bims-proteo Biomed News
on Proteostasis
Issue of 2022–07–03
27 papers selected by
Eric Chevet, INSERM
  1. E2 ubiquitin-conjugating enzymes (UBCs): drivers of ubiquitin signalling in plants.
  2. Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase.
  3. ER-phagy: selective autophagy of the endoplasmic reticulum.
  4. Bro1 family proteins harmonize cargo sorting with vesicle formation.
  5. A heterotypic assembly mechanism regulates CHIP E3 ligase activity.
  6. Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.
  7. The integrated stress response remodels the microtubule organizing center to clear unfolded proteins following proteotoxic stress.
  8. Mechanisms orchestrating the enzymatic activity and cellular functions of deubiquitinases.
  9. Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites.
  10. Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome.
  11. Hsp multichaperone complex buffers pathologically modified Tau.
  12. A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.
  13. P4HB UFMylation regulates mitochondrial function and oxidative stress.
  14. Nuclear protein quality control in yeast: the latest INQuiries.
  15. A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
  16. Functional skewing of TRIM21-SIRT5 interplay dictates IL-1β production in DSS-induced colitis.
  17. Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.
  18. PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.
  19. Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.
  20. DEAD Box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress.
  21. Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.
  22. Autophagy-Related Pathways in Vesicular Unconventional Protein Secretion.
  23. Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1.
  24. LAMP2A mediates the loading of proteins into endosomes and selects exosomal cargo.
  25. Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPN.
  26. A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.
  27. Maintaining soluble protein homeostasis between nuclear and cytoplasmic compartments across mitosis.
  1. Essays Biochem. 2022 Jun 29. pii: EBC20210093. [Epub ahead of print]
    E2 ubiquitin-conjugating enzymes (UBCs): drivers of ubiquitin signalling in plants.
    Carla Brillada, Marco Trujillo.
      Most research in the field of ubiquitination has focused on E3 ubiquitin ligases because they are the specificity determinants of the ubiquitination process. Nevertheless, E2s are responsible for the catalysis during ubiquitin transfer, and are therefore, at the heart of the ubiquitination process. Arabidopsis has 37 ubiquitin E2s with additional ones mediating the attachment of ubiquitin-like proteins (e.g. SUMO, Nedd8 and ATG8). Importantly, E2s largely determine the type of ubiquitin chain built, and therefore, the type of signal that decides over the fate of the modified protein, such as degradation by the proteasome (Lys48-linked ubiquitin chains) or relocalization (Lys63-linked ubiquitin chains). Moreover, new regulatory layers impinging on E2s activity, including post-translational modifications or cofactors, are emerging that highlight the importance of E2s.
    Keywords:  Ubiquitin; plant biology; ubiquitin ligases; ubiquitin signalling; ubiquitin-conjugating enzymes; ubiquitin-like proteins
    DOI:  https://doi.org/10.1042/EBC20210093
  2. Elife. 2022 Jun 30. pii: e77424. [Epub ahead of print]11
    Adaptor linked K63 di-Ubiquitin activates Nedd4/Rsp5 E3 ligase.
    Lu Zhu, Qing Zhang, Ciro D Cordeiro, Sudeep Banjade, Richa Sardana, Yuxin Mao, Scott D Emr.
      Nedd4/Rsp5 family E3 ligases mediate numerous cellular processes, many of which require the E3 ligase to interact with PY-motif containing adaptor proteins. Several Arrestin-Related Trafficking adaptors (ARTs) of Rsp5 were self-ubiquitinated for activation, but the regulation mechanism remains elusive. Remarkably, we demonstrate that Art1, Art4, and Art5 undergo K63 linked di-Ubiquitination by Rsp5. This modification enhances the PM recruitment of Rsp5 by Art1 or Art5 upon substrate induction, required for cargo protein ubiquitination. In agreement with these observations, we find that di-ubiquitin strengthens the interaction between the Pombe orthologs of Rsp5 and Art1, Pub1 and Any1. Further, we discover that the HECT domain exosite protects the K63 linked di-Ubiquitin on the adaptors from cleavage by the deubiquitination enzyme Ubp2. Together, our study uncovers a novel ubiquitination modification implemented by Rsp5 adaptor proteins, underscoring the regulatory mechanism of how adaptor proteins control the recruitment and activity of Rsp5 for the turnover of membrane proteins.
    Keywords:  S. cerevisiae; cell biology
    DOI:  https://doi.org/10.7554/eLife.77424
  3. EMBO Rep. 2022 Jun 27. e55192
    ER-phagy: selective autophagy of the endoplasmic reticulum.
