton, Jolene K Diedrich, Hasan Vatandaslar, Fotis Nikolos, Yanghong Gu, Takao Iwawaki, Elizabeth Tarling, Mireille Ouimet, David L Nelson, John R Yates, Peter Walter, Ebru Erbay.

  Fragile X Mental Retardation protein (FMRP), widely known for its role in hereditary intellectual disability, is an RNA-binding protein (RBP) that controls translation of select mRNAs. We discovered that endoplasmic reticulum (ER) stress induces phosphorylation of FMRP on a site that is known to enhance translation inhibition of FMRP-bound mRNAs. We show ER stress-induced activation of Inositol requiring enzyme-1 (IRE1), an ER-resident stress-sensing kinase/endoribonuclease, leads to FMRP phosphorylation and to suppression of macrophage cholesterol efflux and apoptotic cell clearance (efferocytosis). Conversely, FMRP deficiency and pharmacological inhibition of IRE1 kinase activity enhances cholesterol efflux and efferocytosis, reducing atherosclerosis in mice. Our results provide mechanistic insights into how ER stress-induced IRE1 kinase activity contributes to macrophage cholesterol homeostasis and suggests IRE1 inhibition as a promising new way to counteract atherosclerosis.

Keywords:  ER stress; atherosclerosis; cholesterol homeostasis; efferocytosis; translational regulation

DOI:  https://doi.org/10.15252/emmm.202115344
Discov Oncol. 2021 Oct 08. 12(1): 39

Emerging roles of the HECT-type E3 ubiquitin ligases in hematological malignancies.

Vincenza Simona Delvecchio, Claudia Fierro, Sara Giovannini, Gerry Melino, Francesca Bernassola.

  Ubiquitination-mediated proteolysis or regulation of proteins, ultimately executed by E3 ubiquitin ligases, control a wide array of cellular processes, including transcription, cell cycle, autophagy and apoptotic cell death. HECT-type E3 ubiquitin ligases can be distinguished from other subfamilies of E3 ubiquitin ligases because they have a C-terminal HECT domain that directly catalyzes the covalent attachment of ubiquitin to their substrate proteins. Deregulation of HECT-type E3-mediated ubiquitination plays a prominent role in cancer development and chemoresistance. Several members of this subfamily are indeed frequently deregulated in human cancers as a result of genetic mutations and altered expression or activity. HECT-type E3s contribute to tumorigenesis by regulating the ubiquitination rate of substrates that function as either tumour suppressors or oncogenes. While the pathological roles of the HECT family members in solid tumors are quite well established, their contribution to the pathogenesis of hematological malignancies has only recently emerged. This review aims to provide a comprehensive overview of the involvement of the HECT-type E3s in leukemogenesis.

Keywords:  HECT-type E3 ubiquitin protein ligases; Leukemia; Proteasomal degradation; Ubiquitin; Ubiquitination

DOI:  https://doi.org/10.1007/s12672-021-00435-4
Pharmaceuticals (Basel). 2022 Feb 07. pii: 204. [Epub ahead of print]15(2):

The p97 Inhibitor UPCDC-30245 Blocks Endo-Lysosomal Degradation.

Feng Wang, Shan Li, Kai-Wen Cheng, William M Rosencrans, Tsui-Fen Chou.

  The diverse modes of action of small molecule inhibitors provide versatile tools to investigate basic biology and develop therapeutics. However, it remains a challenging task to evaluate their exact mechanisms of action. We identified two classes of inhibitors for the p97 ATPase: ATP competitive and allosteric. We showed that the allosteric p97 inhibitor, UPCDC-30245, does not affect two well-known cellular functions of p97, endoplasmic-reticulum-associated protein degradation and the unfolded protein response pathway; instead, it strongly increases the lipidated form of microtubule-associated proteins 1A/1B light chain 3B (LC3-II), suggesting an alteration of autophagic pathways. To evaluate the molecular mechanism, we performed proteomic analysis of UPCDC-30245 treated cells. Our results revealed that UPCDC-30245 blocks endo-lysosomal degradation by inhibiting the formation of early endosome and reducing the acidity of the lysosome, an effect not observed with the potent p97 inhibitor CB-5083. This unique effect allows us to demonstrate UPCDC-30245 exhibits antiviral effects against coronavirus by blocking viral entry.

Keywords:  coronavirus; endo-lysosomal degradation; lysomotropic agents; p97 inhibitor; proteomics

DOI:  https://doi.org/10.3390/ph15020204
Biology (Basel). 2022 Jan 27. pii: 199. [Epub ahead of print]11(2):

Oligomannose-Type Glycan Processing in the Endoplasmic Reticulum and Its Importance in Misfolding Diseases.

Taiki Kuribara, Kiichiro Totani.

  Glycoprotein folding plays a critical role in sorting glycoprotein secretion and degradation in the endoplasmic reticulum (ER). Furthermore, relationships between glycoprotein folding and several diseases, such as type 2 diabetes and various neurodegenerative disorders, are indicated. Patients' cells with type 2 diabetes, and various neurodegenerative disorders induce ER stress, against which the cells utilize the unfolded protein response for protection. However, in some cases, chronic and/or massive ER stress causes critical damage to cells, leading to the onset of ER stress-related diseases, which are categorized into misfolding diseases. Accumulation of misfolded proteins may be a cause of ER stress, in this respect, perturbation of oligomannose-type glycan processing in the ER may occur. A great number of studies indicate the relationships between ER stress and misfolding diseases, while little evidence has been reported on the connection between oligomannose-type glycan processing and misfolding diseases. In this review, we summarize alteration of oligomannose-type glycan processing in several ER stress-related diseases, especially misfolding diseases and show the possibility of these alteration of oligomannose-type glycan processing as indicators of diseases.