    Keisuke Mochida, Hitoshi Nakatogawa.
      Eukaryotic cells adequately control the mass and functions of organelles in various situations. Autophagy, an intracellular degradation system, largely contributes to this organelle control by degrading the excess or defective portions of organelles. The endoplasmic reticulum (ER) is an organelle with distinct structural domains associated with specific functions. The ER dynamically changes its mass, components, and shape in response to metabolic, developmental, or proteotoxic cues to maintain or regulate its functions. Therefore, elaborate mechanisms are required for proper degradation of the ER. Here, we review our current knowledge on diverse mechanisms underlying selective autophagy of the ER, which enable efficient degradation of specific ER subdomains according to different demands of cells.
    Keywords:  ER-phagy; autophagy; endoplasmic reticulum; intracellular degradation
    DOI:  https://doi.org/10.15252/embr.202255192
  4. Bioessays. 2022 Jun 30. e2100276
    Bro1 family proteins harmonize cargo sorting with vesicle formation.
    Chun-Che Tseng, Robert C Piper, David J Katzmann.
      The Endosomal Sorting Complexes Required for Transport (ESCRTs) drive membrane remodeling in a variety of cellular processes that include the formation of endosomal intralumenal vesicles (ILVs) during multivesicular body (MVB) biogenesis. During MVB sorting, ESCRTs recognize ubiquitin (Ub) attached to membrane protein cargo and execute ILV formation by controlling the activities of ESCRT-III polymers regulated by the AAA-ATPase Vps4. Exactly how these events are coordinated to ensure proper cargo loading into ILVs remains unclear. Here we discuss recent work documenting the ability of Bro1, an ESCRT-associated Ub-binding protein, to coordinate ESCRT-III and Vps4-dependent ILV biogenesis with upstream events such as cargo recognition.
    DOI:  https://doi.org/10.1002/bies.202100276
  5. EMBO J. 2022 Jun 28. e109566
    A heterotypic assembly mechanism regulates CHIP E3 ligase activity.
    Aniruddha Das, Pankaj Thapa, Ulises Santiago, Nilesh Shanmugam, Katarzyna Banasiak, Katarzyna Dąbrowska, Hendrik Nolte, Natalia A Szulc, Rose M Gathungu, Dominik Cysewski, Marcus Krüger, Michał Dadlez, Marcin Nowotny, Carlos J Camacho, Thorsten Hoppe, Wojciech Pokrzywa.
      CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.
    Keywords:   C. elegans ; CHIP/STUB1/CHN-1; UFD-2; metabolism; ubiquitin ligase
    DOI:  https://doi.org/10.15252/embj.2021109566
  6. EMBO Rep. 2022 Jun 28. e55056
    Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.
    Thuy P Dao, Yiran Yang, Maria F Presti, Michael S Cosgrove, Jesse B Hopkins, Weikang Ma, Stewart N Loh, Carlos A Castañeda.
      Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.
    Keywords:  UBQLN2; liquid-liquid phase separation; polyphasic linkage; polyubiquitin; protein quality control
    DOI:  https://doi.org/10.15252/embr.202255056
  7. Elife. 2022 Jun 27. pii: e77780. [Epub ahead of print]11
    The integrated stress response remodels the microtubule organizing center to clear unfolded proteins following proteotoxic stress.
    Brian Hurwitz, Nicola Guzzi, Anita Gola, Vincent F Fiore, Ataman Sendoel, Maria Nikolova, Douglas Barrows, Thomas S Carroll, H Amalia Pasolli, Elaine Fuchs.
      Cells encountering stressful situations activate the integrated stress response (ISR) pathway to limit protein synthesis and redirect translation to better cope. The ISR has also been implicated in cancers, but redundancies in the stress-sensing kinases that trigger the ISR have posed hurdles to dissecting physiological relevance. To overcome this challenge, we targeted the regulatory node of these kinases, namely the S51 phosphorylation site of eukaryotic translation initiation factor eIF2α and genetically replaced eIF2α with eIF2α-S51A in mouse squamous cell carcinoma (SCC) stem cells of skin. While inconsequential under normal growth conditions, the vulnerability of this ISR-null state was unveiled when SCC stem cells experienced proteotoxic stress. Seeking mechanistic insights into the protective roles of the ISR, we combined ribosome profiling and functional approaches to identify and probe the functional importance of translational differences between ISR-competent and ISR-null SCC stem cells when exposed to proteotoxic stress. In doing so, we learned that the ISR redirects translation to centrosomal proteins that orchestrate the microtubule dynamics needed to efficiently concentrate unfolded proteins at the microtubule organizing center so that they can be cleared by the perinuclear degradation machinery. Thus, rather than merely maintaining survival during proteotoxic stress, the ISR also functions in promoting cellular recovery once the stress has subsided. Remarkably, this molecular program is unique to transformed skin stem cells hence exposing a vulnerability in cancer that could be exploited therapeutically.
    Keywords:  cancer biology; cell biology; mouse
    DOI:  https://doi.org/10.7554/eLife.77780
  8. J Biol Chem. 2022 Jun 25. pii: S0021-9258(22)00640-8. [Epub ahead of print] 102198
    Mechanisms orchestrating the enzymatic activity and cellular functions of deubiquitinases.
    Benjamin Estavoyer, Clémence Messmer, Mohamed Echbicheb, Christopher E Rudd, Eric Milot, El Bachir Affar.
      Deubiquitinases (DUBs) are required for the reverse reaction of ubiquitination and act as major regulators of ubiquitin signaling processes. Emerging evidence suggests that these enzymes are regulated at multiple levels in order to ensure proper and timely substrate targeting, and to prevent the adverse consequences of promiscuous deubiquitination. The importance of DUB regulation is highlighted by disease-associated mutations that inhibit or activate DUBs, deregulating their ability to coordinate cellular processes. Here, we describe the diverse mechanisms governing protein stability, enzymatic activity and function of DUBs. In particular, we outline how DUBs are regulated by their protein domains and interacting partners. Intramolecular interactions can promote protein stability of DUBs, influence their subcellular localization and/or modulate their enzymatic activity. Remarkably, these intramolecular interactions can induce self-deubiquitination to counteract DUB ubiquitination by cognate E3 ubiquitin ligases. In addition to intramolecular interactions, DUBs can also oligomerize and interact with a wide variety of cellular proteins, thereby forming obligate or facultative complexes that regulate their enzymatic activity and function. The importance of signaling and post-translational modifications in the integrated control of DUB function will also be discussed. While several DUBs are described with respect to the multiple layers of their regulation, the tumor suppressor BAP1 will be outlined as a model enzyme whose localization, stability, enzymatic activity and substrate recognition are highly orchestrated by interacting partners and post-translational modifications.
    Keywords:  BAP1; DUB activity; Quality control; deubiquitinase; folding; multi-protein complex; post-translational modifications; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2022.102198
  9. Nat Commun. 2022 Jun 28. 13(1): 3702
    Structural basis for mitoguardin-2 mediated lipid transport at ER-mitochondrial membrane contact sites.
    Hyunwoo Kim, Seowhang Lee, Youngsoo Jun, Changwook Lee.
      The endoplasmic reticulum (ER)-mitochondria contact site (ERMCS) is crucial for exchanging biological molecules such as phospholipids and Ca2+ ions between these organelles. Mitoguardin-2 (MIGA2), a mitochondrial outer membrane protein, forms the ERMCS in higher eukaryotic cells. Here, we report the crystal structures of the MIGA2 Lipid Droplet (LD) targeting domain and the ER membrane protein VAPB bound to the phosphorylated FFAT motif of MIGA2. These structures reveal that the MIGA2 LD targeting domain has a large internal hydrophobic pocket that accommodates phospholipids and that two phosphorylations of the FFAT motif are required for tight interaction of MIGA2 with VAPB, which enhances the rate of lipid transport. Further biochemical studies show that MIGA2 transports phospholipids between membranes with a strong preference for binding and trafficking phosphatidylserine (PS). These results provide a structural and molecular basis for understanding how MIGA2 mediates the formation of ERMCS and facilitates lipid trafficking at the ERMCS.
    DOI:  https://doi.org/10.1038/s41467-022-31462-6
  10. Life Sci Alliance. 2022 Nov;pii: e202101248. [Epub ahead of print]5(11):
    Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome.
    Francisco Bustos, Sunil Mathur, Carmen Espejo-Serrano, Rachel Toth, C James Hastie, Satpal Virdee, Greg M Findlay.