Keywords:  glycan processing; glycoprotein folding; misfolding diseases; reconstructed glycan profile

DOI:  https://doi.org/10.3390/biology11020199
Cell Rep. 2022 Feb 22. pii: S2211-1247(22)00070-5. [Epub ahead of print]38(8): 110354

K48/K63-linked polyubiquitination of ATG9A by TRAF6 E3 ligase regulates oxidative stress-induced autophagy.

Yi-Ting Wang, Ting-Yu Liu, Chia-Hsing Shen, Shu-Yu Lin, Chin-Chun Hung, Li-Chung Hsu, Guang-Chao Chen.

  Excessive generation and accumulation of highly reactive oxidizing molecules causes oxidative stress and oxidative damage to cellular components. Accumulating evidence indicates that autophagy diminishes oxidative damage in cells and maintains redox homeostasis by degrading and recycling intracellular damaged components. Here, we show that TRAF6 E3 ubiquitin ligase and A20 deubiquitinase coordinate to regulate ATG9A ubiquitination and autophagy activation in cells responding to oxidative stress. The ROS-dependent TRAF6-mediated non-proteolytic, K48/63-linked ubiquitination of ATG9A enhances its association with Beclin 1 and the assembly of VPS34-UVRAG complex, thereby stimulating autophagy. Notably, expression of the ATG9A ubiquitination mutants impairs ROS-induced VPS34 activation and autophagy. We further find that lipopolysaccharide (LPS)-induced ROS production also stimulates TRAF6-mediated ATG9A ubiquitination. Ablation of ATG9A causes aberrant TLR4 endosomal trafficking and decreases IRF-3 phosphorylation in LPS-stimulated macrophages. Our findings provide important insights into how K48/K63-linked ubiquitination of ATG9A contributes to the regulation of oxidative stress-induced autophagy.

Keywords:  ATG9A; TRAF6; VPS34 complex; autophagy; oxidative stress

DOI:  https://doi.org/10.1016/j.celrep.2022.110354
J Cell Biol. 2022 04 04. pii: e202112104. [Epub ahead of print]221(4):

Stress-responsive regulation of extracellular proteostasis.

Jaleh S Mesgarzadeh, Joel N Buxbaum, R Luke Wiseman.

Genetic, environmental, and aging-related insults can promote the misfolding and subsequent aggregation of secreted proteins implicated in the pathogenesis of numerous diseases. This has led to considerable interest in understanding the molecular mechanisms responsible for regulating proteostasis in extracellular environments such as the blood and cerebrospinal fluid (CSF). Extracellular proteostasis is largely dictated by biological pathways comprising chaperones, folding enzymes, and degradation factors localized to the ER and extracellular space. These pathways limit the accumulation of nonnative, potentially aggregation-prone proteins in extracellular environments. Many reviews discuss the molecular mechanisms by which these pathways impact the conformational integrity of the secreted proteome. Here, we instead focus on describing the stress-responsive mechanisms responsible for adapting ER and extracellular proteostasis pathways to protect the secreted proteome from pathologic insults that challenge these environments. Further, we highlight new strategies to identify stress-responsive pathways involved in regulating extracellular proteostasis and describe the pathologic and therapeutic implications for these pathways in human disease.

DOI: https://doi.org/10.1083/jcb.202112104
Science. 2022 Feb 25. 375(6583): 839-844

Mechanism of signal sequence handover from NAC to SRP on ribosomes during ER-protein targeting.

Ahmad Jomaa, Martin Gamerdinger, Hao-Hsuan Hsieh, Annalena Wallisch, Viswanathan Chandrasekaran, Zeynel Ulusoy, Alain Scaiola, Ramanujan S Hegde, Shu-Ou Shan, Nenad Ban, Elke Deuerling.

The nascent polypeptide-associated complex (NAC) interacts with newly synthesized proteins at the ribosomal tunnel exit and competes with the signal recognition particle (SRP) to prevent mistargeting of cytosolic and mitochondrial polypeptides to the endoplasmic reticulum (ER). How NAC antagonizes SRP and how this is overcome by ER targeting signals are unknown. Here, we found that NAC uses two domains with opposing effects to control SRP access. The core globular domain prevented SRP from binding to signal-less ribosomes, whereas a flexibly attached domain transiently captured SRP to permit scanning of nascent chains. The emergence of an ER-targeting signal destabilized NAC's globular domain and facilitated SRP access to the nascent chain. These findings elucidate how NAC hands over the signal sequence to SRP and imparts specificity of protein localization.

DOI: https://doi.org/10.1126/science.abl6459
FEBS Lett. 2022 Feb 25.

ABIN1 is a signal-induced autophagy receptor that attenuates NF-κB activation by recognizing linear ubiquitin chains.

Yutaka Shinkawa, Koshi Imami, Yasuhiro Fuseya, Katsuhiro Sasaki, Koichiro Ohmura, Yasushi Ishihama, Akio Morinobu, Kazuhiro Iwai.

  Linear ubiquitin chains play pivotal roles in immune signaling by augmenting NF-κB activation and suppressing programmed cell death induced by various stimuli. A20-binding inhibitor of NF-κB 1 (ABIN1) binds to linear ubiquitin chains and attenuates NF-κB activation and cell death induction. Although interactions with linear ubiquitin chains are thought to play a role in ABIN1-mediated suppression of NF-κB and cell death, the underlying molecular mechanisms remain unclear. Here, we show that upon stimulation by Toll-like receptor (TLR) ligands, ABIN1 is phosphorylated on Ser 83 and functions as a selective autophagy receptor. ABIN1 recognizes components of the MyD88 signaling complex via interaction with linear ubiquitin chains conjugated to components of the complex in TLR signaling, which leads to autophagic degradation of signaling proteins and attenuated NF-κB signaling. Our current findings indicate that phosphorylation and linear ubiquitination also play a role in downregulation of signaling via selective induction of autophagy.