      Ubiquitylation enzymes are involved in all aspects of eukaryotic biology and are frequently disrupted in disease. One example is the E3 ubiquitin ligase RNF12/RLIM, which is mutated in the developmental disorder Tønne-Kalscheuer syndrome (TOKAS). RNF12 TOKAS variants largely disrupt catalytic E3 ubiquitin ligase activity, which presents a pressing need to develop approaches to assess the impact of variants on RNF12 activity in patients. Here, we use photocrosslinking activity-based probes (photoABPs) to monitor RNF12 RING E3 ubiquitin ligase activity in normal and pathogenic contexts. We demonstrate that photoABPs undergo UV-induced labelling of RNF12 that is consistent with its RING E3 ligase activity. Furthermore, photoABPs robustly report the impact of RNF12 TOKAS variants on E3 activity, including variants within the RING domain and distal non-RING regulatory elements. Finally, we show that this technology can be rapidly deployed in human pluripotent stem cells. In summary, photoABPs are versatile tools that can directly identify disruptions to RING E3 ubiquitin ligase activity in human disease, thereby providing new insight into pathogenic mechanisms.
    DOI:  https://doi.org/10.26508/lsa.202101248
  11. Nat Commun. 2022 Jun 27. 13(1): 3668
    Hsp multichaperone complex buffers pathologically modified Tau.
    Antonia Moll, Lisa Marie Ramirez, Momchil Ninov, Juliane Schwarz, Henning Urlaub, Markus Zweckstetter.
      Alzheimer's disease is a neurodegenerative disorder in which misfolding and aggregation of pathologically modified Tau is critical for neuronal dysfunction and degeneration. The two central chaperones Hsp70 and Hsp90 coordinate protein homeostasis, but the nature of the interaction of Tau with the Hsp70/Hsp90 machinery has remained enigmatic. Here we show that Tau is a high-affinity substrate of the human Hsp70/Hsp90 machinery. Complex formation involves extensive intermolecular contacts, blocks Tau aggregation and depends on Tau's aggregation-prone repeat region. The Hsp90 co-chaperone p23 directly binds Tau and stabilizes the multichaperone/substrate complex, whereas the E3 ubiquitin-protein ligase CHIP efficiently disassembles the machinery targeting Tau to proteasomal degradation. Because phosphorylated Tau binds the Hsp70/Hsp90 machinery but is not recognized by Hsp90 alone, the data establish the Hsp70/Hsp90 multichaperone complex as a critical regulator of Tau in neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41467-022-31396-z
  12. PLoS Biol. 2022 Jun 30. 20(6): e3001501
    A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins.
    Mengwen Zhang, Jason M Berk, Adrian B Mehrtash, Jean Kanyo, Mark Hochstrasser.
      Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.
    DOI:  https://doi.org/10.1371/journal.pbio.3001501
  13. Free Radic Biol Med. 2022 Jun 23. pii: S0891-5849(22)00464-6. [Epub ahead of print]
    P4HB UFMylation regulates mitochondrial function and oxidative stress.
    Jing Zhu, Xirui Ma, Yu Jing, Guangya Zhang, Dandan Zhang, Ziming Mao, Xiaowen Ma, Huifang Liu, Fengling Chen.
      UFMylation is a ubiquitin-like modification which attaches the ubiquitin-fold modifier 1 to target proteins. To date, only a few UFMylation targets have been identified. In the current study, we demonstrated that P4HB is a new target protein for UFMylation and it can be UFMylated at three lysine residues in the form of mono-UFMylation. P4HB has oxidoreductase, chaperone and isomerase effects. It presents in the endoplasmic reticulum, mitochondria and cytosol. Next, we generated a stable HepG2 cell line, the hepatocellular cells, with defective P4HB UFMylation. Our data showed that P4HB UFMylation defect promotes P4HB protein degradation via the ubiquitin-proteasome pathway. Defective P4HB UFMylation causes mitochondrial function damage, oxidative stress, and endoplasmic reticulum stress in HepG2 cells. These effects are more obvious when treating HepG2 cells with palmitic acid, which is frequently used as one of the cell models of non-alcoholic fatty liver disease (NAFLD). Our results identify UFMylation as a key post-translational modification for the maintenance of P4HB stability and biological functions in HepG2 cells, and point to P4HB UFMylation as a potential direction in the study of NAFLD.
    Keywords:  Mitochondrial function; Oxidative stress; P4HB; UFM1; UFMylation; Ubiquitination
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2022.06.237
  14. J Biol Chem. 2022 Jun 24. pii: S0021-9258(22)00641-X. [Epub ahead of print] 102199
    Nuclear protein quality control in yeast: the latest INQuiries.
    Arun Kumar, Veena Mathew, Peter C Stirling.
      The nucleus is a highly organized organelle with an intricate substructure of chromatin, RNAs and proteins. This environment represents a challenge for maintaining protein quality control, since non-native proteins may interact inappropriately with other macromolecules and thus interfere with their function. Maintaining a healthy nuclear proteome becomes imperative during times of stress, such as upon DNA damage, heat shock, or starvation, when the proteome must be remodeled to effect cell survival. This is accomplished with the help of nuclear-specific chaperones, degradation pathways, and specialized structures known as protein quality control (PQC) sites that sequester proteins to help rapidly remodel the nuclear proteome. In this review, we focus on the current knowledge of PQC sites in Saccharomyces cerevisiae, particularly on a specialized nuclear PQC site called the intranuclear quality control (INQ) site, a poorly understood nuclear inclusion that coordinates dynamic proteome triage decisions in yeast.
    Keywords:  DNA damage; Nucleus; Protein quality control; proteasome
    DOI:  https://doi.org/10.1016/j.jbc.2022.102199
  15. Nature. 2022 Jun 29.
    A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.
    Peiqiang Feng, Xudong Wu, Satchal K Erramilli, Joao A Paulo, Pawel Knejski, Steven P Gygi, Anthony A Kossiakoff, Tom A Rapoport.
      Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.
    DOI:  https://doi.org/10.1038/s41586-022-04903-x
  16. EMBO Rep. 2022 Jun 30. e54391
    Functional skewing of TRIM21-SIRT5 interplay dictates IL-1β production in DSS-induced colitis.
    Pengbo Yao, Taiqi Chen, Peng Jiang, Li Li, Wenjing Du.
      Macrophage polarization determines the production of pro- or anti-inflammatory cytokines in response to various bacterial and virus infections. Here, we report that pro-inflammatory macrophage polarization induced by lipopolysaccharide (LPS) skews the TRIM21-SIRT5 interplay toward TRIM21 activation and SIRT5 degradation, resulting in an enhancement of interleukin (IL)-1β production in vitro and in vivo. Mechanistically, LPS challenge enhances the interaction between TRIM21 and SIRT5 to promote SIRT5 ubiquitination and degradation, while reducing the binding of SIRT5 to HAUSP, a deubiquitinating enzyme that stabilizes SIRT5. In a feedback loop, SIRT5 degradation sustains the acetylation of TRIM21 at Lys351, thereby increasing its E3 ligase activity in LPS-activated macrophages. Thus, we identify a functional balance between TRIM21 and SIRT5 that is tilted toward SIRT5 suppression in response to LPS stimulation, thereby enhancing IL-1β production during inflammation.
    Keywords:  IL-1β; SIRT5; TRIM21; colitis; ubiquitination
    DOI:  https://doi.org/10.15252/embr.202154391
  17. Life Sci Alliance. 2022 Nov;pii: e202101327. [Epub ahead of print]5(11):
    Multi-omics profiling identifies a deregulated FUS-MAP1B axis in ALS/FTD-associated UBQLN2 mutants.
    Laura Strohm, Zehan Hu, Yongwon Suk, Alina Rühmkorf, Erin Sternburg, Vanessa Gattringer, Henrick Riemenschneider, Riccardo Berutti, Elisabeth Graf, Jochen H Weishaupt, Monika S Brill, Angelika B Harbauer, Dorothee Dormann, Jörn Dengjel, Dieter Edbauer, Christian Behrends.
      Ubiquilin-2 (UBQLN2) is a ubiquitin-binding protein that shuttles ubiquitinated proteins to proteasomal and autophagic degradation. UBQLN2 mutations are genetically linked to the neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). However, it remains elusive how UBQLN2 mutations cause ALS/FTD. Here, we systematically examined proteomic and transcriptomic changes in patient-derived lymphoblasts and CRISPR/Cas9-engineered HeLa cells carrying ALS/FTD UBQLN2 mutations. This analysis revealed a strong up-regulation of the microtubule-associated protein 1B (MAP1B) which was also observed in UBQLN2 knockout cells and primary rodent neurons depleted of UBQLN2, suggesting that a UBQLN2 loss-of-function mechanism is responsible for the elevated MAP1B levels. Consistent with MAP1B's role in microtubule binding, we detected an increase in total and acetylated tubulin. Furthermore, we uncovered that UBQLN2 mutations result in decreased phosphorylation of MAP1B and of the ALS/FTD-linked fused in sarcoma (FUS) protein at S439 which is critical for regulating FUS-RNA binding and MAP1B protein abundance. Together, our findings point to a deregulated UBQLN2-FUS-MAP1B axis that may link protein homeostasis, RNA metabolism, and cytoskeleton dynamics, three molecular pathomechanisms of ALS/FTD.