Keywords:  ABIN1; MyDDosome; NF-κB; Toll-like receptors; autophagy; linear ubiquitination

DOI:  https://doi.org/10.1002/1873-3468.14323
Trends Neurosci. 2022 Feb 21. pii: S0166-2236(22)00018-2. [Epub ahead of print]

How do protein aggregates escape quality control in neurodegeneration?

Margreet B Koopman, Luca Ferrari, Stefan G D Rüdiger.

  Protein aggregates are hallmarks of neurodegenerative diseases. The protein quality control (PQC) system normally prevents proteins from misfolding and accumulation; however, proteins somehow escape this control on disease. Here we review advances in the role of PQC in protein aggregation and neurodegeneration. We focus primarily on the protein Tau, which aggregates in Alzheimer's disease (AD) and other tauopathies. We also examine recent advances in amyloid fibril structures and the process of fibril formation via phase separation, which are shedding new light on the role of PQC in protein aggregation diseases. While specific components of the quality control system appear to be altered in disease, most chaperones and degradation factors are unchanged at the cellular end stage. Advancing the understanding of quality control factors in neurodegeneration, particularly in the early stages of disease, is among the key challenges for neurodegeneration research.

Keywords:  Tau; amyloid fibrils; autophagy; liquid–liquid phase separation; neurodegeneration; protein quality control

DOI:  https://doi.org/10.1016/j.tins.2022.01.006
EMBO J. 2022 Feb 22. e109187

USP33 deubiquitinates and stabilizes HIF-2alpha to promote hypoxia response in glioma stem cells.

Aili Zhang, Zhi Huang, Weiwei Tao, Kui Zhai, Qiulian Wu, Jeremy N Rich, Wenchao Zhou, Shideng Bao.

  Hypoxia regulates tumor angiogenesis, metabolism, and therapeutic response in malignant cancers including glioblastoma, the most lethal primary brain tumor. The regulation of HIF transcriptional factors by the ubiquitin-proteasome system is critical in the hypoxia response, but hypoxia-inducible deubiquitinases that counteract the ubiquitination remain poorly defined. While the activation of ERK1/2 also plays an important role in hypoxia response, the relationship between ERK1/2 activation and HIF regulation remains elusive. Here, we identified USP33 as essential deubiquitinase that stabilizes HIF-2alpha protein in an ERK1/2-dependent manner to promote hypoxia response in cancer cells. USP33 is preferentially induced in glioma stem cells by hypoxia and interacts with HIF-2alpha, leading to its stabilization through deubiquitination. The activation of ERK1/2 upon hypoxia promoted HIF-2alpha phosphorylation, enhancing its interaction with USP33. Silencing of USP33 disrupted glioma stem cells maintenance, reduced tumor vascularization, and inhibited glioblastoma growth. Our findings highlight USP33 as an essential regulator of hypoxia response in cancer stem cells, indicating a novel potential therapeutic target for brain tumor treatment.

Keywords:  ERK1/2; HIF2α; USP33; deubiquitination; glioma stem cell; hypoxia response

DOI:  https://doi.org/10.15252/embj.2021109187
Semin Cell Dev Biol. 2022 Feb 22. pii: S1084-9521(22)00045-3. [Epub ahead of print]

Chemical biology tools to study Deubiquitinases and Ubl proteases.

Magdalena Gorka, Helge Magnus Magnussen, Yogesh Kulathu.

  The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs) for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function at a molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs.

Keywords:  Activity-based protein profiling; Deubiquitinases; Posttranslational modification; Proteases; Signal transduction; UBL; Ubiquitin

DOI:  https://doi.org/10.1016/j.semcdb.2022.02.006
Proc Natl Acad Sci U S A. 2022 Mar 01. pii: e2118919119. [Epub ahead of print]119(9):

Chaperones Skp and SurA dynamically expand unfolded OmpX and synergistically disassemble oligomeric aggregates.

Neharika Chamachi, Andreas Hartmann, Mai Quynh Ma, Anna Svirina, Georg Krainer, Michael Schlierf.

  Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy-entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis.

Keywords:  chaperones; disaggregation; outer membrane protein biogenesis; protein folding; single-molecule FRET

DOI:  https://doi.org/10.1073/pnas.2118919119
Sci Adv. 2022 Feb 25. 8(8): eabl4386

The chaperone Tsr2 regulates Rps26 release and reincorporation from mature ribosomes to enable a reversible, ribosome-mediated response to stress.

Yoon-Mo Yang, Katrin Karbstein.

Although ribosome assembly is quality controlled to maintain protein homeostasis, different ribosome populations have been described. How these form, especially under stress conditions that affect energy levels and stop the energy-intensive production of ribosomes, remains unknown. Here, we demonstrate how a physiologically relevant ribosome population arises during high Na+, sorbitol, or pH stress via dissociation of Rps26 from fully assembled ribosomes to enable a translational response to these stresses. The chaperone Tsr2 releases Rps26 in the presence of high Na+ or pH in vitro and is required for Rps26 release in vivo. Moreover, Tsr2 stores free Rps26 and promotes reincorporation of the protein, thereby repairing the subunit after the Na+ stress subsides. Our data implicate a residue in Rps26 involved in Diamond Blackfan Anemia in mediating the effects of Na+. These data demonstrate how different ribosome populations can arise rapidly, without major energy input and without bypass of quality control mechanisms.

DOI: https://doi.org/10.1126/sciadv.abl4386
J Biol Chem. 2022 Feb 21. pii: S0021-9258(22)00206-X. [Epub ahead of print] 101766

Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer's disease.

Nicha Puangmalai, Urmi Sengupta, Nemil Bhatt, Sagar Gaikwad, Mauro Montalbano, Arijit Bhuyan, Stephanie Garcia, Salome McAllen, Minal Sonawane, Cynthia Jerez, Yingxin Zhao, Rakez Kayed.