    DOI:  https://doi.org/10.26508/lsa.202101327
  18. PLoS Genet. 2022 Jun;18(6): e1010264
    PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.
    Yukako Oe, Keita Kakuda, Shin-Ichiro Yoshimura, Naohiro Hara, Junya Hasegawa, Seigo Terawaki, Yasuyoshi Kimura, Kensuke Ikenaka, Shiro Suetsugu, Hideki Mochizuki, Tamotsu Yoshimori, Shuhei Nakamura.
      Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.
    DOI:  https://doi.org/10.1371/journal.pgen.1010264
  19. ACS Infect Dis. 2022 Jun 29.
    Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.
    Marie O Pohl, Laura Martin-Sancho, Ranjala Ratnayake, Kris M White, Laura Riva, Qi-Yin Chen, Gauthier Lieber, Idoia Busnadiego, Xin Yin, Samuel Lin, Yuan Pu, Lars Pache, Romel Rosales, Marion Déjosez, Yiren Qin, Paul D De Jesus, Anne Beall, Sunnie Yoh, Benjamin G Hale, Thomas P Zwaka, Naoko Matsunaga, Adolfo García-Sastre, Silke Stertz, Sumit K Chanda, Hendrik Luesch.
      There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.
    Keywords:  COVID-19; Sec61; broad-spectrum antivirals; double-membrane vesicles; host-directed therapeutics
    DOI:  https://doi.org/10.1021/acsinfecdis.2c00008
  20. J Biol Chem. 2022 Jun 22. pii: S0021-9258(22)00622-6. [Epub ahead of print] 102180
    DEAD Box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress.
    Lei Li, Mansi Garg, Yixiong Wang, Weiwei Wang, Roseline Godbout.
      The Integrated Stress Response (ISR) is a network of highly-orchestrated pathways activated when cells are exposed to various types of environmental stressors. While global repression of translation is a well-recognized hallmark of the ISR, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD Box 1 (DDX1) protein accumulates at DNA double-strand breaks (DSBs) during genotoxic stress and promotes DSB repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing (RIP-Seq), we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs is increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.
    Keywords:  DEAD Box 1; RNA binding protein; RNA helicase; RNA stability; RNA-protein interaction; oxidative stress; stress granules
    DOI:  https://doi.org/10.1016/j.jbc.2022.102180
  21. Plant Cell. 2022 Jun 29. pii: koac185. [Epub ahead of print]
    Defects in autophagy lead to selective in vivo changes in turnover of cytosolic and organelle proteins in Arabidopsis.
    Lei Li, Chun Pong Lee, Xinxin Ding, Yu Qin, Akila Wijerathna-Yapa, Martyna Broda, Marisa S Otegui, A Harvey Millar.
      Identification of autophagic protein cargo in plants in autophagy related genes (ATG) mutants is complicated by changes in protein synthesis and protein degradation. To detect autophagic cargo, we measured protein degradation rate in shoots and roots of Arabidopsis (Arabidopsis thaliana) atg5 and atg11 mutants. These data show that less than a quarter of proteins changing in abundance are probable cargo and revealed roles of ATG11 and ATG5 in degradation of specific glycolytic enzymes and of other cytosol, chloroplast and ER-resident proteins, and a specialized role for ATG11 in degradation of proteins from mitochondria and chloroplasts. Protein localization in transformed protoplasts and degradation assays in the presence of inhibitors confirm a role for autophagy in degrading glycolytic enzymes. Autophagy induction by Pi limitation changed metabolic profiles and the protein synthesis and degradation rates of atg5 and atg11 plants. A general decrease in the abundance of amino acids and increase in secondary metabolites in autophagy mutants was consistent with altered catabolism and changes in energy conversion caused by reduced degradation rate of specific proteins. Combining measures of changes in protein abundance and degradation rates, we also identify ATG11 and ATG5 associated protein cargo of low Pi induced autophagy in chloroplasts and ER-resident proteins involved in secondary metabolism.
    Keywords:  autophagy; chloroplast; mitochondrion; phosphate starvation; protein degradation
    DOI:  https://doi.org/10.1093/plcell/koac185
  22. Front Cell Dev Biol. 2022 ;10 892450
    Autophagy-Related Pathways in Vesicular Unconventional Protein Secretion.