  Ubiquitin-modified tau aggregates are abundantly found in human brains diagnosed with Alzheimer's disease (AD) and other tauopathies. Soluble tau oligomers (TauO) are the most neurotoxic tau species that propagate pathology and elicit cognitive deficits, but whether ubiquitination contributes to tau formation and spreading is not fully understood. Here, we observe that K63-linked, but not K48- linked, ubiquitinated TauO accumulate at higher levels in AD brains compared to age-matched controls. Using mass spectrometry analyses, we identified 11 ubiquitinated sites on AD brain-derived TauO (AD TauO). We found that K63-linked TauO are associated with enhanced seeding activity and propagation in human tau-expressing primary neuronal and tau biosensor cells. Additionally, exposure of tau-inducible HEK cells to AD TauO with different ubiquitin linkages (wild type, K48, and K63) resulted in enhanced formation and secretion of K63-linked TauO, which was associated with impaired proteasome and lysosome functions. Multi-pathway analysis also revealed the involvement of K63-linked TauO in cell survival pathways, which are impaired in AD. Collectively, our study highlights the significance of selective TauO ubiquitination, which could influence tau aggregation, accumulation, and subsequent pathological propagation. The insights gained from this study hold great promise for targeted therapeutic intervention in AD and related tauopathies.

Keywords:  Alzheimer’s disease; K63 ubiquitination; Tau pathology spreading; Tau secretion; proteasome impairment

DOI:  https://doi.org/10.1016/j.jbc.2022.101766
Elife. 2022 02 24. pii: e70726. [Epub ahead of print]11

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

Wouter Huiting, Suzanne L Dekker, Joris C J van der Lienden, Rafaella Mergener, Maiara K Musskopf, Gabriel V Furtado, Emma Gerrits, David Coit, Mehrnoosh Oghbaie, Luciano H Di Stefano, Hein Schepers, Maria A W H van Waarde-Verhagen, Suzanne Couzijn, Lara Barazzuol, John LaCava, Harm H Kampinga, Steven Bergink.

  A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Keywords:  DNA damage response; cell biology; chaperone; genetics; genomics; genotoxic stress; human; protein aggregation; protein homeostasis

DOI:  https://doi.org/10.7554/eLife.70726
Cell Struct Funct. 2022 ;47(1): 1-18

Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles.

Yusuke Takanashi, Tomoaki Kahyo, Sae Kamamoto, Hengsen Zhang, Bin Chen, Yashuang Ping, Kiyomichi Mizuno, Akikazu Kawase, Kei Koizumi, Masanori Satou, Kazuhito Funai, Norihiko Shiiya, Mitsutoshi Setou.

  Ubiquitin-like 3 (UBL3) is a well-conserved ubiquitin-like protein (UBL) in eukaryotes and regulates the ubiquitin cascade, but the significant roles of UBL3 in cellular processes remained unknown. Recently, UBL3 was elucidated to be a post-translational modification factor that promotes protein sorting to small extracellular vesicles (sEVs). Proteins sorted into sEVs have been studied as etiologies of sEV-related diseases. Also, there have been attempts to construct drug delivery systems (DDSs) by loading proteins into sEVs. In this review, we introduce the new concept that UBL3 has a critical role in the protein-sorting system and compare structure conservation between UBL3 and other UBLs from an evolutionary perspective. We conclude with future perspectives for the utility of UBL3 in sEV-related diseases and DDS.Key words: UBL3, small extracellular vesicles, protein sorting, ubiquitin-like protein, post-translational modification.

Keywords:  UBL3; post-translational modification; protein sorting; small extracellular vesicles; ubiquitin-like protein

DOI:  https://doi.org/10.1247/csf.21078
Cell Discov. 2022 Feb 22. 8(1): 19

Cryo-EM structures of human p97 double hexamer capture potentiated ATPase-competent state.

Haishan Gao, Faxiang Li, Zhejian Ji, Zhubing Shi, Yang Li, Hongtao Yu.

The conserved ATPase p97 (Cdc48 in yeast) and adaptors mediate diverse cellular processes through unfolding polyubiquitinated proteins and extracting them from macromolecular assemblies and membranes for disaggregation and degradation. The tandem ATPase domains (D1 and D2) of the p97/Cdc48 hexamer form stacked rings. p97/Cdc48 can unfold substrates by threading them through the central pore. The pore loops critical for substrate unfolding are, however, not well-ordered in substrate-free p97/Cdc48 conformations. How p97/Cdc48 organizes its pore loops for substrate engagement is unclear. Here we show that p97/Cdc48 can form double hexamers (DH) connected through the D2 ring. Cryo-EM structures of p97 DH reveal an ATPase-competent conformation with ordered pore loops. The C-terminal extension (CTE) links neighboring D2s in each hexamer and expands the central pore of the D2 ring. Mutations of Cdc48 CTE abolish substrate unfolding. We propose that the p97/Cdc48 DH captures a potentiated state poised for substrate engagement.

DOI: https://doi.org/10.1038/s41421-022-00379-1
Mol Immunol. 2022 Feb 17. pii: S0161-5890(22)00028-1. [Epub ahead of print]144 44-48

The role of endoplasmic reticulum stress in the MHC class I antigen presentation pathway of dendritic cells.

Bénédicte Manoury, Lucie Maisonneuve, Katrina Podsypanina.