    Shin Hye Noh, Ye Jin Kim, Min Goo Lee.
      Cellular proteins directed to the plasma membrane or released into the extracellular space can undergo a number of different pathways. Whereas the molecular mechanisms that underlie conventional ER-to-Golgi trafficking are well established, those associated with the unconventional protein secretion (UPS) pathways remain largely elusive. A pathway with an emerging role in UPS is autophagy. Although originally known as a degradative process for maintaining intracellular homeostasis, recent studies suggest that autophagy has diverse biological roles besides its disposal function and that it is mechanistically involved in the UPS of various secretory cargos including both leaderless soluble and Golgi-bypassing transmembrane proteins. Here, we summarize current knowledge of the autophagy-related UPS pathways, describing and comparing diverse features in the autophagy-related UPS cargos and autophagy machineries utilized in UPS. Additionally, we also suggest potential directions that further research in this field can take.
    Keywords:  GRASP; autophagy; golgi bypass; multi-vesicular body; unconventional protein secretion (UPS)
    DOI:  https://doi.org/10.3389/fcell.2022.892450
  23. ACS Cent Sci. 2022 Jun 22. 8(6): 756-762
    Facile Preparation of UFMylation Activity-Based Probes by Chemoselective Installation of Electrophiles at the C-Terminus of Recombinant UFM1.
    Kateryna A Tolmachova, Jakob Farnung, Jin Rui Liang, Jacob E Corn, Jeffrey W Bode.
      Aberrations in protein modification with ubiquitin-fold modifier (UFM1) are associated with a range of diseases, but the biological function and regulation of this post-translational modification, known as UFMylation, remain enigmatic. To provide activity-based probes for UFMylation, we have developed a new method for the installation of electrophilic warheads at the C-terminus of recombinant UFM1. A C-terminal UFM1 acyl hydrazide was readily produced by selective intein cleavage and chemoselectively acylated by a variety of carboxylic acid anhydrides at pH 3, without detriment to the folded protein or reactions at unprotected amino acid side chains. The resulting UFM1 activity-based probes show a range of tunable reactivity and high selectivity for proteins involved in UFMylation processes; structurally related E1s, E2s, and proteases associated with Ub or other Ubls were unreactive. The UFM1 probes were active both in cell lysates and in living cells. A previously inaccessible α-chloroacetyl probe was remarkably selective for covalent modification of the active-site cysteine of de-UFMylase UFSP2 in cellulo.
    DOI:  https://doi.org/10.1021/acscentsci.2c00203
  24. Autophagy. 2022 Jun 27. 1-3
    LAMP2A mediates the loading of proteins into endosomes and selects exosomal cargo.
    João Vasco Ferreira, Ana da Rosa Soares, Paulo Pereira.
      Exosomes are a subtype of extracellular vesicles (EVs), released by all cell types, that originate from the invagination of the endosomal limiting membrane. These EVs can transport biological information in the form of proteins and RNA and have been the focus of intensive research over the last decade. It is becoming apparent that EVs can have important roles in health and disease. EVs are also promising noninvasive biomarkers of disease (liquid biopsies) and valuable vectors for innovative therapies. However, little is known about the mechanisms that regulate the loading of cytosolic proteins into exosomes. We recently showed that soluble proteins containing amino acid sequences biochemically related to the KFERQ motif are loaded into nascent exosomes at the endosomal limiting membrane, in a process mediated by LAMP2A. Because of the subcellular localization and machinery involved, this mechanism has many similarities with chaperone-mediated autophagy (CMA) and endosomal microautophagy (e-Mi), but also some important differences. In this punctum we will focus on the mechanistic details of exosomal LAMP2A loading of cargo (e-LLoC) as well as on its implications for intercellular and interorgan communication.
    Keywords:  LAMP2A; autophagy; chaperones; eLLoC; endosomes; exosomes; inter-organ communication; intercellular communication; lysosome
    DOI:  https://doi.org/10.1080/15548627.2022.2092315
  25. Blood. 2022 Jun 28. pii: blood.2022015629. [Epub ahead of print]
    Whole-genome CRISPR screening identifies N-glycosylation as a genetic and therapeutic vulnerability in CALR-mutant MPN.
    Jonas Samuel Jutzi, Anna E Marneth, Michele Ciboddo, Angel Guerra-Moreno, María José Jiménez-Santos, Anastasia Kosmidou, James W Dressman, Hongyan Liang, Rebecca Svenja Liselotte Hamel, Patricia Rosemary Lozano, Elisa Rumi, John Doench, Jason R Gotlib, Anandi Krishnan, Shannon Elf, Fatima Al-Shahrour, Ann Mullally.
      Calreticulin (CALR) mutations are frequent, disease-initiating events in myeloproliferative neoplasms (MPN). Although the biological mechanism by which CALR mutations cause MPN has been elucidated, there currently are no clonally selective therapies for CALR-mutant MPN. To identify unique genetic dependencies in CALR-mutant MPN, we performed a whole-genome CRISPR knockout depletion screen in mutant CALR-transformed hematopoietic cells. We found that genes in the N-glycosylation pathway (amongst others) were differentially depleted in mutant CALR-transformed cells as compared with control cells. Using a focused pharmacological in vitro screen targeting unique vulnerabilities uncovered in the CRISPR screen, we found that chemical inhibition of N-glycosylation impaired the growth of mutant CALR-transformed cells, through a reduction in MPL cell surface expression. We treated Calr-mutant knockin mice with the N-glycosylation inhibitor, 2-deoxy-glucose (2-DG), and found a preferential sensitivity of Calr-mutant cells to 2-DG as compared to wild-type cells, and normalization of key MPN disease features. To validate our findings in primary human cells, we performed megakaryocyte colony-forming unit (CFU-MK) assays. We found that N-glycosylation inhibition significantly reduced CFU-MK formation in patient-derived CALR-mutant bone marrow, as compared to bone marrow-derived from healthy donors. In aggregate, our findings advance the development of clonally selective treatments for CALR-mutant MPN.
    DOI:  https://doi.org/10.1182/blood.2022015629
  26. Nat Commun. 2022 Jun 28. 13(1): 3720
    A degradative to secretory autophagy switch mediates mitochondria clearance in the absence of the mATG8-conjugation machinery.
    Hayden Weng Siong Tan, Guang Lu, Han Dong, Yik-Lam Cho, Auginia Natalia, Liming Wang, Charlene Chan, Dennis Kappei, Reshma Taneja, Shuo-Chien Ling, Huilin Shao, Shih-Yin Tsai, Wen-Xing Ding, Han-Ming Shen.
      PINK1-Parkin mediated mitophagy, a selective form of autophagy, represents one of the most important mechanisms in mitochondrial quality control (MQC) via the clearance of damaged mitochondria. Although it is well known that the conjugation of mammalian ATG8s (mATG8s) to phosphatidylethanolamine (PE) is a key step in autophagy, its role in mitophagy remains controversial. In this study, we clarify the role of the mATG8-conjugation system in mitophagy by generating knockouts of the mATG8-conjugation machinery. Unexpectedly, we show that mitochondria could still be cleared in the absence of the mATG8-conjugation system, in a process independent of lysosomal degradation. Instead, mitochondria are cleared via extracellular release through a secretory autophagy pathway, in a process we define as Autophagic Secretion of Mitochondria (ASM). Functionally, increased ASM promotes the activation of the innate immune cGAS-STING pathway in recipient cells. Overall, this study reveals ASM as a mechanism in MQC when the cellular mATG8-conjugation machinery is dysfunctional and highlights the critical role of mATG8 lipidation in suppressing inflammatory responses.
    DOI:  https://doi.org/10.1038/s41467-022-31213-7
  27. Trends Cell Biol. 2022 Jun 28. pii: S0962-8924(22)00141-6. [Epub ahead of print]
    Maintaining soluble protein homeostasis between nuclear and cytoplasmic compartments across mitosis.
    Sabina Y van der Zanden, Marlieke L M Jongsma, Anna C M Neefjes, Ilana Berlin, Jacques Neefjes.
      The nuclear envelope (NE) is central to the architecture of eukaryotic cells, both as a physical barrier separating the nucleus from the cytoplasm and as gatekeeper of selective transport between them. However, in open mitosis, the NE fragments to allow for spindle formation and segregation of chromosomes, resulting in intermixing of nuclear and cytoplasmic soluble fractions. Recent studies have shed new light on the mechanisms driving reinstatement of soluble proteome homeostasis following NE reformation in daughter cells. Here, we provide an overview of how mitotic cells confront this challenge to ensure continuity of basic cellular functions across generations and elaborate on the implications for the proteasome - a macromolecular machine that functions in both cytoplasmic and nuclear compartments.
    Keywords:  chromatin condensation; mitosis; nuclear envelope; proteasome; protein homeostasis
    DOI:  https://doi.org/10.1016/j.tcb.2022.06.002
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