  Dendritic cells (DCs) have the unique capacity to link innate to adaptive immunity. While most cells that express major histocompatibility (MHC) molecules are able to present antigens to activated T cells, DCs possess the means for presenting antigens to naïve T cells, and, as such, are able to instruct T cells to initiate immune response. There are two cascades of events necessary for DCs to start their instructive function. First, DCs enzymatically process proteins to make T cells recognize an antigen as unique peptide-MHC complexes. Second, DCs provide secretory cytokines and co-stimulatory functions for T cells to respond to this antigen. Thus, the compartments for protein degradation and for protein synthesis are central to DC function. The endoplasmic reticulum (ER), a vast network of membranes and vesicles, connects these compartments and helps modulate DC-specific performance, such as antigen capture and presentation. However, while the health of ER appears relevant for DC function, the intersection between ER stress and antigen presentation remains to be explored.

Keywords:  Dendritic cells; Endoplasmic reticulum stress; IRE1α; MHCI antigen presentation

DOI:  https://doi.org/10.1016/j.molimm.2022.02.007
Cell Death Differ. 2022 Feb 22.

Human cytomegalovirus hijacks host stress response fueling replication stress and genome instability.

Joanna Maria Merchut-Maya, Jiri Bartek, Jirina Bartkova, Panagiotis Galanos, Mattia Russel Pantalone, MyungHee Lee, Huanhuan L Cui, Patrick J Shilling, Christian Beltoft Brøchner, Helle Broholm, Apolinar Maya-Mendoza, Cecilia Söderberg-Naucler, Jiri Bartek.

Viral infections enhance cancer risk and threaten host genome integrity. Although human cytomegalovirus (HCMV) proteins have been detected in a wide spectrum of human malignancies and HCMV infections have been implicated in tumorigenesis, the underlying mechanisms remain poorly understood. Here, we employed a range of experimental approaches, including single-molecule DNA fiber analysis, and showed that infection by any of the four commonly used HCMV strains: AD169, Towne, TB40E or VR1814 induced replication stress (RS), as documented by host-cell replication fork asymmetry and formation of 53BP1 foci. The HCMV-evoked RS triggered an ensuing host DNA damage response (DDR) and chromosomal instability in both permissive and non-permissive human cells, the latter being particularly relevant in the context of tumorigenesis, as such cells can survive and proliferate after HCMV infection. The viral major immediate early enhancer and promoter (MIEP) that controls expression of the viral genes IE72 (IE-1) and IE86 (IE-2), contains transcription-factor binding sites shared by promoters of cellular stress-response genes. We found that DNA damaging insults, including those relevant for cancer therapy, enhanced IE72/86 expression. Thus, MIEP has been evolutionary shaped to exploit host DDR. Ectopically expressed IE72 and IE86 also induced RS and increased genomic instability. Of clinical relevance, we show that undergoing standard-of-care genotoxic radio-chemotherapy in patients with HCMV-positive glioblastomas correlated with elevated HCMV protein markers after tumor recurrence. Collectively, these results are consistent with our proposed concept of HCMV hijacking transcription-factor binding sites shared with host stress-response genes. We present a model to explain the potential oncomodulatory effects of HCMV infections through enhanced replication stress, subverted DNA damage response and induced genomic instability.

DOI: https://doi.org/10.1038/s41418-022-00953-w
RNA. 2022 Feb 24. pii: rna.079129.122. [Epub ahead of print]

Vault RNA1-1 riboregulates the autophagic function of p62 by binding to K7/R21 that are critical for p62 oligomerisation.

Magdalena Büscher, Rastislav Horos, Ina Huppertz, Kevin Haubrich, Nikolay Dobrev, Florence Baudin, Janosch Hennig, Matthias W Hentze.

  Cellular processes can be regulated at multiple levels, including transcriptional, post-transcriptional and post-translational mechanisms. We have recently shown that the small, non-coding vault RNA1-1 negatively riboregulates p62 oligomerisation in selective autophagy through direct interaction with the autophagic receptor. This function is highly specific for this Pol III transcript, but the determinants of this specificity and a mechanistic explanation of how vault RNA1-1 inhibits p62 oligomerisation are lacking. Here, we combine biochemical and functional experiments to answer these questions. We show that the PB1 domain and adjacent linker region of p62 (aa 1-122) are necessary and sufficient for specific vault RNA1-1 binding, and identify lysine 7 and arginine 21 as key hinges for p62 riboregulation. Chemical structure probing of vault RNA1-1 further reveals a central flexible loop within vault RNA1-1 that is required for the specific interaction with p62. Overall, our data provide molecular insight into how a small RNA riboregulates protein-protein interactions critical to the activation of specific autophagy.

Keywords:  autophagy; p62; riboregulation; small ncRNA; vault RNA

DOI:  https://doi.org/10.1261/rna.079129.122
Biophys J. 2022 Feb 18. pii: S0006-3495(22)00150-3. [Epub ahead of print]

Deciphering the molecular organization of GET pathway chaperones through native intact dissociation of multiprotein complexes.

Fabian Giska, Malaiyalam Mariappan, Moitrayee Bhattacharyya, Kallol Gupta.

Get3/4/5 chaperone complex is responsible for targeting C-terminal tail-anchored membrane proteins (TAPs) to the endoplasmic reticulum (ER). Despite the availability of several crystal structures of independent proteins and partial structures of subcomplexes, different models of oligomeric states and structural organization have been proposed for the protein complexes involved. Here, using native mass spectrometry (Native-MS), coupled with intact dissociation, we show that Get4/5 exclusively forms a tetramer using both Get5/5 and a novel Get4/4 dimerization interface. Addition of Get3 to this leads to a hexameric (Get3)2-(Get4)2-(Get5)2 complex with closed ring cyclic architecture. We further validate our claims through molecular modeling and mutational abrogation of the proposed interfaces. Native-MS has become a principal tool to determine the state of oligomeric organization of proteins. The work demonstrates that for multiprotein complexes, native-MS, coupled with molecular modeling and mutational perturbation, can provide an alternative route to render a detailed view of both the oligomeric states as well as the molecular interfaces involved. This is especially useful for large multiprotein complexes with large unstructured domains that make it recalcitrant to conventional structure determination approaches.

DOI: https://doi.org/10.1016/j.bpj.2022.02.026
Autophagy. 2022 Feb 23. 1-16

Defective autophagy contributes to endometrial epithelial-mesenchymal transition in intrauterine adhesions.

Zhenhua Zhou, Huiyan Wang, Xiwen Zhang, Minmin Song, Simin Yao, Peipei Jiang, Dan Liu, Zhiyin Wang, Haining Lv, Ruotian Li, Ying Hong, Jianwu Dai, Yali Hu, Guangfeng Zhao.

  Intrauterine adhesions (IUA), characterized by endometrial fibrosis, is a common cause of uterine infertility. We previously demonstrated that partial epithelial-mesenchymal transition (EMT) and the loss of epithelial homeostasis play a vital role in the development of endometrial fibrosis. As a pro-survival strategy in maintaining cell and tissue homeostasis, macroautophagy/autophagy, conversely, may participate in this process. However, the role of autophagy in endometrial fibrosis remains unknown. Here, we demonstrated that autophagy is defective in endometria of IUA patients, which aggravates EMT and endometrial fibrosis, and defective autophagy is related to DIO2 (iodothyronine deiodinase 2) downregulation. In endometrial epithelial cells (EECs), pharmacological inhibition of autophagy by chloroquine (CQ) promoted EEC-EMT, whereas enhanced autophagy by rapamycin extenuated this process. Mechanistically, silencing DIO2 in EECs blocked autophagic flux and promoted EMT via the MAPK/ERK-MTOR pathway. Inversely, overexpression of DIO2 or triiodothyronine (T3) treatment could restore autophagy and partly reverse EEC-EMT. Furthermore, in an IUA-like mouse model, the autophagy in endometrium was defective accompanied by EEC-EMT, and CQ could inhibit autophagy and aggravate endometrial fibrosis, whereas rapamycin or T3 treatment could improve the autophagic levels and blunt endometrial fibrosis. Together, we demonstrated that defective autophagy played an important role in EEC-EMT in IUA via the DIO2-MAPK/ERK-MTOR pathway, which provided a potential target for therapeutic implications.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AMPK: adenosine 5'-monophosphate-activated protein kinase; AKT/protein kinase B: AKT serine/threonine kinase; ATG: autophagy related; CDH1/E-cadherin: cadherin 1; CDH2/N-cadherin: cadherin 2; CQ: chloroquine; CTSD: cathepsin D; DIO2: iodothyronine deiodinase 2; DEGs: differentially expressed genes; EECs: endometrial epithelial cells; EMT: epithelial-mesenchymal transition; FN1: fibronectin 1; IUA: intrauterine adhesions; LAMP1: lysosomal associated membrane protein 1; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK: mitogen-activated protein kinase; MTOR: mechanistic target of rapamycin kinase; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; T3: triiodothyronine; T4: tetraiodothyronine; TFEB: transcription factor EB; PBS: phosphate-buffered saline; TEM: transmission electron microscopy; TGFB/TGFβ: transforming growth factor beta.

Keywords:  Autophagy; epithelial-mesenchymal transition; intrauterine adhesions; iodothyronine deiodinase 2; thyroid hormone

DOI:  https://doi.org/10.1080/15548627.2022.2038994
Am J Physiol Cell Physiol. 2022 Feb 23.

PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2.

Andrew L Hellewell, Kate J Heesom, Mark A Jepson, Josephine C Adams.

  The matricellular glycoprotein thrombospondin1 (TSP1) has complex roles in the extracellular matrix and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulphide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalise in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3-/- mouse embryo fibroblasts (Pdia3-/-MEF), led to alterations in the composition of cell-derived ECM, involving changed abundance of fibronectin and TSP1, and was correlated with reduced cell spreading, altered organisation of F-actin and reduced focal adhesions. These cellular phenotypes of Pdia3-/-MEF were normalised by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEF. Rescue depended on PDIA3 activity in WT-MEF, and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEF and Pdia3-/- MEF identified multiple ECM and growth factor proteins to be down-regulated in the CM of Pdia3-/- MEF. Of these, CCN2 was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3-/- MEF and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, pro-adhesive microenvironment, with implications for PDIA3 as a translational target.

Keywords:  Cell Adhesion; Extracellular Matrix; Interactome; Secretome; Thrombospondin-1

DOI:  https://doi.org/10.1152/ajpcell.00258.2021
Nat Commun. 2022 Feb 25. 13(1): 1069

Inhibition of SRP-dependent protein secretion by the bacterial alarmone (p)ppGpp.

Laura Czech, Christopher-Nils Mais, Hanna Kratzat, Pinku Sarmah, Pietro Giammarinaro, Sven-Andreas Freibert, Hanna Folke Esser, Joanna Musial, Otto Berninghausen, Wieland Steinchen, Roland Beckmann, Hans-Georg Koch, Gert Bange.

The stringent response enables bacteria to respond to nutrient limitation and other stress conditions through production of the nucleotide-based second messengers ppGpp and pppGpp, collectively known as (p)ppGpp. Here, we report that (p)ppGpp inhibits the signal recognition particle (SRP)-dependent protein targeting pathway, which is essential for membrane protein biogenesis and protein secretion. More specifically, (p)ppGpp binds to the SRP GTPases Ffh and FtsY, and inhibits the formation of the SRP receptor-targeting complex, which is central for the coordinated binding of the translating ribosome to the SecYEG translocon. Cryo-EM analysis of SRP bound to translating ribosomes suggests that (p)ppGpp may induce a distinct conformational stabilization of the NG domain of Ffh and FtsY in Bacillus subtilis but not in E. coli.

DOI: https://doi.org/10.1038/s41467-022-28675-0
Autophagy. 2022 Feb 23. 1-17

Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E).

Kirstan A Vessey, Andrew I Jobling, Mai X Tran, Anna Y Wang, Ursula Greferath, Erica L Fletcher.

  Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoetm1UncJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.Abbreviations:AMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.

Keywords:  B6.129P2-Apoetm1UncJ; bruch’s membrane; metformin; retina; retinal pigment epithelium; trehalose

DOI:  https://doi.org/10.1080/15548627.2022.2034131
Semin Cell Dev Biol. 2022 Feb 17. pii: S1084-9521(22)00046-5. [Epub ahead of print]

Recent advances in the pharmacological targeting of ubiquitin-regulating enzymes in cancer.

Ana Maria Montagut, Marc Armengol, Gema Gorjón de Pablo, Roger Estrada-Tejedor, José Ignacio Borrell, Gaël Roué.

  As a post-translational modification that has pivotal roles in protein degradation, ubiquitination ensures that intracellular proteins act in a precise spatial and temporal manner to regulate diversified cellular processes. Perturbation of the ubiquitin system contributes directly to the onset and progression of a wide variety of diseases, including various subtypes of cancer. This highly regulated system has been for years an active research area for drug discovery that is exemplified by several approved drugs. In this review, we will provide an update of the main breakthrough scientific discoveries that have been leading the clinical development of ubiquitin-targeting therapies in the last decade, with a special focus on E1 and E3 modulators. We will further discuss the unique challenges of identifying new potential therapeutic targets within this ubiquitous and highly complex machinery, based on available crystallographic structures, and explore chemical approaches by which these challenges might be met.

Keywords:  Cancer; Clinical trial; Deubiquitinase; Preclinical screening; Structure-based drug design; Ubiquitin ligase

DOI:  https://doi.org/10.1016/j.semcdb.2022.02.007
Clin Cancer Res. 2022 Feb 11. pii: clincanres.0344.2021. [Epub ahead of print]

Targeting the ubiquitin-proteasome system using the UBA1 inhibitor TAK-243 is a potential therapeutic strategy for small cell lung cancer.

Safa Majeed, Mansi K Aparnathi, Kevin C J Nixon, Vidhyasagar Venkatasubramanian, Fariha Rahman, Lifang Song, Jessica Weiss, Ranya Barayan, Vijithan Sugumar, Samir H Barghout, Joel D Pearson, Rod Bremner, Aaron D Schimmer, Ming S Tsao, Geoffrey Liu, Benjamin H Lok.

PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with an overall five-year survival rate of <10%. Treatment for SCLC with cisplatin/etoposide chemotherapy (C/E) +/- radiotherapy (RT) has changed modestly over several decades. The ubiquitin-proteasome system is an underexplored therapeutic target for SCLC. We preclinically evaluated TAK-243, a first-in-class small molecule E1 inhibitor against UBA1.
MATERIALS AND METHODS: We assessed TAK-243 in 26 SCLC cell-lines as monotherapy and combined with C/E, the PARP-inhibitor, olaparib, and with radiation (RT) using cell viability assays. We interrogated TAK-243 response with gene expression to identify candidate biomarkers. We evaluated TAK-243 alone and in combination with olaparib or RT with SCLC patient-derived xenografts (PDX).
RESULTS: Most SCLC cell-lines were sensitive to TAK-243 monotherapy (EC50 median 15.8nM, range 10.2nM - 367.3nM). TAK-243 sensitivity was associated with gene-sets involving the cell cycle, DNA and chromatin organization, and DNA damage repair, while resistance associated with cellular respiration, translation, and neurodevelopment. These associations were also observed in SCLC PDXs. TAK-243 synergized with C/E and olaparib <em>in vitro</em> across sensitive and resistant SCLC cell-lines. Considerable TAK-243-olaparib synergy was observed in an SCLC PDX resistant to both drugs individually. TAK-243 radiosensitization was also observed in an SCLC PDX.
CONCLUSIONS: TAK-243 displays efficacy in SCLC preclinical models. Enrichment of gene-sets is associated with TAK-243 sensitivity and resistance. TAK-243 exhibits synergy when combined with genotoxic therapies in cell-lines and PDXs. TAK-243 is a potential therapeutic strategy to improve SCLC patient outcomes, both as a single agent and in combination with existing therapies.

DOI: https://doi.org/10.1158/1078-0432.CCR-21-0344
Nat Chem Biol. 2022 Feb 24.

Deubiquitinase-targeting chimeras for targeted protein stabilization.

Nathaniel J Henning, Lydia Boike, Jessica N Spradlin, Carl C Ward, Gang Liu, Erika Zhang, Bridget P Belcher, Scott M Brittain, Matthew J Hesse, Dustin Dovala, Lynn M McGregor, Rachel Valdez Misiolek, Lindsey W Plasschaert, David J Rowlands, Feng Wang, Andreas O Frank, Daniel Fuller, Abigail R Estes, Katelyn L Randal, Anoohya Panidapu, Jeffrey M McKenna, John A Tallarico, Markus Schirle, Daniel K Nomura.

Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.

DOI: https://doi.org/10.1038/s41589-022-00971-2
EMBO J. 2022 Feb 25. e108272

Reduced ER-mitochondria connectivity promotes neuroblastoma multidrug resistance.

Jorida Çoku, David M Booth, Jan Skoda, Madison C Pedrotty, Jennifer Vogel, Kangning Liu, Annette Vu, Erica L Carpenter, Jamie C Ye, Michelle A Chen, Peter Dunbar, Elizabeth Scadden, Taekyung D Yun, Eiko Nakamaru-Ogiso, Estela Area-Gomez, Yimei Li, Kelly C Goldsmith, C Patrick Reynolds, Gyorgy Hajnoczky, Michael D Hogarty.

  Most cancer deaths result from progression of therapy resistant disease, yet our understanding of this phenotype is limited. Cancer therapies generate stress signals that act upon mitochondria to initiate apoptosis. Mitochondria isolated from neuroblastoma cells were exposed to tBid or Bim, death effectors activated by therapeutic stress. Multidrug-resistant tumor cells obtained from children at relapse had markedly attenuated Bak and Bax oligomerization and cytochrome c release (surrogates for apoptotic commitment) in comparison with patient-matched tumor cells obtained at diagnosis. Electron microscopy identified reduced ER-mitochondria-associated membranes (MAMs; ER-mitochondria contacts, ERMCs) in therapy-resistant cells, and genetically or biochemically reducing MAMs in therapy-sensitive tumors phenocopied resistance. MAMs serve as platforms to transfer Ca2+ and bioactive lipids to mitochondria. Reduced Ca2+ transfer was found in some but not all resistant cells, and inhibiting transfer did not attenuate apoptotic signaling. In contrast, reduced ceramide synthesis and transfer was common to resistant cells and its inhibition induced stress resistance. We identify ER-mitochondria-associated membranes as physiologic regulators of apoptosis via ceramide transfer and uncover a previously unrecognized mechanism for cancer multidrug resistance.

Keywords:  ceramides; inter-organelle contacts; mitochondria-associated membranes; multidrug resistance; sphingolipids

DOI:  https://doi.org/10.15252/embj.2021108272
Autophagy. 2022 Feb 19. 1-2

A dominant-negative regulatory mechanism of SQSTM1 droplets-based autophagy.

Evelina Valionyte, Elizabeth R Barrow, Chris R Baxter, Shouqing Luo.

  SQSTM1/p62 is an autophagy receptor, forming droplets to sequester intracellular polyubiquitinated cargo and mediate its delivery for autophagic clearance. SQSTM1 droplets can function as platforms to allow the formation of autophagosomes at their surfaces. It would be interesting to understand how SQSTM1-droplet formation is regulated. We have shown that inflammatory toxicity induces SQSTM1 cleavage by CASP6 at a novel cleavage site, D256. The C-terminal cleavage product is unlikely to be functional, because it is hardly detectable, possibly due to its rapid turnover. The SQSTM1 N-terminal cleavage product (SQSTM1-N) exerts a dominant-negative effect on SQSTM1-droplet production, in turn attenuating SQSTM1 droplets-based autophagosome formation. Our study suggests that the CASP6-SQSTM1 axis negatively regulates SQSTM1 droplets-based autophagy under certain stress conditions.

Keywords:  Autophagosomes; CASP6; SQSTM1; autophagy; liquid droplets

DOI:  https://doi.org/10.1080/15548627.2022.2029672
Cell Death Differ. 2022 Feb 25.

Prostate-specific oncogene OTUD6A promotes prostatic tumorigenesis via deubiquitinating and stabilizing c-Myc.

Yunhua Peng, Jing Liu, Zhen Wang, Chunping Cui, Tiantian Zhang, Shuangxi Zhang, Peipei Gao, Zhanwu Hou, Huadong Liu, Jianping Guo, Jinfang Zhang, Yurong Wen, Wenyi Wei, Lingqiang Zhang, Jiankang Liu, Jiangang Long.

MYC drives the tumorigenesis of human cancers, including prostate cancer (PrCa), thus deubiquitinase (DUB) that maintains high level of c-Myc oncoprotein is a rational therapeutic target. Several ubiquitin-specific protease (USP) family members of DUB have been reported to deubiquitinate c-Myc, but none of them is the physiological DUB for c-Myc in PrCa. By screening all the DUBs, here we reveal that OTUD6A is exclusively amplified and overexpressed in PrCa but not in other cancers, eliciting a prostatic-specific oncogenic role through deubiquitinating and stabilizing c-Myc oncoprotein. Moreover, genetic ablation of OTUD6A efficiently represses prostatic tumorigenesis of both human PrCa cells and the Hi-Myc transgenic PrCa mice, via reversing the metabolic remodeling caused by c-Myc overexpression in PrCa. These results indicate that OTUD6A is a physiological DUB for c-Myc in PrCa setting and specifically promotes prostatic tumorigenesis through stabilizing c-Myc oncoprotein, suggesting that OTUD6A could be a unique therapeutic target for Myc-driven PrCa.

DOI: https://doi.org/10.1038/s41418-022-00960-x
Nat Rev Immunol. 2022 Feb 23.

Organellar homeostasis and innate immune sensing.

Cassandra R Harapas, Elina Idiiatullina, Mahmoud Al-Azab, Katja Hrovat-Schaale, Thomas Reygaerts, Annemarie Steiner, Pawat Laohamonthonkul, Sophia Davidson, Chien-Hsiung Yu, Lee Booty, Seth L Masters.

A cell is delimited by numerous borders that define specific organelles. The walls of some organelles are particularly robust, such as in mitochondria or endoplasmic reticulum, but some are more fluid such as in phase-separated stress granules. Either way, all organelles can be damaged at times, leading their contents to leak out into the surrounding environment. Therefore, an elegant way to construct an innate immune defence system is to recognize host molecules that do not normally reside within a particular compartment. Here, we provide several examples where organellar homeostasis is lost, leading to the activation of a specific innate immune sensor; these include NLRP3 activation owing to a disrupted trans-Golgi network, Pyrin activation due to cytoskeletal damage, and cGAS-STING activation following the leakage of nuclear or mitochondrial DNA. Frequently, organelle damage is observed downstream of pathogenic infection but it can also occur in sterile settings as associated with auto-inflammatory disease. Therefore, understanding organellar homeostasis is central to efforts that will identify new innate immune pathways, and therapeutics that balance organellar homeostasis, or target the breakdown pathways that trigger innate immune sensors, could be useful treatments for infection and chronic inflammatory diseases.

DOI: https://doi.org/10.1038/s41577-022-00682-8